CN101027079A - Chimeric protein - Google Patents
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- CN101027079A CN101027079A CN200580011525.4A CN200580011525A CN101027079A CN 101027079 A CN101027079 A CN 101027079A CN 200580011525 A CN200580011525 A CN 200580011525A CN 101027079 A CN101027079 A CN 101027079A
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Abstract
A fusion protein containing a first segment that is located at the amino terminus of the fusion protein and specifically binds to and neutralizes a first cytokine or growth factor; and a second segment that is located at the carboxyl terminus of the fusion protein and specifically binds to a second cytokine receptor which is often rich at disease sites such as IL-1 receptor-rich inflammatory site. In addition, the said second segment is usually the receptor antagonist such as IL-1 receptor antagonist and its functional equivalent analogues. Also disclosed are nucleic acids encoding the fusion protein, vectors and host cells having the nucleic acids, and related composition and methods to target inflammatory diseases and indications co-existed with inflammation.
Description
The invention technical field
Chimeric protein pharmaceutical formulation of the present invention is used for the treatment of as various disease such as inflammation, asthma and tumors.
The background of invention technology
Related application: present patent application is in American National Department of Intellectual Property, patent priority application number No.US 60/618,476, on October 12nd, 2004 was put number of patent application US 60/628,994 on record, on November 17th, 2004 put on record, patent title " interleukin 1 receptor antagonist fusion rotein suppresses new vessels and forms ", on February 1st, 2005 put on record, and full content is with reference to detailed description.
Inflammation is a kind of immunoreation that body is resisted damage, and these damages can be by mechanical damage, infect or antigenic stimulus due to.The pathologic inflammatory reaction is meant that the improper stimulation of self antigen causes inflammation, and removes excessive or lasting immunoreation behind the proinflammatory factors reaction that also can cause inflammation.Asthma generates relevant disease with new vessels and often is accompanied by inflammation.Developed multiple human cytokines medicine inflammation-inhibiting reaction, be used for the treatment of the relevant asthma of inflammatory reaction, alleviated the pathologic new vessels and generate.Yet most protein formulation is because weak curative effect has side effect or albumen instability, and therapeutic effect is unsatisfactory.
Summary of the invention:
First fragment that fusion rotein comprises is positioned at aminoterminal, can specificity in conjunction with, first cytokine or somatomedin neutralize; Second fragment be positioned at fusion rotein c-terminus and can specificity in conjunction with the receptor of second cytokine or somatomedin, for example at the interleukin 1 receptor of inflammation part high expressed.These two fragments link together artificially, pairing first or second kind of cytokine at the inflammation part high expressed.
Above-mentioned fusion rotein is a glycosylated protein.It can also comprise one and be used to connect the first and second segmental junction fragments, and this junction fragment can form dimer.For example: junction fragment comprises the Fc fragment or the congenerous fragment of immunoglobulin.Be preferably as follows immunoglobulin: IgA, IgE, IgD, IgG, or IgM.Preferably IgG or its Fc section, for example sequence 2 fragments.Immunoglobulin chain comprises sequence 9,11,12,14,23, or 24; Or derive from their congenerous albumen.
In the above-mentioned fusion rotein, first fragment in conjunction with and in and VEGF, the albumen of angiogenin, tumor necrosis factor, interleukin-18, interleukin-4, interleukin-6 or its congenerous.For example: comprise first fragment of immunoglobulin chain-ordering can specific bond and in and VEGF, angiogenin, tumor necrosis factor, interleukin-18, interleukin-4, interleukin-13 or IgE and other congenerous albumen.First fragment also can comprise VEGF, and new vessels forms the receptor sequence of the factor, tumor necrosis factor, interleukin-18, interleukin-4, interleukin-13 or IgE, and for example sequence 3,6,15, or 19.
In the above-mentioned fusion rotein, second fragment is the receptor of bind interleukin 1 specifically.Second fragment can be the antagonist of interleukin 1, for example comprises the fragment or the proteic fragment of other congenerous of interleukin 1 receptor antagonist sequence (sequence 1).Correspondingly, above-mentioned fusion rotein can comprise sequence 5,8,10,13,17,18,21,22,24, or 25.
What another aspect of the present invention was described is, comprises the nucleic acid of in-vitro separation of the sequence of the above-mentioned fusion rotein of encoding.This nucleic acid fragment can comprise from sequence 1 to sequence a certain sequence 25.
Comprise in the scope that the present invention relates to that (1) above-mentioned fusion rotein or this proteic nucleotide sequence (2) of coding are suitable for medicinal carrier.The present invention also relates to comprise the immunoreactive method of patient of regulating in addition.The method comprises to be identified because excessive inflammatory response, immunne response or new vessels form the patient who causes body injury, and fusion rotein or the above-mentioned disease of this proteic exonuclease treatment of coding of using effective dose.This Drug therapy patient may or prepare to accept allogene or xenotransplantation.The example disease comprises inflammation disease, autoimmune disease, anaphylactic disease or tumor disease.When treatment relied on the tumor disease of new vessels formation, fusion rotein comprised sequence 24.
In addition on the one hand, the method for characteristic feature of an invention half-life of improving recombiant protein in addition in patient's body.This method comprises that with recombiant protein and other protein fragments fusion that comprises sequence 1 fragment or congenerous form the fusion rotein chimera, this method has determined fusion rotein in the intravital half-life of patient.Recombiant protein can be in conjunction with a certain cytokine or somatomedin.
Characteristics of the present invention comprise that also the raising recombiant protein is in the method for the intravital effect of patient.This method comprises recombiant protein is connected with other protein fragments of fragment that comprises sequence 1 or congenerous, forms the fusion rotein chimera.This method has determined fusion rotein in the intravital effect of patient.Specifically, the disease location of inflammation part or high expressed interleukin 1 receptor in patient's body, fusion rotein chimera can be simultaneously in conjunction with and in and interleukin 1 receptor and other cytokines or somatomedin.In addition on the one hand, the disease site of inflammation part or high expressed interleukin 1 receptor in patient's body, fusion rotein chimera can neutralize or the biological activity of antagonism interleukin 1 and other cytokines or somatomedin.
Other one side in the feature of the present invention comprises that treatment albumen can be shipped to the intravital drug target of patient targeting.This method comprises other fragment fusion with fragment that comprises sequence 1 or congenerous of treatment albumen, forms the fusion rotein chimera, and the method for the fusion rotein chimera being injected the patient who treats to needs.Treating proteic target site is the inflammation disease site of high expressed interleukin 1 receptor.Specifically, comprise the proteic fragment of sequence 1 or congenerous and combine with interleukin 1 receptor, recombiant protein be the treatment albumen, can in conjunction with and neutralize a certain cytokine or somatomedin.
Isolating polypeptide refers to be meant and does not contain molecule bonded with it under the naturalness that its purity is at least 75%, just between 75% to 100% (dry weight).Its purity can be by suitable standard method, and for example column chromatography, polyacrylamide gel electrophoresis or high performance liquid chromatography are measured.The isolating polypeptide of indication of the present invention can extract (wild type polypeptide) from natural origin, expresses by the DNA recombinant technique, or obtains by the method for chemosynthesis.Nucleic acid can refer to dna molecular (cDNA or genomic DNA), and the analog of RNA molecule (mRNA) and DNA or RNA, the analog of DNA or RNA can utilize nucleoside analog synthetic.Nucleic acid molecules can be strand or two strands, but general first-selected double-stranded DNA.Isolating nucleic acid is meant that its structure and any natural acid or natural genomic nucleic acids fragment are all different.This notion comprises following several aspect, and for example: a) DNA comprises the partial sequence of natural gene group dna molecular, but does not comprise under the nature situation flanking sequence of this genetic fragment in the biological gene group sequence.B) be incorporated into carrier or protokaryon, the interior nucleic acid molecules of eukaryotic gene group DNA, different with natural carrier or genomic DNA.C) molecular proportion of in-vitro separation such as cDNA, genomic fragment, pcr amplified fragment, the restriction endonuclease digestion fragment, d) recombinant nucleotide sequence is the partial sequence of heterozygous genes, this part gene can encoding fusion protein.Polypeptide protein of the present invention is expressed with said nucleic acid molecule.For this reason, research worker can be by being connected construction of expression vector with said nucleic acid molecule with suitable regulating and controlling sequence.
Carrier is meant the nucleic acid molecules that other coupled nucleotide sequences can be shifted.This carrier can carry out self replication or be integrated in host DNA.Carrier can comprise plasmid vector, Ke Si carrier or viral vector.Carrier comprises one section nucleic acid fragment, and this nucleic acid fragment can be at host cell expression.Comprise one or more regulating and controlling sequences in the carrier, be connected with the nucleotide sequence that will express.Regulating and controlling sequence comprises promoter, enhancer and other expression control element (for example polyadenylic acid signal).But the regulating and controlling sequence bag expands the controlling element sequence of guiding a certain nucleotide sequence constitutive expression, tissue specific expression and/or abduction delivering.Following factor is depended in the design of expression vector: host cell to be transformed, treat expression or other the similar factors of expressed proteins or RNA.Expression vector can be introduced into the polypeptide protein that host cell expression the present invention introduces.Invention also relates to the host cell that contains above-mentioned nucleic acid.Host cell comprises escherichia coli, insect cell (for example using rhabdovirus expression vector), yeast, mammalian cell, referring to articles in the Enzymology method book series such as Goedd, and gene expression technique (nineteen ninety, 185 pages).For expressing polypeptide protein of the present invention, we can use culture medium to cultivate host cell under given conditions, allow the polypeptide protein of host cell expression nucleic acid coding, purification of target albumen from cell or cell culture fluid again.Perhaps the nucleic acid that the present invention is mentioned utilizes T7 promoter and T7 polymerase to carry out vivoexpression.
The congenerous albumen of protein factor refers to come from this proteic polypeptide, and for example this albumen has the combination of one or more point mutation, insertion sudden change, deletion mutation, truncated mutant, fusion rotein or above sudden change.Congenerous albumen has kept this proteic activity fully, for example with cytokine, somatomedin or the bonded ability of its acceptor molecule.
The content that specifies in detail of the present invention is set forth in the following description.Further feature of the present invention, purpose and advantage describe below description and claim description in detail.
The Figure of description explanation
Fig. 1: the first generation Chinese hamster ovary celI clone who expresses TNFRII-Fc and TNFRII-Fc-IL-1ra fusion rotein, cell culture is in 24 well culture plates that contain serum-free medium, direct Coomassie brilliant blue protein staining, all Recombinant Protein Expression obviously as seen, expression 0.5-1.0ug albumen, every hole applied sample amount is 10-15 microlitre/hole.
Fig. 2: affinitive layer purification TNFRII-Fc-IL-1ra chimeric protein, carry out sds polyacrylamide gel electrophoresis, the albumen coomassie brilliant blue staining under reduction and non-reduced condition.
Fig. 3 a: example of our ability to solve problem: by changing the problem that first step purification condition has suppressed chimeric protein TNFRII-Fc-IL-1ra degraded.The TNFRII-Fc-IL-1ra chimeric protein that the efficient liquid phase chromatographic analysis purification is not degraded and part is degraded compares with TNFRII-Fc.
Fig. 4: affinitive layer purification IL-4R-Fc, IL-4R-Fc-IL-1ra, IL-18bp-Fc-IL-1ra.
Fig. 5: in the TNF α cell and experiment show the TNFRII-Fc-IL-1ra chimera protein can in and TNF α to the killing activity of L979 cell.This point is similar to marketed drug TNFRII-Fc (Enbrel).
Fig. 6: in the interleukin 1 cell and experiment show, marketed drug interleukin 1 receptor antagonist (Kineret) and TNFRII-Fc-IL-1ra chimeric protein can both in and the biological activity of interleukin 1 stimulation D10 cell proliferation.
Fig. 7: in the human interleukin 4 by using and the activity of the IL-4R-Fc of measuring IL-4R-Fc-IL-1ra and matched group.
Fig. 8: in the human interleukins-11 and the activity of measuring IL-4R-Fc-IL-1ra.
Fig. 9: in the interleukin-18 and the biological activity of measuring IL-18bp-Fc-IL-1ra..
Figure 10: in the interleukin 1 and the biological activity of measuring IL-18bp-Fc-IL-1ra..
Figure 11: utilize in the D10 cell detection VEGFR1-Fc-IL-1ra fusion rotein and the activity of interleukin 1.
Figure 12: VEGFR1-Fc-IL-1ra albumen is in the HUVE cell and the VEGF activity.
Figure 13: interleukin 1 receptor is in conjunction with experiment.
Detailed content of the present invention is described
Theoretical foundation of the present invention is to find IL-1 R antagonist or its congenerous albumen in a part at least, generate protein fusion with other biological activated protein such as anti-inflammatory albumen, anti-asthma albumen, anti-new vessels, can prolong biological half-life and the biological effectiveness thereof of these cell factors. These functional proteins comprise in the TNF (TNF) and molecule, interleukin-18 in and in the molecule, interleukin-4/interleukin-13 and in the molecule, VEGF and in molecule and the angiogenin and molecule.
Albumen (being positioned at the aminoterminal of fusion) often causes active disappearance with other activated proteins fusions, and especially merging at high molecular weight protein can be more obvious. For example, the existence owing to the aminoterminal polypeptide causes proenzyme and hormone precursor molecule non-activity in proenzyme and hormone precursor molecule. These proenzymes and hormone precursor molecule only just show as bioactive molecule after these aminoterminal polypeptide structures are cut off. And the high molecular weight protein fusion tends to cause low expression. But unexpectedly, the IL-1 R antagonist fusion can pass through mammalian cell expression, can reach the expression of industrialization production. With can not affect IL-1 R antagonist bind interleukin 1 acceptor and its active biological function that neutralizes after other protein fusions, can not affect with combination, the neutralization of other activated protein of its fusion active yet. Equally unexpectedly, IL-1 R antagonist (mammal express glycosylated protein) with and congenerous albumen not only can prolong half-life of activated protein, can also guide fusion to be positioned the inflammation site of high expressed interleukin 1 acceptor.
White interleukin-1 receptor antagonist
Interleukin 1 be a kind of be the cell factor that cell produces by huge biting/monokaryon, it exists with two kinds of forms: interleukin 1 α and interleukin-1 beta. It is by causing its biological effect in conjunction with cell-specific interleukin 1 receptor (IL-1R). Interleukin 1 receptor usually is the surface of cell membrane of expressing at the interleukin 1 sensitive cells.
IL-1 R antagonist (IL-1ra) is albumen in the human body, the natural mortifier of interleukin 1. IL-1 R antagonist has been used for suppressing the biologically active of interleukin 1. Thereby it suppresses interleukin 1 in conjunction with same interleukin 1 receptor in conjunction with cell membrane mating type interleukin 1 receptor. Interleukin 1 receptor is often expressed (Deleuran et al, 1992 in the inflammation site; Laken VD et al, 1997) and lymphocyte (Dower SK et al, 1990). Therefore, IL-1 R antagonist can guide with the human cytokines of its fusion (as below with in the TNF of describing and molecule) to the inflammation part of high expressed interleukin 1 receptor. Because it has the targeting effect, thus the required application dose for the treatment of albumen can be reduced, thus reduce accordingly side effect or increase curative effect of medication. In addition, at least on a part, owing to fusion can make IL-1 R antagonist and arrive simultaneously a certain site with its albumen that merges mutually, because the synergy of the two, the two is used alone or in combination relatively, and fusion can have better result for the treatment of.
The present invention can verify by IL-1 R antagonist and its congenerous albumen. IL-1 R antagonist congenerous albumen refers to derive from the polypeptide (described in brief introduction) of IL-1 R antagonist (sequence 1), it has the biologically active of IL-1 R antagonist, i.e. bind interleukin 1 acceptor and suppress interleukin 1 and the combination of this receptor. IL-1 R antagonist and congenerous albumen thereof comprise the receptor antagonist domain of an interleukin 1 at least, and this domain can specific binding interleukin 1 receptor family member and suppressed the activation of these acceptors that interleukins and family member thereof induce. Interleukin 1 receptor family comprises a plurality of members. Activator and antagonist that several different interleukins families correspondingly, are also just arranged. These interleukin 1 antagonists are not necessarily in conjunction with same interleukin 1 receptor family member. The IL-1 R antagonist of indication of the present invention be used for representing all can bind interleukin 1 receptor family member and/or in and the interleukin 1 antagonist of interleukin 1 family member activity.
IL-1 R antagonist congenerous albumen comprises an IL-1 R antagonist domain. This domain refers to can specific binding interleukin 1 receptor family member, thereby suppresses interleukin 1 and family member thereof to the activation of cell receptor. IL-1 R antagonist can comprise IL-1ra (U.S. Patent number 6,096,728), IL-1 HY1 or interleukins family member 5 (U.S. Patent number 6,541,623), IL-1Hy2 or interleukins family member 10 (U.S. Patent number 6,365,726), IL-1ra β (U.S. Patent number 6,399,573), the albumen of other interleukin 1 antagonist member or its congenerous, namely derive from the polypeptide protein of IL-1 R antagonist, the combination of one or more point mutation, insertion, disappearance, brachymemma or above several sudden changes is for example arranged. They have fully kept the specific binding interleukin 1 receptor and have suppressed the activity that interleukin 1 activates its surface of cell membrane acceptor. These albumen comprise certain fragment of sequence 1 or sequence 1. IL-1 R antagonist preferably derives from the glycosylated polypeptides of mammalian cell. The activity of IL-1 R antagonist can be undertaken in the interleukin 1 cell by the D10 cell (seeing example 3) that uses interleukin 1 to rely on and experiment and other interleukin 1 family member's neutralization tests to detect.
IL-1 R antagonist or its congenerous albumen is glycosylated protein preferably. Natural interleukin 1 receptor antagonist has the glycosylation site (U.S. Patent number 6096728) of two N-link. These two N-link glycosylation sites are for playing an important role activity in vivo, the especially biological half life of IL-1 R antagonist and its haemocyanin binding characteristic. The IL-1 R antagonist Kineret of Bacillus coli expression lacks posttranslational modification. The result has caused it to be combined with the human albumin significantly, shows as lower activity in vivo.
Use interleukin 1 dependent cells D10 cell to carry out in the cell and experiment, or can detect the inhibition active (seeing example 3) of IL-1 R antagonist and congenerous albumen among the other standards interleukin 1 family member with experiment. IL-1 R antagonist merges can be increased the molecular weight of albumen with any protein fusion and improve biological half-life in the body. IL-1 R antagonist is fused on other albumen by the Fc section (for example Fc section of IgG1) of immunoglobulin (Ig), can further increase molecular weight, because the Fc fragment of IgG1 has the dimeric ability of formation, its existence can double to improve albumen at the distribution proportion of a certain specific target site.
TNF
Tumor necrosis factor α and tumor necrosis factor β are the albumen of mammalian cell secretion, and various kinds of cell is had widely effect. These two kinds of cell factors have a lot of similarities at function and structure, so they are called TNF.
TNF brings into play its biological function by the film surface specific TNF acceptor in conjunction with the TNF-sensitive cells. The TNF acceptor has two kinds form at present, and namely molecular weight is the I receptor (TNFRI) of 55 kilodaltons and the II receptor (TNFRII) that molecular weight is 75 kilodaltons. TNFRI and TNFRII can both be in conjunction with TNF α and TNF β.
TNF is very definite in the effect of inflammation disease at present. TNFRII and human IgG1 Fc segment composition (trade name Enbrel) have been used for the treatment of disease such as rheumatoid arthritis and psoriasis that some comprises that TNF relies on. Soluble TNF RI (Onercept, Serono) has carried out clinical trial and has been used for the treatment of psoriasis.
