CN101374862B - The method and composition of targeting polyubiquitin - Google Patents

Abstract

Provide anti-polyubiquitin monoclonal antibody and use the method for this antibody.

Description

The method and composition of targeting polyubiquitin

Invention field

The present invention relates to anti-polyubiquitin antibody art, more particularly relate to and also can not distinguish the anti-polyubiquitin antibody of the polyubiquitin with different isopeptide bond (isopeptidelinkage) from single ubiquitin specific binding.

Background

Ubiquitin (ubiquitin) is a kind of small protein matter in many cellular pathways with important regulative.These are the most on record is on the effect of ubiquitin in proteolytic degradation, this ubiquitin and target protein covalently bound, make target protein be the identification of 26S proteasome and destroyed (see Wilkinson, Semin.CellDevel.Biol.11 (3): 141-148 (2000)).The protein kinase of unlike signal pathway regulates also relevant with ubiquitination effect (see Sun and Chen, Curr.Opin.CellBiol.16:119-126 (2004)).Such as, the I κ B phosphorylation through I kappa b kinase makes the ubiquitination effect of I κ B be able to possibility, and is degraded by 26S proteasome subsequently; Due to the inhibitor that I κ B is NF κ B, therefore the degraded of I κ B have activated NF κ B (Ghosh and Karin, Cell109 (Suppl.): S81-S96 (2002); Palombella etc., Cell78:773-785 (1994)).Ubiquitination effect also mediated dna reparation (see Sun and Chen, Curr.Opin.CellBiol.16:119-126 (2004)).After DNA damage, (PCNA) of proliferating cell nuclear antigen though single ubiquitination effect have activated any DNA damage can resistant to damage polysaccharase (Stelter and Ulrich, the Nature425:188-191 (2003) of synthetic DNA.Other wherein the known physiological process relating to ubiquitination effect comprise cell fission, Growth of Cells, cell movement and apoptosis/necrocytosis (Johnson, Nat.CellBiol.4:E295-E298 (2002); Pickart, Mol.Cell.8:499-504 (2001)).

Ubiquitin (a kind of 76 amino acid whose albumen) is a kind of three step enzymic process (Pickart, Annu.Rev.Biochem.70:503-533 (2001)) with the covalently bound of target protein.First, in ATP-dependent response, ubiquitin activating enzyme E1 forms ubiquitin-E1 thioesters.In second step, ubiquitin by ubiquitin-E1 thioesters be transferred to ubiquitin conjugating enzyme protein (E2) family member.In the third step, under the help of ubiquitin protein ligase (E3), on ubiquitin C-terminal and target protein lysine residue epsilon-amino between form isopeptide bond.Be called the enzyme of ubiquitin protease from target protein by ubiquitin part removing (Guterman and Glickman, Curr.Prot.Pep.Sci.5:201-210 (2004)).The outstanding role of ubiquitin is as important Molecular regulator, human genome contains and manyly relates to ubiquitination effect or remove the different albumen of ubiquitination effect: identified up to now at least 40 kinds of different E2,500 kinds different E3 and 80 kind different remove ubiquitin protease (Wong etc., Drug.Discov.Today8:746-754 (2003)).

Ubiquitin contains 7 lysine residues (Lys6, Lys22, Lys27, Lys33, Lys29, Lys48 and Lys63), therefore ubiquitin self can be used as target protein for ubiquitination effect (Peng etc., Nat.Biotechnol.21:921-926 (2003); Pickart and Fushman, Curr.Opin.Chem.Biol.8:610-616 (2004)).The molecule produced after the ubiquitination effect of ubiquitin albumen is called polyubiquitin molecule, and can comprise two or more ubiquitin parts.In theory, the ubiquitination effect of ubiquitin can there are (Peng etc. in any one in 7 lysine residues, Nat.Biotechnol.21:921-926 (2003)), thus existence has different types of polyubiquitin that isopeptide bond is bonded to different lysine residue in ubiquitin.The single polyubiquitin molecule likely had more than two ubiquitin parts can have more than a kind of Methionin key.There are some researches show that E2 enzyme affects type (Tenno etc., the GenestoCells9:865-875 (2004) of the Methionin key formed between a ubiquitin molecule and another ubiquitin molecule; Deng etc. (2000); Hofmann and Pickart (2001)).Polyubiquitin and ubiquitin can exist as free molecule and the form covalently bound with target protein.

As ubiquitin, find to relate to polyubiquitin in many cell processes, these processes comprise intracellular transport, endocytosis, genetic expression/silence, proteolysis, kinase activator effect, translation and DNA and repair (Hoege etc., Nature419:135-141 (2002); Spence etc., Mol.Cell.Biol.15:1265-1273 (1995); Hofmann and Pickart, Cell96:645-653 (1999).But compare with single ubiquitination effect with single ubiquitin in identical approach, polyubiquitin and polyubiquitin turn into having significantly different physiological roles.Such as, when after DNA damage, single ubiquitination effect of PCNA causes fallibility archaeal dna polymerase to activate, turn into being then observed activation (Stelter and Ulrich, the Nature425:188-191 (2003) that can cause Error-prone DNA repair at the polyubiquitin of the PCNA with single ubiquitination effect identical residue place; Hoege etc., Nature419:135-141 (2002); Spence etc., Mol.Cell.Biol.15:1265-1273 (1995); And Hofmann and Pickart, Cell96:645-653 (1999)).

The polyubiquitin even with different Methionin key all shows and has different physiological roles.The two kinds of polyubiquitins obtaining research are at most polyubiquitins be connected with Lys63 that Lys48 connects, the polyubiquitin structural research of these two kinds of polyubiquitins being proposed to the connection of different Methionin can adopt visibly different conformation, allow, from selected binding partners, there is different interactions (Tenno etc., GenestoCells9:865-875 (2004)) thus.Although the covalent modification of the polyubiquitin connected by Lys48 indicates target protein energy proteolytic degradation usually, but there is some evidences and prove that the polyubiquitin that Lys48 connects also regulates some albumen (Chau etc., Science243:1576-1583 (1989) by non-proteolytic mode; Finley etc., Mol.Cell.Biol.14:5501-5509 (1994); Flick etc., Nat.Cell.Biol.6:634-641 (2004)).In contrast, the polyubiquitin that Lys63 connects is relevant to the intracellular pathway of multiple non-proteolytic, comprise DNA and repair (yeast cell of expressing K 63R-ubiquitin is DNA rectification of defects type), kinase activator effect, intracellular transport and translation (Pickart and Fushman, Curr.Opin.Chem.Biol.8:610-616 (2004); Hicke and Dunn, AnnuRev.CellDev.Biol.19:141-172 (2003); Spece etc., Mol.CellBiol.15:1265-1273 (1995); Ulrich, Eukaryot.Cell1:1-10 (2002); Spence etc., Cell102:67-76 (2000); Seibenhener etc., Mol.Cell.Biol.24 (18): 8055-8068 (2004)).In a specific example, parkin is passed through in the mode not relying on proteasome, the polyubiquitin institute normally ubiquitination that synphilin-1 connects for K63, but the ubiquitination effect institute target that synphilin-1 also can be the polyubiquitin that K48 connects destroys (Lim etc., J.Neurosci.25 (8): 2002-9 (2005)).One turns into relating to and the formation of the Lewy cytorrhyctes (Lewybodyinclusion) of disease-related (Lim etc., J.Neurosci.25 (8): 2002-9 (2005)) the K63-polyubiquitin of the analysis display synphilin-1 suffering from parkinsonian experimenter.

The polyubiquitin that other Methionin connects also do not obtain detailed, study widely, reason is to be difficult between them distinguish.Up to now, research depends on the technology of the cell of the ubiquitin through mutagenesis, the polyubiquitin with specific bonding of enzyme' s catalysis or the such as mass spectroscopy for the polyubiquitin of distinguishing a type and another kind of type expressed wherein one or more Methionins and removed.For the normal physiologic behavior analyzing the polyubiquitin that specific Methionin connects, in those methods above-mentioned, any one is all not suitable for or bothers heavy.Although there are the antibody (Fujimoro etc. being specific to polyubiquitin compared with single ubiquitin, FEBSLett.349:173-180 (1994)), but still there is no the antibody distinguishing the polyubiquitin with different Methionin key up to now.

Not it is shocking, due to their vital role in many cell processes known, therefore ubiquitin and polyubiquitin are also relevant with numerous disease (see Argiles, UbiquitinandDisease, R.G.Landes (1998)).Ubiquitin imbalance (Mitch and Goldberg, NewEngl.J.Med.335:1897-905 (1996) is observed in amyotrophy; Bodine etc., Science294:1704-1708 (2001)).Some heredopathias are associated with abnormal ubiquitin activity, comprise cystic fibrosis (Ward etc., Cell83:121-127 (1995)), Angelman ' s syndrome (Kishino etc., NatureGenet.15:70-73 (1997)) and Liddle syndrome (Staub etc., EMBOJ16:6325-6336 (1997)).In immunity and inflammatory response, ubiquitin also works; Such as, found that extracellular ubiquitin inhibits endotoxic response in TNF α human peripheral blood monocyte as a kind of cytokine, and regulated intracellular toxin low reactivity (Majetschak etc., Blood101:1882-1890 (2003); Ciechanover, EMBOJ17:7151-7160 (1998)).Similarly, find, in human serum, there is ubiquitin and polyubiquitin, in the patients serum with parasitosis and allergic disease, observe these two kinds of molecules there is higher level (Takada etc., ClinicalChem.43:1188-1195 (1997)).

The imbalance of the approach of some ubiquitins mediation also relates to cancer (Spataro etc., Br.J.Cancer77:448-55 (1998); Beckmann etc., Hum.Mutat.25:507-12 (2005)).Such as, sudden change just (Hashizume etc. relevant to mammary cancer in different dimerization ubiquitin protein ligase BRCA1-BARD1, J.Biol.Chem.276:14537-40 (2001)), destroy Myc will by ubiquitin-pathway the sudden change of ability of degrading have activated the carcinogenic potential (Salghetti etc. of c-Myc, EMBOJ.18:717-726 (1999)), and the v-Jun transformed cannot be degraded to the correlative c-Jun of its non-carcinogenic by ubiquitination, cause uncontrolled growth (Ciechanover thus, EMBOJ.17:7151-7160 (1998), Trier etc., Cell78:787-798 (1994)).

Ubiquitin and polyubiquitin (Chung etc., TINS24 (11Suppl.) S7-S14 (2001)) is at large have studied in contextual nervous system disorders.Inclusion, corpusculum and the neurofibrillary tangles accumulated in Huntington Chorea, spinocebellar ataxia, Protein virus encephalopathic, Pick's disease, Lewy corpusculum disease, Parkinson's disease and Alzheimer is positive immunostaining (Alves-Rodrigues etc., TrendsNeurosci.21:516-520 (1998) to single ubiquitin and/or polyubiquitin; Cammarata etc., NeurosciLett.156:96-98 (1993); Kalchman etc., J.Biol.Chem.271:19385-94 (1996); Holmberg etc., HumanMol.Genet.7:913-918 (1998); Yedidia etc., EMBOJ.20:5383-91 (2001); Mori etc., Science235:1641-44 (1987); Leigh etc., ActaNeuropathol. (Berl.) 79:61-72 (1989); With Kuzuhara etc., ActaNeuropathologica75:345-353 (1988)).Although by the Parkinson's disease of some types and ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) gene, the sudden change in ubiquitin protease is gone to be associated (Leroy etc., Nature395:451-452 (1998)), (the Shimura etc. but the Parkinson's disease of other type is but associated with Inactivating mutations in Parkin (a kind of known interact and the E2-dependency ubiquitin protein ligase of ubiquitination synphilin-1 with ubiquitin conjugating enzyme UbcH7), NatureGenet.25:302-305 (2000), Zhang etc., Proc.Natl.Acad.Sci.97:13354-13359 (2000), Lim etc., J.Neurosci.25 (8): 2002-9 (2005)).The sudden change of this two type all causes abnormal proteolysis processing and unsuitable protein aggregation (see McNaught etc., NatureRev.Neurosci.2:589-594 (2001)).It has also been found that UCH-L1 sudden change is isolated (Naze etc., Neurosci.Lett.328:1:1-4 (2002)) with Huntington Chorea.Identify the ubiquitin mutant in Alzheimer human brain, it effectively mixes in polyubiquitin chain, but be just difficult to be removed ubiquitination once formation, the domination causing normal cell protein to be hydrolyzed system of processing potentially suppresses (Lam etc., Proc.Natl.Acad.Sci.97:9902-9906 (2000)).

Obviously not only have the composition and method that can distinguish the polyubiquitin with different Methionin key, and have can target regulate the composition of approach of ubiquitin and polyubiquitin mediation and method should be useful effectively.Above-mentioned composition and method is related in this present invention provided.

Allly to be incorporated herein by reference in full with it at this reference comprising patent application and publication quoted.

Disclosure of the invention content

The invention provides and can combine and/or regulate the bioactive new antibodies relevant with polyubiquitin to polyubiquitin.

In one embodiment, provide the antibody be separated that is a kind of and polyubiquitin specific binding, wherein this antibody not with single ubiquitin specific binding.In one embodiment, provide a kind of antibody be separated of the first polyubiquitin specific binding with comprising the first Methionin key, wherein this antibody not with the second polyubiquitin specific binding comprising the second Methionin key, and wherein the first Methionin key is different from the second Methionin key.In an aspect, the polyubiquitin that the polyubiquitin that the polyubiquitin that the polyubiquitin that the polyubiquitin that antibody is connected with Methionin 6 further polyubiquitin, Methionin 11 connect, Methionin 27 connect, Methionin 29 connect, Methionin 33 connect, Methionin 48 connect or the polyubiquitin specific binding that Methionin 63 connects.

In one embodiment, provide a kind of antibody be separated of the polyubiquitin specific binding connected with a K48, wherein this antibody not from the second polyubiquitin (i.e. non-K48 connect the polyubiquitin) specific binding of Methionin type of attachment comprising different polyubiquitins.In one embodiment, the second polyubiquitin is the polyubiquitin that K63 connects.

In one embodiment, provide a kind of antibody be separated of the polyubiquitin specific binding connected with a K63, wherein this antibody not from the second polyubiquitin (i.e. non-K63 connect the polyubiquitin) specific binding of Methionin type of attachment comprising different polyubiquitins.In one embodiment, the second polyubiquitin is the polyubiquitin that K48 connects.

In one embodiment, provide the antibody be separated of a kind of the first polyubiquitin with comprising the first Methionin key and the second polyubiquitin comprising the second Methionin key all specific binding, wherein the first Methionin key is different from the second Methionin key, wherein this antibody not with single ubiquitin specific binding, and wherein this antibody and the combination of the second polyubiquitin have with this antibody the binding affinity reduced in fact compared with the binding affinity of the first polyubiquitin.

In one embodiment, provide a kind of antibody be separated of the polyubiquitin specific binding connected with Methionin-48, wherein this antibody not with single ubiquitin specific binding.In one embodiment, antibody comprises at least one further and is selected from and is respectively SEQIDNOs:1-25,151-175,265-279,392-459 and 695-704; SEQIDNOs:27-51,177-201,281-295,461-528 and 706-715; SEQIDNOs:53-77,203-227,297-311,530-597 and 717-726; And hypervariable region (HVR) sequence of arbitrary HVR-H1, HVR-H2, HVR-H3 and the HVR-L3 of SEQIDNOs:313-327 and 728-737.In one embodiment, antibody comprises the sequence that at least one is selected from HVR-H1, HVR-H2, HVR-H3 further, and wherein HVR-H1 comprises aminoacid sequence abcdefghij (SEQIDNO:825), and wherein amino acid a is glycine; Amino acid b is phenylalanine; Amino acid c is l-asparagine; Amino acid d is selected from α-amino-isovaleric acid, phenylalanine, leucine and Isoleucine; Amino acid e is selected from Serine and tyrosine; Amino acid f is tyrosine; Amino acid g is selected from Serine and tyrosine; Amino acid h is selected from Serine and tyrosine; Amino acid i is selected from Isoleucine and methionine(Met); Histidine with amino acid j; Wherein HVR-H2 comprises aminoacid sequence klmnopqrstuvwxyza ' (SEQIDNO:826), and wherein amino acid k is Serine; Amino acid l is Isoleucine; Amino acid m is selected from Serine and tyrosine; Amino acid n is selected from proline(Pro) and Serine; Amino acid o is tyrosine; Amino acid p is tyrosine; Amino acid q is selected from Serine and glycine; Amino acid r is selected from Serine and tyrosine; Amino acid s is Threonine, and amino acid t is selected from Serine and tyrosine; Amino acid u is tyrosine; Amino acid v is L-Ala; Amino acid w is aspartic acid; Amino acid x is Serine; Amino acid y is α-amino-isovaleric acid; Amino acid z is Methionin; Glycine with amino acid a '; And wherein HVR-H3 comprises aminoacid sequence b ' c ' d ' e ' f ' g ' h ' i ' j ' k ' l ', wherein amino acid b ' is selected from L-glutamic acid, Serine, glycine and tyrosine; Amino acid c ' is selected from glycine, tyrosine, Serine and l-asparagine; Amino acid d ' is selected from tyrosine, Serine, Methionin, phenylalanine and L-glutamic acid; Amino acid e ' is selected from Serine, tyrosine, glycine and tryptophane; Amino acid f ' is selected from glutamine, tyrosine, Serine and glycine; Amino acid g ' is selected from glycine, Serine, tyrosine, methionine(Met) and L-Ala; Amino acid h ' is selected from glycine, L-Ala, proline(Pro) and Isoleucine; Amino acid i ' is selected from phenylalanine, Isoleucine, methionine(Met), L-Ala and leucine, or does not exist; Amino acid j ' is phenylalanine or does not exist; Amino acid k ' is aspartic acid; Tyrosine with amino acid l '.In one embodiment, antibody comprises HVR-H1, HVR-H2 and HVR-H3 sequence corresponding to those sequences corresponded in Figure 10 A and 10B listed by clone apu01, apu02, apu03, apu04, apu05, apu06, apu07, apu08, apu09, apu10, apu11, apu12, apu13, apu14 or apu15 further.

In one embodiment, antibody comprises the sequence that at least one is selected from HVR-H1, HVR-H2, HVR-H3, and wherein HVR-H1 comprises aminoacid sequence abcdefghij (SEQIDNO:827), and wherein amino acid a is glycine; Amino acid b is phenylalanine; Amino acid c is l-asparagine; Amino acid d is Isoleucine; Amino acid e is selected from Serine and phenylalanine; Amino acid f is tyrosine; Amino acid g is selected from Serine and glycine; Amino acid h is selected from Serine and glycine; Amino acid i is selected from Isoleucine and methionine(Met); Histidine with amino acid j; Wherein HVR-H2 comprises aminoacid sequence klmnopqrstuvwxyza ' (SEQIDNO:828), and wherein amino acid k is Serine; Amino acid l is Isoleucine; Amino acid m is tyrosine; Amino acid n is Serine; Amino acid o is tyrosine; Amino acid p is tyrosine; Amino acid q is Serine; Amino acid r is tyrosine; Amino acid s is Threonine, and amino acid t is Serine; Amino acid u is tyrosine; Amino acid v is L-Ala; Amino acid w is aspartic acid; Amino acid x is Serine; Amino acid y is α-amino-isovaleric acid; Amino acid z ' is Methionin; Glycine with amino acid a '; And wherein HVR-H3 comprises aminoacid sequence b ' c ' d ' e ' f ' g ' h ' i ' j ' k ' (SEQIDNO:829), wherein amino acid b ' is selected from Serine and glycine; Amino acid c ' is tyrosine; Amino acid d ' is Serine; Amino acid e ' is selected from tyrosine and tryptophane; Amino acid f ' is selected from Serine, tyrosine, arginine, phenylalanine and Histidine; Amino acid g ' is selected from L-glutamic acid, Serine, leucine, phenylalanine, methionine(Met), l-asparagine and α-amino-isovaleric acid; Amino acid h ' is selected from L-Ala and glycine; Amino acid i ' is selected from leucine, methionine(Met), phenylalanine and Isoleucine; Amino acid j ' is aspartic acid; Tyrosine with amino acid k '.In one embodiment, antibody comprises HVR-H1, HVR-H2 and HVR-H3 sequence corresponding to those sequences corresponded in Figure 16 A listed by clone apu2.01, apu2.02, apu2.03, apu2.04, apu2.05, apu2.06, apu2.07, apu2.08, apu2.09 or apu2.10 further.

In one embodiment, antibody comprises the HVR-L3 sequence containing aminoacid sequence m ' n ' o ' p ' q ' r ' s ' t ' u ' v ' w ' (SEQIDNO:830) further, and wherein amino acid m ' is glutamine; Amino acid n ' is glutamine; Amino acid o ' is selected from Serine and tyrosine; Amino acid p ' is selected from Serine and tyrosine; Amino acid q ' is selected from Serine and tyrosine; Amino acid r ' is selected from Serine and tyrosine; Amino acid s ' is selected from Serine and tyrosine; Amino acid t ' is selected from leucine, Serine, proline(Pro) and tyrosine; Amino acid u ' is proline(Pro) or does not exist; Amino acid v ' is selected from phenylalanine, Isoleucine, α-amino-isovaleric acid and leucine; Threonine with amino acid w '.In one embodiment, antibody comprises the HVR-L1 sequence of SEQIDNO:79 further, the HVR-L2 sequence of SEQIDNO:80 and the HVR-L3 sequence corresponding to the HVR-L3 sequence corresponded in Figure 10 C listed by clone apu01, apu02, apu03, apu04, apu05, apu06, apu07, apu08, apu09, apu10, apu11, apu12, apu13, apu14 or apu15.In one embodiment, antibody comprises the HVR-L3 sequence containing aminoacid sequence Q-Q-S-S-Y-S-S-L-I-T (SEQIDNO:728) further.In one embodiment, antibody comprises the HVR-L1 sequence of SEQIDNO:79 further, the HVR-L2 sequence of SEQIDNO:80 and the HVR-L3 sequence corresponding to the HVR-L3 sequence corresponded in Figure 16 B listed by clone apu2.01, apu2.02, apu2.03, apu2.04, apu2.05, apu2.06, apu2.07, apu2.08, apu2.09 or apu2.10.

In one embodiment, provide a kind of antibody be separated of the polyubiquitin specific binding connected with Methionin-48, wherein said antibody not with single ubiquitin specific binding, and wherein this antibody comprises the HVR-L3 sequence of the HVR-H1 sequence of SEQIDNO:269, the HVR-H2 sequence of SEQIDNO:285, the HVR-H3 sequence of SEQIDNO:301, the HVR-L1 sequence of SEQIDNO:79, the HVR-L2 sequence of SEQIDNO:80 and SEQIDNO:317.In one embodiment, provide a kind of antibody be separated of the polyubiquitin specific binding connected with Methionin-48, wherein this antibody not with single ubiquitin specific binding, and wherein this antibody comprises the HVR-L3 sequence of the HVR-H1 sequence of SEQIDNO:701, the HVR-H2 sequence of SEQIDNO:712, the HVR-H3 sequence of SEQIDNO:723, the HVR-L1 sequence of SEQIDNO:79, the HVR-L2 sequence of SEQIDNO:80 and SEQIDNO:734.In one embodiment, provide a kind of antibody be separated of the polyubiquitin specific binding connected with Methionin-48, wherein this antibody not with single ubiquitin specific binding, and wherein this antibody comprises the HVR-L3 sequence of the HVR-H1 sequence of SEQIDNO:701, the HVR-H2 sequence of SEQIDNO:712, the HVR-H3 sequence of SEQIDNO:723, the HVR-L1 sequence of SEQIDNO:79, the HVR-L2 sequence of SEQIDNO:80 and SEQIDNO:734.

In one embodiment, provide a kind of antibody be separated of the polyubiquitin specific binding connected with Methionin-63, wherein this antibody not with single ubiquitin specific binding.In one embodiment, antibody comprises at least one further and is selected from and is respectively SEQIDNOs:81-89,229-239,329-336,599-629 and 739-748; SEQIDNOs:91-99,241-251,338-345,631-661 and 750-759; SEQIDNOs:101-109,253-263,347-354,663-693 and 761-770; And hypervariable region (HVR) sequence of arbitrary HVR-H1, HVR-H2, HVR-H3 and the HVR-L3 of SEQIDNOs:356-363 and 772-781.In one embodiment, antibody comprises the sequence that at least one is selected from HVR-H1, HVR-H2, HVR-H3, and wherein HVR-H1 comprises aminoacid sequence abcdefghij (SEQIDNO:831), and wherein amino acid a is glycine; Amino acid b is phenylalanine; Amino acid c is l-asparagine; Amino acid d is selected from α-amino-isovaleric acid, Isoleucine and phenylalanine; Amino acid e is selected from Serine and tyrosine; Amino acid f is selected from Serine and tyrosine; Amino acid g is selected from Serine and tyrosine; Amino acid h is selected from Serine and tyrosine; Amino acid i is selected from Isoleucine and methionine(Met); Histidine with amino acid j; Wherein HVR-H2 comprises aminoacid sequence klmnopqrstuvwxyza ' (SEQIDNO:832), and wherein amino acid k is selected from Serine and tyrosine; Amino acid l is Isoleucine; Amino acid m is selected from Serine and tyrosine; Amino acid n is selected from proline(Pro) and Serine; Amino acid o is selected from Serine and tyrosine; Amino acid p is selected from Serine and tyrosine; Amino acid q is selected from Serine and glycine; Amino acid r is selected from Serine and tyrosine; Amino acid s is Threonine, and amino acid t is selected from Serine and tyrosine; Amino acid u is tyrosine; Amino acid v is L-Ala; Amino acid w is aspartic acid; Amino acid x is Serine; Amino acid y is α-amino-isovaleric acid; Amino acid z is Methionin; Glycine with amino acid a; And wherein HVR-H3 comprises aminoacid sequence b ' c ' d ' e ' f ' g ' h ' i ' j ' k ' l ' m ' n ' o ' p ' q ' r ' s ' t ' u ' v ', wherein amino acid b ' is selected from Serine, L-glutamic acid, glycine and tryptophane; Amino acid c ' is selected from glycine, tyrosine, Isoleucine, glutamine and Serine; Amino acid d ' is selected from tyrosine, methionine(Met), glycine and Isoleucine; Amino acid e ' is selected from tyrosine, arginine, phenylalanine, tryptophane, L-Ala and proline(Pro); Amino acid f ' is selected from tyrosine, tryptophane, Serine and glycine; Amino acid g ' is selected from glutamine, tyrosine, Serine, phenylalanine and α-amino-isovaleric acid; Amino acid h ' is selected from glycine, Threonine, tryptophane, Methionin and proline(Pro); Amino acid i ' is selected from tyrosine, L-Ala, tryptophane, L-glutamic acid, proline(Pro) and Serine; Amino acid j ' is selected from tryptophane, Isoleucine, tyrosine and L-Ala; Amino acid k ' is selected from tryptophane, tyrosine, glycine and aspartic acid, or does not exist; Amino acid l ' is selected from tyrosine, Serine, phenylalanine and tryptophane, or does not exist; Amino acid m ' is selected from tyrosine, aspartic acid and Serine, or does not exist; Amino acid n ' is selected from tyrosine and L-Ala, or does not exist; Amino acid o ' is selected from Threonine, Serine, α-amino-isovaleric acid, glycine and tyrosine, or does not exist; Amino acid p ' is selected from glycine, aspartic acid, Serine, methionine(Met) and tyrosine, or does not exist; Amino acid q ' is selected from tyrosine, L-Ala and glycine, or does not exist; Amino acid r ' is selected from tyrosine, leucine and glycine, or does not exist; Amino acid s ' is glycine or does not exist; Amino acid t ' is selected from methionine(Met) and leucine, or does not exist; Amino acid u ' is aspartic acid; Tyrosine with amino acid v '.In one embodiment, antibody comprises HVR-H1, HVR-H2 and HVR-H3 sequence corresponding to those sequences corresponded in Figure 11 A and 11B listed by clone apu17, apu18, apu19, apu20, apu21, apu22, apu23 and apu24 further.

In one embodiment, antibody comprises the sequence that at least one is selected from HVR-H1, HVR-H2, HVR-H3, and wherein HVR-H1 comprises aminoacid sequence abcdefghij (SEQIDNO:833), and wherein amino acid a is glycine; Amino acid b is phenylalanine; Amino acid c is l-asparagine; Amino acid d is selected from Isoleucine, α-amino-isovaleric acid and leucine; Amino acid e is selected from Serine, Methionin and α-amino-isovaleric acid; Amino acid f is selected from Serine, tryptophane, glycine and Threonine; Amino acid g is selected from Serine, l-asparagine and glycine; Amino acid h is selected from tyrosine, Isoleucine, leucine and phenylalanine; Amino acid i is selected from Isoleucine and methionine(Met); Histidine with amino acid j; Wherein HVR-H2 comprises aminoacid sequence klmnopqrstuvwxyza ' (SEQIDNO:834), and wherein amino acid k is selected from tyrosine, phenylalanine, aspartic acid, Histidine and L-Ala; Amino acid l is Isoleucine; Amino acid m is selected from Serine, L-Ala and glutamine; Amino acid n is proline(Pro); Amino acid o is tyrosine; Amino acid p is selected from leucine, tyrosine and phenylalanine; Amino acid q is selected from Serine and glycine; Amino acid r is selected from Serine, Threonine and tryptophane; Amino acid s is Threonine, and amino acid t is selected from Serine, l-asparagine, Methionin and Isoleucine; Amino acid u is tyrosine; Amino acid v is L-Ala; Amino acid w is aspartic acid; Amino acid x is Serine; Amino acid y is α-amino-isovaleric acid; Amino acid z ' is Methionin; Glycine with amino acid a '; And wherein HVR-H3 comprises aminoacid sequence b ' c ' d ' e ' f ' g ' h ' i ' j ' k ' l ' (SEQIDNO:908), wherein amino acid b ' is L-glutamic acid; Amino acid c ' is tyrosine; Amino acid d ' is tyrosine; Amino acid e ' is arginine; Amino acid f ' is tryptophane; Amino acid g ' is tyrosine; Amino acid h ' is Threonine; Amino acid i ' is L-Ala; Amino acid j ' is Isoleucine; Amino acid k ' is aspartic acid; Tyrosine with amino acid l '.In one embodiment, antibody comprises HVR-H1, HVR-H2 and HVR-H3 sequence corresponding to those sequences corresponded in Figure 17 A listed by clone apu2.11, apu2.12, apu2.13, apu2.14, apu2.15, apu2.16, apu2.17, apu2.18, apu2.19 and apu2.20.

In one embodiment, antibody comprises the sequence that at least one is selected from HVR-H1, HVR-H2, HVR-H3, and wherein HVR-H1 comprises aminoacid sequence abcdefghij (SEQIDNO:835), and wherein amino acid a is glycine; Amino acid b is phenylalanine; Amino acid c is l-asparagine; Amino acid d is selected from Isoleucine, α-amino-isovaleric acid and leucine; Amino acid e is selected from Methionin and methionine(Met); Amino acid f is selected from Threonine, methionine(Met), l-asparagine, arginine and Isoleucine; Amino acid g is selected from glycine, α-amino-isovaleric acid and phenylalanine; Amino acid h is selected from tyrosine, Isoleucine, leucine and phenylalanine; Amino acid i is selected from Isoleucine and methionine(Met); Histidine with amino acid j; Wherein HVR-H2 comprises aminoacid sequence klmnopqrstuvwxyza ' b ' (SEQIDNO:836), and wherein amino acid k is L-Ala; Amino acid l is tyrosine; Amino acid m is Isoleucine; Amino acid n is selected from Serine, Isoleucine and Threonine; Amino acid o is proline(Pro); Amino acid p is tyrosine; Amino acid q is selected from leucine, tyrosine, aspartic acid, Serine and tryptophane; Amino acid r is glycine; Amino acid s is selected from tryptophane, α-amino-isovaleric acid, Serine, l-asparagine, arginine and tyrosine; Amino acid t is Threonine, and amino acid u is selected from arginine, l-asparagine, α-amino-isovaleric acid, Threonine, Serine and Methionin; Amino acid v is tyrosine; Amino acid w is L-Ala; Amino acid x is aspartic acid; Amino acid y is Serine; Amino acid z is α-amino-isovaleric acid; Amino acid a ' is Methionin; Glycine with amino acid b '; And wherein HVR-H3 comprises aminoacid sequence c ' d ' e ' f ' g ' h ' i ' j ' k ' l ' m ' n ' o ' (SEQIDNO:837), wherein amino acid c ' is Serine; Amino acid d ' is arginine; Amino acid e ' is L-glutamic acid; Amino acid f ' is tyrosine; Amino acid g ' is tyrosine; Amino acid h ' is arginine; Amino acid i ' is tryptophane; Amino acid j ' is tyrosine; Amino acid k ' is Threonine; Amino acid l ' is L-Ala; Amino acid m ' is Isoleucine; Amino acid n ' is aspartic acid; Tyrosine with amino acid o '.In one embodiment, antibody comprises HVR-H1, HVR-H2 and HVR-H3 sequence corresponding to those sequences corresponded in Figure 23 A and 23B listed by clone apu3.01, apu3.02, apu3.03, apu3.04, apu3.05, apu3.06, apu3.07, apu3.08, apu3.09, apu3.10 and 3.11.

In one embodiment, antibody comprises the HVR-L3 sequence containing aminoacid sequence w ' x ' y ' z ' ABCDEFG, and wherein amino acid w ' is glutamine; Amino acid x ' is glutamine; Amino acid y ' is selected from Serine and tyrosine; Amino acid z ' is selected from Serine and tyrosine; Amino acid A is selected from Serine and tyrosine; Amino acid B is selected from Serine and tyrosine; Amino acid C is selected from proline(Pro), Serine and leucine; Amino acid D is selected from Serine, proline(Pro) and tyrosine, or does not exist; Amino acid E is selected from leucine and phenylalanine, or does not exist; Amino acid F is selected from phenylalanine, α-amino-isovaleric acid, Threonine and Isoleucine; Arginine, Threonine and phenylalanine is selected from amino acid G.In one embodiment, antibody comprises the HVR-L1 sequence of SEQIDNO:79, the HVR-L2 sequence of SEQIDNO:80 and the HVR-L3 sequence corresponding to the HVR-L3 sequence corresponded in Figure 11 C listed by clone apu17, apu18, apu19, apu20, apu21, apu22, apu23 and apu24.In one embodiment, antibody comprises the HVR-L3 sequence containing aminoacid sequence Q-Q-Y-S-S-Y-S-S-L-F-T (SEQIDNO:772).In one embodiment, antibody comprises the HVR-L1 sequence of SEQIDNO:79, the HVR-L2 sequence of SEQIDNO:80 and the HVR-L3 sequence corresponding to the HVR-L3 sequence corresponded in Figure 17 B listed by clone apu2.11, apu2.12, apu2.13, apu2.14, apu2.15, apu2.16, apu2.17, apu2.18, apu2.19 and apu2.20.In one embodiment, antibody comprises the HVR-L1 sequence of SEQIDNO:79, the HVR-L3 sequence of the HVR-L2 sequence of SEQIDNO:80 and the HVR-L3 sequence corresponding to SEQIDNO:777.

In one embodiment, provide a kind of antibody be separated of the polyubiquitin specific binding connected with Methionin-63, wherein this antibody not with single ubiquitin specific binding, and wherein this antibody comprises the HVR-H1 sequence of SEQIDNO:330, the HVR-H2 sequence of SEQIDNO:339, the HVR-H3 sequence of SEQIDNO:348, the HVR-L1 sequence of SEQIDNO:79, the HVR-L2 sequence of SEQIDNO:80 and the HVR-L3 sequence of SEQIDNO:357.In one embodiment, provide a kind of antibody be separated of the polyubiquitin specific binding connected with Methionin-63, wherein this antibody not with single ubiquitin specific binding, and wherein this antibody comprises the HVR-H1 sequence of SEQIDNO:739, the HVR-H2 sequence of SEQIDNO:750, the HVR-H3 sequence of SEQIDNO:761, the HVR-L1 sequence of SEQIDNO:79, the HVR-L2 sequence of SEQIDNO:80 and the HVR-L3 sequence of SEQIDNO:772.In one embodiment, provide a kind of antibody be separated of the polyubiquitin specific binding connected with Methionin-63, wherein this antibody not with single ubiquitin specific binding, and wherein this antibody comprises the HVR-H1 sequence of SEQIDNO:740, the HVR-H2 sequence of SEQIDNO:751, the HVR-H3 sequence of SEQIDNO:762, the HVR-L1 sequence of SEQIDNO:79, the HVR-L2 sequence of SEQIDNO:80 and the HVR-L3 sequence of SEQIDNO:773.In one embodiment, provide a kind of antibody be separated of the polyubiquitin specific binding connected with Methionin-63, wherein this antibody not with single ubiquitin specific binding, and wherein this antibody comprises the HVR-H1 sequence of SEQIDNO:744, the HVR-H2 sequence of SEQIDNO:755, the HVR-H3 sequence of SEQIDNO:766, the HVR-L1 sequence of SEQIDNO:79, the HVR-L2 sequence of SEQIDNO:80 and the HVR-L3 sequence of SEQIDNO:777.In one embodiment, provide a kind of antibody be separated of the polyubiquitin specific binding connected with Methionin-63, wherein this antibody not with single ubiquitin specific binding, and wherein this antibody comprises the HVR-H1 sequence of SEQIDNO:795, the HVR-H2 sequence of SEQIDNO:807, the HVR-H3 sequence of SEQIDNO:819, the HVR-L1 sequence of SEQIDNO:79, the HVR-L2 sequence of SEQIDNO:80 and the HVR-L3 sequence of SEQIDNO:777.

In an aspect, provide the antibody that is separated of a kind of and arbitrary afore mentioned antibodies in conjunction with antigenic determinant on identical polyubiquitin, wherein this antibody not with single ubiquitin specific binding.In an aspect, provide a kind of and arbitrary afore mentioned antibodies and compete the antibody be separated combined with polyubiquitin, wherein this antibody not with single ubiquitin specific binding.

In an aspect, arbitrary afore mentioned antibodies is combined with polyubiquitin protein-specific.In an aspect, antibody suppresses the degraded of polyubiquitin albumen further.In an aspect, antibody regulates the signal transduction path of at least one polyubiquitin mediation further.In an aspect, antibody suppresses the signal transduction path of at least one polyubiquitin mediation further.In an aspect, antibody stimulates the signal transduction path of at least one polyubiquitin mediation further.

In an aspect, a kind of nucleic acid molecule of antibody of the present invention of encoding is provided.In an aspect, a kind of carrier comprising this nucleic acid is provided.In an aspect, a kind of host cell comprising this carrier is provided.Provide a kind of clone that can produce antibody of the present invention in an aspect.In an aspect, provide a kind of method producing antibody of the present invention, under being included in the condition wherein producing this antibody, cultivate the host cell of the nucleic acid molecule comprising this antibody of coding.In an aspect, provide and a kind ofly include the antibody of the present invention of effective amount and the composition of pharmaceutically acceptable carrier.

In an aspect, provide a kind of method identifying that in sample, polyubiquitin or polyubiquitin albumen exist, comprise the antibody contacts of the present invention by sample and at least one.In an aspect, provide and be a kind ofly used for the treatment of in patient relevant disease or the method for illness of lacking of proper care to polyubiquitin, the method comprises the antibody of the present invention of at least one giving patient effective amounts.In an aspect, patient is mammalian subject.In an aspect, patient is people.In an aspect, disease selected from cancer, disorder of muscle, hereditary conditions, immunity/inflammatory conditions and nervous system disorders that ubiquitin-pathway is relevant.In an aspect, disease selected from cancer (carcinoma), lymphoma (lymphoma), blastoma (blastoma), sarcoma (sarcoma), leukemia (leukemia), muscular dystrophy (musculardystrophy), multiple sclerosis (multiplesclerosis), amyotrophic lateral sclerosis (amyotrophiclateralsclerosis), cystic fibrosis (cysticfibrosis), Angelman ' s syndrome (Angelman ' ssyndrome), Liddle syndrome (Liddlesyndrome), Alzheimer (Alzheimer ' sdisease), Parkinson's disease (Parkinson ' sdisease), Pick's disease (Pick ' sdisease) and Paget's disease (Paget ' sdisease).

In an aspect, provide a kind of method determining to suspect that in the sample containing polyubiquitin or polyubiquitin albumen, polyubiquitin or polyubiquitin albumen exist, comprise the antibody of the present invention that sample is exposed at least one and measure the combination of polyubiquitin or polyubiquitin albumen in this at least one antibody and sample.

In an aspect, provide a kind of method of polyubiquitin albumen and non-poly ubiquitination albumen in sample separation, comprise the antibody contacts of the present invention by sample and at least one.

In an aspect, provide and a kind ofly determine the function of polyubiquitin and/or the method for activity in cell, comprise the antibody contacts of the present invention of cell and at least one and assess the impact of described contact procedure on described cell.

In an aspect, provide and a kind ofly determine the function of polyubiquitin and/or the method for activity in sample, comprise the antibody contacts of the present invention of sample and at least one and assess the impact of described contact procedure on described sample.

In another embodiment, provide a kind of antibody be separated of the polyubiquitin specific binding connected with Methionin-63, the epi-position wherein in the polyubiquitin that connects of this antibodies Methionin-63.In an aspect, described epi-position comprises the residue in the first ubiquitin subunit of polyubiquitin and the second ubiquitin subunit that Methionin-63 connects.State on the other in aspect, described epi-position comprises at least one and is selected from residue in first ubiquitin subunit of Glu-18, Pro-19, Ser-20, Asp-21, Thr-55, Leu-56, Ser-57, Asp-58, Asn-60, Ile-61 and Gln-62.State on the other in aspect, described epi-position comprises at least one and is selected from residue in second ubiquitin subunit of Leu-8, Thr-9, Glu-34, Gly-35, Ile-36, Pro-37, Asp-39, Gln-40, Leu-71, Arg-72, Leu-73, Arg-74 and Gly-75.State on the other in aspect, epi-position comprises at least one and is selected from residue in first ubiquitin subunit of Glu-18, Pro-19, Ser-20, Asp-21, Thr-55, Leu-56, Ser-57, Asp-58, Asn-60, Ile-61 and Gln-62, and at least one is selected from the residue in second ubiquitin subunit of Leu-8, Thr-9, Glu-34, Gly-35, Ile-36, Pro-37, Asp-39, Gln-40, Leu-71, Arg-72, Leu-73, Arg-74 and Gly-75.

In one embodiment, provide a kind of antibody be separated with comprising at least one isopeptide bond and be bonded to the first polyubiquitin specific binding of first lysine residue at the first amino acid position place at ubiquitin molecule, wherein this antibody not with the second polyubiquitin specific binding comprising at least one isopeptide bond and be bonded to second lysine residue at the second amino acid position place at ubiquitin molecule, and wherein the first and second amino acid positions are different.Antibody of the present invention can exist in a variety of forms.Such as, antibody of the present invention can be chimeric antibody, humanized antibody or people's antibody.In one embodiment, antibody of the present invention is people's antibody, and such as it is not the antibody (described in WO96/33735) produced in xenotypic mice (xenomouse).Antibody of the present invention can be total length or its fragment (as comprising the fragment of antigen-binding portion thereof).

In another embodiment, the invention provides a kind of Fab of arbitrary afore mentioned antibodies.

Accompanying drawing is sketched

Fig. 1 shows the primary structure of ubiquitin and the schematic diagram of some polyubiquitin isopeptide bond (polyubiquitinisopeptidelinkage).Fig. 1 a shows the aminoacid sequence (SEQIDNO:377) of people's ubiquitin, and lysine residue is with runic, underlined text display.Fig. 1 b shows the schematic diagram of the key formed between the Methionin-48 of the first ubiquitin molecule (afirstubiquitinmolecule) or the C-terminal glycine residue of Methionin-63 and the second ubiquitin molecule (asecondubiquitinmolecule).

Fig. 2 A-2C shows described in embodiment 1 (A), the heavy chain HVR ring sequence of the anti-polyubiquitin antibody molecule of the polyubiquitin that specific recognition K48 connects.Symbol " 48 " refers to the polyubiquitin that antibody molecule specific recognition K48 connects.Symbol " both " refers to the polyubiquitin be connected with K63 that antibody molecule identification K48 connects.Symbol " all " refers to the polyubiquitin (polyubiquitin) be connected with K63 that antibody molecule identification K48 connects and single ubiquitin (monoubiquitin).Symbol " n.p. " refers to that some is cloned in and indicates that position does not have amino acid.The figure illustrates heavy chain HVR sequence, H1, H2 and H3.Amino acid position is numbered according to following Kabat numbering system.

Fig. 3 A-3B shows described in embodiment 1 (A), the heavy chain HVR ring sequence of the anti-polyubiquitin antibody molecule of the polyubiquitin that specific recognition K63 connects.Symbol " 63 " refers to the polyubiquitin that antibody molecule specific recognition K63 connects.Symbol " both " refers to the polyubiquitin be connected with K48 that antibody molecule identification K63 connects.Symbol " all " refers to the polyubiquitin be connected with K48 that antibody molecule identification K63 connects and single ubiquitin.Symbol " n.p. " refers to that some is cloned in and indicates that position does not have amino acid.The figure illustrates heavy chain HVR sequence, H1, H2 and H3.Amino acid position is numbered according to following Kabat numbering system.

Fig. 4 A and 4B and Fig. 5 depict have following sequence identifier for implement the present invention illustrative acceptor people have framework sequence:

variable region of heavy chain (VH) has framework region (Fig. 4 A and 4B)

The people VH subgroup I removing KabatCDRs has framework region (SEQIDNOs:111,839,858 and 877)

The people VH subgroup I removing the hypervariable region of extension has framework region (SEQIDNOs:112-114,840-842,859-861 and 878-880)

The people VH subgroup II removing KabatCDRs has framework region (SEQIDNOs:115,843,862 and 881)

The people VH subgroup II removing the hypervariable region of extension has framework region (SEQIDNOs:116-118,844-846,863-865 and 882-884)

The people VH subgroup III removing KabatCDRs has framework region (SEQIDNOs:119,847,866 and 885)

The people VH subgroup III removing the hypervariable region of extension has framework region (SEQIDNOs:120-122,848-850,867-869 and 886-888)

Remove the people VH acceptor framework (SEQIDNOs:123,851,870 and 889) of KabatCDRs

Remove the people VH acceptor framework (SEQIDNOs:124-125,852-853,871-872 and 890-891) of the hypervariable region of extension

Remove people VH acceptor 2 framework region (SEQIDNOs:126,854,873 and 892) of KabatCDRs

Remove the people VH acceptor 2 framework region (SEQIDNOs:127-129,855-857,874-876 and 893-895) of the hypervariable region of extension

variable region of light chain (VL) has framework region (Fig. 5)

People VL κ subgroup I has framework region (SEQIDNOs:130,896,900 and 904)

People VL κ subgroup II has framework region (SEQIDNOs:131,897,901 and 905)

People VL κ subgroup III has framework region (SEQIDNOs:132,898,902 and 906)

People VL κ subgroup IV has framework region (SEQIDNOs:133,899,903 and 907)

Fig. 6 depicts the framework sequence of huMAb4D5-8 light chain and heavy chain.The numbering of subscript/runic indicates the amino acid position according to Kabat.

Fig. 7 depict huMAb4D5-8 light chain and heavy chain modified/framework sequence of variation.The numbering of subscript/runic indicates the amino acid position according to Kabat.

Fig. 8 A-8C shows described in embodiment 1A, the heavy chain HVR ring sequence of the anti-polyubiquitin antibody molecule of the polyubiquitin that specific recognition K48 connects.The figure illustrates heavy chain HVR sequence, H1, H2 and H3.Symbol " n.p. " refers to that some is cloned in and indicates that position does not have amino acid.Amino acid position is numbered according to following Kabat numbering system.

Fig. 9 A-9B shows described in embodiment 1A, the heavy chain HVR ring sequence of the anti-polyubiquitin antibody molecule of the polyubiquitin that specific recognition K63 connects.The figure illustrates heavy chain HVR sequence, H1, H2 and H3.Symbol " n.p. " refers to that some is cloned in and indicates that position does not have amino acid.Amino acid position is numbered according to following Kabat numbering system.

Figure 10 A-10C shows described in embodiment 1 (B), the polyubiquitin that specific recognition K48 connects the heavy chain of the anti-polyubiquitin antibody molecule apu01-apu15 identified for the antibody of five Histidines (pentahistidine) for specificity and light chain HVR ring sequence.The figure illustrates heavy chain HVR sequence, H1, H2 and H3, and light chain HVR sequence, L3.Symbol " n.p. " refers to that some is cloned in and indicates that position does not have amino acid.Amino acid position is numbered according to following Kabat numbering system.

Figure 11 A-11C shows described in embodiment 1 (B), the polyubiquitin that specific recognition K63 connects the heavy chain of the anti-polyubiquitin antibody molecule apu17-apu24 identified for the antibody of five Histidines for specificity and light chain HVR ring sequence.The figure illustrates heavy chain HVR sequence, H1, H2 and H3, and light chain HVR sequence, L3.Symbol " n.p. " refers to that some is cloned in and indicates that position does not have amino acid.Amino acid position is numbered according to following Kabat numbering system.

Figure 12 depicts described in embodiment 1 (C), uses BIACORE binding interactions between that anti-polyubiquitin Fabapu09 and K48 of the different concns that analysis and observation arrives connects or that K63 connects polyubiquitin.

Figure 13 A-13B depicts described in embodiment 1 (C), uses BIACORE binding interactions between that anti-polyubiquitin Fabapu18 and K48 of the different concns that analysis and observation arrives connects or that K63 connects polyubiquitin.

Figure 14 A-14F shows as described in Example 2, based on the heavy chain HVR ring sequence of the anti-polyubiquitin antibody molecule of the s-generation of the sequence of the Fabapu05 of the polyubiquitin of specific recognition K48 connection.The figure illustrates heavy chain HVR sequence, H1, H2 and H3.Symbol " n.p. " refers to that some is cloned in and indicates that position does not have amino acid.Amino acid position is numbered according to following Kabat numbering system.Draw shadow words and represent that sequence is identical with the aminoacid sequence of HVR sequence corresponding in Fabapu05.Bold text represents the antibody being proved strong combination in the Phage-ELISA described by embodiment 2 measures.

Figure 15 A-15C shows as described in Example 2, based on the heavy chain HVR ring sequence of the anti-polyubiquitin antibody molecule of the s-generation of the sequence of the Fabapu18 of the polyubiquitin of specific recognition K63 connection.The figure illustrates heavy chain HVR sequence, H1, H2 and H3.Symbol " n.p. " refers to that some is cloned in and indicates that position does not have amino acid.Amino acid position is numbered according to following Kabat numbering system.Draw shadow words and represent that sequence is identical with the aminoacid sequence of HVR sequence corresponding in Fabapu18.Bold text represents the antibody being proved strong combination in the Phage-ELISA described by embodiment 2 measures.

Figure 16 A and 16B shows as described in Example 2, the polyubiquitin that specific recognition K48 connects the aminoacid sequence deriving from the heavy chain hypervariable region of the Fab molecule (apu2.01-apu2.10) through the apu05 of mutagenesis identified for the antibody of five Histidines for specificity.The figure illustrates heavy chain HVR sequence, H1, H2 and H3, and light chain HVR sequence, L3.Symbol " n.p. " refers to that some is cloned in and indicates that position does not have amino acid.Amino acid position is numbered according to following Kabat numbering system.Draw shadow words and represent that sequence is identical with the aminoacid sequence of HVR sequence corresponding in Fabapu05.

Figure 17 A and 17B shows as described in Example 2, the polyubiquitin that specific recognition K63 connects the aminoacid sequence deriving from the heavy chain hypervariable region of the Fab molecule (apu2.11-apu2.20) through the apu18 of mutagenesis identified for the antibody of five Histidines for specificity.The figure illustrates heavy chain HVR sequence, H1, H2 and H3, and light chain HVR sequence, L3.Symbol " n.p. " refers to that some is cloned in and indicates that position does not have amino acid.Amino acid position is numbered according to following Kabat numbering system.Draw shadow words and represent that sequence is identical with the aminoacid sequence of HVR sequence corresponding in Fabapu18.

Figure 18 depicts the result that Phage-ELISA as described in Example 2 measures, and wherein have evaluated the combination of polyubiquitin, the polyubiquitin of K63 connection, single ubiquitin and the bovine serum albumin that each s-generation Fabapu2.01-2.20 and K48 connects.

Figure 19 A and 19B depicts the result of Western blot experiment as described in Example 1.Figure 19 A shows the combination producing the four poly-ubiquitins be connected with immobilized K48 from the Fab of clone apu01-apu15.The polyubiquitin that the Fab that Figure 19 B shows oneself clone apu18-apu24 of generation does not connect with immobilized K63 is combined.

Figure 20 A and 20B depicts the result of the Western blot experiment described in embodiment 2.Figure 20 A shows the combination of the four poly-ubiquitins that apu2.01-apu2.10 is connected with immobilized K48, but the dimerization ubiquitin do not connected with immobilized K63 is combined.Figure 20 B shows the combination of the four poly-ubiquitins that apu2.11-apu2.20 is connected with immobilized K63, but the dimerization ubiquitin do not connected with immobilized K48 is combined.

Figure 21 A and 21B shows as described in Example 3, from the western blotting of the immunoprecipitation experiment to detect RIP ubiquitination active state.Trace in Figure 21 A comprises with apu2.16IgG immunoprecipitation with the sample of the polyubiquitin albumen catching K63 and connect.Trace in Figure 21 B comprises with apu2.07IgG immunoprecipitation with the sample of the polyubiquitin albumen catching K48 and connect.With these two parts of traces of anti-RIP antibody staining.

Figure 22 depicts the result that the Phage-ELISA described in embodiment 4 measures, and wherein have evaluated the combination of polyubiquitin, the polyubiquitin of K63 connection, single ubiquitin and the bovine serum albumin that each third generation clone apu3.01-3.12 and K48 connects.

Figure 23 A and 23B shows as described in Example 4, deriving from through the aminoacid sequence of the heavy chain hypervariable region of the clone (apu3.01-apu3.12) of the apu2.16 of mutagenesis of the polyubiquitin that specific recognition K63 connects.The figure illustrates heavy chain HVR sequence, H1, H2 and H3.Symbol " ND. " represents that sequence is not detected.Amino acid position is numbered according to following Kabat numbering system.Draw shadow words and represent that sequence is identical with the aminoacid sequence of HVR sequence corresponding in apu2.16.

Figure 24 A-24D depicts the result of the Western blot experiment described in embodiment 4.Figure 24 A shows the combination of the poly-ubiquitin of trimerization to seven that apu2.07IgG is connected with immobilized K48, but the poly-ubiquitin of the trimerization to seven do not connected with immobilized K63 or single ubiquitin are combined.Figure 24 B shows the combination of the poly-ubiquitin of trimerization to seven that apu3.07IgG is connected with immobilized K63, but the poly-ubiquitin of the trimerization to seven do not connected with immobilized K48 or single ubiquitin are combined.Figure 24 C shows apu2.07IgG and is combined with four concentration dependents gathering ubiquitins that immobilized K48 connects, but do not connect with immobilized K63 four poly-ubiquitins are combined.Figure 24 D shows apu3.07IgG and is combined with four concentration dependents gathering ubiquitins that immobilized K63 connects, but do not connect with immobilized K48 four poly-ubiquitins are combined.

Figure 25 depicts the result of the Western blot experiment described in embodiment 4.The figure illustrates anti-ubiquitin polyclonal antibody, apu2.07IgG and apu3.07IgG and immobilizedly to use by oneself the combination of the 293T cell lysate of process (+) or untreated (-).

Figure 26 A-26C shows the interaction between polyubiquitin that special fab and the K63 of polyubiquitin that the K63 of the present invention that measured by crystallographic analysis connected connects.Figure 26 A depicts the mixture formed between the dimerization ubiquitin of special fabapu2.16 and the K63 connection of the polyubiquitin of K63 connection.Apu2.16 is shown in the strip-chart bottom this figure, and the dimerization ubiquitin that meanwhile K63 connects then is shown in the ball-like structure at this figure top.Figure 26 B depicts the surface of the dimerization ubiquitin that K63 connects, and those are positioned at fab's within residue lead and marked interested residue.Figure 26 C depicts the surface of apu2.16, and those ubiquitins being positioned at K63 connection are dimeric within residue lead.Marked CDR.

Embodiments of the present invention

General technology

Except as otherwise noted, enforcement of the present invention will adopt molecular biology (comprising recombinant technology), microbiology, cytobiology, biological chemistry and immunologic routine techniques, and these are all in the technical scope of this area.These technology are very full on, such as " MolecularCloning:ALaboratoryManual ", the third edition (Sambrooketal., 2001) in document; " OligonucleotideSynthesis " (M.J.Gait, ed., 1984); " AnimalCellCulture " (R.I.Freshney compiles, 1987); " MethodsinEnzymology " (AcademicPress, Inc.); " CurrentProtocolsinMolecularBiology " (F.M.Ausubeletal. compiles, 1987, andperiodicupdates); " PCR:ThePolymeraseChainReaction ", (Mullisetal. compiles, 1994); PCR2:APracticalApproach (M.J.MacPherson, B.D.HamesandG.R.Taylor compile (1995)); HarlowandLane compiles (1988) Antibodies, ALaboratoryManual; " APracticalGuidetoMolecularCloning " (PerbalBernardV., 1988); And " PhageDisplay:ALaboratoryManual " (Barbasetal., 2001).

Definition

When for this paper, term " ubiquitin (ubiquitin) " (ubiquitin) and " single ubiquitin (monoubiquitin) " are used interchangeably, be defined as the natural people of all kinds with synthesis ubiquitin, substantially similar to 76 amino acid whose protein the 6th, the 22nd, the 27th, the 29th, the 33rd, the 48th and/or the 63rd amino acids place with at least one lysine residue.

When for this paper, term " polyubiquitin (polyubiquitin) " be defined as the natural people of all kinds with synthesis ubiquitin polymeric chain, its fall into ubiquitin difference polymerization mode of connection people with synthesis classification, comprise, but be not limited to, the polyubiquitin that the polyubiquitin that the polyubiquitin that the polyubiquitin that the polyubiquitin that the polyubiquitin that the polyubiquitin that K6-connects, K22-connect, K27-connect, K29-connect, K33-connect, K48-connect is connected with K63-.Polyubiquitin can be any length, comprises at least two ubiquitin modules (moity).Polyubiquitin is different from first as the ubiquitin tandem repeats of single protein expression.

