CN101557817A - Albumin fusion proteins - Google Patents
Albumin fusion proteins Download PDFInfo
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- CN101557817A CN101557817A CNA2007800423220A CN200780042322A CN101557817A CN 101557817 A CN101557817 A CN 101557817A CN A2007800423220 A CNA2007800423220 A CN A2007800423220A CN 200780042322 A CN200780042322 A CN 200780042322A CN 101557817 A CN101557817 A CN 101557817A
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Abstract
The present invention encompasses albumin fusion proteins. Nucleic acid molecules encoding the albumin fusion proteins of the invention are also encompassed by the invention, as are vectors containing these nucleic acids, host cells transformed with these nucleic acids vectors, and methods of making the albumin fusion proteins of the invention and using these nucleic acids, vectors, and/or host cells. Additionally the present invention encompasses pharmaceutical compositions comprising albumin fusion proteins and methods of treating, preventing, or ameliorating diseases, disorders or conditions using albumin fusion proteins of the invention.
Description
With reference to the sequence table on the CD
The application is with reference to following " sequence table " that provides with the e-file on three identical CDs (CD-R), and CD is denoted as " copy 1 ", " copy 2 " and " CRF ".These CDs respectively contain file " PF619PP5 sequence table .txt " (198,000 bytes were created in JIUYUE in 2007 7), and it is incorporated into its integral body by reference at this.
Background of invention
The present invention relates generally to human cytokines (including, but not limited at least a polypeptide, antibody, peptide or its fragment and variant) with albumin or albuminised fragment or variant fusion.The present invention includes polynucleotide, therapeutic albumin fusion proteins, compositions, pharmaceutical composition, preparaton and the medicine box of coding therapeutic albumin fusion proteins.Be also included among the present invention with the polynucleotide transformed host cells of coding therapeutic albumin fusion proteins and the method for using these polynucleotide and/or host cell to prepare albumin fusion proteins of the present invention.
Human serum albumin (HSA or HA) is that a kind of mature form is 585 amino acid whose protein ((SEQ ID NO:1) as shown in Figure 1), is responsible for the great part of serum osmotic pressure, also brings into play the function of the carrier of endogenous and exogenous ligand.At present, the HA that is used for clinical application is from the human blood extraction and prepare.Preparation reorganization HA (rHA) has been disclosed in EP 330451 and EP 361991 in microorganism.
The human cytokines of native state or reorganization preparation, such as interferon and growth hormone, normally unsettled molecule shows the short shelf life, when especially being formulated as aqueous solution.The unstability of these molecules requires the necessary lyophilizing of many these quasi-molecules and keeps freezing if having time in the institute that stores when preparation is used to use, and is difficult to transport and/or store this quasi-molecule thereby cause.When must be outside hospital environment pharmaceutical formulation being stored and when making up a prescription, storage problem is especially serious.
Seldom there is the practical solution of the storage problem that solves unsettled protein molecular to be suggested.Therefore, need the stable permanent preparaton that is easy to make up a prescription of protein for treatment molecule, need the simple preparaton of minimal action after preferred the storage.
After using in the body, the human cytokines of native state or reorganization preparation is such as interferon and growth hormone, owing to remove the plasma stability that performance is short fast from blood flow.Therefore, the therapeutic effect that provides of these albumen also is a short-term.Therefore in order to keep the cylinder therapeutic effect of their expectations, these albumen require from the quick removing of blood must be more continually or with higher dosage administering therapeutic molecule.Yet increase the proteic drug dosage schedule of administering therapeutic and often cause the reaction of patient infusion position, side effect and toxic increase.Similarly, also often cause the increase of patient's toxicity and side effect with higher dosage administering therapeutic albumen.
Proposed to improve the treatment molecule plasma stability practical solution seldom, comprise chemical bond, limited to the benefit that the patient provides.Usually, in most cases, still use the treatment molecule of these chemical modifications, keep patient's remarkable injection site reaction, side effect and toxicity with frequent drug dosage schedule.Therefore, there is demand in the treatment molecule of stable form, it keeps in vivo than the higher plasma stability of therapeutic agent of natural or reorganization preparation only, and can not use more continually, thereby reduces the potential side effect to the patient.
Summary of the invention
The present invention includes albumin fusion proteins, its comprise the human cytokines that merges with albumin or albuminised fragment (part) or variant (as, polypeptide, antibody or peptide or its fragment or variant).The present invention also comprises polynucleotide, and it comprises or is made up of following: the human cytokines that coding and albumin or albuminised fragment (part) or variant merge (as, polypeptide, antibody or peptide or its fragment or variant) nucleic acid molecules.The present invention also comprises polynucleotide, it comprises or is made up of following: coding comprise the human cytokines that merges with albumin or albuminised fragment (part) or variant (as, polypeptide, antibody or peptide, or its fragment or variant) proteic nucleic acid molecules, this albumin or albuminised fragment (part) or variant are enough to the proteic shelf life of extended treatment, be enough to compare the plasma stability that increases human cytokines with the human cytokines that does not merge state, and/or stable human cytokines and/or its external and/or activity in vivo of (or in pharmaceutical composition) in solution.By the albumin fusion proteins of polynucleotide encoding of the present invention and with polynucleotide transformed host cells of the present invention and prepare the method for albumin fusion proteins of the present invention and use the method for these polynucleotide of the present invention and/or host cell to be also included among the present invention.
In one aspect of the invention, albumin fusion proteins includes but not limited to those and this proteic polynucleotide of coding described in the table 2.
The present invention also comprises the pharmaceutical formulation that comprises albumin fusion proteins of the present invention and pharmaceutically acceptable diluent or carrier.This preparaton can be in medicine box or container.This medicine box or container can be packed with the description of the shelf life that prolongs about described human cytokines.This preparaton can be used for treatment, prevention, alleviates or diagnosis patient's (for example mammal or the mankind) the disease or the method for disease symptoms, comprises step from described pharmaceutical formulation to described patient that use.
At other embodiment, the present invention includes the method for prevention, treatment or alleviation disease or illness.At some embodiments, the present invention includes the treatment be listed in table 1 " indication: Y " row disease or the method for illness, comprise to the patient of expectation this treatment, prevention or alleviation and use albumin fusion proteins of the present invention that albumin fusion proteins of the present invention comprises the human cytokines of " human cytokines: the X " row (with the disease to be treated or the same delegation of illness of " indication: the Y " row that are listed in table 1) that are disclosed in table 1 or corresponding to the part (or its fragment or variant) of human cytokines with effective treatment, prevention or the amount of alleviating described disease or illness.
At an embodiment, the albumin fusion proteins described in table 1 or the table 2 has the shelf life of prolongation.
At second kind of embodiment, the albumin fusion proteins described in table 1 or the table 2 is more stable than the corresponding treatment molecule that does not merge described in the table 1.
The present invention also comprises genetically modified organism, and it is modified to comprise nucleic acid molecules of the present invention (including but not limited to the polynucleotide described in table 1 and 2), is for example modified to express albumin fusion proteins of the present invention.
The accompanying drawing summary
Figure 1A-D shows the human albuminised aminoacid sequence (SEQ ID NO:1) of mature form and encodes its polynucleotide (SEQ ID NO:2).The human albumin (SEQ ID NO:1) of nucleotide 1 to the 1755 encoding mature form of SEQ ID NO:2.
Fig. 2 shows the restriction map of pPPC0005 cloning vehicle ATCC preservation thing PTA-3278.
Fig. 3 shows the restriction map (Sleep etc., BioTechnology 8:42 (1990)) of pSAC35 yeast saccharomyces cerevisiae (S.cerevisiae) expression vector.
Fig. 4 is the IFN albumin fusion proteins (CID 3165 albumen) and the antiproliferative activity of reorganization IFNa (rIFNa) to the Hs294T melanoma cells of CID 3165 codings relatively.Mix by BrdU with CID 3165 albumen of variable concentrations or rIFNa cultured cell and after cultivating 3 days and to measure propagation.CID 3165 albumen cause the measurable inhibition of on cell proliferation in the concentration that is higher than 10ng/ml, reach 50% at about 200ng/ml and suppress.(■)=CID 3165 albumen, (◆)=rIFNa.
Fig. 5 shows that the various dilutions of IFNa albumin fusion proteins are to the active effect of SEAP in the ISRE-SEAP/293F report cell.Tested IFNa merge the albumin upstream a kind of prepared product (◆), merge at two kinds of different prepared products (●) in albumin downstream with IFNa and (■).
Fig. 6 shows IFNa albumin fusion proteins (CID2249 albumen) by the dna encoding that comprises in the construct 2249 time and the dosage effect (seeing embodiment 76) to the mRNA level of OAS (p41) in the monkey of treatment.Each time point: article one=vehicle contrast, second=the 1st day iv of 30ug/kg CID 2249 albumen, the 1st day sc of the 3rd=30ug/kg CID 2249 albumen, the 1st, 3 and 5 days sc of the 1st day sc, the 5th=40ug/kg reorganization IFNa of the 4th=300ug/kg CID 2249 albumen.
Fig. 7 is presented at the patient (nonresponder) who infects chronic hepatitis C genotype 1 and fail to unite in response at least one pegylated interferon alfa the therapeutic scheme of ribavirin (PEG-RBV) before, after lasting 0 to 24 week of HSA-IFN α 2b associating ribavirin therapy, change the HCV RNA titre minimizing that (log10IU/ml) measures by intermediate value HCV RNA.
Fig. 8 A shows the patient (nonresponder) who infects chronic hepatitis C genotype 1 and fail to unite in response at least one interferon-ALPHA the therapeutic scheme of ribavirin before, with per two weeks or all around the HSA-IFN α 2b associating ribavirin therapy of 900 μ g, 1200 μ g, 1500 μ g or 1800 μ g continue 48 week the back with real-time quantitative PCR measure reach HCV RNA feminine gender (as, reply or ETR when treatment finishes) percentage ratio.
The percentage ratio that has ETR after failing before Fig. 8 B shows to continue for 48 weeks with the HSA-IFN α 2b associating ribavirin therapy of per two or 900 μ g, 1200 μ g, 1500 μ g or 1800 μ g all around in response to nonresponder patient's subgroup of the therapeutic scheme of at least one pegylated interferon alfa associating ribavirin (PEG+RBV).
Fig. 8 C shows with per two weeks or total virusology that continues that the HSA-IFN α 2b associating ribavirin therapy of 900 μ g, 1200 μ g, 1500 μ g or 1800 μ g continued for 48 weeks, the nonresponder reaches after the follow-up period in 24 weeks is subsequently all around replied (SVR) rate.
Fig. 8 D shows with per two weeks or the SVR that the HSA-IFN α 2b associating ribavirin therapy of 900 μ g, 1200 μ g, 1500 μ g or 1800 μ g continued for 48 weeks, PEG+RBV nonresponder reaches after the follow-up period in 24 weeks subsequently all around leads.
Describe in detail
Definition
Provide as giving a definition so that understand some used in this specification terms.
" polynucleotides " used herein refer to have the nucleic acid molecules of the nucleotide sequence of encoding fusion protein, and described fusion comprises or is made up of following alternatively: at least one molecule albumin (or its fragment or variant) that is connected at least one human cytokines X (or its fragment or variant) in the frame; Finger has the nucleic acid molecules of the nucleotide sequence of encoding fusion protein, and described fusion comprises or is made up of following alternatively: the amino acid sequence of SEQ ID NO:Y (described in the row 6 of table 2) or its fragment or variant; Refer to have the nucleic acid molecules of the nucleotide sequence that comprises sequence that SEQ ID NO:X shows or formed by the sequence shown in the SEQ ID NO:X alternatively; Finger has the nucleic acid molecules of the nucleotide sequence of encoding fusion protein, and described fusion comprises or is made up of following alternatively: the amino acid sequence of SEQ ID NO:Z; Refer to have the nucleic acid molecules of nucleotide sequence of the albumin fusion proteins of the coding generation described in table 2 or embodiment of the present invention; Finger has the nucleic acid molecules of the nucleotide sequence of coding therapeutic albumin fusion proteins of the present invention; Refer to have the nucleic acid molecules of the nucleotide sequence that comprises in the albumin fusion constructs described in the table 2; Or refer to have the nucleic acid molecules of the nucleotide sequence (described in table 3) that comprises in the albumin fusion constructs that is preserved in ATCC.
" albumin fusion constructs " used herein refers to nucleic acid molecules, and it comprises or is made up of following alternatively: the coding that is connected at least one polynucleotides of a part human cytokines (or its fragment or variant) at least of encoding in the frame is the polynucleotides of a part albumin (or its fragment or variant) at least; Refer to nucleic acid molecules, it comprises or is made up of following alternatively: the coding that is connected at least one polynucleotides of a part human cytokines (or its fragment or variant) at least of encoding in the frame of the generation described in table 2 or embodiment is the polynucleotides of a part albumin (or its fragment or variant) at least; Or refer to nucleic acid molecules, it comprises or is made up of following alternatively: the coding that is connected at least one polynucleotides of a part human cytokines (or its fragment or variant) at least of encoding in the frame is the polynucleotides of a part albumin (or its fragment or variant) at least, further comprise for example one or more following elements: (1) functional self-replication carrier (includes but not limited to, shuttle vector, expression vector, integration vector and/or dubbing system), (2) initial zone of transcribing (as, promoter region, such as for example, regulation type or inducible promoter, constitutive promoter), (3) transcription termination region, (4) targeting sequencing and (5) selected marker. The polynucleotides of coding human cytokines and albumin albumen can be called as " partly (portion) ", " zone " or " partly (moiety) of albumin fusion constructs separately when as the part of albumin fusion constructs ".
The present invention relates generally to the polynucleotides of coding albumin fusion proteins; Albumin fusion proteins; Method with polynucleotide therapy, prevention or alleviation disease or the illness of using albumin fusion proteins or coding albumin fusion proteins. " albumin fusion proteins " used herein refers to that at least one molecule albumin (or its fragment or variant) and at least one molecular therapy albumen (or its fragment or variant) merge the albumen that forms. Albumin fusion proteins of the present invention comprises at least one fragment or variant and the albuminised at least one fragment of serum human or the variant of human cytokines, they are connected to each other (namely by the heredity fusion, described albumin fusion proteins is produced by translation nucleic acid, is connected in all or part of albuminised polynucleotides of coding in this nucleic acid in the polynucleotides frame of all or part of human cytokines of coding). Human cytokines and albumin albumen are when as the part of albumin fusion proteins, " partly (portion) ", " zone " or " partly (moiety) that can be called as separately albumin fusion proteins " (as, " human cytokines part " or " albumin protein part "). At a kind of embodiment, albumin fusion proteins of the present invention comprises at least one molecular therapy albumin X or its fragment or variant (including but not limited to the human cytokines X of mature form) and at least one molecule albumin or its fragment or variant (including but not limited to the albumin of mature form).
At further embodiment, albumin fusion proteins of the present invention by host cell processing justacrine in culture medium on every side. The newborn albumin fusion proteins processing that occurs in for the host's who expresses secretory pathway can include but not limited to the signal peptide cutting; Form disulfide bond; Correct folding; Add and processing carbohydrate (such as for example, the glycosylation that N-is connected with O-); Specific proteins hydrolysis cutting; Be assembled into multimeric protein. At another embodiment, albumin fusion proteins of the present invention is form processing. At further embodiment, " form processing of albumin fusion proteins " refers to the albumin fusion proteins product of the terminal signal peptide cutting of process N-, also is called " ripe albumin fusion proteins " herein.
In many situations, the representativeness clone who contains albumin fusion constructs of the present invention be preserved in American type culture collection (referred to herein as
). In addition, may recover the albumin fusion constructs of appointment from the preservation thing by the technology that known in the art and this paper describe elsewhere.
Be positioned at 10801 University Boulevard, Manassas, Virginia 20110-2209, USA. Be used for the clause preparation of the microbial preservation budapest treaty of proprietary program according to international recognition
The preservation thing.
At an embodiment, the invention provides the polynucleotide of coding albumin fusion proteins, described albumin fusion proteins comprises or is made up of following alternatively: human cytokines and serum albumin albumen.At further embodiment, the invention provides a kind of albumin fusion proteins, it comprises or is made up of following alternatively: human cytokines and serum albumin albumen.At another embodiment, the invention provides a kind of albumin fusion proteins by the polynucleotide encoding described in the table 2, it comprises or is made up of following alternatively: human cytokines and serum albumin albumen.At further embodiment, the invention provides a kind of polynucleotide of the albumin fusion proteins of encoding, the sequence of described albumin fusion proteins is shown in the SEQ ID NO:Y in the table 2.At other embodiment, the invention provides albumin fusion proteins, it comprises or is made up of following alternatively: the fragment of the bioactive and/or therapeutic activity of human cytokines and serum albumin albumen.At other embodiment, the invention provides albumin fusion proteins, it comprises or is made up of following alternatively: the variant of the bioactive and/or therapeutic activity of human cytokines and serum albumin albumen.At other embodiment, the serum albumin protein ingredient of albumin fusion proteins is the maturing part of serum albumin.The present invention also comprises the polynucleotide of these albumin fusion proteins of encoding.
At further embodiment, the invention provides albumin fusion proteins, it comprises or is made up of following alternatively: the fragment of the bioactive and/or therapeutic activity of human cytokines and serum albumin.At further embodiment, the invention provides albumin fusion proteins, it comprises or is made up of following alternatively: the variant of the bioactive and/or therapeutic activity of human cytokines and serum albumin.At other embodiment, the human cytokines of albumin fusion proteins partly is the maturing part of human cytokines.At further embodiment, the human cytokines of albumin fusion proteins partly is the extracellular soluble foreign lands of human cytokines.At optional embodiment, the human cytokines of albumin fusion proteins partly is the activity form of human cytokines.The present invention also comprises the polynucleotide of these albumin fusion proteins of encoding.
At further embodiment, the invention provides a kind of albumin fusion proteins, it comprises or is made up of following alternatively: the fragment or the variant of the fragment of the bioactive and/or therapeutic activity of human cytokines or the bioactive and/or therapeutic activity of variant and serum albumin.At other embodiment, the invention provides a kind of albumin fusion proteins, it comprises or is made up of following alternatively: the maturing part of human cytokines and the maturing part of serum albumin.The present invention also comprises the polynucleotide of these albumin fusion proteins of encoding.
Human cytokines
As mentioned above, polynucleotide encoding albumen of the present invention, it comprises or is made up of following alternatively: at least one fragment of human cytokines or at least one fragment or the variant of variant and human serum albumin, they for example merge interconnection by heredity.
Other embodiment comprises the polynucleotide of encoding proteins, and described albumen comprises or is made up of following alternatively: at least one fragment of human cytokines or at least one fragment or the variant of variant and human serum albumin, they are connected to each other by chemical bond.
" human cytokines " used herein refers to have one or more treatments and/or bioactive albumen, polypeptide, antibody, peptide or its fragment or variant.The human cytokines that the present invention includes includes but not limited to, albumen, polypeptide, peptide, antibody and biological substance (biologics).(term peptide, albumen and polypeptide use interchangeably at this paper).Expect that especially term " human cytokines " comprises antibody and its fragment and variant.Therefore albumen of the present invention can contain at least one fragment of human cytokines or at least one fragment or the variant of variant and/or antibody.In addition, term " human cytokines " can refer to the correlative of the endogenous or natural generation of human cytokines.
Polypeptide or " therapeutic activity " albumen of performance " therapeutic activity " refer to polypeptide, and it has one or more known organisms relevant with human cytokines (for example one or more human cytokines as herein described or other treatment albumen known in the art) and learns and/or therapeutic activity.As non-limitative example, " human cytokines " is the albumen that can be used for treating, preventing or alleviate disease, disease or illness.As non-limitative example, " human cytokines " can be specificity in conjunction with the albumen of particular cell types (normal (as lymphocyte) or abnormal as (cancerous cell)) and therefore can be used for specifically target compound (medicine or cytotoxic agent) in the sort of cell type.
At an embodiment, " human cytokines " part that can be comprised by albumin fusion proteins of the present invention is an interferon.At another embodiment, " human cytokines " of albumin fusion proteins of the present invention part is an interferon-ALPHA.At other embodiment, can be included but not limited to interferon beta, interferon gamma or interferon ω by " human cytokines " part that albumin fusion proteins of the present invention comprises.
At other embodiment, the interferon heterozygote also can be blended in albuminised amino or carboxyl terminal to form interferon heterozygote albumin fusion proteins.Interferon heterozygote albumin fusion proteins can have enhanced interferon activity or have the interferon activity of inhibition alternatively, such as antiviral response, the growth of adjusting cell and adjusting immunne response (Lebleu etc., PNAS USA, 73:3107-3111 (1976); Gresser etc., Nature, 251:543-545 (1974); And Johnson, Texas Reports Biol Med, 35:357-369 (1977)).Every kind of interferon heterozygote albumin fusion proteins can be used for treatment, prevention or alleviates viral infection (as, hepatitis (as HCV); Or HIV), multiple sclerosis or cancer.
At an embodiment, the interferon heterozygote of interferon heterozygote albumin fusion proteins partly comprises interferon-ALPHA-interferon-ALPHA heterozygote (this paper is called α-α heterozygote).For example the α of interferon heterozygote albumin fusion proteins-α heterozygote part is formed or is comprised alternatively by following, is blended in the interferon-ALPHA A of interferon-ALPHA D.At further embodiment, the A/D heterozygote merges the common BgIII restriction enzyme site at interferon-ALPHA D, wherein the N-end portion of A/D heterozygote is corresponding to the amino acid/11-62 of interferon-ALPHA A, and the C-end portion is corresponding to the aminoacid 64-166 of interferon-ALPHA D.For example, this A/D heterozygote can comprise following aminoacid sequence:
CDLPQTHSLGSRRTLMLLAQMRX
1ISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFTTKDSSAAWDED LLDKFCTELYQQLNDLEACVMQEERVGETPLMNX
2DSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIMRSLSLSTNLQERLRRKE (SEQ ID NO:99), wherein X
1Be R or K, X
2Be A or V.
At other embodiment, the A/D heterozygote merges at common PvuIII restriction enzyme site, and wherein the N-end portion of A/D heterozygote is corresponding to the amino acid/11-91 of interferon-ALPHA A, and the C-end portion is corresponding to the aminoacid 93-166 of interferon-ALPHA D.For example, this A/D heterozygote can comprise following aminoacid sequence:
CDLPQTHSLGSRRTLMLLAQMRX
1ISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDET LLDKFYTELYQQLNDLEACVMQEERVGETPLMNX
2DSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIMRSLSLSTNLQERLRRKE (SEQ ID NO:100), wherein X
1Be R or K, second X
2Be A or V.These heterozygotes are further described in United States Patent (USP) the 4th, 414, and No. 510, it is incorporated into its integral body by reference at this.
At other embodiment, the α of interferon heterozygote albumin fusion proteins-α heterozygote part is formed or is comprised alternatively by following, is blended in the interferon-ALPHA A of interferon-ALPHA F.At further embodiment, the A/F heterozygote merges at common PvuIII restriction enzyme site, and wherein the N-end portion of A/F heterozygote is corresponding to the amino acid/11-91 of interferon-ALPHA A, and the C-end portion is corresponding to the aminoacid 93-166 of interferon-ALPHA F.For example, this A/F heterozygote can comprise following aminoacid sequence:
CDLPQTHSLGSRRTLMLLAQMRXISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVL HEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDMEACVIQEVGVEETPLMN VDSILAVKKYFQRITLYLTEKKYSPCAWEVVRAEIMRSFSLSKIFQERLRRKE (SEQ ID NO:101), wherein X is R or K.These heterozygotes are further described in United States Patent (USP) the 4th, 414, and No. 510, it is incorporated into its integral body by reference at this.At further embodiment, the α of interferon heterozygote albumin fusion proteins-α heterozygote part is formed or is comprised that alternatively interferon-ALPHA A is blended in interferon-ALPHA B by following.At other embodiment, the A/B heterozygote merges at common PvuIII restriction enzyme site, and wherein the N-end portion of A/B heterozygote is corresponding to the amino acid/11-91 of interferon-ALPHA A, and the C-end portion is corresponding to the aminoacid 93-166 of interferon-ALPHA B.For example, this A/B heterozygote can comprise aminoacid sequence:
CDLPQTHSLGSRRTLMLLAQMRX
1ISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDET LLDKFYTELYQQLNDLEX
2X
3X
4X
5QEVGVIESPLMYEDSILAVRKYFQRITLYLTEKKYSSCAWEVVRAEIMRSFSLSIN LQKRLKSKE (SEQ ID NO:102), wherein X
1Be R or K, X
2To X
5Be SCVM or VLCD.These heterozygotes are further described in United States Patent (USP) the 4th, 414, and No. 510, it is incorporated into its integral body by reference at this.
At another embodiment, the interferon heterozygote of interferon heterozygote albumin fusion proteins partly comprises interferon beta-interferon-ALPHA heterozygote (being called β-α heterozygote herein).For example, the β of interferon heterozygote albumin fusion proteins-α heterozygote part is formed or is comprised alternatively by following, is blended in the interferon beta-1 of interferon-ALPHA D (being also referred to as Alfacon-1).At further embodiment, merge β-1/ α D heterozygote, wherein the N-end portion is corresponding to the amino acid/11-73 of interferon beta-1, and the C-end portion is corresponding to the aminoacid 74-167 of interferon-ALPHA D.For example, this β-1/ α D heterozygote can comprise aminoacid sequence:
MSYNLLGFLQRS SNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFA IFRQDSSAAWDEDLLDKFCTELYQQLNDLEACVMQEERVGETPLMNXDSILAVKKY FRRITLYLTEKKYSPCAWEVVRAEIMRSLSLSTNLQERLRRKE (SEQ ID NO:103), wherein X is A or V.These heterozygotes are further described in United States Patent (USP) the 4th, 758, and No. 428, it is incorporated into its integral body by reference at this.
At another embodiment, the interferon heterozygote of interferon heterozygote albumin fusion proteins partly comprises interferon-ALPHA-interferon beta heterozygote (being called the alpha-beta heterozygote herein).For example, the alpha-beta heterozygote of interferon heterozygote albumin fusion proteins part is formed or is comprised alternatively by following, is blended in the interferon-ALPHA D (being also referred to as Alfacon-1) of interferon beta-1.At further embodiment, merge α D/ β-1 heterozygote, wherein the N-end portion is corresponding to the amino acid/11-73 of interferon-ALPHA D, and the C-end portion is corresponding to the aminoacid 74-166 of interferon beta-1.For example, this α D/ β-1 heterozygote can have aminoacid sequence:
MCDLPETHSLDNRRTLMLLAQMSRISPSSCLMDRHDFGFPQEEFDGNQFQKAPAISVLHELIQQIFNLFTTKDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN(SEQ?ID?NO：104)。These heterozygotes are further described in United States Patent (USP) the 4th, 758, and No. 428, it is incorporated into its integral body by reference at this.
At further embodiment, the interferon heterozygote of interferon heterozygote albumin fusion proteins part can comprise other combination of α-interferon-alpha heterozygote, alpha-beta interferon heterozygote and β-interferon-alpha heterozygote.At other embodiment, the interferon heterozygote of interferon heterozygote albumin fusion proteins part can be modified to comprise sudden change, replacement, disappearance or the interpolation to the aminoacid sequence of interferon heterozygote.Can carry out this modification to interferon heterozygote albumin fusion proteins, for example to improve the preparation level, increase stability, increase or reduce active or to give new biological property.
Above-mentioned interferon heterozygote albumin fusion proteins and the host cell and the carrier that contain the polynucleotide of coding said polypeptide comprise in the present invention.At an embodiment, the interferon heterozygote albumin fusion proteins of aforesaid polynucleotide encoding has the shelf life of prolongation.At other embodiment, the interferon heterozygote albumin fusion proteins of aforesaid polynucleotide encoding has (or in pharmaceutical composition) more stable external and/or activity in vivo in the longer serum half-life and/or solution than the corresponding interferon heterozygote molecule that does not merge.
At another non-limitative example, " human cytokines " is biologically active, is particularly useful for treating, preventing or alleviate the bioactive albumen of disease, illness or infection.Bioactive non-comprising property (non-inclusive) tabulation that human cytokines can have comprises: suppress the HIV-1 infection cell, stimulate enterocyte propagation, reduce the enterocyte permeability, stimulate insulin secretion, induce bronchiectasis and vasodilation, suppress aldosterone and renin secretion, blood pressure regulation, promote neure growth, enhance immunity is replied, strengthen inflammation, appetite-suppressing, or following " biological activity " part described in and/or bioactive any or multiple at table 1 pair TA albumen disclosed (row 2).
At an embodiment, IFN-α-HSA fusant be used for suppressing being divided into category-A-Filovirus (Filo) (Ebola virus) (Ebola), arenavirus (Arena) (Pichende), category-B-batch film virus (Toga) (VEE) or the virokine (viral agent) of C class-Bunyavirus (Bunya) (Pan Tatuoruo virus) (Punto toro), banzi virus (Flavi) (yellow fever virus, west nile virus (West Nile)).For example, CPE suppresses, dimethyl diaminophenazine chloride dyes and the virus yield detection is used to estimate the antiviral activity of fusion at the INF-in HAS downstream α (CID 3165 albumen).Estimate pharmacokinetics and the pharmacodynamics activity of CID 3165 albumen in stump-tailed macaque and human subject.The result represents all RNA viruses of estimating are reached antiviral activity with favourable safety index.During CPE detects the IC50 value from＜0.1ng/ml (Pan Tatuoruo virus of A) to 19ng/ml (VEE).Stump-tailed macaque, the 3165 proteic half-life of CID are 90 hours, and to reach 14 days after administration be detectable.In human subject, CID 3165 albumen are safe and well-tolerated.C behind the single injection dosage
MaxBe and dose proportional.Average C in the 500ug group
MaxBe 22ng/ml, average t
1/2It is 150 hours.Be administered once in every 2-4 week or more by the pharmacokinetics support.Most of curees observe the antiviral response to hepatitis C in single injection group (120-500ug).