Several TNF antagonisies have been identified.These antagonisies can be in conjunction with TNF as soluble TNF RII, and suppresses combining of TNF and TNF receptor.These albumen can be used for suppressing the biologic activity that caused by TNF.Can merge with the albumen of interleukin 1 receptor antagonist or its congenerous with albumen among the TNF.Be similar to interleukin 1, TNF also is a medium important in the inflammatory reaction.Comprise TNF and congenerous albumen thereof with proteic target protein among the above-mentioned TNF.These albumen all comprise among one or more TNF and domain, this domain can in and TNF, just suppress the TNF activity.Comprise the extracellular domain of people TNFRII, the extracellular domain of TNFRI or the variable region of anti-TNF antibodies with domain among the TNF.Such example comprises the chimeric antibody of conjugated protein 1 (rhTBP-1) of ectodomain, the TNF of TNFRII, TNFRI ectodomain, humanized anti-TNF antibodies and anti-TNF.
TNF α and interleukin 1 are two kinds of main media at inflammation disease position, the proteic fusion of TNF antagonist and interleukin 1 receptor antagonist or their congenerous or chimeric molecule both can be used for blocking TNF α plain 1 the path that also can blocking leukocyte be situated between, so more effectively treat acute and chronic inflammation related disease than the two independent use.Can use TNF dependent cells such as L979 cell (ATTC) to detect with the activity of molecule among the TNF in the fusion rotein.The TNF dependent cells can be killed by reorganization TNF α.Among the TNF and albumen can neutralize this TNF rely on active, in and protein active can use also that TNF is external to be detected in conjunction with experiment.
Use interleukin 1 receptor antagonist and TNF receptor 1 (not being receptor 2) once to be used for the treatment of the disease test that interleukin 1 and TNF α cause simultaneously.Yet, Immunex Inc in 2003 and Amgen Inc company are carrying out 242 cases of clinical experiment, the result who treats after 24 weeks shows, use simultaneously Enbrel and Kineret normal dose (Enbrel 25mg biweekly, Kineret 10mg once a day, mol ratio is approximately 1: 12) treatment, the result shows that the associating use can't increase curative effect, and using separately than Enbrel or Kineret has on the contrary increased infection and neutrophilic granulocytopenia odds.
Interleukin-18 and interleukin-4
The above-mentioned interleukin 1 receptor antagonist or the albumen of its congenerous can merge with the albumen of other anti-inflammatory, asthma, angiogenesis inhibitor.Example comprises: (i) in the interleukin-18 and molecule such as interleukin-18 conjugated protein (IL-18bp), interleukin-18 receptor (IL-18R) extracellular region, humanization anti-IL-8 18 antibody.(ii) in the interleukin-4 and molecule such as interleukin-4 receptor (IL-4R) extracellular region (trade name Nuvance, Immunex company), humanization anti-IL-8 4 antibody (Protein Design Labs).(iii) in anti-vascular endothelial cell growth factor antibody and the angiogenin and molecule (soluble T ie2 extracellular region).As discussing at that time, these proteic carbon teminals are introduced the interleukin 1 receptor antagonist: (1) increases their molecular weight (2) when the mammalian host cell expressing protein, increases the inflammation site that two glycosylation sites (3) targeting is shipped to these albumen the high expressed interleukin 1 receptor.(4) with molecular proportion 1: 1 the be situated between biological function of plain 18, interleukin-4, vascular endothelial cell growth factor or angiogenin and interleukin 1 molecule of blocking leukocyte simultaneously.
Recombination human interleukins-11 8 conjugated protein indication psoriasiss of carrying out clinical experiment treatment scytitis by Serono company.Proved the protein-bonded safety of interleukin-18.The interleukin 1 receptor antagonist merges can obviously increase its biological activity persistent period at its carbon teminal.Can increase the targeting in its inflammation site and significantly increase curative effect by fusion with the interleukin 1 receptor antagonist.Have cooperative effect with interleukin 1 receptor antagonist fusion rotein while blocking leukocyte Jie element 18 and interleukin 1, can be used for the treatment of inflammation disease such as psoriasis (Yudoh K etc., result of study in 2004).What is interesting is most that the receptor of interleukin-18 and interleukin 1 belongs to interleukin 1 receptor family together, and almost be to bring into play its biological action by same signal transduction pathway.Double blocking interleukin 1 and interleukin-18 almost can be blocked the caused inflammation process of whole interleukin 1 receptor family.Chimeric protein double blocking interleukin 1 of the present invention and interleukin-18 show as the most effective pharmaceutical formulation of treatment inflammation disease.
The protein-bonded congenerous albumen of interleukin-18 also can be used for the present invention.Conjugated protein or its congenerous albumen of interleukin-18 comprises in the interleukin-18 and domain, this domain can in and interleukin-18, just suppress the biological activity of interleukin-18.For example, in the interleukin-18 and protein structure domain can comprise human interleukin-18 receptor extracellular region (U.S. Patent number 6,589,764), interleukin-18 is conjugated protein, anti-IL-8 18 antibody or interleukin-18 sudden change antagonist albumen.
In the interleukin-18 in the chimeric protein of the present invention and protein active, can use interleukin-18 dependent cells strain KG-1 cell detection.For example, human interleukin-18 is induced KG-1 emiocytosis IFN-g in dose-dependent mode under the condition that TNF α exists.The IFN-g secretion that this interleukin-18 relies on can be suppressed with albumen in the interleukin-18 of effective dose.With proteic activity, also can test and detect in these interleukin-18s by interleukin-18/interleukin-18 receptors bind.
Soluble il-4 receptors or its congenerous albumen can merge with the interleukin 1 receptor antagonist.Interleukin-4 receptor or its congenerous albumen comprise in the interleukin-4 and domain, this domain can in and interleukin-4, just suppress the interleukin-4 activity.For example, comprise interleukin-4 receptor extracellular region with domain in the interleukin-4, anti-IL-8 4 antibody contain pair interleukin-4 mutain antagonist of sudden change R121D/Y124D sequences (Schnarr etc., 1997).What is interesting is that the interleukin-4 receptor subunits not only can bind interleukin 4, also can bind interleukin 13, this is because the receptor of interleukin-4 and interleukin-13 has the natural characteristics of common subunit.
Can detect with the dependent cells TF-1 of interleukin-4 with active in the interleukin-4 of fusion rotein of the present invention.For example, can suppress the TF-1 cell proliferation that interleukin-4 relies on albumen among the IL-4 of adding effective dose.Also can detecting in the interleukin-4 by interleukin-4/interleukin-4 receptor in conjunction with experiment with proteic activity.
VEGF and angiogenin
Said method can be applied to VEGF and angiogenin and their the proteic antagonist of congenerous.VEGF generates for new vessels important effect.Anti-vascular endothelial growth factor antibody (trade name: Avastin, Genentech Inc) can be used for the treatment of tumor disease.Equally, the extracellular region of vascular endothelial growth factor receptor and IgG1 Fc section merge, and have been used for treating new vessels with VEGF and have generated relevant indication.VEGF congenerous albumen comprises in the VEGF and domain, this domain can in and VEGF, just suppress the activity of VEGF.For example, the variable region that comprises human vascular endothelial growth factor receptor extracellular region and anti-vascular endothelial growth factor antibody in the VEGF with protein structure domain.
Can detect with the HUVEC cell that VEGF relies on proteic activity in the chimeric protein medium vessels endothelial cell growth factor (ECGF) that the present invention mentions.For example, human vascular endothelial growth factor can be induced the HUVEC cell proliferation.Can suppress the HUVEC cell proliferation that VEGF relies on albumen in the VEGF of effective dose.With proteic activity, also can detect in conjunction with testing in the VEGF by VEGF/vascular endothelial growth factor receptor.
There is research to point out that soluble vascular generates plain receptor Tie2 and can be used as angiogenesis inhibitor response preparation treatment tumor or the relevant rheumatoid arthritis of new vessels reaction of formation.Clinical observation finds that very early new vessels generates and inflammation is coexistence and complementary.The most general example is exactly in rheumatoid arthritis, and new vessels generates and is accompanied by the inflammation generation.Soluble vascular generates plain receptor Tie2 or its congenerous albumen comprises in the angiogenin and domain, this domain can in and angiogenin, just can suppress the function of angiogenin 1.For example, comprise the plain antibody of people Tie2 extracellular region and anti-Tie2 antibody or angiogenesis inhibitor with domain in the angiogenin.
Can detect with dependent cells HUVEC with proteic activity among the Tie-2 in the chimeric protein that the present invention mentions.For example, human angiogenin 1 can be induced the phosphorylation of HUVEC cell.The phosphorylation that this Tie-2 relies on can be suppressed with albumen among the Tie-2 of effective dose.Also can detect in conjunction with experiment with proteic activity among the Tie-2 with Tie-2/ angiogenin 2.
Known interleukin 1 is the important stimulus factor that pathologic vessels forms.In animal model, can suppress new vessels with interleukin 1 in interleukin 1 receptor antagonist or its congenerous albumen and form and tumor growth, the prompting inflammation may promote new vessels to form.For example, pernicious breast carcinoma is struvite breast carcinoma.Probably aspect treatment tumor or rheumatoid arthritis, the curative effect of interleukin 1 receptor antagonist and inhibition new vessels formation albumen (anti-vascular endothelial growth factor antibody, soluble VEGF-receptor extracellular region, soluble T ie2 extracellular region) fusion rotein will be significantly better than the preparation of the anti-new vessels formation of independent use.
Except above-mentioned treatment preparation, other treatment preparation that is suitable for merging mutually with interleukin 1 receptor antagonist or its congenerous albumen is listed below:
1.E25 (olizumab) .E25 is humanization anti-IgE antibodies (Novartis), is used for the treatment of allergic asthma, seasonal allergic rhinitis.
2.H5G1.1.5G1.1 be the anti-C5 antibody of a kind of humanization (Alexion Pharmaceuticals), can be applied to treating psoriasis and autoimmune disease.
3.TP10.TP10 be a kind of complement receptors (sCR1) of solubility, be used for the treatment of acute respiratory distress syndrome and organ transplantation (AVANT Immunotherapeutics).
4.ABX-IL8.ABX-IL8 be a kind of monoclonal antibody (Abgenix) of anti-IL-8 18, be used for the treatment of psoriasis.
5.CTLA4Ig.CTLA4Ig be a kind of soluble recepter (Bristol-Myers Squibb) of reorganization, be used for immunosuppressant.
In the fusion rotein of the above-mentioned factor and interleukin 1 receptor antagonist or the formation of its congenerous albumen, the function of two kinds of molecules has collaborative or complementary.Interleukin 1 receptor antagonist bind interleukin 1 receptor, the guiding fusion rotein is positioned the inflammation part of high expressed interleukin 1 receptor.It also can in and the interleukin 1 activity.The interleukin 1 receptor antagonist merges with above any albumen can be used for the treatment of the relevant disease of inflammation, asthma, angiogenesis-associated diseases or endothelial cell proliferation.
New vessels generates the pathological symptom that relevant disease is meant needs new vessels to generate or show as the new vessels reaction.Comprising, but be not limited only to cancer, solid tumor, neoplasm metastasis, benign tumor, comprise hemangioma, acoustic neuroma, neurofibroma, trachoma and purulent granuloma piece, rheumatoid arthritis, psoriasis, ocular disease such as diabetic retinopathy, retinopathy of prematurity, macular degeneration, the cornea transplant rejection, neovascular glaucoma, crystal fibre hypertrophy and rubescent, the Osler-Webbe syndrome, the cardiac muscle new vessels generates, the vascularization of speckle shape, telangiectasis, the hemophilia joint, fibrohemangioma and traumatic granulation hamartoplasia.Here, the endothelial cell proliferation reaction symptom include but not limited to adhesion intestinal, atherosclerosis, scleroderma and hypertrophic cicatrix.Fusion rotein described herein also can form by the required new vessels of inhibition embryo nidation and treat the above disease.
The fusion rotein that the present invention mentions preferably comprises a dimerization domain.The dimerization domain refers to can be with two coupled domains together of polypeptide.For example, it comprises IgG Fc section (people's IgG CH just).The example of such Fc section comprises sequence 2.IgG Fc section forms intrachain disulfide bond (covalent bond) by cysteine residues and forms dimer.Sometimes the non-covalent bond dimerization can form under the situation that does not have disulfide bond to participate in.The IgG Fc section of dimerization shows as the fusion rotein chimera, the nitrogen end is two functional TNFII receptors or soluble interleukin-4 receptor, interleukin-18 is conjugated protein or soluble Tie-2 molecule, and one of carbon tip is two functional interleukin 1 receptor antagonist molecules.This arrangement can improve the bonded chance of receptor/ligand in vivo with in more effectively and TNF alpha, interleukin-4, interleukin-18 or angiogenin and interleukin 1 receptor.
The covalency dimerization activity that forms by disulfide bond can detect by reduction and non-reduced sds polyacrylamide gel electrophoresis.Dimer protein molecular weight molecular weight under reducing condition can reduce half.The dimerization of non-covalent bond can detect by electrophoresis under the condition of non-degeneration and degeneration, and the molecular weight of dimer protein can reduce half under the degeneration condition in this case.
The polypeptide that the present invention mentions, among the TNF and domain, or in interleukin-4/interleukin-13 and domain, or in the interleukin-18 and domain, or in the VEGF and domain, or in the angiogenin and domain and interleukin 1 receptor antagonist domain can link together by certain method.Here, connect the conformation that is meant between each domain that merges the back polypeptide and can not influence each other, just keep activity separately.For example in the interleukin-4 and domain keeps in it and the activity of interleukin-4, interleukin 1 receptor antagonist domain keeps its specific bond interleukin 1 receptor, the be situated between activity of plain 1 activating cell receptor of blocking leukocyte.The dimerization domain keeps it to impel two fusion rotein of the present invention to condense together, and promptly two interleukin-4 receptor extracellular region territories are at the nitrogen end, and two interleukin 1 receptor antagonist molecules are in one of carbon tip.
The interleukin 1 receptor antagonist places carbon teminal and above-mentioned TNF and albumen, in the interleukin-18 and in the albumen, interleukin-4 and in the albumen, VEGF and in the albumen, angiogenin and albumen merge and form fusion rotein, have following characteristics: (1) improves molecular weight.When (2) using mammal, two glycosylation sites have additionally been increased on the interleukin 1 receptor antagonist molecules as host cell expression.(3) the molecular targeted inflammation site that is shipped to the high expressed interleukin 1 receptor of neutralization that will merge with the interleukin 1 receptor antagonist.(4) seal interleukin 1 and following arbitrary albumen simultaneously with 1: 1 ratio of molecular proportion: TNF, interleukin-18, interleukin-4, interleukin-13, IgE, VEGF, angiogenin.This double blocking can improve the curative effect of treatment inflammation disease, and can block the development process of inflammation disease more up hill and dale.For some disease, the coexistence of inflammation and asthma or angiogenesis reaction or interdepend plays an important role at disease progression, so double blocking interleukin-4/interleukin-13/VEGF/angiogenin and Bai Jie 1 can treat such disease more effective, more up hill and dale simultaneously.
The peptide material that the present invention mentions can obtain with synthetic or recombinant expressed method.Be the preparation recombinant polypeptide, the nucleotide sequence of this polypeptide of coding merged mutually with the nucleotide sequence of expression fused polypeptide that for example glutathione sulfydryl transferase, 6x-histidine epi-position label or M13 gene 3 proteic nucleotide sequences link together.The integrative nucleic acid sequence expressed fusion protein in appropriate host cell that obtains carries out purification and obtains recombination fusion protein.Many host-expression vector systems can be used for expressed fusion protein.Comprising but be not limited only to, transformed the antibacterial of recombinant phage dna, plasmid DNA, cosmid DNA expression vector; Transformed the yeast of recombinant yeast expression vector; Carry the human archeocyte system of recombinant virus or plasmid expression vector.Utilize traditional method to comprise preparation property chromatograph, immunology separation methods such as monoclonal or polyclonal antibody carry out separation and purification recombinant polypeptide or its fragment.The recombinant polypeptide that is obtained can further be handled, and for example removes the expression tag molecule with enzymic digestion, obtains the recombinant polypeptide of indication of the present invention.
Form and Therapeutic Method
The field of the invention also comprises the method for the treatment of excessive immunoreation disease, angiogenesis reacting phase related disorders by the fusion rotein of the present invention of giving the patient infusion effective dose.The suitable crowd of indication fusion protein formulations of the present invention comprised or the excessively progressive or patient that tire out of reaction that exempts from service unusually, the patient who for example self exempt from service, tumor disease is originated in the repulsion of exempting from service, anaphylactic disease, immunocyte.This method can be used separately or unite use with other medicines or Therapeutic Method.
Treatment is to show symptom, disease syndrome, secondary disease or the susceptible body constitution that disease is cured, alleviates, relaxes, prevents, improved to the patient infusion pharmaceutical formulation.Obtain the dosage of satisfied curative effect in patient's body that the effective dose of treatment is meant in medication.Satisfied curative effect can be objectively (to judge with some inspection or sign), also can be subjective (patient performance or sensation).The scope of treatment disease comprises active chronic inflammation, diabetes, arthritis (comprises rheumatoid arthritis, Rheumatoid Arthritis, osteoarthritis, psoriasis arthropathica), multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupus erythematosus, autoimmune thyroiditis, dermatitis (comprising hereditary allergic dermatitis and eczematoid dermatitis), psoriasis, Sj gren ' s syndrome, segmental enteritis, oral ulcer, iritis, conjunctivitis, keratoconjunctivitis, type i diabetes, inflammatory bowel, ulcerative colitis, asthma, allergic asthma, lupus erythematosus,cutaneous, scleroderma, vaginitis, drug eruption, leprosy is reacted repeatedly, ENL, the autoimmune uveitis, allergic encephalomyelitis, acute cerebral hemorrhage, sensorineural hearing loss, grow anemia, pure red-cell anemia, the constitutional thrombocytopenia, polychondritis, granuloma, chronic hepatitis, Stevens Johnson syndrome, sprue, lichen planus, Graves ' disease, optimum lymph granuloma, early stage biliary cirrhosis, uveitis, the interstitial lung cystic fibrosis, graft versus host disease; Transplant class and comprise allosome or allogene tissue transplantation such as bone marrow transplantation, liver transplantation or the transplanting of other organ or tissue.Allergic disease such as atopic allergy, acquired immune deficiency syndrome (AIDS), lymphocyte tumor such as leukemia, lymphoma, acute hepatitis, new vessels generate relevant disease such as rheumatoid arthritis and tumor, cardiovascular disease..
The patient of above-mentioned one or more symptoms can use pharmaceutical formulation treatment of the present invention.Diagnosing such patient to be provided by patient's sensation or professional medical personnel's judgement, can be subjective (sensation) or objective (result of test or check).
In the approach, the preparation that will contain fusion rotein of the present invention uses to the patient in the body.Usually, albumen is dissolved in pharmaceutical carrier (as normal saline) by injection in oral, venoclysis, subcutaneous implantation or injection, intramuscular injection, intrathecal injection, peritoneal injection, internal rectum injection, nasal injection, the sheath, intrauterine injection, intratracheal injection, filling stomach, pulmonary injection administration.