When for this paper, term " K ^*the polyubiquitin of-connection " and " Lys ^*-the polyubiquitin that connects " be used interchangeably, refer to the in the C-end and another ubiquitin module of a ubiquitin module ^*the polyubiquitin molecule of at least one isopeptide bond is comprised between the Methionin of position.Such as, " K63-connect polyubiquitin " is used interchangeably with " polyubiquitin that Lys63-is connected ", comprises isopeptide bond polyubiquitin molecule between the 63rd Methionin that these two terms all refer to another ubiquitin module in the C-end of a ubiquitin module in the molecule and molecule.

When for this paper, state the first Methionin to connect " difference " and be connected in this first Methionin that the second Methionin connection refers between a ubiquitin module with another ubiquitin module this second Methionin that involved lysine residue (e.g., K6, K22, K27, K29, K33, K48 and/or K63) is different between a ubiquitin module with another ubiquitin module and be connected.

When for this paper, in term " ubiquitin-pathway " phalangeal cell or that rebuild in vitro, to comprise ubiquitin and/or polyubiquitin biochemical route.

" separation " antibody refers to identify and the antibody separating from a kind of composition of its natural surroundings and/or reclaim.The contaminative composition of its natural surroundings refers to disturb the material of the research of this antibody, diagnosis or therepic use, can comprise the solute of enzyme, hormone and other oroteins character or non-proteinaceous.In one embodiment, by antibody purification to (1) according to the mensuration of such as Lowry method, antibody weight is more than 95%, in some embodiment, weight is more than 99%, (2) be enough to by using such as spinning cup sequenator to obtain the N-end of at least 15 residues or the degree of internal amino acid sequence, or (3) are according to the SDS-PAGE under reductibility or non-reducing conditions and use such as Coomassie blue or Silver stain, reach homogeneity.Since at least one composition of antibody natural surroundings can not exist, the antibody be so separated comprises the antibody iM situ in reconstitution cell.But the antibody of separation will be prepared by least one purification step usually.

When for this paper, term " anti-ubiquitin antibody " and " anti-single ubiquitin antibody " are used interchangeably, and referring to can the antibody of specific binding ubiquitin molecule.

When for this paper, term " anti-polyubiquitin antibody " refers to can the antibody of specific binding polyubiquitin molecule.

When for this paper, term " anti-K48-connect polyubiquitin antibody " refers to can the antibody of polyubiquitin that connects of specific binding K48-.

When for this paper, term " the polyubiquitin antibody that anti-K63-connects " refers to the antibody of the polyubiquitin that can connect in conjunction with K63-.

Phrase " substantially similar " " (in fact) is identical substantially ", " quite " or " substantially (in fact) suitable " are for representing two numerical value (such as during this paper, one relevant to certain molecule, another to reference to/to compare molecule relevant) between sufficiently high similarity degree, so that those skilled in the art can think with described numerical value (e.g., Kd value, antiviral effect, etc.) measured by biological characteristics background in difference between two numerical value have very little or there is no biology and/or significance,statistical.Difference between described two numerical value is, such as, is less than about 50%, is less than about 40%, is less than about 30%, is less than about 20% and/or be less than about 10%, as with reference to/compare the function of the numerical value of molecule.

Phrase " substantive reduce (substantiallyreduced) " or " substantive different (substantiallydifferent) " are for representing during this paper that two numerical value (usually, one relevant to certain molecule, another to reference to/to compare molecule relevant) between sufficiently high difference degree, so that those skilled in the art can think to have significance,statistical by the difference in the biological characteristics background measured by described numerical value (e.g., Kd value) between two numerical value.Difference between described two numerical value is such as, be greater than about 10%, be greater than about 20%, be greater than about 30%, be greater than about 40%, be greater than about 50%, as with reference to/compare the function of this numerical value of antibody.

" binding affinity " is often referred to the intensity of whole noncovalent interaction summation between the single binding site of molecule (such as antibody) and its binding partners (such as antigen).Except as otherwise noted, when for this paper, " binding affinity " refers to reflect in conjunction with 1: 1 interactional inherent binding affinity between right member's (such as antibody and antigen).The usual available dissociation constant (Kd) of the avidity of molecule X to its mating partner Y is stated.The common method that avidity is known by this area is measured, and comprises described herein.Low-affinity antibody usually conjugated antigen and be tending towards easily dissociating slowly, and high-affinity antibody conjugated antigen and be tending towards the combination keeping the longer time faster usually.The multiple method measuring binding affinity is known in this area, wherein any one object all used in the present invention.Described below is concrete exemplary.

In one embodiment, that the radio-labelled antigen binding assay (RIA) that object antibody and antigen thereof by using Fab pattern described in following assay method carry out is measured according to " Kd " of the present invention or " Kd value ": by under the condition of titration series that there is unlabelled antigen, with Cmin ¹²⁵i labelled antigen balances, and then catches with the flat board of anti-Fab antibody bag quilt the antigen combined and measures the solution binding affinity (Chen, etal., JMolBiol293:865-881 (1999)) of Fab to antigen.In order to determine condition determination, catch with 5 μ g/ml in 50mM sodium carbonate (pH9.6) and spent the night by microtiter plate (Dynex) with anti-Fab antibody (CappelLabs) bag, use 2% in PBS (w/v) bovine serum albumin(BSA) to close 2-5 hour in room temperature (about 23 DEG C) subsequently.In non-adsorbed flat board (Nunc#269620), by 100pM or 26pM [ ¹²⁵i]-antigen mix with the object Fab of serial dilution (such as with Prestaetal., CancerRes.57:4593-4599 (1997) in VEGF antibody, the assessment of Fab-12 is consistent).Then by object Fab incubated overnight; But, the sustainable longer time (such as 65 hours) is incubated to ensure to reach balance.After this, mixture is transferred to seizure plate to carry out incubation at room temperature (such as 1 hour).Then remove solution, and wash plate 8 times with the PBS containing 0.1%Tween-20.After dull and stereotyped drying, add 150 μ l/ hole scintillation solution (MicroScint-20; Packard), then in Topcount gamma counter (Packard) to plate count 10 minutes.The concentration selecting each Fab to provide to be less than or equal to maximum combined 20% is for competitive binding assay method.According to another embodiment, Kd or Kd value uses BIAcore by surperficial plasmon resonance assays ^tM-2000 or BIAcore ^tM-3000 (BIAcore, Inc., Piscataway, NJ) use immobilized antigen CM5 chip to measure about 10 response units (RU) at 25 DEG C.In brief, carboxymethylation dextran biosensor matrix chip (CM5, BIAcoreInc.) is activated according to specification sheets hydrochloric acid N-ethyl-N '-(3-dimethylaminopropyl)-carbodiimide (EDC) of supplier and N-hydroxy-succinamide (NHS).With 10mM sodium acetate pH4.8 by antigen diluent to 5 μ g/ml (about 0.2 μM), be then injected into the coupling protein matter of acquisition about 10 response units (RU) with the flow velocity of 5 μ l/ minutes.After injecting antigen, inject 1M thanomin with closed unreacted group.In order to carry out kinetic measurement, be infused in the Fab (0.78nM to 500nM) of the middle twice serial dilution of PBS (PBST) containing 0.05%Tween-20 at 25 DEG C with the flow velocity of about 25 μ l/ minutes.Simple Lang Gemiaoer (Langmuir) combination model (BIAcoreEvaluationSoftwareversion3.2) is one to one used to be combined and the sensing figure calculations incorporated speed (k that dissociates by matching simultaneously _on) and dissociation rate (k _off).Equilibrium dissociation constant (Kd) is with ratio k _off/ k _oncalculate.See such as Chen, Y., etal., JMolBiol293:865-881 (1999).If according to surperficial plasmon resonance assays above, association rate is more than 10 ⁶m ^-1s ^-1so association rate can use fluorescent quenching technology to measure, namely spectrophotometer (astop-flowequippedspectrophometer) (AvivInstruments) or the middle measurement with stirring cuvette of 8000 serial SLM-Aminco spectrophotometers (ThermoSpectronic) of cut-off device is such as equipped with according to spectrometer, under the condition of antigen that there is increasing concentration, the anti-antigen-antibody of 20nM (Fab form) measured in PBS, pH7.2 (excites=295nm the fluorescent emission intensity of 25 DEG C; Transmitting=340nm, 16nm band is logical) rising or reduction.

According to " association rate " of the present invention (on-rate, rateofassociation, associationrate) or " k _on" also use BIAcore by identical surperficial plasmon resonance technique mentioned above ^tM-2000 or BIAcore ^tM-3000 (BIAcore, Inc., Piscataway, NJ) use immobilized antigen CM5 chip to measure about 10 response units (RU) at 25 DEG C.In brief, carboxymethylation dextran biosensor matrix chip (CM5, BIAcoreInc.) is activated according to specification sheets hydrochloric acid N-ethyl-N '-(3-dimethylaminopropyl)-carbodiimide (EDC) of supplier and N-hydroxy-succinamide (NHS).With 10mM sodium acetate pH4.8 by antigen diluent to 5 μ g/ml (about 0.2 μM), be then injected into the coupling protein matter of acquisition about 10 response units (RU) with the flow velocity of 5 μ l/ minutes.After injecting antigen, inject 1M thanomin with closed unreacted group.In order to carry out kinetic measurement, be infused in the Fab (0.78nM to 500nM) of the middle twice serial dilution of PBS (PBST) containing 0.05%Tween-20 at 25 DEG C with the flow velocity of about 25 μ l/ minutes.Simple Lang Gemiaoer (Langmuir) combination model (BIAcoreEvaluationSoftwareversion3.2) is one to one used to be combined and the sensing figure calculations incorporated speed (k that dissociates by matching simultaneously _on) and dissociation rate (k _off).Equilibrium dissociation constant (Kd) is with ratio k _off/ k _oncalculate.See such as Chen, Y., etal., JMolBiol293:865-881 (1999).But if according to surperficial plasmon resonance assays above, association rate is more than 10 ⁶m ^-1s ^-1so association rate can use fluorescent quenching technology to measure, namely spectrophotometer (AvivInstruments) or the middle measurement with stirring cuvette of 8000 serial SLM-Aminco spectrophotometers (ThermoSpectronic) of cut-off device is such as equipped with according to spectrometer, under the condition of antigen that there is increasing concentration, the anti-antigen-antibody of 20nM (Fab form) measured in PBS, pH7.2 (excites=295nm the fluorescent emission intensity of 25 DEG C; Transmitting=340nm, 16nm band is logical) rising or reduction.

Term " carrier " is for meaning the nucleic acid molecule that can transport other nucleic acid connected during this paper.One class carrier is " plasmid ", refers to the circular double stranded DNA ring that wherein can connect other region of DNA section.Another kind of carrier is phage vector.Another kind of carrier is virus vector, wherein other region of DNA section can be connected in viral genome.Self-replicating (such as there is bacteria carrier and the episomal mammalian vectors of bacterial origin of replication) in the host cell that some carrier can import at it.Other carrier (such as non-add type mammalian vector) can be incorporated in the genome of host cell, thus along with host genome copies together after importing host cell.In addition, some carrier can instruct the genetic expression be operatively connected with it.Examples of such carriers is referred to herein as " recombinant expression vector " (or referred to as " recombinant vectors ").Usually, useful in recombinant DNA technology expression vector is usually plasmid form.In this manual, " plasmid " and " carrier " is used interchangeably, because plasmid is the most common form of carrier.

" polynucleotide " or " nucleic acid " are used interchangeably in this article, refer to the nucleotide polymer of any length, comprise DNA and RNA.Nucleotide can be deoxyribonucleotide, ribonucleotide, through the Nucleotide modified or base and/or its analogue, or by DNA or RNA polymerase or any substrate being mixed polymkeric substance by building-up reactions.Polynucleotide can comprise the Nucleotide through modifying, such as methylated nucleotide and analogue thereof.If any, can carry out before or after assembling polymkeric substance the modification of nucleotide structure.Nucleotide sequence can be interrupted by non-nucleotide component.Polynucleotide can be modified in post synthesis further, such as by with marker coupling.The modification of other type comprises such as " cap ", one or more naturally occurring Nucleotide analogue is substituted, modify between Nucleotide and such as such as there is neutral connection (such as methyl-phosphonate, phosphotriester, phosphoramidate (phosphoamidate), carbamate etc.) and there is electrically charged connection (such as thiophosphatephosphorothioate, phosphorodithioate etc.) modification, containing pendency module (pendantmoiety) such as such as protein (such as nuclease, toxin, antibody, signal peptide, polylysine etc.) modification, there is intercalator (such as acridine, psoralene etc.) modification, containing sequestrant (such as metal, radioactive metal, boron, oxidisability metal etc.) modification, modification containing alkylating agent, there is the modification of modified connection (such as α anomeric nucleic acid (anomericnucleicacid) etc.), and the polynucleotide of unmodified form.In addition; usually any hydroxyl be present in carbohydrate can be replaced with such as phosphonic acids (phosphonate) group, phosphoric acid (phosphate) group; protect with standard protecting group; or activation is connected with other of other Nucleotide with preparation, or solid or semi-solid support can be coupled to.5 ' and 3 ' end OH can phosphorylation or replace with organic cap group module that adds of amine or 1-20 carbon atom.Other hydroxyl also can be derivatized to standard protecting group.The analogue form of the ribose that polynucleotide also generally can be known containing this area or ribodesose carbohydrate, comprise such as 2 '-oxygen-methyl, 2 '-oxygen-allyl group, 2 '-fluoro-or 2 '-nitrine-ribose, carba sugars, α-anomeric sugar, epimerization sugar is pectinose, wood sugar or lyxose, pyranose, furanose, sedoheptulose such as, acyclic analog and dealkalize yl nucleosides analogue such as methylribonucleotide.Available alternative linking group is replaced one or more phosphodiester and is connected.These alternative linking groups include but not limited to following embodiment, wherein phosphoric acid ester P (O) S (" thioester " (thioate)), P (S) S (" dithioester " (dithioate)), (O) NR ₂(" carboxylic acid amide esters " (amidate)), P (O) R, P (O) OR ', CO or CH ₂(" methylal " (formacetal)) substitutes, wherein R or R ' is independent is separately H or substituted or unsubstituted alkyl (1-20 C), optionally containing ether (-O-) connection, aryl, thiazolinyl, cycloalkyl, cycloalkenyl group or aralkyl (araldyl).All connections not in polynucleotide are all necessarily identical.Aforementioned description is applicable to all polynucleotide mentioned in this article, comprises RNA and DNA.

" oligonucleotide ", for referring generally to short polynucleotide during this paper, is generally strand, and be generally synthesis, length generally but be not to be less than about 200 Nucleotide.Term " oligonucleotide " is not mutually exclusive with " polynucleotide ".Above about polynucleotide description equality and be applicable to oligonucleotide completely.

" antibody " (Ab) and " immunoglobulin (Ig) " (Ig) is the glycoprotein with same structure feature.Although antibody shows the binding specificity to specific antigen, immunoglobulin (Ig) comprises antibody and general other both antibody molecule lacking antigen-specific.A rear class polypeptide is such as generated with low-level by lymphsystem and is generated with the level raised by myelomatosis.

Term " antibody " and " immunoglobulin (Ig) " exchange with most broad sense and use, comprise monoclonal antibody (such as total length or intact monoclonal antibodies), polyclonal antibody, unit price, multivalent antibody, multi-specificity antibody (such as bi-specific antibody, as long as they show the biologic activity of expectation), but also some antibody fragment (as specifically described) can be comprised herein.Antibody can be chimeric, people, humanized and/or affinity maturation.

" variable region " or " variable domain " of antibody refers to the amino terminal domain of heavy chain of antibody or light chain.These structural domains are generally the most variable portion of antibody and comprise antigen binding site.

Term " variable " refer to some part in variable domain between antibody sequence difference extensively and for often kind of specific antibodies to the combination of its specific antigen and specific truth.But variability is not uniformly distributed in the whole variable domain of antibody.It concentrates in light chain and heavy chain variable domain three sections being called complementary determining region (CDR) or hypervariable region.Part comparatively conservative in variable domain is called framework region (FR).Each self-contained four FR of variable domain of native heavy and light chain, they take beta-pleated sheet conformation mostly, by forming loop connecting and three CDR forming a beta-pleated sheet structure part in some situation connect.CDR in every bar chain is by FR keeping together closely, and facilitate the formation of the antigen binding site of antibody (see Kabatetal. together with the CDR of another chain, SequencesofProteinsofImmunologicalInterest, 5th edition, NationalInstitutesofHealth, Bethesda, MD. (1991)).Constant domain does not participate in the combination of antibody and antigen directly, but shows multiple effector functions, the participation of antibody in the cytotoxicity of such as antibody dependent cellular.

Produce two identical Fabs with Papain digestion of antibodies, be called " Fab " fragment, there is an antigen binding site separately, and remaining " Fc " fragment, its title reflects the ability that it is easy to crystallization.Pepsin produces a F (ab ') ₂fragment, it has two antigen binding sites and still can crosslinking antigen.

" Fv " is the minimum antibody fragment comprising intact antigen identification and binding site.In two-chain Fv species, this district is made up of tight a, heavy chain variable domain of Non-covalent binding and the dimer of a light-chain variable domain.In single-chain Fv species, a heavy chain variable domain can be connected by flexible peptide linker covalency with a light-chain variable domain, and light chain and heavy chain are combined in " dimer " structure similar with two-chain Fv species.Just in such configuration, three CDR of each variable domain interact and at V _h-V _ldimer interface determines antigen binding site.Six CDR give antibody jointly with antigen-binding specificity.But even single variable domain (or only comprising half Fv of three CDR to antigen-specific) also has the ability of identification and conjugated antigen, just avidity is lower than entire binding site.

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

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

According to the aminoacid sequence of its heavy-chain constant domains, antibody (immunoglobulin (Ig)) can be included into different classes.Immunoglobulin (Ig) has five large classes: IgA, IgD, IgE, IgG and IgM, wherein some can be further divided into subclass (isotype), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.The heavy-chain constant domains corresponding with inhomogeneous immunoglobulin (Ig) is called α, δ, ε, γ and μ.The subunit structure of inhomogeneous immunoglobulin (Ig) and three-dimensional structure are well-known, are generally recorded in such as Abbasetal., CellularandMol.Immunology, the 4th edition (2000).Antibody can be a part for the larger fusion molecule that antibody covalently or non-covalently associates with one or more other oroteins or peptide and formed.

Term " full length antibody " and " complete antibody " are used interchangeably in this article, refer to complete form substantially antibody but not as hereafter the antibody fragment that defines.This term specifically refers to that heavy chain comprises the antibody in Fc district.

" antibody fragment " only comprises a part for complete antibody, and wherein said part retains at least one item usually relevant with it when this part is present in complete antibody, as many as great majority or all functions.In one embodiment, antibody fragment comprises the antigen binding site of complete antibody, so retains the ability of conjugated antigen.In another embodiment, antibody fragment, such as comprise the antibody fragment in Fc district, retain at least one biological function usually relevant with it when being usually present in complete antibody with Fc district, such as FcRn combination, regulation and control antibody half life, ADCC function and complement combine.In one embodiment, antibody fragment is the Half-life in vivo univalent antibody substantially similar to complete antibody.Such as, such antibody fragment can comprise an antigen binding arm and its with can give this fragment and be connected with the Fc sequence of body internal stability.

Term " monoclonal antibody " is for referring to the antibody obtained from the antibody of a group homogeneity substantially during this paper, each antibody namely forming colony is identical, except may with except the possible natural existence variation of indivisible existence.So, modifier " mono-clonal " indicates the feature that antibody is not the mixture of discrete antibody.This type of monoclonal antibody typically comprises the antibody of the peptide sequence comprised in conjunction with target thing, and its thing Binding peptide sequence that hits the more selects the process of single target thing Binding peptide sequence to obtain in peptide sequence by comprising comforming.Such as, chosen process can be comform polyclone such as hybridoma clone, phage clone or recombinant DNA clone set in select Unique clones.Be to be understood that, selected target thing binding sequence can change further, such as in order to improve avidity to target thing, by target thing binding sequence humanization, improve its output in cell culture, reduce its immunogenicity in vivo, create multi-specificity antibody etc., and the antibody comprising the target thing binding sequence after change is also monoclonal antibody of the present invention.Different from the polyclonal antibody preparations typically comprised for the different antibodies of different determinant (epi-position), often kind of monoclonal antibody of monoclonal antibody preparations is for the single determinant on antigen.Outside their specificity, the advantage of monoclonal antibody preparations is that they are not subject to the pollution of other immunoglobulin (Ig) usually.Modifier " mono-clonal " indicate antibody basically homogeneity antibody population obtain feature, should not be construed as require produce antibody by any ad hoc approach.Such as, the monoclonal antibody used according to the present invention is generated by multiple technologies, comprise such as hybridoma (such as Kohleretal., Nature256:495 (1975); Harlowetal., Antibodies:ALaboratoryManual, ColdSpringHarborLaboratoryPress, the 2nd edition, 1988; Hammerlingetal., in: MonoclonalAntibodiesandT-CellHybridomas, 563-681, Elsevier, N.Y., 1981), recombinant DNA method is (see such as U.S. Patent No. 4,816,567), display technique of bacteriophage is (see such as Clacksonetal., Nature352:624-628 (1991); Marksetal., J.Mol.Biol.222:581-597 (1992); Sidhuetal., J.Mol.Biol.338 (2): 299-310 (2004); Leeetal., J.Mol.Biol.340 (5): 1073-1093 (2004); Fellouse, Proc.Natl.Acad.Sci.USA101 (34): 12467-12472 (2004); Leeetal., J.Immunol.Methods284 (1-2): 119-132 (2004)) and for generating the technology of people or human-like antibodies (see such as WO98/24893 in the animal of gene with part or whole human immunoglobulin gene's seat or encoding human immunoglobulin's sequence; WO96/34096; WO96/33735; WO91/10741; Jakobovitsetal., Proc.Natl.Acad.Sci.USA90:2551 (1993); Jakobovitsetal., Nature362:255-258 (1993); Bruggemannetal., YearinImmunol.7:33 (1993); U.S. Patent No. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016; Marksetal., Bio/Technology10:779-783 (1992); Lonbergetal., Nature368:856-859 (1994); Morrison, Nature368:812-813 (1994); Fishwildetal., NatureBiotechnol.14:845-851 (1996); Neuberger, NatureBiotechnol.14:826 (1996); LonbergandHuszar, Intern.Rev.Immunol.13:65-93 (1995)).

Monoclonal antibody clearly comprises in this article " being fitted together to " antibody, a wherein part for heavy chain and/or light chain or homology identical with derived from the corresponding sequence of Special Thing species or genus 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 isotype or subclass, and the fragment of this antibody-like, as long as they show the biologic activity (U.S. Patent No. 4 of expectation, 816,567; Morrisonetal., Proc.Natl.Acad.Sci.USA81:6851-6855 (1984)).

" humanization " form of inhuman (such as mouse) antibody refers to that bottom line comprises the chimeric antibody of the sequence derived from non-human immunoglobulin.In one embodiment, humanized antibody refers to the immunoglobulin (Ig) that some hypervariable region residues in human normal immunoglobulin (receptor antibody) is replaced with the some hypervariable region residues with non-human species's (donor antibody) such as mouse, rat, rabbit or the non-human primate of expecting specificity, avidity and/or ability.In some situation, framework region (FR) residue of human normal immunoglobulin is replaced with corresponding non-human residues.In addition, humanized antibody can be included in the residue do not found in receptor antibody or in donor antibody.Carrying out that these modify is performance in order to improve antibody further.Generally speaking, humanized antibody by comprise at least one, usual two whole following variable domains substantially, wherein all or substantially all height become ring and correspond to the Gao Bianhuan of non-human immunoglobulin, and all or substantially all FR be the FR of human normal immunoglobulin sequence.Humanized antibody optionally also will comprise at least part of constant region for immunoglobulin (Fc), normally the constant region of human normal immunoglobulin.More details are see Jonesetal., Nature321:522-525 (1986); Riechmannetal., Nature332:323-329 (1988); Presta, Curr.Op.Struct.Biol.2:593-596 (1992).Also can see following summary and the reference quoted thereof: VaswaniandHamilton, Ann.Allergy, Asthma & Immunol.1:105-115 (1998); Harris, Biochem.Soc.Transactions23:1035-1038 (1995); HurleandGross, Curr.Op.Biotech.5:428-433 (1994).

Term " hypervariable region ", " HVR " or " HV " to refer in antibody variable domains alterable height in sequence and/or form the region of the ring that structure defines when for this paper.Usually, antibody comprises six hypervariable regions: three in VH (H1, H2, H3), three in VL (L1, L2, L3).Use herein and contain describing of many hypervariable regions.Kabat complementary determining region (CDR) is based on sequence variability, and be the most frequently used (Kabatetal., SequencesofProteinsofImmunologicalInterest, 5thEd.PublicHealthService, NationalInstitutesofHealth, Bethesda, MD. (1991)).Chothia changes the position (ChothiaandLesk, J.Mol.Biol.196:901-917 (1987)) referring to structure ring into.It is compromise that AbM hypervariable region represents between KabatCDR and Chothia structure ring, and obtain the use of AbM antibody modeling software of OxfordMolecular." contact " hypervariable region is based on the analysis to obtainable complex crystal structure.Hereafter to have recorded in these hypervariable regions the residue of each.

Ring KabatAbMChothia contacts

-----------------------------------------------

L1L24-L34L24-L34L26-L32L30-L36

L2L50-L56L50-L56L50-L52L46-L55

L3L89-L97L89-L97L91-L96L89-L96

H1H31-H35BH26-H35BH26-H32H30-H35B

(Kabat numbering)

H1H31-H35H26-H35H26-H32H30-H35

(Chothia numbering)

H2H50-H65H50-H58H53-H55H47-H58

H3H95-H102H95-H102H96-H101H93-H101

Hypervariable region can comprise " hypervariable region of extension " as follows: the 26-35 (H1) in 24-36 or 24-34 (L1) in VL, 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) and VH, 50-65 or 49-65 (H2) and 93-102,94-102 or 95-102 (H3).For each in these definition, variable domain residue is according to Kabat etc., the numbering that sees above.

" framework " or " FR " residue refers to those residues in variable domain except some hypervariable region residues as defined herein.

Term " the variable domain residue according to Kabat is numbered " or " amino acid position number according to Kabat " and variant thereof refer to Kabatetal., SequencesofProteinsofImmunologicalInterest, 5thEd.PublicHealthService, NationalInstitutesofHealth, antibody editor in Bethesda, MD. (1991) is used for the numbering system of heavy chain variable domain or light-chain variable domain.Use this numbering system, actual linear amino acid sequence can comprise less or other amino acid, corresponding to shortening or the insertion of variable domain FR or HVR.Such as, single amino acid after heavy chain variable domain can comprise H2 residue 52 inserts (be residue 52a according to Kabat) and insertion residue after heavy chain FR residue 82 (be such as residue 82a, 82b and 82c etc. according to Kabat).The Kabat residue numbering of given antibody is by contrasting homologous region to determine by antibody sequence and " standard " Kabat numbered sequence.

" scFv " or " scFv " antibody fragment comprises the V of antibody _hand V _lstructural domain, wherein these structural domains are present on a polypeptide chain.Generally speaking, scFv polypeptide is at V _hwith V _lalso comprise peptide linker between structural domain, make scFv can form the desired structure of conjugated antigen.About the summary of scFv see Pluckthun, in: ThePharmacologyofMonoclonalAntibodies, vol.113, RosenburgandMoore compile, Springer-Verlag, NewYork, pp.269-315 (1994).

Term " double antibody (diabody) " refers to the small antibody fragments with two antigen binding sites, and this fragment is at same polypeptide chain (V _h-V _l) in comprise connected heavy chain variable domain (V _h) and light-chain variable domain (V _l).Making by using too short joint can not match between on same chain two structural domains, forcing the complementary domain of these structural domains and another chain to match, thus produce two antigen binding sites.What double antibody was more complete is recorded in such as EP404, and 097; WO93/1161; Hollingeretal., Proc.Natl.Acad.Sci.USA90:6444-6448 (1993).

" people's antibody " refers to have the aminoacid sequence corresponding with the aminoacid sequence of the antibody generated by people and/or use the antibody generated for any technology of raw human antibodies disclosed herein.This definition clear-cut of people's antibody gets rid of the humanized antibody comprising inhuman antigen binding residues.

" affinity maturation " antibody refers to have in one or more HVR of antibody the antibody that a place or many places change, cause this antibody to improve to some extent compared with the parental antibody not having these to change to the avidity of antigen.In one embodiment, the antibody of affinity maturation has the avidity to target antigen of nmole or even picomole magnitude.The antibody of affinity maturation generates by code known in the art.Marksetal., Bio/Technology10:779-783 (1992) describes and reorganizes by VH and VL structural domain the affinity maturation carried out.The random mutagenesis of CDR and/or Framework residues is described: Barbasetal., Proc.Nat.Acad.Sci.USA91:3809-3813 (1994) with Publication about Document; Schieretal., Gene169:147-155 (1995); Yeltonetal., J.Immunol.155:1994-2004 (1995); Jacksonetal., J.Immunol.154 (7): 3310-9 (1995); Hawkinsetal., J.Mol.Biol.226:889-896 (1992).

" barrier (blocking) " antibody or " Antagonism (antagonist) " antibody refer to the antibody of the biologic activity suppressing or reduce the antigen that it combines.Some blocking antibody or antagonistic antibodies are substantive or suppress the biologic activity of antigen completely.

" agonistic antibody (agonistantibody) " is at the antibody at least one functional activity of finger print plan desired polypeptides during this paper.

" illness " refers to any illness that will benefit from the process using antibody of the present invention to carry out.This comprises chronic and acute disease, or comprises those and make Mammals trend towards the disease of the pathological condition of discussed illness.The non-limitative example of illness to be treated comprises cancer, disorder of muscle, ubiquitin-pathway correlated inheritance illness, immunity/inflammatory conditions, neurological disorders and other ubiquitin-pathway associated conditions herein.

Term " cell proliferative disorders " refers to the illness relevant with abnormal cell proliferation to a certain degree with " proliferative disorders ".In one embodiment, cell proliferative disorders refers to cancer.

No matter " tumour ", for referring to the growth of all neoplastic cell and propagation during this paper, is pernicious or optimum, and (pre-cancerous) and cancerous cells and tissue before all cancers.It is not mutually exclusive when term " cancer ", " carcinous ", " cell proliferative disorders ", " proliferative disorders " and " tumour " are mentioned in this article.

Term " cancer " and " carcinous " are pointed to or describe feature in Mammals and be generally the not modulated physiology illness of Growth of Cells/propagation.The example of cancer includes but not limited to cancer, lymphoma (such as He Jiejinshi (Hodgkin) lymphoma and non_hodgkin lymphoma), blastoma, sarcoma and leukemia.The more specifically example of this type of cancer comprises squamous cell carcinoma, small cell lung cancer, nonsmall-cell lung cancer, the gland cancer of lung, the squama cancer of lung, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, carcinoma of the pancreas, glioblastoma (glioblastoma), cervical cancer, ovarian cancer, liver cancer (livercancer), bladder cancer, hepatoma (hepatoma), mammary cancer, colorectal carcinoma, colorectal carcinoma, carcinoma of endometrium or uterus carcinoma, salivary-gland carcinoma, kidney, prostate cancer, carcinoma vulvae, thyroid carcinoma, liver cancer (hepaticcarcinoma), leukemia and other lympho-proliferative illness, and various types of head and neck cancer.

Term " disorder of muscle " points to or describes containing characteristic feature in muscle animal is that skeletal muscle and/or unstriated muscle fail or reduction makes the significantly reduced physiology illness of normal muscle function.The example of disorder of muscle comprises, but be not limited to, muscular dystrophy (musculardystrophy), multiple sclerosis, amyotrophic lateral sclerosis (amyotrophiclateralsclerosis), Isaac Cotard (Isaac ' ssyndrome), stiff man syndrome (stiff-personsyndrome), familial periodic paralysis (familiarperiodicparalyses), myopathy (myopathy), myotony (myotonia), rhabdomyolysis (rhabdomyolyses), amyotrophy, and various types of myasthenia and muscle rigidity.

Term " ubiquitin-pathway correlated inheritance illness " points to or describes the illness based on heredity that characteristic feature is or contributes to the abnormal function of ubiquitin-pathway.The example of ubiquitin-pathway correlated inheritance illness comprises, but be not limited to, cystic fibrosis (cysticfibrosis), angel's syndrome (Angelman ' ssyndrome) and Liddle syndrome (Liddlesyndrome).

Term " neurological disorders " or " neurological disorder " point to or describe the mammalian central and/or peripheral nervous disease or illness that characteristic feature is the cell-cell communication decline of nervous tissue decline or nervous tissue.The example of neurological disorders includes, but not limited to neurodegenerative disease and (includes, but not limited to Lewy body disease, post poliomyelitis syndrome, Shy-Draeger syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration, τ disease (including, but not limited to degenerative brain disorder and supranuclear paralysis), prion disease (includes, but not limited to bovine spongiform encephalopathy, scrapie, Creutz Fil spy-jacob's syndrome, Kuru disease, Gerstmann-Straussler-Scheinker is sick, chronic wasting disease, and fatal familial insomnia), bulbar paralysis, motor neurone disease, (canavan's disease is included, but not limited to the special-shaped degenerative disease of neural system, Huntington disease, neuronal ceroid lipofuscinosis, Ya Lishan great Shi is sick, Tourette's syndrome, Menkes Menkes Ⅱ syndrome, Cockayne syndrome, Halervorden-Spatz syndrome, myoclonic epilepsy, Rett syndrome, hepatolenticular degeneration, Lesch-Nyhan syndrome, with Unverricht-Lundborg syndrome), dementia (includes, but not limited to Pick's disease, and spinocebellar ataxia.

Term " inflammatory conditions " and " immune disorders " point to or describe the illness caused by abnormal immune mechanism and/or abnormal cytokine intracellular signaling.The example of inflammatory and immune disorders includes, but not limited to autoimmune disease, immunologic defect syndrome and supersensitivity." autoimmune disease " refers to come from and for the nonmalignant disease of individual autologous tissue or illness in this article.Autoimmune disease clearly gets rid of pernicious or Cancerous disease or illness in this article, especially gets rid of B cell lymphoma, Acute Lymphoblastic Leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia and chronic myeloblastic leukemia.The example of autoimmune disorder or illness includes but not limited to inflammatory response, such as inflammatory dermatosis, comprises psoriatic and dermatitis (such as atopic dermatitis); Systemic sclerosis and sclerosis; The response (such as Chron (Crohn) disease and ulcerative colitis) relevant with inflammatory bowel; Respiratory distress syndrome (comprising adult respiratory distress syndrome (ARDS)); Dermatitis; Meningitis; Encephalitis; Uveitis; Colitis; Glomerulonephritis; Allergic condition, such as eczema and asthma and involve T cell infiltrate and chronic inflammatory reply other situation; Atherosclerosis; White corpuscle adhesion defect; Rheumatoid arthritis; Systemic lupus erythematous (SLE) (including but not limited to lupus nephritis, Cutaneous lupus); Diabetes (such as type i diabetes or insulin-dependent diabetes); Multiple sclerosis; Lei Nuoshi (Reynaud) syndrome; Autoimmune thyroiditis; Hashimoto (Hashimoto) thyroiditis; Allergic encephalitis; Si Yegelunshi (Sjogren) syndrome; Children's onset diabetes; Usually the immunne response relevant with cytokine and T-cell mediated acute and delayed hypersensitivity (DH) that be that find in tuberculosis, sarcoidosis, polymyositis, granulomatosis and vasculitis; Pernicious anemia (A Disenshi (Addison) is sick); Involve the disease of leukocyte infiltration; Central nervous system (CNS) inflammatory conditions; Multiple organ injury's syndrome; Hemolytic anemia (including but not limited to cryoglobulinemia or Ku Musishi (Coombs) positive anemia); Myasthenia gravis; The disease of antigen-antibody complex mediation; Anti-glomerular basement membrane is sick; Anti-phospholipid syndrome; Allergic neuritis; Graves (Graves) is sick; Lang-Yi Er Shi (Lambert-Eaton) myasthenic syndrome; Bullous pemphigoid; Pemphigus; The multiple internal secretion adenopathy of autoimmunity; Lay Te Shi (Reiter) is sick; Stiff people's syndromes; Bei Qieteshi (Behcet) is sick; Giant cell arteritis; Immune complex nephritis; IgA nephropathy; IgM polyneuropathy; Immunologic thrombocytopenic purpura (ITP) or AT etc.

The syndromic example of immunologic defect comprises, but be not limited to, the adhesion of ataxia telangiectasia, leukemia deficit syndrome, lymphopenia, dysgammaglobulinemia, HIV or δ retroviral infection, common variable immunodeficiency, agammaglobulinemia, DiGeorge syndrome and Wiskott-Aldrich syndrome.The example of supersensitivity includes, but not limited to transformation reactions, asthma, dermatitis, urticaria, anaphylaxis, Wissler Cotard and thrombopenic purpura.

When for this paper, " treatment " or " process " refers to attempt change the clinical intervention that the nature process of individuality or cell is treated by institute, can be to prevent or carrying out in the process of clinical pathology.The desired effects for the treatment of comprises prophylactic generation or recurrence, relief of symptoms, any direct or indirect pathological consequences of weakening disease, prevention or reduces inflammation and/or tissue/organ damage transfer, slows down the speed of progression of disease, improves or the state and exempt or improve prognosis of palliating a disease.In some embodiment, antibody of the present invention is used for the generation/development postponing disease or illness.

" individuality " refers to vertebrates.In certain embodiments, vertebrates refers to Mammals.Mammals includes, but not limited to livestock (such as ox), motion animal, pet (such as cat, dog and horse), primate, Mouse and rat.In certain embodiments, vertebrates refers to people.

In order to the object for the treatment of, " Mammals " aim enters mammiferous any animal, comprises people, domestic animal and livestock, and zoological park, motion or pet animals, such as dog, horse, cat, ox etc.In certain embodiments, Mammals refers to people.

" significant quantity " refers in required dosage and the amount effectively realizing treatment or the preventive effect expected the time.

" the treatment significant quantity " of substances/molecules of the present invention can cause the factors such as the ability expecting response according to such as individual morbid state, age, sex and body weight and this substances/molecules and change in individuality.Treatment significant quantity also refers to that the treatment beneficial effect of this substances/molecules surpasses the amount of any poisonous or detrimental consequences." prevention significant quantity " refers in required dosage and the amount effectively realizing the preventive effect expected the time.Typically but not necessarily, because preventive dose is before seizure of disease or at disease early stage for experimenter, therefore prevent significant quantity will lower than treatment significant quantity.

Term " cytotoxic agent " for refer to during this paper suppress or prevent the function of cell and/or cause the material of cytoclasis.This term intention comprises: radio isotope, such as At ²¹¹, I ¹³¹, I ¹²⁵, Y ⁹⁰, Re ¹⁸⁶, Re ¹⁸⁸, Sm ¹⁵³, Bi ²¹², P ³², Pb ²¹²with the radio isotope of Lu; Chemotherapeutics, such as methotrexate (methotrexate), Zorubicin (adriamycin), vinca alkaloids (vincaalkaloids) (vincristine(VCR) (vincristine), vinealeucoblastine(VLB) (vinblastine), Etoposide (etoposide)), Dx (doxorubicin), melphalan (melphalan), mitomycin (mitomycin) C, Chlorambucil (chlorambucil), daunorubicin (daunorubicin) or other intercalator; Enzyme and fragment thereof, such as nucleolytic enzyme; Microbiotic; And toxin, the enzyme of such as small molecule toxins or bacterium, fungi, plant or animal origin is lived toxin, comprises its fragment and/or variant; And the various antitumour drug hereafter disclosed or anticarcinogen.Hereafter describe other cytotoxic agent.Kill the destruction that tumour efficacy-enhancing ingredient plays tumour cell.

" chemotherapeutics " refers to the chemical compound that can be used for Therapeutic cancer.The example of chemotherapeutics comprises alkylating agent class (alkylatingagents), such as phosphinothioylidynetrisaziridine (thiotepa) and endoxan (cyclophosphamide), alkyl sulfonate esters class (alkylsulfonates), such as busulfan (busulfan), improsulfan (improsulfan) and piposulfan (piposulfan), aziridines (aziridines), such as benzodepa (benzodepa), carboquone (carboquone), U.S. appropriate replacing send (meturedepa) and uredepa (uredepa), ethyleneimine class (ethylenimines) and methylmelamine class (methylamelamines), comprise altretamine (altretamine), triethylenemelamine (triethylenemelamine), thiotrithylene phosphoramide (triethylenephosphoramide), TESPA (triethylenethiophosphoramide) and trimethylolmelamine (trimethylolomelamine), Annonaceousacetogenicompounds (acetogenin) (especially bullatacin (bullatacin) and bullatacin ketone (bullatacinone)), delta-9-Tetrahydrocannabinol (tetrahydrocannabinol) (dronabinol (dronabinol), ), β-lapachol (lapachone), tecomin (lapachol), colchicine class (colchicines), Betulinic acid (betulinicacid), camptothecine (camptothecin) (comprise synthetic analogues Hycamtin (topotecan) ( ), CPT-11 (irinotecan (irinotecan), ), acetyl camptothecine, Scopoletin (scopoletin) and 9-aminocamptothecin), bryostatin (bryostatin), callystatin, CC-1065 (comprising its U 73975 (adozelesin), U 80244 (carzelesin) and U 77779 (bizelesin) synthetic analogues), podophyllotoxin (podophyllotoxin), Podophyllinic acid (podophyllinicacid), teniposide (teniposide), hidden algae element class (cryptophycins) (particularly hidden algae element 1 and hidden algae element 8), dolastatin (dolastatin), duocarmycin (comprising synthetic analogues, KW-2189 and CB1-TM1), eleutherobin (eleutherobin), pancratistatin, sarcodictyin, sponge chalone (spongistatin), nitrogen mustards (nitrogenmustards), such as Chlorambucil (chlorambucil), Chlornaphazine (chlomaphazine), courage phosphamide (cholophosphamide), estramustine (estramustine), ifosfamide (ifosfamide), chlormethine (mechlorethamine), Nitromin hydrochloride (mechlorethamineoxidehydrochloride), melphalan (melphalan), Novoembichin (novembichin), phenesterin (phenesterine), prednimustine (prednimustine), trofosfamide (trofosfamide), uracil mustard (uracilmustard), nitrosourea (nitrosoureas), such as carmustine (carmustine), chlorozotocin (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine) and ranomustine (ranimustine), antibiotics, such as Enediyne Antibiotic (enediyne) is (as calicheamicin (calicheamicin), especially calicheamicin γ 1I and calicheamicin ω I1 (see such as Agnew, Chem.Intl.Ed.Engl.33:183-186 (1994)), anthracycline antibiotics (dynemicin), comprises dynemicinA, Ai Sibo mycin (esperamicin), and Neocarzinostatin (neocarzinostatin) chromophoric group and related colour albumen Enediyne Antibiotic chromophoric group), aclacinomycin (aclacinomycin), actinomycin (actinomycin), anthramycin (anthramycin), azaserine (azaserine), bleomycin (bleomycin), sanarnycin (cactinomycin), carabicin, carminomycin (carminomycin), cardinophyllin (carzinophilin), Toyomycin (chromomycin), dactinomycin (dactinomycin), daunorubicin (daunorubicin), detorubicin (detorubicin), 6-phenodiazine-5-oxygen-L-nor-leucine, dx (doxorubicin) (comprises morpholino Dx, Cyanomorpholino Dx, 2-pyrroles is for Dx and deoxydoxorubicin), epirubicin (epirubicin), esorubicin (esorubicin), idarubicin (idarubicin), marcellomycin (marcellomycin), mitomycin (mitomycins) such as ametycin, mycophenolic acid (mycophenolicacid), nogalamycin (nogalamycin), Olivomycine (olivomycin), peplomycin (peplomycin), potfiromycin, tetracycline (puromycin), triferricdoxorubicin (quelamycin), rodorubicin (rodorubicin), streptonigrin (streptonigrin), streptozocin (streptozocin), tubercidin (tubercidin), ubenimex (ubenimex), zinostatin (zinostatin), zorubicin (zorubicin), metabolic antagonist class, such as methotrexate and 5 FU 5 fluorouracil (5-FU), folacin, such as N10,9-dimethylfolic acid (denopterin), methotrexate, pteroyltriglutamic acid (pteropterin), trimetrexate (trimetrexate), purine analogue, such as fludarabine (fludarabine), Ismipur (mercaptopurine), ITG (thiamiprine), Tioguanine (thioguanine), pyrimidine analogue, such as Ancitabine (ancitabine), azacitidine (azacitidine), 6-azauridine, carmofur (carmofur), cytosine arabinoside (cytarabine), two deoxyuridine (dideoxyuridine), doxifluridine (doxifluridine), enocitabine (enocitabine), floxuridine (floxuridine), androgens, such as U-22550 (calusterone), dromostanolone propionate (dromostanolonepropionate), S-10275 (epitiostanol), mepitiostane (mepitiostane), testolactone (testolactone), anti-suprarenal gland class, such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), Win-24540 (trilostane), folic acid supplement, such as folinic acid (folinicacid), aceglatone (aceglatone), aldophosphamide glucosides (aldophosphamideglycoside), amino-laevulic acid (aminolevulinicacid), eniluracil (eniluracil), amsacrine (amsacrine), bestrabucil, bisantrene (bisantrene), edatrexate (edatraxate), defosfamide (defosfamide), demecolcine (demecolcine), diaziquone (diaziquone), elfornithine, elliptinium acetate (elliptiniumacetate), esperamicin (epothilone), Etoglucid (etoglucid), gallium nitrate, hydroxyl urea (hydroxyurea), lentinan (lentinan), lonidamine (lonidamine), maytansinoid class (maytansinoids), such as maytenin (maytansine) and maytansinol (maytansinol), ansamitocin (ansamitocin), mitoguazone (mitoguazone), mitoxantrone (mitoxantrone), mopidamol (mopidamol), C-283 (nitracrine), pentostatin (pentostatin), Phenamet (phenamet), pirarubicin (pirarubicin), losoxantrone (losoxantrone), 2-ethyl hydrazides (ethylhydrazide), Procarbazine (procarbazine), polysaccharide compound (JHSNaturalProducts, Eugene, OR), razoxane (razoxane), rhizomycin (rhizoxin), Sizofiran (sizofiran), Spirogermanium (spirogermanium), tenuazonic acid (tenuazonicacid), triaziquone (triaziquone), 2,2 ', 2 "-RA3s, trichothecin class (trichothecenes) (especially T-2 toxin, verrucarine (verrucarin) A, roridin (roridin) A and the rhzomorph (anguidin) that crawls), urethane (urethan), vindesine (vindesine) ( ), Dacarbazine (dacarbazine), mannomustin (mannomustine), mitobronitol (mitobronitol), mitolactol (mitolactol), pipobroman (pipobroman), gacytosine, cytosine arabinoside (arabinoside) (" Ara-C "), phosphinothioylidynetrisaziridine (thiotepa), taxoid (taxoids), such as taxol (paclitaxel) (Bristol-MyersSquibbOncology, Princeton, N.J.), ABRAXANE ^tMnot containing cremophor (Cremophor), the nano particle formulation taxol (AmericanPharmaceuticalPartners, Schaumberg, Illinois) of white protein transformation and taxotere (doxetaxel) ( rorer, Antony, France), Chlorambucil (chlorambucil), gemcitabine (gemcitabine) ( ), 6-Tioguanine (thioguanine), purinethol (mercaptopurine), methotrexate (methotrexate), platinum analogs, such as cis-platinum (cisplatin) and carboplatin (carboplatin), vinealeucoblastine(VLB) (vinblastine) ( ), platinum, Etoposide (etoposide) (VP-16), ifosfamide (ifosfamide), mitoxantrone (mitoxantrone), vincristine(VCR) (vincristine) ( ), oxaliplatin (oxaliplatin), folinic acid (leucovorin), vinorelbine (vinorelbine) ( ), NSC-279836 (novantrone), edatrexate (edatrexate), daunomycin (daunomycin), aminopterin (aminopterin), ibandronate (ibandronate), topoisomerase enzyme inhibitor RFS2000, α-difluorometylornithine (DMFO), class tretinoin (retinoids), such as tretinoin (retinoicacid), capecitabine (capecitabine) ( ), the pharmaceutically acceptable salt of any above-mentioned substance, acid or derivative, and the combination of two or more above-mentioned substances, such as CHOP (abbreviation of endoxan, Dx, vincristine(VCR) and prednisolone conjoint therapy) and FOLFOX (oxaliplatin (ELOXATIN ^tM) abbreviation for the treatment of plan of associating 5-FU and folinic acid).

This definition also comprises the antihormone agent acting as adjustment, reduction, blocking-up or suppress the hormone effect that cancer can be promoted to grow, and is usually the form of system or whole body therapeutic.They self can be hormones.Example comprises anti-estrogens and selective estrogen receptor modulators class (SERM), comprises such as tamoxifen (tamoxifen) and (comprises tamoxifen), raloxifene (raloxifene), droloxifene (droloxifene), 4-hydroxytamoxifen, trioxifene (trioxifene), that Lip river former times sweet smell (keoxifene), LY117018, onapristone (onapristone) and toremifene (toremifene); Mifepristone class; Estrogen receptor down-regulation agent class (ERD); Function is the medicament suppressing or close ovary, such as r-hLH (LHRH) agonist, such as with leuprorelin acetate (leuprolideacetate), goserelin acetate (goserelinacetate), buserelin acetate (buserelinacetate) and triptorelin (triptorelin); Other anti-androgens, such as Drogenil (flutamide), Nilutamide (nilutamide) and than Ka meter Te (bicalutamide); And suppress to regulate the aromatase inhibitor of the aromatase enzyme of estrogen production in suprarenal gland, such as such as 4 (5)-imidazoles, aminoglutethimide (aminoglutethimide), magace (megestrolacetate), exemestane (exemestane), formestane (formestane), fadrozole (fadrozole), r 83842 (vorozole), letrozole (letrozole) and anastrozole (anastrozole).In addition, this definition of chemotherapeutics comprises diphosphonates (bisphosphonates), and such as clodronate (clodronate) (such as or ), etidronate (etidronate), NE-58095, zoledronic acid/zoledronate (zoledronicacid/zoledronate), alendronate (alendronate), pamldronate (pamidronate), tiludronate (tiludronate) or risedronate (risedronate); And troxacitabine (troxacitabine) (DOX nucleosides analogue of cytosine); Antisense oligonucleotide, the signal particularly suppressing to involve adhesive cell propagation by way of in the antisense oligonucleotide of genetic expression, such as such as PKC-α, Raf, H-Ras and EGF-R ELISA (EGF-R); Vaccine, such as vaccine and gene therapy vaccine, such as vaccine, vaccine and vaccine; topoisomerase 1 inhibitor; lapatinibditosylate (ErbB-2 and EGFR dual tyrosine kinase micromolecular inhibitor, also referred to as GW572016); And the pharmaceutically acceptable salt of any above-mentioned substance, acid or derivative.

Composition and prepare the method for above-mentioned substance

The invention provides with polyubiquitin but not the antibody of single ubiquitin specific binding.More particularly, provide can from the antibody of the polyubiquitin specific binding of the polyubiquitin specific binding and not different with comprising second Methionin keys that comprise the first Methionin key.

In an aspect, the invention provides a kind of antibody comprising the HVR-H1 district of sequence containing at least one SEQIDNOs:1-25,81-89,151-175,229-239,265-279,329-336,392-459,599-629,695-704,739-748 and 789-799.In an aspect, the invention provides a kind of comprise be selected from SEQIDNOs:26,90,176,240,280,337,460,630,705, the antibody of the HVR-H1 district consensus sequence of 749 and 800.In an aspect, the invention provides a kind of antibody comprising the HVR-H2 district of sequence containing at least one SEQIDNOs:27-51,91-99,177-201,241-251,281-295,338-345,461-528,631-661,706-715,750-759 and 801-811.In an aspect, the invention provides a kind of comprise be selected from SEQIDNOs:52,100,202,252,296,346,529,662,716, the antibody of the HVR-H2 district consensus sequence of 760 and 812.In an aspect, the invention provides a kind of antibody comprising the HVR-H3 district of sequence containing at least one SEQIDNOs:53-77,101-109,203-227,253-263,297-311,347-354,530-597,663-693,717-726,761-770 and 813-823.In an aspect, the invention provides a kind of comprise be selected from SEQIDNOs:78,110,228,264,312,355,598,694,727, the antibody of the HVR-H3 district consensus sequence of 771 and 824.

In an aspect, the invention provides a kind of antibody comprising the HVR-H2 district of the HVR-H1 district of sequence containing at least one SEQIDNOs:1-26,81-90,151-176,229-240,265-280,329-337,392-460,599-630,695-705,739-749 and 789-800 and the sequence containing at least one SEQIDNOs:27-52,91-100,177-202,241-252,281-296,338-346,461-529,631-662,706-716,750-760 and 801-812.In an aspect, the invention provides a kind of antibody comprising the HVR-H3 district of the HVR-H1 district of sequence containing at least one SEQIDNOs:1-26,81-90,151-176,229-240,265-280,329-337,392-460,599-630,695-705,739-749 and 789-800 and the sequence containing at least one SEQIDNOs:53-78,101-110,203-228,253-264,297-312,347-355,530-598,663-694,717-727,761-771 and 813-824.In an aspect, the invention provides a kind of antibody comprising the HVR-H2 district containing the sequence at least one SEQIDNO:27-52,91-100,177-202,241-252,281-296,338-346,461-529,631-662,706-716,750-760 and 801-812 and the HVR-H3 district containing the sequence at least one SEQIDNOs:53-78,101-110,203-228,253-264,297-312,347-355,530-598,663-694,717-727,761-771 and 813-824.

In an aspect, the invention provides a kind of antibody comprising HVR-L3 district containing the sequence at least one SEQIDNOs:313-327,356-363,728-737 and 772-781.In an aspect, the invention provides a kind of comprise be selected from SEQIDNOs:328,364, the antibody of the HVR-L3 district consensus sequence of 738 and 782.In one embodiment, the invention provides a kind of HVR-L3 district comprised containing the sequence at least one SEQIDNOs:313-328,356-364,728-738 and 772-782, and comprise at least one further and be selected from SEQIDNOs:1-26,81-90,151-176,229-240,265-280,329-337,392-460,599-630,695-705,739-749 and 789-800 respectively; SEQIDNOs:27-52,91-100,177-202,241-252,281-296,338-346,461-529,631-662,706-716,750-760 and 801-812; And the antibody of HVR-H1, HVR-H2 or HVR-H3 sequence of SEQIDNOs:53-78,101-110,203-228,253-264,297-312,347-355,530-598,663-694,717-727,761-771 and 813-824.

In an aspect, the invention provides and a kind ofly comprise at least one, at least two, the antibody of at least three or all four following sequences:

I () comprises the HVR-H1 sequence of the sequence at least one SEQIDNOs:1-26,81-90,151-176,229-240,265-280,329-337,392-460,599-630,695-705,739-749 and 789-800,

(ii) the HVR-H2 sequence of the sequence at least one SEQIDNOs:27-52,91-100,177-202,241-252,281-296,338-346,461-529,631-662,706-716,750-760 and 801-812 is comprised;

(iii) the HVR-H3 sequence of the sequence at least one SEQIDNOs:53-78,101-110,203-228,253-264,297-312,347-355,530-598,663-694,717-727,761-771 and 813-824 is comprised;

(iv) the HVR-L3 sequence of the sequence at least one SEQIDNOs:313-328,356-364,728-738 and 772-782 is comprised.

In an aspect, the invention provides a kind of comprise at least one, at least two, the polyubiquitin be connected with K48 of at least three or all four following sequences has the specific binding of high-affinity but the polyubiquitin be connected with K63 then has the antibody of the combination of the avidity reducing in fact (substantiallyreduced):

I () comprises the HVR-H1 sequence of the sequence at least one SEQIDNOs:1-26,151-176,265-280,392-460 and 695-705;

(ii) the HVR-H2 sequence of the sequence at least one SEQIDNOs:27-52,177-202,281-296,461-529 and 706-716 is comprised;

(iii) the HVR-H3 sequence of the sequence at least one SEQIDNOs:53-78,203-228,297-312,530-598 and 717-727 is comprised; With

(iv) the HVR-L3 sequence of the sequence at least one SEQIDNOs:313-328 and 728-738 is comprised.

In an aspect, the invention provides a kind of comprise at least one, at least two, the polyubiquitin be connected with K63 of at least three or all four following sequences has the specific binding of high-affinity but the polyubiquitin be connected with K48 then has the antibody of the combination of the avidity reduced in fact:

I () comprises the HVR-H1 sequence of the sequence at least one SEQIDNOs:81-90,229-240,329-337,599-630,739-749 and 789-800;

(ii) the HVR-H2 sequence of the sequence at least one SEQIDNOs:91-100,241-252,338-346,631-662,750-760 and 801-812 is comprised;

(iii) the HVR-H3 sequence of the sequence at least one SEQIDNOs:101-110,253-264,347-355,663-694,761-771 and 813-824 is comprised;

(iv) the HVR-L3 sequence of the sequence at least one SEQIDNOs:356-364 and 772-782 is comprised.

As shown in Fig. 2,3,8,9,10,11,14,15,16,17 and 22, the aminoacid sequence of SEQIDNOs:1-78,81-106-149,151-364,392-782 and 789-824 is numbered relative to single HVR (i.e. H1, H2, H3, L3), and this numbering is consistent with following Kabat numbering system.In one embodiment, antibody of the present invention comprises the HVR sequence of, two, three or all above-mentioned (i)-(iv), and HVR-L1 and/or HVR-L2 comprises Kabat consensus sequence (such as SEQIDNO:79 (HVR-L1) and 80 (HVR-L2)).

In an aspect, the invention provides the antibody of the heavy chain HVR sequence comprised as shown in Fig. 2,3,8,9,10,11,14,15,16,17 and 22.In one embodiment, antibody comprises the light chain HVR sequence as shown in Figure 10,11,16 and 17 further.