At further embodiment, IFN-α-HSA fusant is used for the treatment of the patient who suffers from hepatitis C and/or chronic hepatitis C infection (HCV).Interferon-ALPHA, also being called interferon alfa or LeIF is the standard treatment of treatment subject suffering from HCV infection.Term " interferon-ALPHA " refers to have the family of the height homology related polypeptide of antiviral activity.The interferon-ALPHA of IFN-α-HSA fusant part is formed or is comprised alternatively by following: any interferon-ALPHA known in the art or its fragment.The non-limitative example of the interferon-ALPHA of IFN-α of the present invention-HSA fusion rotein part includes but not limited to be disclosed in the interferon-ALPHA albumen of the human cytokines row of table 1.In specific implementations, interferon-ALPHA part is formed or is comprised alternatively by following: any commercially available form that obtains of Intederon Alpha-2a, Interferon Alpha-2b, interferon c, Interferon Alfacon-1 (consensusinterferon), interferon alfacon-1, interferon alfa-n1, Alferon N, interferon-ALPHA, such as for example
A (Schering Corp., Kenilworth, N.J.),
A (Hoffman-La Roche, Nutley, N.J.), the Berofor interferon-ALPHA (BoehringerIngelheim Pharmaceutical, Inc., Ridgefied, Conn.), OMNIFERON
TM(Viragen, Inc., Plantation, FL), MULTIFERON
TM(Viragen, Inc., Plantation, FL),
(GlaxoSmithKline, London, Great Britian),
(Amgen, Inc., Thousands Oaks, CA),
(Sumitomo, Japan),
(Nautilus Biotech, France), MAXY-ALPHA
TM(Maxygen, Redwood City, CA/Hoffman-La Roche, Nutley, N.J.) or the interferon-ALPHA product of any purification or its fragment.At further embodiment, the interferon-ALPHA of IFN-α-HSA fusion rotein part is formed or is comprised alternatively by following: be modified to or be formulated as to be used to prolong and discharge or the interferon-ALPHA of controlled release.For example, interferon-ALPHA part is by the following interferon-ALPHA of forming or comprising commercially available obtainable prolongation release or controlled release alternatively, include but not limited to interferon-' alpha '-XL (Flamel Technologies, France) and LOCTERON
TM(BioLex Therapeutics/OctoPlus, Pittsboro, NC).At other embodiment, the interferon-ALPHA of IFN-α-HSA fusion rotein part can be modified by connecting chemical part.For example, the interferon-ALPHA part can be modified by Pegylation.Therefore, at other embodiment, the interferon-ALPHA of IFN-α-HSA fusion rotein part is formed or is comprised alternatively by following: the Pegylation form of Intederon Alpha-2a, 2b or Interferon Alfacon-1, and include but not limited to commercially available obtainable pegylated interferon alfa, such as for example, PEG-
(Schering Corp., Kenilworth, N.J.),
(Hoffman-La Roche, Nutley, N.J.), PEG-OMNDFERON
TM(Viragen, Inc., Plantation, FL) or its fragment.Yet " IFN-α-HSA " used herein fusant refers to be blended in any interferon-ALPHA albumen known in the art or its segmental HSA.
Subject suffering from HCV infection can be divided into two classes based on the interferon therapy that whether contacted treatment HCV infects before this." patient of first treatment " or " first patient " is the patient who never treated with interferon therapy." live through the patient of treatment " or " patient who lives through " treated or at present just with the patient of interferon therapy treatment with interferon therapy." nonresponder " is the patient who lives through treatment, treated with interferon therapy before this but failed to reach main treatment terminal point, such as early stage virus quantity reduce (early viral loadreduction) (EVR) or treat whole end reply (end-of-treatment response, ETR)." recidivist " is the patient who lives through treatment, treats with interferon therapy before this and has the main treatment terminal point that reaches such as EVR or ETR, but at time point afterwards HCV is become the positive.Yet " HCV patient " used herein refers to subject suffering from HCV infection first treatment or that live through treatment.In addition, " the HCV patient " of " living through " used herein is nonresponder or recidivist.
In addition, hepatitis C virus can be divided into the several genes type, and four kinds of genotype are that genotype 1,2,3 or 4 is the most general.Usually, HCV infection patient's hepatitis C virus comprises the individual gene type.Yet hepatitis virus can comprise two or more genotypic combinations.In addition, the genotype of hepatitis C virus also can be the genotypic variant of known HCV.At further embodiment, HCV patient's hepatitis C virus is genotype 1,2 or 3 or its variant.Yet " HCV " used herein refers to any genotypic hepatitis C virus, or its combination or variant.
The patient's that suffers from HCV standard care scheme comprised with interferon-ALPHA combination anti-viral agent such as ribavirin (ribavirin) treat.Usually, once a day, twice or use interferon-ALPHA once in a week weekly, use that ribavirin continued at least 24 weeks, 48 weeks or longer every day.Yet recent research has also used interferon-ALPHA combination other antiviral agent known in the art with treatment HCV.Therefore at further embodiment, IFN-α-HSA fusant can be separately or with antiviral agent such as ribavirin combined administration for example in HCV patient.At another embodiment, IFN-α-HSA fusant a kind of, two kinds, three kinds, four kinds or more kinds of antiviral agent capable of being combined (such as for example, ribavirin and/or other antiviral agent) are applied to HCV patient.
As mentioned above, the patient's that suffers from HCV standard care is comprised with the agent of interferon combination anti-viral such as ribavirin therapy, with once a day, twice or weekly drug dosage schedule weekly.Yet interferon therapy is with remarkable side effect, includes but not limited to influenza-like symptom, tired, insomnia, headache, feels sick and depressed, and they can seriously limit the toleration of patient to treatment.What at present standard treatment needed frequently uses the reduction quality of life that interferon causes prolonging period, causes the patient to baffle and can not finish treatment.The combination of frequently using and reducing quality of life has caused patient's therapy discontinued of significant number.Therefore clearly need be with the therapeutic scheme of the improvement of frequent drug administration plan more not, thus its quality of life influence to the patient reduces and bigger encouragement to compliance is provided.
The main target of administration therapeutic method is as early as possible virus levels to be reduced to be lower than quantitative limit and to keep the virus feminine gender subsequently after whole treatment neutralization is cured.In the HCV field, it is to continue the strong positive indication signal (positive predictor) that virusology is replied (RVR) that the quick virusology that is lower than quantitative limit definition by the HCV rna level in the 4th week is replied (rapid virologic response).Reaching RVR the 4th week and will after therapeutic scheme is finished, keep the HCVRNA feminine gender probably up to keeping this negative patients the 12nd week.Therefore, need in this area and will reach HCV RNA feminine gender in the 4th week and reply the HCV patient's of (SVR) or healing number maximization to continuing virusology up to keeping this feminine gender the 12nd week.
The invention provides drug dosage schedule, it combines the needs of frequent drug administration more not and at the needs of the 4th week maximization RVR.At an embodiment, the invention provides a kind of drug dosage schedule, comprise that using interferon or combination anti-viral agent more separately to HCV patient uses the interferon short-term, does not use more subsequently more continually.In specific implementations, drug dosage schedule comprise short-term once a day, twice weekly, weekly or per 2 weeks once use interferon separately or interferon is used in the combination anti-viral agent, subsequently the persistent period of therapeutic scheme in per 2 weeks once, per 3 weeks once or per 4 weeks once use the agent of interferon combination anti-viral.At another embodiment, described interferon is an interferon-ALPHA.As mentioned above, the interferon-ALPHA that the present invention includes comprises any interferon-ALPHA known in the art or its fragment or variant.Therefore at another embodiment, use the long lasting interferon-ALPHA of the interferon that includes but not limited to IFN-α-HSA fusion rotein or Pegylation.Defined as the present invention, use the described short-term that interferon-ALPHA or combination anti-viral agent use interferon-ALPHA separately to include but not limited to no more than 1,2,3,4,5,6,7 or 8 weeks.And, describedly include but not limited at least 16 weeks, at least 20 weeks, at least 24 weeks, at least 30 weeks, at least 36 weeks, at least 40 weeks, at least 46 weeks, at least 48 weeks, at least 50 weeks, at least 60 weeks or longer more over a long time.In specific implementations, use interferon-ALPHA or combination anti-viral agent once in a week separately and use interferon-ALPHA and continue 2-4 week, subsequently every month persistent period of therapeutic scheme once.In other specific implementations, once use interferon-ALPHA or combination anti-viral agent per 2 weeks separately and use interferon-ALPHA and continue 2-4 week, subsequently every month persistent period of therapeutic scheme once.At other embodiment, short-term with than low dosage or heavy dose use interferon-ALPHA, over a long time to use than low dosage.
As mentioned above, CID 3165 proteic pharmacokineticss (pharmokinetics) support every 2-4 week once or once drug dosage schedule more of a specified duration.Therefore at further embodiment, every 2-4 week once alone or in combination the antiviral agent of effective dose use IFN-α-HSA fusant and treat HCV patient.At another embodiment, a kind of, two kinds, three kinds, four kinds or the more kinds of antiviral agent that once make up effective dose every 2-4 week are used IFN-α-HSA fusant and are treated HCV patient.At other embodiment, per 4 weeks, an IFN-α-HSA fusant was applied to HCV patient.At other embodiment, be applied to HCV patient more than per 4 all IFN-α-HSA fusants.At other embodiment, to per 4 weeks of HCV patient once or the IFN-α-HSA fusant of using more, wherein treatment also comprises a kind of, two kinds, three kinds, four kinds or the more kinds of antiviral agent of using effective dose.
At another embodiment, whenever biweekly or around every once continued at least 24 weeks with IFN-α-HSA fusant treatment HCV patient.At further embodiment, whenever biweekly or around every once continued for 48 weeks with IFN-α-HSA fusant treatment HCV patient.At other embodiment, described HCV patient infection genotype 1.At another embodiment, described HCV patient infection genotype 2 or 3.At other embodiment, make up at least a, two kinds, three kinds or four kinds antiviral agent that include but not limited to ribavirin and use IFN-α-HSA fusant.
Therefore at further embodiment, with every biweekly and around every once combination with IFN-α-HSA fusant treatment HCV patient.At an embodiment, whenever biweekly with IFN-α-HSA fusant treatment HCV patient twice, every period in the treatment of remainder subsequently when therapeutic scheme begins all around once with EFN-α-HSA fusant treatment.At further embodiment, this treatment with IFN-α-HSA fusant is at least a, two kinds, three kinds or four kinds of antiviral agent that include but not limited to ribavirin of combination.At other embodiment, this treatment continues totally 24 or 48 weeks.
At another embodiment, IFN-α-HSA fusant can be used as the low dosage list treatment of keeping treatment to HCV.At further other embodiment, IFN-α-HSA fusant ribavirin capable of being combined and one or more other antiviral agent use with treatment HCV.Alternatively, at another embodiment, IFN-α-HSA fusant a kind of, two kinds, three kinds or more kinds of antiviral agent that is different from ribavirin capable of being combined are with treatment HCV.
At other embodiment, IFN-α-HSA fusant can be used for treating other viral infection of other viral infection or combination HCV infection.For example, at an embodiment, IFN-α-HSA fusant can be used for treating hepatitis B (HBV).At other embodiment, DFN-α-HSA fusant can be used for treating human papillomavirus (HPV).At further embodiment, IFN-α-HSA fusant can be used for treating cancer, includes but not limited to hairy cell (hairy cell leukemia), malignant melanoma, follicular lymphoma (follicular lymphoma), chronic granulocytic leukemia (chronicmyelogenous leukemia), AIDS relevant Kaposi sarcoma (AIDS related Kaposi ' sSarcoma), multiple myeloma or renal cell carcinoma.
Thereby the main target of any therapeutic scheme is the treatment effectiveness healing patient who treats and eradicate disease or illness as early as possible and keep eradicating described disease or illness.And, to the rapid answer of the medicine positive indication signal of final therapeutic outcome normally.Yet interferon therapy suffers from the patient of disease or illness can be with remarkable side effect, includes but not limited to influenza-like symptom, tired, insomnia, headache, feels sick and depressed.These side effects patient of interferon therapy causes patient's therapy discontinued of significant number to the toleration of therapeutic scheme.Therefore need new design so that the therapeutic scheme of the early stage effectiveness of interferon therapy and toleration maximum.
The invention provides therapeutic scheme, it comprises to the patient who suffers from disease or illness uses interferon, wherein uses interferon with more frequent drug dosage schedule to the patient in short-term, and more over a long time with more not frequent drug dosage schedule.Administration frequency used herein refers to that the patient accepts the number of times that interferon is used in special time period.For example, drug dosage schedule can include but not limited to, once a day, twice of every day, once in a week, twice weekly, on every Wendesdays time, whenever biweekly, per three weeks once, every around once or the like.Therefore more frequent drug dosage schedule used herein is a kind of drug dosage schedule, and wherein the patient is than accepting interferon more frequently with more not frequent drug dosage schedule.When interferon therapy begins, increase administration frequency in order to interferon " preceding using (front-load) " patient so that early stage render a service maximum.Reduce administration frequency then subsequently and increase toleration period in order to the side effect that keeps in all the other of therapeutic scheme rendeing a service times and take place by the result who reduces by interferon therapy.Wherein more frequent drug dosage schedule depends on the disease or the illness of being treated useful period.So as herein defined, " short-term " refers in order to make interferon eradicate the maximum required time period of early stage effectiveness of disease of patient or illness.More " more over a long time " of frequent drug administration do not refer to after interferon maximum effectiveness period remaining treatment period.For example, described short-term, can include but not limited to, 1,2,3,4,5,6,7 or 8 weeks.Describedly can include but not limited at least 16 weeks, at least 20 weeks, at least 24 weeks, at least 30 weeks, at least 36 weeks, at least 40 weeks, at least 46 weeks, at least 48 weeks, at least 50 weeks, at least 60 weeks or longer more over a long time.
Embodiment other the invention provides therapeutic scheme, and it comprises with acceptable safety uses the interferon short-term of maximal dose to the patient who suffers from disease or illness, and the interferon of using maintenance dose subsequently more over a long time.Interferon with maximal dose of acceptable safety used herein is the known interferon that the patient is had the maximum dose level of acceptable safety.In specific implementations, interferon with maximal dose of acceptable safety includes but not limited to, be at least the dosage of standard dose that the present invention gives, or at least one times, twice, three times, four times, five times, six times, seven times, octuple, nine times or ten times of dosage that are higher than standard dose.The dosage of interferon can be determined by standard known in the art.As non-limitative example, dosage can be based on body weight (as, μ g/kg) or based on total compound concentration as described below.And maintenance dose used herein refers to be less than maximal dose with acceptable safety, and can keep the dosage that interferon is renderd a service the patient.For example, maintenance dose can include but not limited to standard dose or at least two/one, 1/3rd, 1/4th, 1/8th, one times, twice, three times, four times, five times, six times of described interferon or be less than the dosage of the maximal dose with acceptable safety more.What as herein defined, " short-term " maximal dose of referring to allow to have acceptable safety made that interferon eradicates disease of patient or illness renders a service the maximum required time period in early days." more over a long time " refers to after interferon maximum effectiveness period remaining treatment period.For example, described short-term, can include but not limited to, 1,2,3,4,5,6,7 or 8 weeks.Describedly can include but not limited at least 16 weeks, at least 20 weeks, at least 24 weeks, at least 30 weeks, at least 36 weeks, at least 40 weeks, at least 46 weeks, at least 48 weeks, at least 50 weeks, at least 60 weeks or longer more over a long time.
At further embodiment, the invention provides a kind of scheme, comprise to the patient who suffers from disease or illness and use maximal dose interferon short-term with acceptable safety, use the interferon of maintenance dose subsequently more over a long time, wherein use maximal dose interferon with acceptable safety with the drug dosage schedule more frequent than the interferon of maintenance dose.
" therapeutic activity " used herein or " activity " can refer to activity, and its effect is with consistent in human desired therapeutic result, or refers to the effect of the expectation in non-human mammal or other species or organism.But body is interior or the in-vitro measurements therapeutic activity.For example, but detect the effect of expectation in the cell culture.This many human cytokines external or that cell culture detects described in this area can get usually.The example that detects includes but not limited in this paper embodiment part or " example is active to be detected " of table 1 is listed as (row 3) described detection.
Corresponding to the human cytokines of the human cytokines of albumin fusion proteins of the present invention part, such as cell surface and secretory protein, normally one or more oligosaccharide groups modify by connecting.But being called the proteic physical features of glycosylated this modification appreciable impact also can be important to protein stability, secretion and location.Glycosylation occurs in along the ad-hoc location of polypeptide main chain.The glycosylation of two kinds of main types is arranged usually: be characterized as the glycosylation of the oligosaccharide of O-connection, oligosaccharide is connected in serine or threonine residues; With the glycosylation that is characterized as the oligosaccharide that N-is connected, oligosaccharide is connected in the asparagine residue in Asn-X-Ser or the Asn-X-Thr sequence, and wherein X can be the outer any aminoacid of proline.N-n acetylneuraminic acid n (being also referred to as sialic acid) is the terminal residue of the oligosaccharide that is connected with O-of N-connection normally.Influence in the chain sugar unit number and character such as variablees such as protein structure and cell types in the different glycosylation site.The also common multiple glycosylation isomer in same site in the designated cell type.
Corresponding to the human cytokines of the human cytokines of albumin fusion proteins of the present invention part with and analog and variant can be modified so that change glycosylation in one or more sites as the result who handles their nucleotide sequences, this manipulation is to be expressed in wherein host cell or because other situation of their expression by them.For example, can or introduce glycosylation site by cancellation and prepare the glycosylation isomer, replace or the disappearance amino acid residue as passing through, replace agedoite such as glutamine, or not glycosylated recombiant protein can prepare by expressing protein in can not their host cell of glycosylation (as escherichia coli (E.coli) or glycosylation-defective yeast).These methods are more detailed description and be known in the art hereinafter.
Human cytokines, especially be disclosed in the human cytokines of table 1, be well known in the art and can derive from public database with its nucleic acid and aminoacid sequence, such as Chemical Abstracts ServicesDatabases (as, CAS registry), GenBank with data base such as the GenSeq (as Derwent) that provides is provided.The Exemplary core nucleotide sequence of human cytokines of polynucleotide of the present invention of can be used for deriving is shown in the row 7 of table 2, " SEQ ID NO:X ".The sequence that is shown as SEQ ID NO:X can be the wild type polynucleotide sequence of coding TA albumen (as total length or sophisticated), or this sequence of certain situation can be described wild type polynucleotide sequence variant (as, the polynucleotide of encoding wild type human cytokines, the DNA sequence of wherein said polynucleotide is optimised, for example is used for expressing at specific species; Or the polynucleotide of this wild type human cytokines variant of encoding (are directed mutants; Allelic variant)).Use is shown as the sequence of SEQ ID NO:X and derives with the construct described in the delegation in those skilled in the art's ability.For example, if SEQ ID NO:X is corresponding to full-length proteins, but only this proteic part is used to produce concrete CID, depends on increase this concrete fragment and it is cloned into suitable carrier in those skilled in the art's ability of Protocols in Molecular Biology such as PCR.
Human cytokines other human cytokines partly corresponding to albumin fusion proteins of the present invention includes but not limited to, is disclosed in one or more human cytokines or the peptide of " human cytokines X " row (row 1) of table 1, or its fragment or variant.
Table 1 provide corresponding to albumin fusion proteins of the present invention or by the albumin fusion proteins of polynucleotide encoding of the present invention the non exhaustive tabulation of human cytokines of human cytokines part.First row " human cytokines X " disclose the human cytokines molecule, and it can have the bracket that contains proteic formal name used at school and trade name subsequently, and this albumen comprises or is made up of following alternatively: human cytokines molecule or its fragment or variant." human cytokines X " used herein can refer to the individual treatment protein molecular, or refers to whole group the human cytokines relevant with the TA protein molecular that is disclosed in these row." biological activity " row (row 2) are described the biological activity relevant with this human cytokines molecule.Row 3 " example is active to be detected " provide a description and can be used for detecting human cytokines: X or comprise the treatment of human cytokines X (or its fragment) albumin fusion proteins partly and/or the list of references of bioactive detection.Incorporate the listed corresponding bioactive corresponding active associated description of especially describing in the list of references (for example seeing wherein method part) that detects of " biological activity " row that are used for detection table 1 by reference into its integral body at this at every piece of list of references that " example is active to be detected " row are quoted.The 4th row " indication: Y " have been described and can or have been comprised albumin fusion proteins treatment, prevention, the diagnosis of human cytokines X (or its fragment) part and/or disease, illness, infection and/or the disease of alleviating by human cytokines X." construct ID " row (row 5) provide the link to the exemplary albumin fusion constructs of the coding albumin fusion proteins that is disclosed in table 2, and this albumin fusion proteins comprises or is made up of following alternatively: the human cytokines X that is mentioned (or its fragment) part.
Table 2 provides the non exhaustive tabulation of polynucleotide of the present invention, and it comprises or is made up of following alternatively: the nucleic acid molecules of coding albumin fusion proteins.First row " fusant No. " provide the fusant numbering to every kind of polynucleotide.Row 2 " construct ID " provide the unique number identifier to every kind of polynucleotide of the present invention.Construct ID can be used for the polynucleotide of identification code albumin fusion proteins, this albumin fusion proteins comprises or is made up of following alternatively: corresponding to the human cytokines part of the TA albumen that is listed in table 1 corresponding line: X, wherein this construct ID is listed in row 5." construct title " row (row 3) provide the title of specifying albumin fusion constructs or polynucleotide.
The 4th row " description " provide the roughly description of specifying the albumin fusion constructs in the table 2, the 5th row " expression vector " are listed the carrier that is cloned into polynucleotide, and these polynucleotide comprise or are made up of following alternatively: coding is specified the nucleic acid molecules of albumin fusion proteins.Carrier is the other places that obtain or be described in known in the art and commercially available.For example, as described in an embodiment, comprise or can be assembled into cloning vehicle easily by following " expression cassette " that one or more are formed alternatively and move into optional carrier such as the expression vector that for example comprises Yeast expression carrier for example or mammalian expression vector subsequently: (1) coding is specified polynucleotide, (2) targeting sequencing, (3) promoter region and (4) transcription terminator of albumin fusion proteins.At an embodiment,, will comprise or the expression cassette be made up of the nucleic acid molecules of coding albumin fusion proteins alternatively is cloned into pSAC35 in saccharomyces cerevisiae (S.cervisiae), expressing.At another embodiment,, will comprise or be cloned into pC4 by the expression cassette formed of nucleic acid molecules of coding albumin fusion proteins alternatively in Chinese hamster ovary celI, expressing.At further embodiment, will comprise or the polynucleotide be made up of the nucleic acid molecules of the human cytokines part of coding albumin fusion proteins alternatively are cloned into pC4:HAS.At further embodiment again,, will comprise or the expression cassette be made up of the nucleic acid molecules of coding albumin fusion proteins alternatively is cloned into pEE12 in the NSO cell, expressing.Other available cloning vehicle and/or expression vector are known and within the scope of the present invention to those skilled in the art.
Row 6 " SEQ ID NO:Y " provide the full length amino acid sequence of albumin fusion proteins of the present invention.In most applications, the undressed form of the albumin fusion proteins that SEQ ID NO:Y demonstration is coded-in other words, SEQ ID NO:Y shows all by particular build body encoded signals sequence, HSA part and therapeutic part.Be all polynucleotide of coding SEQ ID NO:Y by what the present invention includes particularly.When these polynucleotide were used for from the cellular expression encoded protein, the natural secretion of cell and procedure of processing produced the albumen of the signal sequence that lacks the row 4 that are listed in table 2 and/or 11.The concrete aminoacid sequence of listed signal sequence is presented at subsequently in the description or is known in the art.Therefore some embodiments of the present invention comprise the albumin fusion proteins (it will lack the row 4 and/or 11 targeting sequencings that show of table 2) that cell produces.Also comprise in embodiments of the present invention be to comprise SEQ ID NO:Y and be not listed in the row 4 of table 2 and/or the polypeptide of 11 concrete targeting sequencing.Compositions of the present invention and pharmaceutical composition also comprise this two kinds of embodiments.And, with the unlike signal sequence replace being listed in the row 4 of table 2 and/or 11 signal sequence with the secretion of the albumin fusion proteins that promotes processing in those skilled in the art's ability, described unlike signal sequence is such as describing in description subsequently.
The 7th row " SEQ ID NO:X " provide the parent nucleotide sequence of the human cytokines polynucleotide partly of the coding appointment albumin fusion proteins of can therefrom deriving.At an embodiment, the parent nucleotide sequence of the polynucleotide of the human cytokines part of the coding albumin fusion proteins of can therefrom deriving comprises the wild type gene sequence of the human cytokines that shows in the coding schedule 1.At optional embodiment, the parent nucleotide sequence of polynucleotide of human cytokines part of coding albumin fusion proteins of can therefrom deriving comprises the variant or the derivant of the wild type gene sequence of the human cytokines that shows in the coding schedule 1, such as the synthetic codon optimized variant of the wild type gene sequence of the human cytokines of for example encoding.
The 8th row " SEQ ID NO:Z " provide the prediction translation of parent nucleotide sequence (SEQ ID NO:X).This parent sequence can be the concrete construct that is used to derive total length parent albumen, the proteic maturing part of parent, wild-type protein variant or fragment or can be used for producing the artificial sequence of the construct of describing.Those skilled in the art can use the aminoacid sequence that shows among the SEQ ID NO:Z to determine human cytokines provides which amino acid residue of the albumin fusion proteins of specifying the construct coding.And, use the sequence that is shown as SEQ ID NO:Z to derive with the construct described in the delegation fully in those skilled in the art's ability.For example, if SEQ ID NO:Z corresponding to full-length proteins, but only this albumen of part is used to produce concrete CID, relies on increase concrete fragment and it is cloned into suitable carrier in those skilled in the art's ability of Protocols in Molecular Biology such as PCR.
The amplimer that row 9 and 10 provide respectively " SEQ ID NO:A " and " SEQ ID NO:B " are the exemplary primers that is used to produce polynucleotide, and these polynucleotide comprise or specify the nucleic acid molecules of the human cytokines part of albumin fusion proteins to form by coding alternatively.In an embodiment of the invention, oligonucleotide primers with sequences (SEQ ID NO:A and/or B) that row 9 and/or 10 show is used for the polynucleotide of the human cytokines part of pcr amplification coding albumin fusion proteins, uses to comprise or alternatively by the following nucleic acid molecules of forming: the nucleotide sequence that the row 7 of corresponding line provide (SEQ ID NO:X) is as template DNA.PCR method is full-fledged in this area.Those of ordinary skills can be easy to envision and use other useful primer sequence.
At optional embodiment, oligonucleotide primers can be used for overlapping PCR reaction to produce sudden change in the template DNA sequence.PCR method is known in the art.
As shown in table 3, some albumin fusion constructs that are disclosed in the application are preserved in
Table 3
| Construct ID | The construct title | ATCC preserving number/date |
| 2249 | PSAC35:IFNa2-HSA is also referred to as pSAC23:IFN α 2- |
4 days October calendar year 2001 of PTA-3763 |
| 2343 | pSAC35.INV-IFNA2.HSA | PTA-3940 calendar year 2001 December 19 days |
| 2381 | pC4：HSA-IFNa2(C17-E181) | PTA-3942 calendar year 2001 December 19 days |
| 2382 | pC4：IFNa2-HSA | PTA-3939 calendar year 2001 December 19 |
| 3165 | PSAC35:HSA.IFNa is also referred to as |
PTA4670 JIUYUE in 2002 16 days |
May recover specified albumin fusion constructs from the preservation thing by the technology (seeing embodiment 10) that known in the art and this paper describe elsewhere.ATCC is positioned at 10801 University Boulevard, Manassas, Virginia 20110-2209, USA.The clause that is used for the microbial preservation budapest treaty of proprietary program according to international recognition prepares ATCC preservation thing.
At further embodiment of the present invention, comprise or can be moved into or " sub-clone " go into another carrier from carrier by following " expression cassette " that one or more are formed alternatively: (1) coding is specified polynucleotide, (2) targeting sequencing, (3) promoter region and (4) transcription terminator of albumin fusion proteins.Treat can be produced by well known method by the fragment of sub-clone, such as for example, pcr amplification (as, use oligonucleotide primers with the sequence that shows among SEQ IDNO:A or the B), and/or restriction enzyme digestion digestion.
At some embodiments, albumin fusion proteins of the present invention can have corresponding to therapeutic activity and/or bioactive therapeutic activity and/or biological activity corresponding to the human cytokines of the human cytokines of the albumin fusion proteins that is listed in table 1 corresponding line part.At further embodiment, the proteic fragment or the variant of the sequential coding that the SEQ ID NO:X row that this therapeutic activity protein part of albumin fusion proteins of the present invention is a table 2 show, and can have proteic therapeutic activity of corresponding treatment and/or biological activity.