Select dosage according to route of administration, preparation characteristic, patient's symptom and indication, patient's build size, body weight, body surface area, age and sex and the medicine that other are being used, perhaps defer to doctor's judgement.Suitable dosage range is in the 0.01-100.0mg/ kg body weight.Consider the different pharmaceutical availability that different dosage forms constitutes and different administering modes causes, dosage is adjusted according to practical situation.For example, oral administration will be than intravenous dosage height.The change of dosage can be optimized according to the experience of standard.Especially for oral, utilize the suitable preparations carrier (for example poly microparticle or implantable device) of sealing, may improve the efficient of drug delivery.
The pharmaceutical formulation that the present invention also comprises is the fusion rotein that the present invention carries of available pharmaceutical carrier formulation and effective dose.This carrier formulation can be used for treating above-mentioned disease.Pharmaceutical carrier can comprise solvent, dispersant, coating, antibacterial, antifungal, isotonic buffer agent, absorbent slow release agent.Medicine of the present invention is made the treatment preparation of different dosage form by classic methods according to different way of administration.For example, can make capsule, gel, tablet and be used for oral administration.Capsule is made material and is comprised gelatin or the cellulose that standard is medicinal.The making material of tablet comprises that the medicinal solid of standard common is filled, lubricant, and the solid filler comprises starch, sugar, Bentonite.Preparation also can be with the duricrust tablet or contain lactose, mannitol, traditional filler commonly used and the capsule of film-making agent.Medicine is through the parenteral route administration.The example of this classpath comprises liquid drugs injection, normal saline transfusion, 5% glucose active ingredient, or other common pharmaceutical excipients.Other pharmaceutical solutions that cyclodextrin or we are familiar with can be used as excipient and makes medicine become the treatment preparation.
Pharmaceutical preparation of the present invention can be by detecting with external dual mode in the body.Referring to following example.Briefly, external survey principle alive is that can the detection protein formulation vitro inhibition immunne response.Surveying work in the body is by medicine being injected into (animal model just) in the animal body, detecting therapeutic effect.Based on this result, determine suitable dosage scope and route of administration.
Following example only is used for explanation, and is not limited to continue by any way use and exploitation.According to our description at this, the research worker in the field does not need further to explain in detail just can farthest use the method for the invention.The document of being quoted only limits to reference.
Our result also shows the interleukin 1 receptor antagonist fusion molecule of utilizing mammalian host cell to express, comprises glycosylated interleukin 1 receptor antagonist, and the more non-interleukin 1 receptor antagonist of its molecular weight fusion molecule increases to some extent.Their biological activity persistent period also increases to some extent, and injects required effective dose and required medication time number average reduces to some extent.Because it can targeting acts on the characteristics of inflammation morbidity site and low effective dose and low medicine frequency, interleukin 1 receptor antagonist fusion rotein uses with uniting of interleukin 1 receptor antagonist with non-interleukin 1 fusion rotein or corresponding treatment albumen and compares, and side effect is less.
The specific embodiment of the present invention:
Embodiment 1:
We have made up various expression vector, express the following albumen of coding:
A) TNFRII-Fc-IL-1ra (sequence 5), TNFRI-Fc-IL-1ra (sequence 8) and contrast TNFRII-Fc (sequence 4) or TNFRI-Fc (sequence 7);
B) Humira (D2E7)-IL-1ra (sequence 10 and 11), Remicade (cA2)-IL-1ra (sequence 13 and 14) and contrast dimerization Humira (sequence 9 and 11), and Remicade (cA2) (sequence 12 and 14);
C) IL-18bp (sequence 15), dimerization IL-18bp-Fc (sequence 16) and dimerization IL-18bp-Fc-IL-1ra (sequence 17);
D) solubility IL-4R extracellular region domain (sequence 19), IL-4R-Fc (sequence 19), and IL-4R-Fc-IL-1ra (sequence 21);
E) VEGFR1-Fc-IL-1ra and light chain (sequence 24,23), and anti-VEGF heavy chain-IL-1ra and light chain (sequence 25,23).
The recon of most of encoding proteins (serial number 4-25) has carried out determined dna sequence and has expressed at mammal cell line.Sequence 4-25 utilizes codon natural or that optimize, and synthetic or natural secretory signal sequence can express in the mammalian host cell of suspension culture in domestication.Detected the dimerization recombination expression product that contains antibody fragment by non-heating sds gel electrophoresis and Westernblot immunity marking method.
Use serum-free medium, the expression titre of TNFRII-Fc in 24 porocyte culture plates (sequence 4) and TNFRII-Fc-IL-1ra (sequence 5) can reach 50mg-100mg/L respectively.With the method for protein content in the direct coomassie brilliant blue staining method testing conditions culture fluid be presented at domestication can the CHO K1 cell of suspension culture in, the TNFRII-Fc-IL-1ra expression will be higher than TNFRII-Fc.This shows that also expressing the chimeric protein that contains the interleukin 1 receptor antagonist by mammalian cell can reach high level expression, is enough to reach the commodity production requirement.
Embodiment 2:
We have carried out amplification culture, purification TNFRII-Fc-IL-1ra, IL-4R-ECD-Fc-IL-1ra, IL-18bp-Fc-IL-1ra recombination expression product.Chinese hamster ovary celI system carries out serum-free suspension domestication, amplification culture in the 3L cell culture apparatus with CHO-CD4 culture medium (Irvine Scientific) and fed-batch medium commonly used.Fusion rotein TNFRII-Fc-IL-1ra (sequence 5), IL-4R-ECD-Fc-IL-1ra (sequence 20), the output of IL-18bp-Fc-IL-1ra (sequence 17) can reach the level of commodity production.These albumen can be used protein-A affinity chromatograph, ion exchange, hydrophobic chromatography and carry out purification (Fig. 2,3,4).After the albumen that purification obtains added formulation components, the SEC-HPLC liquid phase analysis was used in lyophilizing then.
Embodiment 3:
Utilize biological activity test to measure TNFRII-Fc-IL-1ra, IL-4R-Fc-IL-1ra, the activity of IL-18bp-Fc-IL-1ra and VEGFR1-Fc-IL-1ra.Use interleukin 1 dependent cells D10 and carry out in the interleukin 1 and mensuration, it is active that the D10 cell can be used for detecting following proteic sealing: IL-1ra (Kineret), TNFRII-Fc-IL-1ra, IL-4R-Fc-IL-1ra and IL-18bp-Fc-IL-1ra.They can suppress the propagation of the dependent cells D10 cell of recombination human interleukins-11.
Say that simply people IL-1 α can induce the D10 cell proliferation in dose-dependent mode.Interleukin 1 α induces 50% cell proliferation, this be exactly concentration be exactly EC50 (median effective dose).People IL-1 α generally induces the EC50 scope of D10 cell proliferation at 1-5pg/ml.After the interleukin 1 receptor antagonist of cell and effective dose was hatched altogether, the interleukin 1 receptor antagonist suppressed the inductive cell proliferation of IL-1 α by closing cell surface interleukin 1 receptor.This blocking effect is dose-dependent equally.When the amount of receptor antagonist is low, the receptor that it just can not the closing cell surface.So, the activity of interleukin 1 inducing cell propagation is recovered.It is exactly the median effective dose of receptor antagonist that receptor antagonist makes the concentration of 50% activity inhibited of interleukin 1.
The effect of recombiant protein (TNFRII-Fc-IL-1ra, IL-4R-Fc-IL1ra, IL-18bp-Fc-IL-1ra, or VEGFR1-Fc-IL-1ra) is similar among solvable TNFRII, IL-18, IL-4 or the VEGF and molecule and interleukin 1 receptor antagonist.The cytology measures the biological activity (Fig. 6,8,10,11) of having confirmed these chimeric proteins.
Measure among the TNF and aspect the molecular activity the cytology, L929 cell (mice connective tissue cell system derives from ATCC) is used to detect the activity of TNFRII blocking-up TNF α.Say that simply TNF α induces quick cell death in dose-dependent mode, the medium effective concentration of TNF α (inducing the TNF α concentration of 50% cell death) is lower than 50pg/ml.After the soluble TNF acceptor (sTNFR) of TNF alpha molecule and high concentration was hatched altogether, soluble recepter combined with TNF α, combined with cell surface receptor thereby suppress it.So just block the activity that TNF α causes cell death.This blocking effect also is dose-dependent.The sTNFR concentrated solution is diluted to the following activity of just no longer blocking TNF α of finite concentration, and TNF α continues inducing cell death.So just can determine the medium effective concentration (just blocking the concentration of 50%TNF alpha active) of sTNFR.
Add the L929 cell of some, 10% horse serum, DMEM culture medium (containing glutamine), 1ug/ml actinomycetes D in 96 well culture plates, every hole cumulative volume 150ul adds the human TNF alpha sample (each two multiple hole of every concentration) of serial dilution again.Control wells only contains cell and culture medium.Culture plate places 37 ℃ of incubators, 5%CO
2, cultivated one day under the saturated humidity condition.Every porocyte is fixed with 10% polyformaldehyde, dyes with 1% crystal violet dye liquor.The dyeing back adds 30% acetate dissolution.Measure every hole optical density value (OD), wavelength is located at 540 nanometers, and optical density value and total cellular score are in direct ratio.Draw the cell toxicant curve, the OD value is made as Y-axis, and TNF α concentration is made X-axis.
Serial dilution TNFRII-Fc (Enbrel) and TNFRII-Fc-IL-1ra sample (respectively do two multiple holes) add respectively in the cell plates of actinomycetes D of the human TNF alpha that contains fixed concentration, 10% horse serum, L-glutaminate and 1ug/ml.Hatch altogether for 37 ℃ and change over to after 1 hour in 96 orifice plates that another piece is added with the L929 cell in advance.People TNF final concentration is 500pg/ milliliter (cumulative volume 150ul/ hole).Culture plate places 37 ℃ of incubators, 5%CO
2Cultivated one day under the saturated humidity condition.Cell is fixed with 10% paraformaldehyde, the dyeing of 1% crystal violet dye liquor.The dyeing back adds 30% acetate dissolution.Measure every hole optical density value (OD), wavelength is located at 540 nanometers.Draw neutralization curve respectively, the OD value is made as Y-axis, and TNFRII-Fc and TNFRII-Fc-IL-1ra concentration are made X-axis.
The result shows that people TNF alpha induces the L929 cell death in dose-dependent mode.The background O.D that only contains cell and actinomycin D is 0.5, and people TNF-alpha dose effect curve reduces to 0.1 from 0.5.The O.D. value no longer descended after people TNF-alpha dosage was higher than 100pg/ml.The result shows that people TNF-alpha has reached saturated concentration.2 multiple holes are all done in all experiments.The CV% of each sample concentration is lower than 9%.The medium effective concentration of this condition servant TNF-alpha is 8pg/ml.
Use the L929 cell, find that TNFRII-Fc and TNFRII-Fc-IL-1ra can suppress the activity of people TNF-alpha in dose-dependent mode.The background of OD value was 0.1 (cell culture system contains TNF-alpha (500pg/ml) and actinomycin D).The TNFRII-Fc-IL-1ra that adds variable concentrations, the OD value also is increased to 0.5 from 0.1, shows whole neutralizations.The TNFRII-Fc of 50ng/ml and TNFRII-Fc-IL-1ra can be fully in and people TNF-alpha activity.Each dilution factor of sample is all done two multiple holes, and each dilution factor CV% is lower than 10%, is respectively 3-4ng/ml, 10ng/ml with the active medium effective concentration of people TNF-alpha among TNFRII-Fc and the TNFRII-Fc-IL-1ra.
Human interleukin 4 by using can be induced the TF-1 cell proliferation, can be used in the interleukin-4 in view of the above and experiment.Dilution variable concentrations human interleukin 4 by using adds the 96 porocyte culture plates that contain the TF-1 cell, 37 ℃, 5%CO
2Cultivated 3 days under the condition.Adding MTS in the culture medium continues to cultivate 5 hours.Use enzyme connection instrument to survey each hole OD value of culture plate, wavelength is located at 490 nanometers.Can draw the cell proliferation curve, the OD value is as Y-axis, and interleukin-4 concentration is as X-axis.In neutralization experiment, with the IL-4R-Fc and the IL-4R-Fc-IL-1ra of serial dilution and contain certain density interleukin-4 (2ng/ml) culture medium in 96 well culture plates, 37 ℃ of preincubates 1 hour.The TF-1 cell that adds equal number subsequently in every hole places 37 ℃, 5%CO
2Cultivated 3 days in the incubator.Add MTS, continue to cultivate after 5 hours, use enzyme connection instrument to survey each hole optical density value of culture plate, wavelength 490 nanometers.Draw cell growth inhibited curve, the OD value is as Y-axis, and IL-4R-Fc and IL-4R-Fc-IL-1ra concentration are as X-axis.In this experimental result (chart 7) bind interleukin 1 and result of experiment (Fig. 8), shown that IL-4R-Fc-IL-1ra has biological function, had among people IL-4 receptor and the IL-1 simultaneously and proteic activity.
Human interleukin-18 can dose-dependent mode be induced KG-1 emiocytosis IFN-g (cultivating system contains TNFa), carries out in the interleukin-18 and experiment with this principle.The median effective dose of human interleukin-18 (just be meant interleukin-18 induce KG-1 emiocytosis IFN-g to reach 50% dosage of maximum concentration) is usually at 20-40ng/ml.Before adding cell culture system, human interleukin-18 conjugated protein (IL-18bp) and human interleukin-18 preincubate, so interleukin-18 conjugated protein can in conjunction with, suppress the activity of interleukin-18.This depression effect is dose-dependent.The conjugated protein complete closed IFNg of interleukin-18 expresses 50% dosage of desired concn, is exactly its medium effective concentration.
The IL-18bp-Fc-IL-1ra and the contrast IL-18bp-Fc sample (each concentration is established two multiple holes) that add serial dilution in the Tissue Culture Plate are with the human interleukin-18 (R﹠amp of fixed concentration (50ng/ml); D System) preincubate, 37 ℃ act on 1 hour.The interleukin-18 of serial dilution (each concentration is established two multiple holes) is added in and is made as positive control in the culture plate.Each hole adds the KG-1 cell of same quantity, and (ATCC CCL246), and the TNFa (BioSource Inc.) of same amount, places 37 ℃, 5%CO
2Continue in the incubator to cultivate 24 hours.50ul is taken out in every hole in Tissue Culture Plate, transfers in the another one ELISA check-out console.ELISA detection kit (BioSource Inc.) according to people IFNg illustrates and detects.It is 450 nanometers that optical density value is measured initial wavelength.Draw interleukin-18 abduction delivering IFNg curve, the OD value is a Y-axis, and human interleukin-18 concentration is X-axis.The conjugated protein neutralization curve of interleukin-18 is a Y-axis with the OD value, and IL-18bp-Fc-IL-1ra or matched group IL-18bp-Fc concentration are X-axis.
Show in Fig. 9 with experimental result in the cell.In the bind interleukin 1 and result of experiment (Figure 10), proved that the expression of IL-18bp-Fc-IL-1ra fusion rotein success is successful.It has kept in the while and the activity of interleukin-18 and interleukin 1.
People VEGF (vascular endothelial cell growth factor) induces HUVE (people's endothelium umbilical vein cell proliferation) in dose-dependent mode, medium effective concentration, be exactly it induce the HUVE cell proliferation the highest desired concn 50%, be generally 2-6ng/ml.Will the soluble human vegf receptor 1 with the vascular endothelial cell growth factor preincubate after join in the cell plates, soluble human vegf receptor-1 is in conjunction with people VEGF and seal the activity of its inducing cell propagation, this blocking effect is dose-dependent equally.50% dosage of the required acceptor density of complete closed is exactly its medium effective concentration.Recombiant protein VEGFR1-Fc-IL-1ra is made up of soluble VEGF-receptor 1 and interleukin 1 receptor antagonist two parts.It can bring into play the dual function of soluble VEGFR 1 and interleukin 1 receptor antagonist.
Serial dilution VEGFR1-Fc-IL-1ra sample (respectively do two multiple holes) is respectively with the VEGF (BioSource) of 10ng/ml 37 ℃ of preincubates 1 hour in 96 porocyte culture plates.The people VEGF of serial dilution is made as matched group.The HUVE cell (Cambrex) of every hole adding equal number is cultivated in 96 orifice plates then.At 37 ℃, 5%CO
2Continue in the incubator to cultivate 96 hours.In last 4 hours, add MTS (Promega) to every hole.The wavelength of measuring optical density (OD value) is 490 nanometers.The VEGF that draws variable concentrations makes the OD value curve of cell proliferation.The neutralization curve of VEGF-R is done Y-axis with the OD value, and VEGFR1-Fc-IL-1ra concentration is made X-axis.
People VEGF stimulates the HUVE cell proliferation in dose-dependent mode.Medium effective concentration 3ng/ml.Concentration is after the VEGFR1-Fc-IL-1ra of the VEGF of 10ng/ml and serial dilution is hatched altogether, to add in the cell culture system, and the propagation of VEGF dependent cells is suppressed in dose-dependent mode.The medium effective concentration of VEGFR1-Fc-IL-1ra is 15ng/ml (Figure 12).In the bind interleukin 1 and result of experiment (Figure 11), proved that the VEGFR1-Fc-IL-1ra Expression of Fusion Protein is successful.It has the double activity of neutralize VEGF and IL-1.
Embodiment 4:
Use mouse asthmatic model to detect the IL-4R-Fc-IL-1ra curative effect.Use female BALB/c mouse (6-8 age in week) preparation model.Say that simply these mices were at the 0th day and the 14th day peritoneal injection 40ug OVA (making Emulsion with the emulsifying of 2.25mg aluminium hydroxide, cumulative volume 100ul).
Mice is divided into 8 hours and 48 hours two groups, 8 hours groups comprise 8 hours groups of normal saline contrast, OVA8 hour group, IL-4R-Fc/OVA8 hour group, IL-4R-Fc-IL-1ra/OVA8 hour group, and 48 hours components become 48 hours groups of reason saline control, OVA48 hour group, IL-4R-Fc/OVA48 hour group, IL-4R-Fc-IL-1ra/OVA48 hour group.
The 28th day, all organized equal administration 100ug OVA, were dissolved in the 0.05ml normal saline by intranasal administration, except the normal saline group.Normal saline group the 0th day and the 14th day intraperitoneal are given the normal saline (aluminium hydroxide) of normal amount, give the 0.05ml normal saline at 28 days by intranasal.
At 29 days, organize every group in 48 hours and all give 100ug OVA once more, be dissolved in the 0.05ml normal saline by intranasal administration, except the normal saline group.The normal saline group was given the 0.05ml normal saline at the 29th day by intranasal equally once more.
The drug treatment of IL-4R-Fc and IL-4R-Fc-IL-1ra
At the 28th day, IL-4R-Fc/OVA-8 hour group, IL-4R-Fc-IL-1ra/OVA-8 hour group, IL-4R-Fc/OVA-48 hour group and IL-4R-Fc-IL-1ra/OVA-48 hour group are with 200ug//day dosage administration respectively.OVA excites peritoneal injection administration in preceding 60 minutes.IL-4R-Fc/OVA-48 hour group and IL-4R-Fc-IL-1ra/OVA-48 hour group were at the 29th day dosage rechallenge with 200ug//day.Measure the cell number in the bronchoalveolar lavage fluid.