Some embodiment of antibody of the present invention comprise humanized 4D5 antibody (huMAb4D5-8) as shown in following SEQIDNO:783 ( genentech, Inc., SouthSanFrancisco, CA, USA) variable region of light chain of (also see U.S. Patent number 6,407,213 and Lee etc., J.Mol.Biol. (2004), 340 (5): 1073-93).

1AspIleGlnMetThrGlnSerProSerSerLeuSerAlaSer

ValGlyAspArgValThrIleThrCys ArgAlaSerGlnAspVal

AsnThrAlaValAlaTrpTyrGlnGlnLysProGlyLysAla

ProLysLeuLeuIleTyr SerAlaSerPheLeuTyrSerGlyVal

ProSerArgPheSerGlySerArgSerGlyThrAspPheThr

LeuThrIleSerSerLeuGlnProGluAspPheAlaThrTyrTyr

Cys GlnGlnHisTyrThrThrProProThrPheGlyGlnGly

ThrLysValGluIleLys107 (SEQIDNO:783) (HVR residue is underlined)

In one embodiment, huMAb4D5-8 light-chain variable sequence is modified in one or more positions of the 28th, 30,31,53,66 and 91 (Asp, Asn, Thr, Phe, Arg and His respectively as shown in above bold Italic).In one embodiment, the huMAb4D5-8 sequence of modification comprises Ser at the 28th, comprises Ser at the 30th, comprises Ser at the 31st, comprises Ser at the 53rd, comprises Gly at the 66th and/or comprises Ser at the 91st.Therefore, in one embodiment, antibody of the present invention comprises the variable region of light chain containing sequence shown in following SEQIDNO:784:

1AspIleGlnMetThrGlnSerProSerSerLeuSerAlaSer

ValGlyAspArgValThrIleThrCys ArgAlaSerGlnSerVal

SerSerAlaValAlaTrpTyrGlnGlnLysProGlyLysAlaPro

LysLeuLeuIleTyr SerAlaSerSerLeuTyrSerGlyValPro

SerArgPheSerGlySerGlySerGlyThrAspPheThrLeu

ThrIleSerSerLeuGlnProGluAspPheAlaThrTyrTyrCys

GlnGlnSerTyrThrThrProProThrPheGlyGlnGlyThr

LysValGluIleLys107 (SEQIDNO:784) (HVR residue is underlined)

Relative to huMAb4D5-8, the residue of replacement is indicated with bold Italic as above.

Antibody of the present invention can comprise any suitable framework variable region sequence, and precondition substantially remains the binding activities with the polyubiquitin comprising specific Methionin key.Such as, in certain embodiments, antibody of the present invention comprises people subgroup III heavy chain framework regions consensus sequence.In an embodiment of these antibody, framework region consensus sequence is included in the replacement of the 71st, 73 and/or 78.In some embodiment of these antibody, the 71st be A, the 73rd be T and/or the 78th be A.In one embodiment, these antibody comprise huMAb4D5-8 ( genentech, Inc., SouthSanFrancisco, CA, USA) (also see U.S. Patent number 6,407,213 and 5,821,337, and Lee etc., J.Mol.Biol. (2004), 340 (5): 1073-93) variable region of heavy chain framework sequence.In one embodiment, these antibody comprise people κ I light chain framework region consensus sequence further.In one embodiment, these antibody comprise at least one, two or all SEQIDNOs:79,80, the light chain HVR sequence of 313-328,356-364,728-738 and 772-78.In one embodiment, these antibody comprise as U.S. Patent number 6, the light chain HVR sequence of 407,213 & 5,821, the huMAb4D5-8 described in 337.In one embodiment, these antibody comprise huMAb4D5-8 ( genentech, Inc., SouthSanFrancisco, CA, USA) light-chain variable sequence (SEQIDNO:783 and 784) is (also see U.S. Patent number 6,407,213 & 5,821,337, with Lee etc., J.Mol.Biol. (2004), 340 (5): 1073-93).

In one embodiment, antibody of the present invention comprises variable region of heavy chain, wherein framework sequence comprises the sequence at least one SEQIDNOs:111-129,138-141,146-149 and 839-895, and HVRH1, H2 and H3 sequence to be selected from following three groups at least one: SEQIDNOs:1-26 respectively, 81-90,151-176,229-240,265-280,329-337,392-460,599-630,695-705,739-749 and 789-800; 27-52,91-100,177-202,241-252,281-296,338-346,461-529,631-662,706-716,750-760 and 801-812; And 53-78,101-110,203-228,253-264,297-312,347-355,530-598,663-694,717-727,761-771 and 813-824.In one embodiment, antibody of the present invention comprises variable region of light chain, wherein framework sequence comprises the sequence at least one SEQIDNOs:130-133,134-137,142-145 and 896-907, HVR-L1 sequence to be SEQIDNO:79, HVR-L2 sequence be SEQIDNO:80 and HVR-L3 sequence is selected from following at least one: SEQIDNOs:313-328,356-364,728-738 and 772-782.

In one embodiment, antibody of the present invention comprises variable region of heavy chain, wherein framework sequence comprises the sequence at least one SEQIDNOs:111-129 and 839-895, and HVRH1, H2 and H3 sequence is respectively SEQIDNO:1,27 and 53 (clone 48-1).Similarly, in other embodiments, antibody in each clone 48-2 to 48-118, clone 63-1 to 63-51, Fabapu01-apu24, Fabapu2.01-apu2.20 and clone apu3.01-3.11 all comprises variable region of heavy chain, wherein framework sequence comprises the sequence at least one SEQIDNOs:111-129 and 839-895, and HVR-H1, HVR-H2 and HVR-H3 sequence for for Fig. 2,3, each clone or Fab clearly enumerate in 8-11,14-17 and 22 those sequences.In one embodiment, antibody of the present invention comprises variable region of light chain, wherein framework sequence comprises the sequence at least one SEQIDNOs:130-133 and 896-907, and HVRL1, L2 and L3 sequence is respectively SEQIDNOs:79,80 and 313 (Fabapu01).Similarly, in other embodiments, each antibody in Fabapu01-apu24 and Fabapu2.01-apu2.20 all comprises variable region of light chain, wherein framework sequence comprises the sequence at least one SEQIDNOs:130-133 and 896-907, and HVR-L1 be SEQIDNO:79, HVR-L2 is SEQIDNO:80 and HVR-L3 sequence those sequences for clearly enumerating for each Fab in Figure 10,11C, 16B and 17B.

In one embodiment, antibody of the present invention comprises variable region of heavy chain, wherein framework sequence comprises the sequence at least one SEQIDNOs:138-141, and HVRH1, H2 and H3 sequence is respectively SEQIDNO:1,27 and 53 (clone 48-1).Similarly, in other embodiments, each antibody in clone 48-2 to 48-118, clone 63-1 to 63-51, Fabapu01-apu24, Fabapu2.01-apu2.20 and clone apu3.01-3.11 all comprises variable region of heavy chain, wherein framework sequence comprises at least one sequence of SEQIDNOs:138-141, and HVR-H1, HVR-H2 and HVR-H3 sequence for for Fig. 2,3, each clone or Fab clearly enumerate in 8-11,14-17 and 22 those sequences.In one embodiment, antibody of the present invention comprises variable region of light chain, wherein framework sequence comprises the sequence at least one SEQIDNOs:134-137, and HVRL1, L2 and L3 sequence is respectively SEQIDNOs:79,80 and 313 (Fabapu01).Similarly, in other embodiments, each antibody in Fabapu01-apu24 and Fabapu2.01-apu2.20 all comprises variable region of light chain, wherein framework sequence comprises the sequence at least one SEQIDNOs:134-137, and HVR-L1 be SEQIDNO:79, HVR-L2 is SEQIDNO:80 and HVR-L3 sequence those sequences for clearly enumerating for each Fab in Figure 10,11C, 16B and 17B.

In one embodiment, antibody of the present invention comprises variable region of heavy chain, and wherein framework sequence comprises the sequence at least one SEQIDNOs:146-149, and HVRH1, H2 and H3 sequence is respectively SEQIDNO:1,27 and 53 (clone 48-1).Similarly, in other embodiments, antibody in each clone 48-2 to 48-118, clone 63-1 to 63-51, Fabapu01-apu24, Fabapu2.01-apu2.20 and clone apu3.01-3.11 all comprises variable region of heavy chain, wherein framework sequence comprises the sequence at least one SEQIDNOs:146-149, and HVR-H1, HVR-H2 and HVR-H3 sequence for for Fig. 2,3, each clone or Fab clearly enumerate in 8-11,14-17 and 22 those sequences.In one embodiment, antibody of the present invention comprises variable region of light chain, wherein framework sequence comprises the sequence at least one SEQIDNOs:142-145, and HVRL1, L2 and L3 sequence is respectively SEQIDNOs:79,80 and 313 (Fabapu01).Similarly, in other embodiments, each antibody in Fabapu01-apu24 and Fabapu2.01-apu2.20 all comprises variable region of light chain, wherein framework sequence comprises the sequence at least one SEQIDNOs:142-145, and HVR-L1 be SEQIDNO:79, HVR-L2 is SEQIDNO:80 and HVR-L3 sequence those sequences for clearly enumerating for each Fab in Figure 10,11C, 16B and 17B.

In one embodiment, antibody of the present invention has carried out affinity maturation to obtain the target binding affinity expected.In one example in which, the polyubiquitin be connected with K48 has the specific binding of high-affinity but the antibody that the polyubiquitin be connected with K63 then has an affinity maturation of the present invention of the combination of the avidity reduced in fact is included in the replacement at HVR-H1 the 29th, 30,33 and 34 amino acids place.In another example, the polyubiquitin be connected with K48 has the specific binding of high-affinity but the antibody that the polyubiquitin be connected with K63 then has an affinity maturation of the present invention of the combination of the avidity reduced in fact is included in the replacement at HVR-H2 the 52nd and 52a amino acids place.In another example, the polyubiquitin be connected with K48 there is the specific binding of high-affinity but the antibody that the polyubiquitin be connected with K63 then has an affinity maturation of the present invention of the combination of the avidity reduced in fact be included in HVR-H3 the 99th, 100, the replacement at 100a and 100b amino acids place.In another example, the polyubiquitin be connected with K48 has the specific binding of high-affinity but the antibody that the polyubiquitin be connected with K63 then has an affinity maturation of the present invention of the combination of the avidity reduced in fact is included in the replacement at HVR-H3 95-100,100a and 100b amino acids place.In another example, the polyubiquitin be connected with K48 has the specific binding of high-affinity but the antibody that the polyubiquitin be connected with K63 then has an affinity maturation of the present invention of the combination of the avidity reduced in fact is included in the replacement at HVR-L3 the 91st and 96 amino acids place.In another example, the polyubiquitin be connected with K63 has the specific binding of high-affinity but the antibody that the polyubiquitin be connected with K48 then has an affinity maturation of the present invention of the combination of the avidity reduced in fact is included in the replacement at HVR-H1 29-34 amino acids place.In another example, the polyubiquitin be connected with K63 there is the specific binding of high-affinity but the antibody that the polyubiquitin be connected with K48 then has an affinity maturation of the present invention of the combination of the avidity reduced in fact be included in HVR-H2 the 50th, 52, the replacement at 52a, 53-56 and 58 amino acids places.In another example, the polyubiquitin be connected with K63 has the specific binding of high-affinity but the antibody that the polyubiquitin be connected with K48 then has an affinity maturation of the present invention of the combination of the avidity reduced in fact is included in the replacement at HVR-H3 95-100,100a, 100b and 100c amino acids place.In another example, the polyubiquitin be connected with K63 has the specific binding of high-affinity but the antibody that the polyubiquitin be connected with K48 then has an affinity maturation of the present invention of the combination of the avidity reduced in fact is included in the replacement at HVR-L3 91-95,95a and 95b amino acids place.

In another example, the polyubiquitin be connected with K63 has the specific binding of high-affinity but the antibody that the polyubiquitin be connected with K48 then has an affinity maturation of the present invention of the combination of the avidity reduced in fact is included in the replacement at HVR-H1 29-34 amino acids place.In another example, the polyubiquitin be connected with K63 has the specific binding of high-affinity but the antibody that the polyubiquitin be connected with K48 then has an affinity maturation of the present invention of the combination of the avidity reduced in fact is included in the replacement at HVR-H2 the 50th, 52,54,56 and 58 amino acids place.In another example, the polyubiquitin that K63 connects has the specific binding of high-affinity but the antibody that the polyubiquitin be connected with K48 then has an affinity maturation of the present invention of the combination of the avidity reduced in fact is included in the replacement at HVR-H3 95-100,100a, 100b and 100c amino acids place.

In one embodiment, antibody of the present invention comprise containing SEQIDNOs:265,281 and 297 the variable region of heavy chain of sequence.In one embodiment, antibody of the present invention comprise containing SEQIDNOs:79,80 and 313 the variable region of light chain of sequence.In one embodiment, antibody of the present invention comprise containing SEQIDNOs:265,281 and 297 the variable region of heavy chain of sequence, and also comprise containing SEQIDNOs:79,80 and 313 the variable region of light chain of sequence.In other embodiments, the antibody of the present invention corresponding to specific cloning numbering comprise containing for this clone numbering as Fig. 2,3,8,9,10,11, the HVR-H1 as shown in 14-17 and 22, the variable region of heavy chain of HVR-H2 and HVR-H3 sequence.In other embodiments, the antibody of the present invention corresponding to specific cloning numbering comprises HVR-L1 sequence containing SEQIDNO:79, SEQIDNO:80HVR-L2 sequence and the variable region of light chain for the HVR-L3 sequence of this clone numbering as shown in Figure 10,11,16 and 17.In other embodiments, antibody of the present invention corresponding to specific cloning numbering comprise containing for this clone numbering as Fig. 2,3,8,9,10,11, the variable region of heavy chain of HVR-H1, HVR-H2 and HVR-H3 sequence as shown in 14-17 and 22, and also comprise HVR-L1 sequence containing SEQIDNO:79, SEQIDNO:80HVR-L2 sequence and this clone numbered to the variable region of light chain of the HVR-L3 sequence as shown in Figure 10,11,16 and 17.

In an aspect, the invention provides a kind of and arbitrary afore mentioned antibodies and compete the antibody be combined with polyubiquitin.In an aspect, the invention provides the antibody of a kind of and arbitrary afore mentioned antibodies in conjunction with antigenic determinant on identical polyubiquitin.

As shown in this article, antibody of the present invention and the polyubiquitin specific binding be separated with specific Methionin key.As shown in this article, antibody of the present invention also with the polyubiquitin specific binding (see such as embodiment 3 and 4) when this polyubiquitin is connected with heterologous protein with specific Methionin key.

Provide to comprise at least one anti-polyubiquitin antibody or comprise at least one and to encode the composition of polynucleotide of sequence of anti-polyubiquitin antibody.In certain embodiments, said composition can be pharmaceutical composition.As used herein, described composition comprises one or more antibody be combined with one or more polyubiquitins and/or one or more comprise the polynucleotide of the sequence of one or more antibody be combined with one or more polyubiquitins of encoding.These compositions can comprise suitable carrier further, such as the pharmaceutically acceptable vehicle comprising buffer reagent well-known in the art.

Additionally provide antibody and the polynucleotide of separation.In certain embodiments, the antibody of separation and polynucleotide are substantially pure.

In one embodiment, anti-polyubiquitin antibody is monoclonal antibody.In another embodiment, fragment (as Fab, Fab '-SH and F (ab ') 2 fragments of anti-polyubiquitin antibody are provided).By the such as enzymic digestion or produce these antibody fragments by recombinant technology of traditional method.Above-mentioned antibody fragment can be chimeric, humanized or people.These fragments have following Diagnosis and Treat purposes.

Phage display library is used to produce anti-polyubiquitin antibody

The method of the multiple phage display library for generation of obtaining object antibody is known in the art.A kind of method producing object antibody is by using as Lee etc., J.Mol.Biol. (2004), the phage antibody library described in 340 (5): 1073-93.

There is the antibody cloning of the synthesis expecting activity by using combinatorial library to screen thus prepare anti-polyubiquitin antibody of the present invention.In principle, merged the antibody cloning of the phage library selection synthesis to the phage of the different fragments of the antibody variable region (Fv) of bacteriophage coat protein containing displaying by screening.By the above-mentioned phage library of affinity chromatography elutriation for expectation antigen.Express and in conjunction with the clone of the Fv fragment of expectation antigen by this antigen is adsorbed, and therefore can be separated with non-binding clone in library.Then combining clone is under wash-out antigen, and by the extra further enrichment of Antigen adsorption/elution cycles.By designing suitable antigen selection method to select object phage clone, then the Fv sequence and Kabat etc. from object phage clone is used, SequencesofProteinsofImmunologicalInterest, 5th edition, NIH publication 91-3242, BethesdaMD (1991), the suitable anti-polyubiquitin antibody cloning of constant region (Fc) sequence construct total length described in vols.1-3, thus obtain any one anti-polyubiquitin antibody of the present invention.

The antigen binding domain of antibody is made up of about 110 amino acid coming from two variable (V) districts (respectively from light chain (VL) and heavy chain (VH)), and described two variable regions all exist 3 high change rings or complementary determining region (CDRs).As Winter etc., Ann.Rev.Immunol., described in 12:433-455 (1994), variable region can be used as wherein VH and VL by short, that flexible peptide is covalently bound scFv (scFv) fragment or to merge separately to constant region as wherein VH and VL and the Fab fragment of noncovalent interaction functionally shown on phage.As used herein, scFv encode bacteriophage clone and Fab encode bacteriophage clone are jointly called as " Fv phage clone " or " Fv clone ".

Clone VH and VL gene pool respectively by polymerase chain reaction (PCR) and recombinate at random in phage library, then can as Winter etc., Ann.Rev.Immunol., the search antigen described in 12:433-455 (1994) combines clone.The library of immune origin provides for immunogenic high-affinity antibody, without the need to building hybridoma.Alternatively, natural storehouse can be cloned to provide people's antibody of the single source for various non-self and autoantigen, and without the need to any as Griffiths etc., the immunization described in EMBOJ, 12:725-734 (1993).Finally, also also use the PCR primer containing stochastic sequence to Gao Bian CDR3 district of encoding also as Hoogenboom and Winter by cloning the V-constant gene segment C do not reset from stem cell, J.Mol.Biol., perfect aspect described by 227:381-388 (1992) is reset outward, thus synthesis of natural library.

Filobactivirus is by merging for showing antibody fragment with minor coat protein pIII.As Marks etc., J.Mol.Biol., described by 222:581-597 (1991), antibody fragment can be shown as Single-Chain Fv Fragment of Murine, wherein VH with VL district is connected by flexible spacer polypeptide on same polypeptide chain, or as Hoogenboom etc., Nucl.AcidsRes., described in 19:4133-4137 (1991), antibody can be shown as Fab fragment, wherein a chain merges to pIII, another chain is then secreted in bacterial host cell pericentral siphon, at this by replacing some wild type coat protein, this Fab-coat protein Standard body display is on phage surface.

Generally speaking, the nucleic acid of encoding antibody genes fragment obtains from results from the immunocyte of human or animal.If expect that anti-polyubiquitin clone is partial in library, then with polyubiquitin immunizing subjects to produce antibody response, and reclaim the B cell of splenocyte and/or circulation or other peripheral blood lymphocyte (PBLs) for library construction.In one embodiment, by producing anti-human polyubiquitin antibody response in the transgenic mice carrying functional human immunoglobulin gene array (and lacking functional endogenous antibody generation system), make polyubiquitin immunization cause the B cell produced for people's antibody of polyubiquitin, thus obtain the human immunoglobulin gene frag-ment libraries of the anti-human polyubiquitin clone of deflection.The generation producing the transgenic mice of people's antibody is described in following (III) (b) joint.

By using suitable screening method to be separated the B cell expressing polyubiquitin specific membranes binding antibody, as carried out isolated cell by use polyubiquitin affinity chromatography or then carry out adherent cell by fluorescence-activated cell sorting (FACS) with fluorochrome label polyubiquitin, thus obtain the enrichment of the reactive cell colony of extra anti-polyubiquitin.

Alternatively, provide preferably possible antibody library from the splenocyte of the donor without immunity and/or the use of B cell or other PBLs, and allow that any wherein polyubiquitin of use is that nonantigenic animal species (people or inhuman) builds antibody library.For the library construction integrating antibody gene in vitro, from experimenter, gather in the crops stem cell to provide the nucleic acid of non-rearranged antibody constant gene segment C of encoding.Object immunocyte can obtain from many animals species, as people, mouse, rat, rabbit, luprine, dog, cat, pig, ox, horse and birds species etc.

Reclaim the nucleic acid of encoding antibody variable constant gene segment C (comprising VH and VL section) from object cell and increase.When resetting VH and VL gene library, by such as Orlandi etc., Proc.Natl.Acad.Sci. (USA), described in 86:3833-3837 (1989), by isolation of genomic DNA from lymphocyte or mRNA, then use with 5 ' of restructuring VH and VL gene and 3 ' primer of ends match carry out polymerase chain reaction (PCR) and obtain the DNA expected, make different V gene pools be expressed thus.As (1989) and Ward etc. such as Orlandi, Nature, described in 341:544-546 (1989), can use the exon 5 ' end in the V-district being in encoding mature reverse primer and based on the forward primer in J-district from cDNA and genomic DNA amplification V gene.But, increase for from cDNA, reverse primer also can based on such as Jones etc., Biotechnol., leader exon described in 9:88-89 (1991), and forward primer is as Sastry etc., Proc.Natl.Acad.Sci. (USA), being in constant region described in 86:5728-5732 (1989).In order to make complementary maximization, can degeneracy be incorporated in primer described in (1989) or the Sastry etc. (1989) such as Orlandi.In certain embodiments, as Marks etc., J.Mol.Biol., the method of 222:581-597 (1991) or Orum etc., NucleicAcidsRes., described in the method for 21:4491-4498 (1993), by using the PCR primer of each V-gene family of target to make library diversity be maximized, to increase all available VH and VL arrangements be present in immunocyte nucleic acid samples.In order to the DNA clone of amplification is entered in expression vector, described in (1989) such as Orlandi, rare restriction site can be imported in PCR primer as the label at an end, or as Clackson etc., Nature, described in 352:624-628 (1991), carry out further pcr amplification by the primer of applying marking.

The V gene pool that synthesis is reset can derive from V constant gene segment C in vitro.Most people VH-constant gene segment C has been cloned and has checked order and (has been reported in Tomlinson etc., J.Mol.Biol., in 227:776-798 (1992)), and mapped and (be reported in Matsuda etc., NatureGenet., in 3:88-94 (1993)); As Hoogenboom and Winter, J.Mol.Biol., described in 227:381-388 (1992), use the PCR primer of the H3 ring of the different sequence of coding and length, the section (comprising all major confonnational of H1 with H2 ring) of these clones can be used for producing different VH gene pools.Described in Barbas etc., Proc.Natl.Acad.Sci.USA, 89:4457-4461 (1992), the long H3 ring that all sequence polymorphisms also can be used all to concentrate on single length prepares VH storehouse.People V κ and V λ section have been cloned and have checked order (being reported in Williams and Winter, in Eur.J.Immunol., 23:1456-1461 (1993)) and can be used for preparing the light chain storehouse of synthesis.V gene pool based on the synthesis of different VH and VL folding (fold) and L3 and H3 length can be encoded the antibody of the sizable structure diversity of tool.After V-genes encoding DNAs increases, germline V-gene section can be reset in vitro according to the method for Hoogenboom and Winter, J.Mol.Biol., 227:381-388 (1992).

Antibody fragment storehouse can be built in several ways by being combined by VH and VL gene pool.Each storehouse can be created in different carriers, and as Hogrefe etc., Gene, recombinant vectors in vitro described in 128:119-126 (1993), or as Waterhouse etc., Nucl.AcidsRes., the loxP system described in 21:2265-2266 (1993) infects recombinant vectors by combination in vivo.The restriction to library size that the characteristic that this In vivo recombination method make use of two chains of Fab fragment causes to overcome intestinal bacteria transformation efficiency.Clone natural VH and VL storehouse respectively, a storehouse is cloned in phagemid, and another storehouse is cloned in phage vector.Then by phage-infect containing these two libraries of phagemid bacteria combination, make each cell containing different combinations and library size is only limited to the number (about 10 of existing cell ¹²individual clone).These two carriers all contain In vivo recombination signal, make VH and VL gene recombinate on single replicon and pack altogether into phage particle.These huge libraries provide has good affinity (about 10 in a large number ^-8the K of M _d ^-1) different antibodies.

Alternatively, as Barbas etc., Proc.Natl.Acad.Sci.USA, described in 88:7978-7982 (1991), storehouse order can be cloned in identical carrier, or as Clackson etc., Nature, described in 352:624-628 (1991), be assembled together by PCR and then clone.PCR assembling also can be used for VH with VLDNAs to be connected to form scFv (scFv) storehouse with the DNA of coding flexible spacer peptide.In another technology, described in Embleton etc., Nucl.AcidsRes., 20:3831-3837 (1992), " in cell PCR assembling " for being combined VH and the VL gene in lymphocyte by PCR, and then clones the storehouse of the gene connected.

The screening in library is completed by any technology known in the art.Such as, polyubiquitin can be used for wrapping the hole by absorption flat board, the host cell being attached to absorption flat board is expressed or uses in cell sorting, or be conjugated to vitamin H and catch for the pearl of streptavidin bag quilt, or in method for other elutriation phage display library known in the art any.

Be suitable for phage particle at least partially with under sorbent material conjugation condition, phage library sample is contacted with immobilized polyubiquitin.Usually, select the condition comprising pH, ionic strength, temperature etc. to simulate physiological status.Wash with solid phase in conjunction with phage, then as Barbas etc., Proc.Natl.Acad.SciUSA, described in 88:7978-7982 (1991) by acid or as Marks etc., J.Mol.Biol., described in 222:581-597 (1991) by alkali, or as being similar to Clackson etc., wash-out is carried out by polyubiquitin antigenic competition in the method for the antigenic competition method of Nature, 352:624-628 (1991).Select pnagus medius can enrichment 20-1 in single-wheel, 000 times.In addition, the phage of enrichment can grow and accept the selection of more wheels in bacterial cultures.

The validity selected depends on many factors, and whether the Dissociation comprised in washing process can be combined with antigen with the multiple antibody fragments be on single phage simultaneously.By utilizing antigen coated density high in short washing, multivalent bacteriophage display and solid phase, thus the antibody capable with fast Dissociation (and weak binding avidity) is retained.High-density not merely to be interacted phagus durabilis by multivalence, also helps the phage that recombine has been dissociated.By utilizing long washing and as Bass etc., Proteins, 8:309-314 (1990) and the monovalent phages described in WO92/09690 and as Marks etc., Biotechnol., the low antigen coated density described in 10:779-783 (1992) shows that promotion selection has the antibody of slow Dissociation (and good binding affinity).

Even if to the avidity slightly difference of polyubiquitin, be also the phage antibody likely selecting different avidity.But, through select the random mutation of antibody (as in some affinity maturation technology above-mentioned carry out) many mutant may be produced, most of binding antibody, and small part has more high-affinity.Because polyubiquitin is limited, therefore rare high-affinity phage can be shown one's talent.For retaining the mutant of all more high-affinities, can by phage and excessive biotinylation polyubiquitin incubation, but biotinylation polyubiquitin is then in the concentration of the volumetric molar concentration lower than the target mole affinity costant of polyubiquitin.Then by the phage of the paramagnetic beads seizure high-affinity combination of streptavidin bag quilt.Above-mentioned " balance catch " makes antibody capable be selected according to their binding affinity, has the sensitivity that the mutant clone of allowing the avidity only with 2 times higher is separated with the phage having more low-affinity in a large number.The condition for washing the phage be combined in solid phase of also can controlling is to distinguish based on Dissociation.

Anti-polyubiquitin can be selected to clone based on activity.In one embodiment, the invention provides blocking-up polyubiquitin part and be combined with polyubiquitin, but do not block polyubiquitin part and the protein bound anti-polyubiquitin antibody of the second.Fv clone corresponding to above-mentioned anti-polyubiquitin antibody selects by following steps: (1) phage library as described in saving as above-mentioned B (I) (2) is separated anti-polyubiquitin and clones, and optionally by cultivating be separated phage clone colony thus this colony that increases in suitable host bacterium; (2) polyubiquitin and the second albumen is selected relative to desired blocking-up and non-blacked activity respectively; (3) anti-polyubiquitin phage clone is adsorbed onto on immobilized polyubiquitin; (4) use excessive described the second albumen to carry out overlapping in conjunction with determining area of any unexpected identification of wash-out and the second albumen or share and have this in conjunction with the polyubiquitin of determining area in conjunction with the clone of determining area; (5) clone still adsorbed after elution step (4).Optionally, there is the clone expecting blocking-up/non-blacked characteristic and can obtain enrichment further by repetition one or many selection step described herein.

Routine techniques is used to be easy to be separated and the DNA (Oligonucleolide primers as by utilizing design cause hybridoma or phage DNA template specificity amplification object heavy chain and light chain coding region) of the coding that checks order Fv clone of the present invention.Once be separated, just DNA can be placed in expression vector, then expression vector is transfected into do not produce immunoglobulin (Ig) in addition host cell as in Bacillus coli cells, ape COS cell, Chinese hamster ovary (CHO) cell or myeloma cell, expect the synthesis of monoclonal antibody to obtain in the host cell of restructuring.Review literature for antibody coding DNA recombinant expressed in bacterium comprises Skerra etc., Curr.OpinioninImmunol., 5:256 (1993) and Pluckthun, Immunol.Revs, 130:151 (1992).

The DNA sequence dna of the DNA and known encoding heavy chain and/or constant region of light chain (as obtained from Kabat etc., the same suitable DNA sequence dna) that code book can be invented Fv clone combines with the clone of the heavy chain and/or light chain that form encoding full leng or partial-length.Should be understood that the constant region of any isotype all can be used for this object, comprise IgG, IgM, IgA, IgD and IgE constant region, and above-mentioned constant region can obtain from anyone or animal species.A kind of Fv Clone Origin is in the variable region DNA of a kind of animal (as people) species, and then merged to the constant region DNA of another kind of animal species to form the encoding sequence of " hybrid " antibody, total length heavy chain and/or light chain be included in " be fitted together to " as used herein and " hybrid " antibody definition in.In one embodiment, the Fv clone deriving from people variable region DNA is merged to human constant region DNA with the encoding sequence of the heavy chain and/or light chain that form total man's total length or partial-length.

Produce and can have moderate avidity (about 10 from the antibody of natural (naive) library (natural or synthesis) ⁶-10 ⁷m ^-1k _d ^-1), but also can as (1994) such as Winter, affinity maturation is simulated in the same described also reselection procedure second library of passing through in vitro to build.Such as, at Hawkins etc., J.Mol.Biol., in the method for 226:889-896 (1992) or at Gram etc., in the method for Proc.Natl.Acad.SciUSA, 89:3576-3580 (1992), (be reported in Leung etc. by using fallibility polysaccharase, in Technique, 1:11-15 (1989)) sudden change can be imported at random in vitro.In addition, as cloned at the single Fv through selecting and screen in the clone of more high-affinity, utilize the PCR with the primer carrying the stochastic sequence of crossing over object CDR, carry out affinity maturation by the one or more CDRs of random mutation.WO9607754 (being disclosed on March 14th, 1996) describe a kind of for induced mutation in the complementary determining region of light chain immunoglobulin to create the method in light chain gene library.Another kind of effective means is as Marks etc., Biotechnol., described in 10:779-783 (1992), restructuring obtains from the naturally occurring V region variants storehouse without the donor of immunity through phage display by using, and screens according to VH or the VL district more selected by high-affinity in a few endless chain reorganization.This technology makes the antibody of the avidity had within the scope of 10-9M and antibody fragment produce.

Produce other method of anti-polyubiquitin antibody

Other method producing antibody and assessment affinity of antibody is well-known in the art, and is described in as Kohler etc., Nature256:495 (1975); U.S. Patent number 4,816,567; Goding, MonoclonalAntibodies:PrinciplesandPractice, pp.59-103 (AcademicPress, 1986; Kozbor, J.Immunol., 133:3001 (1984); Brodeur etc., MonoclonalAntibodyProductionTechniquesandApplications, pp.51-63 (MarcelDekker, Inc., NewYork, 1987; Munson etc., Anal.Biochem., 107:220 (1980); Engels etc., Agnew.Chem.Int.Ed.Engl., 28:716-734 (1989); Abrahmsen etc., EMBOJ., 4:3901 (1985); MethodsinEnzymology, vol.44 (1976); In Morrison etc., Proc.Natl.Acad.Sci.USA, 81:6851-6855 (1984).

General method

Generally speaking, the invention provides the anti-polyubiquitin antibody of the disease purposes with the treatment polyubiquitin mediation wherein expecting partly or entirely to close one or more polyubiquitin activity.In one embodiment, anti-polyubiquitin antibody of the present invention is used for the treatment of cancer.In another embodiment, the anti-polyubiquitin antibody provided at this is used for the treatment of disorder of muscle, those illnesss as described above.In another embodiment, the anti-polyubiquitin antibody provided at this is used for the treatment of nervous system disorders, those diseases as described above.In another embodiment, the anti-polyubiquitin antibody provided at this is used for the treatment of heredopathia.In another embodiment, the anti-polyubiquitin antibody provided at this is used for the treatment of immunity/inflammatory conditions.

The unique property of the present invention's anti-polyubiquitin antibody makes it especially can be used for distinguishing the different Methionin types of attachment of polyubiquitin in cell system, and without the need to by means of trouble and the genetic manipulation of costliness or bio-physical method as mass spectroscopy.Anti-polyubiquitin antibody of the present invention is used in external and identifies function and the activity of the polyubiquitin that specific Methionin connects in vivo.Anti-polyubiquitin antibody of the present invention also can be used for the effect of polyubiquitin in disease progression and pathogeny determining that specific Methionin connects.Anti-polyubiquitin antibody of the present invention can be further used for treating the abnormal adjustment of polyubiquitin of wherein one or more specific Methionins connections or the disease of abnormal function, and does not disturb the normal activity for the non-specific polyubiquitin of this anti-polyubiquitin antibody.

In another aspect, anti-polyubiquitin antibody of the present invention has as detecting the effectiveness with the polyubiquitin being separated specific Methionin key, such as detect the polyubiquitin in different cell types and tissue, comprise and measure polyubiquitin density and the distribution in cell colony and given cell, and exist based on polyubiquitin or the cell sorting of quantity.

In in another, anti-polyubiquitin antibody of the present invention is for developing the polyubiquitin antagonist with the blocking-up active patterns being similar to Subject antibodies of the present invention.Such as, the antibody of the polyubiquitin of anti-K48 connection of the present invention can be used for measuring and identifies the antibody of the approach ability that other polyubiquitin binding characteristic with identical K48 connection and/or the polyubiquitin blocking K48 connection mediate.Similarly, the polyubiquitin antibody that anti-K63 of the present invention connects can be used for measuring and identifies the antibody of the approach ability that other polyubiquitin binding characteristic with identical K63 connection and/or the polyubiquitin blocking K63 connection mediate.

As further example, anti-polyubiquitin antibody of the present invention can be used for qualification with herein illustrational antibody substantially in conjunction with other anti-polyubiquitin antibody of identical polyubiquitin antigenic determinant, described antigenic determinant comprises linearly and conformational epitope.

Anti-polyubiquitin antibody of the present invention can be used for the assay method based on physiological routes, wherein polyubiquitin relates to the small molecular antagonists that screening has the polyubiquitin of one or more specific Methionin key, and described small molecular antagonists will have similar pharmacological effect in one or more binding partners of blocking-up are combined with the polyubiquitin with those one or more Methionin keys.Such as, the polyubiquitin that known K48 connects relates to the targeting proteins enzyme body degraded of some albumen (see such as Chau etc., Science243:1576-1583 (1989); Finley etc., Mol.Cell.Biol.14:5501-5509 (1994); Flick etc., Nat.Cell.Biol.6:634-641 (2004)); Therefore by comparing the activity of polyubiquitin antibody in this approach that one or more possible small molecular antagonists are connected with anti-K48, the polyubiquitin antibody that anti-K48 connects can be used for the small molecular antagonists of the targeting proteins enzyme body degraded of the polyubiquitin mediation that Screening and Identification K48 connects.Similarly, in another example, the polyubiquitin that known K63 connects relates to DNA and repairs (see such as Pickart and Fushman, Curr.Opin.Chem.Biol.8:610-616 (2004)), compared with the activity of one or more the possible small molecular antagonists of the polyubiquitin that the activity that the polyubiquitin Antibodies Against DNA that therefore anti-K63 can be connected repairs approach connects with K63 in identical DNA reparation approach.Similarly, in another example, the polyubiquitin that known K63 connects relates to the formation of Lewy corpusculum in Parkinson's disease (see such as Lim etc., J.Neurosci.25 (8): 2002-9 (2005)), compared with the activity of one or more the possible small molecular antagonists of the polyubiquitin that the activity that the polyubiquitin Antibodies Against Lewy corpusculum that therefore anti-K63 can be connected is formed connects with K63 in being formed at antagonism Lewy corpusculum.

This area routine techniques can be used to obtain candidate antibodies, comprise those technology described in this article such as hybridoma technology and use binding molecule (bindermolecules) and screen phage display library.These methods are well-known in the art.

In brief, by utilizing combinatorial library to screen the antibody cloning having and expect active synthesis, anti-polyubiquitin antibody of the present invention is prepared.In principle, by screening containing showing the phage library merged to the phage of different antibody variable region (Fv) fragment of bacteriophage coat protein, the antibody cloning of synthesis is selected.By the above-mentioned phage library of affinity chromatography elutriation for expectation antigen.The clone of the expression Fv fragment that can be combined with expectation antigen is adsorbed on antigen, and is therefore separated with non-binding clone in library.Then from the clone of elution of bound antigen, and by the extra further enrichment of Antigen adsorption/elution cycles.By designing suitable antigen selection method to screen object phage clone, then the Fv sequence and Kabat etc. from object phage clone is used, SequencesofProteinsofImmunologicalInterest, 5th edition, NIH publication 91-3242, BethesdaMD (1991), the suitable anti-polyubiquitin antibody cloning of constant region (Fc) sequence construct total length described in vols.1-3, thus obtain any one anti-polyubiquitin antibody of the present invention.Also see PCT publication number WO03/102157 and the document wherein quoted.

In one embodiment, anti-polyubiquitin antibody of the present invention is monoclonal antibody.Also be included in the scope of the invention be anti-polyubiquitin antibody provided in this article antibody fragment as Fab, Fab ', Fab '-SH and F (ab ') ₂fragment and variant thereof.These antibody fragments are produced as enzymic digestion or by recombinant technology by traditional method.Above-mentioned antibody fragment can be chimeric, people's or humanized.Experiment listed by these fragments have herein, diagnosis and therepic use.

Monoclonal antibody can obtain the antibody from a group homogeneous substantially, and namely except likely naturally occurring a small amount of sudden change, the individual antibody that this colony comprises is identical.Therefore, modifier " mono-clonal " refers to the antibody characteristic of the mixture of same antibody.

Multiple means known in the art can be used to prepare anti-polyubiquitin monoclonal antibody of the present invention, comprise for Kohler etc., Nature, the hybridoma that 256:495 (1975) first describes, or alternatively by recombinant DNA method (as U.S. Patent number 4,816,567) them are prepared.

carrier, host cell and recombination method

In order to recombinant production antibody of the present invention, be separated its nucleic acid of coding, and be inserted into replicable vector, for cloning (DNA cloning) further or expressing.Old process can be used to be easy to be separated the DNA of encoding antibody and to check order (as used the oligonucleotide probe that can be combined with the gene specific of encoding antibody heavy and light chain).Many carriers can be utilized.The selection of carrier depends in part on the host cell that will use.Host cell includes but not limited to what protokaryon or eucaryon (normally Mammals) originated from.Will be appreciated that the constant region of any isotype can be used for this object, comprise IgG, IgM, IgA, IgD and IgE constant region, and this type of constant region can obtain from anyone or animal species.

prokaryotic host cell is used to generate antibody:

Vector construction

Standard recombinant techniques can be used to obtain the polynucleotide sequence of code book invention antibody polypeptides component.Can the polynucleotide sequence of expectation be separated from antibody-producting cell such as hybridoma and check order.Or, nucleotide synthesizer or round pcr synthetic polyribonucleotides can be used.Once obtain, the sequence of coded polypeptide is inserted and can copy in prokaryotic hosts and the recombinant vectors of expressing heterologous polynucleotide.In order to the present invention, this area can be used obtainable and the many carriers known.The selection of appropriate carrier by depend primarily on will insertion vector nucleic acid size and will with the concrete host cell of vector.According to its function (increase or expressing heterologous polynucleotide, or the two furthermore) and the consistency with its concrete host cell resident wherein thereof, often kind of carrier contains multiple component.Support element generally includes but is not limited to replication orgin, selected marker gene, promotor, ribosome bind site (RBS), signal sequence, heterologous nucleic acids Insert Fragment and transcription termination sequence.

Generally speaking, the plasmid vector used together with host cell comprises derived from the replicon and control sequence with these host compatibility species.Carrier carries replication site usually, and can provide the flag sequence of Phenotypic Selection in transformant.Such as, usually with the pBR322 plasmid transformation of E. coli derived from species Escherichia coli.PBR322 comprises the gene of encoding ampicillin (Amp) and tsiklomitsin (Tet) resistance, provides the means of light identification of transformed cell thus.PBR322, its derivative or other microorganism plasmid or phage also can comprise or modified and comprise can by microorganism organism for expressing the promotor of endogenous protein.The people such as Carter, United States Patent (USP) 5,648, describes the example of the pBR322 derivative for expressing specific antibodies in detail in 237.

In addition, the phage vector comprising the replicon compatible with host microorganism and control sequence can be used as the conversion carrier of these hosts.Such as, phage such as λ GEM.TM.-11 can be used to build the recombinant vectors that can be used for transform susceptible host cells such as intestinal bacteria LE392.

Expression vector of the present invention can comprise two or more promotor-cistrons pair, and they are encoded each polypeptide component.Promotor is the untranslated regulating and controlling sequence being positioned at cistron upstream (5 '), the expression of its regulation and control cistron.Prokaryotic promoter is divided into two classes usually, induction type with composition.Inducible promoter refer to respond culture condition change (as nutraceutical existence whether or temperature variation) and start the promotor that the elevated levels by the cistron of its control transcribes.

Be subject to a large amount of promotors of multiple potential host cell identification as everyone knows.Digest by restriction enzyme the promotor that cuts in source DNA and the promoter sequence of separation is inserted carrier of the present invention, the cistron DNA of the promotor of selection with coding light chain or heavy chain can be operatively connected thus.Native promoter sequence and many allogeneic promoters all can be used for amplification and/or the expression of instructing target gene.In some embodiment, use allogeneic promoter, because compared with native target polypeptide promotor, they allow that the higher of expressed target gene is transcribed and higher output yield usually.

The promotor being applicable to prokaryotic hosts comprises PhoA promotor, beta-galactosidase enzymes and lactose promoter system, tryptophane (trp) promoter systems and hybrid promoter such as tac or trc promotor.But, in bacterium, there is other promotor of function (such as other known bacterium or phage promoter) to be also suitable.Their nucleotide sequence is delivered, skilled work personnel can use thus provides the joint of any required restriction site or adapter their cistrons with coding target light chain and heavy chain to be operatively connected (Siebenlistetal., Cell20:269 (1980)).

In one aspect of the invention, each cistron in recombinant vectors comprises the secretory signal sequence component that the expressed polypeptide of guidance wears film transhipment.Generally speaking, signal sequence can be the component of carrier, or it can be a part for the target polypeptid DNA of insertion vector.The signal sequence selected in order to the present invention should be the signal sequence being subject to host cell identification and processing (namely being excised by signal peptidase).The prokaryotic host cell of the signal sequences native of heterologous polypeptide is processed for nonrecognition, the signal sequence prokaryotic signal sequence being selected from such as lower group to be substituted: alkaline phosphatase, penicillinase, Ipp or heat-staple enterotoxin 1 I (STII) leader sequence, LamB, PhoE, PelB, OmpA and MBP.In one embodiment of the invention, the signal sequence all used in two cistrons of expression system is STII signal sequence or its variant.

On the other hand, the generation according to immunoglobulin (Ig) of the present invention can occur in the tenuigenin of host cell, does not therefore need to there is secretory signal sequence in each cistron.In that, light chain immunoglobulin and heavy chain are expressed, are folded and assemble and form Functional immunoglobulin in tenuigenin.Some host strain (as intestinal bacteria trxB-bacterial strain) provides the tenuigenin condition being beneficial to disulfide formation, thus allows the correct folding and assembling of expressed protein subunit.ProbaandPluckthun，Gene159：203(1995))。

Antibody of the present invention can use following expression system to generate, and wherein the quantity ratios of expressed polypeptide component can be regulated and controled, thus will secrete and the maximum production of the antibody of the present invention of correct assembling.This regulation and control are realized by the translation intensity regulating and controlling polypeptide component simultaneously at least partly.

The people such as Simmons, United States Patent (USP) 5,840, discloses a kind of technology for regulating and controlling translation intensity in 523.It utilizes the variant of Translation initiator (TIR) in cistron.For appointment TIR, can create and have certain limit translation a series of amino acid of intensity or nucleotide sequence variants, what provide the expectation expression level for specific chains to regulate this factor thus facilitates means.The codon change that can change aminoacid sequence is caused to generate TIR variant by conventional mutagenesis techniques.In certain embodiments, the change in nucleotide sequence is reticent.Change in TIR can comprise the such as number of Shine-Dalgarno sequence or the change of spacing, and the change in signal sequence.Generate at the beginning of encoding sequence " password word bank " (i.e. change be reticent) not changing signal sequence aminoacid sequence for generating a kind of method of mutant signal sequences.This realizes by the 3rd nucleotide position changing each codon; In addition, some amino acid, such as leucine, Serine and arginine, have multiple first and second position, and this can increase complicacy building in storehouse.Yansuraetal., this mutafacient system is described in detail in METHODS:ACompaniontoMethodsinEnzymol.4:151-158 (1992).

In one embodiment, for each cistron in carrier, generate one group of carrier with certain limit TIR intensity.This finite aggregate provides the expression level of every bar chain and expects the comparison of the output of antibody products under various TIR intensity combination.Expression level by quantizing reporter gene measures TIR intensity, the people such as Simmons, and United States Patent (USP) 5,840, has a detailed description in 523.According to the comparison of translation intensity, the indivedual TIR expected are selected to combine in expression vector establishment thing of the present invention.

The prokaryotic host cell being suitable for expressing antibody of the present invention comprises archeobacteria (Archaebacteria) and eubacterium (Eubacteria), such as Gram-negative or gram-positive organism.The example of useful bacterium comprises Escherichia (Escherichia) (as colon bacillus E.coli), bacillus (Bacillus) (as subtilis B.subtilis), enterobacter (Enterobacteria), Rhodopseudomonas (Pseudomonas) (as Pseudomonas aeruginosa P.aeruginosa) species, Salmonella typhimurium (Salmonellatyphimurium), serratia marcescens (Serratiamarcescans), Klebsiella (Klebsiella), proteus (Proteus), Shigella (Shigella), rhizobium (Rhizobium), Vitreoscilla (Vitreoscilla), or paracoccus (Paracoccus).In one embodiment, gram-negative cells is used.In one embodiment, use Bacillus coli cells as host of the present invention.The example of coli strain comprises bacterial strain W3110 (Bachmann, CellularandMolecularBiology, the 2nd volume, Washington, D.C., American Academy Of Microbiology, 1987,1190-1219 page; ATCC preserving number 27,325) and derivative, comprise the bacterial strain 33D3 (U.S. Patent number 5,639,635) with genotype W3110 Δ fhuA (Δ tonA) ptr3lacIqlacL8 Δ ompT Δ (nmpc-fepE) degP41kanR.Other bacterial strain and derivative thereof, such as intestinal bacteria 294 (ATCC31,446), intestinal bacteria B, intestinal bacteria λ 1776 (ATCC31,537) and intestinal bacteria RV308 (ATCC31,608) are also suitable.These examples just illustrate and unrestricted.This area is known for building the method having and specify genotypic any above-mentioned bacterial derivation thing, see such as Bassetal., Proteins8:309-314 (1990).Usually must consider that the reproducibility of replicon in bacterial cell selects the bacterium be suitable for.Such as, when using well-known plasmid such as pBR322, pBR325, pACYC177 or pKN410 to provide replicon, intestinal bacteria, serratia or Salmonella ssp may be suitable for use as host.Usually, host cell should secrete the proteolytic ferment of minimum, and may wish to mix extra proteinase inhibitor in cell cultures.

Antibody tormation

With above-mentioned expression vector transformed host cell, and cultivate in the conventional nutrient culture suitably changed expecting the gene of sequence in order to evoked promoter, selection transformant or amplification coding.

Transform and import prokaryotic hosts by DNA, DNA can be copied, or as extra-chromosomal element or pass through chromosomal composition.According to host cell used, the standard technique being suitable for these cells is used to transform.The Calcium treatment of calcium chloride is adopted to be generally used for the bacterial cell with firm cell-wall barriers.Another kind of method for transformation adopts polyoxyethylene glycol/DMSO.What use also has a kind of technology to be electroporation.

That know in this area and be suitable for cultivating in the substratum of selected host cell the prokaryotic cell prokaryocyte cultivated for generating polypeptide of the present invention.The example of suitable culture medium comprises the LB substratum (Luriabroth) that with the addition of required nutritional supplement.In some embodiment, the selective agent that substratum is also selected containing the structure of with good grounds expression vector, allows the prokaryotic cell prokaryocyte growth comprising expression vector with selectivity.Such as, in the substratum of the cell for culture expression ampicillin resistance gene, penbritin is added.

Except carbon, nitrogen and inorganic phosphate sources, also can any required fill-in containing proper concn, or to add separately or as the mixture with another kind of fill-in or substratum, such as compound nitrogen source.Optional, substratum can be selected from the reductive agent of lower group containing one or more: gsh, halfcystine, cystamine, thioglycolate salt/ester, dithioerythritol and dithiothreitol (DTT).

Prokaryotic host cell is cultivated in suitable temperature.Such as, for cultivation intestinal bacteria, the temperature range of carrying out cultivating includes but not limited to about 20 DEG C to about 39 DEG C, about 25 DEG C to about 37 DEG C and about 30 DEG C.Depend primarily on host organisms, the pH of substratum can be scope be about 5 to about 9 any pH.For intestinal bacteria, pH can be about 6.8 to about 7.4 or about 7.0.

If use inducible promoter in expression vector of the present invention, be so suitable for induced protein expression under the condition activating promotor.In one aspect of the invention, PhoA promotor is used to control transcribing of polypeptide.Therefore, in order to induce, in phosphoric acid salt restriction substratum, cultivate the host cell through transforming.In one embodiment, phosphoric acid salt restriction substratum is C.R.A.P substratum (see such as Simmonsetal., J.Immunol.Methods263:133-147 (2002)).According to adopted vector construct, other inductor multiple can be adopted, as is known in the art.

In one embodiment, expressed polypeptide of the present invention is secreted in the pericentral siphon of host cell and also therefrom reclaims.Protein recovery involves destroy microorganisms usually, usually by means such as such as osmotic shock (osmoticshock), supersound process or cracking.Once cell is destroyed, by centrifugal or filter clear cell debris or whole cell.Protein can be further purified by such as affine resin chromatography.Or protein may be transported in nutrient solution and also therefrom be separated.From nutrient solution scavenger cell, and culture supernatants can be filtered and concentrates, for being further purified generated protein.Protein expressed by the method such as polyacrylamide gel electrophoresis (PAGE) and the further separation andpreconcentration of western blot analysis generally known can be used.

In one aspect of the invention, antibody producing is carried out in a large number by fermenting process.Multiple extensive fed-batch fermentation flow process can be used for Restruction albumen.Large scale fermentation has the capacity of at least 1000 liters, and such as about 1, the capacity of 000 to 100,000 liter.These fermentor tanks use agitator paddle to distribute oxygen and nutrient, especially glucose (conventional carbon source/energy).On a small scale fermentation is often referred to and is no more than at volume capacity the fermentation carried out in the fermentor tank of about 100 liters, and scope can be about 1 rise to about 100 liters.

During the fermentation, usually expect that density (as OD550 is about 180-220, being in early stage stationary phase at this phase cell) starts the induction of protein expression afterwards being cultured under suitable conditions by cell.According to adopted vector construct, multiple inductor can be used, know as this area with above-described.Can by the time shorter for cell cultures before induction.Usually cell induction is about 12-50 hour, but longer or shorter induction time can be used.

In order to improve the seed output and quality of polypeptide of the present invention, multinomial fermentation condition can be revised.Such as, in order to improve the correct assembling of secreted antibody polypeptides and fold, the additional carrier expressing chaperone such as Dsb albumen (DsbA, DsbB, DsbC, DsbD and/or DsbG) or FkpA (having a kind of peptidyl prolyl-cis of Chaperone Activity, trans-isomerase) that can overuse carrys out cotransformation host prokaryotic cell.Prove that chaperone promotes the correct folding and solubleness of the heterologous protein generated in bacterial host cell.Chenetal., J.Biol.Chem.274:19601-19605 (1999); The people such as Georgiou, United States Patent (USP) 6,083,715; The people such as Georgiou, United States Patent (USP) 6,027,888; BothmannandPluckthun, J.Biol.Chem.275:17100-17105 (2000); RammandPluckthun, J.Biol.Chem.275:17106-17113 (2000); Arieetal., Mol.Microbiol.39:199-210 (2001)).

In order to be down to minimum by the proteolysis of expressed heterologous protein (especially to the heterologous protein of proteolysis sensitivity), some host strain of proteolysis enzyme defect can be used for the present invention.Such as, can host cell strains be modified, in the gene of the known bacteria protease of coding, carry out genetic mutation, such as proteinase II I, OmpT, DegP, Tsp, proteolytic enzyme I, proteolytic enzyme Mi, proteolytic enzyme V, proteolytic enzyme VI and combination thereof.Can obtain some e. coli protein enzyme defect bacterial strain, see such as Jolyetal., (1998) see above; The people such as Georgiou, United States Patent (USP) 5,264,365; The people such as Georgiou, United States Patent (USP) 5,508,192; Haraetal., MicrobialDrugResistance2:63-72 (1996).

In one embodiment, in expression system of the present invention, proteolysis enzyme defect is used and through the coli strain of the Plastid transformation of one or more chaperones of overexpression as host cell.

Antibody purification

In one embodiment, the antibody protein that generates is further purified herein to obtain the goods of homogeneity substantially, for further measuring and using.The Standard protein purification method that this area is known can be adopted.Flow process is below the illustration of appropriate purification flow process: the chromatography on the fractionation on the affine or ion exchange column of immunity, alcohol settling, reversed-phase HPLC, tripoli or Zeo-karb such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation and use the gel-filtration of such as SephadexG-75.

In one aspect, the albumin A be fixed in solid phase is used for the immunoaffinity purification of antibody products of the present invention.Albumin A is the 41kD cell wall protein from streptococcus aureus (Staphylococcusaureas), and it is with high-affinity binding antibody Fc district.Lindmarketal.，J.Immunol.Meth.62：1-13(1983))。The solid phase that albumin A is fixed thereon can be the pillar with glass or quartz surfaces, or controlled pore glass post or silicic acid post.In some applications, pillar with pack quilts such as such as glycerine, in order to the non-specific adhesion of likely preventing pollution thing.

As the first step of purifying, the prepared product derived from cell culture described above can be applied in albumin A immobilization solid phase, make object antibody Specific binding proteins A.Then solid phase is cleaned to remove the pollutent with solid phase non-specific binding.Object antibody is reclaimed from solid phase finally by wash-out.

eukaryotic host cell is used to generate antibody:

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.

(i) signal sequence component

The carrier used in eukaryotic host cell also can comprise signal sequence at the N end of object mature protein or polypeptide or have other polypeptide of special cleavage site.General selection is subject to host cell identification and processes the Heterologous signal sequences of (namely being excised by signal peptidase).In mammalian cell expression, can utilize mammalian signal sequences and viral secretory leading, such as herpes simplex gD signal.

The DNA of these prosomas is connected in the reading frame of the DNA of encoding antibody.

(ii) replication orgin

Usually, mammalian expression vector does not need replication orgin component.Such as, SV40 starting point may only just use because comprising early promoter usually.

(iii) Select gene component

Cloning and expression carrier can comprise Select gene, also referred to as selection marker.Typical Select gene is encoded following protein: (a) gives the resistance to microbiotic or other toxin, as penbritin, Liu Suanyan NEOMYCIN SULPHATE, methotrexate or tsiklomitsin; B () supplies corresponding auxotrophy; Or (c) provides the critical nutrients that can not obtain from complex medium.

An example of selection scheme utilizes medicine to block the growth of host cell.Those Hemapoiesis through heterologous gene successful conversion give the protein of drug resistance, thus survive selection scheme.The example of this type of dominant selection uses drug neomycin, mycophenolic acid and Totomycin.

Another example being suitable for the selection marker of mammalian cell is the selection marker of the cell can identifying picked-up antibody nucleic acids of having the ability, such as DHFR, thymidine kinase, metallothionein(MT) I and II (such as primate metallothionein gene), adenosine deaminase, ornithine decarboxylase etc.

Such as, can first by all transformants being carried out in the substratum containing methotrexate (a kind of competitive antagonist of Mtx, DHFR) cultivate to identify the cell transformed through DHFR Select gene.When adopting wild-type DHFR, suitable host cell comprises Chinese hamster ovary (CHO) clone (as ATCCCRL-9096) of such as DHFR active defects.

Or, by containing for the selective agent such as aminoglycoside antibiotics of selection marker as the substratum of kantlex, Liu Suanyan NEOMYCIN SULPHATE or G418 in culturing cell select the DNA sequence dna of encoded antibody, wild type DHFR protein and another kind of selection marker such as aminoglycoside 3 '-phosphotransferase (APH) to transform or the host cell (particularly comprising the wild-type host of endogenous DHFR) of cotransformation.See United States Patent (USP) 4,965,199.