Polypeptide and polynucleotide passage and variant
Fragment
The invention further relates to the fragment of the human cytokines described in the table 1, albumin albumen and/or albumin fusion proteins of the present invention.
The invention still further relates to the segmental polynucleotide of the human cytokines described in the coding schedule 1, albumin albumen and/or albumin fusion proteins of the present invention.
Even cause modifying or losing one or more biological functions of human cytokines, albumin albumen and/or albumin fusion proteins of the present invention from the one or more aminoacid of albumen N-terminal deletion, still can keep other therapeutic activity and/or functional activity (as, the ability of biological activity, poly, the ability of binding partner).For example, when being less than of complete polypeptide most of residues when the N-end removes, the polypeptide with N-terminal deletion will keep inducing and/or be incorporated into the ability of antibody of the polypeptide of the complete or mature form of identification usually.The specific polypeptide that lacks the N-terminal residue of complete polypeptide whether keep this immunocompetence can be easy to by customary method as herein described and in addition methods known in the art determine.Mutain with-terminal amino acid residue of a large amount of disappearances unlikely can keep some biologys or immunogenicity activity.In fact, mainly can cause immunne response usually by few peptide of forming to six amino acid residues.
Therefore, the polypeptide that comprises full-length proteins and have one or more residues disappearances corresponding to the fragment of the human cytokines of the human cytokines of albumin fusion proteins of the present invention part from the amino terminal of the aminoacid sequence of reference polypeptide (the human cytokines part of the albumin fusion proteins of human cytokines of promptly mentioning the table 1 or the polynucleotide described in the table 2 or albumin fusion constructs coding).Especially, the N-terminal deletion can be described by general formula m to q, wherein q be represent reference polypeptide (as, the human cytokines part of the albumin fusion proteins of polynucleotide described in the human cytokines of mentioning in the table 1 or the human cytokines of albumin fusion proteins of the present invention part or the table 2 or albumin fusion constructs coding) the complete integer (whole integer) of amino acid residue sum in, m is defined as any integer from 2 to q-6.The polynucleotide of these polypeptide of encoding are also included among the present invention.
In addition, the polypeptide that comprises full-length proteins and have one or more residues disappearances corresponding to the fragment of the serum albumin polypeptide of the albumin protein part of albumin fusion proteins of the present invention from the amino terminal of the aminoacid sequence of reference polypeptide (being the serum albumin part of the albumin fusion proteins of polynucleotide described in serum albumin or the table 2 or albumin fusion constructs coding).At some embodiments, the N-terminal deletion can be described by general formula m to 585, and wherein 585 is complete integers of representing amino acid residue total number in mature human's serum albumin (SEQID NO:1), and m is defined as any integer of from 2 to 579.The polynucleotide of these polypeptide of encoding are also included among the present invention.At other embodiment, the N-terminal deletion can be described by general formula m to 609, and wherein 609 is complete integers of representing amino acid residue total number in the human serum albumin of total length (SEQID NO:3), and m is defined as any integer of from 2 to 603.The polynucleotide of these polypeptide of encoding are also included among the present invention.
And, the fragment of albumin fusion proteins of the present invention comprise the total length albumin fusion proteins and from albumin fusion proteins (as the albumin fusion proteins of, polynucleotide described in the table 2 or albumin fusion constructs coding; Or have an albumin fusion proteins of the aminoacid sequence that is disclosed in table 2 row 6) the polypeptide of amino terminal with one or more residues disappearances.Especially, the N-terminal deletion can be described by general formula m to q, and wherein q is a complete integer of representing amino acid residue total number in the albumin fusion proteins, and m is defined as any integer from 2 to q-6.The polynucleotide of these polypeptide of encoding are also included among the present invention.
Also as mentioned above, though from reference polypeptide (as, human cytokines; Serum albumin albumen; Or albumin fusion proteins of the present invention) N-end or the one or more aminoacid of C-terminal deletion cause modifying or losing proteic one or more biological functions, still can keep other functional activity (as, the ability of biological activity, poly, the ability of binding partner) and/or therapeutic activity.For example, when being less than of complete or mature polypeptide most of residues when the C-end removes, the polypeptide with C-terminal deletion will keep inducing and/or be incorporated into the ability of antibody of the polypeptide of the complete or mature form of identification usually.The specific polypeptide that lacks the N-end of reference polypeptide and/or C-terminal residue whether keep therapeutic activity can be easy to by customary method as herein described and in addition methods known in the art determine.
The present invention further provides the polypeptide that the carboxyl terminal of the aminoacid sequence of the human cytokines (the human cytokines part of the albumin fusion proteins of human cytokines of promptly mentioning in the table 1 or the polynucleotide described in the table 2 or albumin fusion constructs coding) corresponding to the human cytokines of albumin fusion proteins of the present invention part has one or more residues disappearances.Especially, the C-terminal deletion can be described by general formula 1 to n, wherein n be from 6 to q-1 any complete integer and wherein q be the complete integer of representing amino acid residue total number in the reference polypeptide (as the human cytokines part of the albumin fusion proteins of the human cytokines mentioned in the table 1 or polynucleotide described in the table 2 or albumin fusion constructs coding).The polynucleotide of these polypeptide of encoding are also included among the present invention.
In addition, the invention provides the polypeptide that the carboxyl terminal of the aminoacid sequence of albumin albumen corresponding to the albumin protein part of albumin fusion proteins of the present invention (being the serum albumin part of the albumin fusion proteins of polynucleotide described in serum albumin or the table 2 or albumin fusion constructs coding) has one or more residues disappearances.Especially, the C-terminal deletion can be described by general formula 1 to n, and wherein n is from 6 to 584 any complete integer, wherein the 584 complete integers of represent the middle amino acid residue total number-1 of mature human's serum albumin (SEQ IDNO:1).The polynucleotide of these polypeptide of encoding are also included among the present invention.Especially, the C-terminal deletion can be described by general formula 1 to n, and wherein n is from 6 to 608 any complete integer, and wherein 608 is complete integers of representing amino acid residue total number-1 in the serum albumin (SEQ ID NO:3).The polynucleotide of these polypeptide of encoding are also included among the present invention.
And, the invention provides the polypeptide that has one or more residue disappearances from the carboxyl terminal of albumin fusion proteins of the present invention.Especially, the C-terminal deletion can be described by general formula 1 to n, and wherein n is any integer from 6 to q-1, and wherein q is the complete integer of amino acid residue total number in the representative albumin fusion proteins of the present invention.The polynucleotide of these polypeptide of encoding are also included among the present invention.
In addition, can be in conjunction with above-mentioned any N-or C-terminal deletion to produce the reference polypeptide of N-and C-terminal deletion.The present invention also provides the polypeptide that has one or more aminoacid deletion from amino and carboxyl terminal both, it can be described as usually and have reference polypeptide (as, the human cytokines of mentioning in the table 1, the human cytokines part of albumin fusion proteins of the present invention, or the human cytokines part of polynucleotide described in the table 2 or albumin fusion constructs coding, or serum albumin (as SEQ ID NO:1), or the albumin protein part of albumin fusion proteins of the present invention, or the albumin protein part of polynucleotide described in the table 2 or albumin fusion constructs coding, or albumin fusion proteins, or the albumin fusion proteins of polynucleotide of the present invention or albumin fusion constructs coding) m to n residue, wherein n and m are aforesaid integers.The polynucleotide of these polypeptide of encoding are also included among the present invention.
The application also relates to albumen, it contains the reference polypeptide sequence listed with this paper (as, the human cytokines of mentioning in the table 1, the human cytokines part of albumin fusion proteins of the present invention, or the human cytokines part of polynucleotide described in the table 2 or albumin fusion constructs coding, or serum albumin (as SEQ ID NO:1), or the albumin protein part of albumin fusion proteins of the present invention, or the albumin protein part of polynucleotide described in the table 2 or albumin fusion constructs coding, or albumin fusion proteins, or the albumin fusion proteins of polynucleotide of the present invention or albumin fusion constructs coding) or its fragment have at least 80%, 85%, 90%, 95%, 96%, 97%, the polypeptide of 98% or 99% homogeneity.At some embodiments, the application relates to albumen, and it comprises the polypeptide that the reference polypeptide with the aminoacid sequence of aforesaid N-of having and C-terminal deletion has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homogeneity.The polynucleotide of these polypeptide of encoding are also included among the present invention.
Polypeptide fragment of the present invention comprises fragment, and it comprises or is made up of following alternatively: the performance aminoacid sequence is the therapeutic activity of segmental human cytokines or the proteic peptide sequence of serum albumin and/or the aminoacid sequence of functional activity (as biological activity).
Other polypeptide fragment comprises bioactive fragment.Bioactive fragment is to show with the active similar of polypeptide of the present invention but active polypeptide fragment that needn't be identical.Segmental biological activity can comprise the expectation activity of raising or the activity of not expecting that reduces.
Variant
" variant " refers to be different from reference nucleic acid or polypeptide but keeps the polynucleotide or the nucleic acid of its essential feature.Usually, variant is totally closely similar to reference nucleic acid or polypeptide, and identical with reference nucleic acid or polypeptide in many zones.
" variant " used herein refers to that sequence is different from human cytokines (as seen in " treatment " row of table 1), albumin and/or albumin fusion proteins respectively, but keeps the human cytokines part of the albumin fusion proteins of the present invention of that this paper describes elsewhere or its at least a function in addition known in the art and/or treatment feature, the albumin part or the albumin fusion proteins of the present invention of albumin fusion proteins of the present invention.Usually, variant to corresponding to the human cytokines of the human cytokines of albumin fusion proteins part, totally very similar corresponding to the aminoacid sequence of the albumin part of the albumin part of albumin fusion proteins and/or albumin fusion proteins, and identical in some zones.The nucleic acid of these variants of encoding is also included among the present invention.
The invention still further relates to albumen, it comprises or is made up of following alternatively: with for example corresponding to the aminoacid sequence of the human cytokines of the human cytokines of albumin fusion proteins of the present invention part (as, be disclosed in the human cytokines of table 1: the aminoacid sequence of X; Or the aminoacid sequence of the human cytokines part of the albumin fusion proteins of polynucleotide described in table 1 and 2 or albumin fusion constructs coding, or its fragment or variant), corresponding to the albuminised aminoacid sequence of the albumin part of albumin fusion proteins of the present invention (as, the aminoacid sequence of the albumin part of the albumin fusion proteins of polynucleotide described in the table 1 and 2 or albumin fusion constructs coding; Be shown in the aminoacid sequence of SEQ ID NO:1; Or its fragment or variant) and/or aminoacid sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homogeneity of the aminoacid sequence of albumin fusion proteins.The fragment (as, those fragments as herein described) of these polypeptide also is provided.Other polypeptide that the present invention includes are polypeptide of polynucleotide encoding, these polynucleotide under the following conditions with the complement hybridization of the nucleic acid molecules of coding albumin fusion proteins of the present invention: stringent hybridization condition (as, in 6X sodium chloride/sodium citrate (SSC), hybridize in the bonded DNA of filter at about 45 degrees centigrade, subsequently at about 50-65 degree centigrade at 0.2X SSC, wash one or many among the 0.1%SDS), high stringent condition (as, in 6X sodium chloride/sodium citrate (SSC), hybridize in the bonded DNA of filter at about 45 degrees centigrade, subsequently about 68 degrees centigrade at 0.1X SSC, wash one or many among the 0.2%SDS), or other stringent hybridization condition well known by persons skilled in the art (for example sees, Ausubel, F.M. wait editor, 1989 Current Protocols inMolecular Biology (up-to-date molecular biology experiment guide), Green publishing associates, Inc. and John Wiley ﹠amp; Sons Inc., New York, 6.3.1-6.3.6 and 2.10.3 page or leaf).The polynucleotide of these polypeptide of encoding are also included among the present invention.
Have with the inquiry aminoacid sequence and have at least for example polypeptide of the aminoacid sequence of 95% " homogeneity ", it is identical with this inquiry sequence (query sequence) to mean the theme amino acid sequence of polypeptide, and per 100 amino acid whose maximum five amino acids that can comprise the inquiry aminoacid sequence except the theme peptide sequence change.In other words, in order to obtain to have the polypeptide that has the aminoacid sequence of at least 95% homogeneity with the inquiry aminoacid sequence, maximum 5% amino acid residue of subject nucleotide sequence (subject sequence) can be inserted into, lack or with another aminoacid replacement.These changes of reference sequences can occur in the amino of reference amino acid sequence-or carboxyl-terminal position or these terminal positions between any position, be dispersed in separately in the reference sequences and be dispersed in the reference sequences between each residue or with one or more continuous group.
Actual, can use the known computer program determine routinely any specific polypeptide whether with for example, the aminoacid sequence of albumin fusion proteins of the present invention or its fragment (such as the human cytokines part of albumin fusion proteins or the albumin part of albumin fusion proteins) has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homogeneity.Determine the method for best total coupling between inquiry sequence (sequence of the present invention) and the subject nucleotide sequence, also be called total sequence alignment, can use based on the FASTDB computer program of (Comp.App.Biosci.6:237-245 (1990)) algorithms such as Brutlag and determine.In sequence alignment, inquiry sequence and subject nucleotide sequence all are nucleotide sequences or all are aminoacid sequences.The result of described total sequence alignment is expressed as homogeneity percentage ratio.The preferred parameter that is used for FASTDB aminoacid comparison is: matrix=PAM 0, k-tuple=2, mispairing point penalty=1, connect the length of point penalty=20, random group length=0, threshold value (Cutoff) mark=1, window size=sequence length, breach point penalty=5, breach size point penalty=0.05, window size=500 or any shorter subject amino acid sequence.
If because N-or C-terminal deletion rather than because inner disappearance, subject nucleotide sequence is shorter than inquiry sequence, then must carry out manual synchronizing to the result.This is because the FASTDB program is not considered the truncate of subject nucleotide sequence N-and C-end when the total homogeneity percentage ratio of calculating.For at N-and C-end subject nucleotide sequence, recently do not proofread and correct homogeneity percentage ratio as the percentage of total base of inquiry sequence at subject nucleotide sequence N-and C-end with the residue number of corresponding theme residue coupling/comparison by calculating inquiry sequence with respect to the inquiry sequence truncate.The result whether residue mates/compare by the FASTDB sequence alignment determines.From using the homogeneity percentage ratio of regulation calculation of parameter to deduct this percentage ratio to obtain final hundred homogeneity proportion by subtraction marks by above-mentioned FASTDB program.This final homogeneity fractions is used for the object of the invention.Have only subject nucleotide sequence N-and C-end are not considered to the purpose of adjusting the homogeneity fractions for artificial with the residue of inquiry sequence coupling/comparison.That is, have only the N-farthest of subject nucleotide sequence and the inquiry residue position outside the C-terminal residue.
For example, the subject nucleotide sequence of 90 amino acid residues and the inquiry sequence of 100 residues are compared to determine homogeneity percentage ratio.Disappearance occurs in the N-end of subject nucleotide sequence, and therefore, the FASTDB comparison does not show the coupling/comparison of preceding 10 residues of N-end.10 unpaired residues are represented 10% (residue total number in N-and the terminal unmatched residue number/inquiry sequence of C-) of sequence, so the homogeneity fractions of calculating from the FASTDB program deducts 10%.If remaining 90 residues mate fully, then final homogeneity percentage ratio will be 90%.At another example, the subject nucleotide sequence of 90 residues and the inquiry sequence of 100 residues are compared.Current disappearance is inner disappearance, thus not with the residue at subject nucleotide sequence N-or C-end of inquiry sequence coupling/comparison.The homogeneity percentage ratio that calculates of manual synchronizing FASTDB not in this example.Again, as performance in FASTDB comparison, have only outside subject nucleotide sequence N-and the C-end not with the residue position of inquiry sequence coupling/comparison by manual synchronizing.For the object of the invention is not carried out other manual synchronizing.
Variant usually and the normal HA of this variant equal length or the length of human cytokines have the sequence homogeneity of at least 75% (for example, at least about 80%, 90%, 95% or 99%).Analyze homology or the homogeneity determined at nucleotide or amino acid sequence level by BLAST (the local comparison in basis research tool), service routine blastp, blastn, blastx, tblastn and tblastx (Karlin etc., Proc.Natl.Acad.Sci.USA 87:2264-2268 (1990) and Altschul, J.Mol.Evol.36:290-300 (1993), incorporate into by reference with its integral body) algorithm that adopts, these programs are modulated to be used for the sequence similarity search.
The method that blast program uses is the similar section of at first considering between inquiry sequence and the database sequence, estimates the statistical significance between all couplings of identifying then, finally summarizes those couplings of the significance threshold value that to satisfy preliminary election.Discussion for underlying issue in the similarity searching of sequence library sees (Nature Genetics 6:119-129 (1994)) such as Altschul, and it is incorporated into by reference with its integral body.Search parameter to block diagram, description, comparison, desired value (i.e. the statistical significance threshold value that report is mated to database sequence), threshold value, matrix and eliminating value (filter) is a default setting.The acquiescence score matrix that blastp, blastx, tblastn and tblastx use is BLOSUM62 matrix (Henikoff etc., Proc.Natl.Acad.Sci.USA 89:10915-10919 (1992) incorporates into by reference with its integral body).For blastn, the score matrix is set the ratio of the N point penalty mark of mispairing residue (promptly to) by the M award mark of a pair of coupling residue (promptly to), and wherein the default value of M and N is respectively 5 and-4.Four blastn parameters can followingly be adjusted: Q=10 (producing the point penalty of breach); R=10 (point penalty that breach extends); Wink=1 (each the wink position generation character along inquiry sequence hits (word hits)); And gapw=16 (window width is set, in this width, produces notched comparison).Suitable Blastp parameter is set to Q=9; R=2; Wink=1; And gapw=32.Best fit (Bestfit) relatively uses the DNA parameter GAP=50 point penalty of breach (produce) and LEN=3 (point penalty of breach extension) between the sequence that can get in GCG software kit 10.0 versions, albumen relatively in suitable setting be GAP=8 and LEN=2.
Polynucleotide variant of the present invention can contain in coding region, noncoding region or both and changes.Embodiments of the present invention comprise the polynucleotide variant, and it contains feature or the active change that produces reticent replacement, interpolation or disappearance but do not change coded polypeptide.Because the degeneracy of genetic codon, replacing the nucleotide variants that produces by silence also is embodiments of the present invention.And, wherein be less than 50, be less than 40, be less than 30, be less than 20, be less than 10 or 5-50,5-25,5-10, a 1-5 or 1-2 aminoacid is replaced with any combination, disappearance or the polypeptide variants that adds also are embodiments of the present invention.For multiple reason can prepare the polynucleotide variant, as for the specific host optimizing codon being expressed (change codon is bacterial host such as yeast or the preferred codon of escherichia coli among the human mRNA).
At an embodiment, the polynucleotide of the present invention of the albumin part of coding albumin fusion proteins are optimized in yeast or mammalian cell expresses.At further embodiment, the polynucleotide of the present invention of the human cytokines part of coding albumin fusion proteins are optimized in yeast or mammalian cell expresses.At further embodiment again, the polynucleotide of the albumin fusion proteins of the present invention of encoding are optimized in yeast or mammalian cell and express.
At optional embodiment, under stringent hybridization condition as herein described, the codon optimized polynucleotide of the human cytokines part of coding albumin fusion proteins not with the wild type multi-nucleotide hybrid of coding human cytokines.At further embodiment, under stringent hybridization condition as herein described, the coding albumin fusion proteins albumin part codon optimized polynucleotide not with the coding albuminised wild type multi-nucleotide hybrid.At another embodiment, under stringent hybridization condition as herein described, the codon optimized polynucleotide of coding albumin fusion proteins not with the wild type multi-nucleotide hybrid of coding human cytokines part or albumin part.
At other embodiment, the polynucleotide of the human cytokines part of coding albumin fusion proteins do not comprise or are made up of following alternatively: the sequence of the natural generation of human cytokines.At further embodiment, the polynucleotide of the albumin part of coding albumin fusion proteins do not comprise or are made up of following alternatively: the sequence of albuminised natural generation.At optional embodiment, the polynucleotide of coding albumin fusion proteins do not comprise or are made up of following alternatively: the sequence of the natural generation of human cytokines part or albumin part.
The variant of natural generation is called " allelic variant ", and refers to occupy one of a plurality of optional forms of the gene of specifying the site on the chromosome of organism.(B. edits for Genes II, Lewin, John Wiley ﹠amp; Sons, New York (1985)).These allelic variants can and comprise in the present invention in polynucleotide and/or the variation of polypeptide level.Alternatively, the variant of non-natural generation can be by induced-mutation technique or by directly synthetic preparation.
Use the known method of protein engineering and recombinant DNA technology, can produce variant to improve or to change the characteristic of polypeptide of the present invention.For example, can be from polypeptide N-end of the present invention or the one or more aminoacid of C-terminal deletion and physical loss biological function not.As an example, Ron etc. (J.Biol.Chem.268:2984-2988 (1993)) have reported even have had the variant KGF albumen of heparin binding activity behind 3,8 or 27 amino-terminal amino acid residues of disappearance.Similarly, interferon gamma shows nearly ten times of higher activity behind the interferon gamma protein carboxyl groups terminal deletion 8-10 amino acid residue.(Dobeli etc., J.Biotechnology 7:199-216 (1988) .)
And, the enough normal maintenances of evidence proof variant and the biological activity of the protein similar of natural generation.For example, Gayle and partner (J.Biol.Chem.268:22105-22111 (1993)) have carried out the mass mutation analysis of human cell's factor IL-1a.They use random mutagenesis to produce above 3,500 independent IL-1a mutants, and average every variant has 2.5 amino acid changes on the whole length of molecule.Check various mutations at each possible amino acid position.Researcher is found " can change most of molecules and [in conjunction with activity or biological activity] almost do not had influence ".In fact, have only 23 unique amino acid sequences to produce the albumen different significantly in 3,500 nucleotide sequences in surpassing of inspection with the activity of wild-type protein.
In addition, if even cause modifying or losing one or more biological functions from polypeptide N-end or the one or more aminoacid of C-terminal deletion, still can keep other biological activity.For example, when being less than of secreted form most of residues when N-end or C-end remove, the disappearance variant keeps inducing and/or in conjunction with the ability of the antibody of identification secreted form probably.Can be easy to determine by customary method as herein described and in addition known in the art whether the specific polypeptide that lacks proteic N-or C-terminal residue keeps this immunogenicity activity.
Therefore the present invention further comprise and have functional activity (as, biological activity and/or therapeutic activity) polypeptide variants.At an embodiment, the invention provides have corresponding to corresponding to one or more biologies of the human cytokines of the human cytokines of albumin fusion proteins part and/or the functional activity of therapeutic activity (as, biological activity and/or therapeutic activity) the albumin fusion proteins variant.At another embodiment, the invention provides corresponding to corresponding to one or more biologies of the human cytokines of the human cytokines of albumin fusion proteins part and/or the functional activity of therapeutic activity (as, biological activity and/or therapeutic activity) the albumin fusion proteins variant.Thereby this variant comprises disappearance, insertion, inversion, repetition and the replacement selected according to universal rule known in the art activity almost there is not influence.The polynucleotide of this variant of encoding are also included among the present invention.
At some embodiments, variant of the present invention has conservative and replaces." conservative replacement " means exchange in the group, such as substituting of aliphatic or hydrophobic aminoacid Ala, Val, Leu and Ile; Hydroxyl residue Ser and Thr substitute; Acidic residues Asp and Glu substitute; Amide residues Asn and Gln substitute, and alkaline residue Lys, Arg and His substitute; Aminoacid Ala, Ser, Thr, Met and the Gly of aromatic moieties Phe, Tyr and Trp alternative and little (small-sized) substitutes.
Guidance about the aminoacid replacement that how to produce the phenotype silence for example is provided in, at Bowie etc., " Deciphering the Message in Protein Sequences:Tolerance to Amino AcidSubstitutions (deciphering the information in the protein sequence: tolerate aminoacid replacement); " Science 247:1306-1310 (1990), wherein the author points out the tolerance of two kinds of main policies research aminoacid sequences to changing.
First kind of strategy adopts the tolerance of the aminoacid replacement that is undertaken by natural selection in the evolutionary process.By comparing the aminoacid sequence in the different plant species, can identify conservative aminoacid.These conservative aminoacid are important to protein function probably.On the contrary, represent that by the amino acid position of the replacement of natural selection tolerance these positions are not crucial to protein function.Therefore, proteic biological activity can still be kept by modification in the position of tolerance aminoacid replacement.
Second kind of strategy uses genetic engineering to introduce amino acid change to identify the zone to the protein function key at the ad-hoc location of clone gene.For example, can use site-directed mutation or alanine-scanning mutagenesis (each residue is introduced single alanine mutation in molecule).See Cunningham and Wells, Science244:1081-1085 (1989).Can test the biological activity of the mutant molecule of gained subsequently.
Described as these authors, these two kinds of strategies have disclosed albumen and have tolerated aminoacid replacement surprisingly.These authors point out that further which amino acid change some amino acid positions in albumen may permit.For example, (in the albumen tertiary structure) amino acid residue that great majority are hidden needs non-polar sidechain, and keeps surface side chains feature seldom usually.And the conservative aminoacid replacement of tolerance comprises substituting of aliphatic or hydrophobic aminoacid Ala, Val, Leu and Ile; Hydroxyl residue Ser and Thr substitute; Acidic residues Asp and Glu substitute; Amide residues Asn and Gln substitute, and alkaline residue Lys, Arg and His substitute; Substituting of aromatic moieties Phe, Tyr and Trp and substituting of undersized aminoacid Ala, Ser, Thr, Met and Gly.Except conservative aminoacid replacement, variant of the present invention also comprises (i) and contains the polypeptide that one or more non-conservative amino acid residues replace, wherein the amino acid residue of this replacement can be genetic codon coding or can be not do not encode for genetic codon, or (ii) contain one or more polypeptide with replacement of substituent amino acid residue, or (iii) merge or chemically combined polypeptide with another kind of chemical compound, this chemical compound such as the chemical compound that increases polypeptide stability and/or dissolubility (for example, Polyethylene Glycol), (iv) contain other amino acid whose polypeptide, such as for example, IgG Fc corresponding circle of sensation peptide.From the instruction of this paper, this variant polypeptide is considered as in those skilled in the art's scope.
For example, contain polypeptide variants that with other charged or neutral amino acid replaces charged amino acid whose aminoacid replacement and can produce the have improved characteristic albumen of (less) such as assembling.Because reducing active and increase, the immunogenicity activity of aggregation, the gathering of pharmaceutical preparation remove.See Pinckard etc., Clin.Exp.Immunol.2:331-340 (1967); Robbins etc., Diabetes 36:838-845 (1987); Cleland etc., Crit.Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993).
In specific implementations, polypeptide of the present invention comprises or is made up of following alternatively: the fragment of the aminoacid sequence of the fragment of the aminoacid sequence of albumin fusion proteins or variant, human cytokines and/or human serum albumin or variant; Wherein this fragment or variant compare with reference amino acid sequence have 1-5,5-10,5-25,5-50,10-50 or 50-150 amino acid residue add, replace and/or disappearance.At other embodiment, aminoacid replacement is guarded.The nucleic acid of these polypeptide of encoding is also included among the present invention.
Polypeptide of the present invention can be mainly be made up of the aminoacid that is connected by the peptide bond (being the peptide isostere) of peptide bond or modification each other, and can contain the aminoacid that is different from 20 kinds of gene-amino acids coding.Polypeptide can be modified by natural process (such as the translation post-treatment) or by chemical modification technology well known in the art.This modification is described in basic reader and more detailed monograph and big research document.Modification can occur in any position of polypeptide, comprises peptide main chain, amino acid side chain and amino or carboxyl terminal.The modification that should be understood that same type can exist with identical or different degree in a plurality of sites of specifying polypeptide.And specify polypeptide can contain the modification of many types.For example because ubiquitinization, polypeptide can be ramose, and polypeptide can be and has or do not have ramose ring-type.Ring-type, ramose and ramose ring type polypeptide can be from translating back natural process acquisition or can being prepared by synthetic method.Modification comprises acetylation; acidylate; the ADP-ribosylation; amidatioon; covalently bound flavin; covalently bound heme moiety; covalently bound nucleotide or nucleotide derivative; covalently bound lipid or lipid derivate; covalently bound phosphatidylinositols; crosslinked; cyclisation; form disulfide bond; demethylation; form covalent crosslink; form cysteine; form pyroglutamic acid; formylated; γ-carboxylated; glycosylation; form the GPI anchor; hydroxylating; iodate; methylate; myristylation; oxidation; Pegylation; Proteolytic enzyme processing; phosphorylation; prenylation; racemization; selenoization (selenoylation); sulfation (sulfation); what transfer-RNA mediated adds aminoacid to albumen, such as arginylization (arginylation) and ubiquitinization.(for example see PROTEINS-STRUCTURE AND MOLECULAR PROPERTIES (albumen-structure and characterization of molecules), second edition, T.E.Creighton, W.H.Freeman and Company, New York (1993); POST-TRANSLATIONAL COVALENT MODIFICATION OFPROTEINS (translation back covalent modification albumen), B.C.Johnson, editor, Academic Press, NewYork, 1-12 page or leaf (1983); Seifter etc., Meth.Enzymol.182:626-646 (1990); Rattan etc., Ann.N.Y.Acad.Sci.663:48-62 (1992)).