8 hours treated animals were at the 28th day, and after OVA of intranasal excited 8 hours, the execution mice was got bronchoalveolar lavage fluid and carries out Histological research.48 hours treated animals are put to death after 28 days and 29 days intranasal OVA excite 48 hours.The lung main bronchus place's ligation of a mice left side, the normal saline of using the 1ml normal concentration is by tracheal intubation lavation right lung.Calculate total (lymph) cell number with blood counting chamber.Sheet is got rid of in preparation, and (fisher Diagnostics, Pittsburgh PA) calculate various noble cells numbers respectively after the dyeing with leukostat.Utilize standard hemocyte calculating method, count macrophage, oxyphil cell, neutrophilic granulocyte, lymphocyte number respectively, should have 200 cells in the visual field of 400 times of amplifications at least.
Lung tissue
Taking out trachea and the upper and lower leaf portion of tissue of left lung uses Carnoy ' s fixative to fix 15 hours under 20 degrees celsius.Use paraffin embedding, section (5um).Every mice left side lung is got 10 airway tissue sections, assessment cellular infiltration and mucus degree of congestion at random.Cellular infiltration intensity around pulmonary vascular and air flue is marked according to sxemiquantitative.
The result
1.IL-4R-Fc-IL-1ra the pneumonia of chimeric protein treatment commitment.
After the mice of 8 hours groups of table 1 once excites through intranasal OVA, the noble cells quantity in the bronchoalveolar lavage fluid.The differential cell counts of the mice normal saline matched group, OVA, IL-4R-Fc/OVA-8hr of 8 hours groups and IL-4R-Fc-IL-1ra/OVA group (5 every group) following (Mean ± SEM).
| All cells quantity x10-3 | Neutrophilic granulocyte x10-3 | |
| 8 hours groups of normal saline matched group | 51±8 | 30±5 |
| OVA-8 |
220±16 | 172±17 |
| IL-4R-Fc/OVA-8 |
200±11 | 165±10 |
| IL-4R-Fc-IL-1ra/OVA-8 hour group | 86±7 | 60±8 |
2. use IL-4R-Fc-IL-1ra blocking-up pneumonia in late period.
After form 2 48 hours group excites through twice of intranasal OVA, the noble cells number in the bronchoalveolar lavage fluid.The noble cells number of the mice normal saline group of 48 hours groups, OVA, IL-4R-Fc/OVA, IL-4R-Fc-IL-1ra/OVA group (5 every group) (Mean ± SEM).
| All cells quantity x10-3 | Oxyphil cell x10-3 | |
| 8 hours groups of normal saline matched group | 44±8 | 3±2 |
| OVA-8 hour group | 180±12 | 52±7 |
| IL-4R-Fc/OVA-8 hour group | 102±10 | 20±5 |
| IL-4R-Fc-IL-1ra/OVA-8 hour group | 68±7 | 10±5 |
Lung tissue research:
The result is presented in OVA-8 hour group and the OVA-48 group experiment pulmonary vascular and airway tissue and observes serious cellular infiltration on every side.With IL-4R-Fc/OVA-48hr group and the comparison of IL-4R-Fc/OVA-8hr group, IL-4R-Fc-IL-1ra/OVA-8 hour treatment group and IL-4R-Fc-IL-1ra/OVA-48 hour treatment group can obviously reduce the cellular infiltration of pulmonary vascular and airway tissue.Result's demonstration, in this animal model in asthma, IL-4R-Fc-IL1ra therapeutic effect the best.
Embodiment 5
Mouse arthritis animal model (CIA) is investigated the therapeutic effect of IL-18bp-IgG1Fc-IL-1ra fusion rotein.Induce the CIA animal model according to the standard method modification (Banada etc., 2002) delivered recently, the DBA/1J mice intradermal injection cattle II collagen type in 8-10 age in week.At 0 day and 21 days, every mice intradermal injection 100ul contained 200ug cattle II collagen type and the incomplete Freund's adjuvant that contains 200ug deactivation Mycobacterium tuberculosis.Mice (5 every group) per 3 days injection for curing between 21 to 36 days once are divided into 3 groups, respectively the contrast of peritoneal injection PBS buffer, 3mg/kg IL-18bp-Fc, 3mg/kg IL-18bp-Fc-IL-1ra.Mice took off neck in 36 days and puts to death.Normal three DBA/1J control mice are condemned to death simultaneously.
Used the disease clinical event scoring of double-blind method evaluation and test CIA at 21 to 36 days per two days, occurring degree with every mice pawl of three grade point systems evaluation and test, marking standard: 0=natural joint, 1=light inflammation and rubescent, serious erythema of 2=and swelling influence whole claw, limitation of activity, 3=deformity joint, ankylosis, ankylosis, afunction.The arthritis occurring degree is calculated by the total points of four claws, and the best result of every animal is 12 minutes.
Got two forelimbs and the right hind of all mices at the 36th day, 10% formalin fixed is made tissue samples according to preceding method (Bendele etc., 2000) and is carried out histologic analysis.The Histological change at positions such as pawl, ankle joint, knee joint is marked with double-blind method by experienced personnel.According to preceding method (Bendele etc., 2000), press the 0-5 scoring, every mice is got the data at 5 positions, and what statistical data showed is the average mark and the whole scoring of inflammation, pannus, cartilage and bone injury.
The result
IL-18bp-Fc-IL-1ra clinical treatment joint tissue disease and joint tissue are learned research.
Each experimental group arthritis sickness rate is near 100%.Compare with the PBS blank, the disease clinical event appraisal result of the protein for treatment group mice of 21 to 36 days injections 3mg/kgIL-18bp-Fc, 3mg/kg IL-18bp-Fc-IL-1ra reduces.Demonstration is analysed in the joint tissue credit, can prevent joint injury with PBS group comparison 3mg/kg IL-18bp-Fc and 3mg/kgIL-18bp-Fc-IL-1ra.Contrast 3mg/kg IL-18bp-Fc and 3mg/kgIL-18bp-Fc-IL-1ra treatment group all have significant difference between scoring of disease clinical event and the histological score.The curative effect of IL-18bp-Fc-IL-1ra obviously is better than IL-18bp-Fc.
The disease clinical event scoring of table 3:IL-18bp-Fc-IL-1ra treatment CIA model mouse
0 day and 21 days DBA/1J injected in mice 200ug cattle II collagen types, incomplete Freund, 200ug tubercule bacillus.Each organizes the mice contrast of per 3 days difference peritoneal injection PBS buffer, the IL-18bp-Fe of 3mg/kg body weight and IL-18bp-Fc-IL-1ra of 3mg/kg body weight between 21 to 36 days.Identify the pathological change degree of every claw of CIA mice by per two days of two experienced research worker respectively with double-blind method, three grades of point systems are given a mark.After following result is initial injection cattle II collagen type, the disease clinical event score data of each experimental mice.((mean±SEM))
| Clinical disease disease condition in the time of 36 days | |
| PBS buffer matched group | 8.8±0.7 |
| IL-18bp-Fc | 6.5±0.6 |
| IL-18bp-Fc-IL-1ra | 3.8±0.5 |
Embodiment 6:
Mice contact hypersensitive test model (CHS) body build-in test IL-18bp-Fc-IL-1ra activity.
The effect experiment of the making of CHS model and interleukin-18 chimeric protein.
Laboratory animal adopts C57BL/6mice (8 weeks and 14 ages in week).Agents useful for same: dinitrofluorobenzene, acetone, azovan blue, Methanamide, bovine serum albumin, acetate, ionomycin, brefeldin A, lipopolysaccharide (Escherichiacoli026:B6) are all available from Sigma-Aldrich company.Acetone/olive oil (4/1) was existing before DNFB (dinitrofluorobenzene) used scrapes off mouse back skin with existing dilution, and the 0.5%DNFB solution of smearing 25 μ l is provided with untreated matched group simultaneously in the mouse back sensitized animal.After 5 days, use 0.2%DNFB (non-irritating dosage) solution of 10 μ l to smear in the mouse right ear both sides, left ear uses and smears with dosage solvent (no DNFB).Before exciting 5 days of disease model, use the thickness of kind of calliper ear every day.Swelling the degree ((T of ear
n-T
5) auris dextra)-(T
n-T
5) left ear)), T
nAnd T
5Mice ear thickness value before representing n days respectively and measuring each mice ear thickness value and excite 5 days.For guarantee in the test swelling of mice ear by DNFB cause inflammation rather than non-specific due to, each experiment is all set up a non-sensitization but is excited animal control groups.Every mice excited administration every day in preceding 60 minute since the 5th day, during respectively the IL-18bp-Fc of peritoneal injection 250 μ g or IL-18bp-Fc-IL-1ra come and interleukin-18 or/and interleukin 1.A control animals injecting normal saline.The first treatment of duration of exciting 7 days was a course of treatment once more.
The result
IL-18bp-Fc-IL-1ra treats contact allergy.
Make the contact allergy animal model, scrape hair, smear hapten DNFB (dinitrofluorobenzene) sensitized animal at mouse back.The contact allergy that DNFB excites mice is smeared by ear after 5 days.The ear swelling increase degree of contrast DNFB and solvent blank processing control animals is given a mark and is determined exciting of inflammation.In the model excitation phase is 5-7 days administration IL-18BP-Fc and IL-18bp-Fc-IL-1ra, can obviously reduce ear's swelling in the meantime.The curative effect of IL-18bp-Fc and IL-18bp-Fc-IL-1ra has significant difference (the results are shown in Table), this result shows that IL-18bp-Fc-IL-1ra has same or be rich in the characteristics of the targeting of IL1 receptor, guarantee double blocking IL-1 and IL-18, its Targeting Performance enough directly guarantees its high efficiency.The curative effect of IL-18bp-Fc-IL-1ra obviously is better than IL-18bp-Fc.
Form 4: the test of pesticide effectiveness of bringing out test I L-18BP in the process at contact anaphylaxis model
The 0th day DNFB sensitization C57BL/6 mice is applied to ear after 5 days and excites.Measure ear's swelling degree every day, excite group to represent than the increase of blank group swelling degree with DNFB.Use every day IL-18bp chimeric protein or blank reagent to handle mice.It below is the meansigma methods of every group of 5 mice data.
| Natural law | 5 | 6 | 7 |
| |
0±0 | 110±12 | 160±10 |
| IL-18bp- |
0±0 | 80±9 | 105±8 |
| II-18BP-Fc-IL- |
0±0 | 50±5 | 70±5 |
Embodiment 7
Say that simply recombination human interleukins-11 receptor extracellular region carries out purification after using expressing cho cell.TNFRII-Fc-IL-1ra, negative control TNFRII-Fc, positive control IL-1ra (Kineret), bag are by in 96 orifice plates, and concentration 1ug/ hole, every hole add the 100ul bag and be cushioned liquid (Sigma).The interleukin 1 receptor of purification (concentration 0.1ug/ hole) in the PBS buffer 37 ℃ the effect 45 minutes.With the anti-human interleukins-11 receptor of rabbit extracellular region antibody (R﹠amp; DSystems) detector ligand/receptors bind reaction.Add the goat anti-rabbit igg (Pierce) of horseradish peroxidase-labeled subsequently, the PBS-T buffer is washed plate, and adding TMB (Sigma, T8665) colour developing, EL800 microplate reader photometry density value, wavelength is 650 nanometers.The testing result mapping, the OD value is a vertical coordinate, extension rate is an abscissa.Figure 13 has shown that TNFRII-Fc-IL-1ra and IL-1ra (Kineret) can both bind interleukin 1 receptors, and TNFRII-Fc (Enbrel) can not.What is interesting is that the interleukin 1 receptor antagonist that mammal is expressed and the interleukin 1 receptor antagonist (Kineret) of escherichia coli expression compare, combine better with interleukin 1 receptor.And the interleukin 1 receptor antagonist that mammal is expressed comprises 2 glycosylated N-link glycosylation sites, so compare with the interleukin 1 receptor antagonist (Kineret) of escherichia coli expression, less in conjunction with serum albumin, also different at external binding characteristic.
Embodiment 8
125-I labelling TNFRII-Fc-IL-1ra, distribution situation in IL-4R-Fc-IL-1ra and the IL-18bp-Fc-IL-1ra test experience animal body, the albumen that merges with non-interleukin 1 receptor antagonist is in contrast.
With the Iodogen method with 125-I labelling TNFRII-Fc-IL-1ra, IL-4R-Fc-IL-1ra and IL-18bp-Fc-IL-1ra, then by the molecular sieve chromatography purification by chromatography (M Hui etc., 1989), the experiment of IL-1 receptors bind is a kind of method commonly used, detects with the mammal express recombinant interleukin 1 receptor extracellular domain fusion rotein that oneself prepares.Interleukin 1 receptor combines with the bonded and cold TNFRII-Fc-IL-1ra of the TNFRI-Fc-IL-1ra of 125-I labelling and carries out parallel comparison.The result shows that the TNFRII-Fc-IL-1ra of 125-I labelling has the function of bind interleukin 1 receptor.
Use 6nmol TPA to be dissolved in 200ul acetone and be applied in mice ear, the generation of inducing scytitis in 2-3 days.At the TNFRII-Fc-IL-1ra of scytitis mouse model injection 125-I labelling and the TNFRII-Fc (Enbrel) of 125-I labelling.Surprisingly, the result shows that the TNFRII-Fc-IL-1ra of 125-I labelling will be more than TNFRII-Fc in the distribution in inflammation site.This is very possible relevant with the combination of interleukin 1 receptor.
IL-4R-Fc-IL-1ra, the IL-18bp-Fc-IL-1ra of 125-I labelling and IL-4R-Fc, the IL-18bp-Fc of 125-I labelling are injected mouse skin inflammatory model animal.Obtained similar result.
The TNFRII-Fc-IL-1ra of form 5:125-I labelling and TNFRII-Fc injected after 4 hours, the distribution in inflammation and non-inflammation skin histology site.This distribution situation is represented (six animals) with the percentage ratio that every gram tissue contains injected dose.
| Treatment | Tissue | Injected dose percentage ratio (n=6) in every gram tissue |
| 125-I labelling TNFRII-Fc-IL-1ra | Inflammation skin | 3.8±0.2 |
| 125-I labelling TNFRII-Fc-IL-1ra | Normal skin | 1.5±0.1 |
| 125-I labelling TNFRII-Fc (Enbrel) | Inflammation skin | 2.8±0.2 |
| 125-I labelling TNFRII-Fc (Enbrel) | Normal skin | 1.4±0.2 |
The IL-4R-Fc-IL-1ra of form 6:125-I labelling and IL-4R-Fc injection are after 4 hours, in the distribution of inflammation and non-inflammation skin histology.Represent (six animals) with the injected dose percentage ratio in every gram tissue.
| Treatment | Tissue | Injected dose percentage ratio (n=6) in every gram tissue |
| 125-I labelling IL-4R-Fc-IL-1ra | Inflammation skin | 4.0±0.2 |
| The 125-I labelling | Normal skin | 1.6±0.1 |
| IL-4R-Fc-IL-1ra | ||
| 125-I labelling IL-4R-Fc | Inflammation skin | 1.4±0.3 |
| 125-I labelling IL-4R-Fc | Normal skin | 1.4±0.2 |
The IL-18bp-Fc-IL-1ra of form 7:125-I labelling and IL-18bp-Fc injection are after 4 hours, in the distribution of inflammation and non-inflammation tissue.Represent (six animals) with the injected dose percentage ratio in every gram tissue.
| Treatment | Tissue | Injected dose percentage ratio (n=6) in every gram tissue |
| 125-I labelling IL-18bp-Fc-IL-1ra | Inflammation skin | 3.9±0.3 |
| 125-I labelling IL-18bp-Fc-IL-1ra | Normal skin | 1.4±0.5 |
| 125-I labelling IL-18bp-Fc | Inflammation skin | 1.5±0.3 |
| 125-I labelling IL-18bp-Fc | Normal skin | 1.4±0.3 |
Embodiment 9
Use 2 macaques to measure the immunogenicity of IL-4R-Fc-IL-1ra.Subcutaneous injection 10mgIL-4R-Fc-IL-1ra injected for 8 weeks altogether weekly.Before the injection, serum sample is collected in back (the 1st and 56 day).Use the neutralization experiment detect whether exist in the blood serum sample can in and IL-4 and the bioactive neutrality antibody of IL-1 at chimeric protein.In order better to detect the neutralizing antibody of low concentration, serum further carries out affinity chromatograph with protein A and anti-IgM antibody.In the IgG of the undiluted serum sample of medicine injection macaque and purification and IgM, all do not detect can in and IL-4 and the bioactive antibody of IL-1 at chimeric protein.Results suggest, chimeric protein IL-4R-Fc-IL-1ra does not have immunogenicity to monkey and people.