(iv) promotor component

Cloning and expression carrier comprises the promotor being subject to host organisms identification usually, and is operatively connected with the nucleic acid of encoding polypeptides of interest (such as antibody).Known eukaryotic promoter sequence.In fact, all eukaryotic genes all have and are rich in AT district, and it is positioned at site upstream about 25 to 30 base places of initiation transcription.Be CNCAAT district permitting the another kind of sequence that 70 to 80 base places, polygenic transcriptional start point upstream find, wherein N can be any Nucleotide.Holding at 3 ' of most of eukaryotic gene is AATAAA sequence, and it may be the signal of 3 ' end interpolation polyadenylic acid (polyA) tail to encoding sequence.In the insertion carrier for expression of eukaryon that all these sequences are suitable.

Can be subject to such as obtaining from virus (such as polyomavirus, fowlpox virus, adenovirus (such as 2 type adenovirus), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and simian virus 40 (SV40)) genome by carrier Antibody transcription polypeptide in mammalian host cell, from heterologous mammal promotor (as actin promoter or immunoglobulin promoter) or the control of promotor from heat-shock promoters, if the words that these promotors are compatible with host cell systems.

Obtain the early stage of SV40 virus and late promoter with the form of SV40 restriction fragment easily, this fragment also comprises SV40 virus origin of replication.The immediate early promoter of human cytomegalic inclusion disease virus is obtained easily with the form of HindIIIE restriction fragment.United States Patent (USP) 4,419, discloses the system using bovine papilloma virus as carrier expressible dna in mammalian hosts in 446.United States Patent (USP) 4,601, describes the one amendment of this system in 978.Also can see Reyesetal., Nature297:598-601 (1982) about the control following table intelligent beta-interferon cDNA at the thymidine kinase promoter from hsv in mouse cell.Or, Rous sarcoma virus long terminal repeat can be used as promotor.

(v) enhancer element component

Usually can improve higher eucaryotic cells transcribing the DNA of code book invention antibody polypeptides by inserting enhancer sequence in the carrier.It is now know that from many enhancer sequence of mammalian genes (sphaeroprotein, elastoser, white protein, α-fetoprotein and Regular Insulin).But, usually use the enhanser from eukaryotic cell virus.Example comprises the enhanser (bp100-270) of SV40 replication orgin side in late period, the sub-enhanser of cytomegalovirus early promoter, the enhanser of polyomavirus replication orgin side in late period and adenovirus cancers.Also can see Yaniv, Nature297:17-18 (1982) about the enhancing element activating eukaryotic promoter.Enhanser can montage in carrier, be positioned at 5 ' or 3 ' position of antibody polypeptides encoding sequence, but be generally positioned at 5 ' site of promotor.

(vi) Transcription Termination component

The expression vector used in eukaryotic host cell usually also comprises termination and transcribes the necessary sequence with stable mRNA.This type of sequence can obtain from 5 ' end of eucaryon or viral DNA or cDNA non-translational region with 3 ' end once in a while usually.The non-translational region transcription that these regions are included in the mRNA of encoding antibody becomes the nucleotide segment of polyadenylated fragments.A kind of useful Transcription Termination component is Trobest polyadenylation district.See WO94/11026 and disclosed in expression vector.

(vii) selection of host cell and conversion

The host cell being suitable for the DNA cloning or express in this paper carrier comprises higher eucaryotic cells described herein, comprises vertebrate host cell.The breeding of vertebrate cells in cultivation (tissue culture) becomes old process.The example of useful mammalian host cell line has monkey kidney CV1 system (COS-7, ATCCCRL1651) transformed through SV40, human embryo kidney (HEK) system (293 cells or for suspension culture and 293 cells of subclone, Grahametal., J.Gen.Virol.36:59 (1977)), baby hamster kidney cell (BHK, ATCCCCL10), Chinese hamster ovary cell/-DHFR (CHO, Urlaubetal., Proc.Natl.Acad.Sci.USA77:4216 (1980)), mouse Sai Tuoli (Sertoli) cell (TM4, Mather, Biol.Reprod.23:243-251 (1980)), monkey-kidney cells (CV1, ATCCCCL70), African green monkey kidney cell (VERO-76, ATCCCRL1587), human cervical carcinoma cell (HELA, ATCCCCL2), Madin-Darby canine kidney(cell line) (MDCK, ATCCCCL34), ox mouse (buffalorat) liver cell (BRL3A, ATCCCRL1442), human pneumonocyte (W138, ATCCCCL75), human liver cell (HepG2, HB8065), mouse mammary tumor (MMT060562, ATCCCCL51), TRI cell (Matheretal., AnnalsN.Y.Acad.Sci.383:44-68 (1982)), MRC5 cell, FS4 cell and human liver cell knurl (hepatoma) are (HepG2).

In order to generate antibody, with expression mentioned above or cloning vector transformed host cell, and cultivate in the conventional nutrient culture suitably changed expecting the gene of sequence in order to evoked promoter, selection transformant or amplification coding.

(viii) cultivation of host cell

The host cell for generating antibody of the present invention can be cultivated in multiple substratum.Commercially available culture medium is HamShi F10 (Sigma), minimum essential medium (MEM such as, Sigma), RPMI-1640 (Sigma) and DulbeccoShi amendment EagleShi substratum (DMEM, Sigma) is suitable for cultivating host cell.In addition, any substratum of recording in following documents can be used as the substratum of host cell: Hametal., Meth.Enz.58:44 (1979); Barnesetal., Anal.Biochem.102:255 (1980); United States Patent (USP) 4,767,704; 4,657,866; 4,927,762; 4,560,655; 5,122,469; WO90/03430; WO87/00195; Or United States Patent (USP) review 30,985.These substratum any can 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 (such as GENTAMYCIN as required ^tMmedicine), trace elements (be defined as usually exist with the final concentration of micro-molar range mineral compound) and glucose or the equivalent energy.Can also suitable concentration contain those skilled in the art will know that any other must fill-in.The host cell that culture condition such as temperature, pH etc. are expression and select is previously used, and this is obvious for those of ordinary skill.

(ix) purifying of antibody

When using recombinant technology, antibody can be generated in cell, or direct secretion is in substratum.If generate antibody in cell, so first generally remove particle debris by such as centrifugal or ultrafiltration, or host cell or crack fragment.If antibody-secreting is in substratum, first commodity in use protein concentration filter (such as Amicon or MilliporePellicon ultra filtration unit) concentrates the supernatant liquor from these expression systems so usually.Proteinase inhibitor such as PMSF can be comprised in any above-mentioned steps to be hydrolyzed with arrestin, and microbiotic can be comprised to prevent the growth of external contaminant.

The antibody compositions that such as hydroxyapatite, gel electrophoresis, dialysis and affinity chromatography (general acceptable purification technique is affinity chromatography) can be used to carry out purifying prepare from cell.Affinity reagent such as albumin A depends on kind and the isotype of any immunoglobulin Fc domain existed in antibody as the suitability of affinity ligand.Albumin A can be used for the antibody (Lindmarketal., J.Immunol.Meth.62:1-13 (1983)) of purifying based on people γ 1, γ 2 or γ 4 heavy chain.Protein G recommends to be used for all mouse isotypes and people γ 3 (Gussetal., EMBOJ.5:1567-1575 (1986)).What the matrix accompanying by affinity ligand was the most frequently used is agarose, but can use other matrix.The matrix of physically stable such as controlled pore glass or poly-(styrene divinyl) benzene can obtain than agarose flow velocity and shorter process period faster.If antibody comprises CH3 structural domain, then BakerbondABX can be used ^tMresin (J.T.Baker, Phillipsburg, NJ) carries out purifying.According to antibody to be recycled, the chromatography on the fractionation on other oroteins purification technique such as ion exchange column, alcohol settling, reversed-phase HPLC, tripoli, heparin SEPHAROSE also can be used ^tMon chromatography, negatively charged ion or Zeo-karb (such as poly aspartic acid post) on chromatography, chromatofocusing, SDS-PAGE and ammonium sulfate precipitation.

After any any preliminary purification step, mixture containing object antibody and pollutent can be carried out further purification step where necessary, such as low pH hydrophobic interaction chromatography, use pH to be about the elution buffer of 2.5-4.5, generally carry out at low salt concn (according to appointment 0-0.25M salt).

It should be noted that generally speaking, is that this area has been improved and set up for the preparation of the techniques and methods of antibody for research, test and Clinical practice, and is consistent above and/or those skilled in the art think that for specific antibody interested be suitable.

activation measurement

The many measure method known by this area is to their physical/chemical properties of Identification of the antibodies of the present invention and biological function.

Characterize the antibody of purifying by a series of assay method further, include but not limited to that N holds order-checking, amino acid analysis, non denatured size exclusion high pressure liquid chromatography (HPLC) (HPLC), mass spectrum, ion exchange chromatography and papain digestion.

Where necessary, antagonist analyzes their biologic activity.In some embodiment, to their antigen-binding activity of antibody test of the present invention.This area is known and the antigen binding assay that can be used for herein include but not limited to use the technology such as such as western blot, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), " sandwich " immunoassay, immunoprecipitation assay, fluorescence immunoassay and protein A immunoassays any directly or competitive binding assay method.

In one embodiment, present invention contemplates and have some but the improvement antibody of not all effector functions, this makes it be important but some effector function (such as complement and ADCC) becomes the material standed for of expectation in being unnecessary or harmful many application at antibody Half-life in vivo.In certain embodiments, the Fc measuring antibody is active in guarantee the characteristic only remaining expectation.External and/or in vivo cytotoxicity assay method can be carried out to confirm the reduction/abatement of CDC and/or ADCC activity.Such as, Fc acceptor (FcR) binding assay can be carried out to confirm antibody deficiency Fc γ R combination (therefore likely lacking ADCC active) but to retain FcRn binding ability.The main cell of mediation ADCC, NK cell, only expresses Fc γ RIII, and monocytes Fc γ RI, Fc γ RII and Fc γ RIII.The FcR that RavetchandKinet, Annu.Rev.Immunol.9:457-92 (1991) the 464th page table 3 summarizes on hematopoietic cell expresses.United States Patent (USP) 5,500,362 or 5,821, describe the example of the vitro assay of the ADCC activity for assessment of molecules of interest in 337.The effector cell that can be used for this type of assay method comprises peripheral blood lymphocytes (PBMC) and NK cell (NK) cell.Or/in addition, can the ADCC of purpose of appraisals molecule in vivo active, as in animal model, disclosed in such as Clynesetal., PNAS (USA) 95:652-656 (1998).Also can carry out C1q binding assay active to confirm that antibody can not lack CDC in conjunction with C1q and therefore.In order to assess complement activation, CDC assay method can be carried out, such as, as described in Gazzano-Santoroetal., J.Immunol.Methods202:163 (1996).The method that this area also can be used to know is carried out FcRn and is combined and removing/transformation period mensuration in body.

Antibody fragment

Antibody fragment is contained in the present invention.In some cases, antibody fragment is used to have superiority, instead of complete antibody.The reduced size of fragment allows quick removing, and can cause being easier to close to solid tumor.

The multiple technologies for generating antibody fragment are developed.Traditionally, these fragments derivative are carried out (see such as Morimotoetal., JournalofBiochemicalandBiophysicalMethods24:107-117 (1992) by proteolytic digestion complete antibody; Brennanetal., Science229:81 (1985)).But, directly can generate these fragments by recombinant host cell now.Fab, Fv and scFv antibody fragment all at expression in escherichia coli and by E. coli secretion, so can be allowed and is easy to generate these a large amount of fragments.Can from phage antibody library discussed above isolated antibody fragment.Or, directly can reclaim Fab '-SH fragment chemical coupling to form F (ab ') 2 fragment (Carteretal., Bio/Technology10:163-167 (1992)) from intestinal bacteria.According to another kind of method, directly F (ab ') 2 fragment can be separated from recombinant host cell culture.Comprise salvage receptor binding epitope residue, there is Fab and F (ab ') 2 fragment of the Half-life in vivo of prolongation be recorded in U.S. Patent No. 5,869,046.Other technology for generating antibody fragment will be apparent for skilled practitioner.In other embodiments, the antibody of selection is Single-Chain Fv Fragment of Murine (scFv).See WO93/16185; U.S. Patent No. 5,571,894; And 5,587,458.Fv and sFv is the unique type having entire binding site, lack constant region; So, non-specific binding is reduced when they are suitable for using in vivo.SFv fusion rotein can be built to generate the amino of effector protein at sFv or the fusion of C-terminal.Compile see AntibodyEngineering, Borrebaeck, supra.Antibody fragment can also be " linear antibodies ", such as, as U.S. Patent No. 5, and 641, described in 870.This type of linear antibody fragments can be monospecific or dual specific.

Humanized antibody

Humanized antibody is contained in the present invention.The multiple method for humanizing non-human antibodies is known in this area.Such as, humanized antibody can have one or more amino-acid residue introduced from inhuman source.These non-human amino acid residues are usually called " input " residue, they are taken from usually " input " variable region.Substantially the method can following Winter and colleague thereof carries out humanization (Jonesetal., Nature321:522-525 (1986); Riechmannetal., Nature332:323-327 (1988); Verhoeyenetal., Science239:1534-1536 (1988)), the corresponding sequence of people's antibody is namely substituted with inhuman hypervariable region sequence.Therefore, this type of " humanization " antibody is chimeric antibody (United States Patent (USP) 4,816,567), is wherein significantly less than the complete people variable region corresponding sequence of non-human species and substitutes.In practice, humanized antibody is normally as servant's antibody, and wherein the residue in some some hypervariable region residues and some possible similar site of FR residue rodent antibodies substitutes.

May be very important for the preparation of people's light chain of humanized antibody and the selection of variable region of heavy chain for reduction antigenicity.According to so-called " the suitableeest (best-fit) " method, screen with the whole library of variable region sequences to known person variable region sequences of rodent antibodies.Then select and people's framework (Simsetal., the J.Immunol.151:2296 (1993) of the immediate human sequence of rodents as humanized antibody; Chothiaetal., J.Mol.Biol.196:901 (1987)).Another kind method uses by the derivative specific frame of the consensus sequence of everyone antibody of specific light chain or heavy chain subclass (subgroup).Identical frames can be used for several different humanized antibody (Carteretal., Proc.Natl.Acad.Sci.USA89:4285 (1992); Prestaetal., J.Immunol.151:2623 (1993)).

General wish that antibody retains high-affinity to antigen and other favourable biological characteristics after humanization further.In order to reach this object, according to a kind of method, prepare humanized antibody by the process using the three-dimensional model of parental array and humanized sequence to analyze parental array and various conceptual humanized products.Usually can adaptive immune sphaeroprotein three-dimensional model, this be those skilled in the art be familiar with.Also can obtain the computer program of the possible three-dimensional conformation structure of diagram and the selected candidate immunoglobulin sequences sequence of display.By checking that these display images can be analyzed residue and play may act in function in candidate immunoglobulin sequences sequence, namely analyzing influence candidate immunoglobulin sequences is in conjunction with the residue of the ability of its antigen.Like this, can from receptor sequence and list entries, select FR residue and combine, thus obtain the antibody characteristic of expectation, such as the avidity of target antigen be raised.Generally speaking, some hypervariable region residues directly and most essence involve the impact that antigen is combined.

People's antibody

People of the present invention anti-polyubiquitin antibody can be selected from people's charon phages and shows that the Fv in storehouse clones variable domain sequence and known people's constant domain sequence and builds by combining as mentioned above.Or, the anti-polyubiquitin antibody of human monoclonal of the present invention can be generated by hybridoma method.Human myeloma and mouse-people's heteromyeloma cell lines for generating human monoclonal antibodies are on the books, such as KozborJ.Immunol., 133:3001 (1984); Brodeuretal., MonoclonalAntibodyProductionTechniquesandApplications, pp.51-63 (MarcelDekker, Inc., NewYork, 1987); And Boerneretal., J.Immunol., 147:86 (1991).

Such as, the transgenic animal (such as mouse) lacking and can give birth to human antibodies's full repertoire when endogenous immunoglobulin generates in immunity afterwards are likely created on now.Such as, the suppression completely of deleting and causing endogenous antibody tormation of isozygotying of antibody heavy chain joining region (JH) gene in chimeric and germ line mutant mice has been described.In this type of germ line mutant mice, shift a large amount of human germline immunoglobulin's gene will cause raw human antibodies after antigen is attacked.See such as Jakobovitsetal., Proc.Natl.Acad.Sci.USA90:2551 (1993); Jakobovitsetal., Nature362:255-258 (1993); Bruggermannetal., YearinImmunol.7:33 (1993).

Gene shuffling is also used in and externally derives people's antibody from inhuman (such as rodents) antibody, and wherein people's antibody has the avidity similar to starting non-human antibody and specificity.According to this method, it is also referred to as " the epi-position marking " (epitopeimprinting), the heavy chain of the non-human antibody fragment obtained as described above by display technique of bacteriophage or variable region of light chain employment V domain gene complete or collected works replace, and produce non-human chain-human chain scFv or Fab block polymer group.Non-human chain/human chain is caused to be fitted together to the separation of scFv or Fab with the selection that antigen carries out, wherein human chain has recovered antigen binding site eliminate corresponding non-human chain in one-level phage display clone after, namely epi-position determines the selection of (marking, imprint) human chain mating partner.When repeating this process to replace the non-human chain of residue, obtain people's antibody (see PCTWO93/06213, being disclosed on April 1st, 1993).From traditional to transplant the humanization of the non-human antibody carried out by CDR different, this technology provides the antibody of complete people, and they are containing FR frame or the CDR residue of non-human origins.

Bi-specific antibody

Bi-specific antibody refer to at least two kinds not synantigen there is the monoclonal antibody of binding specificity.In certain embodiments, bi-specific antibody is people's antibody or humanized antibody.In certain embodiments, one of binding specificity is for the polyubiquitin comprising the connection of specific Methionin, and another of binding specificity is for other antigen any.In certain embodiments, bi-specific antibody can in conjunction with two kinds of different polyubiquitins with the connection of different Methionin.Bi-specific antibody can be prepared into full length antibody or antibody fragment (such as F (ab ') ₂bi-specific antibody).

Known in the art for building the method for bi-specific antibody.Traditional, the recombinant production of bi-specific antibody is based on the coexpression of two pairs of heavy chain immunoglobulin-light chains, and wherein two kinds of heavy chains have different specificitys (MillsteinandCuello, Nature305:537 (1983)).Due to the random assignment of heavy chain immunoglobulin and light chain, these hybridomas (four source hybridomas (quadroma)) generate the potential mixture of 10 kinds of different antibodies molecules, wherein only have a kind ofly to have correct bispecific structure.The purifying of the correct molecule usually undertaken by affinity chromatography step quite bothers and Product yields is low.Similar code is disclosed in WO93/08829 and Trauneckeretal., EMBOJ.10:3655 (1991) disclosed in 13 days Mays in 1993.

According to a kind of different embodiment, antibody variable domains and the immunoglobulin (Ig) constant domain sequence will with expectation binding specificity (antibody-antigene binding site) merge.Such as, with comprise at least part of hinge, the heavy chain immunoglobulin constant domain in CH2 and CH3 district merges.In certain embodiments, at least one fusions, existence comprises first CH (CH1) of light chain in conjunction with necessary site.By encode immunoglobulin heavy fusions and, when needed, the DNA of light chain immunoglobulin inserts expression vector separately, and cotransfection is in suitable host organisms.There is provided in the embodiment of optimum yield when the three peptide species chain ratios for building do not wait, this is that the mutual ratio of adjustment three peptide species fragment provides great handiness.But, when at least two peptide species chains cause high yield with same ratio expression or when there is no special meaning in this ratio, likely the encoding sequence of two kinds or all three peptide species chains is inserted an expression vector.

In an embodiment of the method, bi-specific antibody is by a hybrid immunoglobulin heavy chain arm with the first binding specificity, and the hybrid immunoglobulin heavy chain-light chain on another arm is formed (providing the second binding specificity).Due to the separating pathway that the existence of light chain immunoglobulin only in half bispecific molecule is provided convenience, therefore find that this unsymmetrical structure is convenient to the bispecific compound expected to separate with undesired immunoglobulin chain combinations.The method is disclosed in WO94/04690.About generating the further details of bi-specific antibody see such as Sureshetal., MethodsinEnzymology121:210 (1986).

According to another kind of method, the interface between a pair antibody molecule can be transformed, maximize with the per-cent of the heterodimer will reclaimed from recombinant cell culture thing.Interface comprises at least part of antibody constant territory CH3 structural domain.In the method, one or more p1 amino acid side chain larger side chains (such as tyrosine or tryptophane) of first antibody molecular interface are replaced.By by comparatively p1 amino acid side chain (such as L-Ala or the Threonine) replacement of large amino acid side chain, the interface of second antibody molecule produces compensatory " cavity " with the same or similar size of bulky side chain.This provide and improve the mechanism of heterodimer output than other undesired end product such as homodimer.

Bi-specific antibody comprises crosslinked or " Heteroconjugate " antibody.Such as, a kind of antibody in Heteroconjugate thing can coupling, another kind of antibody and vitamin H coupling plain with affinity.Such as, this antibody-like has been proposed to be used in undesired for immune system cell target cell (U.S. Patent No. 4,676,980), and is used for the treatment of HIV (WO91/00360, WO92/00373 and EP03089).Any cross-linking method easily can be used to prepare Heteroconjugate antibodies.Suitable linking agent is well-known in the art, is disclosed in U.S. Patent No. 4,676,980 together with many crosslinking technologicals.

The technology being generated bi-specific antibody by antibody fragment is also described in document.Such as, chemistry can be used to connect and prepare bi-specific antibody.Brennanetal., Science229:81 (1985) describes by proteolysis cutting complete antibody to generate the code of F (ab ') 2 fragment.These fragments are reduced when existence two mercaptan complexing agent sodium arsenite, prevents the formation of intermolecular disulfide bond with two mercaptan of stable vicinity.Then Fab ' the fragment produced is changed into thionitrobenzoate ester (TNB) derivative.Then one of Fab '-TNB derivative is reverted to Fab '-mercaptan again by the reduction of mercaptoethylamine, and mix, to form bi-specific antibody with the another kind of Fab '-TNB derivative of equimolar amount.The bi-specific antibody produced can be used as the selectivity immobilized reagent of enzyme.

Most new progress is convenient to directly reclaim Fab '-SH fragment from intestinal bacteria, these fragments can chemical coupling to form bi-specific antibody.Shalabyetal., J.Exp.Med.175:217-225 (1992) describes the generation of bi-specific antibody F (ab ') 2 molecule of full-length human.By intestinal bacteria separately secretion often kind of Fab ' fragment, and carry out directed chemical coupling in vitro to form bi-specific antibody.The bi-specific antibody of formation like this in conjunction with the cell of process LAN HER2 acceptor and normal human T cells, and can trigger the lytic activity of people's cytotoxic lymphocyte for people's lacteal tumor target thing.

Also describe and directly generate and the multiple technologies being separated bispecific antibody fragment from recombinant cell culture thing.Such as, leucine zipper has been used to generate bi-specific antibody.Kostelnyetal.，J.Immunol.148(5)：1547-1553(1992)。Leucine zipper peptide from Fos with Jun albumen is connected with the Fab ' part of two kinds of different antibodies by gene fusion.Antibody homodimer, is then oxidized to form antibody heterodimer to form monomer in hinge area reduction again.This method also can be used for generating antibody homodimer.Hollingeretal., " double antibody " technology that Proc.Natl.Acad.Sci.USA90:6444-6448 (1993) records provides the replacement mechanism building bispecific antibody fragment.This fragment comprises the heavy chain variable domain (VH) and light-chain variable domain (VL) that are connected by joint, and described joint too short making can not be matched between on same chain two structural domains.Therefore, force complementary VL and the VH structural domain in VH and the VL structural domain in a fragment and another fragment to match, form two antigen binding sites thus.There was reported the another kind strategy by using scFv (sFv) dimer to build bispecific antibody fragment.See Gruberetal., J.Immunol.152:5368 (1994).

Contemplate the antibody having and tire more than two.Such as, three-specific antibody can be prepared.Tuttetal.，J.Immunol.147：60(1991)。

Multivalent antibody

Multivalent antibody can be subject to the internalization (and/or alienation) of the cell of expressing this antibody institute conjugated antigen faster than bivalent antibody.Antibody of the present invention can be to be easy to multivalent antibody that generated by the recombinant expressed of the nucleic acid of encode antibody polypeptides chain, that have three or more antigen binding site (such as tetravalent antibody) (beyond IgM classification).Multivalent antibody can comprise dimerization domain and three or more antigen binding site.Dimerization domain comprises (or consisting of) such as Fc district or hinge area.In this case, antibody will comprise the aminoterminal three or more antigen binding site in Fc district and Fc district.In one embodiment, multivalent antibody comprises (or consisting of) such as three to about eight, or four antigen binding sites.Multivalent antibody comprises at least one polypeptide chain (such as two polypeptide chains), and wherein said polypeptide chain comprises two or more variable domain.Such as, polypeptide chain can comprise VD1-(X1) n-VD2-(X2) n-Fc, and wherein VD1 is the first variable domain, and VD2 is the second variable domain, and Fc is a polypeptide chain in Fc district, X1 and X2 represented amino acid or polypeptide, and n is 0 or 1.Such as, polypeptide chain can comprise: VH-CH1-flexible joint-VH-CH1-Fc district chain; Or VH-CH1-VH-CH1-Fc district chain.Multivalent antibody herein can comprise at least two (such as four) light chain variable domain polypeptides further.Multivalent antibody herein can comprise such as about two to about eight light chain variable domain polypeptides.The light chain variable domain polypeptide imagined herein comprises light-chain variable domain, and optionally comprises CL structural domain further.

Antibody variants

In some embodiment, contemplate the amino acid sequence modifications of antibody described herein.Such as, the binding affinity and/or other biological characteristics that improve antibody may be wished.The amino acid sequence variation of antibody is by the change of suitable Nucleotide is introduced antibody nucleic acids or prepared by peptide symthesis.This type of is modified the residue comprised in such as antibody amino acids sequence and deletes and/or insert and/or substitute.Any deletion, insertion and alternative combinations can be carried out to obtain final construction, if final construction has the feature of expectation.When preparing sequence, amino acid change can be introduced theme antibody amino acids sequence.

Can be used for identifying in antibody has " alanine scanning mutagenesis ", as described in CunninghamandWells, Science244:1081-1085 (1989) as some residue of preferred mutagenesis position or the method in region.Here, identify that a residue or one group of target residue are (as charged residue, such as arginine, aspartic acid, Histidine, Methionin and L-glutamic acid) and substitute with neutral or electronegative amino acid (such as L-Ala or Polyalanine), to affect the interaction of amino acid and antigen.Then pass through or more or other variant is introduced to alternate site, weighing the amino acid position to alternative display function susceptibility.Thus, although predetermine for the site of introducing variant amino acid sequence, but the essence of sudden change itself need not predetermine.Such as, in order to analyze the consequence of specifying site sudden change, carry out Alanine-scanning or random mutagenesis at target codon or region, and to the activity that expressed immunoglobulin (Ig) screening is expected.

Aminoacid sequence inserts and comprises the fusion of amino and/or C-terminal, and length range, and to be inserted in the sequence of single or multiple amino-acid residue to the polypeptide comprising residue up to a hundred or more by a residue.The example that end inserts comprises the antibody with N end methionyl residue or the antibody merged with cytotoxic polypeptide.Other insertion variant of antibody molecule comprises to be held N or C of antibody and enzyme (as ADEPT) or the peptide fusion extending the antibody serum transformation period.

Another kind of variant is amino acid substitution variant.These variants have the different residue of at least one amino-acid residue to substitute in antibody molecule.The most interesting site of carrying out alternative mutagenesis comprises hypervariable region, but also contemplating FR changes." preferably substitute " hurdle in Table A and show conservative substituting.Cause biologic activity change if this type of substitutes, so can import in Table A and be called the more substantial variations of " illustrating alternative ", or referring below to Amino Acid Classification further described in, and screen product.

Table A

Original Residue	Illustrate and substitute	Preferably substitute
			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)	Tr；Leu；Val；Ile；Ala；Tyr	Tyr
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Val；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 antagonist biological characteristics is modified by selecting to substitute significantly the difference on effect maintaining following aspect to realize: the structure of polypeptide backbone in (a) replacement area, such as (fold) sheet or helical conformation, the electric charge of (b) target site punishment or hydrophobicity, or the volume of (c) side chain.According to the similarity of its side chain properties, amino acid can divide into groups as follows (A.L.Lehninger, " Biochemistry ", the 2nd edition, 73-75 page, WorthPublishers, NewYork, 1975):

(1) nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M)

(2) neutral, polarity: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q)

(3) acid: Asp (D), Glu (E)

(4) alkalescence: Lys (K), Arg (R), His (H)

Or according to common side chain properties, natural generation residue can divide into groups as follows:

(1) hydrophobic: nor-leucine, Met, Ala, Val, Leu, Ile;

(2) neutral, hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acid: Asp, Glu;

(4) alkalescence: His, Lys, Arg;

(5) residue of chain orientation is affected: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substituting needs to replace another classification with the member of one of these classifications.Also this type of alternative residue can be introduced conservative substitution sites, or introduce residue (non-conservative) site.

One class alternative variations involves one or more some hypervariable region residues of alternative parental antibody (such as humanization or people's antibody).Usually, the gained variant being used for exploitation is further selected will to have the biological characteristics of change (such as improving) relative to the parental antibody producing them.A kind of facilitated method for generating this type of alternative variations involves the affinity maturation using phage display.In brief, several hypervariable region sites (such as 6-7 site) is suddenlyd change, produces all possible amino acid replacement in each site.The antibody display of generation like this on filamentous phage particle, as the fusions of at least part of bacteriophage coat protein (such as M13 gene III product) with each particle internal packing.Then the variant to phage display as disclosed herein screens its biologic activity (such as binding affinity).In order to identify the site, candidate hypervariable region for modifying, can carry out scanning (such as Alanine-scanning) mutagenesis and with qualification, antigen being combined to the some hypervariable region residues with significant contribution.Or/in addition, analyze the crystalline structure of antigen-antibody complex to identify that the point of contact between antibody and antigen may be useful.Described contact residues and contiguous residue carry out according to technology known in the art (comprising technology detailed in this article) candidate locus that substitutes.Once produce such variant, use technology known in the art (comprising the techniques described herein) to screen this group variant, the antibody in one or more relevant assay with good characteristic can be selected for further exploitation.

Prepared by the multiple method that the nucleic acid molecule of encoding antibody amino acid sequence variation is known by this area.These methods include but not limited to be separated (when natural generation amino acid sequence variation) from natural origin, or prepare by carrying out oligonucleotide mediated (or fixed point) mutagenesis, PCR mutagenesis and cassette mutagenesis to the comparatively early variant of preparation or the antibody of unmanifest pattern.

May wish to introduce a place in the Fc district of antibody of the present invention or many places amino acid modified, generate Fc region variants thus.Fc region variants can be included in the people Fc region sequence (as human IgG1, IgG2, IgG3 or IgG4Fc district) that one or more amino acid position (comprising hinge cysteine) comprises amino acid modified (as substituted).

Describe and the instruction of this area according to this, contemplate in some embodiment, antibody of the present invention can comprise a place or many places and change compared with the corresponding antibody of wild-type in such as Fc district.Compared with their wild type counterparts, these antibody will identical characteristics substantially required for treatment effect.Such as, think and to carry out in Ke Fc district causing C1q to combine and/or CDC (CDC) changes some change of (namely or strengthen or weaken), such as, described in WO99/51642.Also can see the DuncanandWinter paying close attention to other example of Fc region variants, Nature322:738-40 (1988); United States Patent (USP) 5,648,260; United States Patent (USP) 5,624,821; And WO94/29351.

On the one hand, the invention provides the antibody comprising modification in the interface of the Fc polypeptide forming Fc district, wherein said modification is convenient to and/or is promoted Heterodimerization.These are modified to be included in a Fc polypeptide and introduce protuberance (protuberance), and in the 2nd Fc polypeptide, introduce hole (cavity), wherein said protuberance can be arranged in described hole, thus promotes the compound of the first and second Fc polypeptide.For generating, to have these methods of antibody of modifying be known in the art, such as U.S. Patent No. 5,731, and described in 168.

Immune conjugate

On the one hand, the invention provides and comprise immune conjugate or the antibody-drug conjugates (ADC) that coupling has the antibody of cytotoxic agent, described cytotoxic agent is chemotherapeutics, medicine, growth inhibitor, toxin (enzyme activity toxin or its fragment as bacterium, fungi, plant or animal origin) or radio isotope (namely radiating conjugate) such as.

Antibody-drug conjugates in cancer therapy for purposes (SyrigosandEpenetos, the AnticancerResearch19:605-614 (1999) of the agent of topical delivery Cytotoxic or T suppression cell reagent (namely for killing or the medicine of inhibition tumor cell); Niculescu-DuvazandSpringer, Adv.Drg.Del.Rev.26:151-172 (1997); United States Patent (USP) 4,975,278) allow drug moiety targeting Delivery to tumour, and carry out thin intracellular accumulation there, and these pharmaceutical agents without coupling of systemic application may cause unacceptable to Normocellular toxic level (Baldwinetal., Lancet603-05 (on May 15th, 1986) beyond the tumour cell attempting elimination; Thorpe, " AntibodyCarriersOfCytotoxicAgentsInCancerTherapy:AReview ", in " MonoclonalAntibodies ' 84:BiologicalAndClinicalApplications ", the people such as A.Pinchera compile, 475-506 page, 1985).Attempt thus to obtain maximum effect and minimum toxicity.Polyclonal antibody and monoclonal antibody all have report to can be used for these strategies (Rowlandetal., CancerImmunol.Immunother.21:183-87 (1986)).The medicine used in these methods comprises daunomycin (daunomycin), Dx (doxorubicin), methotrexate (methotrexate) and vindesine (vindesine) (Rowlandetal., 1986, see above).The toxin used in Antibody-toxin conjugate comprises bacteriotoxin such as diphtheria toxin, plant poison such as ricin, small molecule toxins such as geldanamycin (geldanamycin) (Mandleretal., Jour.oftheNat.CancerInst.92 (19): 1573-1581 (2000); Mandleretal., Bioorganic & Med.Chem.Letters10:1025-1028 (2000); Mandleretal., BioconjugateChem.13:786-791 (2002)), maytansinoid class (EP1391213; Liuetal., Proc.Natl.Acad.Sci.USA93:8618-8623 (1996)) and calicheamicin (Lodeetal., CancerRes.58:2928 (1998); Hinmanetal., CancerRes.53:3336-3342 (1993)).Toxin is by comprising tubulin binding, DNA combination or its Cytotoxic of mechanisms play of topoisomerase enzyme level and the effect of T suppression cell.Some cell toxicity medicament be tending towards inactivation when large antibody or protein receptor ligand coupling or active reduce.

(ibritumomabtiuxetan, Biogen/Idec) be antibody-radioisotope element conjugate (Wisemanetal., Eur.Jour.Nucl.Med.27 (7): the 766-77 (2000) be made up of with 111In or the 90Y radio isotope combined by thiourea linker-chelator the mouse IgG1 κ monoclonal antibody for the CD20 antigen found on the surface in normal and malignant B; Wisemanetal., Blood99 (12): 4336-42 (2002); Witzigetal., J.Clin.Oncol.20 (10): 2453-63 (2002); Witzigetal., J.Clin.Oncol.20 (15): 3262-69 (2002)).Although ZEVALIN has the activity for B cell non-Hodgkin's (Hodgkin) lymphoma (NHL), but dispenser causes serious and long hemocytopenia in Most patients.MYLOTARG ^tM(gemtuzumabozogamicin, WyethPharmaceuticals), namely be connected with calicheamicin by people CD33 antibody and the antibody-drug conjugates that forms, be used for through injection for curing acute myelogenous leukemia (DrugsoftheFuture25 (7): 686 (2000) approval in 2000; United States Patent (USP) 4970198; 5079233; 5585089; 5606040; 5693762; 5739116; 5767285; 5773001).Cantuzumabmertansine (ImmunogenInc.), namely be connected with maytansinoid drugs part DM1 through disulfde linker SPP by huC242 antibody and the antibody-drug conjugates that forms, be used for the treatment of cancer such as colorectal carcinoma, carcinoma of the pancreas, cancer of the stomach and other cancer of expressing CanAg in test.MLN-2704 (MillenniumPharm., BZLBiologics, ImmunogenInc.), namely be connected with maytansinoid drugs part DM1 by anti-prostatic specific membrane antigen (PSMA) monoclonal antibody and the antibody-drug conjugates that forms, be used for the potential treatment of tumor of prostate in test.By the synthetic analogues auristatin peptide of dolastatin (dolastatin), auristatinE (AE) and monomethyl auristatin (MMAE) and chimeric mAb cBR96 (special to the LewisY in cancer) and cAC10 (special to the CD30 on Hematological malignancies) coupling (Doroninaetal., NatureBiotechnology21 (7): 778-784 (2003)), and carry out therapeutic exploitation.

(above) describes the chemotherapeutics that can be used for generating immune conjugate herein.Spendable enzyme activity toxin and fragment thereof comprise diphtheria toxin A chain, the nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa Pseudomonasaeruginosa), ricin (ricin) A chain, toxalbumin (abrin) A chain, capsule lotus root toxalbumin (modeccin) A chain, α-broom aspergillin (sarcin), tung oil tree (Aleutitesfordii) toxalbumin, Dianthus caryophyllus L. (dianthin) toxalbumin, dyers' grapes (Phytolacaamericana) toxalbumin (PAPI, PAPII and PAP-S), balsam pear (Momordicacharantia) inhibition, curcin (curcin), crotin (crotin), Saponaria officinalis (sapaonariaofficinalis) inhibition, gelonin (gelonin), NSC-69529 (mitogellin), restrictocin (restrictocin), phenomycin (phenomycin), enomycin (enomycin) and trichothecin (trichothecenes).See such as WO93/21232 disclosed in 28 days October in 1993.Multiple radionuclide can be used for generating radiation coupled antibody.Example comprises 212Bi, 131I, 131In, 90Y and 186Re.The conjugate of antibody and cytotoxic agent can use multiple bifunctional protein coupling agent to prepare, such as N-succinimido-3-(2-pyridyl dithio) propionic ester (SPDP), iminothiolane (IT), imido-ester (all example hydrochloric acid hexanedioyl imido acid dimethyl esters), active ester class (such as suberic acid two succinimido ester), aldehydes (such as glutaraldehyde), double azido compound (such as two (p-azido benzoyl base) hexanediamine), dual azepine derivatives (such as two (p-diazoniumbenzoyl) hexanediamine), vulcabond (such as toluene 2, 6-vulcabond), with double activated fluorine cpd (such as 1, 5-bis-fluoro-2, 4-dinitrobenzene) dual-function derivative.Such as, ricin immunotoxin can be prepared as described in Vitettaetal., Science238:1098 (1987).The 1-isothiocyanic acid phenmethyl-3-methyl diethylene triaminepentaacetic acid(DTPA) (MX-DTPA) of carbon-14 mark is for the exemplary sequestrant by radioactive nucleotides and antibody coupling.See WO94/11026.

Also contemplate antibody herein and one or more small molecule toxins such as calicheamicin (calicheamicin), maytansinoid class (maytansinoids), dolastatin class (dolostatins), aurostatins, trichothecin (trichothecene) and CC1065 and these toxin have the conjugate of the fragment of neurotoxin active.

maytenin and maytansinoid class

In some embodiment, immune conjugate comprises the antibody of the present invention that coupling has one or more maytansinoid molecule.

Maytansinoid class is the mitotic inhibitor by suppressing tubulin polymerization to play a role.Maytenin is separated from East Africa shrub tingia Caulis Mayteni (Maytenusserrata) at first and obtains (United States Patent (USP) 3,896,111).Find that certain micro-organisms also generates maytansinoid class, such as maytansinol and C-3 maytansinol ester (United States Patent (USP) 4,151,042) subsequently.Such as followingly U.S. patents disclose synthesis maytansinol and derivative and analogue: 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; And 4,371,533.

Maytansinoid class drug moiety is attractive drug moiety in antibody drug conjugates, because they: (i) is relatively prepared easily through the chemically modified of fermentation or tunning, derivatize; (ii) the functional group's derivatize with the coupling be suitable for by non-disulfde linker is easy to; (iii) stable in blood plasma; And (iv) is effectively for kinds of tumor cells system.

The exemplary embodiment of maytansinoid class drug moiety comprises DM1, DM3 and DM4.Such as following patent discloses immune conjugate comprising maytansinoid class and preparation method thereof and therepic use: United States Patent (USP) 5,208,020; 5,416,064; And European patent EP 0425235B1, clearly its disclosure is collected herein by reference.Liuetal., Proc.Natl.Acad.Sci.USA93:8618-8623 (1996) describes the immune conjugate being called the maytansinoid of DM1 comprising and be connected with the monoclonal antibody C242 for human colorectal cancer.Find that this conjugate has the high cell toxicity of colon cancer cell for cultivating, and show anti-tumor activity in tumor growth assay method in vivo.Charietal., CancerResearch52:127-131 (1992) describes the wherein maytansinoid immune conjugate through disulfde linker and the murine antibody A7 in conjunction with antigen in CCL188 or the another kind of mouse monoclonal antibody TA.1 coupling in conjunction with HER-2/neu oncogene.The cytotoxicity of TA.1-maytansinoid conjugate is tested in vitro, each cell expressing of this clone 3 × 105 HER-2 surface antigens on human breast cancer cell system SK-BR-3.Drug conjugates reaches the cytotoxicity to a certain degree similar to free maytansinoid drugs, and this improves by the maytansinoid molecule amount increasing each antibody molecule coupling.A7-maytansinoid conjugate shows low systemic cellular toxicity in mouse.

Antibody-maytansinoid conjugate is by be connected antibody with maytansinoid molecular chemistry and prepared by the biologic activity significantly not weakening antibody or maytansinoid molecule.See such as U.S. Patent No. 5,208,020, clearly its disclosure is collected herein by reference.Each antibody molecule coupling average 3-4 maytansinoid molecule shows effect in enhancing is for the cytotoxicity of target cell, and the function of antagonist or solubleness do not have negative impact, although estimate that the use than naked antibody is also strengthened cytotoxicity by the toxin/antibody of an even molecule.Maytansinoid class is well known in the art, and synthesizes by known technology or be separated from natural origin.Such as United States Patent (USP) 5,208,020 and other patent mentioned above and non-Patent Publication thing in disclose suitable maytansinoid class.Maytansinoid class includes but not limited to aromatic nucleus or other position maytansinol analogue through modifying of maytansinol and maytansinol molecule, such as various maytansinol ester.

This area knows that many linking groups can be used for Dispersal risk-maytansinoid conjugate, comprises such as United States Patent (USP) 5,208,020 or European patent 0425235B1; Charietal., CancerResearch52:127-131 (1992); The U.S. Patent application No.10/960 that on October 9th, 2004 submits to, disclosed in 602, clearly its disclosure is collected herein by reference.The U.S. Patent application No.10/960 that the antibody-maytansinoid class conjugate comprising joint member SMCC can be submitted to as on October 8th, 2004, preparing disclosed in 602.Linking group comprises disulphide group, sulfide group, acid-unstable group, photo-labile group, the unstable group of peptase or the unstable group of esterase, disclosed in as mentioned previously in patent.Herein describe and exemplified with other linking group.

Multiple bifunctional protein coupling agent can be used to carry out the conjugate of Dispersal risk and maytansinoid, such as N-succinimido-3-(2-pyridyl dithio) propionic ester (SPDP), succinimido-4-(N-Maleimidomethyl) hexanaphthene-1-carboxylicesters (SMCC), iminothiolane (IT), imido-ester (all example hydrochloric acid hexanedioyl imido acid dimethyl esters), active ester class (such as suberic acid two succinimido ester), aldehydes (such as glutaraldehyde), double azido compound (such as two (p-azido benzoyl base) hexanediamine), dual azepine derivatives (such as two (p-diazoniumbenzoyl)-quadrol), vulcabond (such as toluene 2, 6-vulcabond), with double activated fluorine cpd (such as 1, 5-bis-fluoro-2, 4-dinitrobenzene) dual-function derivative.Coupling agent includes but not limited to N-succinimido-3-(2-pyridyl dithio) propionic ester (SPDP) (Carlssonetal., Biochem.J.173:723-737 (1978)) and N-succinimido-4-(2-pyridylthio) valerate (SPP), provide disulfide linkage to connect thus.

According to the type connected, joint can be attached to multiple positions of maytansinoid molecule.Such as, conventional coupling techniques can be used by forming ester bond with the reaction of hydroxyl.Reaction can occur in there is hydroxyl C-3 position, through methylol modify C-14 position, through the C-15 position of hydroxyl modified and the C-20 position with hydroxyl.In one embodiment, key connection is formed in the C-3 position of maytansinol or maytansinol analogue.

auristatin and dolastatin

In some embodiment, immune conjugate comprises antibody of the present invention (U.S. Patent No. 5,635,483 with dolastatin class (dolastatins) or dolastatin peptide analogs and derivative, the coupling of auristatin class; 5,780,588).Dolastatin class and auristatin class have demonstrated to be disturbed microtubule dynamics, GTP hydrolysis and core and cell fission (Woykeetal (2001) Antimicrob.AgentsandChemother.45 (12): 3580-3584) and has anticancer (US5,663,149) and anti-mycotic activity (Pettitetal (1998) Antimicrob.AgentsChemother.42:2961-2965).Dolastatin or auristatin drug moiety can be attached to antibody (WO02/088172) via the N of peptide drug moiety (amino) end or C (carboxyl) end.

Exemplary auristatin embodiment comprises monomethyl auristatin drug moiety DE and DF that N-end connects, be disclosed in " MonomethylvalineCompoundsCapableofConjugationtoLigands ", U.S.Ser.No.10/983,340, filedNov.5,2004, be clearly collected herein by reference complete for its disclosure.

Exemplary auristatin embodiment comprises MMAE and MMAF.Other the exemplary embodiment comprising MMAE or MMAF and various terminal component (further describing) herein comprises Ab-MC-vc-PAB-MMAF, Ab-MC-vc-PAB-MMAE, Ab-MC-MMAE and Ab-MC-MMAF.

Typically, based on the drug moiety of peptide by forming peptide bond to prepare between two or more amino acid and/or peptide fragment.This type of peptide bond can be prepared according to the such as well-known liquid phase synthesizing method in chemistry of peptides field (see andK.L ü bke, ThePeptides, volume1, pp76-136,1965, AcademicPress).Auristatin/ dolastatin drug moiety can be prepared according to the method in Publication about Document: US5,635,483; US5,780,588; Pettitetal (1989) J.Am.Chem.Soc.111:5463-5465; Pettitetal (1998) Anti-CancerDrugDesign13:243-277; Pettit, G.R., etal.Synthesis, 1996,719-725; Pettitetal (1996) J.Chem.Soc.PerkinTrans.15:859-863; And Doronina (2003) NatBiotechnol21 (7): 778-784; " MonomethylvalineCompoundsCapableofConjugationtoLigands ", U.S. Serial No.10/983,340, on November 5th, 2004 submits to, by its complete be collected herein by reference (disclose and such as prepare joint and the method that such as MMAE and MMAF is coupled to the monomethyl α-amino-isovaleric acid compound of joint).

calicheamicin

In other embodiments, immune conjugate comprises the antibody of the present invention that coupling has one or more calicheamicin molecules.Calicheamicin microbiotic family can generate double-strand DNA cleavage at sub-picomolar concentrations.About the preparation of calicheamicin family conjugate see United States Patent (USP) 5,712,374; 5,714,586; 5,739,116; 5,767,285; 5,770,701; 5,770,710; 5,773,001; With 5,877,296 (all authorizing Cyanamid company of the U.S.).Available calicheamicin analog includes but not limited to γ 1I, α 2I, α 3I, N-ethanoyl-γ 1I, PSAG and θ I1 (Hinmanetal., CancerResearch53:3336-3342 (1993); Lodeetal., CancerResearch58:2925-2928 (1998); And the above-mentioned United States Patent (USP) authorizing Cyanamid company of the U.S.).Can be QFA with the another kind of antitumor drug of antibody coupling, it be a kind of antifolic thing.Calicheamicin and QFA have action site in born of the same parents, and not easily through plasma membrane.Therefore, these reagent greatly strengthen their cytotoxic effect via the cellular uptake of antibody-mediated internalization.

other cytotoxic agent

BCNU, streptozocin (streptozoicin), vincristine(VCR) (vincristine), 5 FU 5 fluorouracil, United States Patent (USP) 5 can be comprised with other antineoplastic agent of antibody coupling of the present invention, 053,394,5,770, the reagent family being referred to as LL-E33288 mixture recorded in 710 and Ai Sibo mycin class (esperamicins) (United States Patent (USP) 5,877,296).

Available enzyme activity toxin and fragment thereof comprise diphtheria toxin A chain, the nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa Pseudomonasaeruginosa), ricin (ricin) A chain, toxalbumin (abrin) A chain, capsule lotus root toxalbumin (modeccin) A chain, α-broom aspergillin (sarcin), tung oil tree (Aleutitesfordii) toxalbumin, Dianthus caryophyllus L. (dianthin) toxalbumin, dyers' grapes (Phytolacaamericana) toxalbumin (PAPI, PAPII and PAP-S), balsam pear (Momordicacharantia) inhibition, curcin (curcin), crotin (crotin), Saponaria officinalis (sapaonariaofficinalis) inhibition, gelonin (gelonin), NSC-69529 (mitogellin), restrictocin (restrictocin), phenomycin (phenomycin), enomycin (enomycin) and trichothecin (trichothecenes).See the WO93/21232 such as announced on October 28th, 1993.

Present invention further contemplates antibody and there is the compound of nucleolysis activity (as rnase or DNA endonuclease, such as deoxyribonuclease; DNA enzymatic) between formed immune conjugate.

In order to selective destruction tumour, antibody can comprise high radioactive atom.Multiple radio isotope can be used for generating radiation coupled antibody.Example comprises At ²¹¹, I ¹³¹, I ¹²⁵, Y ⁹⁰, Re ¹⁸⁶, Re ¹⁸⁸, Sm ¹⁵³, Bi ²¹², P ³², Pb ²¹²with the radio isotope of Lu.When conjugate is used for detecting, radioactive atom can be comprised and study for scitiphotograph, such as Tc ^99mor I ¹²³, or comprise spin label for nucleus magnetic resonance (NMR) imaging (also referred to as nuclear magnetic resonance, MRI), such as iodo-123, iodine-131, indium-111, fluoro-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

In a known way radioactivity or other marker can be mixed conjugate.Such as, can biosynthesizing peptide, or by chemical amino acid synthesis method synthetic peptide, wherein use and involve the suitable amino group acid precursor that such as fluoro-19 replace hydrogen.Marker can be adhered to by cysteine residues in peptide, such as Tc ^99mor I ¹²³, Re ¹⁸⁶, Re ¹⁸⁸and In ¹¹¹.Yttrium-90 can be adhered to through lysine residue.IODOGEN method (Frakeretal. (1978) Biochem.Biophys.Res.Commun.80:49-57) can be used for mixing iodo-123." MonoclonalAntibodiesinImmunoscintigraphy " (Chatal, CRCPress, 1989) describe other method in detail.

Multiple bifunctional protein coupling agent can be used to carry out the conjugate of Dispersal risk and cytotoxic agent, such as N-succinimido-3-(2-pyridyl dithio) propionic ester (SPDP), succinimido-4-(N-Maleimidomethyl) hexanaphthene-1-carboxylicesters (SMCC), iminothiolane (IT), imido-ester (all example hydrochloric acid hexanedioyl imido acid dimethyl esters), active ester class (such as suberic acid two succinimido ester), aldehydes (such as glutaraldehyde), double azido compound (such as two (p-azido benzoyl base) hexanediamine), dual azepine derivatives (such as two (p-diazoniumbenzoyl)-quadrol), diisothio-cyanate (such as toluene 2, 6-vulcabond), with double activated fluorine cpd (such as 1, 5-bis-fluoro-2, 4-dinitrobenzene) dual-function derivative.Such as, ricin immunotoxin can be prepared as described in Vitettaetal., Science238:1098 (1987).The 1-isothiocyanic acid phenmethyl-3-methyl diethylene triaminepentaacetic acid(DTPA) (MX-DTPA) of carbon-14 mark is for the exemplary sequestrant by radioactive nucleotides and antibody coupling.See WO94/11026.Joint can be convenient to release cells cytotoxic drug in cell " can cut joint ".Such as, sour unstable joint, peptidase-sensitive linker, photo-labile joint, dimethyl linker can be used or contain disulfde linker (Charietal., CancerResearch52:127-131 (1992); United States Patent (USP) 5,208,020).

Compound of the present invention clearly contains but the ADC commercialization being not limited to prepare with following linking agent (as purchased from PierceBiotechnologyInc., Rockford, IL, U.S.A.) BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC and sulfo-SMPB and SVSB (succinimido-(4-vinyl sulphone) benzoic ether).See 2003-2004 year application manual and products catalogue (ApplicationsHandbookandCatalog) 467-498 page.

the preparation of antibody-drug conjugates

In antibody-drug conjugates of the present invention (ADC), antibody (Ab) is puted together through joint (L) and one or more drug moiety (D), such as each antibody coupling about 1 to about 20 drug moiety.Organic chemical reactions, condition and the reagent that those skilled in the art will know that can be adopted by several paths to be prepared the ADC of general formula I, comprise: the nucleophilic group of (1) antibody is through covalent linkage and bivalent linker reagent react, form Ab-L, react with drug moiety D subsequently; (2) nucleophilic group of drug moiety is through covalent linkage and bivalent linker reagent react, forms D-L, reacts subsequently with the nucleophilic group of antibody.There is described herein the method for distinguishing for the preparation of ADC.

Ab-(L-D) _pI

Joint can be made up of one or more joint member.Exemplary joint member comprises 6-maleimidocaproyl (" MC "), maleimide propionyl (" MP "), valine-citrulline (" val-cit "), alanine-phenylalanine (" ala-phe "), to aminobenzyloxycarbonyl (" PAB "), 4-(2-pyridylthio) valeric acid N-succinimido ester (" SPP "), 4-(N-maleimidomehyl) hexanaphthene-1 carboxylic acid N-succinimido ester (" SMCC '), (the iodo-ethanoyl of 4-) benzaminic acid N-succinimido ester (" SIAB ").Other joint member is known in this area, also illustrates herein.Also can see " MonomethylvalineCompoundsCapableofConjugationtoLigands ", U.S. Serial No.10/983, on November 5th, 340,2004 submits to, is collected herein by reference by its complete content.

In some embodiment, joint can comprise amino-acid residue.Exemplary Amino acid linker component comprises dipeptides, tripeptides, tetrapeptide or pentapeptide.Exemplary dipeptides comprises: valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe).Exemplary tripeptides comprises: glycine-valine-citrulline (gly-val-cit) and Gly-Gly-Gly (gly-gly-gly).The amino-acid residue forming Amino acid linker component comprises those naturally occurring amino acid, and the amino acid analogue of secondary amino acid and non-natural existence, such as citrulline.The selectivity aspect that Amino acid linker component can cut at the enzymatic of their certain enzyme (such as tumor correlated albumen enzyme, cathepsin B, C and D, or fibrinolytic enzyme enzyme) carries out designing and optimizing.

Exemplary joint member structure shows below (wherein wavy line instruction covalent attachment is to the site of other component of ADC):

Other exemplary joint member and abbreviation comprise (wherein depict antibody (Ab) and joint, and p being 1 to about 8):

The nucleophilic group of antibody includes but not limited to: (i) N-terminal is amino; (ii) side-chain amino group, as Methionin; (iii) side chain thiol, as halfcystine; (iv) sugared in glycosylated antibodies hydroxyl or amino.Amino, sulfydryl and hydroxyl are nucleophilics, can react with the electrophilic group on shank and form covalent linkage, and linker reagents comprises: (i) active ester class, such as NHS ester, HOBt ester, haloformate and acid halide; (ii) alkyl and benzyl halide, such as Haloacetamide; (iii) aldehydes, ketone, carboxyl and maleimide base group.Some antibody has reducible interchain disulfide bond, i.e. halfcystine bridge.Antibody is made to have the reactive behavior of puting together with linker reagents by reductive agent such as DTT (dithiothreitol (DTT)) process.Each halfcystine bridge is in theory by reactive for formation two nucleophilic thiol body.Can, via the reaction of Methionin and 2-iminothiolane (TrautShi reagent), cause amine to change mercaptan into, thus extra nucleophilic group is introduced antibody.Can pass through importing one, two, three, four, or more cysteine residues (such as preparation comprises the Mutant Antibodies of one or more non-natural cysteine amino) and reactive thiol group is imported antibody (or its fragment).

Also generate antibody-drug conjugates of the present invention by modified antibodies, namely introduce can with the electrophilic part of the nucleophilic substitution radical reaction on linker reagents or medicine.The sugar of available such as periodate oxidation agent oxidative glycosylation antibody, thus form the aldehydes or ketones group that can react with the amine groups of linker reagents or drug moiety.Gained imines Schiff base can form stable key, or available such as borohydride reagent reduces and forms stable amine connection.In one embodiment, the reaction of the carbohydrate portions of glycosylated antibodies and galactose oxidase or sodium metaperiodate can generate carbonyl (aldehyde and ketone) group in protein, it can react (Hermanson, BioconjugateTechniques) with the suitable groups on medicine.In another embodiment, the protein comprising N-terminal Serine or threonine residues can react with sodium metaperiodate, cause generating aldehyde (Geoghegan & Stroh, BioconjugateChem.3:138-146 (1992) at first amino acid place; US5362852).This type of aldehyde can react with drug moiety or joint nucleophile.

Equally, nucleophilic group on drug moiety includes but not limited to: amine, mercaptan, hydroxyl, hydrazides, oxime, hydrazine, thiosemicarbazone, hydrazinecarboxylate and aryl hydrazide group, they can react with the electrophilic group on shank and form covalent linkage, and linker reagents comprises: (i) active ester class, such as NHS ester, HOBt ester, haloformate and acid halide; (ii) alkyl and benzyl halide, such as Haloacetamide; (iii) aldehydes, ketone, carboxyl and maleimide base group.

Or, the fusion rotein comprising antibody and cytotoxic agent is prepared by such as recombinant technology or peptide symthesis.The length of DNA can comprise the region of each own coding conjugate two parts, or abuts one another or separated by the region of encoding linker peptide, and this joint peptide does not destroy the desired characteristic of conjugate.

In still another embodiment, can by antibody with " acceptor " (such as Streptavidin) coupling thus for tumour target in advance, wherein to patient's administration of antibodies-receptor conjugate, then use scavenging agent to remove unconjugated conjugate in circulating, then use and " part " of cytotoxic agent (as radioactive nucleotides) coupling (as affinity element).