Functional activity
" polypeptide with functional activity " refers to show the relevant active polypeptide of one or more known functions of the total length of human cytokines, preceding albumen and/or mature form.This functional activity includes but not limited to, biological activity, antigenicity [in conjunction with (or combine with the polypeptide competition) in the ability of anti--polypeptide antibody], immunogenicity (generation is incorporated into the ability of the antibody of concrete polypeptide of the present invention), with the ability of polypeptide formation polymer of the present invention be incorporated into the ability of the receptor or the part of polypeptide.
" polypeptide of biologically active " refers to performance with the active similar of human cytokines of the present invention (comprising mature form) but active polypeptide that needn't be identical, as measurement in the particular biological detection, has or do not have dose dependent.There is the situation of dose dependent, dose dependent must be with polypeptide identical, but compare roughly similar at the dosage-dependency of specified activity with polypeptide of the present invention (is that candidate's polypeptide shows bigger activity or is no more than about 25 times of few activity with respect to polypeptide of the present invention, at an embodiment, be no more than about 10 times of few activity, with at further embodiment, be no more than about 3 times of few activity).
At some embodiments, albumin fusion proteins of the present invention has human cytokines part (or its fragment or variant) relevant at least a biology and/or the therapeutic activity when not merging with albumin.
At other embodiment, albumin fusion proteins of the present invention is compared the plasma stability with increase with the human cytokines part (or its fragment or variant) that does not merge state.The detection method known in the art that can use detection method known in the art or revise routinely detects the plasma stability of the human cytokines part (or its fragment or variant) of albumin fusion proteins of the present invention or not fusion.
Can use the functional activity that detection method known in the art or the detection method of revising routinely known in the art and detection method as herein described detect albumin fusion proteins of the present invention (as, biological activity).In addition, use table 1 respective column (as, at the row 3 of table 1) detection method mentioned, those skilled in the art can detect the segmental activity of human cytokines corresponding to the human cytokines part of albumin fusion proteins routinely.Further, those skilled in the art can use the detection method described in the known in the art and/or following embodiment part to detect activity corresponding to the albumin protein fragments of the albumin protein part of albumin fusion proteins routinely.
For example, at an embodiment that detects the albumin fusion proteins combination or combine the ability of anti--therapeutical peptide antibody and/or anti--albumin antibody with the human cytokines competition, can use various immune detection known in the art, including but not limited to, competitive and non--the competitive assay system, use detects such as radioimmunity, ELISA (enzyme linked immunological absorption detects), " sandwich " immune detection, immune radiating detects, gel diffusion precipitation reaction, immunodiffusion detects, the original position immune detection (is for example used gold colloidal, enzyme or labelled with radioisotope), the western trace, precipitation, the coagulation detection (as, the gel coagulation detects, blood coagulation detects), complement is in conjunction with detection, immunofluorescence detects, technology such as protein A detection and immunoelectrophoresis detection.At an embodiment, detect antibodies by detecting the labelling on the first antibody.At another embodiment, detect first antibody with combining of first antibody by detecting second antibody or reagent.At further embodiment, second antibody is a labelling.Many methods are known in the artly to be used for detecting the bonded of immune detection and within the scope of the present invention.
At an embodiment, when the binding partners of having identified human cytokines (as, receptor or part) time, can detect the combination of the albumin fusion proteins that comprises this human cytokines (as the human cytokines part of fusant) to binding partners, as, by method well known in the art, such as for example, reproducibility and irreducibility gel chromatography, albumen affinity chromatography and affinity blotting.Usually see Phizicky etc., Microbiol.Rev.59:94-123 (1995).At another embodiment, can use the ability of the physiology correlative combination of technology for detection albumin fusion proteins known in the art routinely corresponding to the substrate (substrate) of the therapeutical peptide of the human cytokines part of fusant.
At optional embodiment, when estimating the ability of albumin fusion proteins poly, can be as detecting the association with other component of polymer by method well known in the art, described method is such as for example, reproducibility and irreducibility gel chromatography, albumen affinity chromatography and affinity blotting.Usually see above-mentioned Phizicky etc.
At some embodiments, comprise with all or part of albumin fusion proteins of the bonded antibody of human cytokines has Yu the antibody of combined treatment albumen (or its fragment or variant) is relevant when human cytokines does not merge with albumin at least a biology and/or therapeutic activity (as, specific binding polypeptide or epi-position).At other embodiment, comprise with the biological activity of all or part of albumin fusion proteins of the bonded antibody of human cytokines and/or therapeutic activity be suppress (being antagonism) or activation (promptly exciting) with by the proteic antibody specificity of combined treatment relevant one or more biological activitys and/or the therapeutic activity of bonded polypeptide.
Comprise that the fragment at least of the proteic antibody of combined treatment or the albumin fusion proteins of variant can characterize in many ways.Especially, use the techniques described herein or albumin fusion proteins specificity that the technology known in the art revised routinely can detect the fragment at least that comprises the proteic antibody of combined treatment or variant in conjunction with ability corresponding to the bonded same antigen in antibody specificity ground of the human cytokines of the human cytokines part of albumin fusion proteins.
To albumin fusion proteins (as, the fragment at least or the variant that comprise the proteic antibody of combined treatment) (specifically) in conjunction with the detection of the ability of concrete albumen or epi-position can in solution, carry out (as, Houghten, Bio/Techniques 13:412-421 (1992)), on microballon, carry out (as, Lam, Nature354:82-84 (1991)), on chip, carry out (as, Fodor, Nature 364:555-556 (1993)), on antibacterial (as, United States Patent (USP) the 5th, 223, No. 409), on spore, carry out (as, patent the 5th, 571,698; 5,403,484; With 5,223, No. 409), on plasmid, carry out (as, Cull etc., Proc.Natl.Acad.Sci.USA 89:1865-1869 (1992)) or on phage, carry out (as, Scott and Smith, Science 249:386-390 (1990); Devlin, Science 249:404-406 (1990); Cwirla etc., Proc.Natl.Acad.Sci.USA 87:6378-6382 (1990); And Felici, J.Mol.Biol.222:301-310 (1991)) (each comfortable this of these lists of references is incorporated into by reference with its integral body).Can use or customary revise technology for detection described herein or in addition known in the art and comprise specificity and the affinity of the albumin fusion proteins of the fragment at least of therapeutic antibodies or variant concrete albumen or epi-position.
The albumin fusion proteins that can detect the fragment at least that comprises the proteic antibody of combined treatment or variant by any method known in the art and other antigen (as, with corresponding to the bonded antibody specificity of human cytokines (or its fragment or variant) of the human cytokines part of albumin fusion proteins of the present invention the molecule of bonded molecule with sequence/structure conservative) cross reactivity.
The immune detection that can be used for analysis (immunologic opsonin) combination and cross reactivity includes but not limited to competitive and non--competitive assay system, technology such as use such as western trace, radioimmunity detection, ELISA (enzyme linked immunological absorption detects), " sandwich " immune detection, immunoprecipitation detection, precipitin reaction, gel diffusion precipitation reaction, immunodiffusion detection, coagulation detection, complement fixation detection, immune radiating detection, fluorescence immunoassay detection and protein A immune detection only give some instances.This detection be conventional and well known in the art (see as, editors such as Ausubel, 1994, Current Protocols in MolecularBiology (up-to-date molecular biology experiment guide), the 1st volume, John Wiley ﹠amp; Sons, Inc., New York incorporates into its integral body by reference at this).Exemplary immunization detects and briefly is described in following (but unexpectedly being restriction).
The immunoprecipitation scheme generally comprise be added with phosphoprotein phosphatase and/or protease inhibitor (as, EDTA, PMSF, aprotinin, vanadic acid sodium) lysis buffer such as RIPA buffer (1%NP-40 or Triton X-100,1% NaTDC, 0.1%SDS, 0.15M NaCl, 0.01M sodium phosphate, at pH 7.2, cell lysis colony 1%Trasylol), to cell lysate add albumin fusion proteins of the present invention (as, the fragment at least or the variant that comprise the proteic antibody of combined treatment), hatch a period of time at 40 ℃ (as, 1 to 4 hour), for example add the agarose microballon that is coupled to anti--albumin antibody to cell lysate, hatched about one hour or longer at 40 ℃, in lysis buffer, clean microballon and resuspended microballon in the SDS/ sample buffer.Can be by the ability of estimating albumin fusion proteins immunoprecipitation specific antigen as the western engram analysis.One skilled in the art will know that can be modified with increase albumin fusion proteins to antigenic combination and reduce background (as, with agarose microballon presettling cell lysate) parameter.Further discussion about the immunoprecipitation scheme sees, as, editors such as Ausubel, 1994, CurrentProtocols in Molecular Biology (up-to-date molecular biology experiment guide), the 10.16.1 of the 1st volume, John Wiley ﹠amp; Sons, Inc., New York.
The Western engram analysis generally comprises the preparation protein sample, polyacrylamide gel (as, depend on antigen molecular, electrophoresis protein sample 8%-20%SDS-PAGE), from polyacrylamide gel transfer protein sample to film such as celluloid, PVD or nylon, lock solution (as, the PBS that contains 3%BSA or defatted milk powder) closing membrane in, cleaning buffer solution (as, PBS-Tween 20) the middle film that cleans, add albumin fusion proteins of the present invention (being diluted in the sealing buffer) to film, in cleaning buffer solution, clean film, apply be diluted in the sealing buffer yoke close in zymolyte (as, horseradish peroxidase or alkali phosphatase) or the radioactivity molecule (as
32P or
125I) second antibody (it discerns albumin fusion proteins, as Anti-Human's class serum albumin antibody) is cleaned film, and is detected antigenic existence in cleaning buffer solution.One skilled in the art will know that and to be modified with signal that increases detection and the parameter that reduces background noise.Further discussion about western trace scheme sees, as, editors such as Ausubel, 1994, CurrentProtocols in Molecular Biology (up-to-date molecular biology experiment guide), the 10.8.1 of the 1st volume, John Wiley ﹠amp; Sons, Inc., New York.
ELISA comprises preparation antigen, hole with antigen coated 96-hole microtitration plate, flush away is the antigen of combined hole not, to the hole add yoke close in detectable chemical compound such as zymolyte (as, horseradish peroxidase or alkali phosphatase) albumin fusion proteins of the present invention (as, the fragment at least or the variant that comprise the proteic antibody of combined treatment) and hatch a period of time, flush away is unconjugated or non--bonded specifically albumin fusion proteins, and detection specificity ground conjugated antigen bag is by the existence of the albumin fusion proteins in hole.Albumin fusion proteins needn't close in detectable chemical compound by yoke among the ELISA; On the contrary, can add yoke to the hole and close second antibody (it discerns albumin fusion proteins) in detectable chemical compound.Further, replace with antigen coated hole, can the albumin fusion proteins bag by the hole.This situation, detectable molecule can be yoke close in detectable chemical compound such as zymolyte (as, horseradish peroxidase or alkali phosphatase) antigen.One skilled in the art will know that and to be modified with the parameter of the signal that increase to detect and other variable of ELISA known in the art.About the further discussion of ELISA see as, editors such as Ausubel, 1994, Current Protocols in Molecular Biology (up-to-date molecular biology experiment guide), the 11.2.1 of the 1st volume, John Wiley ﹠amp; Sons, Inc., New York.
Can determine binding affinity and the albumin fusion proteins-protein/antigen/epi-position interactional dissociation yield of albumin fusion proteins in conjunction with detecting by competitive to albumen, antigen or epi-position.Competitive is that radioimmunity detects in conjunction with an example that detects, the unlabelled antigen that is included in recruitment exist the antigen of hatching labelling down (as,
3H or
125I), and detect the antigenic antibody that is incorporated into labelling with albumin fusion proteins of the present invention.Can determine that albumin fusion proteins is to the affinity of concrete albumen, antigen or epi-position with in conjunction with dissociation yield from the data of Scatchard tracing analysis.The second proteic competition that is incorporated into same albumen, antigen or epi-position with albumin fusion proteins also can use the radioimmunity detection to determine.This situation, recruitment with albumin fusion proteins of the present invention be incorporated into hatch in the presence of unlabelled second albumen of same albumen, antigen or epi-position albumen, antigen or epi-position and yoke close chemical compound in labelling (as,
3H or
125I) albumin fusion proteins.
At an embodiment, the BIAcore dynamic analysis is used for determining combination rate and the dissociation yield of albumin fusion proteins of the present invention to albumen, antigen or epi-position.The BIAcore dynamic analysis comprises from the combination of the analysis albumin fusion proteins of the chip that has immobilized concrete polypeptide, antigen or epi-position or albumin fusion proteins on its surface respectively or concrete polypeptide, antigen or epi-position and dissociates.
In conjunction with also can they the binding affinity of specifying albumen or antigen (for example, the bonded antigen of their specificitys) be described or illustrating corresponding to the antibody of the human cytokines of the human cytokines of albumin fusion proteins part.Binding affinity comprises dissociation constant or Kd is less than 5 * 10
-2M, 10
-2M, 5 * 10
-3M, 10
-3M, 5 * 10
-4M, 10
-4The binding affinity of M.Other binding affinity comprises dissociation constant or Kd is less than 5 * 10
-5M, 10
-5M, 5 * 10
-6M, 10
-6M, 5 * 10
-7M, 10
-7M, 5 * 10
-8M or 10
-8The binding affinity of M.Other binding affinity comprises dissociation constant or Kd is less than 5 * 10
-9M, 10
-9M, 5 * 10
-10M, 10
-10M, 5 * 10
-11M, 10
-11M, 5 * 10
-12M, 10
-12M, 5 * 10
-13M, 10
-13M, 5 * 10
-14M, 10
-14M, 5 * 10
-15M or 10
-15The binding affinity of M.At some embodiments, the albumin fusion proteins that comprises the fragment at least of the proteic antibody of combined treatment or variant is similar to the affinity of specifying albumen or epi-position and having and the proteic corresponding antibodies of combined treatment (merging to albumin), considers tiring of albumin fusion proteins (fragment at least or the variant that comprise the proteic antibody of combined treatment) and tiring of corresponding antibodies.In addition, can adopt detection as herein described (seeing embodiment and table 1) and detection known in the art in addition to measure the ability that albumin fusion proteins and its fragment, variant and derivant cause the biological activity relevant with the human cytokines part of albumin fusion proteins and/or albumin part and/or therapeutic activity (external or body interior) routinely.Other method is known and within the scope of the present invention to those skilled in the art.
Albumin
As mentioned above, albumin fusion proteins of the present invention comprises the fragment at least of human cytokines or the fragment at least or the variant of variant and human serum albumin, and they for example merge interconnection by heredity.
Other embodiment comprises the fragment at least of human cytokines or the fragment at least or the variant of variant and human serum albumin, and they are connected to each other by chemical bond.
Human serum albumin (HSA) of term and human albumin (HA) use interchangeably at this paper.Term " albumin " and " serum albumin " are more wide in range, comprise human serum albumin (with its fragment and variant) and from the albumin (with its fragment and variant) of other species.
" albumin " used herein refers to albumin albumen or albumin aminoacid sequence jointly or has albuminised one or more functional activities the albumin fragment or the variant of (as, biological activity).Especially, " albumin " refers to that human albumin or its fragment (for example see, EP 201239, EP 322094, WO97/24445, WO95/23857), especially be presented at the human albumin mature form of Fig. 1 and SEQ ID NO:1, or from other vertebrate albumin or its fragment, or these molecules or its segmental analog or variant.
At some embodiments, the human serum albumin albumen that is used for albumin fusion proteins of the present invention contains with next group or two groups of point mutation: Leu-407 to Ala, Leu-408 to Val, Val-409 to Ala and Arg-410 to Ala with respect to SEQ ID NO:1; Or Arg-410 to A, Lys-413 to Gln and Lys-414 to Gln (see as, international publication WO95/23857 number is incorporated into its integral body by reference at this).At other embodiment, the albumin fusion proteins that contains above-mentioned one group or two groups point mutation of the present invention has the stability/resistance of raising to yeast Yap3p proteolytic cleavage, allows to increase the reorganization albumin fusion proteins that preparation is expressed in yeast host cell.
Used hereinly be enough to the proteic therapeutic activity of extended treatment or the albumin part of plasma stability or shelf life refers to, thereby be enough to stable on length or the structure or prolong proteic therapeutic activity or plasma stability is compared the shelf life of the human cytokines part that prolongs or extend albumin fusion proteins or the albumin part of plasma stability with the shelf life or the plasma stability of non--fusant state.The albumin part of albumin fusion proteins can comprise the total length of aforesaid HA sequence, maybe can comprise it and can stablize or the active one or more fragments of extended treatment.This segmental length can be 10 or more amino acids maybe can comprise the part or all of concrete structure territory that maybe can comprise HA from about 15,20,25,30,50 or more continuous amino acids of HA sequence.For example, can use one or more fragments of striding preceding two immunoglobulins-class formation territory of HA.At an embodiment, the HA fragment is the mature form of HA.
The albumin part of albumin fusion proteins of the present invention can be the variant of normal HA.The human cytokines part of albumin fusion proteins of the present invention also can be the variant of human cytokines as described herein.Term " variant " comprises conservative or nonconservative insertion, disappearance and replacement, wherein this variation not the albuminised colloid infiltration of material change (oncotic), useful part-combination and non--immunogenicity feature one or more or give the avtive spot or the active structure domain of the therapeutic activity of human cytokines.
Especially, albumin fusion proteins of the present invention can comprise the polymorphism variant and the human albuminised fragment of human albuminised natural generation, for example is disclosed in the fragment (be called HA (Pn), wherein n is 369 to 419) of EP 322094.Albumin can be derived from any vertebrates, especially any mammal, for example mankind, cattle, sheep or pig.The albumin of nonmammalian includes but not limited to the albumin of hen and salmon.The albumin part of albumin fusion proteins can with human cytokines part from different animals.
In general, HA fragment or variant at least 100 amino acid longs, for example, at least 150 amino acid longs.The HA variant can be formed or comprised alternatively by following: the HA of at least one full domain, for example domain 1 (amino acid/11-194 of SEQ ID NO:1), domain 2 (the amino acid/11 95-387 of SEQ ID NO:1), domain 3 (the aminoacid 388-585 of SEQ ID NO:1), domain 1 and 2 (1-387 of SEQID NO:1), domain 2 and 3 (195-585 of SEQ ID NO:1) or domain 1 and 3 (the aminoacid 388-585 of the amino acid/11-194 of SEQ ID NO:1 and SEQ ID NO:1).Each domain itself is made up of two homology subdomains, be 1-105,120-194,195-291,316-387,388-491 and 512-585, have the flexible interior-subdomain that comprises residue Lys106 to Glu119, Glu292 to Val315 and Glu492 to Ala511 and be connected the tagma.
On the one hand, the albumin part of albumin fusion proteins of the present invention comprises at least one subdomain or domain or its conservative modification of HA.If fusant is based on subdomain, some or all of adjacent connectors can be used for being connected to the human cytokines part.
The proteic antibody of specificity combined treatment also is human cytokines
The present invention also comprises albumin fusion proteins, and it comprises at least fragment or the variant of specificity in conjunction with the antibody of the human cytokines that is disclosed in table 1.Comprise particularly, term " human cytokines " comprise combined treatment albumen (as, described in the row I of table 1) antibody and its fragment and variant.Therefore albumin fusion proteins of the present invention can contain the fragment at least or the variant of human cytokines, and/or the fragment at least or the variant of the proteic antibody of combined treatment.
Antibody structure and background
Known basic antibody structure unit comprises tetramer.Each tetramer mainly is made up of following: two pairs of identical polypeptide chains, every pair has one " light " (about 25kDa) and one " weight " chain (about 50-70kDa).Amino-the end portion of every chain comprises about 100 to 110 or the variable region of amino acids more, mainly is responsible for antigen recognition.The carboxyl of every chain-end portion limits constant region, mainly is responsible for effector function.Human light chain is divided into κ and lambda light chain.Heavy chain is divided into μ, δ, γ, α or ε, and the isotype that limits antibody respectively is IgM, IgD, IgG, IgA and IgE.Usually see Fundamental Immunology (basic immunology) 3-5 chapter (Paul, W. edits, the 4th edition .Raven Press, N.Y. (1998)) (incorporating into by reference with its integral body at this) for all purposes.The variable region of every pair of light/heavy chain forms antibody combining site.
Therefore complete IgG antibody has two binding sites.Except in difunctional or bi-specific antibody, these two binding sites are identical.
Chain all shows identical general structure: Bao Shou framework region (FR) is connected in three hypervariable regions relatively, also is called complementary determining region or CDR.The CDR district is antibody contact antigen and definite its specific part normally.Every pair the weight and the CDR of light chain align with framework region, make to be bonded to defined epitope.To the C-end, light and variable region of heavy chain all comprises domain FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from the N-end.The variable region is connected in heavy or constant region of light chain.Aminoacid is according to Kabat Sequences of Proteins of Immunological Interest (the Kabat protein sequence of immunology aspect) (National Institutes of Health to the distribution of each domain, Bethesda, Md. (1987 and 1991)) definition, or Chothia ﹠amp; Lesk J Mol.Biol.196:901-917 (1987); Nature342:878-883 such as Chothia (1989).
" antibody " used herein refers to the immunocompetence part of immunoglobulin molecules and immunoglobulin molecules, promptly contain the antigen binding site of specificity conjugated antigen molecule (as, contain the molecule in one or more antibody CDR district).Can include but not limited to corresponding to the human cytokines antibody partly of albumin fusion proteins, monoclonal, polyspecific, the mankind, humanization or chimeric antibody, single-chain antibody (as, strand Fv), the fragment of Fab fragment, F (ab ') fragment, the preparation of Fab expression library, anti--idiotype (anti--Id) antibody (comprise as, specifically to anti--Id antibody of antibody specificity of the present invention), with the epi-position-binding fragment of any above-mentioned antibody (as, VH domain, VL domain or one or more CDR district).
The proteic antibody of combined treatment
The present invention includes comprise combined treatment albumen (as, be disclosed in table 1) or the fragment at least of the antibody of its fragment or variant or the albumin fusion proteins of variant.
The antibody of combined treatment albumen (or its fragment or variant) can comprise birds and mammal from any animal origin.Antibody can be the mankind, Mus (as, mice and rat), donkey, sheep, rabbit, goat, Cavia porcellus, camel, horse or chicken antibody.At an embodiment, this antibody is human antibodies." mankind " used herein antibody comprises the antibody of the aminoacid sequence with human immunoglobulin and comprises from the human immunoglobulin library with by xenogenesis mice (xenomice) or other the organism isolated antibody of genetic modification with the preparation human antibodies.
Be incorporated into human cytokines and can be corresponding to the antibody molecule of the human cytokines of albumin fusion proteins of the present invention part immunoglobulin molecules any type (as, IgG, IgE, IgM, IgD, IgA and IgY), any class (as, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.At some embodiments, be incorporated into human cytokines and can be IgG1 corresponding to the antibody molecule of the human cytokines of albumin fusion proteins part.At other embodiment, be incorporated into human cytokines and can be IgG2 corresponding to the immunoglobulin molecules of the human cytokines of albumin fusion proteins part.At other embodiment, be incorporated into human cytokines and can be IgG4 corresponding to the immunoglobulin molecules of the human cytokines of albumin fusion proteins part.
At an embodiment, be incorporated into human cytokines and can be human antigen of the present invention-binding antibody fragment and include but not limited to, the Fv (sdFv) that Fab, Fab ' are connected with F (ab ') 2, Fd, strand Fv (scFv), single-chain antibody, disulfide bond and comprise VL or the fragment of VH domain corresponding to the antibody of the human cytokines of albumin fusion proteins part.Antigen-binding antibody the fragment that comprises single-chain antibody can comprise separately or with following bonded in whole or in part variable region: hinge region, CH1, CH2 and CH3 domain.Other antigen-binding proteins that can be incorporated into human cytokines or albumin fusion proteins comprises, as, Small Modular ImmunoPharmaceuticals (SMIPSs
TM) (see as, No. the 20050202534th, U.S. published patent application).
Be incorporated into human cytokines and can be monospecific, bispecific, tri-specific corresponding to the antibody of the human cytokines of albumin fusion proteins part or polyspecific more.Multi-specificity antibody can be specific or can be that both are specific to human cytokines and allos epi-position to the different epi-positions of human cytokines, such as heterologous polypeptide or solid phase support material.See as, PCT announces WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt etc., J.Immunol.147:60-69 (1991); United States Patent (USP) the 4th, 474,893; 4,714,681; 4,925,648; 5,573,920; 5,601, No. 819; Kostelny etc., J.Immunol.148:1547-1553 (1992).
The antibody of combined treatment albumen (or its fragment or variant) can be bispecific or bifunctional, and this expression antibody is artificial hybrid antibody, have two differences heavy/light chain to two different binding sites.The antibody that can be prepared bispecific by several different methods comprises and merges hybridoma or connect Fab ' fragment.See as, Songsivilai ﹠ amp; J Immunol.148:15471553 (1992) such as Lachmann Clin.Exp.Immunol.79:315-321 (1990); Kostelny.,"" ( Holliger′"Diabodies′：small bivalent and bispecific antibody fragments ( ′′： ) "PNAS USA 90：6444-6448 ( 1993 ) ) "Janusins" ( Traunecker"Bispecific single chain molecules ( Janusins ) target cytotoxic lymphocytes on HIV infected cells ( ( Janusins ) HIV ) "EMBO J 10：3655-3659 ( 1991 ) Traunecker"Janusin：new molecular design for bispecificreagents ( Janusin： ) "Int J Cancer Suppl 7：51-52 ( 1992 ) ) 。
The present invention also provides the fragment that comprises the known antibody in other places in as herein described or this area or the albumin fusion proteins of variant (comprising derivant).The standard technique of introducing sudden change in the nucleotide sequence that is used in coding molecule of the present invention well known by persons skilled in the art is comprised, for example, cause the site-directed mutation and the PCR-mediated mutagenesis of aminoacid replacement.At an embodiment, variant (comprising derivant) is less than 50 aminoacid replacement, is less than 40 aminoacid replacement, is less than 30 aminoacid replacement, is less than 25 aminoacid replacement, is less than 20 aminoacid replacement, is less than the fifteen amino acid replacement, is less than the ten amino acid replacement, is less than 5 aminoacid replacement, is less than 4 aminoacid replacement, is less than 3 aminoacid replacement or is less than 2 aminoacid replacement with respect to reference VH domain, VHCDR1, VHCDR2, VHCDR3, VL domain, VLCDR1, VLCDR2 or VLCDR3 coding.In specific implementations, the replacement of variant coding VHCDR3.At an embodiment, variant has conservative aminoacid replacement at the non essential amino acid residue of one or more predictions.
Be incorporated into human cytokines and can corresponding to the antibody of the human cytokines of albumin fusion proteins part can their identification or the epi-position or the part of the bonded human cytokines of specificity describe or explanation.The antibody that also can not comprise the defined epitope of specificity combined treatment albumen or human cytokines.Therefore, the present invention includes the proteic antibody of specificity combined treatment, and allow not comprise them.At some embodiments, comprise that fragment that the albumin fusion proteins and antibody of the fragment at least of the proteic antibody of combined treatment or variant do not merge itself or variant are in conjunction with identical epi-position.
Be incorporated into human cytokines and can also can describe or explanation their cross reactivity corresponding to the antibody of the human cytokines of albumin fusion proteins part.Comprise the not antibody of proteic any other analog of combined treatment, lineal congener or congener.The present invention also comprises in conjunction with having the antibody of the polypeptide of 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55% and 50% sequence homogeneity (using method calculating known in the art and as herein described) with human cytokines at least at least at least at least at least at least at least at least at least at least.In specific implementations, be incorporated into human cytokines and can be corresponding to the antibody of the human cytokines of albumin fusion proteins part and the corresponding epi-position cross reaction of Mus, rat and/or rabbit congener of human protein with it.The present invention also do not comprise in conjunction with having with human cytokines and is less than 95%, be less than 90%, be less than 85%, be less than 80%, be less than 75%, be less than 70%, be less than 65%, be less than 60%, be less than 55% and be less than the antibody of the polypeptide of 50% sequence homogeneity (using method calculating known in the art and as herein described).In specific implementations, above-mentioned cross reactivity is about any single specific antigenic disclosed herein or immunogenic polypeptide, or 2,3,4,5 or the more combination of polyspecific antigenicity and/or immunogenic polypeptide.At other embodiment, the albumin fusion proteins that comprises the fragment at least of the proteic antibody of combined treatment or variant is compared with the fragment of specific antibodies itself or variant has similar or roughly the same cross reaction characteristic.
The present invention further comprises antibody, and it is combined in the polypeptide of (as described herein) under the stringent hybridization condition and the polynucleotide encoding of the multi-nucleotide hybrid of coding human cytokines.Combined treatment albumen also can also can they be described the binding affinity of polypeptide of the present invention or illustrate corresponding to the antibody of the human cytokines of albumin fusion proteins of the present invention part.Binding affinity comprises dissociation constant or Kd is less than 5 * 10
-2M, 10
-2M, 5 * 10
-3M, 10
-3M, 5 * 10
-4M, 10
-4The binding affinity of M.Other binding affinity comprises dissociation constant or Kd is less than 5 * 10
-5M, 10
-5M, 5 * 10
-6M, 10
-6M, 5 * 10
-7M, 10
-7M, 5 * 10
-8M or 10
-8The binding affinity of M.Further, binding affinity comprises dissociation constant or Kd is less than 5 * 10
-9M, 10
-9M, 5 * 10
-10M, 10
-10M, 5 * 10
-11M, 10
-11M, 5 * 10
-12M, 10
-12M, 5 * 10
-13M, 10
-13M, 5 * 10
-14M, 10
-14M, 5 * 10
-15M or 10
-15The binding affinity of M.At some embodiments, the albumin fusion proteins that comprises the fragment at least of the proteic antibody of combined treatment or variant is similar to the affinity of specifying albumen or epi-position and having and the proteic corresponding antibodies of combined treatment (not merging to albumin), has considered tiring of albumin fusion proteins (fragment at least or the variant that comprise the proteic antibody of combined treatment) and tiring of corresponding antibodies.