Sequence table
The chimeric protein sequence
＜110〉Amprotein Corp., Biological Engineering Co., Ltd., Hayao Group
＜120〉chimeric protein
<130>2006010
<140>200580011525.4
<141>2004-04-08
<150>PCT/US2005/012194
<151>2004-10-12
<160>25
<210>1
<211>152
<212>PRT
＜213〉artificial sequence
<310>WO2006/043972 A1
<311>2006-04-27
<400>1
Arg Pro Ser Gly Arg Lys Ser Ser Lys Met Gln Ala Phe Arg Ile
1 5 10 15
Trp Asp Val Asn Gln Lys Thr Phe Tyr leu Arg Asn Asn Gln leu
20 25 30
Val Ala Gly Tyr leu Gln Gly Pro Asn Val Asn leu Lys Glu Lys
35 40 45
Ile Asp Val Val Pro Ile Glu Pro His Ala leu Phe leu Gly Ile
50 55 60
His Gly Gly Lys Met Cys leu Ser Cys Val Lys Ser Gly Asp Glu
65 70 75
Thr Arg leu Gln leu Glu Ala Val Asn Ile Thr Asp leu Ser Glu
80 85 90
Asn Arg Lys Gln Asp Lys Arg Phe Ala Phe Ile Arg Ser Asp Ser
95 100 105
Gly Pro Thr Thr Ser Phe Glu Ser Ala Ala Cys Pro Gly Trp Phe
110 115 120
leu Cys Thr Ala Met Glu Ala Asp Gln Pro Val Ser leu Thr Asn
125 130 135
Met Pro Asp Glu Gly Val Met Val Thr Lys Phe Tyr Phe Gln Glu
140 145 150
Asp Glu
<210>2
<211>232
<212>PRT
＜213〉artificial sequence
<310>WO2006/043972 A1
<311>2006-04-27
<400>2
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
5 10 15
Ala Pro Glu Lue Lue Gly Gly Pro Ser Val Phe Lue Phe Pro Pro
20 25 30
Lys Pro Lys Asp Thr Lue Met Ile Ser Arg Thr Pro Glu Val Thr
35 40 45
Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
50 55 60
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
65 70 75
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
80 85 90
Lue Thr Val Lue His Gln Asp Trp Lue Asn Gly Lys Glu Tyr Lys
95 100 105
Cys Lys Val Ser Asn Lys Ala Lue Pro Ala Pro Ile Glu Lys Thr
110 115 120
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
125 130 135
Lue Pro Pro Ser Arg Asp Glu Lue Thr Lys Asn Gln Val Ser Lue
140 145 150
Thr Cys Lue Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
155 160 165
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
170 175 180
Pro Val Lue Asp Ser Asp Gly Ser Phe Phe Lue Tyr Ser Lys Lue
185 190 195
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
200 205 210
Ser Val Met His Glu Ala Lue His Asn His Tyr Thr Gln Lys Ser
215 220 225
Lue Ser Lue Ser Pro Gly
230
<210>3
<211>235
<212>PRT
＜213〉artificial sequence
<310>WO2006/043972 A1
<311>2006-04-27
<400>3
Lue Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly
5 10 15
Ser Thr Cys Arg Lue Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met
20 25 30
Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys
35 40 45
Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr
50 55 60
Tyr Thr Gln Lue Trp Asn Trp Val Pro Glu Cys Lue Ser Cys Gly
65 70 75
Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg
80 85 90
Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala
95 100 105
Lue Ser Lys Gln Glu Gly Cys Arg Lue Cys Ala Pro Lue Arg Lys
110 115 120
Cys Arg Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser
125 130 135
Asp Val Val Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr
140 145 150
Thr Ser Ser Thr Asp Ile Cys Arg Pro His Gln Ile Cys Asn Val
155 160 165
Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala Val Cys Thr Ser
170 175 180
Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val His Lue Pro
185 190 195
Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr Pro Glu
200 205 210
Pro Ser Thr Ala Pro Ser Thr Ser Phe Lue Lue Pro Met Gly Pro
215 220 225
Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp
230 235
<210>4
<211>467
<212>PRT
＜213〉artificial sequence
<310>WO2006/043972 A1
<311>2006-04-27
<400>4
Lue Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly
5 10 15
Ser Thr Cys Arg Lue Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met
20 25 30
Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys
35 40 45
Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr
50 55 60
Tyr Thr Gln Lue Trp Asn Trp Val Pro Glu Cys Lue Ser Cys Gly
65 70 75
Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg
80 85 90
Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala
95 100 105
Lue Ser Lys Gln Glu Gly Cys Arg Lue Cys Ala Pro Lue Arg Lys
110 115 120
Cys Arg Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser
125 130 135
Asp Val Val Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr
140 145 150
Thr Ser Ser Thr Asp Ile Cys Arg Pro His Gln Ile Cys Asn Val
155 160 165
Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala Val Cys Thr Ser
170 175 180
Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val His Lue Pro
185 190 195
Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr Pro Glu
200 205 210
Pro Ser Thr Ala Pro Ser Thr Ser Phe Lue Lue Pro Met Gly Pro
215 220 225
Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp Glu Pro Lys Ser Cys
230 235 240
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lue Lue
245 250 255
Gly Gly Pro Ser Val Phe Lue Phe Pro Pro Lys Pro Lys Asp Thr
260 265 270
Lue Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
275 280 285
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
290 295 300
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
305 310 315
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Lue Thr Val Lue His
320 325 330
Gln Asp Trp Lue Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
335 340 345
Lys Ala Lue Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
350 355 360
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Lue Pro Pro Ser Arg
365 370 375
Asp Glu Lue Thr Lys Asn Gln Val Ser Lue Thr Cys Lue Val Lys
380 385 390
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
395 400 405
Gln Pro Glu Asn Ash Tyr Lys Thr Thr Pro Pro Val Lue Asp Ser
410 415 420
Asp Gly Ser Phe Phe Lue Tyr Ser Lys Lue Thr Val Asp Lys Ser
425 430 435
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
440 445 450
Ala Lue His Asn His Tyr Thr Gln Lys Ser Lue Ser Lue Ser Pro
455 460 465
Gly Lys
<210>5
<211>619
<212>PRT
＜213〉artificial sequence
<310>WO2006/043972 A1
<311>2006-04-27
<400>5
Lue Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly
5 10 15
Ser Thr Cys Arg Lue Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met
20 25 30
Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys
35 40 45
Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr
50 55 60
Tyr Thr Gln Lue Trp Asn Trp Val Pro Glu Cys Lue Ser Cys Gly
65 70 75
Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg
80 85 90
Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala
95 100 105
Lue Ser Lys Gln Glu Gly Cys Arg Lue Cys Ala Pro Lue Arg Lys
110 115 120
Cys Arg Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser
125 130 135
Asp Val Val Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr
140 145 150
Thr Ser Ser Thr Asp Ile Cys Arg Pro His Gln Ile Cys Asn Val
155 160 165
Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala Val Cys Thr Ser
170 175 180
Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val His Lue Pro
185 190 195
Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr Pro Glu
200 205 210
Pro Ser Thr Ala Pro Ser Thr Ser Phe Lue Lue Pro Met Gly Pro
215 220 225
Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp Glu Pro Lys Ser Cys
230 235 240
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lue Lue
245 250 255
Gly Gly Pro Ser Val Phe Lue Phe Pro Pro Lys Pro Lys Asp Thr
260 265 270
Lue Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
275 280 285
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
290 295 300
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
305 310 315
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Lue Thr Val Lue His
320 325 330
Gln Asp Trp Lue Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
335 340 345
Lys Ala Lue Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
350 355 360
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Lue Pro Pro Ser Arg
365 370 375
Asp Glu Lue Thr Lys Asn Gln Val Ser Lue Thr Cys Lue Val Lys
380 385 390
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
395 400 405
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Lue Asp Ser
410 415 420
Asp Gly Ser Phe Phe Lue Tyr Ser Lys Lue Thr Val Asp Lys Ser
425 430 435
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
440 445 450
Ala Lue His Asn His Tyr Thr Gln Lys Ser Lue Ser Lue Ser Pro
455 460 465
Gly Lys Arg Pro Ser Gly Arg Lys Ser Ser Lys Met Gln Ala Phe
470 475 480
Arg Ile Trp Asp Val Asn Gln Lys Thr Phe Tyr Lue Arg Asn Asn
485 490 495
Gln Lue Val Ala Gly Tyr Lue Gln Gly Pro Asn Val Asn Lue Lys
500 505 510
Glu Lys Ile Asp Val Val Pro Ile Glu Pro His Ala Lue Phe Lue
515 520 525
Gly Ile His Gly Gly Lys Met Cys Lue Ser Cys Val Lys Ser Gly
530 535 540
Asp Glu Thr Arg Lue Gln Lue Glu Ala Val Asn Ile Thr Asp Lue
545 550 555
Ser Glu Asn Arg Lys Gln Asp Lys Arg Phe Ala Phe Ile Arg Ser
560 565 570
Asp Ser Gly Pro Thr Thr Ser Phe Glu Ser Ala Ala Cys Pro Gly
575 580 585
Trp Phe Lue Cys Thr Ala Met Glu Ala Asp Gln Pro Val Ser Lue
590 595 600
Thr Asn Met Pro Asp Glu Gly Val Met Val Thr Lys Phe Tyr Phe
605 610 615
Gln Glu Asp Glu
<210>6
<211>249
<212>PRT
＜213〉artificial sequence
<310>WO2006/043972 A1
<311>2006-04-27
<400>6
Met Gly Lue Ser Thr Val Pro Asp Lue Lue Lue Pro Lue Val Lue
5 10 15
Lue Glu Lue Lue Val Gly Ile Tyr Pro Ser Gly Val Ile Gly Lue
20 25 30
Val Pro His Lue Gly Asp Arg Glu Lys Arg Asp Ser Val Cys Pro
35 40 45
Gln Gly Lys Tyr Ile His Pro Gln Asn Asn Ser Ile Cys Cys Thr
50 55 60
Lys Cys His Lys Gly Thr Tyr Lue Tyr Asn Asp Cys Pro Gly Pro
65 70 75
Gly Gln Asp Thr Asp Cys Arg Glu Cys Glu Ser Gly Ser Phe Thr
80 85 90
Ala Ser Glu Asn His Lue Arg His Cys Lue Ser Cys Ser Lys Cys
95 100 105
Arg Lys Glu Met Gly Gln Val Glu Ile Ser Ser Cys Thr Val Asp
110 115 120
Arg Asp Thr Val Cys Gly Cys Arg Lys Asn Gln Tyr Arg His Tyr
125 130 135
Trp Ser Glu Asn Lue Phe Gln Cys Phe Asn Cys Ser Lue Cys Lue
140 145 150
Asn Gly Thr Val His Lue Ser Cys Gln Glu Lys Gln Asn Thr Val
155 160 165
Cys Thr Cys His Ala Gly Phe Phe Lue Arg Glu Asn Glu Cys Val
170 175 180
Ser Cys Ser Asn Cys Lys Lys Ser Lue Glu Cys Thr Lys Lue Cys
185 190 195
Lue Pro Gln Ile Glu Asn
200
<210>7
<211>482
<212>PRT
＜213〉artificial sequence
<310>WO2006/043972 A1
<311>2006-04-27
<400>7
Met Gly Lue Ser Thr Val Pro Asp Lue Lue Lue Pro Lue Val Lue
5 10 15
Lue Glu Lue Lue Val Gly Ile Tyr Pro Ser Gly Val Ile Gly Lue
20 25 30
Val Pro His Lue Gly Asp Arg Glu Lys Arg Asp Ser Val Cys Pro
35 40 45
Gln Gly Lys Tyr Ile His Pro Gln Asn Asn Ser Ile Cys Cys Thr
50 55 60
Lys Cys His Lys Gly Thr Tyr Lue Tyr Asn Asp Cys Pro Gly Pro
65 70 75
Gly Gln Asp Thr Asp Cys Arg Glu Cys Glu Ser Gly Ser Phe Thr
80 85 90
Ala Ser Glu Asn His Lue Arg His Cys Lue Ser Cys Ser Lys Cys
95 100 105
Arg Lys Glu Met Gly Gln Val Glu Ile Ser Ser Cys Thr Val Asp
110 115 120
Arg Asp Thr Val Cys Gly Cys Arg Lys Asn Gln Tyr Arg His Tyr
125 130 135
Trp Ser Glu Asn Lue Phe Gln Cys Phe Asn Cys Ser Lue Cys Lue
140 145 150
Asn Gly Thr Val His Lue Ser Cys Gln Glu Lys Gln Asn Thr Val
155 160 165
Cys Thr Cys His Ala Gly Phe Phe Lue Arg Glu Asn Glu Cys Val
170 175 180
Ser Cys Ser Asn Cys Lys Lys Ser Lue Glu Cys Thr Lys Lue Cys
185 190 195
Lue Pro Gln Ile Glu Asn Glu Pro Lys Ser Cys Asp Lys Thr His
200 205 210
Thr Cys Pro Pro Cys Pro Ala Pro Glu Lue Lue Gly Gly Pro Ser
215 220 225
Val Phe Lue Phe Pro Pro Lys Pro Lys Asp Thr Lue Met Ile Ser
230 235 240
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
245 250 255
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
260 265 270
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
275 280 285
Tyr Arg Val Val Ser Val Lue Thr Val Lue His Gln Asp Trp Lue
290 295 300
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Lue Pro
305 310 315
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
320 325 330
Glu Pro Gln Val Tyr Thr Lue Pro Pro Ser Arg Asp Glu Lue Thr
335 340 345
Lys Asn Gln Val Ser Lue Thr Cys Lue Val Lys Gly Phe Tyr Pro
350 355 360
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
365 370 375
Asn Tyr Lys Thr Thr Pro Pro Val Lue Asp Ser Asp Gly Ser Phe
380 385 390
Phe Lue Tyr Ser Lys Lue Thr Val Asp Lys Ser Arg Trp Gln Gln
395 400 405
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Lue His Asn
410 415 420
His Tyr Thr Gln Lys Ser Lue Ser Lue Ser Pro Gly Lys
425 430
<210>8
<211>634
<212>PRT
＜213〉artificial sequence
<310>WO2006/043972 A1
<311>2006-04-27
<400>8
Met Gly Lue Ser Thr Val Pro Asp Lue Lue Lue Pro Lue Val Lue
5 10 15
Lue Glu Lue Lue Val Gly Ile Tyr Pro Ser Gly Val Ile Gly Lue
20 25 30
Val Pro His Lue Gly Asp Arg Glu Lys Arg Asp Ser Val Cys Pro
35 40 45
Gln Gly Lys Tyr Ile His Pro Gln Asn Asn Ser Ile Cys Cys Thr
50 55 60
Lys Cys His Lys Gly Thr Tyr Lue Tyr Asn Asp Cys Pro Gly Pro
65 70 75
Gly Gln Asp Thr Asp Cys Arg Glu Cys Glu Ser Gly Ser Phe Thr
80 85 90
Ala Ser Glu Asn His Lue Arg His Cys Lue Ser Cys Ser Lys Cys
95 100 105
Arg Lys Glu Met Gly Gln Val Glu Ile Ser Ser Cys Thr Val Asp
110 115 120
Arg Asp Thr Val Cys Gly Cys Arg Lys Asn Gln Tyr Arg His Tyr
125 130 135
Trp Ser Glu Asn Lue Phe Gln Cys Phe Asn Cys Ser Lue Cys Lue
140 145 150
Asn Gly Thr Val His Lue Ser Cys Gln Glu Lys Gln Asn Thr Val
155 160 165
Cys Thr Cys His Ala Gly Phe Phe Lue Arg Glu Asn Glu Cys Val
170 175 180
Ser Cys Ser Asn Cys Lys Lys Ser Lue Glu Cys Thr Lys Lue Cys
185 190 195
Lue Pro Gln Ile Glu Asn Glu Pro Lys Ser Cys Asp Lys Thr His
200 205 210
Thr Cys Pro Pro Cys Pro Ala Pro Glu Lue Lue Gly Gly Pro Ser
215 220 225
Val Phe Lue Phe Pro Pro Lys Pro Lys Asp Thr Lue Met Ile Ser
230 235 240
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
245 250 255
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
260 265 270
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
275 280 285
Tyr Arg Val Val Ser Val Lue Thr Val Lue His Gln Asp Trp Lue
290 295 300
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Lue Pro
305 310 315
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
320 325 330
Glu Pro Gln Val Tyr Thr Lue Pro Pro Ser Arg Asp Glu Lue Thr
335 340 345
Lys Asn Gln Val Ser Lue Thr Cys Lue Val Lys Gly Phe Tyr Pro
350 355 360
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
365 370 375
Asn Tyr Lys Thr Thr Pro Pro Val Lue Asp Ser Asp Gly Ser Phe
380 385 390
Phe Lue Tyr Ser Lys Lue Thr Val Asp Lys Ser Arg Trp Gln Gln
395 400 405
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Lue His Asn
410 415 420
His Tyr Thr Gln Lys Ser Lue Ser Lue Ser Pro Gly Lys Arg Pro
425 430 435
Ser Gly Arg Lys Ser Ser Lys Met Gln Ala Phe Arg Ile Trp Asp
440 445 450
Val Asn Gln Lys Thr Phe Tyr Lue Arg Asn Asn Gln Lue Val Ala
455 460 465
Gly Tyr Lue Gln Gly Pro Asn Val Asn Lue Lys Glu Lys Ile Asp
470 475 480
Val Val Pro Ile Glu Pro His Ala Lue Phe Lue Gly Ile His Gly
485 490 495
Gly Lys Met Cys Lue Ser Cys Val Lys Ser Gly Asp Glu Thr Arg
500 505 510
Lue Gln Lue Glu Ala Val Asn Ile Thr Asp Lue Ser Glu Asn Arg
515 520 525
Lys Gln Asp Lys Arg Phe Ala Phe Ile Arg Ser Asp Ser Gly Pro
530 535 540
Thr Thr Ser Phe Glu Ser Ala Ala Cys Pro Gly Trp Phe Lue Cys
545 550 555
Thr Ala Met Glu Ala Asp Gln Pro Val Ser Lue Thr Asn Met Pro
560 565 570
Asp Glu Gly Val Met Val Thr Lys Phe Tyr Phe Gln Glu Asp Glu
575 580 585
<210>9
<211>430
<212>PRT
＜213〉artificial sequence
<310>WO2006/043972 A1
<311>2006-04-27
<400>9
Glu Val Gln Lue Val Glu Ser Gly Gly Gly Lue Val Gln Pro Gly
5 10 15
Arg Ser Lue Arg Lue Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp
20 25 30
Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Lue
35 40 45
Glu Trp Val Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr
50 55 60
Ala Asp Ser Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
65 70 75
Lys Asn Ser Lue Tyr Lue Gln Met Asn Ser Lue Arg Ala Glu Asp
80 85 90
Thr Ala Val Tyr Tyr Cys Ala Lys Val Ser Ala Ser Thr Lys Gly
95 100 105
Pro Ser Val Phe Pro Lue Ala Pro Ser Ser Lys Ser Thr Ser Gly
110 115 120
Gly Thr Ala Ala Lue Gly Cys Lue Val Lys Asp Tyr Phe Pro Glu
125 130 135
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Lue Thr Ser Gly Val
140 145 150
His Thr Phe Pro Ala Val Lue Gln Ser Ser Gly Lue Tyr Ser Lue
155 160 165
Ser Ser Val Val Thr Val Pro Ser Ser Ser Lue Gly Thr Gln Thr
170 175 180
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
185 190 195
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
200 205 210
Pro Cys Pro Ala Pro Glu Lue Lue Gly Gly Pro Ser Val Phe Lue
215 220 225
Phe Pro Pro Lys Pro Lys Asp Thr Lue Met Ile Ser Arg Thr Pro
230 235 240
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
245 250 255
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