Following MC-MMAE carrys out Dispersal risk (Ab)-MC-MMAE by any antibody that coupling provides herein.The antibody of 500mM Sodium Tetraborate and 500mM sodium-chlor pH8.0 is dissolved in the process of excessive 100mM dithiothreitol (DTT) (DTT).In 37 DEG C of incubations after about 30 minutes, by the wash-out exchange buffering liquid on SephadexG25 resin also with the PBS wash-out containing 1mMDTPA.By solution at 280nm place absorbance measurement reduction antibody concentration, and by with the reaction of DTNB (Aldrich, Milwaukee, WI) and the absorbance measurement concentrations of mercaptans at 412nm place, check mercaptan/value for antibody thus.The antibody of the reduction be dissolved in PBS is turned cold on ice.By medicine linker reagents maleimidocaproyl-MMAE (MMAE) i.e. MC-MMAE of being dissolved in DMSO at acetonitrile and dilution with water to concentration known, and add to the antibody 2H9 of the reduction in the PBS of cooling.After about 1 hour, add excessive maleimide and react with cancellation and cover any unreacted antibody thiol group.By centrifugal ultrafiltration, reaction mixture is concentrated, by the wash-out of the G25 resin in PBS by 2H9-MC-MMAE purifying and desalination, aseptically by 0.2 μm of frit, and freezing for storage.

The scheme that antibody-MC-MMAF can follow to be provided for preparing Ab-MC-MMAE is prepared by any antibody provided herein with MC-MMAF coupling.

The scheme that antibody-MC-val-cit-PAB-MMAE can follow to be provided for preparing Ab-MC-MMAE is prepared by any antibody provided herein with MC-val-cit-PAB-MMAE coupling.

The scheme that antibody-MC-val-cit-PAB-MMAF can follow to be provided for preparing Ab-MC-MMAE is prepared by any antibody provided herein with MC-val-cit-PAB-MMAF coupling.

Any antibody that following SMCC-DM1 is provided herein by coupling carrys out Dispersal risk-SMCC-DM1.By the antibody of purifying with 4-(N-maleimidomehyl) hexanaphthene-1-carboxylic acid succinimido ester (SMCC, PierceBiotechnology, Inc) derivatize to introduce SMCC joint.Specifically, the 20mg/ml antibody in 50mM potassiumphosphate/50mM sodium-chlor/2mMEDTA, pH6.5 is processed with the SMCC of 7.5 molar equivalents (20mM, in DMSO, 6.7mg/ml).Under argon gas after envrionment temperature stirs 2 hours, by reaction mixture by filtering with the SephadexG25 post of 50mM potassiumphosphate/50mM sodium-chlor/2mMEDTA, pH6.5 balance.Merge and measure the fraction containing antibody.

Antibody-SMCC 50mM potassiumphosphate/50mM sodium-chlor/2mMEDTA, the pH6.5 so prepared is diluted to final concentration and is about 10mg/ml, and with the 10mM solution reaction of DM1 in N,N-DIMETHYLACETAMIDE.Reaction solution is stirred 16.5 hours in envrionment temperature under argon gas.Then linked reaction mixed solution is filtered by the SephadexG25 gel-filtration column (1.5 × 4.9cm) balanced with 1xPBSpH6.5.According to the measurement of the absorbancy at 252nm and 280nm place, DM1 medicine/antibody ratio (p) can be about 2-5.

Any antibody that following SPP-DM1 is provided herein by coupling prepares Ab-SPP-DM1.By the antibody of purifying with 4-(2-pyridylthio) valeric acid N-succinimido ester derivatize to introduce dithiopyridines base.44.7ml is processed containing the antibody (376.0mg, 8mg/mL) in the 50mM potassium phosphate buffer (pH6.5) of NaCl (50mM) and EDTA (1mM) with SPP (5.3 molar equivalents in 2.3ml ethanol)., after 90 minutes reaction mixture is passed through to use 35mM Trisodium Citrate in envrionment temperature incubation under argon gas, the SephadexG25 post gel-filtration of 154mMNaCl and 2mMEDTA damping fluid balance.Then merge and measure the fraction containing antibody.The degree of modification of antibody measures as mentioned above.

Antibody-SPP-Py (about 10 μm of ol releasable 2-thiopyridine groups) is diluted to final concentration with 35mM sodium citrate buffer solution pH6.5 above and is about 2.5mg/ml.Then in antibody-solutions, add the DM1 (1.7 equivalents, 17 μm of ol) in 3.0mM N,N-DIMETHYLACETAMIDE (DMA is 3%v/v in final reaction mixture).Reaction is allowed to carry out about 20 hours in envrionment temperature under argon gas.Reaction solution is loaded on and uses 35mM Trisodium Citrate, the SephacrylS300 gel-filtration column (5.0cm × 90.0cm, 1.77L) of 154mMNaCl, pH6.5 balance.Flow velocity can be about 5.0ml/min, have collected 65 parts of fractions (each 20.0ml).Merge and detect each fraction, the absorbancy wherein by measuring 252nm with 280nm place measures the number (p ') of the DM1 drug molecule that each antibody molecule is connected, and can be that each 2H9 antibody is about 2-4 DM1 drug moiety.

Any antibody that following BMPEO-DM1 is provided herein by coupling carrys out Dispersal risk-BMPEO-DM1.Can bismaleimides reagent BM (PEO) 4 (PierceChemical) modified antibodies be used, the surface of antibody leaves unreacted maleimide base group.This can realize as follows, BM (PEO) 4 is dissolved to concentration 10mM in 50% ethanol/water mixed solution, the solution containing antibody in phosphate buffered saline (PBS) with the concentration of about 1.6mg/ml (10 micromole) is added to 10 times of molar excess, and allow it react 1 hour to form antibody-linker intermediate, 2H9-BMPEO.Excessive BM (PEO) 4 is removed by gel-filtration (HiTrapcolumn, Pharmacia) in 30mM Citrate trianion pH6 and 150mMNaCl damping fluid.The DM1 of about 10 times of molar excess is dissolved in N,N-DIMETHYLACETAMIDE (DMA) and adds to 2H9-BMPEO intermediate.Also dimethyl formamide (DMF) can be adopted to carry out dissolved substance modular reagent.Allow reaction mixture react spend the night, then in PBS gel-filtration or dialysis to remove unreacted DM1.Remove high molecular weight aggregates by gel-filtration on the S200 post in PBS and supply the 2H9-BMPEO-DM1 of purifying.

antibody derivatives

Can modify further antibody of the present invention with comprise that this area is known and be easy to obtain extra non-proteinaceous part.In one embodiment, the part being suitable for antibody derivatize is water-soluble polymers.The limiting examples of water-soluble polymers includes but not limited to polyoxyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethene/copolymer-maleic anhydride, polyamino acid (homopolymer or randomcopolymer), with dextran or poly-(n-VP) polyoxyethylene glycol, propropylene glycol homopolymers, propylene oxide/ethylene oxide copolymer, polyoxyethylated polyols (as glycerine), polyvinyl alcohol and composition thereof.Due to its stability in water, methoxy PEG-propionaldehyde may have advantage aborning.Polymkeric substance can be any molecular weight, and can be branch or unbranched.The polymkeric substance number be attached on antibody can change, and if attached to more than a polymkeric substance, so they can be identical or different molecules.Generally speaking, number and/or the type of the polymkeric substance of derivatize can be determined according to following consideration, include but not limited to the concrete property of antibody to be modified or function, treatment etc. that whether antibody derivatives will be used to specify under condition.

In another embodiment, antibody and the conjugate of the non-proteinaceous module that selectivity heats by being exposed to radiation is provided.In one embodiment, this non-proteinaceous module is carbon nanotube (Kametal., Proc.Natl.Acad.Sci.102:11600-11605 (2005)).Radiation can be any wavelength, includes but not limited to harmless to ordinary cells but non-proteinaceous module is heated to the wavelength of the killed temperature of cell close to antibody-non-proteinaceous module.

medicinal proportional preparation

By expecting that the antibody of purity can accept carrier, vehicle or stablizer with optional physiology and mix the treatment preparaton prepared and comprise antibody of the present invention by having, with the form of the aqueous solution, freeze-drying or other dry formulation storage (" Remington ' sPharmaceuticalSciences ", 16th edition, Osol, A. compile, 1980).Acceptable carrier, vehicle or stablizer are nontoxic in adopted dosage and concentration to recipient, and comprise buffer reagent, such as phosphoric acid salt, Citrate trianion and other organic acid; Antioxidant, comprises xitix and methionine(Met); Sanitas (such as octadecyl dimethyl benzyl ammonium chloride; Hexamethonium chloride; Benzalkonium chloride, benzethonium chloride; Phenol, butanols or phenylcarbinol; Alkyl paraben, such as methyl p-hydroxybenzoate or propyl ester; Pyrocatechol; Resorcinol; Hexalin; 3-amylalcohol; And meta-cresol); Lower molecular weight (being less than about 10 residues) polypeptide; Protein, such as serum albumin, gelatin or immunoglobulin (Ig); Hydrophilic polymer, such as polyvinylpyrrolidone; Amino acid, such as glycine, glutamine, l-asparagine, Histidine, arginine or Methionin; Monose, disaccharides and other carbohydrate, comprise glucose, seminose or dextrin; Sequestrant, such as EDTA; Carbohydrate, such as sucrose, N.F,USP MANNITOL, trehalose or sorbyl alcohol; Salify counter ion, such as sodium; Metal composite (as Zn-protein complex); And/or nonionogenic tenside, such as TWEEN ^tM, PLURONICS ^tMor polyoxyethylene glycol (PEG).

Preparaton herein also a kind of can treat the necessary active compound of concrete indication containing exceeding, and includes but not limited to those complementary activities and do not have disadvantageous effect each other.It is suitable that, this quasi-molecule is effectively to measure combination for predetermined object.

Activeconstituents also can wrap be loaded in such as by (being such as Walocel MT 20.000PV or gelatin-microcapsule and poly-(methyl methacrylate) microcapsule respectively) in condensation technique or the microcapsule prepared by interfacial polymerization, in colloidal drug delivery systems (such as liposome, albumin microspheres, microemulsion, nano particle and Nano capsule) or in macro emulsion.This type of technology is disclosed in such as " Remington ' sPharmaceuticalSciences ", the 16th edition, and Osol, A. compile, and 1980.

Preparaton for using in body must be aseptic.This can be easy to realize by using sterilised membrane filter to filter.

Sustained release preparaton can be prepared.The suitable example of sustained release preparaton comprises the solid hydrophobic polymers semipermeable matrices containing immunoglobulin (Ig) of the present invention, and this matrix exists with the form of standardized product, as film or microcapsule.The example of sustained-release matrix comprises polyester, hydrogel (such as poly-(2-hydroxyethyl-methacrylic ester) or poly-(vinyl alcohol)), polylactide (United States Patent (USP) 3,773,919), multipolymer, nondegradable ethane-acetic acid ethyenyl, the degradable lactic acid-ethanol copolymer such as LUPRONDEPOT of Pidolidone and γ-ethyl-L-glutamate ester ^tM(the Injectable microspheres body be made up of lactic acid-ethanol copolymer and leuprorelin acetate) and poly-D-(-)-3-hydroxybutyrate.Although the such as polymkeric substance such as ethane-acetic acid ethyenyl and lactic acid-ethanol can sustained release molecule 1 more than 00 day, the time of some hydrogel release protein is shorter.When encapsulated antibodies maintains in vivo for a long time, they may, owing to being exposed to the wet environment of 37 DEG C and sex change or gathering, cause loss of biological activity and immunogenicity to change.Stabilization strategy reasonable in design can be carried out according to related mechanism.Such as, if find that aggregation of multiple is exchanged via thio-disulfide and forms intermolecular S-S key, so by modifying sulfhydryl residue, being suitable for additive and developing specific polymer matrix composition to realize stablizing by acidic solution freeze-drying, controlling moisture, employing.

Purposes

Antibody of the present invention can be used for such as external, in vitro and interior therapeutic method.Antibody of the present invention can be used as a kind of antagonist partially or completely to close specific antigen activity in vitro in vitro, and/or in body.In addition, the antibody of the present invention of at least some can neutralize the antigenic activity from other species.Therefore, antibody of the present invention can be used for such as containing in the cell culture of antigen, have with the people experimenter of the antigen of antibody cross reaction of the present invention or other mammalian subject (as chimpanzee, baboon, marmoset monkey, macaque and rhesus monkey, pig or mouse) in suppress specific antigen activity.In one embodiment, by by antibody and antigen contact so that antigenic activity suppressed, antibody of the present invention can be used for suppressing antigenic activity.In one embodiment, described antigen is people's protein molecular.

In one embodiment, antibody of the present invention can be used for suppressing to suffer from the method that wherein antigenic activity is this antigen in the experimenter of harmful illness, and described method comprises and gives experimenter antibody of the present invention, and this antigenic activity in experimenter is suppressed.In one embodiment, antigen is people's protein molecular and experimenter is people experimenter.Alternatively, experimenter can be the Mammals of expressing antibody institute of the present invention conjugated antigen.Further, experimenter can be a kind of antigen import (as by give antigen or by antigen expressed transgenosis) Mammals.People experimenter can be given by antibody of the present invention and be used for the treatment of object.In addition, antibody of the present invention can be expressed the non-human mammal (as primate, pig or mouse) of the antigen of this antibody cross reaction for animal doctor's object or the animal model as human disease.About the latter, above-mentioned animal model can be used for the result for the treatment of (as test dosage and time-histories) evaluating antibody of the present invention.Antibody of the present invention can be used for treating, suppress, postpone progress, prevention/postpone recurrence, improve or prevention is expressed with polyubiquitin and polyubiquitin abnormal protein and/or activity is relevant disease, illness or situation, includes but not limited to hereditary conditions, immunity/inflammatory conditions, nervous system disorders and other ubiquitin-pathway associated conditions that cancer, disorder of muscle, ubiquitin-pathway are correlated with.

In an aspect, blocking antibody of the present invention is specific to the polyubiquitin with specific Methionin key, and by close or interference have specific Methionin key polyubiquitin and and the interactional albumen of this polyubiquitin between interaction suppress normal polyubiquitin active, suppress corresponding signal transduction path and other relevant molecule or cell event thus.

In certain embodiments, the immunoconjugates that patient comprises the antibody being conjugated with cytotoxic agent is given.In certain embodiments, immunoconjugates and/or its antigen combined are the internalization of cell institute, thus are killing the therapeutic efficacy enhancing this immunoconjugates in the target cell that this immunoconjugates combines.In one embodiment, cytotoxic agent target or interference target cell in nucleic acid.The example of above-mentioned cytotoxic agent comprises any one chemotherapeutic mentioned in this article (as maytenin (maytansinoid) or calicheamycin (calicheamicin)), radio isotope or rnase or DNA endonuclease.

In the treatment antibody of the present invention can separately or with other composition conbined usage.Such as, antibody of the present invention can with another kind of antibody and/or adjuvant/therapeutical agent (such as steroid) Combined Preparation.Such as, in the illness that treatment plan is as relevant with other ubiquitin-pathway in the hereditary conditions comprising cancer in treatment any one disease described herein, disorder of muscle, ubiquitin-pathway are correlated with, immunity/inflammatory conditions, nervous system disorders, antibody of the present invention can with anti-inflammatory agent and/or sterilant Combined Preparation.Mark and comprise Combined Preparation (wherein two or more medicines are included in same or different preparations) and individually dosed in upper such combination therapy, under these circumstances, antibody of the present invention can be given before or after additional treatment gives.

Antibody of the present invention (and additional therapeutical agent), by any applicable mode administration, comprises parenteral, subcutaneous, intraperitoneal, lung interior and intranasal administration, and look topical therapeutic needs, damage zone administration.Parenteral infusions comprises intramuscular, intravenously, intra-arterial, intraperitoneal or subcutaneous administration.In addition, antibody is suitable for, by pulsatile infusion administration, particularly using the antibody of attenuated dosage.Whether be short-term or long-term, can be that any applicable approach such as, as passed through injection, vein or subcutaneous injection if depending in part on administration.Whether be short-term or long-term, give dosage by any applicable approach if depending in part on administration, as by injecting such as intravenously or subcutaneous injection.

Preparing and giving the position in conjunction with target will considering antibody of the present invention in antibody.When being intracellular molecules in conjunction with target, wherein provide certain embodiments of the present invention in conjunction with the antibody in the cell residing for target or its Fab for being imported into.In one embodiment, antibody of the present invention can as intracellular antibody at cell inner expression.As used herein, term " intracellular antibody " refers to as Marasco, GeneTherapy4:11-15 (1997); Kontermann, Methods34:163-170 (2004); U.S. Patent number 6,004,940 and 6,329,173; Described in U.S. Patent Application Publication No. 2003/0104402 and PCT publication number WO2003/077945, a kind of at cell inner expression and can with the antibody of target molecule selective binding or its antigen-binding portion thereof.The cell inner expression of intracellular antibody by coding is expected antibody or its antigen-binding portion thereof nucleic acid (lack wild-type leader sequence and usually and the secretion signal of the gene-correlation of encoding antibody or Fab) import impact in target cell.Can use any by the standard method in nucleic acid into cells, include but not limited to microinjection, ballistic injection (ballisticinjection), electroporation, calcium phosphate precipitation, liposome and use carry the retrovirus of object nucleic acid, adenovirus, adeno associated virus and vaccinia virus vector and carry out transfection.One or more can be encoded all or part of delivery of nucleic acids of anti-polyubiquitin antibody of the present invention to target cell, one or more intracellular antibodies (intrabody) are expressed, thus can be combined with polyubiquitin in cell and regulate the cellular pathways that one or more polyubiquitin mediates.

In another embodiment, the antibody of internalization is provided.Antibody can have some and strengthen the characteristic entered by antibody delivery in cell, or can be modified to have above-mentioned characteristic.Technology for realizing it is known in the art.Such as, the cationization of known antibodies can promote that it is by cellular uptake (see such as U.S. Patent number 6,703,019).Lipofection or liposome also can be used for antibody delivery to enter in cell.When using antibody fragment, normally favourable with the minimum inhibition fragment of target protein binding domain specific binding.Such as, based on the variable region sequences of antibody, can design retain with target protein sequence binding ability peptide molecule.Above-mentioned peptide can chemosynthesis and/or produced by recombinant DNA technology.See such as Marasco etc., proc.Natl.Acad.Sci.USA, 90: 7889-7893 (1993).

Increasing by methods known in the art regulates polypeptide to enter target cell.Such as, some sequence those sequence directs heterologous proteins as derived from HIVTat or feeler foot (Antennapedia) homeodomain protein pass cytolemma thus effectively take in.See such as Chen etc., Proc.Natl.Acad.Sci.USA (1999), 96:4325-4329.

When the target combined is arranged in brain, some embodiment of the present invention provides antibody through hemato encephalic barrier or its Fab.Some neurodegenerative disease is relevant with the blood-brain barrier permeability of increase, makes antibody or Fab easily to import in brain.When hemato encephalic barrier keeps complete, there are some for transport molecule through the methods known in the art of this hemato encephalic barrier, include but not limited to physical method, the method based on lipid and the method based on acceptor and passage.

Physical method antibody or Fab being transported through hemato encephalic barrier includes but not limited to walk around hemato encephalic barrier completely or pass through to produce opening in hemato encephalic barrier.The method of walking around includes but not limited to be injected directly in brain (see such as Papanastassiou etc., GeneTherapy9:398-406 (2002)), interstitial infusion/convection current strengthen send (convection-enhanceddelivery) (see such as Bobo etc., Proc.Natl.Acad.Sci.USA91:2076-2080 (1994)) and in brain, insert delivery apparatus (see such as Gill etc., NatureMed.9:589-595 (2003); And GliadelWafers ^tM, GuildfordPharmaceutical).The method producing opening in barrier includes but not limited to ultrasonic wave (see such as U.S. Patent Publication No. 2002/0038086), osmotic pressure is (as by giving the N.F,USP MANNITOL (Neuwelt that height oozes, E.A., ImplicationoftheBlood-BrainBarrieranditsManipulation, Vols1 & 2, PlenumPress, N.Y. (1989))), by such as bradykinin or thoroughly agent A-7 thoroughly change (see such as U.S. Patent number 5, 112, 596, 5, 268, 164, 5, 506, 206 and 5, 686, 416) and cross over the neurocyte (see such as U.S. Patent Publication No. 2003/0083299) of hemato encephalic barrier with the carrier transfection of the gene containing encoding antibody or Fab.

Method antibody or Fab being transported through hemato encephalic barrier based on lipid includes but not limited in the liposome of the antibody binding fragment entering to be connected with receptors bind on the blood vessel endothelium with hemato encephalic barrier by antibody or Fab packing (see such as U.S. Patent Application Publication No. 20020025313), and antibody or Fab is coated in low-density lipoprotein particle (see such as U.S. Patent Application Publication No. 20040204354) or apo E (see such as U.S. Patent Application Publication No. 20040131692).

Method antibody or Fab being transported through hemato encephalic barrier based on acceptor and passage includes but not limited to use glucocorticosteroid blocker to increase the perviousness (see such as U.S. Patent Application Publication No. 2002/0065259,2003/0162695 and 2005/0124533) of hemato encephalic barrier; Activating potassium channel (see such as U.S. Patent Application Publication No. 2005/0089473), suppresses abc drug vehicle (see such as U.S. Patent Application Publication No. 2003/0073713); The activity (see such as U.S. Patent Application Publication No. 2003/0129186) of one or more TfRs is regulated with Transferrins,iron complexes coated antibody, and cationized antibodies (see such as U.S. Patent number 5,004,697).

Antibody compositions of the present invention should be prepared, determine dosage and administration in a kind of mode meeting good medical practice.The factor considered about this point is included in the particular condition for the treatment of, specific Mammals in treatment, the clinical state of individual patients, the cause of disease, drug delivery position, medication, administration schedule and other factor known to practitioner.Antibody without the need to but optionally prepare together with the medicine that prevents or treat described illness at present with one or more.The significant quantity of above-mentioned other medicines depend on antibody of the present invention existing in formula amount, the factor of sanatory type and other above-mentioned discussion.These medicines usually use with identical dosage and have route of administration described herein, or use with the dosage described herein of about 1-99%, or used by any approach with any dosage, described dosage and approach be to determine by rule of thumb/suitable through clinical assays.

In order to prevent or disease therapy, antibody of the present invention (when separately or with other medicines as chemotherapeutic conbined usage time) suitable dose should depend on the type of the disease that will treat, the kind of antibody, the seriousness of disease and the course of disease, give the prevention of antibody or therapeutic purpose, treatment before, the clinical history of patient and the response of antagonist and attending doctor consideration determine.Antibody is suitable for once or in a series for the treatment of giving patient.Depend on type and the seriousness of disease, the antibody of about 1 μ g/kg-15mg/kg (such as 0.1mg/kg-10mg/kg) can be used as candidate's consumption first and gives patient, no matter is such as independent by one or many administration or passes through continuous infusion.Depend on and give the above-mentioned factor mentioned, a typical per daily dose can in the scope of about 1 μ g/kg-100mg/kg or more.For several days or repeat administration for more time, depend on the state of an illness, treatment should continue usually till occurring that illness obtains the suppression expected.An illustrative dosage of antibody should be about 0.05mg/kg-and is about 10mg/kg.Therefore, patient can be given by the dosage of one or more about 0.5mg/kg, 2.0mg/kg, 4.0mg/kg or 10mg/kg (or their any combination).Above-mentioned dosage can intermittently give, as weekly or within every three weeks, give once (as make patient obtain about 2-about 20, or the antibody of such as about 6 dosage).Initial higher loading dose can be given, then give one or more lower dosage.An illustrative dosage regimen comprises the initial loading dose giving about 4mg/kg, succeeded by all maintenance doses of about 2mg/kg antibody.But, other dosage regimen can be used.The progress of monitoring this treatment is easy to by routine techniques and measuring method.

Goods

In another aspect of the present invention, provide the goods comprising and can be used for the material treating, prevent and/or diagnose disorder mentioned above.Goods comprise container and label on the container or with its associated or package insert.Suitable container comprises such as bottle, tubule, syringe etc.Container can be made from a variety of materials, such as glass or plastics.Himself is housed in container or effectively treats, prevent and/or diagnose the composition of illness when combining other composition, and sterile access port (such as container can be intravenous solution bag or the tubule with the stopper that hypodermic needle can pierce through) can be had.At least one promoting agent in composition is antibody of the present invention.Label or package insert instruction said composition are used for the treatment of the illness of selection.In addition, goods can comprise the first container that (a) is wherein equipped with composition, and wherein said composition comprises antibody of the present invention; (b) second container of composition is wherein housed, and wherein said composition comprises the therapeutical agent of other cytotoxic agent or other type.Goods in this embodiment of the present invention also can comprise the package insert that instruction said composition can be used for treating specific illness.Or/in addition, goods also can comprise second (or 3rd) container, the acceptable buffer reagent of pharmacopedics is wherein housed, such as injection bacteriostatic water (BWFI), phosphate buffered saline (PBS), woods grignard (Ringer) solution and dextrose solution.It also can comprise other material required in business and user's position, comprises other buffer reagent, thinner, filter, syringe needle and syringe.

It is below the embodiment of method and composition of the present invention.Should be appreciated that according to general description provided above, other embodiment various can be implemented.

embodiment

Embodiment 1: be separated and identify the anti-polyubiquitin antibody of the first-generation

A) library sorting

Allow from natural the phage in body library stands the combination selection comprising the synthetic peptide of isopeptide bond of the polyubiquitin of polyubiquitin or the K63 connection connected for immobilized simulation K48.Enrichment is not observed after taking turns selection 6.Lengthen synthetic peptide, and again screen natural antibody library.Again, select not observe enrichment in 6 combinations taken turns.

Allow the phage from natural YS-B antibody library stand 4 combinations taken turns for the polyubiquitin chain with different known ubiquitin isopeptide bonds select.YS-B antibody library contains randomized in all three heavy chain CDR and light chain CDR3 and is the amino acid (see U.S. Patent Application Publication No. 2005-0106667) based on humanized antibody 4D5.

That by length be the total length K48 connection of the enzymic synthesis of 3-7 unit or that K63 connects polyubiquitin chain (BostonBiochem) is fixed on the Maxisorp immuno plate of 96-hole (NUNC).With being dissolved in 50mM carbonate buffer solution, the polyubiquitin bag that K48 or K63 of 5 μ g/ml in pH9.6 connects is spent the night by flat board.The flat board of bag quilt washs by phosphate buffered saline(PBS) (PBS) and uses the bovine serum albumin (BSA) of 0.2% or casein in PBS to close.Then dull and stereotyped with PBS (PBST) washing containing 0.05%Tween20 at 25 DEG C.In room temperature, 10 of each Kong Douyong 100 μ l ¹²sorting damping fluid (0.2%BSA or the casein PBST solution) incubation of individual phage/ml 2 hours.8 times are washed to remove unconjugated phage in each Kong Douzai PBST.By the phage be combined with the extracting in 10 minutes of 0.1MHCl incubation, and with in 2MTris alkali and extract.By the phage of institute's extracting propagation in the E.colistrain XL1 blue (Stratagene) adding M13-KO7 helper phage (NewEnglandBiolabs).

The phage of amplification is used for the selection for the identical target used in round before of additional rounds.2-4 wheel is selected, 10 μ g/ml ubiquitins are included in sorting damping fluid to take turns as anti-(counterselection) the 3rd and 4 of selection and also use simultaneously or do not use extra anti-selection to carry out sorting: the polyubiquitin that the polyubiquitin is connected at K48 connects with 10 μ g/mlK63 in selecting is counter to be selected, or the polyubiquitin connected with 10 μ g/mlK48 in the polyubiquitin selection of K63 connection is counter selects.

For those clones lacked wherein in the anti-2-4 wheel selection selected of polyubiquitin, being supplemented with in the 2YT meat soup of Pyocianil and M13-KO7 helper phage of 400 μ l in 96-orifice plate cultivates single clone.The clone that polyubiquitin, ubiquitin and BSA that polyubiquitin supernatant liquor from those cultures being used for high-throughput Phage-ELISA to screen connect with K48, K63 connect are combined.DNA sequence analysis is carried out to all clones.A part for the heavy chain comprising HVR is checked order, for the analysis of heavy chain hypervariable region.Identify the polyubiquitin that K48 connects or both identified that the polyubiquitin that K48 connects also identified that the heavy chain hypervariable region of the clone of the polyubiquitin that K63 connects is shown in Fig. 2 A-C.Identify the polyubiquitin that K63 connects or both identified that the polyubiquitin that K48 connects also identified that the heavy chain hypervariable region of the clone of the polyubiquitin that K63 connects is shown in Fig. 3 A and 3B.Light chain HVR for each clone does not check order, but based on the characteristic in YS-B library, estimates that the sequence of HVR-L1 and HVR-L2 is constant, and HVR-L3 sequence then estimates it is that clone is special.According to library designs, HVR-L1 sequence is RASQSVSSAVA (SEQIDNO:79), and HVR-L2 sequence is SASSLYS (SEQIDNO:80).All clones have identical heavy chain and light chain framework region sequence (see Fig. 6).

A different set of clone includes in 2-4 wheel is selected with the anti-selection that the polyubiquitin (polyubiquitin that K48 connects or the polyubiquitin that K63 connects) of the different Methionin key of 10 μ g/ml carries out.10 μ l wash-outs are used for infecting the intestinal bacteria CJ23620 minute grown from the phage that fourth round is selected, then containing overnight incubation on the solid agar of Pyocianil.2YT meat soup 15ml being supplemented with Pyocianil and paraxin adds in flat board with the resuspended CJ236 cell containing phagemid.Add M13-KO7 helper phage.Stir incubation after 1 hour at 37 DEG C, add the suspension of 2.5ml in the 2YT meat soup of 250ml being supplemented with Pyocianil and kantlex.Allow suspension overnight incubation.

Gather in the crops phage by polyethylene glycol precipitation, and use M13spin test kit (Qiagen) to be separated KunkelDNA.Use oligonucleotide F1560-2 (TCTTGTGACAAAACTCACCATCACCATCACCATCACTAGGGCGGTGGCTCTGGTTC CGGTGATTTT) (SEQIDNO:150), the Kunkel template of 1 μ g is used for Kunkel mutagenic treatment (see Kunkel, Proc.Natl.Acad.Sci.USA82:488 (1985)).Mutagenesis reaction thing to be transformed in E.colistrain XL1 blue and containing overnight incubation on the solid agar of Pyocianil.Then as above select and cultivate single clone, and in Phage-ELISA, screening as mentioned above the polyubiquitin that anti-K48 with K63 be connected, single ubiquitin, BSA and resist the clone of five polyhistidine antibody (anti-pentaHisantibody) (Qiagen).DNA sequence analysis is carried out to the clone of the polyubiquitin being accredited as polyubiquitin or the K63 connection being specific to K48 connection.Hypervariable region HVR-H1, HVR-H2 and the aminoacid sequence of HVR-H3 are shown in Fig. 8 A-C (being specific to the polyubiquitin that K48 is connected) and 9A with 9B (being specific to the polyubiquitin that K63 is connected).Light chain HVR for each clone does not check order, but based on the characteristic in YS-B library, estimates that the sequence of HVR-L1 and HVR-L2 is constant, and HVR-L3 sequence then estimates it is that clone is special.According to library designs, HVR-L1 sequence is RASQSVSSAVA (SEQIDNO:79), and HVR-L2 sequence is SASSLYS (SEQIDNO:80).All clones have identical heavy chain and light chain framework region sequence (see Fig. 6).

(B) Fab is produced

In order to produce Fab, the phage supernatants from the Unique clones for the five histidine-tagged positives is used for ehec infection FF34B8 (one is wherein by cultivating with XL1 mating and in Selective solid culture medium thus adding F ' episome to clone in 34B8 bacterial strain).The cell infected is being rule and overnight incubation containing on the solid agar of Pyocianil.Monospecific polyclonal containing the FF34B8 of phagemid is chosen and in 37 DEG C of overnight incubation in containing the LB of Pyocianil from flat board.Then those cultures are used for inoculating the complete CRAP substratum (3.57g (NH of 500ml containing Pyocianil ₄) ₂sO ₄, 0.71g Trisodium Citrate 2H ₂o, 1.07gKCl, 5.36g yeast extract (certified), 5.36gHycaseSF (Sheffield), adjust pH to 7.3 by adding KOH and adjust volume to 872mL with ultrapure water, autoclaved, is cooled to 55 DEG C, adds 110mL1MMOPSpH7.3,11mL50% glucose and 7mL1MMgSO to its (every L) ₄), and 30 DEG C of stir culture 24 hours.Cell precipitation is stored in-20 DEG C by centrifugation harvested cell.By each cell precipitation being resuspended in the middle purifying Fab of the cold lavation buffer solution (PBS+150mMNaCl) containing 0.2mg/ml N,O-Diacetylmuramidase and 0.3U/mLDNA enzyme I of 35ml.Resuspended cell precipitation to be transferred in 50ml centrifuge tube and 25 DEG C of quick vortexs 45 minutes.Lysate is also loaded into 1ml at 4 DEG C of protein G-Sepharose posts with lavation buffer solution pre-equilibration by centrifugation.With the lavation buffer solution washing pillar that 50ml is cold, with 3ml0.1M acetic acid wash-out, and neutralize with the 2MTris alkali of 150 μ l.The Fab buffer-exchanged of wash-out to be entered in PBS and to use Centriprep10 centrifugal filter (Millipore) to concentrate.Fab concentration (the 1OD obtained by spectrophotometry _280nm=1.55mg/mL).Concentrated Fab is stored in 4 DEG C.

As mentioned above, each Fab is included in measure the relative affinity of the polyubiquitin is connected with K63 that it connects K48 in ELSA protein determination, and in order to confirm Fab reacting with single ubiquitin or BSA.Compared with the polyubiquitin that K63 is connected, the specificity that the polyubiquitin tool that Fabapu01-15 connects K48 is higher.In ELISA, Fabapu17-apu24 is proved and compares the higher specificity of polyubiquitin tool that K63 connects with the polyubiquitin be connected K48.Apu16 does not produce as Fab.

DNA sequence analysis is carried out to all Fab.The aminoacid sequence of hypervariable region HVR-H1, HVR-H2, HVR-H3 and HVR-L3 of the Fab of each polyubiquitin specific binding be connected with K48 is shown in Figure 10 A-C.The aminoacid sequence of hypervariable region HVR-H1, HVR-H2, HVR-H3 and HVR-L3 of the Fab of each polyubiquitin specific binding be connected with K63 is shown in Figure 11 A-C.For each Fab, heavy chain and light chain framework region sequence shown in Figure 6.According to library designs, be identical (see above SEQIDNOs:79 and 80) for each clone two light chain hypervariable region HVR-L1 and HVR-L2.

(C) the avidity analysis of the Fab be separated

Use 3000 systems (Biacore) measure Fab (see above-mentioned (B) joint) avidity to the form that ubiquitin and Methionin thereof connect through selecting by surperficial plasmon resonance.Use the amine coupling protocols that manufacturer provides, the polyubiquitin (chain length 3-7) that the dimerization ubiquitin connect the ubiquitin of about 100 resonance units, K48 or K63 or K48 or K63 connect is fixed on the different flow cells (flowcell) of CM5 chip.In each experiment, activate and close the flow cell 1 not having ankyrin with thanomin, being used as with reference to deduction.The Fab albumen (1.6-100nM) injection (altogether injecting 50 μ l with the flow velocity of 25 μ l/min) of each in the apu01-apu24 of serial dilution is overflowed each flow cell (flowcell).Record the signal of each flow cell and deduct reference signal.After the phase of dissociating (5 minutes), with the 20mMHCl regeneration chip surface of 13 μ l.The binding curve of illustrative Fabapu09 and apu18 is shown in Figure 12 and 13.As shown in Figure 12, the polyubiquitin that Apu09 and K48 connects combines, but the polyubiquitin do not connected with K63 is combined.Figure 13 A shows the binding curve of the polyubiquitin that apu18 and K48 connects.Although observed some to combine, it is less than the viewed combination of polyubiquitin (Figure 13 B) connected K63 substantially.Similar analysis is carried out to each Fab.

The software utilizing manufacturer to provide by nonlinear regression analysis computational dynamics constant and binding constant simultaneously, and is shown in table B.In table B, word " NB " refers to combination do not detected for indicated interaction.In table B, word " nd " refers to do not have take off data for indicated interaction.Result shows the kinetic constant that specific Fab is combined with dimerization ubiquitin and is very similar to those kinetic constants be combined with polyubiquitin.Therefore, Fab appears the specific isopeptide bond (isopeptidelinkage) between identification two ubiquitin parts.

Table B: pass through analyze the kinetic constant of the anti-polyubiquitin Fab measured

^*the interactional kinetic constant of dimerization ubiquitin that K63 is connected that the second time Biacore of Apu18 obtains before analyzing and confirming.

(D) western blotting

Be separated in polyacrylamide gel four poly-ubiquitins (in the appropriate case K48 connect or K63 connects) and dimerization ubiquitin (K48 be connected or K63 connection) (BostonBiochem) transferred on nitrocellulose membrane by electroblotting.By the nonspecific binding site spent the night on closing membrane at 4 DEG C of incubation films in 0.5%Qiagen closed reagent (Qiagen).Closed film is positioned in miniblotter device.Fab clone (1 μ g/ml) is put in the film serial section be in 0.5%Qiagen closed reagent (Qiagen).At incubation after 1 hour, washing film.According to manufacturers instruction, anti-five polyhistidine antibody (Qiagen) puting together HRP are used to develop conjunctival anti-ubiquitin antibody.

The four poly-ubiquitin specific bindings (see Figure 19 B) that the polyubiquitin specificity Fab that the K48 that clone apu01-apu15 produces connects is connected with the K48 be fixed on nitrocellulose membrane.The combination (see Figure 19 A) of the polyubiquitin that the polyubiquitin specificity Fab not observing the K63 connection that clone apu17-apu24 produces is connected with the K63 be fixed on nitrocellulose membrane.

Embodiment 2: be separated and identify the anti-polyubiquitin antibody of the s-generation

Build with apu18 (polyubiquitin that K63 is connected optionally) phagemid the s-generation library (see Figure 10 and 11) being used for Fab and showing by the clone apu05 identified before coding (polyubiquitin that K48 connects optionally).The phage cloned from those is used for infect CJ236 cell to prepare KunkelDNA template.Subsequently, the template containing terminator codon to insert terminator codon, and is used for following library construction by those templates of mutagenic treatment.

According to two different schemes mutagenic treatment Fabapu05 to create the derivative library of two different apu05.In first library, only mutagenic treatment HVR-H3.First modify HVR-H3 terminator codon to be included in Kunkel template, then utilize the oligonucleotide of four mutagenesis to carry out mutagenesis.In all cases, terminator codon oligonucleotides coding is CGTCTATTATTGTGCTCGCTAATAAGACTACTGGGGTCAAGG (SEQIDNO:365).The oligonucleotide of three mutagenesis is three kinds of changes (permutation) of identical expectation sequence, one of them tyrosine residues is fixed, and utilize NNS mixed cipher subgroup (wherein N is corresponding to G, C, A or T, and S-phase should in G or C) each remaining tyrosine residues of randomization; The amino acid of 100b position is selected from phenylalanine, methionine(Met), leucine and Isoleucine; The amino acid of 100a position is selected from glycine and L-Ala; And remaining amino acid is carried out gentle randomization.Gentle randomization refers to that the time of some nucleotide position 70% is occupied by indicated base within a context, and each of other three kinds of bases then occupies the time of 10%.Wherein above-mentioned gentleness those oligonucleotide randomized are comprised at particular bases place for those, by indicating gentle randomized existence in the existence of this base positions place numeral.Numeral " 5 " refers to the time occupying 70% in this position bases adenine, and base guanine, cytosine(Cyt) and thymus pyrimidine then occupy the time of 10% respectively.Similarly, numeral " 6 " refers to guanine, and " 7 " refer to cytosine(Cyt), and " 8 " refer to thymus pyrimidine, and in all cases, each in other three kinds of bases only occupies the time of 10%.The oligonucleotide sequence of three mutagenesis is: CGTCTATTATTGTGCTCGC567TAC567NNSNNS567GSTWTSGACTACTGGGGTC AAGG (SEQIDNO:367), CGTCTATTATTGTGCTCGC567NNS567TACNNS567GSTWTSGACTACTGGGGTC AAGG (SEQIDNO:368) and CGTCTATTATTGTGCTCGC567NNS567NNSTAC567GSTWTSGACTACTGGGGTC AAGG (SEQIDNO:369).The oligonucleotide of Article 4 mutagenesis comprises the randomization utilizing NNS mixed cipher subgroup to the 96th, 98 and 99 tyrosine; The amino acid of 100b position is selected from phenylalanine, methionine(Met), leucine and Isoleucine; From glycine and L-Ala, select the amino acid of 100a position, and consistently carry out gentle randomization in other position each with above-mentioned gentle randomization nomenclature.The sequence of Article 4 oligonucleotide is CGTCTATTATTGTGCTCGC567NNS567NNSNNS567GSTWTSGACTACTGGGGTC AAGG (SEQIDNO:370).

In second apu05 library, mutagenic treatment HVR-H1, HVR-H2, HVR-H3 and HVR-L3.Utilize NNS mixed cipher subgroup (wherein N is corresponding to G, C, A or T, and S-phase should in G or C) modify HVR-H1 make the 30th and the Serine of 33 be randomized; The amino acid of the 29th is selected from amino acid phenylalanine, leucine, Isoleucine and α-amino-isovaleric acid; And from Isoleucine and methionine(Met), select the amino acid of the 34th.Oligonucleotide for mutagenic treatment apu05HVR-H1 is GCAGCTTCTGGCTTCAACTAATAACACTGGGTGCGTCAGG (SEQIDNO:371) and GCAGCTTCTGGCTTCAACNTCNNSTACTCTNNSATSCACTGGGTGCGTCAGG (SEQIDNO:372).Utilizing NNS mixed cipher subgroup to modify HVR-H2 makes the tyrosine of the 52nd be randomized, and selects the amino acid of 52a position from proline(Pro) and Serine.Oligonucleotide for mutagenic treatment HVR-H2 is: GGCCTGGAATGGGTTGCATAATAATATGCCGATAGCGTCAAGG (SEQIDNO:373) and GGCCTGGAATGGGTTGCATCTATCNNSYCTTACTACTCTTACACCTCTTATGCCGA TAGCGTCAAGG (SEQIDNO:374).Utilizing NNS mixed cipher subgroup to modify HVR-H3 makes the Serine of the tyrosine of the 99th and the 100th be randomized; The amino acid of 100a position is selected from glycine and L-Ala; And from phenylalanine, methionine(Met), leucine and Isoleucine, select the amino acid of 100b position.Oligonucleotide for mutagenic treatment HVR-H3 is: CGTCTATTATTGTGCTCGCTAATAAGACTACTGGGGTCAAGG (SEQIDNO:365) and CGTCTATTATTGTGCTCGCTCTTACTCTTACNNSNNSGSTWTSGACTACTGGGGTC AAGG (SEQIDNO:375).Modifying HVR-L3 according to NNS mixed cipher subgroup makes S91 position be randomized, and selects I96 position from phenylalanine, Isoleucine and α-amino-isovaleric acid.Oligonucleotide for mutagenic treatment HVR-L3 is: CGCAACTTATTACTGTCAGCAATAATAAACGTTCGGACAGGGTACC (SEQIDNO:376) and CGCAACTTATTACTGTCAGCAANNSTCTTACTCTTCTCTGDTTACGTTCGGACAGG GTACC (SEQIDNO:378).

The derivative library of six different apu18-is prepared by six different apu18 mutagenesis program.In first library, only mutagenic treatment HVR-H3.First modify HVR-H3 terminator codon to be included in Kunkel template, then utilize the oligonucleotide of seven mutagenesis to carry out mutagenesis.In all cases, terminator codon oligonucleotides coding is CGTCTATTATTGTGCTCGCTAATAAGACTACTGGGGTCAAGG (SEQIDNO:366).The oligonucleotide of six mutagenesis is six kinds of changes of identical expectation sequence, wherein two tyrosine or tryptophan residue are fixed, and utilize NNS mixed cipher subgroup (wherein N is corresponding to G, C, A or T, and S-phase should in G or C) each remaining tyrosine of randomization and tryptophan residue; The amino acid of 100c position is selected from phenylalanine, methionine(Met), leucine and Isoleucine; The amino acid of 100b position is selected from glycine and L-Ala; And with the above-mentioned gentle randomization nomenclature consistently remaining amino acid of gentle randomization.The oligonucleotide sequence of six mutagenesis is: CGTCTATTATTGTGCTCGC655TACTAC565NNSNNS577GSTWTSGACTACTGGG GTCAAGG (SEQIDNO:379), CGTCTATTATTGTGCTCGC655NNSNNS565TGGTAC577GSTWTSGACTACTGGG GTCAAGG (SEQIDNO:380), CGTCTATTATTGTGCTCGC655TACBBS565NNSTAC577GSTWTSGACTACTGGG GTCAAGG (SEQIDNO:381), CGTCTATTATTGTGCTCGC655NNSTAC565TGGNNS577GSTWTSGACTACTGGG GTCAAGG (SEQIDNO:382), CGTCTATTATTGTGCTCGC655TACNNS565TGGNNS577GSTWTSGACTACTGGG GTCAAGG (SEQIDNO:383) and CGTCTATTATTGTGCTCGC655NNSTAC565NNSTAC577GSTWTSGACTACTGGG GTCAAGG (SEQIDNO:384).The oligonucleotide of Article 7 mutagenesis comprises and utilizes the tyrosine of NNS mixed cipher subgroup randomization the 96th, 97 and 100 and the tryptophane of the 99th; The amino acid of 100b position is selected from phenylalanine, methionine(Met), leucine and Isoleucine; The amino acid of 100c position is selected from phenylalanine, methionine(Met), leucine and Isoleucine; From glycine and L-Ala, select the amino acid of 100b position, and consistently carry out gentle randomization in other position each with above-mentioned gentle randomization nomenclature.The sequence of Article 7 oligonucleotide is: CGTCTATTATTGTGCTCGC655NNSNNS565NNSNNS577GSTWTSGACTACTGGG GTCAAGG (SEQIDNO:385).

In second apu18 library, only mutagenic treatment HVR-H2.First modify HVR-H2 terminator codon to be included in Kunkel template, then utilize the oligonucleotide of four mutagenesis to carry out mutagenesis.In all cases, terminator codon oligonucleotides coding is GGCCTGGAATGGGTTGCATAATAATATGCCGATAGCGTCAAGG (SEQIDNO:373).The oligonucleotide of three mutagenesis is three kinds of changes of identical expectation sequence, one of them tyrosine residues is fixed, and utilize the Serine of NNS mixed cipher subgroup (wherein N is corresponding to G, C, A or T, and S-phase should in G or C) each remaining tyrosine of randomization and the 52nd; The amino acid of 52a position is selected from proline(Pro) and Serine; The amino acid of the 55th is selected from glycine and Serine; Fix the Isoleucine of the 51st and the Threonine of the 57th; And with the above-mentioned gentle randomization nomenclature consistently remaining amino acid of gentle randomization.The oligonucleotide sequence of three mutagenesis is: GGCCTGGAATGGGTTGCATACATCNNSYCTNNSNNSRGC567ACC567TATGCCGA TAGCGTCAAGG (SEQIDNO:386), GGCCTGGAATGGGTTGCANNSATCNNSYCTTACNNSRGC567ACC567TATGCCGA TAGCGTCAAGG (SEQIDNO:387) and GGCCTGGAATGGGTTGCANNSATCNNSYCTNNSTACRGC567ACC567TATGCCGA TAGCGTCAAGG (SEQIDNO:388).The oligonucleotide of Article 4 mutagenesis comprises the tyrosine utilizing NNS mixed cipher subgroup randomization the 50th, 53 and 54; The amino acid of 52a position is selected from phenylalanine and Serine; The amino acid of the 55th is selected from glycine and Serine; Fix the 51st and the Isoleucine of 57 and threonine residues respectively; And consistently carry out gentle randomization in other position each with above-mentioned gentle randomization nomenclature.The sequence of Article 4 oligonucleotide is: GGCCTGGAATGGGTTGCANNSATCNNSYCTNNSNNSRGC567ACC567TATGCCGA TAGCGTCAAGG (SEQIDNO:389).

In the 3rd apu18 library, mutagenic treatment HVR-H2 and HVR-H3.The same with the modification carried out HVR-H2 in second apu18 library, utilize the oligonucleotides-modified HVR-H2 of four identical mutagenesis.The same with the modification carried out HVR-H3 in first apu18 library, utilize the oligonucleotides-modified HVR-H3 of identical six mutagenesis.

In the 4th apu18 library, mutagenic treatment HVR-H3 and HVR-L3.The same with the modification carried out HVR-H3 in first apu18 library, utilize the oligonucleotides-modified HVR-H3 of identical six mutagenesis.First modify HVR-L3 terminator codon to be included in Kunkel template, what then utilize mutagenic oligonucleotide carries out mutagenesis.In HVR-L3, utilize NNS mixed cipher subgroup randomization the 91st and the tyrosine of 94 and the Serine of 95a position; The leucine of 95b position is selected from phenylalanine, Isoleucine and α-amino-isovaleric acid; And with the Serine of above-mentioned gentle randomization nomenclature consistently gentle randomization the 92nd, 93 and 95.Oligonucleotide for HVR-L3 mutagenic treatment is CGCAACTTATTACTGTCAGCAATAATAAACGTTCGGACAGGGTACC (SEQIDNO:376) and CGCAACTTATTACTGTCAGCAANNS567567NNS567NNSCTGDTTACGTTCGGAC AGGGTACC (SEQIDNO:390).

In the 5th apu18 library, mutagenic treatment HVR-H1 and HVR-H2.The same with the modification carried out HVR-H2 in second apu18 library, utilize the oligonucleotides-modified HVR-H2 of four identical mutagenesis.Modify HVR-H1 to comprise terminator codon; Utilize the Serine of NNS mixed cipher subgroup randomization the 30th and the tyrosine of the 33rd; The amino acid of the 29th is selected from phenylalanine, leucine, Isoleucine and α-amino-isovaleric acid; From Isoleucine and methionine(Met), select the amino acid of the 34th, and with the amino acid of above-mentioned gentle randomization nomenclature consistently gentle randomization the 31st and 32.Oligonucleotide for HVR-H1 mutagenic treatment is: GCAGCTTCTGGCTTCAACTAATAACACTGGGTGCGTCAGG (SEQIDNO:371) and GCAGCTTCTGGCTTCAACNTCNNS567567NNSATSCACTGGGTGCGTCAGG (SEQIDNO:391).

In the 6th apu18 library, mutagenic treatment HVR-H1, HVR-H2 and HVR-L3.The same with the modification carried out HVR-H1 in the 5th apu18 library, utilize the oligonucleotides-modified HVR-H1 of identical mutagenesis.The same with the modification carried out HVR-H2 in second apu18 library, utilize the oligonucleotides-modified HVR-H2 of four identical mutagenesis.The same with the modification carried out HVR-L3 in the 4th apu18 library, utilize the oligonucleotides-modified HVR-L3 of identical mutagenesis.

The mutagenesis reaction of each in the library derived in the library derived two apu5 and six apu18 by electroporation is transformed in the intestinal bacteria XL-1 of Electrocompetent.In SOC substratum, allow cell stir recovery 30 minutes at 37 DEG C.Retaining the celliferous SOC substratum of 20 μ L for measuring the number of transformant, then remaining SOC media transfer being contained Pyocianil and every milliliter 10 to 500ml ¹⁰in the 2YT of individual M13K07 helper phage.After stirring 45 minutes at 37 DEG C, supplement kantlex to meat soup and spend the night 37 DEG C of stir culture.The transformant number in each library is > 10 ⁹.Gather in the crops from meat soup by centrifugal and PEG precipitation and concentrate phage, and in selection cycles subsequently.

Described in above embodiment 1 (A), the polyubiquitin that the polyubiquitin that K48 connects is connected with K63 is fixed on different Maxisorp flat boards (NUNC).For its corresponding target, (library derived for two apu05 is the polyubiquitin that K48 connects respectively in each library, the library derived for six apu18 is the polyubiquitin that K63 connects) sorting one takes turns, additional 3 μMs of single ubiquitins in sorting damping fluid.Increase and the phage (two storehouses, the polyubiquitin target that often kind of Methionin connects respectively) collecting wash-out for further sorting round.

Selection cycles is subsequently the sorting of solution phase.Room temperature in solution sorting damping fluid (PBST containing 0.5%Superblock (Pierce)) by phage library and biotinylated (sulfo-NHS-biotin, Pierce) polyubiquitin chain incubation 1-2 hour.Mixture is diluted in solution sorting damping fluid 5-10 doubly and add neutravidin (neutravidin) to and wrap in the hole of quilt and catch for of short duration (5 minutes) of biotinylated polyubiquitin.Reaction containing not biotinylated polyubiquitin chain monitors background phage combination with comparing.Also 0.1MHCl wash-out 10 minutes are used with PBST washing is dull and stereotyped.

Regulate stringency in three ways: by the concentration of biotinylated polyubiquitin; Before the hole being neutravidin bag quilt catches, carry out competition binding by adding excessive not biotinylated polyubiquitin; And the time length by competing.For often taking turns sorting, during first time incubation step, add single ubiquitin with other key and polyubiquitin in sorting damping fluid with the concentration of 30 μ g/ml.First round solution sorting employs the biotinylated polyubiquitin of 20nM and phage incubation at room temperature 1 hour.Then in solution sorting damping fluid by mixture diluted 10 times, and use the hole of neutravidin bag quilt to catch 5 minutes.Take turns for second, phage is balanced with the biotinylated polyubiquitin of the same 20nM in the first round, but (K48 is selected to connect with K48 containing 30 μ g/ml not biotinylated polyubiquitin, K63 is selected to connect with K63) solution sorting damping fluid in dilution 10 times for the dissociation rate selection of 15 minutes, catch on the hole of neutravidin bag quilt subsequently.Third round solution sorting and second is taken turns described the same, but the dissociation rate comprising the biotinylated polyubiquitin of 5nM and 30 minutes is further selected.After taking turns dull and stereotyped sorting and the sorting of three-wheel solution one, each being selected from the s-generation is cloned in 96 described orifice plates and cultivates.Screen each by Phage-ELISA clone and check order.

After first round solution sorting, the library based on apu05 and the library based on apu18 be observed respectively to the enrichment (see table C) reaching 40 times and reach 7 times.After taking turns selection (the solution sorting to slow dissociation rate) second, for K48 specificity, clone obtains 11 times of extra enrichments, then obtains extra 3 times of enrichments (see table C) for K63 specificity clone.Third round selects (avidity sorting and dissociation rate (off-rate) sorting) to K48 specificity clone's generation 18 times of enrichments, then produces 4 times of enrichments (see table C) to K63 specificity clone.

Table C: the s-generation anti-polyubiquitin antibody library solution separation results

Qualification obtains the difference clone of the polyubiquitin that 68 specific binding K48 connect; Also DNA sequence analysis is carried out to those clones.HVR-H1, HVR-H2 and HVR-H3 sequence of those clones is shown in Figure 14 A-F.Qualification obtains the difference clone of the polyubiquitin that 31 specific binding K63 connect; Also sequential analysis is carried out to those clones.HVR-H1, HVR-H2 and HVR-H3 sequence of those clones is shown in Figure 15 A-C.The polyubiquitin specificity clone that the polyubiquitin connected for each K48 is connected with K63, light chain HVR does not check order, but be constant with regard to the sequence expection of apu05 and apu18, HVR-L1 and HVR-L2, HVR-L3 sequence expection is simultaneously that clone is special.According to library designs, HVR-L1 sequence is RASQSVSSAVA (SEQIDNO:79) and HVR-L2 sequence is SASSLYS (SEQIDNO:80).All clones have identical heavy chain and light chain framework region sequence (see Fig. 6).

As described in Example 1,20 have the clone (the specific polyubiquitin be connected with 10 K63 of polyubiquitin that 10 K48 connect is specific) observing maximum combined and are produced by as Fab.DNA sequence analysis is carried out to Fabapu2.01-apu2.20.HVR-H1, HVR-H2, HVR-H3 and HVR-L3 sequence of those clones is shown in Figure 16 A and B (Fab that K48 is special) and Figure 17 A and B (Fab that K63 is special).

Fabapu2.01-apu2.20 is included in measure the relative affinity of the polyubiquitin is connected with K63 that they connect K48 in ELISA protein determination, and in order to confirm Fab reacting (see Figure 18) with single ubiquitin or BSA.In this mensuration, Apu clone 2.11 and 2.12 is proved compared with the polyubiquitin that is connected with to K48 respectively, and the polyubiquitin connected K63 has the avidity of height about 300 times.Compare in the avidity of the polyubiquitin that K63 connects at the polyubiquitin be connected with K48, Apu2.20 and 2.16 has less, but still significant difference (being respectively height about 30 times and height about 10 times).The combination of the polyubiquitin that clone apu2.01-apu2.10 and K63 connects or dimerization ubiquitin do not detected.

Also analyze each Fab by the BIAcore such as before described in embodiment 1 (C).The kinetic constant obtained and binding constant are shown in table D.In table D, word " NB " refers to combination do not detected for indicated interaction.

Table D: the kinetic constant being analyzed the anti-polyubiquitin Fab measured by BIAcore

Several Fab based on apu05 have the K lower compared with the Fab corresponding to apu05 to the dimerization ubiquitin that K48 connects _d, show to be combined with polyubiquitin tightr.Each polyubiquitin not only connected with K63 in apu2.11-2.20Fab is combined, and the dimerization ubiquitin also connected with K48 in less degree is combined.Although only apu2.13 has lower Kd compared with apu18, compared with apu18, it wants large to the Kd of the dimerization ubiquitin that K48 connects.Apu2.11,2.12,2.16 has the better ratio to the Kd of the dimerization ubiquitin that the Kd of the polyubiquitin that K63 is connected is connected with to K48 with each in 2.20 compared with apu18.The polyubiquitin that Fab and K63 based on apu18 is connected be similar to for the dimerization ubiquitin be connected with K63 observed by combination to those kinetic constants in conjunction with viewed kinetic constant.