The present invention also provides the bonded antibody of competitive inhibition antibody to the human cytokines epi-position, as being used for determining that by known in the art competitive bonded any method immune detection for example as herein described is definite.At some embodiments, the antibody competition inhibition is to bonded at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60% or at least 50% of epi-position.At other embodiment, comprise of the combination of the albumin fusion proteins competitive inhibition second antibody of the fragment at least of the proteic antibody of combined treatment or variant to the human cytokines epi-position.At other embodiment, the albumin fusion proteins competitive inhibition second antibody that comprises the fragment at least of the proteic antibody of combined treatment or variant is to bonded at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60% or at least 50% of human cytokines epi-position.
Be incorporated into human cytokines and can be used as the agonist or the antagonist of human cytokines corresponding to the antibody of the human cytokines of albumin fusion proteins of the present invention part.For example, the present invention comprises and partially or even wholly destroys receptor/ligand and the interactional antibody of polypeptide of the present invention.The present invention is characterized as receptor-specific antibody and part-specific antibody.The present invention also is characterized as receptor-specific antibody, and it does not prevent the part combination but prevents receptor activation.Can determine receptor activation (being signal transduction) by technology as herein described or in addition known in the art.For example, can by immunoprecipitation, subsequently the phosphorylation of western engram analysis (for example) detection receptor or its substrate as above-mentioned (as, tyrosine or serine/threonine) and determine receptor activation.In specific implementations, provide that to suppress ligand activity or receptor active be active at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60% or at least 50% antibody when not having antibody.At some embodiments, the albumin fusion proteins that comprises the fragment at least of the proteic antibody of combined treatment or variant compare with the fragment that does not merge of the proteic antibody of combined treatment or variant about prevent part in conjunction with and/or prevent that the receptor activation aspect has similar or roughly similar characteristic.
The present invention also is characterized as and not only prevents the part combination but also prevent the receptor-specific antibody of receptor activation and the antibody that identification receptor-part complex is not discerned unconjugated receptor or unconjugated part specifically.Similarly, the present invention comprises neutralizing antibody, and its binding partner also prevents the combination of part to receptor, and the antibody of binding partner, thereby prevents receptor activation but do not prevent the part bind receptor.The present invention further comprises the antibody of activated receptor.These antibody can act as receptor stimulating agent, promptly strengthen or the receptor activation of activation part-mediation bioactive all or part of, for example, by inducing receptor dimerizationization.Can illustrate that antibody is to comprising concrete bioactive agonist, antagonist or the inverse agonist of human cytokines (as being disclosed in table 1).Can use means known in the art to prepare above-mentioned antibody agonist.See as, PCT announces WO 96/40281; United States Patent (USP) the 5th, 811, No. 097; Deng etc., Blood 92 (6): 1981-1988 (1998); Chen etc., Cancer Res.58 (16): 3668-3678 (1998); Harrop etc., J.Immunol.161 (4): 1786-1794 (1998); Zhu etc., Cancer Res.58 (15): 3209-3214 (1998); Yoon etc., J.Immunol.160 (7): 3170-3179 (1998); Prat etc., J.Cell.Sci.111 (Pt2): 237-247 (1998); Pitard etc., J.Immunol.Methods205 (2): 177-190 (1997); Liautard etc., Cytokine 9 (4): 233-241 (1997); Carlson etc., J.Biol.Chem.272 (17): 11295-11301 (1997); Taryman etc., Neuron 14 (4): 755-762 (1995); Muller etc., Structure 6 (9): 1153-1167 (1998); Bartunek etc., Cytokine 8 (1): 14-20 (1996) (all incorporating into by reference with its integral body at this).At some embodiments, comprise that the fragment at least of the proteic antibody of combined treatment or the albumin fusion proteins of variant have similar or roughly the same excitement or antagonism feature to the fragment or the variant that do not merge of the proteic antibody of combined treatment.
Be incorporated into human cytokines and can be used in occlusion body outer and intravital diagnosis and the Therapeutic Method for example purification, detection and targeted therapeutic albumen corresponding to the antibody of the human cytokines of albumin fusion proteins of the present invention part.For example, antibody can be used for immune detection to measure the level of human cytokines in the biological sample qualitatively and quantitatively.See as, Harlow etc., Antibodies:A LaboratoryManual (antibody laboratory manual), (Cold Spring Harbor Laboratory Press, the 2nd edition .1988); Incorporate into by reference with its integral body at this.Similarly, the albumin fusion proteins that comprises the fragment at least of the proteic antibody of combined treatment or variant is used in occlusion body outer and intravital diagnosis and the Therapeutic Method for example purification, detection and targeted therapeutic albumen.
Be incorporated into human cytokines and can comprise the derivant of modified corresponding to the antibody of the human cytokines of albumin fusion proteins part, promptly by covalently bound any kind molecule in antibody.Such as but not limited to, antibody derivatives comprises the antibody of modified, as, by glycosylation, acetylation, Pegylation, phosphorylation, amidatioon, known protection/blocking groups derivatization, proteolytic cleavage, be connected in cell ligand or other albumen etc.Can carry out any number of chemical modification by known technology, include but not limited to concrete chemical cracking, acetylation, formylated, the synthetic tunicamycin of metabolism etc.In addition, derivant can contain one or more nonclassical amino acids.Also can aforesaid modification albumin fusion proteins of the present invention.
The preparation method of the proteic antibody of combined treatment
Can be incorporated into human cytokines by any appropriate method generation known in the art also can be corresponding to the human cytokines antibody partly of albumin fusion proteins of the present invention.Can be by the polyclonal antibody of various scheme preparations well known in the art to target antigen.For example, administering therapeutic albumen includes but not limited to that in various host animals rabbit, mice, rat etc. contain the serum of specificity to this antigenic polyclonal antibody to induce to produce.Depend on host species, various adjuvants can be used for increasing immune response, and including but not limited to, Freund adjuvant (fully with incomplete), mineral rubber such as aluminium hydroxide, surfactant such as LYSOLECITHIN SUNLECITHIN A, Pluronic polyols (pluronic polyols), polyanion, peptide class, oiliness Emulsion, keyhole limpet hemocyanin, dinitrophenol and useful potentially human adjuvant such as BCG (bacillus calmette-guerin vaccine) and coryne bacterium parvum.This adjuvant also is well known in the art.
Can use multiple technologies known in the art to prepare monoclonal antibody, comprise and use hybridoma, reorganization and display technique of bacteriophage or its combination.For example, can use and comprise known in the art and instruct in for example Harlow etc., Antibodies:A Laboratory Manual (antibody laboratory manual), (Cold SpringHarbor Laboratory Press, the 2nd edition .1988); Hammerling etc., Monoclonal Antibodiesand T-Cell Hybridomas (monoclonal antibody and T quadroma) 563-681 (Elsevier, N.Y., 1981) hybridoma technology of (described list of references is incorporated into its integral body by reference at this) prepares monoclonal antibody.Term used herein " monoclonal antibody " is not limited to the antibody by the hybridoma technology preparation.Term " monoclonal antibody " refers to derived from monoclonal, comprises the antibody of any eucaryon, protokaryon or phage clone, is not meant the method for preparing it.
It is customary and well known in the art using the method for hybridoma technology generation and screening antibody specific.At non-limitative example, can human cytokines or its fragment or variant, albumin fusion proteins or express this human cytokines or the cellular immunization mice of its fragment or variant or albumin fusion proteins.After detecting immunne response, as, in mice serum, detect specially to this antigenic antibody, then collect mouse spleen and separating Morr. cell.By knowing technology splenocyte is blended in any suitable myeloma cell subsequently, for example the cell of the cell line SP20 that can obtain from ATCC.Select hybridoma and by limited dilution cloning.Detecting secretion among the hybridoma clone by means known in the art subsequently can be in conjunction with the cell of the antibody of polypeptide of the present invention.Can produce the ascites fluid that generally contains high-level antibody with positive hybridoma clone immune mouse.
Therefore, the antibody that the invention provides the preparation monoclonal antibody method and prepare by this method, this method comprises the hybridoma of cultivating secretory antibody, wherein, for example, the following generation of this hybridoma: merge from the isolating splenocyte of the mice of antigen immune of the present invention and myeloma cell and subsequently can be in conjunction with the hybridoma clone of the antibody of polypeptide of the present invention from the hybridoma screening secretion of merging gained.
The generation polyclone that another kind is known and the method for monoclonal human B cell line are to use Epstein-Barr virus (EBV) to transform.The scheme of the B cell line that preparation EBV-transforms is that this area is known usually, such as for example, be listed in the scheme of the 7.22nd chapter of Current Protocols in Immunology (the up-to-date experimental program of immunology), editors such as Coligan, 1994, John Wiley ﹠amp; Sons, NY, it is incorporated into its integral body by reference at this.The normally human peripheral blood in the source of the B cell that is used to transform, but the B cell that is used to transform also can originate from other, includes but not limited to lymph node, tonsil, spleen, tumor tissues and infected tissue.Transforming as last at EBV is single-cell suspension liquid with tissue preparation.In addition, can take steps with in the sample that contains the B cell, physically remove or inactivation T cell (as, handle with Ciclosporin A) because can suppress the B cell immortalityization of EBV from the T cell of the male individual acquisition of antagonism-EBV antibody serum.
Usually, contain the sample of human B cell with EBV inoculation and cultivate 3-4 week.The typical case source of EBV is the culture supernatant of B95-8 cell line (ATCC#VR-1492).Generally can be observed the physiology sign that EBV transforms in the latter stage of 3-4 week culture period.Under the phase contrast microscope, that cell transformed can show is big, transparent, hair shape and be tending towards assembling for cell tight bunch.At first, EBV cell line generally is polyclonal.Yet through behind the long-time cell culture, as the result of selectivity development particular B cell clone, EBV cell line can be changed into monoclonal or polyclone.Alternatively, polyclone EBV cell transformed system can by sub-clone (as, cultivate by limiting dilution) or merge with suitable fusion partner and with the limiting dilution bed board to obtain monoclonal B cell line.To the EBV cell transformed be suitable fusion partner comprise mouse myeloma cell line (as, SP2/0, X63-Ag8.653), heterozygosis myeloma cell line (human x mice; As, SPAM-8, SBC-H20 and CB-F7) and human cell system (as, GM 1500, SKO-007, RPMI 8226 and KR-4).Therefore the present invention also provides the method for preparation at polypeptide of the present invention or its segmental polyclone or monoclonal human antibody-like, comprises that EBV-transforms human B cell.
Can produce the antibody fragment of the concrete epi-position of identification by known technology.For example, can prepare Fab of the present invention and F (ab ') 2 fragments, use enzyme such as papain (to produce the Fab fragment) or pepsin (to produce F (ab ') 2 fragments) by the proteolytic cleavage immunoglobulin molecules.F (ab ') 2 fragments contain the CH1 domain of variable region, constant region of light chain and heavy chain.
For example, also can use various phage display method known in the art to produce the antibody that is incorporated into human cytokines.In the phage display method, the function antibody domain is illustrated on the phage particle surface of the polynucleotide sequence that carries encoding function antibody structure territory.In specific implementations, this phage can be used for showing from repertoire (repertoire) or combinatorial antibody library (as, the mankind or Mus) the antigen binding structural domain of expressing.Can selection of antigen or identify the phage of expressing in conjunction with the antigenic antigen binding structural domain of being paid close attention to, as, the antigen of usage flag or solid phase surface or microballon in conjunction with or the antigen of catching.The phage that is used for these methods is filobactivirus normally, comprises fd and M13 binding structural domain from phage expression, having reorganization and being blended in the stable Fv antibody structure territory of phage gene III or the proteic Fab of gene VIII, Fv or disulfide bond.The example that can be used for preparing the phage display method of the antibody that is incorporated into human cytokines comprises and is disclosed in following methods of exhibiting: Brinkman etc., J.Immunol.Methods 182:41-50 (1995); Ames etc., J.Immunol.Methods 184:177-186 (1995); Kettleborough etc., Eur.J.Immunol.24:952-958 (1994); Persic etc., Gene1879-18 (1997); Burton etc., Advances in Immunology (immunology progress) 57:191-280 (1994); PCT applies for PCT/GB91/01134 number; PCT announces WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; With United States Patent (USP) the 5th, 698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969, No. 108; Its each comfortable this is incorporated into by reference with its integral body.
Described in above-mentioned list of references, after selecting phage, can and be used to produce the full length antibody that comprises human antibodies from phage separation antibody coding region, or the Fab of any other expectation, and at any desired host expresses, comprise mammalian cell, insect cell, plant cell, yeast and antibacterial, as detailed below.For example, reorganization produces Fab, Fab ' and F (ab ') 2 segmental technology also can use methods known in the art to adopt, and announces WO92/22324 such as being disclosed in following method: PCT; Mullinax etc., BioTechniques 12 (6): 864-869 (1992); With Sawai etc., AJRI34:26-34 (1995); With Better etc., Science 240:1041-1043 (1988) (described list of references is incorporated into its integral body by reference at this).
The example that can be used for producing the technology of strand Fv and antibody comprises the following technology that is described in: United States Patent (USP) the 4th, 946,778 and 5,258, No. 498; Huston etc., Methods in Enzymology 203:46-88 (1991); Shu etc., PNAS 90:7995-7999 (1993); With Skerra etc., Science 240:1038-1040 (1988).Some are included in the human body application of using antibody and vitro detection analysis can use chimeric, humanization or human antibodies.Chimeric antibody is the molecule of the different piece of wherein antibody derived from the different animals species, such as having derived from the variable region of mouse monoclonal antibody and the antibody of human immunoglobulin constant region.The method that produces chimeric antibody is known in the art.See as, Morrison, Science 229:1202 (1985); Oi etc., BioTechniques 4:214 (1986); Gillies etc., (1989) J.Immunol.Methods 125:191-202; United States Patent (USP) the 5th, 807,715; 4,816,567; With 4, No. 816397, it is incorporated into its integral body by reference at this.Humanized antibody is the antigenic antibody molecule from non-human species's antibody in conjunction with expectation, and it has from one or more complementary determining regions (CDR) of non-human species with from the framework region of human immunoglobulin molecule.Usually, the framework residue in the human framework region will be replaced by the corresponding residue of CDR donor antibody, with Change Example as, improve the antigen combination.These frameworks replace by well known method to be identified, as, by the interaction of simulation (modeling) CDR and framework residue with identify to antigen combine important framework residue and comparative sequences with evaluation at the uncommon framework residue of ad-hoc location.(see as, Queen etc., United States Patent (USP) the 5th, 585, No. 089; Riechmann etc., Nature 332:323 (1988), it is incorporated into its integral body by reference at this.) can use multiple technologies humanized antibody known in the art, for example comprise, (EP 239,400 in the CDR-transplanting; PCT announces WO 91/09967; United States Patent (USP) the 5th, 225,539; 5,530,101 and 5,585, No. 089), (EP 592,106 for gummed (veneering) or resurfacing (resurfacing); EP 519,596; Padlan, Molecular Immunology 28 (4/5): 489-498 (1991); Studnicka etc., ProteinEngineering 7 (6): 805-814 (1994); Roguska etc., PNAS 91:969-973 (1994)) and chain reorganization (United States Patent (USP) the 5th, 565, No. 332).
Human antibodies is special expectation to the therapeutic treatment human patients fully.Can produce human antibodies by several different methods known in the art, comprise aforesaid phage display method, use from the deutero-antibody library of human immunoglobulin sequence.Also see United States Patent (USP) the 4th, 444,887 and 4,716, No. 111; Announce WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735 and WO 91/10741 with PCT; Its each comfortable this is incorporated into by reference with its integral body.
Also can use the transgenic mice of can not the endogenous immunoglobulin of expressive function but can express human immunoglobulin gene to prepare human antibodies.For example, the mankind are heavy and light chain immunoglobulin gene complex can be introduced randomly or introduce mouse embryo stem cell by homologous recombination.Alternatively, heavy and light chain gene also can be with human variable region, constant region and variable region introducing mouse embryo stem cell except the mankind.Introducing the human immunoglobulin site by homologous recombination can be respectively or side by side cause the heavy and light chain immunoglobulin gene of mice not have function.The disappearance JH district of especially, isozygotying stops endogenous antibody to produce.Embryonic stem cell amplification and the microinjection modified are gone into blastocyst to produce gomphosis mouse.Breed gomphosis mouse subsequently to produce the offspring of isozygotying of express human antibody.With selected antigen all or part of as, polypeptide of the present invention with normal mode immune transgenic mice.Use conventional hybridization tumor technology to obtain at antigenic monoclonal antibody from the transgenic mice of immunity.The human immunoglobulin transgenic that transgenic mice has is reset during the B cell differentiation, and experiences class conversion and somatic mutation subsequently.Therefore use this technology may produce treatment useful IgG, IgA, IgM and IgE antibody.The general introduction that produces this technology of human antibodies sees Lonberg and Huszar, Int.Rev.Immunol.13:65-93 (1995).Produce human antibodies and human monoclonal antibody's this technology and produce this antibody scheme go through see as, PCT announces WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; No. the 0598877th, European patent; United States Patent (USP) the 5th, 413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; 5,939,598; 6,075,181; With 6,114, No. 598, it is incorporated into its integral body by reference at this.In addition, such as Abgenix, Inc. (Freemont, CA) and Genpharm (San Jose, CA) etc. company can use similar in appearance to the technology of aforesaid technology and be devoted to provide at selected antigenic human antibodies.
Can use the technology that is called " guiding is selected " to produce the complete human antibodies of the selected epi-position of identification.Selected non-human monoclonal antibody in this method, as, mouse antibodies is used for instructing and selects to discern the complete human antibodies of same epi-position.(Jespers etc., Bio/technology 12:899-903 (1988)).
The polynucleotide of encoding antibody
The present invention further provides the polynucleotide that comprise encoding antibody and its segmental nucleotide sequence.The present invention also be included under the stringent condition as defined above or alternatively under low strict hybridization conditions with encode for example proteic antibody of specificity combined treatment, further, in conjunction with the polynucleotide of multi-nucleotide hybrid that have as the antibody of the polypeptide of the aminoacid sequence of " human cytokines: X " of " SEQ ID NO:Z " row of being disclosed in table 2.
Can obtain polynucleotide by any method known in the art, and determine the nucleotide sequence of these polynucleotide.For example, if the nucleotide sequence of antibody is known, encode this antibody polynucleotide can from the assembling of the oligonucleotide of chemosynthesis (as, Kutmeier etc., described in the BioTechniques 17:242 (1994)), briefly comprise eclipsed oligonucleotide, the annealing of the part of synthesizing the sequence that contains this antibody of encoding and the oligonucleotide that connects these oligonucleotide, connects by pcr amplification subsequently.
Alternatively, can produce the polynucleotide of encoding antibody from the nucleic acid in suitable source.If contain clone's non-availability of the nucleic acid of the specific antibodies of encoding, but the sequence of this antibody molecule is known, but the synthetic primer pcr amplification of then chemosynthesis or can hybridize in this sequence 3 ' and 5 ' terminal by using or by use to the specific oligonucleotide probe clone of specific gene sequence from suitable source (as, the antibody cDNA library or cDNA library or the isolating nucleic acid that produce such as the hybridoma of selecting with expressing antibodies from any tissue of expressing antibodies or cell, for example, polyadenylic acid+RNA) obtains the nucleic acid of this immunoglobulin of coding, to identify, as, the cDNA clone who comes the cDNA library of this antibody of own coding.The nucleic acid of the amplification that is produced by PCR can use any method well known in the art to be cloned into reproducible cloning vehicle (seeing embodiment 65) subsequently.
After determining the nucleotide sequence and amino acid sequence corresponding of antibody, can use the well known method that is used to handle nucleotide sequence to handle the nucleotide sequence of antibody, as recombinant DNA technology, site-directed mutation, PCR etc. (for example see, the technology of the following stated: Sambrook etc., 1990, MolecularCloning, A Laboratory Manual (molecular cloning laboratory manual), the 2nd edition, Cold SpringHarbor Laboratory, Cold Spring Harbor, editor such as NY and Ausubel, 1998, CurrentProtocols in Molecular Biology (up-to-date molecular biology experiment guide), John Wiley ﹠amp; Sons, NY, it is incorporated into its integral body by reference at this), the antibody so that generation has the different aminoacids sequence for example produces aminoacid replacement, disappearance and/or interpolation.
In specific implementations, the aminoacid sequence that can check heavy and/or light chain variable domain to be being identified the sequence of complementary determining region (CDR) by the method for knowing in this area, as by the known amino acid sequence of other heavy and variable region of light chain relatively to determine the high variable district of sequence.Use the routine recombinant dna technology, one or more CDR can insert framework region, as, insert human framework region as described above with the humanization non-human antibody.Framework region can be framework region natural generation or total and for example human framework region (for example seeing Chothia etc., the human framework region tabulation of J.Mol.Biol.278:457-479 (1998)).On the one hand, the polynucleotide encoding specificity of group frame district and CDR generation is in conjunction with the antibody of polypeptide of the present invention.On the other hand, as mentioned above, can in framework region, produce one or more aminoacid replacement, and for example, this aminoacid replacement improves antibody to its antigenic combination.In addition, this method can be used for producing the aminoacid replacement or the disappearance of the variable region cysteine residue of one or more participation intrachain disulfide bonds, lacks the antibody molecule of one or more intrachain disulfide bonds with generation.Other changes of polynucleotide is comprised in the present invention and in those skilled in the art's ability.
In addition, can use by montage and have the gene of mouse antibodies molecule of suitable antigenic specificity and technology (Morrison etc., the Proc.Natl.Acad.Sci.81:851-855 (1984) that gene exploitation with suitable bioactive human antibody molecule is used for producing " chimeric antibody "; Neuberger etc., Nature312:604-608 (1984); Takeda etc., Nature 314:452-454 (1985)).As mentioned above, chimeric antibody be wherein different piece derived from the molecule of different animals species, such as having, as, humanized antibody derived from the variable region of Mus mAb and the molecule of human immunoglobulin constant region.
Alternatively, can revise technology (United States Patent (USP) the 4th, 946, No. 778 that description is used to produce single-chain antibody; Bird, Science 242:423-42 (1988); Huston etc., Proc.Natl.Acad.Sci.USA 85:5879-5883 (1988); With Ward etc., Nature 334:544-54 (1989)) to produce single-chain antibody.The weight and the light chain segments that are met the Fv district by the aminoacid bridging form single chain polypeptide and form single-chain antibody.Also can use the segmental technology of assembling function Fv in escherichia coli (Skerra etc., Science 242:1038-1041 (1988)).
Recombinant expressed antibody
Recombinant expressed antibody or its fragment, derivant or analog (as, the weight of antibody or light chain or single-chain antibody), require structure to contain the expression vector of the polynucleotide of encoding antibody.Behind the polynucleotide of the weight of acquisition coding antibody molecule of the present invention or antibody or light chain or its part (for example containing heavy or light chain variable domain), can use well known technology to produce the carrier of antibody molecule by the recombinant DNA technology preparation.Therefore this paper has described by expression and has contained the polynucleotide of antibody coding nucleotide sequence and prepare proteic method.Method well known to those skilled in the art can be used for making up and contains antibody coding sequence and the suitable expression vector of transcribing and translate control signal.These methods comprise, for example, and genetic recombination in extracorporeal recombinant DNA technology, synthetic technology and the body.Therefore the invention provides and comprise that coding antibody molecule of the present invention or its nucleotide sequence heavy or light chain or heavy or light chain variable domain are operably connected to the replicable vector of promoter.This carrier can comprise encoding antibody molecule constant region nucleotide sequence (see as, PCT announces WO 86/05807; PCT announces WO 89/01036; With United States Patent (USP) the 5th, 122, No. 464) and the variable domains of antibody can be cloned into this carrier with The expressed heavy or light chain.
By routine techniques expression vector is transferred to host cell and cultivated cells transfected to produce antibody by routine techniques subsequently.Therefore the present invention comprises and contains the host cell that coding antibody of the present invention or its polynucleotide heavy or light chain or single-chain antibody are operably connected to allogeneic promoter.Express the embodiment of double-stranded antibody at some, the carrier of the heavy and light chain of coding can be in host cell coexpression with the The expressed immunoglobulin molecules, as detailed below.
Multiple host-expression vector system can be used for expressing antibody molecule of the present invention.This host-expression system represents to prepare the carrier of the coded sequence of paying close attention to subsequent purificn, but but the cell of expression expressed in situ antibody molecule of the present invention when the time also with conversion of suitable nucleotide coding sequence or transfection.These are including but not limited to microorganism, such as with the recombinant phage dna, plasmid DNA or the cosmid DNA expression vector microorganism transformed that contain antibody coding sequence (as, escherichia coli, bacillus subtilis); The yeast that transforms with the recombinant yeast expression vector that contains antibody coding sequence (as, saccharomyces cerevisiae, Pichia sp.); Infection contains the insect cell system of the recombinant virus expression vector (as, baculovirus) of antibody coding sequence; The infection recombinant virus expression vector (as, cauliflower mosaic virus CaMV; Tobacco mosaic virus (TMV) TMV) or with the recombinant plasmid expression vector that contains antibody coding sequence (as, Ti-plasmids) plant transformed cell system; Or comprise and contain derived from mammalian cell genome (as, metallothionein promoter) or mammalian virus (as, gland virus stage starting; The mammal cell line system of the recombinant expression construct body of promoter vaccinia virus 7.5K promoter) (as, COS, CHO, BHK, 293,3T3 cell).On the one hand, bacterial cell such as escherichia coli and eukaryotic cell further in particular for expressing the cell of complete recombinant antibody molecule, are used for the expressing recombinant antibody molecule.For example, early stage (major intermediate early) gene promoter sub-element in main centre of mammalian cell such as Chinese hamster ovary cell (CHO) combination carrier such as human cytomegalovirus is effective expression system (Foecking etc., the Gene 45:101 (1986) of antagonist; Cockett etc., Bio/Technology 8:2 (1990)).
In bacterial system, can be according to advantageously being selected the great expression carrier by the application of the antibody molecule plan expressed.For example, when a large amount of this albumen to be prepared are used to prepare the pharmaceutical composition of antibody molecule, can expect to guide the expression high level to be easy to the carrier of the fusion protein product of purification.This carrier is including but not limited to coli expression carrier pUR278 (Ruther etc., EMBO are (1983) J.2:1791), thereby wherein antibody coding sequence can connect into carrier preparation fusion rotein in the frame individually with lac Z coding region; PIN carrier (Inouye ﹠amp; Inouye, Nucleic Acids Res.13:3101-3109 (1985); VanHeeke ﹠amp; Schuster, J.Biol.Chem.24:5503-5509 (1989)); And analog.The pGEX carrier also can be used for expressing allogenic polypeptide for the fusion rotein of glutathione S-transferase (GST).Usually, this fusion rotein is solvable and can be easy to from cracked cell by absorption be incorporated into substrate glutathion-agarose microballon, eluting and purification in the presence of free glutathione subsequently.Thereby design pGEX carrier is to comprise the target gene product that thrombin or factor Xa protease cracking site can partly discharge the clone from GST.
In the insecticide system, Autographa californica multicapsid nucleopolyhedrosisvirus (Autographa californica) nuclear polyhedral virus (AcNPV) is as the carrier of expression alien gene.Viral growth is in voracious noctuid (Spodopterafrugiperda) cell.Antibody coding sequence can be cloned into the nonessential region (for example polyhedron gene) of virus separately and place under the control of AcNPV promoter (for example polyhedrin promoter).
In mammalian host cell, can use a large amount of expression systems based on virus.In the situation of adenovirus as expression vector, the antibody coding sequence of being paid close attention to can be connected in adenovirus and transcribe/translate the control complex, as, late promoter and tripartite leader[.This mosaic gene can insert the adenoviral gene group by reorganization in external or the body subsequently.Insert virus genomic nonessential region (as, district E1 or E3) will produce can survive and can be in the host of infection the recombinant virus of expressed antibody molecule.(as, see Logan ﹠amp; Shenk, Proc.Natl.Acad.Sci.USA 81:355-359 (1984).The antibody coding sequence that effective translation is inserted also can require the specificity initial signal.These signals comprise ATG start codon and flanking sequence.In addition, start codon must be in same reading frame to guarantee to translate complete insertion sequence with the coded sequence of expectation.These external translation control signals and start codon can be multiple source, and be natural and synthetic.Can strengthen expression efficiency (seeing Bittner etc., Methods in Enzymol.153:51-544 (1987)) by comprising suitable transcriptional enhancer element, transcription terminator etc.