260 265 270
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
275 280 285
Val Ser Val Lue Thr Val Lue His Gln Asp Trp Lue Asn Gly Lys
290 295 300
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Lue Pro Ala Pro Ile
305 310 315
Glu Lyr Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
320 325 330
Val Tyr Thr Lue Pro Pro Ser Arg Asp Glu Lue Thr Lys Asn Gln
335 340 345
Val Ser Lue Thr Cys Lue Val Lys Gly Phe Tyr Pro Ser Asp Ile
350 355 360
Ala Val Glu Trp Glu Ser Asn Gly Glr Pro Glu Asn Asn Tyr Lys
365 370 375
Thr Thr Pro Pro Val Lue Asp Ser Asp Gly Ser Phe Phe Lue Tyr
380 385 390
Ser Lys Lue Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
395 400 405
Phe Ser Cys Ser Val Met His Glu Ala Lue His Asn His Tyr Thr
410 415 420
Gln Lys Ser Lue Ser Lue Ser Pro Gly Lys
425 430
<210>10
<211>582
<212>PRT
＜213〉artificial sequence
<310>WO2006/043972 A1
<311>2006-04-27
<400>10
Glu Val Gln Lue Val Glu Ser Gly Gly Gly Lue Val Gln Pro Gly
5 10 15
Arg Ser Lue Arg Lue Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp
20 25 30
Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Lue
35 40 45
Glu Trp Val Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr
50 55 60
Ala Asp Ser Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
65 70 75
Lys Asn Ser Lue Tyr Lue Gln Met Asn Ser Lue Arg Ala Glu Asp
80 85 90
Thr Ala Val Tyr Tyr Cys Ala Lys Val Ser Ala Ser Thr Lys Gly
95 100 105
Pro Ser Val Phe Pro Lue Ala Pro Ser Ser Lys Ser Thr Ser Gly
110 115 120
Gly Thr Ala Ala Lue Gly Cys Lue Val Lys Asp Tyr Phe Pro Glu
125 130 135
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Lue Thr Ser Gly Val
140 145 150
His Thr Phe Pro Ala Val Lue Gln Ser Ser Gly Lue Tyr Ser Lue
155 160 165
Ser Ser Val Val Thr Val Pro Ser Ser Ser Lue Gly Thr Gln Thr
170 175 180
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
185 190 195
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
200 205 210
Pro Cys Pro Ala Pro Glu Lue Lue Gly Gly Pro Ser Val Phe Lue
215 220 225
Phe Pro Pro Lys Pro Lys Asp Thr Lue Met Ile Ser Arg Thr Pro
230 235 240
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
245 250 255
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
260 265 270
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
275 280 285
Val Ser Val Lue Thr Val Lue His Gln Asp Trp Lue Asn Gly Lys
290 295 300
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Lue Pro Ala Pro Ile
305 310 315
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
320 325 330
Val Tyr Thr Lue Pro Pro Ser Arg Asp Glu Lue Thr Lys Asn Gln
335 340 345
Val Ser Lue Thr Cys Lue Val Lys Gly Phe Tyr Pro Ser Asp Ile
350 355 360
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
365 370 375
Thr Thr Pro Pro Val Lue Asp Ser Asp Gly Ser Phe Phe Lue Tyr
380 385 390
Ser Lys Lue Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
395 400 405
Phe Ser Cys Ser Val Met His Glu Ala Lue His Asn His Tyr Thr
410 415 420
Gln Lys Ser Lue Ser Lue Ser Pro Gly Lys Arg Pro Ser Gly Arg
425 430 435
Lys Ser Ser Lys Met Gln Ala Phe Arg Ile Trp Asp Val Asn Gln
440 445 450
Lys Thr Phe Tyr Lue Arg Asn Asn Gln Lue Val Ala Gly Tyr Lue
455 460 465
Gln Gly Pro Asn Val Asn Lue Lys Glu Lys Ile Asp Val Val Pro
470 475 480
Ile Glu Pro His Ala Lue Phe Lue Gly Ile His Gly Gly Lys Met
485 490 495
Cys Lue Ser Cys Val Lys Ser Gly Asp Glu Thr Arg Lue Gln Lue
500 505 510
Glu Ala Val Asn Ile Thr Asp Lue Ser Glu Asn Arg Lys Gln Asp
515 520 525
Lys Arg Phe Ala Phe Ile Arg Ser Asp Ser Gly Pro Thr Thr Ser
530 535 540
Phe Glu Ser Ala Ala Cys Pro Gly Trp Phe Lue Cys Thr Ala Met
545 550 555
Glu Ala Asp Gln Pro Val Ser Lue Thr Asn Met Pro Asp Glu Gly
560 565 570
Val Met Val Thr Lys Phe Tyr Phe Gln Glu Asp Glu
575 580
<210>11
<211>214
<212>PRT
＜213〉artificial sequence
<310>WO2006/043972 A1
<311>2006-04-27
<400>11
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Lue Ser Ala Ser Val
5 10 15
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg
20 25 30
Asn Tyr Lue Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
35 40 45
Lue Lue Ile Tyr Ala Ala Ser Thr Lue Gln Ser Gly Val Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Lue Thr Ile
65 70 75
Ser Ser Lue Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg
80 85 90
Tyr Asn Arg Ala Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu
95 100 105
Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
110 115 120
Ser Asp Glu Gln Lue Lys Ser Gly Thr Ala Ser Val Val Cys Lue
125 130 135
Lue Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val
140 145 150
Asp Asn Ala Lue Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
155 160 165
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Lue Ser Ser Thr Lue Thr
170 175 180
Lue Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu
185 190 195
Val Thr His Gln Gly Lue Ser Ser Pro Val Thr Lys Ser Phe Asn
200 205 210
Arg Gly Glu Cys
<210>12
<211>449
<212>PRT
＜213〉artificial sequence
<310>WO2006/043972 A1
<311>2006-04-27
<400>12
Glu Val Lys Lue Glu Glu Ser Gly Gly Gly Lue Val Gln Pro Gly
5 10 15
Gly Ser Met Lys Lue Ser Cys Val Ala Ser Gly Phe Ile Phe Ser
20 25 30
Asn His Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Lue
35 40 45
Glu Trp Val Ala Glu Ile Arg Ser Lys Ser Ile Ash Ser Ala Thr
50 55 60
His Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
65 70 75
Asp Ser Lys Ser Ala Val Tyr Lue Gln Met Thr Asp Lue Arg Thr
80 85 90
Glu Asp Thr Gly Val Tyr Tyr Cys Ser Arg Asn Tyr Tyr Gly Ser
95 100 105
Thr Tyr Asp Tyr Trp Gly Gln Gly Thr Thr Lue Thr Val Ser Ala
110 115 120
Ser Thr Lys Gly Pro Ser Val Phe Pro Lue Ala Pro Ser Ser Lys
125 130 135
Ser Thr Ser Gly Gly Thr Ala Ala Lue Gly Cys Lue Val Lys Asp
140 145 150
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Lue
155 160 165
Thr Ser Gly Val His Thr Phe Pro Ala Val Lue Gln Ser Ser Gly
170 175 180
Lue Tyr Ser Lue Ser Ser Val Val Thr Val Pro Ser Ser Ser Lue
185 190 195
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
200 205 210
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
215 220 225
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lue Lue Gly Gly Pro
230 235 240
Ser Val Phe Lue Phe Pro Pro Lys Pro Lys Asp Thr Lue Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
260 265 270
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
275 280 285
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
290 295 300
Thr Tyr Arg Val Val Ser Val Lue Thr Val Lue His Gln Asp Trp
305 310 315
Lue Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Lue
320 325 330
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
335 340 345
Arg Glu Pro Gln Val Tyr Thr Lue Pro Pro Ser Arg Asp Glu Lue
350 355 360
Thr Lys Asn Gln Val Ser Lue Thr Cys Lue Val Lys Gly Phe Tyr
365 370 375
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
380 385 390
Asn Asn Tyr Lys Thr Thr Pro Pro Val Lue Asp Ser Asp Gly Ser
395 400 405
Phe Phe Lue Tyr Ser Lys Lue Thr Val Asp Lys Ser Arg Trp Gln
410 415 420
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Lue His
425 430 435
Asn His Tyr Thr Gln Lys Ser Lue Ser Lue Ser Pro Gly Lys
440 445
<210>13
<211>601
<212>PRT
＜213〉artificial sequence
<310>WO2006/043972 A1
<311>2006-04-27
<400>13
Glu Val Lys Lue Glu Glu Ser Gly Gly Gly Lue Val Gln Pro Gly
5 10 15
Gly Ser Met Lys Lue Ser Cys Val Ala Ser Gly Phe Ile Phe Ser
20 25 30
Asn His Trp Met Asn Trp Val Arg Gln Ser Pro Glu Lys Gly Lue
35 40 45
Glu Trp Val Ala Glu Ile Arg Ser Lys Ser Ile Asn Ser Ala Thr
50 55 60
His Tyr Ala Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
65 70 75
Asp Ser Lys Ser Ala Val Tyr Lue Gln Met Thr Asp Lue Arg Thr
80 85 90
Glu Asp Thr Gly Val Tyr Tyr Cys Ser Arg Asn Tyr Tyr Gly Ser
95 100 105
Thr Tyr Asp Tyr Trp Gly Gln Gly Thr Thr Lue Thr Val Ser Ala
110 115 120
Ser Thr Lys Gly Pro Ser Val Phe Pro Lue Ala Pro Ser Ser Lys
125 130 135
Ser Thr Ser Gly Gly Thr Ala Ala Lue Gly Cys Lue Val Lys Asp
140 145 150
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Lue
155 160 165
Thr Ser Gly Val His Thr Phe Pro Ala Val Lue Gln Ser Ser Gly
170 175 180
Lue Tyr Ser Lue Ser Ser Val Val Thr Val Pro Ser Ser Ser Lue
185 190 195
Gly Thr Gln Thr Tyr Ile Cys Asn Val Ash Hi s Lys Pro Ser Asn
200 205 210
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
215 220 225
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lue Lue Gly Gly Pro
230 235 240
Ser Val Phe Lue Phe Pro Pro Lys Pro Lys Asp Thr Lue Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
260 265 270
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
275 280 285
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
290 295 300
Thr Tyr Arg Val Val Ser Val Lue Thr Val Lue His Gln Asp Trp
305 310 315
Lue Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys ALa Lue
320 325 330
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
335 340 345
Arg Glu Pro Gln Val Tyr Thr Lue Pro Pro Ser Arg Asp Glu Lue
350 355 360
Thr Lys Asn Gln Val Ser Lue Thr Cys Lue Val Lys Gly Phe Tyr
365 370 375
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
380 385 390
Asn Asn Tyr Lys Thr Thr Pro Pro Val Lue Asp Ser Asp Gly Ser
395 400 405
Phe Phe Lue Tyr Ser Lys Lue Thr Val Asp Lys Ser Arg Trp Gln
410 415 420
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Lue His
425 430 435
Asn His Tyr Thr Gln Lys Ser Lue Ser Lue Ser Pro Gly Lys Arg
440 445 450
Pro Ser Gly Arg Lys Ser Ser Lys Met Gln Ala Phe Arg Ile Trp
455 460 465
Asp Val Asn Gln Lys Thr Phe Tyr Lue Arg Asn Asn Gln Lue Val
470 475 480
Ala Gly Tyr Lue Gln Gly Pro Asn Val Asn Lue Lys Glu Lys Ile
485 490 495
Asp Val Val Pro Ile Glu Pro His Ala Lue Phe Lue Gly Ile His
500 505 510
Gly Gly Lys Met Cys Lue Ser Cys Val Lys Ser Gly Asp Glu Thr
515 520 525
Arg Lue Gln Lue Glu Ala Val Asn Ile Thr Asp Lue Ser Glu Asn
530 535 540
Arg Lys Gln Asp Lys Arg Phe Ala Phe Ile Arg Ser Asp Ser Gly
545 550 555
Pro Thr Thr Ser Phe Glu Ser Ala Ala Cys Pro Gly Trp Phe Lue
560 565 570
Cys Thr Ala Met Glu Ala Asp Gln Pro Val Ser Lue Thr Asn Met
575 580 585
Pro Asp Glu Gly Val Met Val Thr Lys Phe Tyr Phe Gln Glu Asp
590 595 600
Glu
<210>14
<211>214
<212>PRT
＜213〉artificial sequence
<310>WO2006/043972 A1
<311>2006-04-27
<400>14
Asp Ile Lue Lue Thr Gln Ser Pro Ala Ile Lue Ser Val Ser Pro
5 10 15
Gly Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Phe Val Gly
20 25 30
Ser Ser Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg
35 40 45
Lue Lue Ile Lys Tyr Ala Ser Glu Ser Met Ser Gly Ile Pro Ser
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Lue Ser Ile
65 70 75
Asn Thr Val Glu Ser Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln
80 85 90
Ser His Ser Trp Pro Phe Thr Phe Gly Ser Gly Thr Asn Lue Glu
95 100 105
Val Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
110 115 120
Ser Asp Glu Gln Lue Lys Ser Gly Thr Ala Ser Val Val Cys Lue
125 130 135
Lue Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val
140 145 150
Asp Asn Ala Lue Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
155 160 165
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Lue Ser Ser Thr Lue Thr
170 175 180
Lue Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu
185 190 195
Val Thr His Gln Gly Lue Ser Ser Pro Val Thr Lys Ser Phe Asn
200 205 210
Arg Gly Glu Cys
<210>15
<211>192
<212>PRT
＜213〉artificial sequence
<310>WO2006/043972 A1
<311>2006-04-27
<400>15
Met Arg His Asn Trp Thr Pro Asp Lue Ser Pro Lue Trp Val Lue
5 10 15
Lue Lue Cys Ala His Val Val Thr Lue Lue Val Arg Ala Thr Pro
20 25 30
Val Ser Gln Thr Thr Thr Ala Ala Thr Ala Ser Val Arg Ser Thr
35 40 45
Lys Asp Pro Cys Pro Ser Gln Pro Pro Val Phe Pro Ala Ala Lys
50 55 60
Gln Cys Pro Ala Lue Glu Val Thr Trp Pro Glu Val Glu Val Pro
65 70 75
Lue Asn Gly Thr Lue Ser Lue Ser Cys Val Ala Cys Ser Arg Phe
80 85 90
Pro Asn Phe Ser Ile Lue Tyr Trp Lue Gly Asn Gly Ser Phe Ile
95 100 105
Glu His Lue Pro Gly Arg Lue Trp Glu Gly Ser Thr Ser Arg Glu
110 115 120
Arg Gly Ser Thr Gly Thr Gln Lue Cys Lys Ala Lue Val Lue Glu
125 130 135
Gln Lue Thr Pro Ala Lue His Ser Thr Asn Phe Ser Cys Val Lue
140 145 150
Val Asp Pro Glu Gln Val Val Gln Arg His Val Val Lue Ala Gln
155 160 165
Lue Trp Ala Gly Lue Arg Ala Thr Lue Pro Pro Thr Gln Glu Ala
170 175 180
Lue Pro Ser Ser His Ser Ser Pro Gln Gln Gln Gly
185 190
<210>16
<211>424
<212>PRT
＜213〉artificial sequence
<310>WO2006/043972 A1
<311>2006-04-27
<400>16
Met Arg His Asn Trp Thr Pro Asp Lue Ser Pro Lue Trp Val Lue
5 10 15
Lue Lue Cys Ala His Val Val Thr Lue Lue Val Arg Ala Thr Pro
20 25 30
Val Ser Gln Thr Thr Thr Ala Ala Thr Ala Ser Val Arg Ser Thr
35 40 45
Lys Asp Pro Cys Pro Ser Gln Pro Pro Val Phe Pro Ala Ala Lys
50 55 60
Gln Cys Pro Ala Lue Glu Val Thr Trp Pro Glu Val Glu Val Pro
65 70 75
Lue Asn Gly Thr Lue Ser Lue Ser Cys Val Ala Cys Ser Arg Phe
80 85 90
Pro Asn Phe Ser Ile Lue Tyr Trp Lue Gly Asn Gly Ser Phe Ile
95 100 105
Glu His Lue Pro Gly Arg Lue Trp Glu Gly Ser Thr Ser Arg Glu
110 115 120
Arg Gly Ser Thr Gly Thr Gln Lue Cys Lys Ala Lue Val Lue Glu
125 130 135
Gln Lue Thr Pro Ala Lue His Ser Thr Asn Phe Ser Cys Val Lue
140 145 150
Val Asp Pro Glu Gln Val Val Gln Arg His Val Val Lue Ala Gln
155 160 165
Lue Trp Ala Gly Lue Arg Ala Thr Lue Pro Pro Thr Gln Glu Ala
170 175 180
Lue Pro Ser Ser His Ser Ser Pro Gln Gln Gln Gly Glu Pro Lys
185 190 195
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
200 205 210
Lue Lue Gly Gly Pro Ser Val Phe Lue Phe Pro Pro Lys Pro Lys
215 220 225
Asp Thr Lue Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
230 235 240
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
245 250 255
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
260 265 270
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Lue Thr Val
275 280 285
Lue His Gln Asp Trp Lue Asn Gly Lys Glu Tyr Lys Cys Lys Val
290 295 300
Ser Asn Lys Ala Lue Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
305 310 315
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Lue Pro Pro
320 325 330
Ser Arg Asp Glu Lue Thr Lys Asn Gln Val Ser Lue Thr Cys Lue
335 340 345
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
350 355 360
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Lue
365 370 375
Asp Ser Asp Gly Ser Phe Phe Lue Tyr Ser Lys Lue Thr Val Asp
380 385 390
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
395 400 405
His Glu Ala Lue His Asn His Tyr Thr Gln Lys Ser Lue Ser Lue
410 415 420
Ser Pro Gly Lys
<210>17
<211>576
<212>PRT
＜213〉artificial sequence
<310>WO2006/043972 A1
<311>2006-04-27
<400>17
Met Arg His Asn Trp Thr Pro Asp Lue Ser Pro Lue Trp Val Lue
5 10 15
Lue Lue Cys Ala His Val Val Thr Lue Lue Val Arg Ala Thr Pro
20 25 30
Val Ser Gln Thr Thr Thr Ala Ala Thr Ala Ser Val Arg Ser Thr
35 40 45
Lys Asp Pro Cys Pro Ser Gln Pro Pro Val Phe Pro Ala Ala Lys
50 55 60
Gln Cys Pro Ala Lue Glu Val Thr Trp Pro Glu Val Glu Val Pro
65 70 75
Lue Ash Gly Thr Lue Ser Lue Ser Cys Val Ala Cys Ser Arg Phe
80 85 90
Pro Asn Phe Ser Ile Lue Tyr Trp Lue Gly Asn Gly Ser Phe Ile
95 100 105
Glu His Lue Pro Gly Arg Lue Trp Glu Gly Ser Thr Ser Arg Glu
110 115 120
Arg Gly Ser Thr Gly Thr Gln Lue Cys Lys Ala Lue Val Lue Glu
125 130 135
Gln Lue Thr Pro Ala Lue His Ser Thr Asn Phe Ser Cys Val Lue
140 145 150
Val Asp Pro Glu Gln Val Val Gln Arg His Val Val Lue Ala Gln
155 160 165
Lue Trp Ala Gly Lue Arg Ala Thr Lue Pro Pro Thr Gln Glu Ala
170 175 180
Lue Pro Ser Ser His Ser Ser Pro Gln Gln Gln Gly Glu Pro Lys
185 190 195
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
200 205 210
Lue Lue Gly Gly Pro Ser Val Phe Lue Phe Pro Pro Lys Pro Lys
215 220 225
Asp Thr Lue Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
230 235 240
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
245 250 255
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
260 265 270
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Lue Thr Val
275 280 285
Lue His Gln Asp Trp Lue Asn Gly Lys Glu Tyr Lys Cys Lys Val
290 295 300
Ser Asn Lys Ala Lue Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
305 310 315
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Lue Pro Pro
320 325 330
Ser Arg Asp Glu Lue Thr Lys Asn Gln Val Ser Lue Thr Cys Lue
335 340 345
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
350 355 360
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Lue
365 370 375
Asp Ser Asp Gly Ser Phe Phe Lue Tyr Ser Lys Lue Thr Val Asp
380 385 390
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
395 400 405
His Glu Ala Lue His Asn His Tyr Thr Gln Lys Ser Lue Ser Lue
410 415 420
Ser Pro G1y Lys Arg Pro Ser Gly Arg Lys Ser Ser Lys Met Gln