Also have rated by the western blotting such as before described in embodiment 1 (D) ability that each Fab specific binding is fixed on the polyubiquitin on nitrocellulose membrane.By the four poly-ubiquitins containing K48 key or K63 key, dimerization ubiquitin (the dimerization ubiquitin connected with K63 when the four poly-ubiquitins such as connected as K48 use containing the contrary Methionin key of ubiquitins poly-with four, or the dimerization ubiquitin connected with K48 when the four poly-ubiquitins that K63 connects use) and single ubiquitin be fixed on nitrocellulose membrane, and evaluate the ability (Figure 20 A and 20B) of all three kinds of immobilized molecules of Fabapu2.01-2.20 identification.There is no a kind of Fab specific recognition list ubiquitin.Each all four poly-ubiquitin of connecting of the immobilized K48 of specific recognition in Apu2.01-apu2.10, but all dimerization ubiquitins (see Figure 20 A) of connecting of the immobilized K63 of nonrecognition.Some other bands seen on trace represent pollutent trimerizations, five poly-and eight poly-ubiquitin materials in four poly-ubiquitin prepared products of K48 connection.The four poly-ubiquitins that the immobilized K63 of the equal specific recognition of Apu2.11-apu2.20 connects, and the dimerization ubiquitin (see Figure 20 B) of all immobilized K48 connections of nonrecognition.

Embodiment 3: the combination of anti-polyubiquitin antibody and endogenous polyubiquitin albumen

Previous experiment has confirmed that the activity of receptor interacting protein (RIP) (the required medium of TNF acceptor 1 (TNFR1) the intracellular signaling mixture of the vicinity of a kind of 140kD) turns into regulating (Wertz etc., Nature430:694-699 (2004)) by polyubiquitin.When the polyubiquitin chain institute polyubiquitin that RIP connects for K63, facilitate the intracellular signaling by TNFR1.By going the N-end of ubiquitination A20 to remove from RIP the polyubiquitin chain that K63 connects, and by the polyubiquitin chain replacement that the ubiquitin protein ligase function of A20C end connects with K48, thus inactivation RIP its targeting proteins enzyme body is degraded.This mechanism is by using the clone of the sudden change ubiquitin of expressing that only can form K48 connection or that K63 connects polyubiquitin to be illustrated.

Have rated in different time points place after TNF process, the polyubiquitin associated proteins specific recognition that two kinds of anti-K48 of the present invention are connected with anti-K63 is present in the ability of the RIP of the different polyubiquitin form in HeLaS3 cell.With 21 μMs of MG-132 process 4 liters about 1.5 × 10 ⁶the HeLaS3 cell of individual cells/ml.After treatment, from total culture, pipette the cell of 1 liter immediately, by harvested by centrifugation, with 200mLPBS washing, and recentrifuge.This sample is used as time zero time point.With the remaining 3 liters of cell cultures of 100ng/mLTNF process.Pipette, gather in the crops 1 liter of cell and with 200mLPBS washing, and with recentrifuge respective after TNF process 5,15 and 25 minutes.At IP lysis buffer (the LB) (20mMTrispH7.5 of 30mL, 150mMNaCl, 1%Tritonx-100,1mMEDTA, 25 μMs of MG-132, often kind of protease inhibitor cocktail 1 and 2 (Sigma) of 10mMNEM, 30 μ L and 1 adequate proteins enzyme inhibitors (Completeproteaseinhibitor) (Roche)) in cracking from each time point cell and at 4 DEG C, rotate incubation 20-60 minute.Often kind of lysate to be transferred in 50mL centrifuge tube and again to precipitate for centrifugal 5 minutes under 10,000xg.Estimate the protein concentration from the lysate of each time point.Will often kind of lysate (there is the 30mL of normalized protein concentration for each time point) and the untight albumin A of 1mL/G pearl incubation 1.25-1.5 hour at 4 DEG C.By centrifugal 5 minutes under 2000rpm, pearl and chip and lysate are separated.From often kind of lysate, sampling is used for direct western blot analysis, and by remaining freezing at-80 DEG C.

Gather four parts of 16mL samples of often kind of lysate.25mMMG-132 and 20 μ LNEM is added to often kind of sample.By two increment product separately with the anti-TNFR1 Antibody Combination of 2.4 μ g/mL.Other two increment product are combined with the control antibodies (anti-myc) of 2.4 μ g/mL separately.All samples all rotates incubation 2 hours at 4 DEG C, then adds the 50% untight albumin A pearl slurry of 150 μ L.By extra for rotary sample incubation 5 hours at 4 DEG C.By centrifugation sample, pearl 15mLLB washs once, contain the LB of 1MNaCl with 10mL washes twice and washes twice with 10mLLB.The pearl of washing is resuspended in 1.25mLLB, and transfers in Eppendorf tube.To often kind of sample drawing liquid, make this pipe contain the cumulative volume of 950 μ L, and add in 360mg solid urea to often kind of sample and make the final concentration of urea in often kind of sample be 6M.Room temperature gentle agitation sample incubation 15 minutes.The pearl in sample is often planted by centrifugation.The part retaining often kind of supernatant liquor is used for western blot analysis, and with the dilution buffer (1%Triton-X100 that dissociates, 0.5% deoxycholate salt, 120mMNaCl, 50mMHEPESpH7.2 and adequate proteins Protease Inhibitor Cocktail (Completeproteaseinhibitorcocktail) (Roche)) dilution from the remaining supernatant liquor (approximately often kind of sample 1mL) of often kind of sample to 10mL.

Often kind of sample is divided into the part of two 5mL.The part apu2.16 process of the IgG form that is rearranged to from fab form of 2.5 μ g, another part apu2.07 process of the IgG form that is rearranged to from fab form of 2.5 μ g.In these two samples, add 50 μ L albumin A pearls, and at 4 DEG C incubated samples 5 hours.Precipitation also washs albumin A pearl 3 times in TNFR1LB, is transferred in Eppendorf tube by pearl in carrying out washing treatment process.Added to by sample buffer in all samples, often kind of sample (before comprising for sample that western blot analysis is preserved) is all reduced and in 10%tris/gly1.5mm10-hole gel (Invitrogen) runs glue.Run after glue, according to the working instructions of manufacturer by the protein delivery in gel to Invitrolon ^tMon pvdf membrane (Invitrogen).To spend the night with the detection of anti-RIP monoclonal antibody (BectonDickinson) at room temperature 5%PBS-T closing membrane.Then wash trace, with goat anti-mouse second antibody (Cappel) detection of puting together HRP, washing, is exposed to reagent to excite chemoluminescence, and is exposed to film.Result is shown in Figure 21 A and 21B.

Figure 21 A depicts and contains first with TNFR1 or anti-myc immunoprecipitation and then with the trace of the sample of apu2.16IgG (polyubiquitin that K63 connects optionally) immunoprecipitation.As shown in figure 21 a, in anti-myc control sample or time zero sample, RIP is not seen.RIP band is the strongest in 5 minutes samples, significantly weakens subsequently at 15 and 25 minutes in sample.Figure 21 B depicts and contains first with TNFR1 or anti-myc immunoprecipitation and then with the trace of the sample of apu2.07IgG (polyubiquitin that K48 connects optionally) immunoprecipitation.As shown in figure 21b, in anti-myc contrast swimming lane, RIP is not observed.In this trace, RIP level is pass by time and increases, and the strongest in 25 minutes point samples.These data are relevant to discovery comparatively early, and described discovery is firm by TNFR1 conducted signal, and RIP, just first by the polyubiquitin that K63 connects, removes ubiquitination and the polyubiquitin connected by K48 by A20 subsequently.Therefore, the polyubiquitin polypeptide that the polyubiquitin polypeptide that the K63 that antibody capable specific binding of the present invention also distinguishes polyubiquitin in cell connects is connected with K48.

Embodiment 4: be separated and identify the anti-polyubiquitin antibody of the third generation

The third generation library (see embodiment 2 and Figure 17 A and 17B) being used for Fab and showing is built by the phagemid of the clone apu2.16 identified before coding (polyubiquitin that K63 connects optionally).Phage from this clone is used for infect CJ236 cell to prepare KunkelDNA template.This template of mutagenic treatment is to insert terminator codon subsequently, and the template containing terminator codon is used for following library construction.

According to three different scheme mutagenic treatment Fabapu2.16 to create the derivative library of three different apu2.16-.In first library, mutagenic treatment HVR-H1 and HVR-H2.Mutagenic treatment HVR-H1, terminator codon to be included in Kunkel template, then utilizes the oligonucleotide of a mutagenesis to carry out mutagenic treatment.Terminator codon encoded oligonucleotide acid sequence is: GCAGCTTCTGGCTTCAACTAATAACACTGGGTGCGTCAGG (SEQIDNO:371).The oligonucleotide of mutagenesis makes the Isoleucine of the 29th can be selected from phenylalanine, leucine, Isoleucine and α-amino-isovaleric acid; Utilize NNS mixed cipher subgroup (wherein N is corresponding to G, C, A or T, and S-phase should in G or C), the Isoleucine of the 34th can be selected from methionine(Met) and Isoleucine; And gentle randomised amino acid K30, T31, G32 and L33.Gentle randomization refers to that the time of some nucleotide position 70% is occupied by indicated base within a context, and each in other three kinds of bases then occupies the time of 10%.Wherein above-mentioned gentleness those oligonucleotide randomized are comprised at particular bases place for those, by indicating gentle randomized existence in the existence of this base positions place numeral.Numeral " 5 " refers to the time occupying 70% in this position bases adenine, and base guanine, cytosine(Cyt) and thymus pyrimidine then occupy the time of 10% respectively.Similarly, numeral " 6 " refers to guanine, and " 7 " refer to cytosine(Cyt), and " 8 " refer to thymus pyrimidine, and in all cases, each in other three kinds of bases only occupies the time of 10%.Oligonucleotide sequence for mutagenic treatment apu3.16HVR-H1 is: GCAGCTTCTGGCTTCAACNTC556577668788ATSCACTGGGTGCGTCAGG (SEQIDNO:785).

Also modify HVR-H2 terminator codon to be included in Kunke template, then utilize the oligonucleotide of three mutagenesis to carry out mutagenesis.In all cases, terminator codon oligonucleotides coding is GGCCTGGAATGGGTTGCATAATAATATGCCGATAGCGTCAAGG (SEQIDNO:373).Article three, the oligonucleotide of mutagenesis is two kinds of changes (permutation) that Article 1 expects sequence, and Article 2 expects that the one of sequence changes (permutation).Article 1, the sequence expected comprises: fix one the 50th and 54 arbitrary tyrosine residuess, utilize another tyrosine residues of NNS mixed cipher subgroup (above-mentioned) randomization; Residue is fixed in the 51st (Isoleucine), 52a (proline(Pro)), 53 (tyrosine), 55 (glycine) and 57 (Threonine) position; And gentle randomization S52, S56 and S58 position (according to above-mentioned gentle randoming scheme).The sequence that Article 2 is expected comprises: at the 51st (Isoleucine), 52a position (proline(Pro)), the 53rd (tyrosine), the 55th (glycine) and the 57th residue that (Threonine) is fixing; Utilize the tyrosine of the fierce mutagenesis the 50th of NNS mixed cipher subgroup (above-mentioned) and 54, and gentle randomization S52, S56 and S58 position (according to above-mentioned gentle mutagenesis program).Oligonucleotide for mutagenic treatment apu2.16HVR-H2 is: GGCCTGGAATGGGTTGCANNSATC567CCGTACTACGGT567ACC567TATGCCGA TAGCGTCAAGG (SEQIDNO:786), GGCCTGGAATGGGTTGCATACATC567CCGTACNNSGGT567ACC567TATGCCGA TAGCGTCAAGG (SEQIDNO:787) and GGCCTGGAATGGGTTGCANNSATC567CCGTACNNSGGT567ACC567TATGCCGA TAGCGTCAAGG (SEQIDNO:788).

In second apu2.16 library, mutagenic treatment HVR-H2 and HVR-H3.The same with the modification carried out HVR-H2 in first apu2.16 library, utilize identical containing the oligonucleotide of terminator codon and the oligonucleotide mutagenic treatment HVR-H2 of identical three mutagenesis.With in first apu18 library to the modification the same (being described in embodiment 2) that HVR-H3 carries out, utilize identical containing the oligonucleotide of terminator codon and the oligonucleotide mutagenic treatment HVR-H3 of identical six mutagenesis.

In the 3rd apu2.16 library, mutagenic treatment HVR-H1 and HVR-H3.The same with the modification carried out HVR-H1 in first apu2.16 library, utilize identical containing the oligonucleotide of terminator codon and the oligonucleotide mutagenic treatment HVR-H1 of identical mutagenesis.With in first apu18 library to the modification the same (being described in embodiment 2) that HVR-H3 carries out, utilize the identical oligonucleotide containing terminator codon and identical six mutagenic oligonucleotide mutagenic treatment HVR-H3.

By electroporation, the mutagenesis reaction of each in the library derived three apu2.16 is transformed in the intestinal bacteria XL-1 of Electrocompetent.In SOC substratum, allow cell stir recovery 30 minutes at 37 DEG C.Retain 20 μ l and contain the SOC substratum of cell for measuring transformant quantity, then remaining media transfer is contained Pyocianil and every milliliter 10 to 500ml ¹⁰in the 2YT of individual M13K07 helper phage.At 37 DEG C, stir incubation after 45 minutes, supplement kantlex and stir culture is spent the night at 37 DEG C to meat soup.The transformant number in each library is > 10 ⁹.Gather in the crops from meat soup by centrifugal and PEG precipitation and concentrate phage, and in selection cycles subsequently.

Described in embodiment 1 (A), the polyubiquitin connected for the K63 be fixed on Maxisorp flat board (NUNC) respectively carries out respective sorting to three third generation libraries.Regulate stringency in three ways: by the concentration of biotinylated polyubiquitin; Before the hole being neutravidin bag quilt catches, carry out competition binding by adding excessive not biotinylated polyubiquitin; And the time length by competing.During incubation step, each solution of taking turns is sorted in sorting damping fluid the polyubiquitin all comprising 3 μMs of single ubiquitins and be connected with 30 μ g/mLK48.Increase and the phage collecting wash-out for further sorting round.Selection cycles is subsequently the sorting of solution phase.First round solution sorting is included in room temperature by biotinylated for 100nM (sulfo-NHS-biotin, Pierce) polyubiquitin and phage library incubation 1 hour.Then in solution sorting damping fluid (PBST containing 0.5%Superblock (Pierce)) by mixture diluted 10 times, and use the hole of neutravidin bag quilt to catch 5 minutes.Reaction containing not biotinylated polyubiquitin chain is used as contrast and combines to monitor background phage.Also 0.1MHCl wash-out 10 minutes are used with PBST washing is dull and stereotyped.Solution sorting is taken turns for the 2nd, as the 1st take turns in 30nM biotinylated polyubiquitin balance phage, but the dissociation rates of 5 minutes are selected to dilute 10 times in containing the solution sorting damping fluid of the not biotinylated polyubiquitin of 30 μ g/mL (K63 connection), then catches on the hole of neutrophilous organism fibroin bag quilt.

After the first round molten sorting, 6.5 times of enrichments (see table E) be observed for the combinatorial library based on apu2.16.For slow dissociation rate, after taking turns solution sorting second, obtain extra 10 times of enrichments (see table E).

Table E: the third generation anti-polyubiquitin antibody library solution separation results

After taking turns dull and stereotyped sorting and the sorting of two-wheeled solution one, in 96-orifice plate, cultivate the single clone being selected from the third generation and screened by the Phage-ELISA such as described in above-described embodiment 2.72 different clones are identified by order-checking.In those clones, in Phage-ELISA measures, there are 12 clones to be proved to have the specificity (Figure 22) of the polyubiquitin connected for K63 of highest level.Those 12 clones are named as apu3.01-3.12, and their HVR-H1, HVR-H2 and HVR-H3 sequence is shown in Figure 23 A and 23B.The light chain HVR of the polyubiquitin specificity clone connected for each K63 does not check order, but HVR-L1 and HVR-L2 sequence should be constant, and HVR-L3 sequence should be identical with apu2.16 simultaneously.According to library designs, HVR-L1 sequence is RASQSVSSAVA (SEQIDNO:79), and HVR-L2 sequence is SASSLYS (SEQIDNO:80).All clones have identical heavy chain and light chain framework region sequence (see Fig. 6).

Using Apu2.07 (see embodiment 2 and Figure 16 A and 16B) in CHO or 293 cells) and apu3.07 (see above and Figure 23 A and 23B) express as human IgG.By the Kunkel mutagenesis preparation table expression constructs (Gorman etc., DNAProt.Eng.Tech.2:3-10 (1990)) of the heavy chain of suitable coding human IgG and the pRK mammalian expression vector of light chain.Use standard method by affinitive layer purification IgG.

The ability of the polyubiquitin specific binding of each IgG key suitable with the tool be fixed on nitrocellulose membrane is evaluated by western blotting.The polyubiquitin connect K48 or K63 and single ubiquitin (BostonBiochem) carry out race glue on 4-20%Tris-Glycine polyacrylamide gel (Invitrogen).By electroblotting, gel inclusion is transferred to nitrocellulose membrane.Dissolving 5% skim-milk containing the Tris-buffer saline (TBST) of 0.1%Tween-20 in close on nitrocellulose membrane nonspecific binding site 1 hour.Then K48 specificity or K63 specific antibody are added in trace with 2 μ g/mL (apu2.07IgG) or 1 μ g/mL (apu3.07IgG) concentration, and incubation is occurred to make combination for 1 hour.As positive control, a trace and rabbit anti-ubiquitin antibody (Sigma) incubation.In TBST, wash trace and pass through with 1: 10 in the TBST containing 5% skim-milk, the antibody that the 000 Goat anti human IgFc (ICN) puting together peroxidase diluted or anti-rabbit Ig (Amersham) detection of puting together peroxidase combine.After 1 hour, in TBST, wash trace and use SuperSignalWestDura reagent (Pierce) to develop to disclose peroxidase activity.Result is shown in Figure 24 A-24D.

As expected, the poly-ubiquitin (Figure 24 A) of trimerization to seven that the four poly-ubiquitins that the immobilized K48 of apu2.07IgG specific recognition connects are connected with K48, but the polyubiquitin sample that not immobilized with any one K63 connects is combined.Similarly, the poly-ubiquitin (Figure 24 B) of trimerization to seven that the four poly-ubiquitins that the immobilized K63 of apu3.07IgG specific recognition connects are connected with K63, but the polyubiquitin sample that not immobilized with any one K48 connects is combined.These two kinds of IgG are not combined with immobilized single ubiquitin.For evaluating the sensitivity of often kind of IgG, be connected with K63 the four poly-ubiquitins (25-1000ng/ swimming lane) using the immobilized K48 of different concns to connect have carried out additional protein engram analysis (Figure 24 C and 24D).Apu2.07IgG detects the four poly-ubiquitins that the immobilized K48 of only 50ng connects, and be not again connected with immobilized K63 four poly-ubiquitin specific bindings (Figure 24 C).Apu3.07IgG detects the four poly-ubiquitins that the immobilized K63 of only 50ng connects, and be not again connected with immobilized K48 four poly-ubiquitin specific bindings (Figure 24 D).In both cases, the quantity increasing by immobilized four poly-ubiquitins just causes the increase of viewed combination.

For determining whether IgG can detect endogenic polyubiquitin albumen, by using or do not use 20 μMs of proteasome inhibitors (bortezomib) the human embryonic kidney cell line 293T processing 4 hours prepares albumen lysate.In 4-20%Tris-Glycine polyacrylamide gel (Invitrogen), differentiate lysate by SDS-PAGE, and carry out western blotting as mentioned above.Result is shown in Figure 25.Exist and do not exist when process, Anti-TNF-α ubiquitin antibody (Sigma) all detects a large amount of high-molecular-weight protein (the most left swimming lane).Apu2.07IgG combines numerous albumen (the rightest swimming lane) with different molecular weight, and compared with immobilized untreated lysate, the viewed combination of lysate for immobilized velcade process is more.Use should observed significantly less combination generally (middle swimming lane) in conjunction with the apu3.07IgG of K63-polyubiquitin albumen, and does not have significant difference between velcade process and untreated swimming lane.The polyubiquitin that known K48 connects turns into making intracellular protein targeting proteins hydrolytic deterioration (Chau etc., Science243:1576-1583 (1989); Finley etc., Mol.Cell.Biol.14:5501-5509 (1994); Flick etc., Nat.Cell.Biol.6:634-641 (2004)).Therefore, a kind of explanation for apu2.07IgG result is that the K48-polyubiquitin albumen of increasing amount is just retained in lysate, causes the apu2.07IgG of the increase exceeding untreated samples to combine when proteolysis processing is prevented from.And do not know that polyubiquitin targeting proteins that K63 connects is for degraded (Pickart and Fushman, Curr.Opin.Chem.Biol.8:610-616 (2004); Hicke and Dunn, Annu.Rev.CellDev.Biol.19:141-172 (2003); Spece etc., Mol.CellBiol.15:1265-1273 (1995); Ulrich, Eukaryot.Cell1:1-10 (2002); Spence etc., Cell102:67-76 (2000); Seibenhener etc., Mol.Cell.Biol.24 (18): 8055-8068 (2004)).Therefore, a kind of explanation for apu3.07IgG result is that the suppression of proteasome does not cause gathering of K63-polyubiquitin albumen.

Embodiment 5: with the structural analysis of the FAB that the polyubiquitin that anti-K63-connects is combined

In order to understand the interaction between the polyubiquitin fab of anti-K63 connection and polyubiquitin better, the dimerization ubiquitin cocrystallization that polyubiquitin fabapu2.16 and the K63 connected by anti-K63 connects.Use 1 μ Lapu2.16 solution (15mg/mL, be dissolved in 10mMTris, in 75mMNaClpH8.0) and 1 μ L pond liquid (cellsolution) (0.1MLiCl, 0.1MTrispH8.2,1M Citrate trianion), growing crystal in hanging drop.Add 0.5 μ L0.1M cupric chloride to often dripping hanging drop, and in the process of several days, grow druse go forward side by side line operate to obtain diffraction monocrystalline to often dripping andeachdropwasstreakseeded. in hanging drop.Structure is measured by molecular replacement.Natural data are collected and with HKL2000 process under 100K.Crystal belongs to C2 spacer, has unit cell dimension with β=107, containing two mixtures (complexes) in an asymmetry unit.The coordinate of service routine Phaser and humanized 4d5fab fragment variant (4d5:DBcode1FVE in PDB) is by molecular replacement analytic structure.In program Coot, carry out mould build and use Refmac5 refined structure.The resolving power of this structure is this mixture is by the R of the R of refine to 24.5% and 30.4% _free.

Interaction between the dimerization ubiquitin that apu2.16 and K63 connects is shown in Figure 26 A-26C.Structure epi-position be when with fab in conjunction with embed at least 25% its Solvent accessible surface and/or at the heavy chain of fab or light chain within there is the combination of the residue of more than one atom.The ubiquitin chain of contribution K63 is chain A and the ubiquitin chain contributing C-end is chain B.Fab light chain residues belongs to chain L and fab heavy chain residues belongs to chain H.Be chain numbering in the following table before residue numbering, and fab residue serial number.

Table F: the residue being arranged in the dimerization ubiquitin bonding interface that apu2.16-K63 connects

Ubiquitin residue	Fab residue
		A 18Glu
A 19Pro	L 31Ser
		A 20Ser	L 49Tyr
A 21Asp	L 50Ser
		A 55Thr	L 51Ala
A 56Leu	L 52Ser
		A 57Ser	L 53Ser
A 58Asp	L 66Arg
		A 60Asn	L 98Phe
A 61Ile
		A 62Gln	H 30Lys
	H 31Thr
		B 8Leu	H 32Gly
B 9Thr	H 33Leu
		B 34Glu	H 50Tyr
B 35Gly	H 52Ser
		B 36Ile	H 54Tyr
B 37Pro	H 55Tyr
		B 39Asp	H 99Glu
B 40Gln	H 100Tyr
		B 71Leu	H 101Tyr
B 72Arg	H 102Arg

B 73Leu	H 104Tyr
		B 74Arg	H 105Thr
B 75Gly

As shown in F and Figure 26 B shown in lead region, when with apu2.16 in conjunction with time, be positioned at apu2.16's within there is the residue in 11 K63-dimerization ubiquitin A chains and the residue in 13 K63-dimerization ubiquitin B chains.As shown as shown in F, and as shown in lead region in Figure 26 C, when with dimerization ubiquitin that K63 connects in conjunction with time, the dimerization ubiquitin being positioned at K63 and connecting within there is the residue in 8 apu2.16 light chains and the residue in 14 apu2.16 heavy chains.Based on these data, the dimerization ubiquitin that apu2.16 with K63 may be regulated the to be connected interactional residue be between the two arranged on the dimerization ubiquitin of K63 connection comprises Glu-18, Ser-20, Leu-57 and Asp-58 of A chain and Pro-37, Arg-74 and Gly-75 of B chain.What is interesting is, this antibody does not interact nearly with K63-Gly76 key, but obtains specificity by the interaction of the dimerization ubiquitin composite surface with this key adjacent.

Sequence table

<110> Genentech Inc (GENENTECH, INC.)

The method and composition of <120> targeting polyubiquitin

<130>P2260R1-PCT

<140>

<141>

<150>60/751,081

<151>2005-12-15

<150>60/793,980

<151>2006-04-21

<160>908

<170>PatentInversion3.3

<210>1

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>1

GlyPheAsnLeuSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>2

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>2

GlyPheAsnValSerTyrSerSerIleHis

1　　　　　　　5　　　　　　10

<210>3

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>3

GlyPheAsnIleTyrTyrSerSerIleHis

1　　　　　　　5　　　　　　10

<210>4

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>4

GlyPheAsnIleSerTyrTyrTyrIleHis

1　　　　　　5　　　　　　　10

<210>5

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>5

GlyPheAsnValSerTyrTyrTyrMetHis

1　　　　　　5　　　　　　　10

<210>6

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>6

GlyPheAsnPheTyrSerSerTyrMetHis

1　　　　　　　5　　　　　　　10

<210>7

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>7

GlyPheAsnLeuTyrTyrSerTyrMetHis

1　　　　　　　5　　　　　　　10

<210>8

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>8

GlyPheAsnValTyrTyrSerSerIleHis

1　　　　　　5　　　　　　　10

<210>9

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>9

GlyPheAsnIleSerTyrSerTyrMetHis

1　　　　　　5　　　　　　　10

<210>10

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>10

GlyPheAsnValTyrTyrSerSerIleHis

1　　　　　　5　　　　　　　10

<210>11

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>11

GlyPheAsnValSerTyrSerTyrMetHis

1　　　　　　5　　　　　　　10

<210>12

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>12

GlyPheAsnLeuTyrTyrSerTyrMetHis

1　　　　　　　5　　　　　　　10

<210>13

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>13

GlyPheAsnValTyrTyrSerSerIleHis

1　　　　　　5　　　　　　　10

<210>14

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>14

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　5　　　　　　　10

<210>15

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>15

GlyPheAsnValSerTyrSerSerIleHis

1　　　　　　5　　　　　　　10

<210>16

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>16

GlyPheAsnPheTyrTyrTyrTyrIleHis

1　　　　　　5　　　　　　　10

<210>17

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>17

GlyPheAsnValSerSerTyrSerIleHis

1　　　　　　5　　　　　　　10

<210>18

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>18

GlyPheAsnValSerSerTyrSerMetHis

1　　　　　　5　　　　　　　10

<210>19

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>19

GlyPheAsnLeuSerTyrTyrSerIleHis

1　　　　　　5　　　　　　　10

<210>20

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>20

GlyPheAsnLeuSerTyrTyrSerIleHis

1　　　　　　5　　　　　　　10

<210>21

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>21

GlyPheAsnValSerTyrSerTyrMetHis

1　　　　　　5　　　　　　　10

<210>22

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>22

GlyPheAsnValSerTyrTyrSerIleHis

1　　　　　　5　　　　　　　10

<210>23

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>23

GlyPheAsnLeuSerTyrSerSerIleHis

1　　　　　　5　　　　　　　10

<210>24

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>24

GlyPheAsnValSerTyrSerSerIleHis

1　　　　　　5　　　　　　　10

<210>25

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>25

GlyPheAsnLeuSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>26

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(4)..(4)

<223> leucine, α-amino-isovaleric acid, Isoleucine or phenylalanine

<220>

<221>MOD_RES

<222>(5)..(8)

<223> Serine or tyrosine

<220>

<221>MOD_RES

<222>(9)..(9)

<223> methionine(Met) or Isoleucine

<400>26

GlyPheAsnXaaXaaXaaXaaXaaXaaHis

1　　　　　　　5　　　　　　　10

<210>27

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>27

SerIleTyrProTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>28

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>28

SerIleSerProTyrTyrGlyTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>29

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>29

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>30

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>30

SerIleSerSerTyrTyrGlyTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>31

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>31

SerIleSerSerTyrTyrGlyTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>32

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>32

SerIleSerProTyrTyrGlyTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>33

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>33

SerIleSerSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>34

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>34

SerIleTyrProTyrTyrGlyTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>35

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>35

SerIleSerSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>36

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>36

SerIleSerProTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>37

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>37

SerIleSerProTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>38

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>38

SerIleSerProTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>39

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>39

SerIleSerProTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>40

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>40

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>41

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>41

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>42

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>42

SerIleSerProTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>43

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>43

SerIleSerSerSerTyrSerSerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>44

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>44

SerIleTyrSerSerSerGlySerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>45

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>45

SerIleTyrSerSerTyrSerSerThrTyrTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>46

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>46

SerIleTyrProTyrTyrGlyTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>47

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>47

SerIleSerSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>48

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>48

SerIleTyrProTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>49

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>49

SerIleTyrProTyrTyrGlyTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>50

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>50

SerIleTyrProTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>51

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>51

SerIleTyrProTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>52

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(3)..(3)

<223> tyrosine or Serine

<220>

<221>MOD_RES

<222>(4)..(4)

<223> proline(Pro) or Serine

<220>

<221>MOD_RES

<222>(5)..(6)

<223> tyrosine or Serine

<220>

<221>MOD_RES

<222>(7)..(7)

<223> Serine or glycine

<220>

<221>MOD_RES

<222>(8)..(8)

<223> tyrosine or Serine

<220>

<221>MOD_RES

<222>(10)..(10)

<223> Serine or tyrosine

<400>52

SerIleXaaXaaXaaXaaXaaXaaThrXaaTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>53

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>53

GlyTyrGluGlyGlyMetAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>54

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>54

AspGlyTyrAlaMetAspAlaLeuAspTyr

1　　　　　　　5　　　　　　　10

<210>55

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>55

LeuTyrHisAsnThrLeuGlyMetAspTyr

1　　　　　　　5　　　　　　　10

<210>56

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>56

ProTyrSerTyrSerGluAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>57

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>57

GluTyrTyrMetTyrAspAlaLeuAspTyr

1　　　　　　　5　　　　　　　10

<210>58

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>58

AspTyrTyrTyrIleSerAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>59

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>59

SerTyrSerTyrSerSerAlaLeuAspTyr

1　　　　　　　5　　　　　　　10

<210>60

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>60

GlyTyrLysTyrTrpSerAlaPheAspTyr

1　　　　　　5　　　　　　　10

<210>61

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>61

SerTyrSerSerTyrSerAlaIleAspTyr

1　　　　　　5　　　　　　　10

<210>62

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>62

GluGlyTyrSerGlnGlyGlyPheAspTyr

1　　　　　　　5　　　　　　　10

<210>63

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>63

SerTyrGlyTyrTyrValAlaPheAspTyr

1　　　　　　5　　　　　　　10

<210>64

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>64

AspTyrLysPheGlyTyrAlaIleAspTyr

1　　　　　　　5　　　　　　10

<210>65

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>65

GluGlyTyrSerGlnGlyGlyPheAspTyr

1　　　　　　　5　　　　　　10

<210>66

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>66

SerTyrSerTyrTyrSerAlaMetAspTyr

1　　　　　　5　　　　　　　10

<210>67

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>67

GlyTyrMetTrpTyrGlyGlyIleAspTyr

1　　　　　　5　　　　　　　10

<210>68

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>68

GluTyrTyrSerTyrLeuGlyAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>69

<211>21

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>69

HisThrLysTyrValTyrLeuTyrThrTyrTrpGluAspSerMetAsp

1　　　　　　5　　　　　　　10　　　　　　　15

TyrGlyLeuAspTyr

　　　　　20

<210>70

<211>21

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>70

SerSerIleSerGluTrpTyrGlySerTrpTyrTyrPheTrpGluSer

1　　　　　　5　　　　　　　10　　　　　　　15

SerGlyIleAspTyr

　　　　　20

<210>71

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>71

GluSerTyrTrpSerTyrAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>72

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>72

SerTyrSerTyrSerTyrGlyIleAspTyr

1　　　　　　5　　　　　　　10

<210>73

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>73

SerTyrSerTyrTyrSerAlaIleAspTyr

1　　　　　　5　　　　　　　10

<210>74

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>74

SerTyrSerTyrSerTyrGlyLeuAspTyr

1　　　　　　5　　　　　　　10

<210>75

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>75

GlyTyrIleHisTrpGluAlaLeuAspTyr

1　　　　　　5　　　　　　　10

<210>76

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>76

SerTyrSerTyrSerSerGlyLeuAspTyr

1　　　　　　5　　　　　　　10

<210>77

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>77

SerTyrSerTyrSerTyrGlyMetAspTyr

1　　　　　　5　　　　　　　10

<210>78

<211>21

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(1)..(1)

<223> glycine, aspartic acid, leucine, proline(Pro), L-glutamic acid, Serine or Histidine

<220>

<221>MOD_RES

<222>(2)..(2)

<223> tyrosine, glycine, Threonine or Serine

<220>

<221>MOD_RES

<222>(3)..(3)

<223> L-glutamic acid, tyrosine, Histidine, Serine, Methionin, glycine, methionine(Met) or Isoleucine

<220>

<221>MOD_RES

<222>(4)..(4)

<223> glycine, L-Ala, the acid of asparagus fern acyl, tyrosine, methionine(Met), Serine, phenylalanine, tryptophane or Histidine

<220>

<221>MOD_RES

<222>(5)..(5)

<223> glycine, methionine(Met), Threonine, Serine, tyrosine, Isoleucine, tryptophane, glutamine, α-amino-isovaleric acid or L-glutamic acid

<220>

<221>MOD_RES

<222>(6)..(6)

<223> methionine(Met), aspartic acid, leucine, L-glutamic acid, Serine, glycine, α-amino-isovaleric acid, tyrosine or tryptophane

<220>

<221>MOD_RES

<222>(7)..(7)

<223> L-Ala, glycine, leucine or tyrosine

<220>

<221>MOD_RES

<222>(8)..(8)

<223> methionine(Met), leucine, Isoleucine, phenylalanine, L-Ala, tyrosine or glycine

<220>

<221>MOD_RES

<222>(9)..(9)

<223> Isoleucine, Threonine, Serine or do not exist

<220>

<221>MOD_RES

<222>(10)..(11)

<223> tyrosine, tryptophane or do not exist

<220>

<221>MOD_RES

<222>(12)..(12)

<223> L-glutamic acid, tyrosine or do not exist

<220>

<221>MOD_RES

<222>(13)..(13)

<223> aspartic acid, phenylalanine or do not exist

<220>

<221>MOD_RES

<222>(14)..(14)

<223> Serine, tryptophane or do not exist

<220>

<221>MOD_RES

<222>(15)..(15)

<223> methionine(Met), L-glutamic acid or do not exist

<220>

<221>MOD_RES

<222>(16)..(16)

<223> aspartic acid, Serine or do not exist

<220>

<221>MOD_RES

<222>(17)..(17)

<223> tyrosine, Serine or do not exist

<220>

<221>MOD_RES

<222>(18)..(18)

This residue presence or absence of <223>

<220>

<221>MOD_RES

<222>(19)..(19)

<223> leucine, Isoleucine or do not exist

<400>78

XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa

1　　　　　　　5　　　　　　　　10　　　　　　　　15

XaaGlyXaaAspTyr

　　　　　20

<210>79

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>79

ArgAlaSerGlnSerValSerSerAlaValAla

1　　　　　　　5　　　　　　　10

<210>80

<211>7

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>80

SerAlaSerSerLeuTyrSer

1　　　　　　5

<210>81

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>81

GlyPheAsnIleSerSerSerTyrIleHis

1　　　　　　5　　　　　　　10

<210>82

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>82

GlyPheAsnPheSerTyrSerTyrIleHis

1　　　　　　5　　　　　　　10

<210>83

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>83

GlyPheAsnLeuSerTyrTyrTyrIleHis

1　　　　　　5　　　　　　　10

<210>84

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>84

GlyPheAsnPheTyrSerSerTyrIleHis

1　　　　　　5　　　　　　　10

<210>85

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>85

GlyPheAsnPheTyrSerSerSerIleHis

1　　　　　　5　　　　　　　10

<210>86

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>86

GlyPheAsnIleSerSerSerSerIleHis

1　　　　　　5　　　　　　　10

<210>87

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>87

GlyPheAsnPheTyrTyrSerSerIleHis

1　　　　　　　5　　　　　　10

<210>88

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>88

GlyPheAsnPheSerTyrSerSerIleHis

1　　　　　　5　　　　　　　10

<210>89

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>89

GlyPheAsnValSerSerTyrSerIleHis

1　　　　　　5　　　　　　　10

<210>90

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(4)..(4)

<223> Isoleucine, phenylalanine, leucine or α-amino-isovaleric acid

<220>

<221>MOD_RES

<222>(5)..(8)

<223> Serine or tyrosine

<400>90

GlyPheAsnXaaXaaXaaXaaXaaIleHis

1　　　　　　　5　　　　　　　10

<210>91

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>91

TyrIleSerProTyrTyrGlySerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>92

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>92

SerIleSerSerSerTyrGlyTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>93

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>93

SerIleSerSerTyrTyrGlyTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>94

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>94

SerIleSerSerSerTyrGlySerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>95

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>95

SerIleSerSerTyrTyrGlySerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>96

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>96

SerIleSerProSerTyrSerSerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>97

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>97

SerIleTyrSerSerTyrGlyTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>98

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>98

SerIleTyrSerSerTyrSerSerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>99

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>99

SerIleTyrProSerSerGlySerThrTyrTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>100

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(1)..(1)

<223> tyrosine or Serine

<220>

<221>MOD_RES

<222>(3)..(3)

<223> Serine or tyrosine

<220>

<221>MOD_RES

<222>(4)..(4)

<223> proline(Pro) or Serine

<220>

<221>MOD_RES

<222>(5)..(6)

<223> tyrosine or Serine

<220>

<221>MOD_RES

<222>(7)..(7)

<223> glycine or Serine

<220>

<221>MOD_RES

<222>(8)..(8)

<223> Serine or tyrosine

<220>

<221>MOD_RES

<222>(10)..(10)

<223> Serine or tyrosine

<400>100

XaaIleXaaXaaXaaXaaXaaXaaThrXaaTyrAlaAspSerValLys

1　　　　　　5　　　　　　　　10　　　　　　　　15

Gly

<210>101

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>101

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>102

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>102

GluLysMetTyrTyrSerTyrGlyPheAspTyr

1　　　　　　　5　　　　　　　10

<210>103

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>103

GluSerTyrSerIleHisPheGlyPheAspTyr

1　　　　　　　5　　　　　　　10

<210>104

<211>16

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>104

MetTyrTyrSerTyrTyrTrpArgProTyrGlyAsnAlaIleAspTyr

1　　　　　　5　　　　　　　10　　　　　　　15

<210>105

<211>19

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>105

GlySerIleProSerTyrTrpSerAlaAspTrpTyrTyrTyrTyrGly

1　　　　　　5　　　　　　　10　　　　　　　15

LeuAspTyr

<210>106

<211>14

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>106

TyrLysTyrAsnTyrTyrTyrPheGluSerGlyMetAspTyr

1　　　　　　　5　　　　　　　10

<210>107

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>107

GluTyrTyrTrpTrpTyrLysGluAlaTrpTyrSerAlaGlyMetAsp

1　　　　　　5　　　　　　　10　　　　　　　15

Tyr

<210>108

<211>19

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>108

GlyIleMetPheSerSerTrpTrpTrpTyrTyrAspTyrSerAspAla

1　　　　　　5　　　　　　　10　　　　　　　15

LeuAspTyr

<210>109

<211>21

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>109

SerGlyTyrTyrTyrGlnGlyTyrTrpTrpTyrTyrTyrThrGlyTyr

1　　　　　　5　　　　　　　10　　　　　　　15

TyrGlyMetAspTyr

　　　　20

<210>110

<211>21

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(1)..(1)

<223> L-glutamic acid, methionine(Met), glycine, tyrosine or Serine

<220>

<221>MOD_RES

<222>(2)..(2)

<223> tyrosine, Methionin, Serine, Isoleucine or glycine

<220>

<221>MOD_RES

<222>(3)..(3)

<223> tyrosine, methionine(Met) or Isoleucine

<220>

<221>MOD_RES

<222>(4)..(4)

<223> arginine, tyrosine, Serine, proline(Pro), the acid of asparagus fern acyl, tryptophane or phenylalanine

<220>

<221>MOD_RES

<222>(5)..(5)

<223> tryptophane, tyrosine, Isoleucine or Serine

<220>

<221>MOD_RES

<222>(6)..(6)

<223> tyrosine, Serine, Histidine or glutamine

<220>

<221>MOD_RES

<222>(7)..(7)

<223> Threonine, tyrosine, phenylalanine, tryptophane, Methionin or glycine

<220>

<221>MOD_RES

<222>(8)..(8)

<223> L-Ala, glycine, arginine, Serine, phenylalanine, L-glutamic acid, tryptophane or tyrosine

<220>

<221>MOD_RES

<222>(9)..(9)

<223> Isoleucine, phenylalanine, proline(Pro), L-Ala, L-glutamic acid or tryptophane

<220>

<221>MOD_RES

<222>(10)..(10)

<223> tyrosine, aspartic acid, Serine, tryptophane or do not exist

<220>

<221>MOD_RES

<222>(11)..(11)

<223> glycine, tryptophane, tyrosine or do not exist

<220>

<221>MOD_RES

<222>(12)..(12)

The acid of <223> asparagus fern acyl, tyrosine, methionine(Met), Serine, aspartic acid or do not exist

<220>

<221>MOD_RES

<222>(13)..(13)

<223> L-Ala, tyrosine or do not exist

<220>

<221>MOD_RES

<222>(14)..(14)

<223> Isoleucine, tyrosine, glycine, Serine, Threonine or do not exist

<220>

<221>MOD_RES

<222>(15)..(15)

<223> tyrosine, methionine(Met), aspartic acid, glycine or do not exist

<220>

<221>MOD_RES

<222>(16)..(16)

<223> glycine, L-Ala, tyrosine or do not exist

<220>

<221>MOD_RES

<222>(17)..(17)

<223> leucine, tyrosine or do not exist

<220>

<221>MOD_RES

<222>(18)..(18)

This residue of <223> can be presence or absence

<220>

<221>MOD_RES

<222>(19)..(19)

This residue of <223> can be presence or absence

<400>110

XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa

1　　　　　　5　　　　　　　10　　　　　　　15

XaaGlyMetAspTyr

　　　　　20

<210>111

<211>30

<212>PRT

The <213> mankind (Homosapiens)

<400>111

GlnValGlnLeuValGlnSerGlyAlaGluValLysLysProGlyAla

1　　　　　　5　　　　　　　10　　　　　　　15

SerValLysValSerCysLysAlaSerGlyTyrThrPheThr

　　　　20　　　　　　　25　　　　　　　30

<210>112

<211>25

<212>PRT

The <213> mankind (Homosapiens)

<400>112

GlnValGlnLeuValGlnSerGlyAlaGluValLysLysProGlyAla

1　　　　　　5　　　　　　　10　　　　　　　15

SerValLysValSerCysLysAlaSer

　　　　　　20　　　　　　25

<210>113

<211>25

<212>PRT

The <213> mankind (Homosapiens)

<400>113

GlnValGlnLeuValGlnSerGlyAlaGluValLysLysProGlyAla

1　　　　　　5　　　　　　　10　　　　　　　15

SerValLysValSerCysLysAlaSer

　　　　　20　　　　　　25

<210>114

<211>25

<212>PRT

The <213> mankind (Homosapiens)

<400>114

GlnValGlnLeuValGlnSerGlyAlaGluValLysLysProGlyAla

1　　　　　　5　　　　　　　10　　　　　　　15

SerValLysValSerCysLysAlaSer

　　　　　20　　　　　　25

<210>115

<211>30

<212>PRT

The <213> mankind (Homosapiens)

<400>115

GlnValGlnLeuGlnGluSerGlyProGlyLeuValLysProSerGln

1　　　　　　5　　　　　　　10　　　　　　　15

ThrLeuSerLeuThrCysThrValSerGlyGlySerValSer

　　　　20　　　　　　　25　　　　　　　30

<210>116

<211>25

<212>PRT

The <213> mankind (Homosapiens)

<400>116

GlnValGlnLeuGlnGluSerGlyProGlyLeuValLysProSerGln

1　　　　　　5　　　　　　　10　　　　　　　15

ThrLeuSerLeuThrCysThrValSer

　　　　　20　　　　　　　25

<210>117

<211>25

<212>PRT

The <213> mankind (Homosapiens)

<400>117

GlnValGlnLeuGlnGluSerGlyProGlyLeuValLysProSerGln

1　　　　　　5　　　　　　　10　　　　　　　15

ThrLeuSerLeuThrCysThrValSer

　　　　　20　　　　　　　25

<210>118

<211>25

<212>PRT

The <213> mankind (Homosapiens)

<400>118

GlnValGlnLeuGlnGluSerGlyProGlyLeuValLysProSerGln

1　　　　　　5　　　　　　　10　　　　　　　15

ThrLeuSerLeuThrCysThrValSer

　　　　　20　　　　　　25

<210>119

<211>30

<212>PRT

The <213> mankind (Homosapiens)

<400>119

GluValGlnLeuValGluSerGlyGlyGlyLeuValGlnProGlyGly

1　　　　　　5　　　　　　　10　　　　　　　15

SerLeuArgLeuSerCysAlaAlaSerGlyPheThrPheSer

　　　　　20　　　　　　　25　　　　　　　30

<210>120

<211>25

<212>PRT

The <213> mankind (Homosapiens)

<400>120

GluValGlnLeuValGluSerGlyGlyGlyLeuValGlnProGlyGly

1　　　　　　5　　　　　　　10　　　　　　　15

SerLeuArgLeuSerCysAlaAlaSer

　　　　　20　　　　　　25

<210>121

<211>25

<212>PRT

The <213> mankind (Homosapiens)

<400>121

GluValGlnLeuValGluSerGlyGlyGlyLeuValGlnProGlyGly

1　　　　　　5　　　　　　　10　　　　　　　15

SerLeuArgLeuSerCysAlaAlaSer

　　　　　20　　　　　　　25

<210>122

<211>25

<212>PRT

The <213> mankind (Homosapiens)

<400>122

GluValGlnLeuValGluSerGlyGlyGlyLeuValGlnProGlyGly

1　　　　　　5　　　　　　　10　　　　　　　15

SerLeuArgLeuSerCysAlaAlaSer

　　　　　20　　　　　　　25

<210>123

<211>30

<212>PRT

The <213> mankind (Homosapiens)

<400>123

GluValGlnLeuValGluSerGlyGlyGlyLeuValGlnProGlyGly

1　　　　　　5　　　　　　　10　　　　　　　15

SerLeuArgLeuSerCysAlaAlaSerGlyPheAsnIleLys

　　　　20　　　　　　　25　　　　　　　30

<210>124

<211>25

<212>PRT

The <213> mankind (Homosapiens)

<400>124

GluValGlnLeuValGluSerGlyGlyGlyLeuValGlnProGlyGly

1　　　　　　5　　　　　　　10　　　　　　　15

SerLeuArgLeuSerCysAlaAlaSer

　　　　　20　　　　　　25

<210>125

<211>25

<212>PRT

The <213> mankind (Homosapiens)

<400>125

GluValGlnLeuValGluSerGlyGlyGlyLeuValGlnProGlyGly

1　　　　　　5　　　　　　　10　　　　　　　15

SerLeuArgLeuSerCysAlaAlaSer

　　　　　　20　　　　　　25

<210>126

<211>30

<212>PRT

The <213> mankind (Homosapiens)

<400>126

GluValGlnLeuValGluSerGlyGlyGlyLeuValGlnProGlyGly

1　　　　　　5　　　　　　　10　　　　　　　15

SerLeuArgLeuSerCysAlaAlaSerGlyPheAsnIleLys

　　　　20　　　　　　　25　　　　　　　30

<210>127

<211>25

<212>PRT

The <213> mankind (Homosapiens)

<400>127

GluValGlnLeuValGluSerGlyGlyGlyLeuValGlnProGlyGly

1　　　　　　5　　　　　　　10　　　　　　　15

SerLeuArgLeuSerCysAlaAlaSer

　　　　　　20　　　　　　25

<210>128

<211>25

<212>PRT

The <213> mankind (Homosapiens)

<400>128

GluValGlnLeuValGluSerGlyGlyGlyLeuValGlnProGlyGly

1　　　　　　　5　　　　　　　10　　　　　　　15

SerLeuArgLeuSerCysAlaAlaSer

　　　　　　20　　　　　　25

<210>129

<211>25

<212>PRT

The <213> mankind (Homosapiens)

<400>129

GluValGlnLeuValGluSerGlyGlyGlyLeuValGlnProGlyGly

1　　　　　　　5　　　　　　　10　　　　　　15

SerLeuArgLeuSerCysAlaAlaSer

　　　　　　20　　　　　　25

<210>130

<211>23

<212>PRT

The <213> mankind (Homosapiens)

<400>130

AspIleGlnMetThrGlnSerProSerSerLeuSerAlaSerValGly

1　　　　　　5　　　　　　　10　　　　　　　15

AspArgValThrIleThrCys

　　　　　20

<210>131

<211>23

<212>PRT

The <213> mankind (Homosapiens)

<400>131

AspIleValMetThrGlnSerProLeuSerLeuProValThrProGly

1　　　　　　5　　　　　　　10　　　　　　　15

GluProAlaSerIleSerCys

　　　　　20

<210>132

<211>23

<212>PRT

The <213> mankind (Homosapiens)

<400>132

GluIleValLeuThrGlnSerProGlyThrLeuSerLeuSerProGly

1　　　　　　5　　　　　　　10　　　　　　　15

GluArgAlaThrLeuSerCys

　　　　　20

<210>133

<211>23

<212>PRT

The <213> mankind (Homosapiens)

<400>133

AspIleValMetThrGlnSerProAspSerLeuAlaValSerLeuGly

1　　　　　　5　　　　　　　10　　　　　　　15

GluArgAlaThrIleAsnCys

　　　　　20

<210>134

<211>23

<212>PRT

The <213> mankind (Homosapiens)

<400>134

AspIleGlnMetThrGlnSerProSerSerLeuSerAlaSerValGly

1　　　　　　5　　　　　　　10　　　　　　　15

AspArgValThrIleThrCys

　　　　　20

<210>135

<211>15

<212>PRT

The <213> mankind (Homosapiens)

<400>135

TrpTyrGlnGlnLysProGlyLysAlaProLysLeuLeuIleTyr

1　　　　　　5　　　　　　　10　　　　　　　15

<210>136

<211>32

<212>PRT

The <213> mankind (Homosapiens)

<400>136

GlyValProSerArgPheSerGlySerArgSerGlyThrAspPheThr

1　　　　　　5　　　　　　　10　　　　　　　15

LeuThrIleSerSerLeuGlnProGluAspPheAlaThrTyrTyrCys

　　　　20　　　　　　　25　　　　　　　30

<210>137

<211>10

<212>PRT

The <213> mankind (Homosapiens)

<400>137

PheGlyGlnGlyThrLysValGluIleLys

1　　　　　　5　　　　　　　10

<210>138

<211>25

<212>PRT

The <213> mankind (Homosapiens)

<400>138

GluValGlnLeuValGluSerGlyGlyGlyLeuValGlnProGlyGly

1　　　　　　5　　　　　　　10　　　　　　　15

SerLeuArgLeuSerCysAlaAlaSer

　　　　　　20　　　　　　25

<210>139

<211>13

<212>PRT

The <213> mankind (Homosapiens)

<400>139

TrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal

1　　　　　　　5　　　　　　　10

<210>140

<211>30

<212>PRT

The <213> mankind (Homosapiens)

<400>140

ArgPheThrIleSerAlaAspThrSerLysAsnThrAlaTyrLeuGln

1　　　　　　5　　　　　　　10　　　　　　　15

MetAsnSerLeuArgAlaGluAspThrAlaValTyrTyrCys

　　　　　20　　　　　　　25　　　　　　　30

<210>141

<211>11

<212>PRT

The <213> mankind (Homosapiens)

<400>141

TrpGlyGlnGlyThrLeuValThrValSerSer

1　　　　　　5　　　　　　　10

<210>142

<211>23

<212>PRT

The <213> mankind (Homosapiens)

<400>142

AspIleGlnMetThrGlnSerProSerSerLeuSerAlaSerValGly

1　　　　　　5　　　　　　　10　　　　　　　15

AspArgValThrIleThrCys

　　　　　20

<210>143

<211>15

<212>PRT

The <213> mankind (Homosapiens)

<400>143

TrpTyrGlnGlnLysProGlyLysAlaProLysLeuLeuIleTyr

1　　　　　　5　　　　　　　10　　　　　　　15

<210>144

<211>32

<212>PRT

The <213> mankind (Homosapiens)

<400>144

GlyValProSerArgPheSerGlySerGlySerGlyThrAspPheThr

1　　　　　　5　　　　　　　10　　　　　　　15

LeuThrIleSerSerLeuGlnProGluAspPheAlaThrTyrTyrCys

　　　　　20　　　　　　　25　　　　　　　30

<210>145

<211>10

<212>PRT

The <213> mankind (Homosapiens)

<400>145

PheArgGlnGlyThrLysValGluIleLys

1　　　　　　5　　　　　　　10

<210>146

<211>25

<212>PRT

The <213> mankind (Homosapiens)

<400>146

GluValGlnLeuValGluSerGlyGlyGlyLeuValGlnProGlyGly

1　　　　　　5　　　　　　　10　　　　　　　15

SerLeuArgLeuSerCysAlaAlaSer

　　　　　　20　　　　　　25

<210>147

<211>13

<212>PRT

The <213> mankind (Homosapiens)

<400>147

TrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal

1　　　　　　　5　　　　　　　10

<210>148

<211>30

<212>PRT

The <213> mankind (Homosapiens)

<400>148

ArgPheThrIleSerArgAspAsnSerLysAsnThrLeuTyrLeuGln

1　　　　　　5　　　　　　　10　　　　　　　15

MetAsnSerLeuArgAlaGluAspThrAlaValTyrTyrCys

　　　　20　　　　　　　25　　　　　　　30

<210>149

<211>11

<212>PRT

The <213> mankind (Homosapiens)

<400>149

TrpGlyGlnGlyThrLeuValThrValSerSer

1　　　　　　　5　　　　　　　10

<210>150

<211>66

<212>DNA

<213> artificial sequence

<220>

The description of <223> artificial sequence: the oligonucleotide of synthesis

<400>150

tcttgtgacaaaactcaccatcaccatcaccatcactagggcggtggctctggttccggt　60

gatttt　　　　　　　　　　　　　　　　　　　　　　　　66

<210>151

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>151

GlyPheAsnValTyrTyrSerSerIleHis

1　　　　　　5　　　　　　　10

<210>152

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>152

GlyPheAsnValSerTyrSerTyrMetHis

1　　　　　　5　　　　　　　10

<210>153

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>153

GlyPheAsnPheSerTyrTyrSerMetHis

1　　　　　　5　　　　　　　10

<210>154

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>154

GlyPheAsnLeuSerTyrTyrSerIleHis

1　　　　　　5　　　　　　　10

<210>155

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>155

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　5　　　　　　　10

<210>156

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>156

GlyPheAsnValTyrTyrSerSerIleHis

1　　　　　　5　　　　　　　10

<210>157

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>157

GlyPheAsnLeuSerTyrSerTyrIleHis

1　　　　　　5　　　　　　　10

<210>158

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>158

GlyPheAsnPheTyrTyrTyrTyrIleHis

1　　　　　　5　　　　　　　10

<210>159

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>159

GlyPheAsnLeuSerTyrSerSerIleHis

1　　　　　　　5　　　　　　10

<210>160

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>160

GlyPheAsnValSerTyrSerSerIleHis

1　　　　　　5　　　　　　　10

<210>161

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>161

GlyPheAsnValSerTyrSerSerIleHis

1　　　　　　5　　　　　　　10

<210>162

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>162

GlyPheAsnValTyrTyrSerSerIleHis

1　　　　　　5　　　　　　　10

<210>163

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>163

GlyPheAsnValSerTyrTyrTyrIleHis

1　　　　　　5　　　　　　　10

<210>164

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>164

GlyPheAsnLeuSerTyrSerSerIleHis

1　　　　　　　5　　　　　　10

<210>165

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>165

GlyPheAsnIleSerTyrSerTyrMetHis

1　　　　　　　5　　　　　　10

<210>166

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>166

GlyPheAsnLeuTyrTyrSerTyrMetHis

1　　　　　　　5　　　　　　　10

<210>167

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>167

GlyPheAsnValSerTyrTyrTyrMetHis

1　　　　　　5　　　　　　　10

<210>168

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>168

GlyPheAsnIleSerTyrSerTyrMetHis

1　　　　　　5　　　　　　　10

<210>169

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>169

GlyPheAsnIleSerTyrSerSerIleHis

1　　　　　　5　　　　　　　10

<210>170

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>170

GlyPheAsnValSerTyrSerSerMetHis

1　　　　　　5　　　　　　　10

<210>171

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>171

GlyPheAsnLeuSerTyrTyrSerIleHis

1　　　　　　5　　　　　　　10

<210>172

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>172

GlyPheAsnValSerTyrTyrSerIleHis

1　　　　　　5　　　　　　　10

<210>173

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>173

GlyPheAsnIleSerTyrSerSerIleHis

1　　　　　　5　　　　　　　10

<210>174

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>174

GlyPheAsnPheSerTyrTyrSerIleHis

1　　　　　　　5　　　　　　10

<210>175

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>175

GlyPheAsnLeuSerTyrSerSerMetHis

1　　　　　　5　　　　　　　10

<210>176

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(4)..(4)