In addition, can select to regulate the expression of insertion sequence or the host cell bacterial strain of modification and processed gene product in the hope of the specificity mode of hoping.This modification (as, glycosylation) and processing (as, cracking) protein product can be important to protein function.Different host cells have peculiar and specific mechanism to translating post-treatment and modified protein and gene outcome.Can select correct modification and the processing of suitable cell line or host system with the foreign protein guaranteeing to express.For this purpose, can use and have the initial transcripton of correct processing, the eukaryotic host cell of the cell mechanism of glycosylation and phosphorylation gene outcome.This mammalian host cell is including but not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293,3T3, WI38, and especially, breast cancer cell line is such as for example, BT483, Hs578T, HTB2, BT20 and T47D, with MCF-10 such as for example, CRL7030 and Hs578Bst.
At an embodiment, for the extended high rate rate produces recombiant protein, but stably express albumen.For example, can transform the cell line of stably express antibody molecule.Be different from and use the expression vector contain the virus replication starting point, can by suitable expression control element (as, promoter, enhancer, sequence, transcription terminator, polyadenylation site etc.) DNA and the selected marker transformed host cell of control.After introducing foreign DNA, can allow engineered cells on enrichment medium, to grow 1-2 days, forward the selection culture medium subsequently to.Selected marker in the recombiant plasmid is given resistance and is entered its chromosome and growth formation focus (foci) to select and to allow the cytotostatic integrated plasmid, and it can be cloned conversely and increase into cell line.This method can be advantageously used in the cell line of transforming expressed antibody molecule.The cell line of this transformation can be particularly useful for screening and estimate directly or indirectly and the interactional chemical compound of antibody molecule.
Can use a large amount of selective systems, including but not limited to herpes simplex virus thymidine kinase (Wigler etc., Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyl transferase (Szybalska ﹠amp; Szybalski, Proc.Natl.Acad.Sci.USA 48:202 (1992)) and adenine phosphoribosyl transferase (Lowy etc., Cell 22:817 (1980)) gene, can be used for tk-, hgprt-or aprt-cell respectively.Equally, the antimetabolite resistance can be used as the selection basis to following gene: dhfr, gives the resistance to methotrexate (Wigler etc., Natl.Acad.Sci.USA 77:357 (1980); O ' Hare etc., Proc.Natl.Acad.Sci.USA78:1527 (1981)); Gpt gives (the Mulligan ﹠amp of the resistance to mycophenolic acid; Berg, Proc.Natl.Acad.Sci.USA 78:2072 (1981)); Neo gives resistance (the ClinicalPharmacy 12:488-505 to aminoglycoside G-418; Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann.Rev.Pharmacol.Toxicol.32:573-596 (1993); Mulligan, Science 260:926-932 (1993); With Morgan and Anderson, Ann.Rev.Biochem.62:191-217 (1993); May, 1993, TIB TECH 11 (5): 155-215 (1993)); And hygro, give the resistance to hygromycin (Santerre etc., Gene 30:147 (1984)).The common known method in administered recombinant dna technique field is selected the recombinant clone expected routinely, and this method for example is described in, Ausubel etc. (editor), Current Protocols in Molecular Biology (up-to-date molecular biology experiment guide), John Wiley ﹠amp; Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual (gene transfer and expression, lab scenario), Stockton Press, NY (1990); With (editors) such as Dracopoli, 12 and 13 chapters of Current Protocols in Human Genetics (up-to-date human genetics experiment guide), John Wiley ﹠amp; Sons, NY (1994); Colberre-Garapin etc., J.Mol.Biol.150:1 (1981), it is incorporated into its integral body by reference at this.
(summary sees Bebbington and Hentschel.The use of vectors based on gene amplification for the expression ofcloned gene in mammalian cells in DNA cloning (using the carrier based on gene amplification to be used for the gene of dna clone at the mammalian cell expression cloning) can to increase the expression of antibody molecule by carrier amplification, the 3rd volume. (Academic Press, New York, 1987)).When the labelling in the carrier system of expressing antibodies can increase, the level increase of the inhibitor that exists in the host cell culture will increase the copy number of marker gene.Because the district of amplification is relevant with antibody gene, production of antibodies also will increase (Crouse etc., Mol.Cell.Biol.3:257 (1983)).
Use glutamine synthase (GS) or DHFR can when having medicine methionine sulphoximine or methotrexate, increase respectively as the carrier of selected marker.Based on the advantage of the carrier of glutamine synthase is to utilize the cell line of glutamine synthase feminine gender (as, rat bone marrow tumour cell system, NSO).The glutamine synthase expression system also can work in glutamine synthase express cell (as Chinese hamster ovary (CHO) cell), by providing other inhibitor to prevent endogenous gene performance function.Glutamine synthase expression system and its component are specified in PCT and announce: WO87/04462; WO86/05807; WO89/01036; WO89/10404 and WO91/06657, it is incorporated into its integral body by reference at this.In addition, spendable glutamine synthase expression vector is from comprising for example Lonza Biologies according to the present invention, and (Portsmouth, supplier NH) is commercially available obtainable for Inc..Use the GS expression system in rat bone marrow tumour cell, to express and produce monoclonal antibody and be described in Bebbington etc., Bio/Technology 10:169 (1992) and Biblia and Robinson Biotechnol.Prog.11:1 (1995), it is incorporated into its integral body by reference at this.
Host cell can two expression vector cotransfections of the present invention, the first vector encoded heavy chain polypeptide of deriving, the second vector encoded derived light chain polypeptide.These two carriers can contain identical selected marker, make to express heavy and light chain polypeptide equably.Alternatively, can use coding also can express the single carrier of heavy and light chain polypeptide.In this situation, light chain should place heavy chain before to avoid excessive poisonous free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc.Natl.Acad.Sci.USA 77:2197 (1980)).Coded sequence heavy and light chain can comprise cDNA or genomic DNA.
Behind animal, chemosynthesis or recombinant expressed preparation antibody molecule of the present invention, can be by any this antibody molecule of method purification that is used for the purification immunoglobulin molecules known in the art, for example, by chromatograph (as, ion exchange, affinity, especially by behind the protein A to the affinity and the screening column chromatography (sizing column chromatography) of specific antigen), centrifugal, dissolubility difference or by any other standard technique that is used for purifying protein.In addition, being incorporated into human cytokines also can merge so that purification with allogeneic polypeptide sequence as herein described or in addition known in the art corresponding to the antibody of albumin fusion proteins of the present invention or its segmental human cytokines part.
The modification of antibody
The antibody of combined treatment albumen or fragment or variant can merge so that purification with labelled sequence such as peptide.In other embodiments, marker amino acid sequence is six-histidine peptide, such as the label that provides in the pQE carrier (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), and other, wherein many is commercially available getting.As Gentz etc., described in the Proc.Natl.Acad.Sci.USA 86:821-824 (1989), for example, six-histidine provides the purification that makes things convenient for to fusion rotein.Other peptide tag that is used for purification includes but not limited to hemagglutinin label (being also referred to as " HA label "), and it is corresponding to epi-position (Wilson etc., Cell 37:767 (1984)) and " flag (flag) " label derived from influenza hemagglutinin protein.
The present invention further comprises antibody or its fragment of puting together in diagnosis or therapeutic agent.Can use antibody with for example in diagnosis, the development or the progress of monitoring tumor with for example, are determined the effectiveness of TA scheme as the part of Clinical detection scheme.Coupling antibody can help to detect in detectable material.Detectable examples of substances comprises the positron emitting metal and the on-radiation paramagnetic metal ion of various enzymes, prothetic group, fluorescent material, luminescent material, bioluminescent material, active material, the various positron emission tomographies of use.The directly coupling or put together of detectable material in antibody (or its fragment), or use technology known in the art by intermedium (such as for example, joint known in the art) coupling or put together indirectly in antibody (or its fragment).For example see, United States Patent (USP) the 4th, 741, No. 900 metal ion, it can be puted together according to the present invention in antibody to be used as diagnostic agent.The example of suitable enzyme comprises horseradish peroxidase, alkali phosphatase, beta galactosidase or acetylcholinesterase; The example of suitable prothetic group complex comprises Streptavidin/biotin and avidin/biotin; The example of suitable fluorescent material comprises umbelliferone, fluorescein, Fluorescein isothiocyanate, rhodamine, dichlorotriazine amine (dichlorotriazinylamine) fluorescein, dansyl chloride or phycoerythrin; The example of luminescent material comprises luminol; The example of bioluminescent material comprises luciferase, luciferin and aequorin; The example of suitable active material comprises 125I, 131I, 111In or 99Tc.Other example of detectable material is described in the other places of this paper.
Further, antibody of the present invention can be puted together in therapeutic part such as cytotoxin, as, cytostatics or cytocide, therapeutic agent or radioactive metal ion, as, alpha emitter such as for example, 213Bi.Cytotoxin or cytotoxic agent comprise deleterious dose of any pair cell.Example comprises Pa Litasai (paclitaxol), Cytochalasin B, Gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin diketone, mitoxantrone, mithramycin, actinomycin D, 1-boldenone, glucocorticoid, procaine, tetracaine, lignocaine, Propranolol and puromycin and its analog or homologue.Therapeutic agent includes but not limited to, antimetabolite (as, methotrexate, Ismipur, the 6-thioguanine, cytosine arabinoside, the 5-fluorouracil decarbazine), alkylating agent (as, chlormethine, Thiotef (thioepa) chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, mitobronitol, streptozotocin, ametycin, with along (II) (DDP) cisplatin of dichloro diamidogen platinum (cis-dichlorodiamine platinum)), anthracycline antibiotics (as, daunorubicin (daunomycin in the past) and doxorubicin), antibiotic (as, dactinomycin (D actinomycin D in the past), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agent (as, vincristine and vinblastine).
Conjugate of the present invention can be used for modifying specifies biological answer-reply, and described therapeutic agent or drug moiety are not interpreted as and are limited to classical chemical therapeutic agent.For example, drug moiety can be protein or the polypeptide with desired biological activity.This protein can comprise, for example, and toxin such as abrin, ricin A, Rhodopseudomonas extracellular toxin or diphtheria toxin, diphtherotoxin; Albumen such as tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, apoptosis agent, as TNF-α, TNF-β, AIM I (seeing No. 97/33899, international publication WO), AIM II (seeing No. 97/34911, international publication WO), Fas part (Takahashi etc., Int.Immunol, 6:1567-1574 (1994)), VEGI (seeing No. 99/23105, international publication WO), thrombosis agent or anti--blood vessel propellant, as angiostatin or endostatin; Or the biological answer-reply dressing agent is such as for example, lymphokine, il-1 (" IL-1 "), interleukin-2 (" IL-2 "), interleukin-6 (" IL-6 "), granulocyte macrophage colony stimulating factor (" GM-CSF "), granulocyte colony-stimulating factor (" G-CSF ") or other somatomedin.
Antibody also can be attached to solid support, and this immunoassay or purification to target antigen is particularly useful.This solid support includes but not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polrvinyl chloride or polypropylene.
This therapeutic part is known with the technology that antibody is puted together.For example see, Arnon etc., " Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy (monoclonal antibody that in treatment of cancer, is used for immune targeted drug) ", in Monoclonal Antibodies AndCancer Therapy (monoclonal antibody and treatment of cancer), Reisfeld etc. (editor), the 243-56 page or leaf (Alan R.Liss, Inc.1985); Hellstrom etc., " Antibodies For Drug Delivery (antibody that is used for delivering drugs) ", in Controlled Drug Delivery (controlled medicine is sent) (the 2nd edition), Robinson etc. (editor), the 623-53 page or leaf (Marcel Dekker, Inc.1987); Thorpe, " Antibodies Carriers Of Cytotoxic Agents In Cancer Therapy:A Review (summary of the antibody carrier of cytotoxic agent in the treatment of cancer) ", in Monoclonal Antibodies ' 84:BiologicalAnd Clinical Applications (monoclonal antibody ' 84: biology and clinical practice), Pinchera etc. (editor), 475-506 page or leaf (1985); " Analysis; Results; And Future Prospective Of TheTherapy Use Of Radiolabeled Antibodies In Cancer Therapy (analysis of the therapeutic use of radiolabeled antibody in treatment of cancer; result and vision of the future) ", at Monoclonal AntibodiesFor Cancer Detection And Therapy (monoclonal antibody that is used for cancer detection and treatment), Baldwin etc. (editor), 303-16 page or leaf (Academic Press 1985), with Thorpe etc., " ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates (preparation of antibody-toxin conjugate and cytotoxicity feature) ", Immunol.Rev.62:119-58 (1982).
Alternatively, antibody can be puted together in second antibody forming antibody heterozygosis conjugate (heteroconjugate), as by Segal at United States Patent (USP) the 4th, 676, described in No. 980, it is incorporated into its integral body by reference at this.
No matter whether have the therapeutic part of puting together, separately or the antibody useful as therapeutics used of associational cells virulence factor and/or cytokine in antibody.
Antibody-albumin merges
Be incorporated into human cytokines and also can include but not limited to, in conjunction with the human cytokines of " human cytokines X " row that are disclosed in table 1 or the antibody of its fragment or variant corresponding to the human cytokines antibody partly of albumin fusion proteins of the present invention.
In specific implementations, immunologic opsonin combined treatment albumen also comprises the VH domain or is made up of the VH domain alternatively corresponding to the fragment of the antibody of the human cytokines of albumin fusion proteins part or variant.In other embodiments, immunologic opsonin combined treatment albumen and comprise one, two or three VHCDR or form by it alternatively corresponding to the fragment of the antibody of the human cytokines of albumin fusion proteins part or variant.In other embodiments, immunologic opsonin combined treatment albumen and comprise VHCDR1 or form by VH CDR1 alternatively corresponding to the fragment of the antibody of the human cytokines of albumin fusion proteins part or variant.In other embodiments, immunologic opsonin combined treatment albumen and comprise VH CDR2 or form by VH CDR2 alternatively corresponding to the fragment of the antibody of the human cytokines of albumin fusion proteins part or variant.In other embodiments, immunologic opsonin combined treatment albumen and comprise VH CDR3 or form by VH CDR3 alternatively corresponding to the fragment of the antibody of the human cytokines of albumin fusion proteins part or variant.
In specific implementations, immunologic opsonin combined treatment albumen also comprises the VL domain or is made up of the VL domain alternatively corresponding to the fragment of the antibody of the human cytokines of albumin fusion proteins part or variant.In other embodiments, immunologic opsonin combined treatment albumen and comprise one, two or three VLCDR or form by it alternatively corresponding to the fragment of the antibody of the human cytokines of albumin fusion proteins part or variant.In other embodiments, immunologic opsonin combined treatment albumen and comprise VLCDR1 or form by VL CDR1 alternatively corresponding to the fragment of the antibody of the human cytokines of albumin fusion proteins part or variant.In other embodiments, immunologic opsonin combined treatment albumen and comprise VL CDR2 or form by VL CDR2 alternatively corresponding to the fragment of the antibody of the human cytokines of albumin fusion proteins part or variant.In other embodiments, immunologic opsonin combined treatment albumen and comprise VL CDR3 or form by VL CDR3 alternatively corresponding to the fragment of the antibody of the human cytokines of albumin fusion proteins part or variant.
In other embodiments, immunologic opsonin combined treatment albumen and comprise one, two, three, four, five or six VH and/or VL CDR or form by it alternatively corresponding to the fragment of the antibody of the human cytokines of albumin fusion proteins part or variant.
In some embodiments, immunologic opsonin combined treatment albumen also comprises scFv or is made up of scFv alternatively corresponding to the fragment of the antibody of the human cytokines of albumin fusion proteins part or variant, described scFv comprises the VH domain of therapeutic antibodies, and it passes through such as (Gly
4Ser)
3The peptide linker of (SEQ ID NO:4) etc. is connected in the VL domain of therapeutic antibodies.
Immunophenotyping
At least one fragment of antibody of the present invention or the antibody that comprises combined treatment albumen (or its fragment or variant) of the present invention or the albumin fusion proteins of variant can be used for the immunophenotyping of cell line and biological sample.Human cytokines of the present invention can be used as cell-specific marker, or more specifically as the cell marking at particular cell types differentiation and/or sophisticated different phase differential expression.To allow the cell colony of this labelling of screening expression at the monoclonal antibody of concrete epi-position or epi-position combination (or comprise at least one fragment of the proteic antibody of combined treatment or the albumin fusion proteins of variant).Can adopt multiple technologies to use monoclonal antibody (or comprise at least one fragment of the proteic antibody of combined treatment or the albumin fusion proteins of variant) to screen the cell colony of expressing this labelling, and comprise the magnetic bead that uses antibody sandwich Magnetic Isolation, with attach to solid-phase matrix (being plate) antibody " elutriation " and flow cytometry (see as, United States Patent (USP) the 5th, 985, No. 660; With Morrison etc., Cell, 96:137-49 (1999)).
These technology allow screening specific cells colonies, and all " non-self " cells as seen in haematological malignancies (i.e. minimal residual disease in acute leukemic patient (MRD)) and in transplanting are to prevent graft versus host disease (GVHD).Alternatively, hematopoietic stem cell and CFU-GM that these technology allow screening to breed and/or to break up are as finding in human Cord blood.
Characterize the proteic antibody of combined treatment and comprise the fragment of the proteic antibody of combined treatment or the albumin fusion proteins of variant
At least one fragment of antibody of the present invention or the antibody that comprises combined treatment albumen (or its fragment or variant) of the present invention or the albumin fusion proteins of variant can characterize in many ways.Particularly, the technology known in the art of using the techniques described herein or revising routinely can be measured the albumin fusion proteins specificity of at least one fragment that comprises the proteic antibody of combined treatment or variant in conjunction with the ability by the bonded same antigen in following antibody specificity ground, described antibodies and the human cytokines corresponding human cytokines of antibody partly in conjunction with albumin fusion proteins.
To the albumin fusion proteins (specifically) of at least one fragment of antibody of the present invention or the antibody that comprises combined treatment albumen (or its fragment or variant) of the present invention or variant in conjunction with the mensuration of the ability of specified protein or epi-position can in solution, carry out (as, Houghten, Bio/Techniques 13:412-421 (1992)), on pearl, carry out (as, Lam, Nature 354:82-84 (1991)), on chip, carry out (as, Fodor, Nature 364:555-556 (1993)), on antibacterial, carry out (as, United States Patent (USP) the 5th, 223, No. 409), on spore, carry out (as, patent the 5th, 571,698; 5,403,484; With 5,223, No. 409), on plasmid, carry out (as, Cull etc., Proc.Natl.Acad.Sci.USA 89:1865-1869 (1992)) or on phage, carry out (as, Scott and Smith, Science 249:386-390 (1990); Devlin, Science 249:404-406 (1990); Cwirla etc., Proc.Natl.Acad.Sci.USA 87:6378-6382 (1990); And Felici, J.Mol.Biol.222:301-310 (1991)) (each comfortable this of these lists of references is incorporated into by reference with its integral body).Also can use or revise routinely the techniques described herein or technology in addition known in the art and measure specificity and the affinity of the albumin fusion proteins of at least one fragment of antibody of the present invention or the antibody that comprises combined treatment albumen (or its fragment or variant) of the present invention or variant specified protein or epi-position.
Can by any method known in the art measure the albumin fusion proteins of at least one fragment that comprises the proteic antibody of combined treatment of the present invention or variant and other antigen (as, with the molecule that has sequence/structure conservative by the bonded molecule in following antibody specificity ground, described antibodies is corresponding to the human cytokines (or its fragment or variant) of the human cytokines part of albumin fusion proteins of the present invention) cross reactivity.
The immunoassay that can be used for analysis (immunologic opsonin) combination and cross reactivity includes but not limited to, competitive and the non--competitive assay system of technology such as use such as western trace, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), " sandwich " immunoassay, immunoprecipitation assay, precipitin reaction, GDP reaction, immunodiffusion algoscopy, agglutination assay, complement fixation assay, immunoradiometry, fluorescence immunoassay and protein A immunoassay only gives some instances.Such algoscopy be conventional and well known in the art (see as, editors such as Ausubel, 1994, Current Protocols in Molecular Biology (up-to-date molecular biology experiment guide), the 1st volume, John Wiley ﹠amp; Sons, Inc., New York incorporates into its integral body by reference at this).The exemplary immunization algoscopy briefly is described in hereinafter (but unexpectedly being restriction).
The immunoprecipitation scheme generally comprise lysis buffer such as be added with protein phosphatase and/or protease inhibitor (as, EDTA, PMSF, aprotinin, vanadic acid sodium) RIPA buffer (1%NP-40 or Triton X-100,1% NaTDC, 0.1%SDS, 0.15M NaCl, 0.01M sodium phosphate, at pH 7.2, cell lysis colony 1%Trasylol), add antibody of the present invention or comprise at least one fragment of antibody of combined treatment albumen (or its fragment or variant) or the albumin fusion proteins of the present invention of variant to cell lysate, 40 ℃ of incubation a period of times (as, 1 to 4 hour), adding protein A and/or Protein G sepharose 4B to cell lysate (or is comprising under the situation of albumin fusion proteins of at least one fragment of therapeutic antibodies or variant, add pearl) with suitable anti-idiotype antibody or anti-albumin antibody sandwich, at about one hour of 40 ℃ of incubations or longer, in lysis buffer, clean pearl and resuspended pearl in the SDS/ sample buffer.Can be by the ability of assessing antibody of the present invention or albumin fusion proteins immunoprecipitation specific antigen as the western engram analysis.One skilled in the art will know that can be modified with increase antibody or albumin fusion proteins to antigenic combination and reduce background parameter (as, with sepharose 4B presettling cell lysate).Further discussion about the immunoprecipitation scheme sees, as, editors such as Ausubel, 1994, Current Protocols in Molecular Biology (up-to-date molecular biology experiment guide), the 10.16.1 of the 1st volume, John Wiley ﹠amp; Sons, Inc., New York.
The Western engram analysis generally comprises the preparation protein example, polyacrylamide gel (as, 8%-20%SDS-PAGE, it depends on antigen molecular) in protein example is carried out electrophoresis, protein example is transferred to such as celluloid from polyacrylamide gel, on the film such as PVD or nylon, lock solution (as, the PBS that contains 3%BSA or defatted milk) closing membrane in, cleaning buffer solution (as, PBS-Tween 20) the middle film that cleans, antibody of the present invention or albumin fusion proteins (being diluted in the sealing buffer) are put on the film, in cleaning buffer solution, clean film, apply be diluted in sealing in the buffer put together in zymolyte (as, horseradish peroxidase or alkali phosphatase) or Geigers (as
32P or
125I) second antibody (it discerns albumin fusion proteins, as Anti-Human's serum albumin antibody) is cleaned film, and is detected antigenic existence in cleaning buffer solution.One skilled in the art will know that and to be modified with signal that improves detection and the parameter that reduces background noise.Further discussion about western trace scheme sees, as, editors such as Ausubel, 1994, Current Protocols in Molecular Biology (up-to-date molecular biology experiment guide), the 10.8.1 of the 1st volume, John Wiley ﹠amp; Sons, Inc., New York.
ELISA comprises preparation antigen, hole with antigen coated 96-hole microtitration plate, flush away is not attached to the antigen on the hole, to the hole add put together in detectable chemical compound such as zymolyte (as, horseradish peroxidase or alkali phosphatase) antibody of the present invention or albumin fusion proteins (at least one fragment or the variant that comprise the proteic antibody of combined treatment) and incubation a period of time, flush away is unconjugated or non--bonded specifically albumin fusion proteins, and detection specificity ground in conjunction with bag by the antigenic antibody in hole or the existence of albumin fusion proteins.Antibody or albumin fusion proteins needn't be puted together in detectable chemical compound among the ELISA; On the contrary, can add the second antibody (its identification antibody or albumin fusion proteins) of puting together to the hole in detectable chemical compound.Further, replace with antigen coated hole, can be with antibody or albumin fusion proteins bag by to the hole.In this case, detectable molecule can be put together in detectable chemical compound such as zymolyte (as, horseradish peroxidase or alkali phosphatase) antigen.One skilled in the art will know that and to be modified with the parameter of the signal that improve to detect and other modification of ELISA known in the art.About the further discussion of ELISA see as, editors such as Ausubel, 1994, CurrentProtocols in Molecular Biology (up-to-date molecular biology experiment guide), the 11.2.1 of the 1st volume, John Wiley ﹠amp; Sons, Inc., New York.
Can determine that albumin fusion proteins is to the binding affinity of albumen, antigen or epi-position and antibody-or interactional dissociation yield of albumin fusion proteins-protein/antigen/epi-position (off-rate) by the competitive binding assay method.An example of competitive binding assay method is a radioimmunoassay, the unlabelled antigen that is included in cumulative amount exist down with the antigen of labelling (as,
3H or
125I) with antibody of the present invention or albumin fusion proteins incubation, and the bonded antibody of the antigen of detection and labelling.Can determine that antibody or albumin fusion proteins are to the affinity of specified protein, antigen or epi-position with in conjunction with dissociation yield from the data of Scatchard tracing analysis.The second proteinic competition that combines same protein, antigen or epi-position with antibody or albumin fusion proteins also can use the radioimmunity detection to determine.In this case, in the presence of unlabelled second protein that combines same protein, antigen or epi-position with albumin fusion proteins of the present invention of cumulative amount with protein, antigen or epi-position and put together chemical compound in labelling (as,
3H or
125I) antibody of the present invention or albumin fusion proteins be incubation together.
In one embodiment, the BIAcore dynamic analysis is used for definite antibody of the present invention or albumin fusion proteins combination and dissociation yield to protein, antigen or epi-position.The BIAcore dynamic analysis comprises analyzes combining and dissociating of antibody, albumin fusion proteins or specific polypeptide, antigen or epi-position and chip, has immobilized specific polypeptide, antigen or epi-position, antibody or albumin fusion proteins on the surface of its chips respectively.
Therapeutic use
The invention further relates to therapy based on antibody, it comprises antibody of the present invention or the present invention is comprised at least one fragment of the proteic antibody of combined treatment or the albumin fusion proteins of variant (or its compositions, as, pharmaceutical composition) is applied to animal, for example, mammal or human patients are to treat one or more disclosed diseases, illness or disease.Therapeutic compound of the present invention includes but not limited to, nucleic acid, at least one fragment or the albumin fusion proteins of variant and the nucleic acid of this albumin fusion proteins of encoding that comprises the proteic antibody of combined treatment of the present invention of antibody of the present invention (comprising its fragment, analog and derivant, as described herein), the antibody of the present invention of encoding (comprising its fragment, analog and derivant and anti--idiotype antibody as described herein).The albumin fusion proteins of antibody of the present invention or at least one fragment that comprises the proteic antibody of combined treatment of the present invention or variant can be used for treating, suppress or unconventionality expression and/or active relevant disease, illness or the disease of prevention and human cytokines, includes but not limited to any or multiple disease as herein described, illness or disease.Include but not limited to alleviate the symptom of being correlated with the unconventionality expression of human cytokines and/or active relevant disease, illness or treatment of conditions and/or prevention with these diseases, illness or disease.Antibody of the present invention or of the present inventionly comprise at least one fragment of the proteic antibody of combined treatment or the albumin fusion proteins of variant can provide in pharmacy acceptable composition known in the art or as described herein.
In specific implementations, the present invention relates to therapy based on antibody, it comprises antibody of the present invention or of the present inventionly comprises at least one fragment of the proteic antibody of combined treatment or the albumin fusion proteins of variant is applied to animal, for example, mammal or human patients are to treat one or more diseases, illness or disease, including but not limited to: neuropathy, the immune system illness, the muscle illness, the reproduction illness, gastrointestinal tract disease, pulmonary disease, cardiovascular disease, the kidney illness, the propagation illness, and/or Cancerous disease and disease, and/or this paper describes elsewhere.Therapeutic compound of the present invention includes but not limited to that antibody of the present invention is (as, the antibody of the full-length proteins of expressing on the cell surface at mammalian cell; At the antibody of the epi-position of human cytokines and coding antibody of the present invention (comprise its fragment, analog and derivant and as herein described anti--idiotype antibody) nucleic acid.Antibody of the present invention can be used for treating, suppress or unconventionality expression and/or active relevant disease, illness or the disease of prevention and human cytokines, includes but not limited to any or multiple disease as herein described, illness or disease.Include but not limited to unconventionality expression and/or active relevant disease, illness or treatment of conditions and/or the prevention of human cytokines, alleviate the symptom relevant with these diseases, illness or disease.Antibody of the present invention or of the present inventionly comprise at least one fragment of the proteic antibody of combined treatment or the albumin fusion proteins of variant can provide in pharmacy acceptable composition known in the art or as described herein.
Wherein can in treatment, use the summary of mode of the albumin fusion proteins of antibody of the present invention or at least one fragment that comprises the proteic antibody of combined treatment of the present invention or variant to comprise local in vivo or systematically combined treatment albumen or the direct cytotoxicity by antibody, as mediating by complement (CDC) or by effector lymphocyte (ADCC).In these methods some are described in more detail following.Utilize instruction provided by the invention, those of ordinary skills will know for diagnose, how monitoring or therapeutic purposes use antibody of the present invention or the albumin fusion proteins of at least one fragment that comprises the proteic antibody of combined treatment of the present invention or variant and do not need undo experimentation.
The albumin fusion proteins of antibody of the present invention or at least one fragment that comprises the proteic antibody of combined treatment of the present invention or variant can advantageously unite other monoclonal or chimeric antibody or lymphokine or hemopoietic growth factor (such as, as, IL-2, IL-3 and IL-7) use together, for example, be used to increase number or activity with the interactional effector lymphocyte of antibody.