425 430 435
Ala Phe Arg Ile Trp Asp Val Asn Gln Lys Thr Phe Tyr Lue Arg
440 445 450
Asn Asn Gln Lue Val Ala Gly Tyr Lue Gln Gly Pro Asn Val Asn
455 460 465
Lue Lys Glu Lys Ile Asp Val Val Pro Ile Glu Pro His Ala Lue
470 475 480
Phe Lue Gly Ile His Gly Gly Lys Met Cys Lue Ser Cys Val Lys
485 490 495
Ser Gly Asp Glu Thr Arg Lue Gln Lue Glu Ala Val Asn Ile Thr
500 505 510
Asp Lue Ser Glu Asn Arg Lys Gln Asp Lys Arg Phe Ala phe Ile
515 520 525
Arg Ser Asp Ser Gly Pro Thr Thr Ser Phe Glu Ser Ala Ala Cys
530 535 540
Pro Gly Trp Phe Lue Cys Thr Ala Met Glu Ala Asp Gln Pro Val
545 550 555
Ser Lue Thr Asn Met Pro Asp Glu Gly Val Met Val Thr Lys Phe
560 565 570
Tyr Phe Gln Glu Asp Glu
575
<210>18
<211>576
<212>PRT
＜213〉artificial sequence
<310>WO2006/043972 A1
<311>2006-04-27
<400>18
Met Asn Cys Arg Glu Lue Pro Lue Thr Lue Trp Val Lue Ile Ser
5 10 15
Val Ser Thr Ala Glu Ser Cys Thr Ser Arg Pro His Ile Thr Val
20 25 30
Val Glu Gly Glu Pro Phe Tyr Lue Lys His Cys Ser CyS Ser Lue
35 40 45
Ala His Glu Ile Glu Thr Thr Thr Lys Ser Trp Tyr Lys Ser Ser
50 55 60
Gly Ser Gln Glu His Val Glu Lue Asn Pro Arg Ser Ser Ser Arg
65 70 75
Ile Ala Lue His Asp Cys Val Lue Glu Phe Trp Pro Val Glu Lue
80 85 90
Asn Asp Thr Gly Ser Tyr Phe Phe Gln Met Lys Asn Tyr Thr Gln
95 100 105
Lys Trp Lys Lue Asn Val Ile Arg Arg Asn Lys His Ser Cys Phe
110 115 120
Thr Glu Arg Gln Val Thr Ser Lys Ile Val Glu Val Lys Lys Phe
125 130 135
Phe Gln Ile Thr Cys Glu Asn Ser Tyr Tyr Gln Thr Lue Val Asn
140 145 150
Ser Thr Ser Lue Tyr Lys Asn Cys Lys Lys Lue Lue Lue Glu Asn
155 160 165
Asn Lys Asn Pro Thr Ile Lys Lys Asn Ala Glu Phe Glu Asp Gln
170 175 180
Gly Tyr Tyr Ser Cys Val His Phe Lue His His Asn Gly Lys Lue
185 190 195
Phe Asn Ile Thr Lys Thr Phe Asn Ile Thr Ile Val Glu Asp Arg
200 205 210
Ser Asn Ile Val Pro Val Lue Lue Gly Pro Lys Lue Asn His Val
215 220 225
Ala Val Glu Lue Gly Lys Asn Val Arg Lue Asn Cys Ser Ala Lue
230 235 240
Lue Asn Glu Glu Asp Val Ile Tyr Trp Met Phe Gly Glu Glu Asn
245 250 255
Gly Ser Asp Pro Asn Ile His Glu Glu Lys Glu Met Arg Ile Met
260 265 270
Thr Pro Glu Gly Lys Trp His Ala Ser Lys Val Lue Arg Ile Glu
275 280 285
Asn Ile Gly Glu Ser Asn Lue Asn Val Lue Tyr Asn Cys Thr Val
290 295 300
Ala Ser Thr Gly Gly Thr Asp Thr Lys Ser Phe Ile Lue Val Arg
305 310 315
Lys Ala Asp Met Ala Asp Ile Pro Gly His Val Phe Thr Arg Glu
320 325 330
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
335 340 345
Pro Glu Lue Lue Gly Gly Pro Ser Val Phe Lue Phe Pro Pro Lys
350 355 360
Pro Lys Asp Thr Lue Met Ile Ser Arg Thr Pro Glu Val Thr Cys
365 370 375
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
380 385 390
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
395 400 405
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Lue
410 415 420
Thr Val Lue His Gln Asp Trp Lue Asn Gly Lys Glu Tyr Lys Cys
425 430 435
Lys Val Ser Asn Lys Ala Lue Pro Ala Pro Ile Glu Lys Thr Ile
440 445 450
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Lue
455 460 465
Pro Pro Ser Arg Asp Glu Lue Thr Lys Asn Gln Val Ser Lue Thr
470 475 480
Cys Lue Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
485 490 495
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
500 505 510
Val Lue Asp Ser Asp Gly Ser Phe Phe Lue Tyr Ser Lys Lue Thr
515 520 525
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
530 535 540
Val Met His Glu Ala Lue His Asn His Tyr Thr Gln Lys Ser Lue
545 550 555
Ser Lue Ser Pro Gly Lys Arg Pro Ser Gly Arg Lys Ser Ser Lys
560 565 570
Met Gln Ala Phe Arg Ile Trp Asp Val Asn Gln Lys Thr Phe Tyr
575 580 585
Lue Arg Asn Asn Gln Lue Val Ala Gly Tyr Lue Gln Gly Pro Asn
590 595 600
Val Asn Lue Lys Glu Lys Ile Asp Val Val Pro Ile Glu Pro His
605 610 615
Ala Lue Phe Lue Gly Ile His Gly Gly Lys Met Cys Lue Ser Cys
620 625 630
Val Lys Ser Gly Asp Glu Thr Arg Lue Gln Lue Glu Ala Val Asn
635 640 645
Ile Thr Asp Lue Ser Glu Asn Arg Lys Gln Asp Lys Arg Phe Ala
650 655 660
Phe Ile Arg Ser Asp Ser Gly Pro Thr Thr Ser Phe Glu Ser Ala
665 670 675
Ala Cys Pro Gly Trp Phe Lue Cys Thr Ala Met Glu Ala Asp Gln
680 685 690
Pro Val Ser Lue Thr Asn Met Pro Asp Glu Gly Val Met Val Thr
695 700 705
Lys Phe Tyr Phe Gln Glu Asp Glu
710
<210>19
<211>232
<212>PRT
＜213〉artificial sequence
<310>WO2006/043972 A1
<311>2006-04-27
<400>19
Met Gly Trp Lue Cys Ser Gly Lue Lue Phe Pro Val Ser Cys Lue
5 10 15
Val Lue Lue Gln Val Ala Ser Ser Gly Asn Met Lys Val Lue Gln
20 25 30
Glu Pro Thr Cys Val Ser Asp Tyr Met Ser Ile Ser Thr Cys Glu
35 40 45
Trp Lys Met Asn Gly Pro Thr Asn Cys Ser Thr Glu Lue Arg Lue
50 55 60
Lue Tyr Gln Lue Val Phe Lue Lue Ser Glu Ala His Thr Cys Ile
65 70 75
Pro Glu Asn Asn Gly Gly Ala Gly Cys Val Cys His Lue Lue Met
80 85 90
Asp Asp Val Val Ser Ala Asp Asn Tyr Thr Lue Asp Lue Trp Ala
95 100 105
Gly Gln Gln Lue Lue Trp Lys Gly Ser Phe Lys Pro Ser Glu His
110 115 120
Val Lys Pro Arg Ala Pro Gly Asn Lue Thr Val His Thr Asn Val
125 130 135
Ser Asp Thr Lue Lue Lue Thr Trp Ser Asn Pro Tyr Pro Pro Asp
140 145 150
Asn Tyr Lue Tyr Asn His Lue Thr Tyr Ala Val Asn Ile Trp Ser
155 160 165
Glu Asn Asp Pro Ala Asp Phe Arg Ile Tyr Asn Val Thr Tyr Lue
170 175 180
Glu Pro Ser Lue Arg Ile Ala Ala Ser Thr Lue Lys Ser Gly Ile
185 190 195
Ser Tyr Arg Ala Arg Val Arg Ala Trp Ala Gln Cys Tyr Asn Thr
200 205 210
Thr Trp Ser Glu Trp Ser Pro Ser Thr Lys Trp His Asn Ser Tyr
215 220 225
Arg Glu Pro Phe Glu Gln His
230
<210>20
<211>464
<212>PRT
＜213〉artificial sequence
<310>WO2006/043972 A1
<311>2006-04-27
<400>20
Met Gly Trp Lue Cys Ser Gly Lue Lue Phe Pro Val Ser Cys Lue
5 10 15
Val Lue Lue Gln Val Ala Ser Ser Gly Asn Met Lys Val Lue Gln
20 25 30
Glu Pro Thr Cys Val Ser Asp Tyr Met Ser Ile Ser Thr Cys Glu
35 40 45
Trp Lys Met Asn Gly Pro Thr Asn Cys Ser Thr Glu Lue Arg Lue
50 55 60
Lue Tyr Gln Lue Val Phe Lue Lue Ser Glu Ala His Thr Cys Ile
65 70 75
Pro Glu Asn Asn Gly Gly Ala Gly Cys Val Cys His Lue Lue Met
80 85 90
Asp Asp Val Val Ser Ala Asp Asn Tyr Thr Lue Asp Lue Trp Ala
95 100 105
Gly Gln Gln Lue Lue Trp Lys Gly Ser Phe Lys Pro Ser Glu His
110 115 120
Val Lys Pro Arg Ala Pro Gly Asn Lue Thr Val His Thr Asn Val
125 130 135
Ser Asp Thr Lue Lue Lue Thr Trp Ser Asn Pro Tyr Pro Pro Asp
140 145 150
Asn Tyr Lue Tyr Asn His Lue Thr Tyr Ala Val Asn Ile Trp Ser
155 160 165
Glu Asn Asp Pro Ala Asp Phe Arg Ile Tyr Asn Val Thr Tyr Lue
170 175 180
Glu Pro Ser Lue Arg Ile Ala Ala Ser Thr Lue Lys Ser Gly Ile
185 190 195
Ser Tyr Arg Ala Arg Val Arg Ala Trp Ala Gln Cys Tyr Asn Thr
200 205 210
Thr Trp Ser Glu Trp Ser Pro Ser Thr Lys Trp His Asn Ser Tyr
215 220 225
Arg Glu Pro Phe Glu Gln His Glu Pro Lys Ser Cys Asp Lys Thr
230 235 240
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lue Lue Gly Gly Pro
245 250 255
Ser Val Phe Lue Phe Pro Pro Lys Pro Lys Asp Thr Lue Met Ile
260 265 270
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
275 280 285
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
290 295 300
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
305 310 315
Thr Tyr Arg Val Val Ser Val Lue Thr Val Lue His Gln Asp Trp
320 325 330
Lue Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Lue
335 340 345
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
350 355 360
Arg Glu Pro Gln Val Tyr Thr Lue Pro Pro Ser Arg Asp Glu Lue
365 370 375
Thr Lys Asn Gln Val Ser Lue Thr Cys Lue Val Lys Gly Phe Tyr
380 385 390
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
395 400 405
Asn Asn Tyr Lys Thr Thr Pro Pro Val Lue Asp Ser Asp Gly Ser
410 415 420
Phe Phe Lue Tyr Ser Lys Lue Thr Val Asp Lys Ser Arg Trp Gln
425 430 435
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Lue His
440 445 450
Asn His Tyr Thr Gln Lys Ser Lue Ser Lue Ser Pro Gly Lys
455 460
<210>21
<211>616
<212>PRT
＜213〉artificial sequence
<310>WO2006/043972 A1
<311>2006-04-27
<400>21
Met Gly Trp Lue Cys Ser Gly Lue Lue Phe Pro Val Ser Cys Lue
5 10 15
Val Lue Lue Gln Val Ala Ser Ser Gly Asn Met Lys Val Lue Gln
20 25 30
Glu Pro Thr Cys Val Ser Asp Tyr Met Ser Ile Ser Thr Cys Glu
35 40 45
Trp Lys Met Asn Gly Pro Thr Asn Cys Ser Thr Glu Lue Arg Lue
50 55 60
Lue Tyr Gln Lue Val Phe Lue Lue Set Glu Ala His Thr Cys Ile
65 70 75
Pro Glu Asn Asn Gly Gly Ala Gly Cys Val Cys His Lue Lue Met
80 85 90
Asp Asp Val Val Ser Ala Asp Asn Tyr Thr Lue Asp Lue Trp Ala
95 100 105
Gly Gln Gln Lue Lue Trp Lys Gly Ser Phe Lys Pro Ser Glu His
110 115 120
Val Lys Pro Arg Ala Pro Gly Asn Lue Thr Val His Thr Asn Val
125 130 135
Ser Asp Thr Lue Lue Lue Thr Trp Ser Asn Pro Tyr Pro Pro Asp
140 145 150
Asn Tyr Lue Tyr Asn His Lue Thr Tyr Ala Val Asn Ile Trp Ser
155 160 165
Glu Asn Asp Pro Ala Asp Phe Arg Ile Tyr Asn Val Thr Tyr Lue
170 175 180
Glu Pro Ser Lue Arg Ile Ala Ala Ser Thr Lue Lys Ser Gly Ile
185 190 195
Ser Tyr Arg Ala Arg Val Arg Ala Trp Ala Gln Cys Tyr Asn Thr
200 205 210
Thr Trp Ser Glu Trp Ser Pro Ser Thr Lys Trp His Asn Ser Tyr
215 220 225
Arg Glu Pro Phe Glu Gln His Glu Pro Lys Ser Cys Asp Lys Thr
230 235 240
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lue Lue Gly Gly Pro
245 250 255
Ser Val Phe Lue Phe Pro Pro Lys Pro Lys Asp Thr Lue Met Ile
260 265 270
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
275 280 285
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
290 295 300
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
305 310 315
Thr Tyr Arg Val Val Ser Val Lue Thr Val Lue His Gln Asp Trp
320 325 330
Lue Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Lue
335 340 345
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
350 355 360
Arg Glu Pro Gln Val Tyr Thr Lue Pro Pro Ser Arg Asp Glu Lue
365 370 375
Thr Lys Asn Gln Val Ser Lue Thr Cys Lue Val Lys Gly Phe Tyr
380 385 390
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
395 400 405
Asn Asn Tyr Lys Thr Thr Pro Pro Val Lue Asp Ser Asp Gly Ser
410 415 420
Phe Phe Lue Tyr Ser Lys Lue Thr Val Asp Lys Ser Arg Trp Gln
425 430 435
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Lue His
440 445 450
Asn His Tyr Thr Gln Lys Ser Lue Ser Lue Ser Pro Gly Lys Arg
455 460 465
Pro Ser Gly Arg Lys Ser Ser Lys Met Gln Ala Phe Arg Ile Trp
470 475 480
Asp Val Asn Gln Lys Thr Phe Tyr Lue Arg Asn Asn Gln Lue Val
485 490 495
Ala Gly Tyr Lue Gln Gly Pro Asn Val Asn Lue Lys Glu Lys Ile
500 505 510
Asp Val Val Pro Ile Glu Pro His Ala Lue Phe Lue Gly Ile His
515 520 525
Gly Gly Lys Met Cys Lue Ser Cys Val Lys Ser Gly Asp Glu Thr
530 535 540
Arg Lue Gln Lue Glu Ala Val Asn Ile Thr Asp Lue Ser Glu Asn
545 550 555
Arg Lys Gln Asp Lys Arg Phe Ala Phe Ile Arg Ser Asp Ser Gly
560 565 570
Pro Thr Thr Ser Phe Glu Ser Ala Ala Cys Pro Gly Trp Phe Lue
575 580 585
Cys Thr Ala Met Glu Ala Asp Gln Pro Val Ser Lue Thr Asn Met
590 595 600
Pro Asp Glu Gly Val Met Val Thr Lys Phe Tyr Phe Gln Glu Asp
605 610 615
<210>22
<211>856
<212>PRT
＜213〉artificial sequence
<310>WO2006/043972 A1
<311>2006-04-27
<400>22
Met Asp Ser Lue Ala Ser Lue Val Lue Cys Gly Val Ser Lue Lue
5 10 15
Lue Ser Gly Thr Val Glu Gly Ala Met Asp Lue Ile Lue Ile Asn
20 25 30
Ser Lue Pro Lue Val Ser Asp Ala Glu Thr Ser Lue Thr Cys Ile
35 40 45
Ala Ser Gly Trp Arg Pro His Glu Pro Ile Thr Ile Gly Arg Asp
50 55 60
Phe Glu Ala Lue Met Asn Gln His Gln Asp Pro Lue Glu Val Thr
65 70 75
Gln Asp Val Thr Arg Glu Trp Ala Lys Lys Val Val Trp Lys Arg
80 85 90
Glu Lys Ala Ser Lys Ile Asn Gly Ala Tyr Phe Cys Glu Gly Arg
95 100 105
Val Arg Gly Glu Ala Ile Arg Ile Arg Thr Met Lys Met Arg Gln
110 115 120
Gln Ala Ser Phe Lue Pro Ala Thr Lue Thr Met Thr Val Asp Lys
125 130 135
Gly Asp Asn Val Asn Ile Ser Phe Lys Lys Val Lue Ile Lys Glu
140 145 150
Glu Asp Ala Val Ile Tyr Lys Asn Gly Ser Phe Ile His Ser Val
155 160 165
Pro Arg His Glu Val Pro Asp Ile Lue Glu Val His Lue Pro His
170 175 180
Ala Gln Pro Gln Asp Ala Gly Val Tyr Ser Ala Arg Tyr Ile Gly
185 190 195
Gly Asn Lue Phe Thr Ser Ala Phe Thr Arg Lue Ile Val Arg Arg
200 205 210
Cys Glu Ala Gln Lys Trp Gly Pro Glu Cys Asn His Lue Cys Thr
215 220 225
Ala Cys Met Asn Asn Gly Val Cys His Glu Asp Thr Gly Glu Cys
230 235 240
Ile Cys Pro Pro Gly Phe Met Gly Arg Thr Cys Glu Lys Ala Cys
245 250 255
Glu Lue His Thr Phe Gly Arg Thr Cys Lys Glu Arg Cys Ser Gly
260 265 270
Gln Glu Gly Cys Lys Ser Tyr Val Phe Cys Lue Pro Asp Pro Tyr
275 280 285
Gly Cys Ser Cys Ala Thr Gly Trp Lys Gly Lue Gln Cys Asn Glu
290 295 300
Ala Cys His Pro Gly Phe Tyr Gly Pro Asp Cys Lys Lue Arg Cys
305 310 315
Ser Cys Asn Asn Gly Glu Met Cys Asp Arg Phe Gln Gly Cys Lue
320 325 330
Cys Ser Pro Gly Trp Gln Gly Lue Gln Cys Glu Arg Glu Gly Ile
335 340 345
Pro Arg Met Thr Pro Lys Ile Val Asp Lue Pro Asp His Ile Glu
350 355 360
Val Asn Ser Gly Lys Phe Asn Pro Ile Cys Lys Ala Ser Gly Trp
365 370 375
Pro Lue Pro Thr Asn Glu Glu Met Thr Lue Val Lys Pro Asp Gly
380 385 390
Thr Val Lue His Pro Lys Asp Phe Asn His Thr Asp His Phe Ser
395 400 405
Val Ala Ile Phe Thr Ile His Arg Ile Lue Pro Pro Asp Ser Gly
410 415 420
Val Trp Val Cys Ser Val Asn Thr Val Ala Gly Met Val Glu Lys
425 430 435
Pro Phe Asn Ile Ser Val Lys Val Lue Pro Lys Pro Lue Asn Ala
440 445 450
Pro Asn Val Ile Asp Thr Gly His Asn Phe Ala Val Ile Asn Ile
455 460 465
Ser Ser Glu Pro Tyr Phe Gly Glu Pro Lys Ser Cys Asp Lys Thr
470 475 480
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lue Lue Gly Gly Pro
485 490 495
Ser Val Phe Lue Phe Pro Pro Lys Pro Lys Asp Thr Lue Met Ile
500 505 510
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
515 520 525
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
530 535 540
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
545 550 555
Thr Tyr Arg Val Val Ser Val Lue Thr Val Lue His Gln Asp Trp
560 565 570
Lue Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Lue
575 580 585
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
590 595 600
Arg Glu Pro Gln Val Tyr Thr Lue Pro Pro Ser Arg Asp Glu Lue
605 610 615
Thr Lys Asn Gln Val Ser Lue Thr Cys Lue Val Lys Gly Phe Tyr
620 625 630
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
635 640 645
Asn Asn Tyr Lys Thr Thr Pro Pro Val Lue Asp Ser Asp Gly Ser
650 655 660
Phe Phe Lue Tyr Ser Lys Lue Thr Val Asp Lys Ser Arg Trp Gln
665 670 675
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Lue His
680 685 690
Asn His Tyr Thr Gln Lys Ser Lue Ser Lue Ser Pro Gly Lys Arg
695 700 705
Pro Ser Gly Arg Lys Ser Ser Lys Met Gln Ala Phe Arg Ile Trp
710 715 720
Asp Val Asn Gln Lys Thr Phe Tyr Lue Arg Asn Asn Gln Lue Val
725 730 735
Ala Gly Tyr Lue Gln Gly Pro Asn Val Asn Lue Lys Glu Lys Ile
740 745 750
Asp Val Val Pro Ile Glu Pro His Ala Lue Phe Lue Gly Ile His
755 760 765
Gly Gly Lys Met Cys Lue Ser Cys Val Lys Ser Gly Asp Glu Thr
770 775 780
Arg Lue Gln Lue Glu Ala Val Asn Ile Thr Asp Lue Ser Glu Asn
785 790 795
Arg Lys Gln Asp Lys Arg Phe Ala Phe Ile Arg Ser Asp Ser Gly
800 805 810
Pro Thr Thr Ser Phe Glu Ser Ala Ala Cys Pro Gly Tro Phe Lue
815 820 825
Cys Thr Ala Met Glu Ala Asp Gln Pro Val Ser Lue Thr Asn Met
830 835 840
Pro Asp Glu Gly Val Met Val Thr Lys Phe Tyr Phe Gln Glu Asp
845 850 855
Glu
<210>23
<211>215
<212>PRT
＜213〉artificial sequence
<310>WO2006/043972 A1
<311>2006-04-27
<400>23
Asp Ile Gln Lue Ile Thr Gln Ser Pro Ser Ser Lue Ser Ala Ser
5 10 15
Val Gly Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile
20 25 30
Ser Asn Tyr Lue Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
35 40 45
Lys Val Lue Ile Tyr Phe Thr Ser Ser Lue His Ser Gly Val Pro
50 55 60
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Lue Thr
65 70 75
Ile Ser Ser Lue Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
80 85 90
Gln Tyr Ser Thr Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val
95 100 105
Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro
110 115 120
Pro Ser Asp Glu Gln Lue Lys Ser Gly Thr Ala Ser Val Val Cys
125 130 135
Lue Lue Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys
140 145 150
Val Asp Asn Ala Lue Gln Ser Gly Asn Ser Gln Glu Ser Val Thr
155 160 165
Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Lue Ser Ser Thr Lue
170 175 180
Thr Lue Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys
185 190 195
Glu Val Thr His Gln Gly Lue Ser Ser Pro Val Thr Lys Ser Phe
200 205 210
Asn Arg Gly Glu Cys
215
<210>24
<211>718
<212>PRT
＜213〉artificial sequence
<310>WO2006/043972 A1
<311>2006-04-27
<400>24
Met Val Ser Tyr Trp Asp Thr Gly Val Lue Lue Cys Ala Lue Lue
5 10 15
Ser Cys Lue Lue Lue Thr Gly Ser Ser Ser Gly Ser Lys Lue Lys
20 25 30
Asp Pro Glu Lue Ser Lue Lys Gly Thr Gln His Ile Met Gln Ala
35 40 45
Gly Gln Thr Lue His Lue Gln Cys Arg Gly Glu Ala Ala His Lys