<223> α-amino-isovaleric acid, phenylalanine, leucine or Isoleucine

<220>

<221>MOD_RES

<222>(5)..(5)

<223> tyrosine or Serine

<220>

<221>MOD_RES

<222>(7)..(8)

<223> Serine or tyrosine

<220>

<221>MOD_RES

<222>(9)..(9)

<223> Isoleucine or methionine(Met)

<400>176

GlyPheAsnXaaXaaTyrXaaXaaXaaHis

1　　　　　　　5　　　　　　　10

<210>177

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>177

SerIleSerProTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>178

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>178

SerIleSerSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>179

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>179

SerIleTyrProTyrTyrGlyTyrThrTyrTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>180

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>180

SerIleTyrProTyrTyrGlyTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>181

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>181

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>182

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>182

SerIleTyrProTyrTyrGlyTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>183

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>183

SerIleSerSerTyrTyrGlySerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>184

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>184

SerIleSerProTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>185

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>185

SerIleTyrProTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>186

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>186

SerIleTyrProTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>187

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>187

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>188

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>188

SerIleTyrProTyrTyrGlyTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>189

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>189

SerIleSerSerTyrTyrGlyTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>190

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>190

SerIleTyrSerTyrTyrGlyTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>191

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>191

SerIleSerProTyrTyrGlyTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>192

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>192

SerIleSerSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>193

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>193

SerIleSerSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>194

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>194

SerIleSerSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>195

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>195

SerIleSerSerSerTyrSerSerThrTyrTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>196

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>196

SerIleSerProTyrTyrGlyTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>197

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>197

SerIleTyrSerSerTyrSerSerThrTyrTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>198

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>198

SerIleTyrProTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>199

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>199

SerIleTyrProTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>200

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>200

SerIleTyrProTyrTyrSerTyrThrTyrTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>201

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>201

SerIleTyrProTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>202

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(3)..(3)

<223> Serine or tyrosine

<220>

<221>MOD_RES

<222>(4)..(4)

<223> proline(Pro) or Serine

<220>

<221>MOD_RES

<222>(5)..(5)

<223> tyrosine or Serine

<220>

<221>MOD_RES

<222>(7)..(7)

<223> Serine or glycine

<220>

<221>MOD_RES

<222>(8)..(8)

<223> tyrosine or Serine

<220>

<221>MOD_RES

<222>(10)..(10)

<223> Serine or tyrosine

<400>202

SerIleXaaXaaXaaTyrXaaXaaThrXaaTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>203

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>203

GluGlyTyrSerGlnGlyGlyPheAspTyr

1　　　　　　5　　　　　　　10

<210>204

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>204

SerTyrSerTyrTyrSerAlaIleAspTyr

1　　　　　　5　　　　　　　10

<210>205

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>205

SerTyrSerTyrSerTyrGlyMetAspTyr

1　　　　　　5　　　　　　　10

<210>206

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>206

SerTyrSerTyrSerTyrGlyIleAspTyr

1　　　　　　5　　　　　　　10

<210>207

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>207

SerTyrSerTyrTyrSerAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>208

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>208

GlyTyrLysTyrTrpSerAlaPheAspTyr

1　　　　　　5　　　　　　　10

<210>209

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>209

GluSerPheTyrTyrSerProAlaPheAspTyr

1　　　　　　5　　　　　　　10

<210>210

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>210

GluTyrTyrSerTyrLeuGlyAlaIleAspTyr

1　　　　　　5　　　　　　　10

<210>211

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>211

GlyTyrGluGlyGlyMetAlaMetAspTyr

1　　　　　　5　　　　　　　10

<210>212

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>212

SerTyrSerTyrSerSerGlyLeuAspTyr

1　　　　　　5　　　　　　　10

<210>213

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>213

GlyTyrMetTrpTyrGlyGlyIleAspTyr

1　　　　　　　5　　　　　　　10

<210>214

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(5)..(5)

The amino acid that <223> is variable

<400>214

AspCysTyrTyrXaaAlaAlaPheAspTyr

1　　　　　　　5　　　　　　　10

<210>215

<211>9

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>215

GluAsnTyrTrpTrpAlaIleAspTyr

1　　　　　　5

<210>216

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>216

SerTyrSerTyrTyrSerAlaPheAspTyr

1　　　　　　5　　　　　　　10

<210>217

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>217

AspTyrTyrPhePheSerAlaIleAspTyr

1　　　　　　5　　　　　　　10

<210>218

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>218

SerTyrSerTyrSerSerAlaLeuAspTyr

1　　　　　　5　　　　　　　10

<210>219

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>219

GluGlyTyrIleSerGlyAspAlaIleAspTyr

1　　　　　　5　　　　　　　10

<210>220

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>220

SerTyrSerSerTyrSerAlaIleAspTyr

1　　　　　　5　　　　　　　10

<210>221

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>221

GlyTyrPheGluGlyTrpTyrGlyLeuAspTyr

1　　　　　　5　　　　　　　10

<210>222

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>222

GluTyrSerTyrTyrGlyGlyPheAspTyr

1　　　　　　5　　　　　　　10

<210>223

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>223

GluSerTyrTrpSerTyrAlaMetAspTyr

1　　　　　　5　　　　　　　10

<210>224

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>224

SerTyrSerTyrSerTyrGlyLeuAspTyr

1　　　　　　5　　　　　　　10

<210>225

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>225

TyrTyrSerTyrSerSerGlyLeuAspTyr

1　　　　　　5　　　　　　　10

<210>226

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>226

SerTyrSerTyrSerTyrGlyLeuAspTyr

1　　　　　　5　　　　　　　10

<210>227

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>227

SerTyrSerTyrSerTyrGlyMetAspTyr

1　　　　　　5　　　　　　　10

<210>228

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(1)..(1)

<223> L-glutamic acid, Serine, glycine, aspartic acid or tyrosine

<220>

<221>MOD_RES

<222>(2)..(2)

<223> glycine, tyrosine, Serine, halfcystine or the acid of asparagus fern acyl

<220>

<221>MOD_RES

<222>(3)..(3)

<223> tyrosine, Serine, Methionin, phenylalanine, L-glutamic acid or methionine(Met)

<220>

<221>MOD_RES

<222>(4)..(4)

<223> Serine, tyrosine, glycine, tryptophane, phenylalanine, Isoleucine or L-glutamic acid

<220>

<221>MOD_RES

<222>(5)..(5)

The amino acid that <223> is variable

<220>

<221>MOD_RES

<222>(6)..(6)

<223> glycine, Serine, tyrosine, leucine, methionine(Met), glycine, L-Ala or tryptophane

<220>

<221>MOD_RES

<222>(7)..(7)

<223> glycine, L-Ala, proline(Pro), Isoleucine, aspartic acid or tyrosine

<220>

<221>MOD_RES

<222>(8)..(8)

<223> phenylalanine, Isoleucine, methionine(Met), L-Ala, leucine, glycine or do not exist

<220>

<221>MOD_RES

<222>(9)..(9)

<223> phenylalanine, Isoleucine, leucine or do not exist

<400>228

XaaXaaXaaXaaXaaXaaXaaXaaXaaAspTyr

1　　　　　　　5　　　　　　　10

<210>229

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>229

GlyPheAsnIleSerSerSerTyrIleHis

1　　　　　　5　　　　　　　10

<210>230

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>230

GlyPheAsnValSerSerTyrSerIleHis

1　　　　　　5　　　　　　　10

<210>231

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>231

GlyPheAsnPheSerTyrSerSerIleHis

1　　　　　　5　　　　　　　10

<210>232

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>232

GlyPheAsnPheTyrSerSerSerIleHis

1　　　　　　5　　　　　　　10

<210>233

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>233

GlyPheAsnPheTyrTyrSerSerIleHis

1　　　　　　5　　　　　　　10

<210>234

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>234

GlyPheAsnValTyrTyrSerSerIleHis

1　　　　　　5　　　　　　　10

<210>235

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>235

GlyPheAsnPheTyrTyrSerTyrIleHis

1　　　　　　5　　　　　　　10

<210>236

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>236

GlyPheAsnValSerSerSerTyrIleHis

1　　　　　　5　　　　　　　10

<210>237

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>237

GlyPheAsnPheTyrTyrSerSerIleHis

1　　　　　　5　　　　　　　10

<210>238

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>238

GlyPheAsnPheTyrSerSerTyrMetHis

1　　　　　　5　　　　　　　10

<210>239

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>239

GlyPheAsnValSerSerTyrSerIleHis

1　　　　　　5　　　　　　　10

<210>240

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(4)..(4)

<223> Isoleucine, α-amino-isovaleric acid or phenylalanine

<220>

<221>MOD_RES

<222>(5)..(8)

<223> Serine or tyrosine

<220>

<221>MOD_RES

<222>(9)..(9)

<223> Isoleucine or methionine(Met)

<400>240

GlyPheAsnXaaXaaXaaXaaXaaXaaHis

1　　　　　　　5　　　　　　　10

<210>241

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>241

TyrIleSerProTyrTyrGlySerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>242

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>242

SerIleTyrProSerSerGlySerThrTyrTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>243

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>243

SerIleTyrSerSerTyrSerSerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>244

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>244

SerIleSerSerTyrTyrGlySerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>245

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>245

SerIleTyrSerSerTyrGlyTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>246

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>246

SerIleSerProSerTyrGlyTyrThrTyrTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>247

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>247

TyrIleSerProSerTyrSerSerThrTyrTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>248

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>248

SerIleSerSerSerTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>249

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>249

SerIleTyrSerSerTyrSerSerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>250

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>250

TyrIleSerSerTyrSerGlyTyrThrTyrTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>251

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>251

SerIleTyrProSerSerGlySerThrTyrTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>252

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(1)..(1)

<223> tyrosine or Serine

<220>

<221>MOD_RES

<222>(3)..(3)

<223> Serine or tyrosine

<220>

<221>MOD_RES

<222>(4)..(4)

<223> proline(Pro) or Serine

<220>

<221>MOD_RES

<222>(5)..(6)

<223> tyrosine or Serine

<220>

<221>MOD_RES

<222>(7)..(7)

<223> glycine or Serine

<220>

<221>MOD_RES

<222>(8)..(8)

<223> Serine or tyrosine

<220>

<221>MOD_RES

<222>(10)..(10)

<223> Serine or tyrosine

<400>252

XaaIleXaaXaaXaaXaaXaaXaaThrXaaTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>253

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>253

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　5　　　　　　　10

<210>254

<211>21

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>254

SerGlyTyrTyrTyrGlnGlyTyrTrpTrpTyrTyrTyrThrGlyTyr

1　　　　　　5　　　　　　　10　　　　　　　15

TyrGlyMetAspTyr

　　　　　20

<210>255

<211>19

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>255

GlyIleMetPheSerSerTrpTrpTrpTyrTyrAspTyrSerAspAla

1　　　　　　5　　　　　　　10　　　　　　　15

LeuAspTyr

<210>256

<211>19

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>256

GlySerIleProSerTyrTrpSerAlaAspTrpTyrTyrTyrTyrGly

1　　　　　　5　　　　　　　10　　　　　　　15

LeuAspTyr

<210>257

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>257

GluTyrTyrTrpTrpTyrLysGluAlaTrpTyrSerAlaGlyMetAsp

1　　　　　　5　　　　　　　10　　　　　　　15

Tyr

<210>258

<211>21

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>258

TrpGlnGlyTyrGlyPheLysTyrTyrTrpSerTyrTyrValSerTyr

1　　　　　　5　　　　　　　10　　　　　　　15

GlyGlyLeuAspTyr

　　　　　20

<210>259

<211>14

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>259

SerTyrSerTyrTyrTyrTyrSerSerTyrGlyPheAspTyr

1　　　　　　　5　　　　　　　10

<210>260

<211>13

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>260

GluSerTyrAlaGlyValProProTyrGlyPheAspTyr

1　　　　　　　5　　　　　　　10

<210>261

<211>19

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>261

GlyIleMetPheSerSerTrpTrpTrpTyrTyrAspTyrSerAspAla

1　　　　　　5　　　　　　　10　　　　　　　15

LeuAspTyr

<210>262

<211>19

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>262

GlyIleMetPheSerSerTrpTrpTrpTyrTyrAspTyrSerAspAla

1　　　　　　5　　　　　　　10　　　　　　　15

LeuAspTyr

<210>263

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>263

GluTyrTyrTrpTrpTyrLysGluAlaTrpTyrSerAlaGlyMetAsp

1　　　　　　5　　　　　　　10　　　　　　　15

Tyr

<210>264

<211>21

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(1)..(1)

<223> L-glutamic acid, Serine, glycine or tryptophane

<220>

<221>MOD_RES

<222>(2)..(2)

<223> tyrosine, glycine, Isoleucine, Serine or glutamine

<220>

<221>MOD_RES

<222>(3)..(3)

<223> tyrosine, methionine(Met), Isoleucine, glycine or Serine

<220>

<221>MOD_RES

<222>(4)..(4)

<223> arginine, tyrosine, phenylalanine, proline(Pro) or tryptophane

<220>

<221>MOD_RES

<222>(5)..(5)

<223> tryptophane, tyrosine, Serine or glycine

<220>

<221>MOD_RES

<222>(6)..(6)

<223> tyrosine, glutamine, Serine, phenylalanine or α-amino-isovaleric acid

<220>

<221>MOD_RES

<222>(7)..(7)

<223> Threonine, glycine, tryptophane, Methionin, tyrosine or proline(Pro)

<220>

<221>MOD_RES

<222>(8)..(8)

<223> L-Ala, tyrosine, tryptophane, Serine, L-glutamic acid or proline(Pro)

<220>

<221>MOD_RES

<222>(9)..(9)

<223> Isoleucine, tryptophane, L-Ala, tyrosine or Serine

<220>

<221>MOD_RES

<222>(10)..(10)

<223> tryptophane, tyrosine, aspartic acid or glycine

<220>

<221>MOD_RES

<222>(11)..(11)

<223> tyrosine, tryptophane, Serine, glycine or phenylalanine

<220>

<221>MOD_RES

<222>(12)..(12)

<223> tyrosine, aspartic acid, Serine, phenylalanine or do not exist

<220>

<221>MOD_RES

<222>(13)..(13)

<223> tyrosine, L-Ala or do not exist

<220>

<221>MOD_RES

<222>(14)..(14)

<223> Threonine, Serine, tyrosine, glycine, α-amino-isovaleric acid or do not exist

<220>

<221>MOD_RES

<222>(15)..(15)

<223> glycine, aspartic acid, tyrosine, methionine(Met), Serine or do not exist

<220>

<221>MOD_RES

<222>(16)..(16)

<223> tyrosine, L-Ala, glycine or do not exist

<220>

<221>MOD_RES

<222>(17)..(17)

<223> tyrosine, leucine, glycine or do not exist

<220>

<221>MOD_RES

<222>(18)..(18)

This residue of <223> can be presence or absence

<220>

<221>MOD_RES

<222>(19)..(19)

<223> methionine(Met), leucine or do not exist

<400>264

XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa

1　　　　　　5　　　　　　　10　　　　　　　15

XaaGlyXaaAspTyr

　　　　　20

<210>265

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>265

GlyPheAsnValTyrTyrSerSerIleHis

1　　　　　　5　　　　　　　10

<210>266

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>266

GlyPheAsnValSerTyrSerTyrMetHis

1　　　　　　5　　　　　　　10

<210>267

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>267

GlyPheAsnPheSerTyrTyrSerMetHis

1　　　　　　5　　　　　　　10

<210>268

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>268

GlyPheAsnLeuSerTyrTyrSerIleHis

1　　　　　　5　　　　　　　10

<210>269

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>269

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　5　　　　　　　10

<210>270

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>270

GlyPheAsnValTyrTyrSerSerIleHis

1　　　　　　5　　　　　　　10

<210>271

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>271

GlyPheAsnLeuSerTyrSerTyrIleHis

1　　　　　　5　　　　　　　10

<210>272

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>272

GlyPheAsnLeuSerTyrSerSerMetHis

1　　　　　　5　　　　　　　10

<210>273

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>273

GlyPheAsnValSerTyrSerSerIleHis

1　　　　　　5　　　　　　　10

<210>274

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>274

GlyPheAsnValSerTyrSerSerIleHis

1　　　　　　5　　　　　　　10

<210>275

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>275

GlyPheAsnValSerTyrTyrTyrIleHis

1　　　　　　5　　　　　　　10

<210>276

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>276

GlyPheAsnLeuTyrTyrSerTyrMetHis

1　　　　　　5　　　　　　　10

<210>277

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>277

GlyPheAsnValSerTyrTyrSerIleHis

1　　　　　　5　　　　　　　10

<210>278

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>278

GlyPheAsnIleSerTyrSerSerIleHis

1　　　　　　5　　　　　　　10

<210>279

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>279

GlyPheAsnPheSerTyrTyrSerIleHis

1　　　　　　5　　　　　　　10

<210>280

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(4)..(4)

<223> α-amino-isovaleric acid, phenylalanine, leucine or Isoleucine

<220>

<221>MOD_RES

<222>(5)..(5)

<223> tyrosine or Serine

<220>

<221>MOD_RES

<222>(7)..(8)

<223> Serine or tyrosine

<220>

<221>MOD_RES

<222>(9)..(9)

<223> Isoleucine or methionine(Met)

<400>280

GlyPheAsnXaaXaaTyrXaaXaaXaaHis

1　　　　　　　5　　　　　　　10

<210>281

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>281

SerIleSerProTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>282

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>282

SerIleSerSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>283

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>283

SerIleTyrProTyrTyrGlyTyrThrTyrTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>284

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>284

SerIleTyrProTyrTyrGlyTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>285

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>285

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>286

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>286

SerIleTyrProTyrTyrGlyTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>287

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>287

SerIleSerSerTyrTyrGlySerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>288

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>288

SerIleTyrProTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>289

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>289

SerIleTyrProTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>290

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>290

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>291

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>291

SerIleSerSerTyrTyrGlyTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>292

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>292

SerIleSerSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>293

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>293

SerIleTyrProTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>294

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>294

SerIleTyrProTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>295

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>295

SerIleTyrProTyrTyrSerTyrThrTyrTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>296

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(3)..(3)

<223> Serine or tyrosine

<220>

<221>MOD_RES

<222>(4)..(4)

<223> proline(Pro) or Serine

<220>

<221>MOD_RES

<222>(7)..(7)

<223> Serine or glycine

<220>

<221>MOD_RES

<222>(8)..(8)

<223> tyrosine or Serine

<220>

<221>MOD_RES

<222>(10)..(10)

<223> Serine or tyrosine

<400>296

SerIleXaaXaaTyrTyrXaaXaaThrXaaTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>297

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>297

GluGlyTyrSerGlnGlyGlyPheAspTyr

1　　　　　　5　　　　　　　10

<210>298

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>298

SerTyrSerTyrTyrSerAlaIleAspTyr

1　　　　　　5　　　　　　　10

<210>299

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>299

SerTyrSerTyrSerTyrGlyMetAspTyr

1　　　　　　5　　　　　　　10

<210>300

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>300

SerTyrSerTyrSerTyrGlyIleAspTyr

1　　　　　　5　　　　　　　10

<210>301

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>301

SerTyrSerTyrTyrSerAlaMetAspTyr

1　　　　　　5　　　　　　　10

<210>302

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>302

GlyTyrLysTyrTrpSerAlaPheAspTyr

1　　　　　　5　　　　　　　10

<210>303

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>303

GluSerPheTyrTyrSerProAlaPheAspTyr

1　　　　　　5　　　　　　　10

<210>304

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>304

GlyTyrGluGlyGlyMetAlaMetAspTyr

1　　　　　　5　　　　　　　10

<210>305

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>305

SerTyrSerTyrSerSerGlyLeuAspTyr

1　　　　　　5　　　　　　　10

<210>306

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>306

GlyTyrMetTrpTyrGlyGlyIleAspTyr

1　　　　　　5　　　　　　　10

<210>307

<211>9

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>307

GluAsnTyrTrpTrpAlaIleAspTyr

1　　　　　　5

<210>308

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>308

SerTyrSerTyrSerSerAlaLeuAspTyr

1　　　　　　5　　　　　　　10

<210>309

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>309

SerTyrSerTyrSerTyrGlyLeuAspTyr

1　　　　　　5　　　　　　　10

<210>310

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>310

TyrTyrSerTyrSerSerGlyLeuAspTyr

1　　　　　　5　　　　　　　10

<210>311

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>311

SerTyrSerTyrSerTyrGlyLeuAspTyr

1　　　　　　5　　　　　　　10

<210>312

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(1)..(1)

<223> L-glutamic acid, Serine, glycine or tyrosine

<220>

<221>MOD_RES

<222>(2)..(2)

<223> glycine, tyrosine, Serine or the acid of asparagus fern acyl

<220>

<221>MOD_RES

<222>(3)..(3)

<223> tyrosine, Serine, Methionin, phenylalanine or L-glutamic acid

<220>

<221>MOD_RES

<222>(4)..(4)

<223> Serine, tyrosine, glycine or tryptophane

<220>

<221>MOD_RES

<222>(5)..(5)

<223> glutamine, tyrosine, Serine or glycine

<220>

<221>MOD_RES

<222>(6)..(6)

<223> glycine, Serine, tyrosine, methionine(Met) or L-Ala

<220>

<221>MOD_RES

<222>(7)..(7)

<223> glycine, L-Ala, proline(Pro) or Isoleucine

<220>

<221>MOD_RES

<222>(8)..(8)

<223> phenylalanine, Isoleucine, methionine(Met), L-Ala, leucine or do not exist

<220>

<221>MOD_RES

<222>(9)..(9)

This residue of <223> can be presence or absence

<400>312

XaaXaaXaaXaaXaaXaaXaaXaaPheAspTyr

1　　　　　　　5　　　　　　　10

<210>313

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>313

GlnGlnSerSerTyrSerSerLeuPheThr

1　　　　　　　5　　　　　　　10

<210>314

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>314

GlnGlnSerSerTyrSerSerLeuPheThr

1　　　　　　　5　　　　　　　10

<210>315

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>315

GlnGlnTyrSerSerTyrSerSerProIleThr

1　　　　　　5　　　　　　　10

<210>316

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>316

GlnGlnTyrSerSerTyrTyrSerProValThr

1　　　　　　5　　　　　　　10

<210>317

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>317

GlnGlnSerSerTyrSerSerLeuIleThr

1　　　　　　5　　　　　　　10

<210>318

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>318

GlnGlnSerSerTyrSerSerLeuValThr

1　　　　　　5　　　　　　　10

<210>319

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>319

GlnGlnSerTyrTyrTyrSerLeuPheThr

1　　　　　　5　　　　　　　10

<210>320

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>320

GlnGlnSerSerTyrSerSerLeuValThr

1　　　　　　5　　　　　　　10

<210>321

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>321

GlnGlnSerSerTyrSerSerLeuPheThr

1　　　　　　5　　　　　　　10

<210>322

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>322

GlnGlnTyrSerTyrSerSerLeuPheThr

1　　　　　　5　　　　　　　10

<210>323

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>323

GlnGlnSerTyrTyrTyrTyrProIleThr

1　　　　　　5　　　　　　　10

<210>324

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>324

GlnGlnSerSerTyrSerSerLeuValThr

1　　　　　　5　　　　　　　10

<210>325

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>325

GlnGlnTyrSerSerSerTyrTyrProPheThr

1　　　　　　5　　　　　　　10

<210>326

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>326

GlnGlnSerSerTyrSerSerLeuLeuThr

1　　　　　　5　　　　　　　10

<210>327

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>327

GlnGlnTyrTyrTyrTyrTyrTyrProIleThr

1　　　　　　5　　　　　　　10

<210>328

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(3)..(7)

<223> Serine or tyrosine

<220>

<221>MOD_RES

<222>(8)..(8)

<223> leucine, Serine, proline(Pro) or tyrosine

<220>

<221>MOD_RES

<222>(9)..(9)

This residue of <223> can be presence or absence

<220>

<221>MOD_RES

<222>(10)..(10)

<223> phenylalanine, Isoleucine, α-amino-isovaleric acid or leucine

<400>328

GlnGlnXaaXaaXaaXaaXaaXaaProXaaThr

1　　　　　　　5　　　　　　　10

<210>329

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>329

GlyPheAsnValSerSerTyrSerIleHis

1　　　　　　5　　　　　　　10

<210>330

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>330

GlyPheAsnIleSerSerSerTyrIleHis

1　　　　　　5　　　　　　　10

<210>331

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>331

GlyPheAsnPheSerTyrSerSerIleHis

1　　　　　　5　　　　　　　10

<210>332

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>332

GlyPheAsnValTyrTyrSerSerIleHis

1　　　　　　5　　　　　　　10

<210>333

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>333

GlyPheAsnPheTyrTyrSerSerIleHis

1　　　　　　5　　　　　　　10

<210>334

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>334

GlyPheAsnValSerSerSerTyrIleHis

1　　　　　　5　　　　　　　10

<210>335

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>335

GlyPheAsnPheTyrSerSerTyrMetHis

1　　　　　　5　　　　　　　10

<210>336

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>336

GlyPheAsnPheTyrSerSerSerIleHis

1　　　　　　　5　　　　　　　10

<210>337

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(4)..(4)

<223> α-amino-isovaleric acid, Isoleucine or phenylalanine

<220>

<221>MOD_RES

<222>(5)..(8)

<223> Serine or tyrosine

<220>

<221>MOD_RES

<222>(9)..(9)

<223> Isoleucine or methionine(Met)

<400>337

GlyPheAsnXaaXaaXaaXaaXaaXaaHis

1　　　　　　　5　　　　　　　10

<210>338

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>338

SerIleTyrProSerSerGlySerThrTyrTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>339

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>339

TyrIleSerProTyrTyrGlySerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>340

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>340

SerIleTyrSerSerTyrSerSerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>341

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>341

SerIleSerProSerTyrGlyTyrThrTyrTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>342

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>342

SerIleTyrSerSerTyrGlyTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>343

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>343

SerIleSerSerSerTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>344

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>344

TyrIleSerSerTyrSerGlyTyrThrTyrTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>345

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>345

SerIleSerSerTyrTyrGlySerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>346

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(1)..(1)

<223> Serine or tyrosine

<220>

<221>MOD_RES

<222>(3)..(3)

<223> tyrosine or Serine

<220>

<221>MOD_RES

<222>(4)..(4)

<223> proline(Pro) or Serine

<220>

<221>MOD_RES

<222>(5)..(6)

<223> Serine or tyrosine

<220>

<221>MOD_RES

<222>(7)..(7)

<223> glycine or Serine

<220>

<221>MOD_RES

<222>(8)..(8)

<223> Serine or tyrosine

<220>

<221>MOD_RES

<222>(10)..(10)

<223> tyrosine or Serine

<400>346

XaaIleXaaXaaXaaXaaXaaXaaThrXaaTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>347

<211>21

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>347

SerGlyTyrTyrTyrGlnGlyTyrTrpTrpTyrTyrTyrThrGlyTyr

1　　　　　　5　　　　　　　10　　　　　　　15

TyrGlyMetAspTyr

　　　　　20

<210>348

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>348

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>349

<211>19

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>349

GlyIleMetPheSerSerTrpTrpTrpTyrTyrAspTyrSerAspAla

1　　　　　　5　　　　　　　10　　　　　　　15

LeuAspTyr

<210>350

<211>21

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>350

TrpGlnGlyTyrGlyPheLysTyrTyrTrpSerTyrTyrValSerTyr

1　　　　　　5　　　　　　　10　　　　　　　15

GlyGlyLeuAspTyr

　　　　　20

<210>351

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>351

GluTyrTyrTrpTrpTyrLysGluAlaTrpTyrSerAlaGlyMetAsp

1　　　　　　5　　　　　　　10　　　　　　　15

Tyr

<210>352

<211>13

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>352

GluSerTyrAlaGlyValProProTyrGlyPheAspTyr

1　　　　　　　5　　　　　　　10

<210>353

<211>19

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>353

GlyIleMetPheSerSerTrpTrpTrpTyrTyrAspTyrSerAspAla

1　　　　　　5　　　　　　　10　　　　　　　15

LeuAspTyr

<210>354

<211>19

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>354

GlySerIleProSerTyrTrpSerAlaAspTrpTyrTyrTyrTyrGly

1　　　　　　5　　　　　　　10　　　　　　　15

LeuAspTyr

<210>355

<211>21

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(1)..(1)

<223> Serine, L-glutamic acid, glycine or tryptophane

<220>

<221>MOD_RES

<222>(2)..(2)

<223> glycine, tyrosine, Isoleucine, glutamine or Serine

<220>

<221>MOD_RES

<222>(3)..(3)

<223> tyrosine, methionine(Met), glycine or Isoleucine

<220>

<221>MOD_RES

<222>(4)..(4)

<223> tyrosine, arginine, phenylalanine, tryptophane, L-Ala or proline(Pro)

<220>

<221>MOD_RES

<222>(5)..(5)

<223> tyrosine, tryptophane, Serine or glycine

<220>

<221>MOD_RES

<222>(6)..(6)

<223> glutamine, tyrosine, Serine, phenylalanine or α-amino-isovaleric acid

<220>

<221>MOD_RES

<222>(7)..(7)

<223> glycine, Threonine, tryptophane, Methionin or proline(Pro)

<220>

<221>MOD_RES

<222>(8)..(8)

<223> tyrosine, L-Ala, tryptophane, L-glutamic acid, proline(Pro) or Serine

<220>

<221>MOD_RES

<222>(9)..(9)

<223> tryptophane, Isoleucine, tyrosine or L-Ala

<220>

<221>MOD_RES

<222>(10)..(10)

<223> tryptophane, tyrosine, glycine, aspartic acid or do not exist

<220>

<221>MOD_RES

<222>(11)..(11)

<223> tyrosine, Serine, phenylalanine, tryptophane or do not exist

<220>

<221>MOD_RES

<222>(12)..(12)

<223> tyrosine, aspartic acid, Serine or do not exist

<220>

<221>MOD_RES

<222>(13)..(13)

<223> tyrosine, L-Ala or do not exist

<220>

<221>MOD_RES

<222>(14)..(14)

<223> Threonine, Serine, α-amino-isovaleric acid, glycine, tyrosine or do not exist

<220>

<221>MOD_RES

<222>(15)..(15)

<223> glycine, aspartic acid, Serine, methionine(Met), tyrosine or do not exist

<220>

<221>MOD_RES

<222>(16)..(16)

<223> tyrosine, L-Ala, glycine or do not exist

<220>

<221>MOD_RES

<222>(17)..(17)

<223> tyrosine, leucine, glycine or do not exist

<220>

<221>MOD_RES

<222>(18)..(18)

This residue of <223> can be presence or absence

<220>

<221>MOD_RES

<222>(19)..(19)

<223> methionine(Met), leucine or do not exist

<400>355

XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa

1　　　　　　5　　　　　　　10　　　　　　　15

XaaGlyXaaAspTyr

　　　　　20

<210>356

<211>9

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>356

GlnGlnTyrSerTyrTyrProPheArg

1　　　　　　5

<210>357

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>357

GlnGlnTyrSerSerTyrSerSerLeuPheThr

1　　　　　　　5　　　　　　　10

<210>358

<211>9

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>358

GlnGlnTyrSerSerSerLeuValThr

1　　　　　　5

<210>359

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>359

GlnGlnTyrSerSerSerSerProPheThrPhe

1　　　　　　　5　　　　　　　10

<210>360

<211>9

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>360

GlnGlnSerSerTyrSerProIleThr

1　　　　　　5

<210>361

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>361

GlnGlnTyrSerTyrSerSerTyrLeuIleThr

1　　　　　　　5　　　　　　　10

<210>362

<211>9

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>362

GlnGlnSerTyrTyrSerProPheThr

1　　　　　　5

<210>363

<211>9

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>363

GlnGlnTyrTyrSerSerLeuValThr

1　　　　　　5

<210>364

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(3)..(6)

<223> tyrosine or Serine

<220>

<221>MOD_RES

<222>(7)..(7)

<223> proline(Pro), Serine or leucine

<220>

<221>MOD_RES

<222>(8)..(8)

<223> Serine, proline(Pro), tyrosine or do not exist

<220>

<221>MOD_RES

<222>(9)..(9)

<223> leucine, phenylalanine or do not exist

<220>

<221>MOD_RES

<222>(10)..(10)

<223> phenylalanine, α-amino-isovaleric acid, Threonine or Isoleucine

<220>

<221>MOD_RES

<222>(11)..(11)

<223> arginine, Threonine or phenylalanine

<400>364

GlnGlnXaaXaaXaaXaaXaaXaaXaaXaaXaa

1　　　　　　　5　　　　　　　10

<210>365

<211>42

<212>DNA

<213> artificial sequence

<220>

The description of <223> artificial sequence: the oligonucleotide of synthesis

<400>365

cgtctattattgtgctcgctaataagactactggggtcaagg42

<210>366

<211>42

<212>DNA

<213> artificial sequence

<220>

The description of <223> artificial sequence: the oligonucleotide of synthesis

<400>366

cgtctattattgtgctcgctaataagactactggggtcaagg42

<210>367

<211>60

<212>DNA

<213> artificial sequence

<220>

The description of <223> artificial sequence: the oligonucleotide of synthesis

<220>

The base of <221> through modifying

<222>(20)..(22)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(26)..(30)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(32)..(33)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(35)..(37)

<223>a, c, g or t

<220>

<223> details refer in specification sheets the description being substituted and preferably replacing

<400>367

cgtctattattgtgctcgcnnntacnnnnnsnnsnnngstwtsgactactggggtcaagg60

<210>368

<211>60

<212>DNA

<213> artificial sequence

<220>

The description of <223> artificial sequence: the oligonucleotide of synthesis

<220>

The base of <221> through modifying

<222>(20)..(24)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(26)..(28)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(32)..(33)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(35)..(37)

<223>a, c, g or t

<220>

<223> details refer in specification sheets the description being substituted and preferably replacing

<400>368

cgtctattattgtgctcgcnnnnnsnnntacnnsnnngstwtsgactactggggtcaagg60

<210>369

<211>60

<212>DNA

<213> artificial sequence

<220>

The description of <223> artificial sequence: the oligonucleotide of synthesis

<220>

The base of <221> through modifying

<222>(20)..(24)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(26)..(30)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(35)..(37)

<223>a, c, g or t

<220>

<223> details refer in specification sheets the description being substituted and preferably replacing

<400>369

cgtctattattgtgctcgcnnnnnsnnnnnstacnnngstwtsgactactggggtcaagg60

<210>370

<211>60

<212>DNA

<213> artificial sequence

<220>

The description of <223> artificial sequence: the oligonucleotide of synthesis

<220>

The base of <221> through modifying

<222>(20)..(24)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(26)..(30)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(32)..(33)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(35)..(37)

<223>a, c, g or t

<220>

<223> details refer in specification sheets the description being substituted and preferably replacing

<400>370

cgtctattattgtgctcgcnnnnnsnnnnnsnnsnnngstwtsgactactggggtcaagg60

<210>371

<211>40

<212>DNA

<213> artificial sequence

<220>

The description of <223> artificial sequence: the oligonucleotide of synthesis

<400>371

gcagcttctggcttcaactaataacactgggtgcgtcagg40

<210>372

<211>52

<212>DNA

<213> artificial sequence

<220>

The description of <223> artificial sequence: the oligonucleotide of synthesis

<220>

The base of <221> through modifying

<222>(19)..(19)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(22)..(23)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(31)..(32)

<223>a, c, g or t

<400>372

gcagcttctggcttcaacntcnnstactctnnsatscactgggtgcgtcagg52

<210>373

<211>43

<212>DNA

<213> artificial sequence

<220>

The description of <223> artificial sequence: the oligonucleotide of synthesis

<400>373

ggcctggaatgggttgcataataatatgccgatagcgtcaagg43

<210>374

<211>67

<212>DNA

<213> artificial sequence

<220>

The description of <223> artificial sequence: the oligonucleotide of synthesis

<220>

The base of <221> through modifying

<222>(25)..(26)

<223>a, c, g or t

<400>374

ggcctggaatgggttgcatctatcnnsycttactactcttacacctcttatgccgatagc60

gtcaagg67

<210>375

<211>60

<212>DNA

<213> artificial sequence

<220>

The description of <223> artificial sequence: the oligonucleotide of synthesis

<220>

The base of <221> through modifying

<222>(32)..(33)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(35)..(36)

<223>a, c, g or t

<400>375

cgtctattattgtgctcgctcttactcttacnnsnnsgstwtsgactactggggtcaagg60

<210>376

<211>46

<212>DNA

<213> artificial sequence

<220>

The description of <223> artificial sequence: the oligonucleotide of synthesis

<400>376

cgcaacttattactgtcagcaataataaacgttcggacagggtacc46

<210>377

<211>76

<212>PRT

The <213> mankind (Homosapiens)

<400>377

MetGlnIlePheValLysThrLeuThrGlyLysThrIleThrLeuGlu

1　　　　　　5　　　　　　　10　　　　　　　15

ValGluProSerAspThrIleGluAsnValLysAlaLysIleGlnAsp

　　　　20　　　　　　　25　　　　　　　30

LysGluGlyIleProProAspGlnGlnArgLeuIlePheAlaGlyLys

　　　35　　　　　　　40　　　　　　　45

GlnLeuGluAspGlyArgThrLeuSerAspTyrAsnIleGlnLysGlu

　　50　　　　　　　55　　　　　　　60

SerThrLeuHisLeuValLeuArgLeuArgGlyGly

65　　　　　70　　　　　　　75

<210>378

<211>61

<212>DNA

<213> artificial sequence

<220>

The description of <223> artificial sequence: the oligonucleotide of synthesis

<220>

The base of <221> through modifying

<222>(23)..(24)

<223>a, c, g or t

<400>378

cgcaacttattactgtcagcaannstcttactcttctctgdttacgttcggacagggtac60

c61

<210>379

<211>63

<212>DNA

<213> artificial sequence

<220>

The description of <223> artificial sequence: the oligonucleotide of synthesis

<220>

The base of <221> through modifying

<222>(20)..(22)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(29)..(33)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(35)..(36)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(38)..(40)

<223>a, c, g or t

<220>

<223> details refer in specification sheets the description being substituted and preferably replacing

<400>379

cgtctattattgtgctcgcnnntactacnnnnnsnnsnnngstwtsgactactggggtca60

agg63

<210>380

<211>63

<212>DNA

<213> artificial sequence

<220>

The description of <223> artificial sequence: the oligonucleotide of synthesis

<220>

The base of <221> through modifying

<222>(20)..(24)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(26)..(27)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(29)..(31)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(38)..(40)

<223>a, c, g or t

<220>

<223> details refer in specification sheets the description being substituted and preferably replacing

<400>380

cgtctattattgtgctcgcnnnnnsnnsnnntggtacnnngstwtsgactactggggtca60

agg63

<210>381

<211>63

<212>DNA

<213> artificial sequence

<220>

The description of <223> artificial sequence: the oligonucleotide of synthesis

<220>

The base of <221> through modifying

<222>(20)..(22)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(29)..(33)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(38)..(40)

<223>a, c, g or t

<220>

<223> details refer in specification sheets the description being substituted and preferably replacing

<400>381

cgtctattattgtgctcgcnnntacbbsnnnnnstacnnngstwtsgactactggggtca60

agg63

<210>382

<211>63

<212>DNA

<213> artificial sequence

<220>

The description of <223> artificial sequence: the oligonucleotide of synthesis

<220>

The base of <221> through modifying

<222>(20)..(24)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(29)..(31)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(35)..(36)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(38)..(40)

<223>a, c, g or t

<220>

<223> details refer in specification sheets the description being substituted and preferably replacing

<400>382

cgtctattattgtgctcgcnnnnnstacnnntggnnsnnngstwtsgactactggggtca60

agg63

<210>383

<211>63

<212>DNA

<213> artificial sequence

<220>

The description of <223> artificial sequence: the oligonucleotide of synthesis

<220>

The base of <221> through modifying

<222>(20)..(22)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(26)..(27)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(29)..(31)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(35)..(36)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(38)..(40)

<223>a, c, g or t

<220>

<223> details refer in specification sheets the description being substituted and preferably replacing

<400>383

cgtctattattgtgctcgcnnntacnnsnnntggnnsnnngstwtsgactactggggtca60

agg63

<210>384

<211>63

<212>DNA

<213> artificial sequence

<220>

The description of <223> artificial sequence: the oligonucleotide of synthesis

<220>

The base of <221> through modifying

<222>(20)..(24)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(29)..(33)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(38)..(40)

<223>a, c, g or t

<220>

<223> details refer in specification sheets the description being substituted and preferably replacing

<400>384

cgtctattattgtgctcgcnnnnnstacnnnnnstacnnngstwtsgactactggggtca60

agg63

<210>385

<211>63

<212>DNA

<213> artificial sequence

<220>

The description of <223> artificial sequence: the oligonucleotide of synthesis

<220>

The base of <221> through modifying

<222>(20)..(24)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(26)..(27)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(29)..(33)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(35)..(36)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(38)..(40)

<223>a, c, g or t

<220>

<223> details refer in specification sheets the description being substituted and preferably replacing

<400>385

cgtctattattgtgctcgcnnnnnsnnsnnnnnsnnsnnngstwtsgactactggggtca60

agg63

<210>386

<211>67

<212>DNA

<213> artificial sequence

<220>

The description of <223> artificial sequence: the oligonucleotide of synthesis

<220>

The base of <221> through modifying

<222>(25)..(26)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(31)..(32)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(34)..(35)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(40)..(42)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(46)..(48)

<223>a, c, g or t

<220>

<223> details refer in specification sheets the description being substituted and preferably replacing

<400>386

ggcctggaatgggttgcatacatcnnsyctnnsnnsrgcnnnaccnnntatgccgatagc60

gtcaagg67

<210>387

<211>67

<212>DNA

<213> artificial sequence

<220>

The description of <223> artificial sequence: the oligonucleotide of synthesis

<220>

The base of <221> through modifying

<222>(19)..(20)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(25)..(26)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(34)..(35)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(40)..(42)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(46)..(48)

<223>a, c, g or t

<220>

<223> details refer in specification sheets the description being substituted and preferably replacing

<400>387

ggcctggaatgggttgcannsatcnnsycttacnnsrgcnnnaccnnntatgccgatagc60

gtcaagg67

<210>388

<211>67

<212>DNA

<213> artificial sequence

<220>

The description of <223> artificial sequence: the oligonucleotide of synthesis

<220>

The base of <221> through modifying

<222>(19)..(20)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(25)..(26)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(31)..(32)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(40)..(42)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(46)..(48)

<223>a, c, g or t

<220>

<223> details refer in specification sheets the description being substituted and preferably replacing

<400>388

ggcctggaatgggttgcannsatcnnsyctnnstacrgcnnnaccnnntatgccgatagc60

gtcaagg67

<210>389

<211>67

<212>DNA

<213> artificial sequence

<220>

The description of <223> artificial sequence: the oligonucleotide of synthesis

<220>

The base of <221> through modifying

<222>(19)..(20)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(25)..(26)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(31)..(32)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(34)..(35)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(40)..(42)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(46)..(48)

<223>a, c, g or t

<220>

<223> details refer in specification sheets the description being substituted and preferably replacing

<400>389

ggcctggaatgggttgcannsatcnnsyctnnsnnsrgcnnnaccnnntatgccgatagc60

gtcaagg67

<210>390

<211>64

<212>DNA

<213> artificial sequence

<220>

The description of <223> artificial sequence: the oligonucleotide of synthesis

<220>

The base of <221> through modifying

<222>(23)..(24)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(26)..(33)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(35)..(39)

<223>a, c, g or t

<220>

<223> details refer in specification sheets the description being substituted and preferably replacing

<400>390

cgcaacttattactgtcagcaannsnnnnnnnnsnnnnnsctgdttacgttcggacaggg60

tacc64

<210>391

<211>52

<212>DNA

<213> artificial sequence

<220>

The description of <223> artificial sequence: the oligonucleotide of synthesis

<220>

The base of <221> through modifying

<222>(19)..(19)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(22)..(23)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(25)..(32)

<223>a, c, g or t

<220>

<223> details refer in specification sheets the description being substituted and preferably replacing

<400>391

gcagcttctggcttcaacntcnnsnnnnnnnnsatscactgggtgcgtcagg52

<210>392

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>392

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　5　　　　　　　10

<210>393

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>393

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　5　　　　　　　10

<210>394

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>394

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　5　　　　　　　10

<210>395

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>395

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　5　　　　　　　10

<210>396

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>396

GlyPheAsnIleGlyTyrSerPheMetHis

1　　　　　　5　　　　　　　10

<210>397

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>397

GlyPheAsnValAspTyrSerTyrMetHis

1　　　　　　5　　　　　　　10

<210>398

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>398

GlyPheAsnValAspTyrSerTyrMetHis

1　　　　　　5　　　　　　　10

<210>399

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>399

GlyPheAsnPheSerTyrSerPheMetHis

1　　　　　　5　　　　　　　10

<210>400

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>400

GlyPheAsnIleValTyrSerPheMetHis

1　　　　　　5　　　　　　　10

<210>401

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>401

GlyPheAsnIleIleTyrSerPheMetHis

1　　　　　　5　　　　　　　10

<210>402

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>402

GlyPheAsnIleValTyrSerPheIleHis

1　　　　　　5　　　　　　　10

<210>403

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>403

GlyPheAsnLeuSerTyrSerPheMetHis

1　　　　　　　5　　　　　　　10

<210>404

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>404

GlyPheAsnValAspTyrSerPheMetHis

1　　　　　　5　　　　　　　10

<210>405

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>405

GlyPheAsnValIleTyrSerPheMetHis

1　　　　　　5　　　　　　　10

<210>406

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>406

GlyPheAsnValAlaTyrSerLeuMetHis

1　　　　　　5　　　　　　　10

<210>407

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>407

GlyPheAsnIleSerTyrSerTrpMetHis

1　　　　　　5　　　　　　　10

<210>408

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>408

GlyPheAsnLeuAspTyrSerPheMetHis

1　　　　　　5　　　　　　　10

<210>409

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>409

GlyPheAsnPheLeuTyrSerGlyIleHis

1　　　　　　5　　　　　　　10

<210>410

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>410

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　5　　　　　　　10

<210>411

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>411

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　5　　　　　　　10

<210>412

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>412

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　5　　　　　　　10

<210>413

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>413

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>414

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>414

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>415

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>415

GlyPheAsnIleLeuTyrSerGlyIleHis

1　　　　　　5　　　　　　　10

<210>416

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>416

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　5　　　　　　　10

<210>417

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>417

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>418

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>418

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>419

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>419

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>420

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>420

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>421

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>421

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>422

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>422

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>423

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>423

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>424

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>424

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>425

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>425

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>426

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>426

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　5　　　　　　　10

<210>427

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>427

GlyPheAsnIlePheTyrSerGlyIleHis

1　　　　　　　5　　　　　　　10

<210>428

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>428

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>429

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>429

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>430

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>430

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>431

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>431

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>432

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>432

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>433

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>433

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>434

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>434

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>435

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>435

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>436

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>436

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>437

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>437

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>438

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>438

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>439

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>439

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>440

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>440

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>441

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>441

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>442

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>442

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>443

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>443

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>444

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>444

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>445

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>445

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>446

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>446

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>447

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>447

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>448

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>448

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>449

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>449

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>450

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>450

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>451

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>451

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>452

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>452

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>453

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>453

GlyPheAsnLeuSerTyrSerGlyMetHis

1　　　　　　　5　　　　　　　10

<210>454

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>454

GlyPheAsnLeuLeuTyrSerGlyMetHis

1　　　　　　　5　　　　　　　10

<210>455

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>455

GlyPheAsnValAlaTyrSerGlyIleHis

1　　　　　　　5　　　　　　　10

<210>456

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>456

GlyPheAsnValAspTyrSerGlyMetHis

1　　　　　　　5　　　　　　　10

<210>457

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>457

GlyPheAsnValAspTyrSerGlyMetHis

1　　　　　　　5　　　　　　　10

<210>458

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>458

GlyPheAsnValSerTyrSerSerIleHis

1　　　　　　　5　　　　　　　10

<210>459

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>459

GlyPheAsnValValTyrSerGlyIleHis

1　　　　　　　5　　　　　　　10

<210>460

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(4)..(4)

<223> Isoleucine, α-amino-isovaleric acid, phenylalanine or leucine

<220>

<221>MOD_RES

<222>(5)..(5)

<223> Serine, glycine, aspartic acid, α-amino-isovaleric acid, Isoleucine, leucine, phenylalanine or L-Ala

<220>

<221>MOD_RES

<222>(8)..(8)

<223> Serine, phenylalanine, tyrosine, leucine, tryptophane or glycine

<220>

<221>MOD_RES

<222>(9)..(9)

<223> methionine(Met) or Isoleucine

<400>460

GlyPheAsnXaaXaaTyrSerXaaXaaHis

1　　　　　　　5　　　　　　　10

<210>461

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>461

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>462

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>462

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>463

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>463

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>464

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>464

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>465

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>465

SerIleAlaSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>466

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>466

SerIleAlaProTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>467

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>467

SerIleAlaProTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>468

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>468

SerIleSerSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>469

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>469

SerIleSerProTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>470

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>470

SerIleSerSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>471

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>471

SerIleSerSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>472

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>472

SerIleSerSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>473

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>473

SerIleSerSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>474

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>474

SerIleSerSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>475

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>475

SerIleSerSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>476

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>476

SerIleThrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>477

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>477

SerIleThrProTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>478

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>478

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>479

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>479

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>480

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>480

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>481

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>481

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>482

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>482

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>483

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>483

SerIleTyrProTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>484

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>484

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>485

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>485

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>486

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>486

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>487

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>487

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>488

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>488

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>489

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>489

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>490

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>490

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>491

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>491

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>492

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>492

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>493

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>493

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>494

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>494

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>495

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>495

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>496

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>496

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>497

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>497

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>498

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>498

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>499

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>499

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>500

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>500

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>501

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>501

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>502

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>502

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>503

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>503

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>504

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>504

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>505

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>505

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>506

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>506

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>507

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>507

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>508

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>508

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>509

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>509

SerIleTyrProTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>510

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>510

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>511

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>511

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>512

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>512

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>513

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>513

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>514

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>514

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>515

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>515

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>516

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>516

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>517

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>517

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>518

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>518

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>519

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>519

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>520

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>520

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>521

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>521

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>522

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>522

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>523

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>523

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>524

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>524

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>525

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>525

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>526

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>526

SerIleTyrSerTyrTyrThrTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>527

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>527

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>528

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>528

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>529

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(3)..(3)

<223> tyrosine, L-Ala, Serine or Threonine

<220>

<221>MOD_RES

<222>(4)..(4)

<223> Serine or proline(Pro)

<220>

<221>MOD_RES

<222>(7)..(7)

<223> Serine or Threonine

<400>529

SerIleXaaXaaTyrTyrXaaTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>530

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>530

SerTyrAsnAsnThrThrSerIleAspTyr

1　　　　　　　5　　　　　　　10

<210>531

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>531

GlyTyrSerTrpTyrAsnAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>532

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>532

GlyTyrSerTrpPheAsnAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>533

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>533

GlyTyrTyrTrpPheAspAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>534

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>534

SerTyrSerTyrTyrSerAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>535

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>535

SerTyrSerTyrArgGluThrMetAspTyr

1　　　　　　　5　　　　　　　10

<210>536

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>536

SerTyrSerTyrArgGluThrMetAspTyr

1　　　　　　　5　　　　　　　10

<210>537

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>537

SerTyrSerTyrTyrSerAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>538

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>538

SerTyrSerTyrTyrSerAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>539

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>539

SerTyrSerTyrTyrSerAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>540

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>540

SerTyrSerTyrTyrSerAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>541

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>541

SerTyrSerTyrTyrSerAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>542

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>542

SerTyrSerTyrTyrSerAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>543

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>543

SerTyrSerTyrTyrSerAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>544

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>544

SerTyrSerTyrTyrSerAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>545

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>545

SerTyrSerTyrTyrSerAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>546

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>546

SerTyrSerTyrTyrSerAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>547

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>547

SerTyrSerTyrTyrSerAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>548

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>548

SerTyrSerTyrTyrSerAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>549

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>549

SerTyrSerTyrSerPheGlyMetAspTyr

1　　　　　　　5　　　　　　　10

<210>550

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>550

SerTyrSerTyrPheMetGlyMetAspTyr

1　　　　　　　5　　　　　　　10

<210>551

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>551

SerTyrSerTyrHisValAlaPheAspTyr

1　　　　　　　5　　　　　　　10

<210>552

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>552

SerTyrSerTyrTyrSerAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>553

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>553

SerTyrSerTyrTyrSerAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>554

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>554

SerTyrSerTyrHisLeuAlaPheAspTyr

1　　　　　　　5　　　　　　　10

<210>555

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>555

SerTyrSerTyrSerLeuAlaPheAspTyr

1　　　　　　　5　　　　　　　10

<210>556

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>556

SerTyrSerTyrTyrIleAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>557

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>557

SerTyrSerTyrTyrMetGlyMetAspTyr

1　　　　　　　5　　　　　　　10

<210>558

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>558

SerTyrSerTyrSerMetGlyMetAspTyr

1　　　　　　　5　　　　　　　10

<210>559

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>559

SerTyrSerTyrHisValAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>560

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>560

SerTyrSerTyrHisMetGlyMetAspTyr

1　　　　　　　5　　　　　　　10

<210>561

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>561

SerTyrSerTyrHisLeuGlyMetAspTyr

1　　　　　　　5　　　　　　　10

<210>562

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>562

SerTyrSerTyrTyrGlnGlyPheAspTyr

1　　　　　　　5　　　　　　　10

<210>563

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>563

SerTyrSerTyrSerMetGlyMetAspTyr

1　　　　　　　5　　　　　　　10

<210>564

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>564

SerTyrSerTyrPheLeuAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>565

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>565

SerTyrSerTyrTyrSerAlaMetAspTyr

1　　　　　　5　　　　　　　10

<210>566

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>566

SerTyrSerTyrSerGluAlaLeuAspTyr

1　　　　　　　5　　　　　　　10

<210>567

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>567

SerTyrSerTyrSerLeuGlyMetAspTyr

1　　　　　　　5　　　　　　　10

<210>568

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>568

SerTyrSerTyrTyrSerAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>569

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>569

SerTyrSerTyrPheMetGlyMetAspTyr

1　　　　　　　5　　　　　　　10

<210>570

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>570

SerTyrSerTyrPheLeuAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>571

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>571

SerTyrSerTyrPheLeuAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>572

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>572

SerTyrSerTyrTyrLeuAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>573

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>573

SerTyrSerTyrPheIleGlyMetAspTyr

1　　　　　　　5　　　　　　　10

<210>574

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>574

SerTyrSerTyrHisLeuGlyMetAspTyr

1　　　　　　　5　　　　　　　10

<210>575

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>575

SerTyrSerTyrThrGluAlaPheAspTyr

1　　　　　　　5　　　　　　　10

<210>576

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>576

SerTyrSerTyrTyrSerAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>577

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>577

SerTyrSerTyrThrTyrGlyLeuAspTyr

1　　　　　　　5　　　　　　　10

<210>578

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>578

SerTyrSerTyrTyrSerAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>579

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>579

SerTyrSerTyrSerLeuGlyMetAspTyr

1　　　　　　　5　　　　　　　10

<210>580

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>580

SerTyrSerTyrTrpValGlyMetAspTyr

1　　　　　　　5　　　　　　　10

<210>581

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>581

SerTyrSerTyrThrLeuAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>582

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>582

SerTyrSerTyrThrMetGlyLeuAspTyr

1　　　　　　　5　　　　　　　10

<210>583

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>583

SerTyrSerTyrPheLeuGlyMetAspTyr

1　　　　　　　5　　　　　　　10

<210>584

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>584

SerTyrSerTyrHisMetGlyPheAspTyr

1　　　　　　　5　　　　　　　10

<210>585

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>585

SerTyrSerTyrSerLeuGlyMetAspTyr

1　　　　　　　5　　　　　　　10

<210>586

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>586

SerTyrSerTyrTyrGluAlaPheAspTyr

1　　　　　　　5　　　　　　　10

<210>587

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>587

SerTyrSerTyrArgMetAlaPheAspTyr

1　　　　　　　5　　　　　　　10

<210>588

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>588

SerTyrSerTyrHisIleAlaPheAspTyr

1　　　　　　　5　　　　　　　10

<210>589

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>589

SerTyrSerTyrSerValGlyMetAspTyr

1　　　　　　　5　　　　　　　10

<210>590

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>590

SerTyrSerTyrThrLeuGlyMetAspTyr

1　　　　　　　5　　　　　　　10

<210>591

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>591

SerTyrSerTyrTyrSerAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>592

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>592

SerTyrSerTyrTyrSerAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>593

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>593

SerTyrSerTyrTyrSerAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>594

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>594

SerTyrSerTyrTyrSerAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>595

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>595

SerTyrSerTyrTyrSerAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>596

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>596

SerTyrSerTyrTyrSerAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>597

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>597

SerTyrSerTyrTyrSerAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>598

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(1)..(1)

<223> Serine or glycine

<220>

<221>MOD_RES

<222>(3)..(3)

<223> Serine, the acid of asparagus fern acyl or tyrosine

<220>

<221>MOD_RES

<222>(4)..(4)

<223> tyrosine, the acid of asparagus fern acyl or tryptophane

<220>

<221>MOD_RES

<222>(5)..(5)

<223> Threonine, tyrosine, phenylalanine, arginine, Serine, Histidine or tryptophane

<220>

<221>MOD_RES

<222>(6)..(6)

<223> Threonine, the acid of asparagus fern acyl, aspartic acid, Serine, L-glutamic acid, phenylalanine, methionine(Met), α-amino-isovaleric acid, leucine, Isoleucine or tyrosine

<220>

<221>MOD_RES

<222>(7)..(7)

<223> Serine, L-Ala, Threonine or glycine

<220>

<221>MOD_RES

<222>(8)..(8)