The albumin fusion proteins of antibody of the present invention or at least one fragment that comprises the proteic antibody of combined treatment of the present invention or variant can use separately or unite other type treatment (as, radiotherapy, chemotherapy, hormone therapy, immunization therapy and antitumor agent) use together.Usually, can use the product that belongs to the On the Origin of Species or the species reactivity (under the situation of antibody) of same species with the patient.Therefore in one embodiment, people's antibody, fragment derivant, analog or nucleic acid being applied to people patient is used for the treatment of or prevents.
In one embodiment, with at the high-affinity of human cytokines and/or suppress effectively in vivo and/or neutral antibody, its fragment or district (or albumin fusion proteins correlative of this antibody) is used for treatment at the immunoassay of polynucleotide of the present invention or polypeptide (comprising its fragment) and the illness relevant with polynucleotide of the present invention or polypeptide (comprising its fragment).Such antibody, fragment or district can have the affinity to polynucleotide of the present invention or polypeptide (comprising its fragment).Binding affinity comprises that dissociation constant or Kd are less than 5 * 10
-2M, 10
-2M, 5 * 10
-3M, 10
-3M, 5 * 10
-4M, 10
-4The binding affinity of M.Other binding affinity comprises that dissociation constant or Kd are less than 5 * 10
-5M, 10
-5M, 5 * 10
-6M, 10
-6M, 5 * 10
-7M, 10
-7M, 5 * 10
-8M or 10
-8The binding affinity of M.Other binding affinity comprises that also dissociation constant or Kd are less than 5 * 10
-9M, 10
-9M, 5 * 10
-10M, 10
-10M, 5 * 10
-11M, 10
-11M, 5 * 10
-12M, 10
-12M, 5 * 10
-13M, 10
-13M, 5 * 10
-14M, 10
-14M, 5 * 10
-15M or 10
-15The binding affinity of M.
Gene therapy
In specific implementations, by the mode of gene therapy, use and comprise the coding proteic antibody of combined treatment or comprise at least one fragment of the proteic antibody of combined treatment or the nucleic acid of the sequence of the albumin fusion proteins of variant is treated, suppressed or prevent and treats abnormal exprssion and/or relevant disease or the illness of activity.Gene therapy points to that the curee uses nucleic acid expression or effable and the treatment carried out.In this embodiment of the invention, this nucleic acid produces the protein of the mediation therapeutical effect of its coding.
Any gene therapy methods that is used for that can get in this area can be used according to the invention.Illustrative methods is described in greater detail in the application other places.
Confirm treatment or prophylactic activity
Before being used for the mankind, can be external and body build-in test chemical compound of the present invention or pharmaceutical composition desired therapeutic or prophylactic activity subsequently.For example, prove that the treatment of chemical compound or pharmaceutical composition or the external test method of preventive use comprise, the effect of chemical compound pair cell system or patient tissue samples.Can use technology well known by persons skilled in the art to determine the effect of chemical compound or compositions pair cell system and/or tissue sample, include but not limited to that rosette forms algoscopy and cytolysis algoscopy.According to the present invention, can be used for determining whether to show that the external test method of using particular compound comprises cell in vitro cultivation algoscopy, wherein patient tissue samples is grown in culture, and be exposed to chemical compound or administered compound otherwise, and observe of the effect of this chemical compound to tissue sample.
Therapeutic/preventative using and compositions
The invention provides by using the chemical compound of the present invention or the pharmaceutical composition of effective dose to the curee, as albumin fusion proteins or its compositions (as, pharmaceutical composition) and the method for the treatment of, suppressing and prevent.In one embodiment, this chemical compound be basically purification (as, do not contain the material that limits its effect or produce the side effect do not expect substantially).This curee can be animal, including but not limited to animal such as cattle, pig, horse, chicken, cat, dog etc. be mammal in one embodiment and be the people in another embodiment.
Adoptable preparation and application process are aforesaid when chemical compound comprises nucleic acid or immunoglobulin; Other appropriate formulation and route of administration can be selected from following those of this paper.
Various delivery systems are known and can be used for using chemical compound of the present invention, as, be encapsulated in the liposome, in the microparticle, microcapsule, can express the reconstitution cell of chemical compound, the endocytosis of receptor-mediation (see as, Wu and Wu, J.Biol.Chem.262:4429-4432 (1987)), make up as the nucleic acid of the part of retrovirus or other carrier etc.The method of introducing includes but not limited to Intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral route.Can be by any administered compound of approach easily or compositions, for example by infusion or bolus injection, by through epithelium or mucocutaneous lining (as, oral mucosa, rectum and intestinal mucosa, etc.) absorb and can use with other bioactivator.Use the system of can be or partial.In addition, may expect medical compounds of the present invention or compositions to be incorporated into the central nervous system, comprise Intraventricular and intrathecal injection by any suitable way; Can be convenient to intracerebral ventricle injection by intraventricular catheter, for example, be connected in the intraventricular catheter of storage such as the Ommaya storage.Also can adopt lung to use, as, by using inhaler or aerosol apparatus and the preparation that has propellant.
In specific implementations, may expect position local application medical compounds of the present invention or compositions to the needs treatment; This can be as being issued to, such as but not limited to, by the local infusion in operation process, surface applied as in conjunction with postoperative wound dressing, by injection, by the conduit mode, by the suppository mode or by the implant mode, described implant is porous, non-porous or gel-like material, comprise film, such as sialastic film or fiber.In one embodiment, when using the protein that comprises antibody of the present invention, must be noted that use does not absorb proteinic material.
In another embodiment, can be in vesicle (vesicle), particularly send chemical compound in the liposome or compositions (sees Langer, Science 249:1527-1533 (1990); Treat etc., at Liposomes in the Therapy of Infectious Disease and Cancer (liposome in infectious disease and the treatment of cancer), Lopez-Berestein and Fidler (editor), Liss, New York, 353-365 page or leaf (1989); Lopez-Berestein, the same, the 317-327 page or leaf; Generally see the same).
In another embodiment, can in controlled release system, send chemical compound or compositions.In one embodiment, can use pump (to see above-mentioned Langer; Sefton, CRC Crit.Ref.Biomed.Eng.14:201 (1987); Buchwald etc., Surgery 88:507 (1980); Saudek etc., N.Engl.J.Med.321:574 (1989)).In another embodiment, can use polymeric material (to see MedicalApplications of Controlled Release (medical application of controlled release), Langer and Wise (editor), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, DrugProduct Design And Performance (drug bioavailability of control, pharmaceutical product design and performance), Smolen and Ball (editor), Wiley, New York (1984); Ranger and Peppas, J., Macromol.Sci.Rev.Macromol.Chem.23:61 (1983); Also see Levy etc., Science228:190 (1985); During etc., Ann.Neurol.25:351 (1989); Howard etc., J.Neurosurg.71:105 (1989)).In another embodiment, controlled release system can be placed treatment target such as brain near, thereby only need system's dosage a part (see as, Goodson, at above-mentioned MedicalApplications of Controlled Release (medical application of controlled release), the 2nd volume, pp.115-138 (1984)).
Other controlled release system is discussed at the summary (Science 249:1527-1533 (1990)) of Langer.
At chemical compound of the present invention is in the specific implementations of nucleic acid of coded protein, but administration of nucleic acid is to promote the expression of its coded protein in the body, that it is become is intracellular thereby by it being configured to the part of suitable nucleic acid expression vector and using this carrier, as, by using retroviral vector (seeing United States Patent (USP) the 4th, 980, No. 286), or pass through direct injection, or bombard (as particle gun by microparticle; Biolistic, Dupont), or with lipid or cell surface receptor or transfection agents bag quilt, or by be connected to known enter nuclear homeobox class peptide use it (see as, Joliot etc., Proc.Natl.Acad.Sci.USA 88:1864-1868 (1991)) etc.Alternatively, can introduce nucleic acid in the cell and be integrated into host cell DNA by homologous recombination to express.
The present invention also provides pharmaceutical composition, as, the pharmaceutical composition that comprises albumin fusion proteins, forms or form by albumin fusion proteins basically by albumin fusion proteins.Such compositions comprises the chemical compound for the treatment of effective dose (as, albumin fusion proteins), and pharmaceutically acceptable carrier.In specific implementations, term " pharmacy is acceptable " refer to federal government or state government approved by management or be listed in and be used for animal in American Pharmacopeia or other the generally acknowledged pharmacopeia and more specifically be used for human.Term " carrier " refers to diluent, adjuvant, excipient or the vehicle (vehicle) used with therapeutic agent.Such pharmaceutical carrier can be sterile liquid, such as water and oil, comprises those of oil, animal, plant or synthetic source, such as Oleum Arachidis hypogaeae semen, soybean oil, mineral oil, Oleum sesami and analog.In one embodiment, water is carrier when intravenous drug administration compositions.Also can adopt saline solution and aqueous dextrose and glycerite is liquid carrier, in particular for injection solution.Suitable drug excipient comprises starch, glucose, lactose, sucrose, gelatin, Fructus Hordei Germinatus, rice, flour, Chalk, silica gel, sodium stearate, glyceryl monostearate, Talcum, sodium chloride, defatted milk powder, glycerol, propylene, ethylene glycol, water, ethanol and analog.If expectation, compositions also can contain a spot of wetting agent or emulsifying agent or pH buffer agent.These compositionss can be taked forms such as solution, suspension, emulsion, tablet, pill, capsule, powder, sustained release forms.Compositions can be formulated as suppository such as triglyceride with conventional adhesive and carrier.Peroral dosage form can comprise mannitol, lactose, starch, magnesium stearate, saccharin sodium, cellulose, magnesium carbonate of standard vector such as pharmaceutical grade etc.The example of suitable pharmaceutical carrier be described in E.W.Martin's " Remington ' s Pharmaceutical Sciences (Lei Shi pharmacy principle) ".Such compositions will contain the chemical compound for the treatment of effective dose, for example, with purified form, with the carrier of suitable amount so that the form that is fit to be applied to the patient to be provided.Dosage form should adapt to the mode of using.
In one embodiment, compositions is formulated as according to customary scheme is suitable for intravenous and is applied to human pharmaceutical composition.Usually, being used for the compositions that intravenous uses is the solution of sterile isotonic water-containing buffering liquid.In case of necessity, compositions also can comprise solubilizing agent and local anesthetic such as lignocaine to alleviate the pain of injection site.Usually, each composition provides or is mixed into unit dosage form separately, and for example, as the dry freeze-dried powder in sealed container or there is not aqueous concentrate, sealed container is such as the ampoule or the medicated bag (sachette) of the amount of indication activating agent.When infusion is used compositions, can be with containing aseptic medicine level water or brinish infusion bottle is made up a prescription.When compositions is used in injection, thereby can provide ampoule Injectable sterile water or saline can before using, mix each composition.
Chemical compound of the present invention can be formulated as neutrality or salt form.The acceptable salt of pharmacy comprises the salt that forms with anion, such as salt derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid etc., with the salt that forms with cation, such as salt derived from sodium, potassium, ammonium, calcium, hydrated ferric oxide., 2-aminopropane., triethylamine, 2-ethylaminoethanol, histidine, procaine etc.
The amount of the disease that effective treatment, inhibition and prevention are relevant with treatment abnormal exprssion and/or activity or the chemical compound of the present invention of illness can be determined by standard clinical techniques.In addition, the external test method can be chosen wantonly and be used for helping to identify the optimal dose scope.The accurate dosage of taking in the preparation also depends on the severity of route of administration and disease or illness, and should determine according to practitioner's judgement and each patient's situation.Can be from dose-response curve extrapolation effective dose derived from external or animal model test macro.
For antibody, the dosage that is applied to the patient is 0.1mg/kg to 100mg/kg weight in patients normally.In one embodiment, the dosage that is applied to the patient is 0.1mg/kg to 20mg/kg weight in patients.In another embodiment, dosage is 1mg/kg to 10mg/kg weight in patients.Usually, because to the immunne response of allogenic polypeptide, people's antibody has the longer half-life in the human body internal ratio from the antibody of other species.Therefore people's antibody and more not frequent the using than low dosage usually is possible.Further, can by modify such as the picked-up of for example lipid increase antibody and tissue penetration (as, enter brain) and reduce dosage and the frequency of using antibody of the present invention.
Diagnosis and imaging
The antibody of the labelling of combined treatment albumen (or its fragment or variant) and its derivant and analog (comprising at least one fragment that comprises the proteic antibody of combined treatment or the albumin fusion proteins of variant) can be used for diagnostic purpose with detect, diagnosis or monitoring and treatment abnormal exprssion and/or relevant disease, illness and/or the disease of activity.The invention provides detection to the treatment abnormal exprssion, comprise that (a) uses to specific one or more TPPA treatment albumen of target polypeptides in the cell of individuality or the expression in the body fluid and (b) with gene expression dose and standard gene expression level relatively, thus the increase that the proteic expression of the treatment that records is compared with the standard expression or reduce and represent unconventionality expression.
The invention provides a kind of diagnostic algoscopy that is used to diagnose illness, comprising that (a) uses one or more antibody of treatment protein-specific or comprise measures treatment albumen in the cell of individuality or the expression in the body fluid and (b) with gene expression dose and standard gene expression level relatively to the albumin fusion proteins of at least one fragment of the antibody of treatment protein-specific or variant, thus the increase that the expression of the treatment protein gene that records is compared with the standard expression or reduce and represent specific illness.For cancer, exist the transcript of relative a large amount can indicate easy the to be ill body constitution that develops disease in the individual biological biopsy, maybe can be provided at the means that disease appears detecting before in actual clinical symptom.Thereby such more reliable diagnostic can allow fitness guru earlier to adopt preventive measure or initiative treatment to prevent the development of cancer or further make progress.
Antibody of the present invention or comprise albumin fusion proteins at least one fragment of antibody of treatment protein-specific or variant can be used for using to classical immunohistology method well known by persons skilled in the art measure protein level in the biological sample (as, see Jalkanen etc., J.Cell.Biol.101:976-985 (1985); Jalkanen etc., J.Cell.Biol.105:3087-3096 (1987)).Other can be used for detecting the method based on antibody that protein gene expresses and comprises immunoassay, such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA).Suitable TPPA labelling is known in the art and comprises enzyme labelling, such as, glucoseoxidase; Radiosiotope is such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (112In) and technetium (99Tc); Luminescent marking is such as luminol; With fluorescent labeling such as fluorescein and rhodamine and biotin.
One aspect of the present invention (facet) is detection and for example diagnoses animal, disease or illness that mammal or philtrum are relevant with the treatment abnormal exprssion.In one embodiment, diagnosis comprises: a) use the molecule of the specificity of (for example, parenteral, subcutaneous or intraperitoneal) effective dose in conjunction with the labelling of the polypeptide of being paid close attention to the curee; B) use the back wait for certain hour at interval with the molecule that allows labelling preferentially in the curee the proteic site of expression treatment concentrate (and allow to be eliminated to background level to the molecule of unconjugated labelling); C) determine background level; And d) molecule of labelling among the detection curee, the molecule that detects labelling thus is higher than background level and indicates the curee to suffer from and relevant specified disease or the illness of treatment abnormal exprssion.Can determine background level by the whole bag of tricks, comprise the labelling that will detect molecule amount with to the predetermined standard value of particular system relatively.
Should be understood that the build size of curee in this area and the amount that used imaging system will determine to produce the required imaging moiety of diagnostic image.In the situation of radiosiotope part, for human subject, radioactive amount of injection usually will be from the 99mTc of about 5 to 20 millicuries.The antibody of labelling, antibody fragment or comprise at least one fragment of the proteic antibody of combined treatment or the albumin fusion proteins of variant will preferentially contain the accumulation of the proteic cell of particular treatment position subsequently.The in-vivo tumour imaging is described in S.W.Burchiel etc., " Immunopharmacokinetics of Radiolabeled Antibodies and TheirFragments (radio-labeled antibody and its segmental immune pharmacokinetics)." (the 13rd chapter of Tumor Imaging:TheRadiochemical Detection of Cancer (tumor imaging: radiochemistry detects cancer), S.W.Burchiel and B.A.Rhodes edit, Masson Publishing Inc. (1982)).
Depend on the type that comprises used labelling and a plurality of variablees of method of application, use molecule that the back allows labelling preferentially the position in the curee concentrate and allow the molecule of unconjugated labelling be eliminated to the interval of background level be 6 to 48 hours or 6 to 24 hours or 6 to 12 hours.In another embodiment, using the back interval is 5 to 20 days or 5 to 10 days.
In one embodiment, diagnose the illness or the method for disease is carried out the monitoring of disease or illness by being recycled and reused for, for example, back one month of initial diagnosis, diagnose back six months at first, diagnose back 1 year at first etc.
Use the method that is used for the body interscan known in the art can detect the get the bid existence of molecule of note of patient.These methods depend on the type of used labelling.Those skilled in the art can determine to detect the appropriate method of specific markers.The method and apparatus that can be used for diagnostic method of the present invention includes but not limited to, computer tomography (CT), body scan such as position emission tomography (positon emission tomography) (PET), NMR (Nuclear Magnetic Resonance)-imaging (MRI) and Ultrasonography.
In specific implementations, molecule is with labelled with radioisotope and use radiation response surgery instrument to detect (Thurston etc., United States Patent (USP) the 5th, 441, No. 050) in the patient.In another embodiment, molecule is with the fluorescent chemicals labelling and use the fluorescence response scanner to detect in the patient.In another embodiment, molecule is with the positron emitting metal labelling and use positron emission tomography to detect in patient (patent).In another embodiment, molecule is with paramagnetism labelling labelling and use NMR (Nuclear Magnetic Resonance)-imaging (MRI) to detect in the patient.Detecting albumin fusion proteins specifically but not detecting independent albumin or treat proteic antibody is a kind of embodiment.These can be used for detecting as the described albumin fusion proteins of description entire chapter.
Test kit
The invention provides the test kit that can be used for said method.In one embodiment, test kit is included in the antibody in one or more containers, for example, and antibody purified.In specific implementations, test kit of the present invention contains isolating basically polypeptide, described polypeptide comprise with test kit in the antibody that comprises play the epi-position of specific immune response.In another embodiment, test kit of the present invention further comprises not the control antibodies with the polypeptide reaction of being paid close attention to.In another specific implementations, test kit of the present invention contain detect antibody combine with the polypeptide of being paid close attention to (as, antibody can be puted together in the substrate of detectable substrate such as fluorescent chemicals, enzyme, radioactive compound or luminophor, or identification first antibody second antibody can put together in detectable substrate) means.
In another specific implementations of the present invention, this test kit is to be used to screen contain the diagnostic kit of specificity at the serum of the antibody of proliferative and/or carcinous polynucleotide and polypeptide.This test kit can comprise not the control antibodies with the polypeptide reaction of being paid close attention to.This test kit can comprise isolating basically polypeptide antigen, its comprise with at least a anti--polypeptide antigen antibody plays the epi-position of specific immune response.Further, this test kit comprises described antibody and antigenic combination means of (can put together in can be by fluorescent chemicals such as the fluorescein or the rhodamine of Flow cytometry as, this antibody) that detect.In specific implementations, this test kit can comprise the polypeptide antigen of reorganization preparation or chemosynthesis.The polypeptide antigen of test kit also can attach to solid support.
In embodiment more specifically, the detection means of mentioned reagent box comprises the solid support that is attached with described polypeptide antigen.This test kit also can comprise Anti-Human's antibody of non-attached reporter molecules labelling.In this embodiment, combining of antibody and polypeptide antigen can be by the combination of the antibody of described reporter molecules labelling and detect.
In other embodiment, the present invention comprises the diagnostic kit that is used to screen the antigenic serum that contains polypeptide of the present invention.This diagnostic kit comprises the bonded means that play the isolated antibody basically of specific immune response with polypeptide or polynucleotide antigen and be used to detect polynucleotide or polypeptide antigen and antibody.In one embodiment, this antibody attaches to solid support.In specific implementations, this antibody can be monoclonal antibody.The detection means of test kit can comprise the monoclonal antibody of second kind of labelling.Alternatively, or in addition, detection means can comprise the competition antigen of labelling.
In a kind of diagnosis configuration, make test sera and have the antigenic solid-phase reagent reaction of the surface combination that obtains with method of the present invention.After specific antigen-antibody combines with reagent and removes unconjugated serum composition by cleaning, the Anti-Human's antibody response that makes reagent and reporter molecules labelling with reporter molecules by with solid support on bonded anti--certain proportion of the amount of antigen-antibody combines with reagent.Cleaning reagent to be removing the antibody of unconjugated labelling once more, and determines and the amount of the bonded reporter molecules of reagent.Usually, reporter molecules is an enzyme, and it is by (Sigma, St.Louis carry out incubation and detect in the time of MO) having suitable fluorescence, luminous or colorimetric substrates with solid phase.
Solid phase surface reagent in the said determination method is by protein material being attached to the known technology preparation of solid phase support material, such as polymeric beads, dipping bar (dip sticks), 96-orifice plate or filtering material.These attachment methods generally comprise the non--specific adsorption of protein to support, or protein attaches to chemically reactive group on the solid support by the free amino covalency usually, such as activatory carboxyl, hydroxyl or aldehyde radical.Alternatively, the antigen of the plate biotin-bindingization of Streptavidin bag quilt uses.
Therefore the invention provides a kind of mensuration system or test kit that is used to carry out this diagnostic method.Test kit generally comprises Anti-Human's antibody of reporter molecules labelling that surface combination has the support of recombinant antigen and is used to detect anti--antigen-antibody of surface combination.
Albumin fusion proteins
The present invention briefly relates to albumin fusion proteins and treatment, prevention or alleviates the method for disease or illness." albumin fusion proteins " used herein refers to that at least one molecule albumin (or its fragment or variant) and at least one molecular therapy albumen (or its fragment or variant) merge the protein that forms.Albumin fusion proteins of the present invention comprises proteic at least one fragment of treatment or variant and human serum albumin's at least one fragment or variant, they are by for example the heredity fusion is interconnection (promptly, described albumin fusion proteins is produced by translated nucleic acid, and the proteic polynucleotide of all or part of treatment of coding connect with identical reading frame with all or part of albuminised polynucleotide of coding in this nucleic acid).Human cytokines and albumin be in case during as the part of albumin fusion proteins, can be called as " partly (portion) ", " zone " or " partly (moiety) of albumin fusion proteins separately ".
In one embodiment, the invention provides a kind of albumin fusion proteins by polynucleotide described in table 1 or the table 2 or albumin fusion constructs coding.The polynucleotide of these albumin fusion proteins of encoding are also included among the present invention.
The embodiment of albumin fusion proteins of the present invention includes but not limited to, albumin fusion proteins by following nucleic acid molecule encoding: a kind of nucleic acid molecules, its comprise the coding that is connected with at least one section polynucleotide of a part human cytokines (or its fragment or variant) at least of encoding with identical reading frame at least a part albumin (or its fragment or variant) polynucleotide or form by it alternatively; A kind of nucleic acid molecules, its comprise the coding that is connected with at least one section polynucleotide of the human cytokines (or its fragment or variant) of the generation described in a part at least such as table 1, table 2 or the embodiment of encoding with identical reading frame at least a part albumin (or its fragment or variant) polynucleotide or form by it alternatively; Or a kind of nucleic acid molecules, its comprise the coding that is connected with at least one section polynucleotide of a part human cytokines (or its fragment or variant) at least of encoding with identical reading frame at least a part albumin (or its fragment or variant) polynucleotide or form by it alternatively, further comprise for example one or more following elements: (1) functional self-replication carrier (includes but not limited to, shuttle vector, expression vector, integration vector and/or dubbing system), (2) initial zone of transcribing (as, promoter region, such as for example, regulation and control type or inducible promoter, constitutive promoter), (3) transcription termination region, (4) targeting sequencing and (5) selected marker.
In one embodiment, the invention provides a kind of albumin fusion proteins, it comprises human cytokines (described in table 1) and serum albumin albumen or is made up of it alternatively.In other embodiments, the invention provides a kind of albumin fusion proteins, it comprises fragment biologic activity and/or therapeutic activity and the serum albumin albumen of human cytokines or is made up of it alternatively.In other embodiments, the invention provides a kind of albumin fusion proteins, it comprises variant biologic activity and/or therapeutic activity and the serum albumin albumen of human cytokines or is made up of it alternatively.In some embodiments, the serum albumin protein ingredient of albumin fusion proteins is the maturing part of serum albumin.
In further embodiment, the invention provides a kind of albumin fusion proteins, it comprises fragment biologic activity and/or therapeutic activity of human cytokines and serum albumin or is made up of it alternatively.In further embodiment, the invention provides a kind of albumin fusion proteins, it comprises variant biologic activity and/or therapeutic activity of human cytokines and serum albumin or is made up of it alternatively.In some embodiments, the human cytokines of albumin fusion proteins partly is the maturing part of human cytokines.
In further embodiment, the invention provides a kind of albumin fusion proteins, it comprises fragment biologic activity and/or therapeutic activity of human cytokines or fragment biologic activity and/or therapeutic activity or the variant of variant and serum albumin, or is made up of it alternatively.In other embodiments, the invention provides a kind of albumin fusion proteins, it contains the maturing part of the maturing part of human cytokines and serum albumin or is made up of it alternatively.
On the one hand, albumin fusion proteins comprise HA as N-end portion and human cytokines as the C-end portion.Alternatively, also can use and comprise HA as C-end portion and human cytokines albumin fusion proteins as the N-end portion.
In other embodiments, albumin fusion proteins has human cytokines terminal with albuminised N-and that the C-end all merges.In one embodiment, the human cytokines that merges at N-and C-end is identical human cytokines.In optional embodiment, the human cytokines that merges at N-and C-end is different human cytokines.In another embodiment, the human cytokines that merges at N-and C-end is different human cytokines, and it can be used for treating or prevents identical or relevant disease, illness or disease (as be listed in table 1 " indication Y " row).In another embodiment, fusion is different human cytokines at the human cytokines of N-and C-end, it can be used for treatment, alleviation or prevent disease or illness (as be listed in table 1 " indication Y " row), these diseases or illness be known in the art usually side by side, in the patient, exist concomitantly or continuously, or in the patient, exist with being relative to each other usually.
Albumin fusion proteins of the present invention comprises such protein, and it contains and the N-of albumin fusion proteins of the present invention or the N-and/or terminal, two, three, four or more polymolecular TA albumin X or its variant that merges of C-of C-end and/or albumin or its variant.The molecule of TA albumin X or its variant can be the direction of any number, include but not limited to, ' head to head ' direction (as, wherein the N-of the human cytokines X of a part end merges with the N-of the human cytokines X of another molecule is terminal), or ' head is to tail ' direction (as, the terminal fusion of N-of the terminal human cytokines X with another molecule of the C-of the human cytokines X of a part wherein).
In one embodiment, one, two, three or the N-of the N-of the directed human cytokines X polypeptide (or its fragment or variant) of more a plurality of series connection and albumin fusion proteins of the present invention or C-end and/or albumin or its variant and/or C-is terminal merges.
Albumin fusion proteins of the present invention further comprises such protein, it contains N-or the N-of C-end and/or albumin or its variant and/or TA albumin X or its variant of terminal, two, three, four or the more a plurality of molecules that merge of C-with albumin fusion proteins of the present invention, and wherein each molecule connects by peptide linker.Example comprises and is described in United States Patent (USP) the 5th, 073, the peptide linker of No. 627 (incorporating into by reference at this).Can use conventional recombinant DNA technology preparation to comprise the albumin fusion proteins of a plurality of human cytokines X polypeptide of separating by peptide linker.Divide period of the day from 11 p.m. to 1 a.m joint to be even more important when merging little peptide in big HSA.By the tandem copy of fusogenic peptide, itself can be joint peptide, maybe can use other known joint.Incorporating the construct of joint into describes maybe apparent when checking SEQ ID NO:Y in table 2.
Further, albumin fusion proteins of the present invention also can prepare by N-end and/or terminal fusion of C-with human cytokines X or its variant and albumin or its variant, and the mode of its fusion allows to form intramolecularly and/or intermolecular polymer form.In an embodiment of the invention, albumin fusion proteins can be monomer or polymer form (being dimer, trimer, tetramer and Geng Gao polymer).In further embodiment of the present invention, the human cytokines of albumin fusion proteins partly can be monomeric form or polymer form (being dimer, trimer, tetramer and Geng Gao polymer).In specific implementations, the human cytokines of albumin fusion proteins partly is polymer form (being dimer, trimer, tetramer and Geng Gao polymer), and the albumin protein part is a monomeric form.
Except albumin part wherein is blended in the N-end of human cytokines part and/or the albumin fusion proteins of C-end, albumin fusion proteins of the present invention also can by the human cytokines that will be paid close attention to or peptide (as, be disclosed in the human cytokines X of table 1 or the antibody of combined treatment albumen or its fragment or variant) be inserted into the interior zone of HA and prepare.For example, the alpha-helix end in the protein sequence of HA molecule and exist a large amount of between the beginning by disulfide bond stable ring or revolution (turn).Open from the Subject Extension of molecule from the major part of the definite ring (PDB identifier 1AO6,1BJ5, IBKE, IBM0,1E7E to 1E7I and 1UOR) of the crystal structure of HA.These rings can be used for inserting or the inner peptide that merges therapeutic activity, especially need secondary structure so that the peptide of function to be arranged, or human cytokines, have the active albumin molecule of particular biological to produce in fact.