50 55 60
Trp Ser Lue Pro Glu Met Val Ser Lys Glu Ser Glu Arg Lue Ser
65 70 75
Ile Thr Lys Ser Ala Cys Gly Arg Asn Gly Lys Gln Phe Cys Ser
80 85 90
Thr Lue Thr Lue Asn Thr Ala Gln Ala Asn His Thr Gly Phe Tyr
95 100 105
Ser Cys Lys Tyr Lue Ala Val Pro Thr Ser Lys Lys Lys Glu Thr
110 115 120
Glu Ser Ala Ile Tyr Ile Phe Ile Ser Asp Thr Gly Arg Pro Phe
125 130 135
Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu
140 145 150
Gly Arg Glu Lue Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile
155 160 165
Thr Val Thr Lue Lys Lys Phe Pro Lue Asp Thr Lue Ile Pro Asp
170 175 180
Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser
185 190 195
Asn Ala Thr Tyr Lys Glu Ile Gly Lue Lue Thr Cys Glu Ala Thr
200 205 210
Val Asn Gly His Lue Tyr Lys Thr Asn Tyr Lue Thr His Arg Gln
215 220 225
Thr Asn Thr Ile Ile Asp Val Gln Ile Ser Thr Pro Arg Pro Val
230 235 240
Lys Lue Lue Arg Gly His Thr Lue Val Lue Asn Cys Thr Ala Thr
245 250 255
Thr Pro Lue Asn Thr Arg Val Gln Met Thr Trp Ser Tyr Pro Asp
260 265 270
Glu Lys Asn Lys Arg Ala Ser Val Arg Arg Arg Ile Asp Gln Ser
275 280 285
Asn Ser His Ala Asn Ile Phe Tyr Ser Val Lue Thr Ile Asp Lys
290 295 300
Met Gln Asn Lys Asp Lys Gly Lue Tyr Thr Cys Arg Val Arg Ser
305 310 315
Gly Pro Ser Phe Lys Ser Val Asn Thr Ser Val His Ile Tyr Asp
320 325 330
Lys Ala Phe Ile Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
335 340 345
Pro Pro Cys Pro Ala Pro Glu Lue Lue Gly Gly Pro Ser Val Phe
350 355 360
Lue Phe Pro Pro Lys Pro Lys Asp Thr Lue Met Ile Ser Arg Thr
365 370 375
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
380 385 390
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
395 400 405
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
410 415 420
Val Val Ser Val Lue Thr Val Lue His Gln Asp Trp Lue Asn Gly
425 430 435
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Lue Pro Ala Pro
440 445 450
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
455 460 465
Gln Val Tyr Thr Lue Pro Pro Ser Arg Asp Glu Lue Thr Lys Asn
470 475 480
Gln Val Ser Lue Thr Cys Lue Val Lys Gly Phe Tyr Pro Ser Asp
485 490 495
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
500 505 510
Lys Thr Thr Pro Pro Val Lue Asp Ser Asp Gly Ser Phe Phe Lue
515 520 525
Tyr Ser Lys Lue Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
530 535 540
Val Phe Ser Cys Ser Val Met His Glu Ala Lue His Asn His Tyr
545 550 555
Thr Gln Lys Ser Lue Ser Lue Ser Pro Gly Lys Arg Pro Ser Gly
560 565 570
Arg Lys Ser Ser Lys Met Gln Ala Phe Arg Ile Trp Asp Val Asn
575 580 585
Gln Lys Thr Phe Tyr Lue Arg Asn Asn Gln Lue Val Ala Gly Tyr
590 595 600
Lue Gln Gly Pro Asn Val Asn Lue Lys Glu Lys Ile Asp Val Val
605 610 615
Pro Ile Glu Pro His Ala Lue Phe Lue Gly Ile His Gly Gly Lys
620 625 630
Met Cys Lue Ser Cys Val Lys Ser Gly Asp Glu Thr Arg Lue Gln
635 640 645
Lue Glu Ala Val Asn Ile Thr Asp Lue Ser Glu Asn Arg Lys Gln
650 655 660
Asp Lys Arg Phe Ala Phe Ile Arg Ser Asp Ser Gly Pro Thr Thr
665 670 675
Ser Phe Glu Ser Ala Ala Cys Pro Gly Trp Phe Lue Cys Thr Ala
680 685 690
Met Glu Ala Asp Gln Pro Val Ser Lue Thr Asn Met Pro Asp Glu
695 700 705
Gly Val Met Val Thr Lys Phe Tyr Phe Gln Glu Asp Glu
710 715
<210>25
<211>605
<212>PRT
＜213〉artificial sequence
<310>WO2006/043972 A1
<311>2006-04-27
<400>25
Glu Val Gln Lue Val Glu Ser Gly Gly Gly Lue Val Gln Pro Gly
5 10 15
Gly Ser Lue Arg Lue Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr
20 25 30
Asn Tyr Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Lue
35 40 45
Glu Trp Val Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr
50 55 60
Ala Ala Asp Phe Lys Arg Arg Phe Thr Phe Ser Lue Asp Thr Ser
65 70 75
Lys Ser Thr Ala Tyr Lue Gln Met Asn Ser Lue Arg Ala Glu Asp
80 85 90
Thr Ala Val Tyr Tyr Cys Ala Lys Tyr Pro Tyr Tyr Tyr Gly Ser
95 100 105
Ser His Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Lue Val Thr
110 115 120
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Lue Ala
125 130 135
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Lue Gly Cys
140 145 150
Lue Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
155 160 165
Ser Gly Ala Lue Thr Ser Gly Val His Thr Phe Pro Ala Val Lue
170 175 180
Gln Ser Ser Gly Lue Tyr Ser Lue Ser Ser Val Val Thr Val Pro
185 190 195
Ser Ser Ser Lue Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
200 205 210
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
215 220 225
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Lue
230 235 240
Lue Gly Gly Pro Ser Val Phe Lue Phe Pro Pro Lys Pro Lys Asp
245 250 255
Thr Lue Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
260 265 270
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
275 280 285
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
290 295 300
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Lue Thr Val Lue
305 310 315
His Gln Asp Trp Lue Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
320 325 330
Asn Lys Ala Lue Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
335 340 345
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Lue Pro Pro Ser
350 355 360
Arg Asp Glu Lue Thr Lys Asn Gln Val Ser Lue Thr Cys Lue Val
365 370 375
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
380 385 390
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Lue Asp
395 400 405
Ser Asp Gly Ser Phe Phe Lue Tyr Ser Lys Lue Thr Val Asp Lys
410 415 420
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
425 430 435
Glu Ala Lue His Asn His Tyr Thr Gln Lys Ser Lue Ser Lue Ser
440 445 450
Pro Gly Lys Arg Pro Ser Gly Arg Lys Ser Ser Lys Met Gln Ala
455 460 465
Phe Arg Ile Trp Asp Val Asn Gln Lys Thr Phe Tyr Lue Arg Asn
470 475 480
Asn Gln Lue Val Ala Gly Tyr Lue Gln Gly Pro Asn Val Asn Lue
485 490 495
Lys Glu Lys Ile Asp Val Val Pro Ile Glu Pro His Ala Lue Phe
500 505 510
Lue Gly Ile His Gly Gly Lys Met Cys Lue Ser Cys Val Lys Ser
515 520 525
Gly Asp Glu Thr Arg Lue Gln Lue Glu Ala Val Asn Ile Thr Asp
530 535 540
Lue Ser Glu Asn Arg Lys Gln Asp Lys Arg Phe Ala Phe Ile Arg
545 550 555
Ser Asp Ser Gly Pro Thr Thr Ser Phe Glu Ser Ala Ala Cys Pro
560 565 570
Gly Trp Phe Lue Cys Thr Ala Met Glu Ala Asp Gln Pro Val Ser
575 580 585
Lue Thr Asn Met Pro Asp Glu Gly Val Met Val Thr Lys Phe Tyr
590 595 600
Phe Gln Glu Asp Glu
605
Claims (32)
1. fusion rotein, it is characterized in that first fragment is positioned at the amino terminal of fusion rotein, and combination specifically, and the function of neutralize a cytokine or somatomedin, second fragment is positioned at the carboxyl terminal of fusion rotein, and the receptor of a kind of somatomedin of specific bond or a kind of cytokine, these two fragments are artificial connections, and the receptor of second cytokine is at inflammation or disease site high expressed.
2. according to the fusion rotein described in the claim 1, it is characterized in that: described protein contains junction fragment, and this junction fragment will connect first fragment and second fragment connects together, and wherein junction fragment has Dimerized function.
3. according to fusion rotein described in the claim 2, it is characterized in that: described proteic junction fragment comprises Fc fragment or its congenerous fragment of immunoglobulin.
4. the fusion rotein described in the claim 3, it is characterized in that: described immunoglobulin is meant IgA, IgE, IgD, IgG or IgM.
5. according to the fusion rotein described in the claim 3, it is characterized in that: described immunoglobulin is meant IgG.
6. according to the fusion rotein described in the claim 5, it is characterized in that: described Fc fragment comprises sequence 2.
7. according to the fusion rotein described in the claim 1, it is characterized in that: described albumen, its first fragment can in conjunction with and in and vascular endothelial cell growth factor, angiogenin, tumor necrosis factor, interleukin-18, interleukin-4 or interleukin-13 or their congenerous albumen.
8. according to the fusion rotein described in the claim 7, it is characterized in that: described albumen, wherein first fragment comprises an immunoglobulin chain-ordering, can be specifically in conjunction with and in and vascular endothelial cell growth factor, new vessels generates plain, interleukin-18, interleukin-4, interleukin-13 or IgE or its congenerous albumen.
9. the fusion rotein described in according to Claim 8 is characterized in that: described albumen, and its immunoglobulin chain comprises sequence 9,11,12,14,23, or 24 or congenerous albumen.
10. according to the fusion rotein described in the claim 7, it is characterized in that: described albumen, its first fragment comprise the receptor or the conjugated protein sequence of VEGF, angiogenin, tumor necrosis factor, interleukin-18, interleukin-4 and interleukin-13.
11. the fusion rotein according to described in the claim 10 is characterized in that: described albumen, first fragment comprises sequence 3,6,15, or 19.
12. the fusion rotein according to described in the claim 1 is characterized in that: described albumen is glycosylated protein.
13. the fusion rotein according to described in the claim 1 is characterized in that: described second cytokine is meant interleukin 1.
14. the fusion rotein according to described in the claim 13 is characterized in that: described albumen, second fragment are the antagonisies of interleukin 1.
15. the fusion rotein according to described in the claim 14 is characterized in that: described albumen, second fragment comprise sequence (sequence 1) or its congenerous protein sequence of interleukin 1 receptor antagonist.
16. the fusion rotein according to described in the claim 14 is characterized in that: described albumen comprises sequence 5,8,10,13,17,18,21,22,24, or 25.
17. the nucleic acid of an in-vitro separation is characterized in that: the sequence that wherein comprises the fusion rotein described in the coding claim 1.
18., comprise a certain sequence of coded sequence 1 to 25 according to the nucleotide sequence described in the claim 17.
19. a carrier is characterized in that: wherein comprise the nucleotide sequence described in the claim 17.
20. a host cell is characterized in that: wherein comprise the nucleic acid described in the claim 17.
21. a method of producing polypeptide comprises and utilizes cell culture medium to cultivate the cell described in the claim 20, the polypeptide of nucleic acid sequence encoding is expressed, then these polypeptide of purification from cultured cells or cell culture fluid in cell.
22. a pharmaceutical preparation component is characterized in that: wherein comprise the fusion rotein that claim 1 is mentioned, or the nucleic acid molecules of this fusion rotein of encoding, and can be used for medicinal carrier
23. regulate the immunoreactive method of patient for one kind, this method comprises:
Determine or patient who is tiring out and the claim 1 that gives this patient's effective dose for excessive immunoreation in the fusion rotein mentioned or the nucleic acid molecules of this fusion rotein of encoding
24. according to the method for being mentioned in the claim 23, it is characterized in that: described patient or prepare to accept homogenic or allotransplantation.
25. according to the method for being mentioned in the claim 23, it is characterized in that: disease wherein is that inflammation, autoimmune disease, anaphylactic disease or new vessels form the tumor disease that relies on.
26. the method according to claim 25 is characterized in that: described disease is a tumor disease, and fusion rotein comprises sequence 22,24 and 25.
27. the method for half-life that prolongs recombiant protein in patient's body, it is characterized in that: this method comprises: with certain recombiant protein with comprise sequence 1, or the sequence fragment of its same function connects, form the fusion rotein chimera, determine this fusion rotein in the intravital half-life of patient, recombiant protein is combination and neutralize a certain cytokine or somatomedin in vivo.
28. a method that prolongs recombiant protein in the intravital effect of patient, it is characterized in that: this method comprises:
With certain recombiant protein with comprise sequence 1, or the sequence fragment of other same function connects, and forms the fusion rotein chimera, determines this fusion rotein in the intravital effect of patient,
29. a method of in patient's body human cytokines being delivered to a certain target position, it is characterized in that: this method comprises:
To treat albumen and comprise sequence 1, or the fragment of its congenerous connects, form the fusion rotein chimera, and this fusion rotein is applied to suitable patient, human cytokines wherein can targeting be positioned the inflammation site of high expressed interleukin 1 receptor.
30. the method for in patient's body, human cytokines being delivered to a certain target position according to claim 28, it is characterized in that: described fragment comprises the fragment of sequence 1 or other same function, this fragment bind interleukin 1 receptor, and recombiant protein be a kind of can in conjunction with and also the human cytokines of neutralize a certain cytokine or somatomedin.
31. the method for in patient's body, human cytokines being delivered to a certain target position according to claim 30, it is characterized in that: described fusion rotein chimera, the patient intravital can the disease site of inflammation or high expressed interleukin 1 receptor simultaneously in conjunction with and in and interleukin 1 receptor and another cytokine or somatomedin.
32. the described method of in patient's body human cytokines being delivered to a certain target position of claim 30 is characterized in that: described fusion rotein chimera can neutralize or antagonism interleukin 1 and other cytokines or somatomedin simultaneously at the disease site of patient intravital inflammation site or high expressed interleukin 1 receptor.
Applications Claiming Priority (7)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US61847604P | 2004-10-12 | 2004-10-12 | |
| US60/618,476 | 2004-10-12 | ||
| US62899404P | 2004-11-17 | 2004-11-17 | |
| US60/628,994 | 2004-11-17 | ||
| US65073405P | 2005-02-01 | 2005-02-01 | |
| US60/650,734 | 2005-02-01 | ||
| PCT/US2005/012194 WO2006043972A1 (en) | 2004-10-12 | 2005-04-08 | Chimeric protein |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101027079A true CN101027079A (en) | 2007-08-29 |
| CN101027079B CN101027079B (en) | 2011-08-17 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2005800115254A Active CN101027079B (en) | 2004-10-12 | 2005-04-08 | Chimeric protein |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN101027079B (en) |
| ZA (1) | ZA200702957B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102906119A (en) * | 2010-03-19 | 2013-01-30 | 韩国生命工学研究院 | Dual antagonist for TNF-A and Il-21 for preventing and treating autoimmune diseases |
| CN104479026A (en) * | 2010-08-31 | 2015-04-01 | 健能隆医药技术（上海）有限公司 | Application of interleukin-22 to treat viral hepatitis |
| CN110724200A (en) * | 2015-08-20 | 2020-01-24 | 奥托路斯有限公司 | Chimeric cytokine receptors |
-
2005
- 2005-04-08 CN CN2005800115254A patent/CN101027079B/en active Active
-
2007
- 2007-04-11 ZA ZA200702957A patent/ZA200702957B/en unknown
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102906119A (en) * | 2010-03-19 | 2013-01-30 | 韩国生命工学研究院 | Dual antagonist for TNF-A and Il-21 for preventing and treating autoimmune diseases |
| CN102906119B (en) * | 2010-03-19 | 2015-07-01 | 韩国生命工学研究院 | Dual antagonist for TNF-A and IL-21 for preventing and treating autoimmune diseases |
| CN104479026A (en) * | 2010-08-31 | 2015-04-01 | 健能隆医药技术（上海）有限公司 | Application of interleukin-22 to treat viral hepatitis |
| CN104479026B (en) * | 2010-08-31 | 2019-02-12 | 健能隆医药技术（上海）有限公司 | Application of the interleukin-22 in treatment virus hepatitis |
| CN110724200A (en) * | 2015-08-20 | 2020-01-24 | 奥托路斯有限公司 | Chimeric cytokine receptors |
| CN110724200B (en) * | 2015-08-20 | 2024-01-26 | 奥托路斯有限公司 | Chimeric cytokine receptors |
Also Published As
| Publication number | Publication date |
|---|---|
| ZA200702957B (en) | 2008-11-26 |
| CN101027079B (en) | 2011-08-17 |
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