<223> Isoleucine, methionine(Met), phenylalanine or leucine

<400>598

XaaTyrXaaXaaXaaXaaXaaXaaAspTyr

1　　　　　　　5　　　　　　　10

<210>599

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>599

GlyPheAsnIleSerSerSerTyrIleHis

1　　　　　　　5　　　　　　　10

<210>600

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>600

GlyPheAsnIleSerSerSerTyrIleHis

1　　　　　　　5　　　　　　　10

<210>601

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>601

GlyPheAsnIleSerSerSerTyrIleHis

1　　　　　　　5　　　　　　　10

<210>602

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>602

GlyPheAsnIleSerSerSerTyrIleHis

1　　　　　　　5　　　　　　　10

<210>603

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>603

GlyPheAsnIleSerSerSerTyrIleHis

1　　　　　　　5　　　　　　　10

<210>604

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>604

GlyPheAsnIleSerSerSerTyrIleHis

1　　　　　　　5　　　　　　　10

<210>605

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>605

GlyPheAsnIleSerSerSerTyrIleHis

1　　　　　　　5　　　　　　　10

<210>606

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>606

GlyPheAsnIleSerSerSerTyrIleHis

1　　　　　　　5　　　　　　　10

<210>607

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>607

GlyPheAsnIleSerSerSerTyrIleHis

1　　　　　　　5　　　　　　　10

<210>608

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>608

GlyPheAsnIleSerSerSerTyrIleHis

1　　　　　　　5　　　　　　　10

<210>609

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>609

GlyPheAsnIleSerSerSerTyrIleHis

1　　　　　　　5　　　　　　　10

<210>610

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>610

GlyPheAsnIleSerSerSerTyrIleHis

1　　　　　　　5　　　　　　　10

<210>611

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>611

GlyPheAsnIleSerSerSerTyrIleHis

1　　　　　　　5　　　　　　　10

<210>612

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>612

GlyPheAsnIleSerSerSerTyrIleHis

1　　　　　　　5　　　　　　　10

<210>613

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>613

GlyPheAsnPheSerSerSerTyrIleHis

1　　　　　　　5　　　　　　　10

<210>614

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>614

GlyPheAsnIleLysGlySerLeuIleHis

1　　　　　　　5　　　　　　　10

<210>615

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>615

GlyPheAsnIleLysGlySerIleMetHis

1　　　　　　　5　　　　　　　10

<210>616

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>616

GlyPheAsnIleLysSerSerIleMetHis

1　　　　　　　5　　　　　　　10

<210>617

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>617

GlyPheAsnIleSerSerSerTyrIleHis

1　　　　　　　5　　　　　　　10

<210>618

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>618

GlyPheAsnIleSerSerSerTyrIleHis

1　　　　　　　5　　　　　　　10

<210>619

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>619

GlyPheAsnIleSerSerSerTyrIleHis

1　　　　　　　5　　　　　　　10

<210>620

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>620

GlyPheAsnIleSerSerSerTyrIleHis

1　　　　　　　5　　　　　　　10

<210>621

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>621

GlyPheAsnIleSerSerSerTyrIleHis

1　　　　　　　5　　　　　　　10

<210>622

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>622

GlyPheAsnIleSerSerSerTyrIleHis

1　　　　　　　5　　　　　　　10

<210>623

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>623

GlyPheAsnIleSerSerSerTyrIleHis

1　　　　　　　5　　　　　　　10

<210>624

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>624

GlyPheAsnIleSerSerSerTyrIleHis

1　　　　　　　5　　　　　　　10

<210>625

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>625

GlyPheAsnLeuAlaSerSerPheMetHis

1　　　　　　　5　　　　　　　10

<210>626

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>626

GlyPheAsnLeuValSerSerLeuMetHis

1　　　　　　　5　　　　　　　10

<210>627

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>627

GlyPheAsnValLysThrGlyLeuIleHis

1　　　　　　　5　　　　　　　10

<210>628

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>628

GlyPheAsnValLysTrpAsnTyrIleHis

1　　　　　　　5　　　　　　　10

<210>629

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>629

GlyPheAsnValValSerSerPheIleHis

1　　　　　　　5　　　　　　　10

<210>630

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(4)..(4)

<223> Isoleucine, phenylalanine, leucine or α-amino-isovaleric acid

<220>

<221>MOD_RES

<222>(5)..(5)

<223> Serine, Methionin, L-Ala or α-amino-isovaleric acid

<220>

<221>MOD_RES

<222>(6)..(6)

<223> Serine, glycine, Threonine or tryptophane

<220>

<221>MOD_RES

<222>(7)..(7)

<223> Serine, glycine or the acid of asparagus fern acyl

<220>

<221>MOD_RES

<222>(8)..(8)

<223> tyrosine, leucine, Isoleucine or phenylalanine

<220>

<221>MOD_RES

<222>(9)..(9)

<223> Isoleucine or methionine(Met)

<400>630

GlyPheAsnXaaXaaXaaXaaXaaXaaHis

1　　　　　　　5　　　　　　　10

<210>631

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>631

AlaIleAlaProTyrLeuGlySerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>632

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>632

AlaIleProProPheTyrGlyTrpThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>633

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>633

AlaIleGlnProTyrPheGlyTrpThrIleTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>634

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>634

AlaIleSerProTyrLeuGlySerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>635

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>635

AspIleAlaProTyrLeuGlyThrThrLysTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>636

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>636

AspIleSerProTrpTyrGlyGlyThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>637

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>637

AspIleSerSerTyrThrGlySerThrAspTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>638

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>638

PheIleGlnProTyrTyrGlySerThrIleTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>639

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>639

PheIleSerProTyrLeuGlySerThrAsnTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>640

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>640

GlyIleThrProTyrLeuGlyTrpThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>641

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>641

HisIleSerProTyrLeuGlySerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>642

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>642

IleIleSerProTyrLeuGlySerThrGlyTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>643

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>643

SerIleThrProTyrTyrGlyTrpThrArgTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>644

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>644

TrpIleSerProTyrLeuGlyArgThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>645

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>645

TyrIleSerProTyrTyrGlySerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>646

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>646

TyrIleSerProTyrTyrGlySerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>647

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>647

TyrIleSerProTyrTyrGlySerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>648

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>648

TyrIleSerProTyrTyrGlySerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>649

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>649

TyrIleGlyProPheThrGlySerThrAsnTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>650

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>650

TyrIleSerProPheLeuSerThrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>651

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>651

TyrIleSerProTyrTyrGlySerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>652

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>652

TyrIleSerProTyrSerGlySerThrLysTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>653

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>653

TyrIleSerProTyrTyrSerSerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>654

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>654

TyrIleSerProTyrLeuGlySerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>655

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>655

TyrIleSerProTyrLeuSerSerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>656

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>656

TyrIleSerProTyrTyrGlySerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>657

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>657

TyrIleSerProTyrTyrGlySerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>658

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>658

TyrIleSerProTyrTyrGlySerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>659

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>659

TyrIleSerProTyrTyrGlySerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>660

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>660

TyrIleSerProTyrTyrGlySerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>661

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>661

TyrIleSerProTyrTyrGlySerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>662

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(1)..(1)

<223> L-Ala, aspartic acid, phenylalanine, glycine, Histidine, Isoleucine, Serine, tryptophane or tyrosine

<220>

<221>MOD_RES

<222>(3)..(3)

<223> L-Ala, proline(Pro), glutamine, Serine, Threonine or glycine

<220>

<221>MOD_RES

<222>(4)..(4)

<223> proline(Pro) or Serine

<220>

<221>MOD_RES

<222>(5)..(5)

<223> tyrosine or phenylalanine

<220>

<221>MOD_RES

<222>(6)..(6)

<223> leucine, tyrosine, phenylalanine or Threonine

<220>

<221>MOD_RES

<222>(7)..(7)

<223> glycine or Serine

<220>

<221>MOD_RES

<222>(8)..(8)

<223> Serine, tryptophane, Threonine, glycine or arginine

<220>

<221>MOD_RES

<222>(10)..(10)

<223> Serine, Isoleucine, Methionin, aspartic acid, the acid of asparagus fern acyl, glycine or arginine

<400>662

XaaIleXaaXaaXaaXaaXaaXaaThrXaaTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>663

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>663

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>664

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>664

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>665

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>665

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>666

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>666

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>667

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>667

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>668

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>668

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>669

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>669

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>670

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>670

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>671

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>671

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>672

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>672

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>673

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>673

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>674

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>674

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>675

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>675

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>676

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>676

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>677

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>677

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>678

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>678

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>679

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>679

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>680

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>680

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>681

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>681

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>682

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>682

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>683

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>683

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>684

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>684

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>685

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>685

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>686

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>686

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>687

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>687

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>688

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>688

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>689

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>689

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>690

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>690

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>691

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>691

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>692

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>692

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>693

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>693

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>694

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>694

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>695

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>695

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>696

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>696

GlyPheAsnIlePheTyrGlyGlyIleHis

1　　　　　　　5　　　　　　　10

<210>697

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>697

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>698

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>698

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>699

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>699

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>700

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>700

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>701

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>701

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>702

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>702

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>703

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>703

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>704

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>704

GlyPheAsnIleSerTyrSerSerMetHis

1　　　　　　　5　　　　　　　10

<210>705

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(5)..(5)

<223> Serine or phenylalanine

<220>

<221>MOD_RES

<222>(7)..(8)

<223> Serine or glycine

<220>

<221>MOD_RES

<222>(9)..(9)

<223> methionine(Met) or Isoleucine

<400>705

GlyPheAsnIleXaaTyrXaaXaaXaaHis

1　　　　　　　5　　　　　　　10

<210>706

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>706

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>707

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>707

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>708

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>708

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>709

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>709

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>710

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>710

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>711

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>711

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>712

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>712

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>713

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>713

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>714

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>714

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>715

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>715

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>716

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>716

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>717

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>717

SerTyrSerTyrSerGluAlaLeuAspTyr

1　　　　　　5　　　　　　　10

<210>718

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>718

SerTyrSerTyrTyrSerAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>719

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>719

SerTyrSerTyrSerLeuAlaPheAspTyr

1　　　　　　　5　　　　　　　10

<210>720

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>720

SerTyrSerTyrSerPheGlyMetAspTyr

1　　　　　　　5　　　　　　　10

<210>721

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>721

SerTyrSerTyrArgMetAlaPheAspTyr

1　　　　　　　5　　　　　　　10

<210>722

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>722

GlyTyrSerTrpPheAsnAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>723

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>723

SerTyrSerTyrHisLeuGlyMetAspTyr

1　　　　　　　5　　　　　　　10

<210>724

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>724

SerTyrSerTyrSerValGlyMetAspTyr

1　　　　　　　5　　　　　　　10

<210>725

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>725

SerTyrSerTyrHisValAlaPheAspTyr

1　　　　　　　5　　　　　　　10

<210>726

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>726

SerTyrSerTyrPheLeuAlaMetAspTyr

1　　　　　　　5　　　　　　　10

<210>727

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(1)..(1)

<223> Serine or glycine

<220>

<221>MOD_RES

<222>(4)..(4)

<223> tyrosine or tryptophane

<220>

<221>MOD_RES

<222>(5)..(5)

<223> Serine, tyrosine, arginine, phenylalanine or Histidine

<220>

<221>MOD_RES

<222>(6)..(6)

<223> L-glutamic acid, Serine, leucine, phenylalanine, methionine(Met), the acid of asparagus fern acyl or α-amino-isovaleric acid

<220>

<221>MOD_RES

<222>(7)..(7)

<223> L-Ala or glycine

<220>

<221>MOD_RES

<222>(8)..(8)

<223> leucine, methionine(Met), phenylalanine or Isoleucine

<400>727

XaaTyrSerXaaXaaXaaXaaXaaAspTyr

1　　　　　　　5　　　　　　　10

<210>728

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>728

GlnGlnSerSerTyrSerSerLeuIleThr

1　　　　　　　5　　　　　　　10

<210>729

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>729

GlnGlnSerSerTyrSerSerLeuIleThr

1　　　　　　　5　　　　　　　10

<210>730

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>730

GlnGlnSerSerTyrSerSerLeuIleThr

1　　　　　　　5　　　　　　　10

<210>731

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>731

GlnGlnSerSerTyrSerSerLeuIleThr

1　　　　　　　5　　　　　　　10

<210>732

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>732

GlnGlnSerSerTyrSerSerLeuIleThr

1　　　　　　　5　　　　　　　10

<210>733

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>733

GlnGlnSerSerTyrSerSerLeuIleThr

1　　　　　　　5　　　　　　　10

<210>734

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>734

GlnGlnSerSerTyrSerSerLeuIleThr

1　　　　　　　5　　　　　　　10

<210>735

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>735

GlnGlnSerSerTyrSerSerLeuIleThr

1　　　　　　　5　　　　　　　10

<210>736

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>736

GlnGlnSerSerTyrSerSerLeuIleThr

1　　　　　　　5　　　　　　　10

<210>737

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>737

GlnGlnSerSerTyrSerSerLeuIleThr

1　　　　　　　5　　　　　　　10

<210>738

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>738

GlnGlnSerSerTyrSerSerLeuIleThr

1　　　　　　　5　　　　　　　10

<210>739

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>739

GlyPheAsnIleSerSerSerTyrIleHis

1　　　　　　　5　　　　　　　10

<210>740

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>740

GlyPheAsnIleSerSerSerTyrIleHis

1　　　　　　　5　　　　　　　10

<210>741

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>741

GlyPheAsnValLysTrpAsnTyrIleHis

1　　　　　　　5　　　　　　　10

<210>742

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>742

GlyPheAsnIleSerSerSerTyrIleHis

1　　　　　　　5　　　　　　　10

<210>743

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>743

GlyPheAsnIleLysGlySerIleMetHis

1　　　　　　　5　　　　　　　10

<210>744

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>744

GlyPheAsnValLysThrGlyLeuIleHis

1　　　　　　　5　　　　　　　10

<210>745

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>745

GlyPheAsnIleSerSerSerTyrIleHis

1　　　　　　　5　　　　　　　10

<210>746

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>746

GlyPheAsnLeuValSerSerLeuMetHis

1　　　　　　　5　　　　　　　10

<210>747

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>747

GlyPheAsnValValSerSerPheIleHis

1　　　　　　　5　　　　　　10

<210>748

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>748

GlyPheAsnIleSerSerSerTyrIleHis

1　　　　　　5　　　　　　　10

<210>749

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(4)..(4)

<223> Isoleucine, α-amino-isovaleric acid or leucine

<220>

<221>MOD_RES

<222>(5)..(5)

<223> Serine, Methionin or α-amino-isovaleric acid

<220>

<221>MOD_RES

<222>(6)..(6)

<223> Serine, tryptophane, glycine or Threonine

<220>

<221>MOD_RES

<222>(7)..(7)

<223> Serine, the acid of asparagus fern acyl or glycine

<220>

<221>MOD_RES

<222>(8)..(8)

<223> tyrosine, Isoleucine, leucine or phenylalanine

<220>

<221>MOD_RES

<222>(9)..(9)

<223> Isoleucine or methionine(Met)

<400>749

GlyPheAsnXaaXaaXaaXaaXaaXaaHis

1　　　　　　　5　　　　　　　10

<210>750

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>750

TyrIleSerProTyrLeuSerSerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>751

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>751

PheIleSerProTyrLeuGlySerThrAsnTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>752

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>752

TyrIleSerProTyrTyrGlySerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>753

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>753

AspIleAlaProTyrLeuGlyThrThrLysTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>754

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>754

TyrIleSerProTyrTyrGlySerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>755

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>755

TyrIleSerProTyrTyrGlySerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>756

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>756

HisIleSerProTyrLeuGlySerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>757

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>757

TyrIleSerProTyrTyrGlySerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>758

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>758

TyrIleSerProTyrTyrGlySerThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>759

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>759

AlaIleGlnProTyrPheGlyTrpThrIleTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>760

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(1)..(1)

<223> tyrosine, phenylalanine, aspartic acid, Histidine or L-Ala

<220>

<221>MOD_RES

<222>(3)..(3)

<223> Serine, L-Ala or glutamine

<220>

<221>MOD_RES

<222>(6)..(6)

<223> leucine, tyrosine or phenylalanine

<220>

<221>MOD_RES

<222>(7)..(7)

<223> Serine or glycine

<220>

<221>MOD_RES

<222>(8)..(8)

<223> Serine, Threonine or tryptophane

<220>

<221>MOD_RES

<222>(10)..(10)

<223> Serine, the acid of asparagus fern acyl, Methionin or Isoleucine

<400>760

XaaIleXaaProTyrXaaXaaXaaThrXaaTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>761

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>761

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>762

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>762

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>763

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>763

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>764

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>764

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>765

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>765

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>766

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>766

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>767

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>767

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>768

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>768

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>769

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>769

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>770

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>770

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>771

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>771

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>772

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>772

GlnGlnTyrSerSerTyrSerSerLeuPheThr

1　　　　　　　5　　　　　　　10

<210>773

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>773

GlnGlnTyrSerSerTyrSerSerLeuPheThr

1　　　　　　　5　　　　　　　10

<210>774

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>774

GlnGlnTyrSerSerTyrSerSerLeuPheThr

1　　　　　　　5　　　　　　　10

<210>775

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>775

GlnGlnTyrSerSerTyrSerSerLeuPheThr

1　　　　　　　5　　　　　　　10

<210>776

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>776

GlnGlnTyrSerSerTyrSerSerLeuPheThr

1　　　　　　　5　　　　　　　10

<210>777

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>777

GlnGlnTyrSerSerTyrSerSerLeuPheThr

1　　　　　　　5　　　　　　　10

<210>778

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>778

GlnGlnTyrSerSerTyrSerSerLeuPheThr

1　　　　　　　5　　　　　　　10

<210>779

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>779

GlnGlnTyrSerSerTyrSerSerLeuPheThr

1　　　　　　　5　　　　　　　10

<210>780

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>780

GlnGlnTyrSerSerTyrSerSerLeuPheThr

1　　　　　　　5　　　　　　　10

<210>781

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>781

GlnGlnTyrSerSerTyrSerSerLeuPheThr

1　　　　　　　5　　　　　　　10

<210>782

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>782

GlnGlnTyrSerSerTyrSerSerLeuPheThr

1　　　　　　　5　　　　　　　10

<210>783

<211>107

<212>PRT

The <213> mankind (Homosapiens)

<400>783

AspIleGlnMetThrGlnSerProSerSerLeuSerAlaSerValGly

1　　　　　　5　　　　　　　10　　　　　　　15

AspArgValThrIleThrCysArgAlaSerGlnAspValAsnThrAla

　　　　20　　　　　　　25　　　　　　　30

ValAlaTrpTyrGlnGlnLysProGlyLysAlaProLysLeuLeuIle

　　　35　　　　　　　40　　　　　　　45

TyrSerAlaSerPheLeuTyrSerGlyValProSerArgPheSerGly

　　50　　　　　　　55　　　　　　　60

SerArgSerGlyThrAspPheThrLeuThrIleSerSerLeuGlnPro

65　　　　　70　　　　　　　75　　　　　　　　80

GluAspPheAlaThrTyrTyrCysGlnGlnHisTyrThrThrProPro

　　　　　　　85　　　　　　　　90　　　　　　　95

ThrPheGlyGlnGlyThrLysValGluIleLys

　　　　　　100　　　　　　　105

<210>784

<211>107

<212>PRT

The <213> mankind (Homosapiens)

<400>784

AspIleGlnMetThrGlnSerProSerSerLeuSerAlaSerValGly

1　　　　　　5　　　　　　　10　　　　　　　15

AspArgValThrIleThrCysArgAlaSerGlnSerValSerSerAla

　　　　20　　　　　　　25　　　　　　　30

ValAlaTrpTyrGlnGlnLysProGlyLysAlaProLysLeuLeuIle

　　　35　　　　　　　40　　　　　　　45

TyrSerAlaSerSerLeuTyrSerGlyValProSerArgPheSerGly

　　50　　　　　　　55　　　　　　　60

SerGlySerGlyThrAspPheThrLeuThrIleSerSerLeuGlnPro

65　　　　　70　　　　　　　75　　　　　　　　80

GluAspPheAlaThrTyrTyrCysGlnGlnSerTyrThrThrProPro

　　　　　　　85　　　　　　　　90　　　　　　　95

ThrPheGlyGlnGlyThrLysValGluIleLys

　　　　　　100　　　　　　　105

<210>785

<211>52

<212>DNA

<213> artificial sequence

<220>

The description of <223> artificial sequence: the oligonucleotide of synthesis

<220>

The base that <221> modifies

<222>(19)..(19)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(22)..(33)

<223>a, c, g or t

<220>

<223> details refer in specification sheets the description being substituted and preferably replacing

<400>785

gcagcttctggcttcaacntcnnnnnnnnnnnnatscactgggtgcgtcagg　　　52

<210>786

<211>67

<212>DNA

<213> artificial sequence

<220>

The description of <223> artificial sequence: the oligonucleotide of synthesis

<220>

The base of <221> through modifying

<222>(19)..(20)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(25)..(27)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(40)..(42)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(46)..(48)

<223>a, c, g or t

<220>

<223> details refer in specification sheets the description being substituted and preferably replacing

<400>786

ggcctggaatgggttgcannsatcnnnccgtactacggtnnnaccnnntatgccgatagc　　　60

gtcaagg　　　　　　　　　　　　　　　　　　　　　　　　　67

<210>787

<211>67

<212>DNA

<213> artificial sequence

<220>

The description of <223> artificial sequence: the oligonucleotide of synthesis

<220>

The base of <221> through modifying

<222>(25)..(27)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(34)..(35)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(40)..(42)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(46)..(48)

<223>a, c, g or t

<220>

<223> details refer in specification sheets the description being substituted and preferably replacing

<400>787

ggcctggaatgggttgcatacatcnnnccgtacnnsggtnnnaccnnntatgccgatagc　　60

gtcaagg　　　　　　　　　　　　　　　　　　　　　　　　67

<210>788

<211>67

<212>DNA

<213> artificial sequence

<220>

The description of <223> artificial sequence: the oligonucleotide of synthesis

<220>

The base of <221> through modifying

<222>(19)..(20)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(25)..(27)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(34)..(35)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(40)..(42)

<223>a, c, g or t

<220>

The base of <221> through modifying

<222>(46)..(48)

<223>a, c, g or t

<220>

<223> details refer in specification sheets the description being substituted and preferably replacing

<400>788

ggcctggaatgggttgcannsatcnnnccgtacnnsggtnnnaccnnntatgccgatagc　　60

gtcaagg　　　　　　　　　　　　　　　　　　　　　　　　67

<210>789

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>789

GlyPheAsnValLysThrGlyPheMetHis

1　　　　　　　5　　　　　　　10

<210>790

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>790

GlyPheAsnValLysThrGlyPheIleHis

1　　　　　　　5　　　　　　　10

<210>791

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>791

GlyPheAsnIleLysMetValPheMetHis

1　　　　　　5　　　　　　　10

<210>792

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>792

GlyPheAsnValLysAsnPheIleIleHis

1　　　　　　5　　　　　　　10

<210>793

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>793

GlyPheAsnValLysThrGlyPheMetHis

1　　　　　　　5　　　　　　　10

<210>794

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>794

GlyPheAsnValLysArgGlyPheMetHis

1　　　　　　　5　　　　　　　10

<210>795

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>795

GlyPheAsnLeuLysThrGlyPheIleHis

1　　　　　　5　　　　　　　10

<210>796

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>796

GlyPheAsnValLysThrGlyTyrMetHis

1　　　　　　5　　　　　　　10

<210>797

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>797

GlyPheAsnValLysThrGlyLeuIleHis

1　　　　　　5　　　　　　　10

<210>798

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>798

GlyPheAsnValMetIleGlyIleIleHis

1　　　　　　5　　　　　　10

<210>799

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>799

GlyPheAsnIleLysThrGlyPheMetHis

1　　　　　　5　　　　　　　10

<210>800

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(4)..(4)

<223> α-amino-isovaleric acid, Isoleucine or leucine

<220>

<221>MOD_RES

<222>(5)..(5)

<223> Methionin or methionine(Met)

<220>

<221>MOD_RES

<222>(6)..(6)

<223> Threonine, methionine(Met), the acid of asparagus fern acyl, arginine or Isoleucine

<220>

<221>MOD_RES

<222>(7)..(7)

<223> glycine, α-amino-isovaleric acid or phenylalanine

<220>

<221>MOD_RES

<222>(8)..(8)

<223> phenylalanine, Isoleucine, tyrosine or leucine

<220>

<221>MOD_RES

<222>(9)..(9)

<223> methionine(Met) or Isoleucine

<400>800

GlyPheAsnXaaXaaXaaXaaXaaXaaHis

1　　　　　　　5　　　　　　　10

<210>801

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>801

TyrIleSerProTyrTyrGlyTrpThrArgTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>802

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>802

TyrIleSerProTyrLeuGlyValThrArgTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>803

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>803

TyrIleSerProTyrAspGlySerThrAsnTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>804

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>804

TyrIleIleProTyrSerGlyAsnThrValTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>805

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>805

TyrIleSerProTyrSerGlyArgThrArgTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>806

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>806

TyrIleSerProTyrLeuGlySerThrArgTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>807

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>807

TyrIleSerProTyrTrpGlySerThrThrTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>808

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>808

TyrIleSerProTyrTyrGlySerThrArgTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>809

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>809

TyrIleSerProTyrPheGlyTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>810

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>810

TyrIleIleProTyrSerGlySerThrLysTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>811

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>811

TyrIleThrProTyrTrpGlySerThrLysTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>812

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(3)..(3)

<223> Serine, Isoleucine or Threonine

<220>

<221>MOD_RES

<222>(6)..(6)

<223> tyrosine, leucine, aspartic acid, Serine or tryptophane

<220>

<221>MOD_RES

<222>(8)..(8)

<223> tryptophane, α-amino-isovaleric acid, Serine, the acid of asparagus fern acyl, arginine or tyrosine

<220>

<221>MOD_RES

<222>(10)..(10)

<223> arginine, the acid of asparagus fern acyl, α-amino-isovaleric acid, Threonine, Serine or Methionin

<400>812

TyrIleXaaProTyrXaaGlyXaaThrXaaTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>813

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>813

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>814

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>814

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>815

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>815

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>816

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>816

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>817

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>817

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>818

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>818

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>819

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>819

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>820

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>820

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>821

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>821

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　5　　　　　　　10

<210>822

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>822

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>823

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>823

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>824

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>824

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>825

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(4)..(4)

<223> α-amino-isovaleric acid, phenylalanine, leucine or Isoleucine

<220>

<221>MOD_RES

<222>(5)..(5)

<223> Serine or tyrosine

<220>

<221>MOD_RES

<222>(7)..(8)

<223> Serine or tyrosine

<220>

<221>MOD_RES

<222>(9)..(9)

<223> Isoleucine or methionine(Met)

<400>825

GlyPheAsnXaaXaaTyrXaaXaaXaaHis

1　　　　　　　5　　　　　　　10

<210>826

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(3)..(3)

<223> Serine or tyrosine

<220>

<221>MOD_RES

<222>(4)..(4)

<223> proline(Pro) or Serine

<220>

<221>MOD_RES

<222>(7)..(7)

<223> Serine or glycine

<220>

<221>MOD_RES

<222>(8)..(8)

<223> Serine or tyrosine

<220>

<221>MOD_RES

<222>(10)..(10)

<223> Serine or tyrosine

<400>826

SerIleXaaXaaTyrTyrXaaXaaThrXaaTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>827

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(5)..(5)

<223> Serine or phenylalanine

<220>

<221>MOD_RES

<222>(7)..(8)

<223> Serine or glycine

<220>

<221>MOD_RES

<222>(9)..(9)

<223> Isoleucine or methionine(Met)

<400>827

GlyPheAsnIleXaaTyrXaaXaaXaaHis

1　　　　　　　5　　　　　　　10

<210>828

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>828

SerIleTyrSerTyrTyrSerTyrThrSerTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>829

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(1)..(1)

<223> Serine or glycine

<220>

<221>MOD_RES

<222>(4)..(4)

<223> tyrosine or tryptophane

<220>

<221>MOD_RES

<222>(5)..(5)

<223> Serine, tyrosine, arginine, phenylalanine or Histidine

<220>

<221>MOD_RES

<222>(6)..(6)

<223> L-glutamic acid, Serine, leucine, phenylalanine, methionine(Met), the acid of asparagus fern acyl or α-amino-isovaleric acid

<220>

<221>MOD_RES

<222>(7)..(7)

<223> L-Ala or glycine

<220>

<221>MOD_RES

<222>(8)..(8)

<223> leucine, methionine(Met), phenylalanine or Isoleucine

<400>829

XaaTyrSerXaaXaaXaaXaaXaaAspTyr

1　　　　　　　5　　　　　　　10

<210>830

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(3)..(7)

<223> Serine or tyrosine

<220>

<221>MOD_RES

<222>(8)..(8)

<223> leucine, Serine, proline(Pro) or tyrosine

<220>

<221>MOD_RES

<222>(9)..(9)

This residue of <223> can be presence or absence

<220>

<221>MOD_RES

<222>(10)..(10)

<223> phenylalanine, Isoleucine, α-amino-isovaleric acid or leucine

<400>830

GlnGlnXaaXaaXaaXaaXaaXaaProXaaThr

1　　　　　　　5　　　　　　　10

<210>831

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(4)..(4)

<223> α-amino-isovaleric acid, Isoleucine or phenylalanine

<220>

<221>MOD_RES

<222>(5)..(8)

<223> Serine or tyrosine

<220>

<221>MOD_RES

<222>(9)..(9)

<223> Isoleucine or methionine(Met)

<400>831

GlyPheAsnXaaXaaXaaXaaXaaXaaHis

1　　　　　　　5　　　　　　　10

<210>832

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(1)..(1)

<223> Serine or tyrosine

<220>

<221>MOD_RES

<222>(3)..(3)

<223> Serine or tyrosine

<220>

<221>MOD_RES

<222>(4)..(4)

<223> proline(Pro) or Serine

<220>

<221>MOD_RES

<222>(5)..(6)

<223> Serine or tyrosine

<220>

<221>MOD_RES

<222>(7)..(7)

<223> Serine or glycine

<220>

<221>MOD_RES

<222>(8)..(8)

<223> Serine or tyrosine

<220>

<221>MOD_RES

<222>(10)..(10)

<223> Serine or tyrosine

<400>832

XaaIleXaaXaaXaaXaaXaaXaaThrXaaTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>833

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(4)..(4)

<223> Isoleucine, α-amino-isovaleric acid or leucine

<220>

<221>MOD_RES

<222>(5)..(5)

<223> Serine, Methionin or α-amino-isovaleric acid

<220>

<221>MOD_RES

<222>(6)..(6)

<223> Serine, tryptophane, glycine or Threonine

<220>

<221>MOD_RES

<222>(7)..(7)

<223> Serine, the acid of asparagus fern acyl or glycine

<220>

<221>MOD_RES

<222>(8)..(8)

<223> tyrosine, Isoleucine, leucine or phenylalanine

<220>

<221>MOD_RES

<222>(9)..(9)

<223> Isoleucine or methionine(Met)

<400>833

GlyPheAsnXaaXaaXaaXaaXaaXaaHis

1　　　　　　　5　　　　　　　10

<210>834

<211>17

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(1)..(1)

<223> tyrosine, phenylalanine, aspartic acid, Histidine or L-Ala

<220>

<221>MOD_RES

<222>(3)..(3)

<223> Serine, L-Ala or glutamine

<220>

<221>MOD_RES

<222>(6)..(6)

<223> leucine, tyrosine or phenylalanine

<220>

<221>MOD_RES

<222>(7)..(7)

<223> Serine or glycine

<220>

<221>MOD_RES

<222>(8)..(8)

<223> Serine, Threonine or tryptophane

<220>

<221>MOD_RES

<222>(10)..(10)

<223> Serine, the acid of asparagus fern acyl, Methionin or Isoleucine

<400>834

XaaIleXaaProTyrXaaXaaXaaThrXaaTyrAlaAspSerValLys

1　　　　　　5　　　　　　　10　　　　　　　15

Gly

<210>835

<211>10

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(4)..(4)

<223> Isoleucine, α-amino-isovaleric acid or leucine

<220>

<221>MOD_RES

<222>(5)..(5)

<223> Methionin or methionine(Met)

<220>

<221>MOD_RES

<222>(6)..(6)

<223> Threonine, methionine(Met), the acid of asparagus fern acyl, arginine or Isoleucine

<220>

<221>MOD_RES

<222>(7)..(7)

<223> glycine, α-amino-isovaleric acid or phenylalanine

<220>

<221>MOD_RES

<222>(8)..(8)

<223> tyrosine, Isoleucine, leucine or phenylalanine

<220>

<221>MOD_RES

<222>(9)..(9)

<223> Isoleucine or methionine(Met)

<400>835

GlyPheAsnXaaXaaXaaXaaXaaXaaHis

1　　　　　　　5　　　　　　　10

<210>836

<211>18

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(4)..(4)

<223> Serine, Isoleucine or Threonine

<220>

<221>MOD_RES

<222>(7)..(7)

<223> leucine, tyrosine, aspartic acid, Serine or tryptophane

<220>

<221>MOD_RES

<222>(9)..(9)

<223> tryptophane, α-amino-isovaleric acid, Serine, the acid of asparagus fern acyl, arginine or tyrosine

<220>

<221>MOD_RES

<222>(11)..(11)

<223> arginine, aspartic acid, α-amino-isovaleric acid, Threonine, Serine or Methionin

<400>836

AlaTyrIleXaaProTyrXaaGlyXaaThrXaaTyrAlaAspSerVal

1　　　　　　5　　　　　　　10　　　　　　　15

LysGly

<210>837

<211>13

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>837

SerArgGluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　　5　　　　　　　10

<210>838

<211>18

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<220>

<221>MOD_RES

<222>(2)..(2)

<223> tyrosine, phenylalanine, aspartic acid, Histidine or L-Ala

<220>

<221>MOD_RES

<222>(4)..(4)

<223> Serine, L-Ala or glutamine

<220>

<221>MOD_RES

<222>(7)..(7)

<223> leucine, tyrosine and phenylalanine

<220>

<221>MOD_RES

<222>(8)..(8)

<223> Serine or glycine

<220>

<221>MOD_RES

<222>(9)..(9)

<223> Serine, Threonine or tryptophane

<220>

<221>MOD_RES

<222>(11)..(11)

<223> Serine, the acid of asparagus fern acyl, Methionin or Isoleucine

<400>838

AlaXaaIleXaaProTyrXaaXaaXaaThrXaaTyrAlaAspSerVal

1　　　　　　5　　　　　　　10　　　　　　　15

LysGly

<210>839

<211>14

<212>PRT

The <213> mankind (Homosapiens)

<400>839

TrpValArgGlnAlaProGlyGlnGlyLeuGluTrpMetGly

1　　　　　　　5　　　　　　　10

<210>840

<211>13

<212>PRT

The <213> mankind (Homosapiens)

<400>840

TrpValArgGlnAlaProGlyGlnGlyLeuGluTrpMet

1　　　　　　　5　　　　　　　10

<210>841

<211>13

<212>PRT

The <213> mankind (Homosapiens)

<400>841

TrpValArgGlnAlaProGlyGlnGlyLeuGluTrpMet

1　　　　　　　5　　　　　　　10

<210>842

<211>13

<212>PRT

The <213> mankind (Homosapiens)

<400>842

TrpValArgGlnAlaProGlyGlnGlyLeuGluTrpMet

1　　　　　　　5　　　　　　　10

<210>843

<211>14

<212>PRT

The <213> mankind (Homosapiens)

<400>843

TrpIleArgGlnProProGlyLysGlyLeuGluTrpIleGly

1　　　　　　　5　　　　　　　10

<210>844

<211>13

<212>PRT

The <213> mankind (Homosapiens)

<400>844

TrpIleArgGlnProProGlyLysGlyLeuGluTrpIle

1　　　　　　　5　　　　　　　10

<210>845

<211>13

<212>PRT

The <213> mankind (Homosapiens)

<400>845

TrpIleArgGlnProProGlyLysGlyLeuGluTrpIle

1　　　　　　　5　　　　　　　10

<210>846

<211>13

<212>PRT

The <213> mankind (Homosapiens)

<400>846

TrpIleArgGlnProProGlyLysGlyLeuGluTrpIle

1　　　　　　　5　　　　　　　10

<210>847

<211>14

<212>PRT

The <213> mankind (Homosapiens)

<400>847

TrpValArgGlnAlaProGlyLysGlyLeuGluTrpValSer

1　　　　　　　5　　　　　　　10

<210>848

<211>13

<212>PRT

The <213> mankind (Homosapiens)

<400>848

TrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal

1　　　　　　　5　　　　　　　10

<210>849

<211>13

<212>PRT

The <213> mankind (Homosapiens)

<400>849

TrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal

1　　　　　　　5　　　　　　　10

<210>850

<211>13

<212>PRT

The <213> mankind (Homosapiens)

<400>850

TrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal

1　　　　　　　5　　　　　　　10

<210>851

<211>14

<212>PRT

The <213> mankind (Homosapiens)

<400>851

TrpValArgGlnAlaProGlyLysGlyLeuGluTrpValSer

1　　　　　　　5　　　　　　　10

<210>852

<211>13

<212>PRT

The <213> mankind (Homosapiens)

<400>852

TrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal

1　　　　　　　5　　　　　　　10

<210>853

<211>13

<212>PRT

The <213> mankind (Homosapiens)

<400>853

TrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal

1　　　　　　　5　　　　　　　10

<210>854

<211>14

<212>PRT

The <213> mankind (Homosapiens)

<400>854

TrpValArgGlnAlaProGlyLysGlyLeuGluTrpValSer

1　　　　　　　5　　　　　　　10

<210>855

<211>13

<212>PRT

The <213> mankind (Homosapiens)

<400>855

TrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal

1　　　　　　　5　　　　　　　10

<210>856

<211>13

<212>PRT

The <213> mankind (Homosapiens)

<400>856

TrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal

1　　　　　　　5　　　　　　　10

<210>857

<211>13

<212>PRT

The <213> mankind (Homosapiens)

<400>857

TrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal

1　　　　　　　5　　　　　　　10

<210>858

<211>32

<212>PRT

The <213> mankind (Homosapiens)

<400>858

ArgValThrIleThrAlaAspThrSerThrSerThrAlaTyrMetGlu

1　　　　　　5　　　　　　　10　　　　　　　15

LeuSerSerLeuArgSerGluAspThrAlaValTyrTyrCysAlaArg

　　　　20　　　　　　　25　　　　　　　30

<210>859

<211>32

<212>PRT

The <213> mankind (Homosapiens)

<400>859

ArgValThrIleThrAlaAspThrSerThrSerThrAlaTyrMetGlu

1　　　　　　5　　　　　　　10　　　　　　　15

LeuSerSerLeuArgSerGluAspThrAlaValTyrTyrCysAlaArg

　　　　20　　　　　　　25　　　　　　　30

<210>860

<211>31

<212>PRT

The <213> mankind (Homosapiens)

<400>860

ArgValThrIleThrAlaAspThrSerThrSerThrAlaTyrMetGlu

1　　　　　　5　　　　　　　10　　　　　　　15

LeuSerSerLeuArgSerGluAspThrAlaValTyrTyrCysAla

　　　　20　　　　　　　25　　　　　　　30

<210>861

<211>30

<212>PRT

The <213> mankind (Homosapiens)

<400>861

ArgValThrIleThrAlaAspThrSerThrSerThrAlaTyrMetGlu

1　　　　　　5　　　　　　　10　　　　　　　15

LeuSerSerLeuArgSerGluAspThrAlaValTyrTyrCys

　　　　20　　　　　　　25　　　　　　　30

<210>862

<211>32

<212>PRT

The <213> mankind (Homosapiens)

<400>862

ArgValThrIleSerValAspThrSerLysAsnGlnPheSerLeuLys

1　　　　　　5　　　　　　　10　　　　　　　15

LeuSerSerValThrAlaAlaAspThrAlaValTyrTyrCysAlaArg

　　　　20　　　　　　　25　　　　　　　30

<210>863

<211>32

<212>PRT

The <213> mankind (Homosapiens)

<400>863

ArgValThrIleSerValAspThrSerLysAsnGlnPheSerLeuLys

1　　　　　　5　　　　　　　10　　　　　　　15

LeuSerSerValThrAlaAlaAspThrAlaValTyrTyrCysAlaArg

　　　　20　　　　　　　25　　　　　　　30

<210>864

<211>31

<212>PRT

The <213> mankind (Homosapiens)

<400>864

ArgValThrIleSerValAspThrSerLysAsnGlnPheSerLeuLys

1　　　　　　5　　　　　　　10　　　　　　　15

LeuSerSerValThrAlaAlaAspThrAlaValTyrTyrCysAla

　　　　20　　　　　　　25　　　　　　　30

<210>865

<211>30

<212>PRT

The <213> mankind (Homosapiens)

<400>865

ArgValThrIleSerValAspThrSerLysAsnGlnPheSerLeuLys

1　　　　　　5　　　　　　　10　　　　　　　15

LeuSerSerValThrAlaAlaAspThrAlaValTyrTyrCys

　　　　20　　　　　　　25　　　　　　　30

<210>866

<211>32

<212>PRT

The <213> mankind (Homosapiens)

<400>866

ArgPheThrIleSerArgAspAsnSerLysAsnThrLeuTyrLeuGln

1　　　　　　5　　　　　　　10　　　　　　　15

MetAsnSerLeuArgAlaGluAspThrAlaValTyrTyrCysAlaArg

　　　　20　　　　　　　25　　　　　　　30

<210>867

<211>32

<212>PRT

The <213> mankind (Homosapiens)

<400>867

ArgPheThrIleSerArgAspAsnSerLysAsnThrLeuTyrLeuGln

1　　　　　　5　　　　　　　10　　　　　　　15

MetAsnSerLeuArgAlaGluAspThrAlaValTyrTyrCysAlaArg

　　　　20　　　　　　　25　　　　　　　30

<210>868

<211>31

<212>PRT

The <213> mankind (Homosapiens)

<400>868

ArgPheThrIleSerArgAspAsnSerLysAsnThrLeuTyrLeuGln

1　　　　　　5　　　　　　　10　　　　　　　15

MetAsnSerLeuArgAlaGluAspThrAlaValTyrTyrCysAla

　　　　20　　　　　　　25　　　　　　　30

<210>869

<211>30

<212>PRT

The <213> mankind (Homosapiens)

<400>869

ArgPheThrIleSerArgAspAsnSerLysAsnThrLeuTyrLeuGln

1　　　　　　5　　　　　　　10　　　　　　　15

MetAsnSerLeuArgAlaGluAspThrAlaValTyrTyrCys

　　　　20　　　　　　　25　　　　　　　30

<210>870

<211>32

<212>PRT

The <213> mankind (Homosapiens)

<400>870

ArgPheThrIleSerAlaAspThrSerLysAsnThrAlaTyrLeuGln

1　　　　　　5　　　　　　　10　　　　　　　15

MetAsnSerLeuArgAlaGluAspThrAlaValTyrTyrCysSerArg

　　　　20　　　　　　　25　　　　　　　30

<210>871

<211>32

<212>PRT

The <213> mankind (Homosapiens)

<400>871

ArgPheThrIleSerAlaAspThrSerLysAsnThrAlaTyrLeuGln

1　　　　　　5　　　　　　　10　　　　　　　15

MetAsnSerLeuArgAlaGluAspThrAlaValTyrTyrCysSerArg

　　　　20　　　　　　　25　　　　　　　30

<210>872

<211>31

<212>PRT

The <213> mankind (Homosapiens)

<400>872

ArgPheThrIleSerAlaAspThrSerLysAsnThrAlaTyrLeuGln

1　　　　　　5　　　　　　　10　　　　　　　15

MetAsnSerLeuArgAlaGluAspThrAlaValTyrTyrCysSer

　　　　20　　　　　　　25　　　　　　　30

<210>873

<211>32

<212>PRT

The <213> mankind (Homosapiens)

<400>873

ArgPheThrIleSerAlaAspThrSerLysAsnThrAlaTyrLeuGln

1　　　　　　5　　　　　　　10　　　　　　　15

MetAsnSerLeuArgAlaGluAspThrAlaValTyrTyrCysAlaArg

　　　　20　　　　　　　25　　　　　　　30

<210>874

<211>32

<212>PRT

The <213> mankind (Homosapiens)

<400>874

ArgPheThrIleSerAlaAspThrSerLysAsnThrAlaTyrLeuGln

1　　　　　　5　　　　　　　10　　　　　　　15

MetAsnSerLeuArgAlaGluAspThrAlaValTyrTyrCysAlaArg

　　　　20　　　　　　　25　　　　　　　30

<210>875

<211>31

<212>PRT

The <213> mankind (Homosapiens)

<400>875

ArgPheThrIleSerAlaAspThrSerLysAsnThrAlaTyrLeuGln

1　　　　　　5　　　　　　　10　　　　　　　15

MetAsnSerLeuArgAlaGluAspThrAlaValTyrTyrCysAla

　　　　20　　　　　　　25　　　　　　　30

<210>876

<211>30

<212>PRT

The <213> mankind (Homosapiens)

<400>876

ArgPheThrIleSerAlaAspThrSerLysAsnThrAlaTyrLeuGln

1　　　　　　5　　　　　　　10　　　　　　　15

MetAsnSerLeuArgAlaGluAspThrAlaValTyrTyrCys

　　　　20　　　　　　　25　　　　　　　30

<210>877

<211>11

<212>PRT

The <213> mankind (Homosapiens)

<400>877

TrpGlyGlnGlyThrLeuValThrValSerSer

1　　　　　　　5　　　　　　　10

<210>878

<211>11

<212>PRT

The <213> mankind (Homosapiens)

<400>878

TrpGlyGlnGlyThrLeuValThrValSerSer

1　　　　　　　5　　　　　　　10

<210>879

<211>11

<212>PRT

The <213> mankind (Homosapiens)

<400>879

TrpGlyGlnGlyThrLeuValThrValSerSer

1　　　　　　　5　　　　　　　10

<210>880

<211>11

<212>PRT

The <213> mankind (Homosapiens)

<400>880

TrpGlyGlnGlyThrLeuValThrValSerSer

1　　　　　　　5　　　　　　　10

<210>881

<211>11

<212>PRT

The <213> mankind (Homosapiens)

<400>881

TrpGlyGlnGlyThrLeuValThrValSerSer

1　　　　　　　5　　　　　　　10

<210>882

<211>11

<212>PRT

The <213> mankind (Homosapiens)

<400>882

TrpGlyGlnGlyThrLeuValThrValSerSer

1　　　　　　　5　　　　　　　10

<210>883

<211>11

<212>PRT

The <213> mankind (Homosapiens)

<400>883

TrpGlyGlnGlyThrLeuValThrValSerSer

1　　　　　　　5　　　　　　　10

<210>884

<211>11

<212>PRT

The <213> mankind (Homosapiens)

<400>884

TrpGlyGlnGlyThrLeuValThrValSerSer

1　　　　　　　5　　　　　　　10

<210>885

<211>11

<212>PRT

The <213> mankind (Homosapiens)

<400>885

TrpGlyGlnGlyThrLeuValThrValSerSer

1　　　　　　　5　　　　　　　10

<210>886

<211>11

<212>PRT

The <213> mankind (Homosapiens)

<400>886

TrpGlyGlnGlyThrLeuValThrValSerSer

1　　　　　　　5　　　　　　　10

<210>887

<211>11

<212>PRT

The <213> mankind (Homosapiens)

<400>887

TrpGlyGlnGlyThrLeuValThrValSerSer

1　　　　　　　5　　　　　　　10

<210>888

<211>11

<212>PRT

The <213> mankind (Homosapiens)

<400>888

TrpGlyGlnGlyThrLeuValThrValSerSer

1　　　　　　　5　　　　　　　10

<210>889

<211>11

<212>PRT

The <213> mankind (Homosapiens)

<400>889

TrpGlyGlnGlyThrLeuValThrValSerSer

1　　　　　　　5　　　　　　　10

<210>890

<211>11

<212>PRT

The <213> mankind (Homosapiens)

<400>890

TrpGlyGlnGlyThrLeuValThrValSerSer

1　　　　　　　5　　　　　　　10

<210>891

<211>11

<212>PRT

The <213> mankind (Homosapiens)

<400>891

TrpGlyGlnGlyThrLeuValThrValSerSer

1　　　　　　　5　　　　　　　10

<210>892

<211>11

<212>PRT

The <213> mankind (Homosapiens)

<400>892

TrpGlyGlnGlyThrLeuValThrValSerSer

1　　　　　　　5　　　　　　　10

<210>893

<211>11

<212>PRT

The <213> mankind (Homosapiens)

<400>893

TrpGlyGlnGlyThrLeuValThrValSerSer

1　　　　　　　5　　　　　　　10

<210>894

<211>11

<212>PRT

The <213> mankind (Homosapiens)

<400>894

TrpGlyGlnGlyThrLeuValThrValSerSer

1　　　　　　　5　　　　　　　10

<210>895

<211>11

<212>PRT

The <213> mankind (Homosapiens)

<400>895

TrpGlyGlnGlyThrLeuValThrValSerSer

1　　　　　　　5　　　　　　　10

<210>896

<211>15

<212>PRT

The <213> mankind (Homosapiens)

<400>896

TrpTyrGlnGlnLysProGlyLysAlaProLysLeuLeuIleTyr

1　　　　　　5　　　　　　　10　　　　　　　15

<210>897

<211>15

<212>PRT

The <213> mankind (Homosapiens)

<400>897

TrpTyrLeuGlnLysProGlyGlnSerProGlnLeuLeuIleTyr

1　　　　　　5　　　　　　　10　　　　　　　15

<210>898

<211>15

<212>PRT

The <213> mankind (Homosapiens)

<400>898

TrpTyrGlnGlnLysProGlyGlnAlaProArgLeuLeuIleTyr

1　　　　　　5　　　　　　　10　　　　　　　15

<210>899

<211>15

<212>PRT

The <213> mankind (Homosapiens)

<400>899

TrpTyrGlnGlnLysProGlyGlnProProLysLeuLeuIleTyr

1　　　　　　5　　　　　　　10　　　　　　　15

<210>900

<211>32

<212>PRT

The <213> mankind (Homosapiens)

<400>900

GlyValProSerArgPheSerGlySerGlySerGlyThrAspPheThr

1　　　　　　5　　　　　　　10　　　　　　　15

LeuThrIleSerSerLeuGlnProGluAspPheAlaThrTyrTyrCys

　　　　20　　　　　　　25　　　　　　　30

<210>901

<211>32

<212>PRT

The <213> mankind (Homosapiens)

<400>901

GlyValProAspArgPheSerGlySerGlySerGlyThrAspPheThr

1　　　　　　5　　　　　　　10　　　　　　　15

LeuLysIleSerArgValGluAlaGluAspValGlyValTyrTyrCys

　　　　20　　　　　　　25　　　　　　　30

<210>902

<211>32

<212>PRT

The <213> mankind (Homosapiens)

<400>902

GlyIleProAspArgPheSerGlySerGlySerGlyThrAspPheThr

1　　　　　　5　　　　　　　10　　　　　　　15

LeuThrIleSerArgLeuGluProGluAspPheAlaValTyrTyrCys

　　　　20　　　　　　　25　　　　　　　30

<210>903

<211>32

<212>PRT

The <213> mankind (Homosapiens)

<400>903

GlyValProAspArgPheSerGlySerGlySerGlyThrAspPheThr

1　　　　　　5　　　　　　　10　　　　　　　15

LeuThrIleSerSerLeuGlnAlaGluAspValAlaValTyrTyrCys

　　　　　20　　　　　　　25　　　　　　　30

<210>904

<211>10

<212>PRT

The <213> mankind (Homosapiens)

<400>904

PheGlyGlnGlyThrLysValGluIleLys

1　　　　　　5　　　　　　　10

<210>905

<211>10

<212>PRT

The <213> mankind (Homosapiens)

<400>905

PheGlyGlnGlyThrLysValGluIleLys

1　　　　　　5　　　　　　　10

<210>906

<211>10

<212>PRT

The <213> mankind (Homosapiens)

<400>906

PheGlyGlnGlyThrLysValGluIleLys

1　　　　　　5　　　　　　　10

<210>907

<211>10

<212>PRT

The <213> mankind (Homosapiens)

<400>907

PheGlyGlnGlyThrLysValGluIleLys

1　　　　　　5　　　　　　10

<210>908

<211>11

<212>PRT

<213> artificial sequence

<220>

The description of <223> artificial sequence: synthetic peptide

<400>908

GluTyrTyrArgTrpTyrThrAlaIleAspTyr

1　　　　　　5　　　　　　10

261

Claims (26)

1. the antibody be separated of the polyubiquitin specific binding connected with Methionin-48, wherein this antibody comprises six hypervariable region (HVR) sequences, wherein HVR-H1, HVR-H2, HVR-H3 and HVR-L3 sequence corresponds in Figure 10 A-10C apu01, apu02, apu03, apu04, apu05, apu06, apu07, apu08, apu09, apu10, apu11, apu12, apu13, to apu2.01 in listed or Figure 16 A and 16B of apu14 or apu15, apu2.02, apu2.03, apu2.04, apu2.05, apu2.06, apu2.07, apu2.08, HVR-H1 listed by apu2.09 or apu2.10, HVR-H2, HVR-H3 and HVR-L3 sequence, HVR-L1 sequence corresponds to SEQIDNO:79, and HVR-L2 sequence corresponds to SEQIDNO:80, wherein said HVR-H1, HVR-H2, the aminoacid sequence of HVR-H3 with HVR-L3 is from identical clone.

2. the antibody of claim 1, it comprises the HVR-H1 sequence of SEQIDNO:269, and the HVR-H2 sequence of SEQIDNO:285 is bad, the HVR-H3 sequence of SEQIDNO:301, the HVR-L1 sequence of SEQIDNO:79, the HVR-L2 sequence of SEQIDNO:80 and the HVR-L3 sequence of SEQIDNO:317.

3. the antibody of claim 1, it comprises the HVR-H1 sequence of SEQIDNO:701, the HVR-H2 sequence of SEQIDNO:712, the HVR-H3 sequence of SEQIDNO:723, the HVR-L1 sequence of SEQIDNO:79, the HVR-L2 sequence of SEQIDNO:80 and the HVR-L3 sequence of SEQIDNO:734.

4. the antibody of any one of claim 1-3, wherein this antibody is combined with polyubiquitin protein-specific.

5. the antibody of claim 4, the wherein degraded of this antibody suppression polyubiquitin albumen.

6. the antibody of claim 4, wherein this antibody regulates the signal transduction path of at least one polyubiquitin mediation.

7. the antibody of claim 4, the signal transduction path of wherein this antibody suppression at least one polyubiquitin mediation.

8. the antibody of claim 4, wherein this antibody stimulates the signal transduction path of at least one polyubiquitin mediation.

9. the nucleic acid molecule of the antibody of an any one of claim 1-3 of encoding.

10. one kind comprises the carrier of the nucleic acid of claim 9.

11. 1 kinds of host cells comprising the carrier of claim 10.

12. 1 kinds of clones that can produce the antibody of any one of claim 1-3.

13. 1 kinds of methods producing the antibody of any one of claim 1-3, cultivate the host cell of the nucleic acid molecule comprising this antibody of coding under being included in the condition wherein producing this antibody.

14. the antibody of any one of claim 1-3 and the composition of pharmaceutically acceptable carrier including effective amount.

15. 1 kinds include the antibody of any one of claim 1-3 of effective amount or the goods of the composition of claim 14.

Identify the polyubiquitin that Methionin-48 in sample connects or the method that the polyubiquitin albumen that Methionin-48 connects exists for 16. 1 kinds, comprise the antibody contacts of any one of claim 1-3 of sample and at least one.

Determine to suspect polyubiquitin that in the sample of the polyubiquitin albumen that the polyubiquitin that connects containing Methionin-48 or Methionin-48 connect, Methionin-48 connects or the method that the polyubiquitin albumen that Methionin-48 connects exists for 17. 1 kinds, comprise the antibody of any one of claim 1-3 sample being exposed at least one and measure the combination of polyubiquitin that this at least one antibody is connected with Methionin in sample-48 or the polyubiquitin albumen that Methionin-48 connects.

The polyubiquitin albumen that in 18. 1 kinds of sample separation, Methionin-48 connects and the method for non-poly ubiquitination albumen, comprise the antibody contacts of any one of claim 1-3 of sample and at least one.

19. determine the function of polyubiquitin that Methionin-48 in cell connects and/or a method for activity, comprise the antibody contacts of any one of claim 1-3 of cell and at least one and assess the impact of described contact procedure on cell.

20. determine the function of polyubiquitin that Methionin-48 in sample connects and/or a method for activity, comprise the antibody contacts of any one of claim 1-3 of sample and at least one and assess the impact of described contact procedure on sample.

The Fab of the antibody of 21. 1 kinds of any one of claim 1-3, wherein this Fab is Fab, F (ab') ₂, Fv, scFv or Fab ' fragment.

Purposes in the polyubiquitin that the antibody of any one of claim 1-3 of 22. at least one Methionin-48 in for the preparation of qualification sample connects or the goods that the polyubiquitin albumen that Methionin-48 connects exists, described qualification comprises the antibody contacts of any one of claim 1-3 of sample and at least one.

Purposes in the polyubiquitin that the antibody of any one of claim 1-3 of 23. at least one Methionin-48 in for the preparation of the sample determining to suspect the polyubiquitin albumen that the polyubiquitin that connects containing Methionin-48 or Methionin-48 connect connects or the goods that the polyubiquitin albumen that Methionin-48 connects exists, described determine to comprise any one of claim 1-3 sample being exposed at least one antibody and measure the combination of polyubiquitin that this at least one antibody is connected with Methionin in sample-48 or the polyubiquitin albumen that Methionin-48 connects.

Purposes in the goods of the polyubiquitin albumen that the antibody of any one of claim 1-3 of 24. at least one Methionin-48 in for the preparation of sample separation connects and non-poly ubiquitination albumen, described separation comprises the antibody contacts of any one of claim 1-3 of sample and at least one.

The antibody of any one of claim 1-3 of 25. at least one for the preparation of the purposes determined in the function of polyubiquitin that in cell, Methionin-48 connects and/or the goods of activity, comprises the antibody contacts of any one of claim 1-3 of cell and at least one and assesses the impact of described contact procedure on cell.

The antibody of any one of claim 1-3 of 26. at least one for the preparation of the purposes determined in the function of polyubiquitin that in sample, Methionin-48 connects and/or the goods of activity, is describedly determined to comprise by the antibody contacts of any one of claim 1-3 of sample and at least one and assesses the impact of described contact procedure on sample.