Can insert peptide or polypeptide comprises with the ring in the people's albumin structure that produces albumin fusion proteins of the present invention: Val54-Asn61, Thr76-Asp89, Ala92-Glu100, Gln170-Ala176, His247-Glu252, Glu266-Glu277, Glu280-His288, Ala362-Glu368, Lys439-Pro447, Val462-Lys475, Thr478-Pro486 and Lys560-Thr566.In other embodiment, peptide or polypeptide are inserted into Val54-Asn61, Gln170-Ala176 and/or the Lys560-Thr566 ring of acquaintance's albumin (SEQ ID NO:1).
The peptide that is inserted into can be derived from the phage display that the particular organisms activity is screened or synthetic peptide library or derived from the active part of the molecule with desired function.In addition, can in specific ring, generate random peptide library or by producing random peptide library in the specific ring that the randomization peptide is inserted into the HA molecule, and wherein represent amino acid whose institute to make up.
Such library is one of can be by the following method sternly living on the domain fragment of HA or HA:
Aminoacid in randomization sudden change HA or the segmental one or more peptide rings of HA domain.In the ring one, a plurality of or all residues can suddenly change by this way;
With length X
nRandomized peptide (wherein X be aminoacid and n is the residue number) replace HA or the segmental one or more rings of HA domain or insert HA or the segmental one or more rings of HA domain (promptly inner fusion);
Except (a) and/or (b), merge N-, C-or N-and C-terminal peptide/albumen.
Also can be by being grafted to same HA derived from the peptide of the difference screening of different rings being carried out at different targets or HA domain fragment makes HA or HA domain fragment become multi-functional.
In some embodiments, the peptide of insertion human serum albumin's ring is fragments of peptides or the peptide variant that is disclosed in the human cytokines of table 1.More specifically, the present invention includes the length that comprises the ring that inserts the human serum albumin is the albumin fusion proteins of at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 35 or at least 40 amino acid whose fragments of peptides or peptide variant.The present invention also comprises the albumin fusion proteins that comprises with terminal at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 35 or at least 40 amino acid whose fragments of peptides that merge of human serum albumin's N-or peptide variant.The present invention also comprises the albumin fusion proteins that comprises with terminal at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 35 or at least 40 amino acid whose fragments of peptides that merge of human serum albumin's C-or peptide variant.For example, the small peptide described in table 1 and 2 (as, therapeutic agent Y) can be inserted in the albumin ring.
Usually, albumin fusion proteins of the present invention can have a HA-derive the district and the human cytokines-district of deriving.Yet each proteic a plurality of district can be used for preparing albumin fusion proteins of the present invention.Similarly, can be used for preparing albumin fusion proteins of the present invention more than a kind of human cytokines.For example, human cytokines can with the N-of HA and terminal the two fusion of C-.In this configuration, human cytokines partly can be identical or different human cytokines molecule.The structure of bifunctional albumin fusion proteins can be expressed as: X-HA-Y or Y-HA-X.
For example, can prepare anti--BLyS
TMScFv-HA-IFN α-2b fusant is anti-to regulate-BLyS
TMScFv is to the immunne response of IFN α-2b.A kind of selectable mode is two (or even many) functional agents of preparation HA-fusant, as resisting-BLyS with various ratios and the HA-that depends on function, half-life etc.
TMThe blended HA-IFN α of scFv fusant or other HA-fusant-2b fusant.
Also can prepare two-or human cytokines part targeting that many-function albumin fusion proteins makes fusant with protein or peptide by the HA opposite ends in Target organ or cell type.
As the alternative of the fusant of known treatment molecule, can obtain peptide by the screening library, be common 6,8,12,20 or 25 or X with described library construction
nThe fusant of (wherein X is that aminoacid (aa) and n equal the residue number) individual randomized aminoacid and HA or the segmental N-of HA domain, C-or N-and C-end, and wherein represented amino acid whose institute to make up.A special advantage of this method be can be on the HA molecule original position select peptide, and therefore potential change and can not take place just as selecting in the feature of peptide as situation about being attached to then by any other method derived peptide on the HA.
In addition, thereby the accessibility maximum of joint peptide to provide physical separation bigger between the each several part to make the human cytokines part can be provided between the each several part that merges albumin fusion proteins of the present invention, for example, and to be incorporated into its associated receptor.The joint peptide can be made up of to be flexible or rigidity more aminoacid.
Joint sequence can be by protease cracking or chemical cracking to produce the relevant part of growth hormone.On the one hand, protease is by the natural generation of host, for example saccharomyces cerevisiae (S.cerevisiae) protease kex2 or equivalent protease.
Therefore, as mentioned above, albumin fusion proteins of the present invention can have following formula R1-L-R2; R2-L-R1; Or R1-L-R2-L-R1, wherein R1 is at least a human cytokines, peptide or peptide sequence, and needs not to be identical human cytokines, L is that joint and R2 are the serum albumin sequences.
In some embodiments, comprise the albumin fusion proteins of the present invention of human cytokines and have higher plasma stability with not comparing with the proteic plasma stability of identical treatment that albumin merges.When plasma stability is often referred in body administering therapeutic albumen and is written into blood flow and human cytokines is degraded and the time period between blood flow is removed when entering organ such as kidney that human cytokines the most at last removes in body or the liver.Plasma stability calculated according to the half-life of human cytokines in the blood flow.The half-life of human cytokines can easily be determined by the common known algoscopy in this area in the blood flow.
The proteic shelf life of identical treatment when in some embodiments, comprising the albumin fusion proteins of the present invention of human cytokines and not merging with albumin is compared the shelf life with prolongation.It is stable and excessively do not lose therapeutic activity through this time period that shelf life is often referred in solution or some other depot formulations the human cytokines therapeutic activity.Many human cytokines its not the fusion state be highly unsettled.As described below, the typical shelf life of these human cytokines prolongs after mixing albumin fusion proteins of the present invention significantly.
Of the present inventionly have " prolongation " or " extension " the albumin fusion proteins of shelf life with respect to the bigger therapeutic activity of standard substance performance that stands after identical storage and the operating condition.Standard substance can be the total length human cytokines that does not merge.When the human cytokines of albumin fusion proteins partly be this proteinic analog, variant or otherwise change or when not comprising the protein complete sequence, the prolongation of therapeutic activity can be compared with the peptide of this analog, variant, change or the equivalent that does not merge of imperfect sequence alternatively.As an example, when standing identical storage and operating condition with standard substance, when at the appointed time comparing, albumin fusion proteins of the present invention can maintain the standard product therapeutic activity greater than about 100% or greater than about 105%, 110%, 120%, 130%, 150% or 200% of therapeutic activity.
Shelf life also can the standardized therapeutic activity of therapeutic activity that the back keeps according to storing, when beginning at storage be estimated.Of the present invention have the albumin fusion proteins (being shown as the therapeutic activity that prolongs or extend) that prolongs or extend the shelf life can keep the human cytokines that equivalence do not merge when standing the same terms therapeutic activity greater than about 50%, about 60%, 70%, 80% or 90% or more.
Expressed fusion protein
Albumin fusion proteins of the present invention can be by producing as recombinant molecule from yeast, microorganism such as antibacterial or the secretion of human or animal's cell line.On the one hand, polypeptide is from secretory host cell.
Specific implementations of the present invention comprises DNA construct, its coding is guiding excretory signal sequence fusion molecule of yeast-derivative signal sequence (especially homologous with yeast host) and first aspect present invention especially in yeast effectively, does not wherein have yeast-deutero-former sequence (pro sequence) between signal and the mature polypeptide.
Saccharomyces cerevisiae invertase signal is an example of yeast-derivative signal sequence.
That class conjugate that does not comprise Poznansky etc. (FEBS Lett.239:18 (1988)) preparation, wherein the polypeptide of preparation links together by chemical crosslinking respectively.
The present invention also comprises cell, for example, and through transforming to express the yeast cells of albumin fusion proteins of the present invention.Except institute's transformed host cells itself, the present invention also comprises the culture of these cells in Nutrient medium, for example, and monoclonal (homogeneity on the clone) culture or derived from the culture of monoclonal culture.If polypeptide is excretory, then culture medium will contain this polypeptide, together with cell, or not together with cell (if having filtered or the centrifugal cell of removing).Many expression systems are known and can use, and comprise antibacterial (for example escherichia coli and bacillus subtilis (Bacillum subtilis)), yeast (for example saccharomyces cerevisiae, Kluyveromyces lactis (Kluyveromyces lactis) and pichia pastoris phaff (Pichiapastoris)), filamentous fungi (for example aspergillosis (Aspergillus)), plant cell, zooblast and insect cell.
The example that is used to produce the yeast strain of albumin fusion proteins is D88, DXY1 and BXP10.D88[leu2-3, leu2-122, can1, pral, ubc4] be that parent strain AH22his+ (is also referred to as DB1; See as, Biotechnology 8:42-46 (1990) such as Sleep) derivant.This bacterial strain contains the leu2 sudden change, and this allows that the plasmid based on 2 microns that contains the LEU2 gene is carried out auxotrophy (auxotropic) and selects.D88 also shows derepressing of PRB1 when glucose is excessive.This PRB1 promoter is controlled by two checkpoints of the gentle growth stage of monitoring G/W usually.This promoter is activated when going forward side by side into resting stage when glucose exhausts in wild-type yeast.Bacterial strain D88 shows and is subjected to glucose repression, induces but keep when entering resting stage.PRA1 gene code yeast vacuole protease YscA endo protease-A, it is positioned among the ER.The UBC4 gene is present in the ubiquitin approach and involves makes short-lived and paraprotein targeting degrade in the ubiquitin dependency.The separation of finding this ubc4 sudden change increase expression plasmid in cell the copy religion and cause increasing (see as, international publication WO99/00504 number is incorporated into its integral body by reference at this) from the proteic expression of the expectation of plasmid expression.
DXY1 is the derivant of D88, has following genotype: [leu2-3, leu2-122, can1, pra1, ubc4, ura3::yap3].Except isolating sudden change among the D88, this bacterial strain also has YAP3 protease and knocks out.This protease causes the cracking of most of two alkaline residues (RR, RK, KR, KK), but also can promote in the albumen cracking at single alkaline residue.The separation of this yap3 sudden change causes higher levels of total length HSA to produce (see as, United States Patent (USP) the 5th, 965, No. 386 and Kerry-Williams etc., Yeast 14:161-169 (1998) incorporates into its integral body by reference at this).
BXP10 has following genotype: leu2-3, leu2-122, can1, pra1, ubc4, ura3, yap3::URA3, lys2, hsp150::LYS2, pmt1::URA3.Except in the isolating sudden change of DXY1, this bacterial strain also has knocking out of PMT1 gene and HSP 150 genes.The PMT1 gene is to evolve to go up the member of conservative polyterpene base phosphoric acid-D-mannosylglycoprotein O-mannose transferase (Pmts) family.The Transmembrane Topology of Pmt1p represents that it is the integral protein of endoplasmic reticulum, works in the O-linked glycosylation.This sudden change is used to reduce/eliminate the O-linked glycosylation (see as, international publication WO00/44772 number is incorporated into its integral body by reference at this) of HSA fusant.Research discloses Hsp150 albumen and can not separate from rHA effectively by ion-exchange chromatography.Sudden change in HSP 150 genes can be removed possible pollutant, has proved that these pollutant are difficult to be removed by the standard purification technology.See as, United States Patent (USP) the 5th, 783 No. 423, is incorporated into its integral body by reference at this.
Prepare desirable protein in a usual manner, for example from being inserted into the coded sequence on host chromosome or the free plasmid.With any usual way for example electroporation come with the proteinic coded sequence transformed yeast of expectation.Method by the electroporation transformed yeast is disclosed in Becker ﹠amp; Guarente (1990) MethodsEnzymol.194,182.
Can promptly contain the cell of DNA construct of the present invention by knowing technical appraisement success cell transformed.For example, can cultivate the cell of introducing expression construct gained to produce the polypeptide of expectation.Can collect and cell lysis and using such as by Southern (1975) J.Mol.Biol.98,503 or (1985) Biotech.3 such as Berent, 208 methods of describing check in its DNA composition whether have this DNA.Alternatively, proteinic existence can be used antibody test in the supernatant.
Available yeast plasmid carrier comprises that pRS403-406 and pRS413-416 also generally can be from Stratagene Cloning Systems, La Jolla, and CA 92037, and USA obtains.Plasmid pRS403, pRS404, pRS405 and pRS406 are yeast integrated plasmid (YIp), and incorporate yeast selected marker HIS3,7RP1, LEU2 and URA3 into.Plasmid pRS413-416 is yeast centromeric plasmid (Ycp).
The example that is used for express producing at yeast the carrier of albumin fusion proteins comprises pPPC0005, pScCHSA, pScNHSA and pC4:HSA, and they are described in detail in embodiment 1.Fig. 2 shows the collection of illustrative plates of pPPC0005 plasmid, and this plasmid can be used as and the polynucleotide of coding human cytokines can be cloned into wherein to form the carrier is carrier of HA-fusant.It contains DNA (rHA) and the ADH1 saccharomyces cerevisiae terminator sequence of PRB1 saccharomyces cerevisiae promoter (PRB1p), fusion targeting sequencing (FL), coding HA.The sequence that merges targeting sequencing is made up of following: preceding 19 aminoacid of the signal peptide of human serum albumin (SEQ ID NO:3) and the last five amino acid (SLDKR of mating factor α 1 promoter, see EP-A-387319, it is incorporated into its integral body by reference at this).
Plasmid pPPC0005, pScCHSA, pScNHSA and pC4:HSA are preserved in American type culture collection in April 11 calendar year 2001,10801 University Boulevard, Manassas, Virginia 20110-2209 gives accession number ATCC PTA-3278, PTA-3276, PTA-3279 and PTA-3277 respectively.Be used for being described in Sleep etc. at another carrier pSAC35 carrier of yeast expression albumin fusion proteins, Biotechnology 8:42 (1990), it is incorporated into its integral body by reference at this.
The Yeast promoter that can be used for expressing albumin fusion proteins is the MET25 promoter.For example see Dominik Mumburg, Rolf Muller and Martin Funk.Nucleic Acids Research (nucleic acids research), 1994, the 22 volumes, No.25,5767-5768 page or leaf.Long 383 bases (base-382 is to-1) of Met25 promoter also are called Met15, Met17 and YLR303W by the gene of this promoter expression.
An embodiment uses following sequence, wherein at 5 of following sequence ' end, and for Not 1 site that is used to clone underlines, and at 3 ' end, for the ATG start codon underlines:
GCGGCCGCCGGATGCAAGGGTTCGAATCCCTTAGCTCTCATTATTTTTTGCTTTTTCTCTTGAGGTCACATGATCGCA
AAATGGCAAATGGCACGTGAAGCTGTCGATATTGGGGAACTGTGGTGGTTGGCAAATGACTAATTAAGTTAGTCAAG
GCGCCATCCTCATGAAAACTGTGTAACATAATAACCGAAGTGTCGAAAAGGTGGCACCTTGTCCAATTGAACACGCT
CGATGAAAAAAATAAGATATATATAAGGTTAAGTAAAGCGTCTGTTAGAAAGGAAGTTTTTCCTTTTTCTTGCTCTCT
TGTCTTTTCATCTACTATTTCCTTCGTGTAATACAGGGTCGTCAGATACATAGATACAATTCTATTACCCCCATCCATA
CA
ATG(SEQ?ID?NO：5)
Be used in other promoter of expressing albumin fusion proteins in the yeast and comprise following promoter:
A) cbh1 promoter:
TCTAGAGTTGTGAAGTCGGTAATCCCGCTGTATAGTAATACGAGTCGCATCTAAATACTCCGAAGCTGCTG
CGAACCCGGAGAATCGAGATGTGCTGGAAAGCTTCTAGCGAGCGGCTAAATTAGCATGAAAGGCTATGAG
AAATTCTGGAGACGGCTTGTTGAATCATGGCGTTCCATTCTTCGACAAGCAAAGCGTTCCGTCGCAGTAGC
AGGCACTCATTCCCGAAAAAACTCGGAGATTCCTAAGTAGCGATGGAACCGGAATAATATAATAGGCAAT
ACATTGAGTTGCCTCGACGGTTGCAATGCAGGGGTACTGAGCTTGGACATAACTGTTCCGTACCCCACCTC
TTCTCAACCTTTGGCGTTTCCCTGATTCAGCGTACCCGTACAAGTCGTAATCACTATTAACCCAGACTGAC
CGGACGTGTTTTGCCCTTCATTTGGAGAAATAATGTCATTGCGATGTGTAATTTGCCTGCTTGACCGACTG
GGGCTGTTCGAAGCCCGAATGTAGGATTGTTATCCGAACTCTGCTCGTAGAGGCATGTTGTGAATCTGTGT
CGGGCAGGACACGCCTCGAAGGTTCACGGCAAGGGAAACCACCGATAGCAGTGTCTAGTAGCAACCTGT
AAAGCCGCAATGCAGCATCACTGGAAAATACAAACCAATGGCTAAAAGTACATAAGTTAATGCCTAAAG
AAGTCATATACCAGCGGCTAATAATTGTACAATCAAGTGGCTAAACGTACCGTAATTTGCCAACGGCTTGT
GGGGTTGCAGAAGCAACGGCAAAGCCCCACTTCCCCACGTTTGTTTCTTCACTCAGTCCAATCTCAGCTGG
TGATCCCCCAATTGGGTCGCTTGTTTGTTCCGGTGAAGTGAAAGAAGACAGAGGTAAGAATGTCTGACTC
GGAGCGTTTTGCATACAACCAAGGGCAGTGATGGAAGACAGTGAAATGTTGACATTCAAGGAGTATTTAG
CCAGGGATGCTTGAGTGTATCGTGTAAGGAGGTTTGTCTGCCGATACGACGAATACTGTATAGTCACTTCT
GGTGAAGTGGTCCATATTGAAATGTAAGTCGGCACTGAACAGGCAAAAGATTGAGTTGAAACTGCCTAAG
ATCTCGGGCCCTCGGGCCTTCGGCCTTTGGGTGTACATGTTTGTGCTCCGGGCAAATGCAAAGTGTGGTAG
GATCGAACACACTGCTGCCTTTACCAAGCAGCTGAGGGTATGTGATAGGCAAATGTTCAGGGGCCACTGC
ATGGTTTCGAATAGAAAGAGAAGCTTAGCCAAGAACAATAGCCGATAAAGATAGCCTCATTAAACGGAAT
GAGCTAGTAGGCAAAGTCAGCGAATGTGTATATATAAAGGTTCGAGGTCCGTGCCTCCCTCATGCTCTCCC
CATCTACTCATCAACTCAGATCCTCCAGGAGACTTGTACACCATCTTTTGAGGCACAGAAACCCAATAGTC
AACCGCGGACTGGCATC(SEQ?ID?NO：113)
B) from the cysD promoter of aspergillus nidulans (Aspergillus nidulans):
AGATCTGGTTCCTGAGTACATCTACCGATGCGCCTCGATCCCCCTCTTAGCCGCATGAGATTCCTACCATTT
ATGTCCTATCGTTCAGGGTCCTATTTGGACCGCTAGAAATAGACTCTGCTCGATTTGTTTCCATTATTCACGC
AATTACGATAGTATTTGGCTCTTTTCGTTTGGCCCAGGTCAATTCGGGTAAGACGCGATCACGCCATTGTGG
CCGCCGGCGTTGTGCTGCTGCTATTCCCCGCATATAAACAACCCCTCCACCAGTTCGTTGGGCTTTGCGAAT
GCTGTACTCTATTTCAAGTTGTCAAAAGAGAGGATTCAAAAAATTATACCCCAGATATCAAAGATATCAAA
GCCATC(SEQ?ID?NO：114)
C) have the modified cbh1 promoter of following sequence:
TCTAGAGTTGTGAAGTCGGTAATCCCGCTGTATAGTAATACGAGTCGCATCTAAATACTCCGAAGCTGCTG
CGAACCCGGAGAATCGAGATGTGCTGGAAAGCTTCTAGCGAGCGGCTAAATTAGCATGAAAGGCTATGAG
AAATTCTGGAGACGGCTTGTTGAATCATGGCGTTCCATTCTTCGACAAGCAAAGCGTTCCGTCGCAGTAGC
AGGCACTCATTCCCGAAAAAACTCGGAGATTCCTAAGTAGCGATGGAACCGGAATAATATAATAGGCAAT
ACATTGAGTTGCCTCGACGGTTGCAATGCAGGGGTACTGAGCTTGGACATAACTGTTCCGTACCCCACCTC
TTCTCAACCTTTGGCGTTTCCCTGATTCAGCGTACCCGTACAAGTCGTAATCACTATTAACCCAGACTGAC
CGGACGTGTTTTGCCCTTCATTTGGAGAAATAATGTCATTGCGATGTGTAATTTGCCTGCTTGACCGACTG
GGGCTGTTCGAAGCCCGAATGTAGGATTGTTATCCGAACTCTGCTCGTAGAGGCATGTTGTGAATCTGTGT
CGGGCAGGACACGCCTCGAAGGTTCACGGCAAGGGAAACCACCGATAGCAGTGTCTAGTAGCAACCTGT
AAAGCCGCAATGCAGCATCACTGGAAAATACAAACCAATGGCTAAAAGTACATAAGTTAATGCCTAAAG
AAGTCATATACCAGCGGCTAATAATTGTACAATCAAGTGGCTAAACGTACCGTAATTTGCCAACGGCTTGT
GGGGTTGCAGAAGCAACGGCAAAGCCCCACTTCCCCACGTTTGTTTCTTCACTCAGTCCAATCTCAGCTGG
TGATCCCCCAATTGGGTCGCTTGTTTGTTCCGGTGAAGTGAAAGAAGACAGAGGTAAGAATGTCTGACTC
GGAGCGTTTTGCATACAACCAAGGGCAGTGATGGAAGACAGTGAAATGTTGACATTCAAGGAGTATTTAG
CCAGGGATGCTTGAGTGTATCGTGTAAGGAGGTTTGTCTGCCGATACGACGAATACTGTATAGTCACTTCT
GGTGAAGTGGTCCATATTGAAATGTAAGTCGGCACTGAACAGGCAAAAGATTGAGTTGAAACTGCCTAAG
ATCTCGGGCCCTCGGGCCTTCGGCCTTTGGGTGTACATGTTTGTGCTCCGGGCAAATGCAAAGTGTGGTAG
GATCGAACACACTGCTGCCTTTACCAAGCAGCTGAGGGTATGTGATAGGCAAATGTTCAGGGGCCACTGC
ATGGTTTCGAATAGAAAGAGAAGCTTAGCCTGCAGCCTCTTATCGAGAAAGAAATTACCGTCGCTCGTGA
TTTGTTTGCAAAAAGAACAAAACTGAAAAAACCCAGACACGCTCGACTTCCTGTCTTCC?TATTGATTGCAG
CTTCCAATTTCGTCACACAACAAGGTCCTAGCTTAGCCAAGAACAATAGCCGATAAAGATAGCCTCATTA
AACGGAATGAGCTAGTAGGCAAAGTCAGCGAATGTGTATATATAAAGGTTCGAGGTCCGTGCCTCCCTCA
TGCTCTCCCCATCTACTCATCAACTCAGATCCTCCAGGAGACTTGTACACCATCTTTTGAGGCACAGAAAC
CCAATAGTCAACCGCGGACTGGCATC(SEQ?ID?NO：115)
D) have the cysD promoter of following sequence from aspergillus nidulans:
AGATCTGGTTCCTGAGTACATCTACCGATGCGCCTCGATCCCCCTCTTAGCCGCATGAGATTCCTACCATT
TATGTCCTATCGTTCAGGGTCCTATTTGGACCGCTAGAAATAGACTCTGCTCGATTTGTTTCCATTATTCAC
GCAATTACGATAGTATTTGGCTCTTTTCGTTTGGCCCAGGTCAATTCGGGTAAGACGCGATCACGCCATTG
TGGCCGCCGGCGCTGCAGCCTCTTATCGAGAAAGAAATTACCGTCGCTCGTGATTTGTTTGCAAAAAGAA
CAAAACTGAAAAAACCCAGACACGCTCGACTTCCTGTCTTCCTATTGATTGCAGCTTCCAATTTCGTCACA
CAACAAGGTCCTACGCCGGCGTTGTGCTGCTGCTATTCCCCGCATATAAACAACCCCTCCACCAGTTCGTT
GGGCTTTGCGAATGCTGTACTCTATTTCAAGTTGTCAAAAGAGAGGATTCAAAAAATTATACCCCAGATAT
CAAAGATATCAAAGCCATC(SEQ?ID?NO：116)
Developed several different methods DNA is operably connected with carrier by the sticking end of complementation.For example, can add complementary homopolymer bundle (tract) to the DNA section that is inserted into carrier DNA.Come connection carrier and DNA section to form recombinant DNA molecules by the hydrogen bond between the complementary homopolymer tail subsequently.
The synthetic linker that contains one or more restriction sites provides a kind of optional method that the DNA section is connected with carrier.To handle with phage T4DNA polymerase or e. coli dna polymerase I by the DNA section that the endonuclease restrictive diges-tion produces, these enzymes with its 3 ' 5 '-exonucleolytic activity removes outstanding γ-strand end, and with its polymerization activity fill 3 of depression '-end.
Therefore these active combination results are put down terminal DNA section.Subsequently enzyme that can catalysis flat terminal dna molecular connects such as phage T4DNA ligase in the presence of, section that will flat end is with the linkers incubation of big molar excess.Therefore product is to carry the DNA section of polylinker sequence at its end.With these DNA sections of suitable Restriction Enzyme cracking and be connected to expression vector, this expression vector is with the enzymatic lysis that produces the end compatible with this DNA segment ends subsequently.
The synthetic linker that contains multiple restriction endonuclease site is can be from comprising InternationalBiotechnologies Inc, New Haven, CT, the commercially available acquisition in a large amount of sources of USA.
If for example will prepare the HA variant, the mode of a kind of ideal modification DNA according to the present invention is to use as (1988) Science 239 such as Saiki, the disclosed polymerase chain reaction of 487-491.The DNA flank for the treatment of enzymatic amplification in this method is two specific oligonucleotide primers, itself incorporates the DNA of amplification into.Auele Specific Primer can contain the restriction endonuclease recognition site, and it can be used for using means known in the art to be cloned into expression vector.
It is pichia (Pichia) (Hansenula (Hansenula)) that expection can be used as the zymic exemplary genus that the host who expresses albumin fusion proteins is used for the present invention's practice, Saccharomyces, Kluyveromyces, mycocandida (Candida), Torulopsis (Torulopsis), spore torulopsis (Torulaspora) is arranged, Schizosaccharomyces (Schizosaccharomyces), Citeromycesbaodingensis belongs to (Citeromyces), pipe capsule Saccharomyces (Pachysolen), Debaryomyces (Debaromyces), the neat Saccharomyces of prunus mume (sieb.) sieb.et zucc. (Metschunikowia), Rhodosporidium (Rhodosporidium), Leucosporidium (Leucosporidium), Botryoascus, lock is thrown Saccharomyces (Sporidiobolus), Endomycopsis (Endomycopsis) or the like.Exemplary genus is selected from down group: Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia sp. and the spore torulopsis is arranged.The example of Saccharomyces kind is saccharomyces cerevisiae, Italian yeast (S.italicus) and Lu Shi yeast (S.rouxii).
The example of Kluyveromyces kind is Kluyveromyces fragilis (K.fragilis), Kluyveromyces lactis (K.lactis) and marxianus yeast (K.marxianus).The suitable kind that the spore torulopsis is arranged is that Dell has spore torula (T.delbrueckii).The example that pichia (Hansenula) is planted is Angus Pichia sp. (P.angusta) (being called multiple-shaped nuohan inferior yeast (H.polymorpha) in the past), unusual Pichia sp. (P.anomala) (being called unusual Hansenula yeast (H.anomala) in the past) and pichia pastoris phaff (P.pastoris).The method summary of transformed saccharomyces cerevisiae is instructed in EP 251744, EP 258067 and WO90/01063, and these lists of references are all incorporated into by reference at this.
The exemplary kind of Saccharomyces comprises saccharomyces cerevisiae, Italian yeast, saccharifying yeast (S.diastaticus) and Lu Shi zygosaccharomyces (Zygosaccharomyces rouxii).The exemplary kind of Kluyveromyces comprises Kluyveromyces fragilis and Kluyveromyces lactis.The exemplary kind of Hansenula comprises multiple-shaped nuohan inferior yeast (being called the Angus Pichia sp. now), unusual Hansenula yeast (being called unusual Pichia sp. now) and pod membrane Pichia sp. (Pichia capsulata).The exemplary kind of other of pichia comprises pichia pastoris phaff.The exemplary kind of aspergillus (Aspergillus) comprises aspergillus niger (A.niger) and aspergillus nidulans (A.nidulans).The exemplary kind of Ye Shi Saccharomyces (Yarrowia) comprises separates fat Ye Shi yeast (Y.lipolytics).Many yeast species can obtain from ATCC.For example, following yeast species can and be used to express albumin fusion proteins from the ATCC acquisition: Chinese Sen Shi saccharomyces cerevisiae (Saccharomyces cerevisiae Hansen), epigamous bacterial strain BY4743 yap3 mutant (ATCC searching number 4022731); Chinese Sen Shi saccharomyces cerevisiae, epigamous bacterial strain BY4743 hsp150 mutant (ATCC searching number 4021266); Chinese Sen Shi saccharomyces cerevisiae, epigamous bacterial strain BY4743 pmt1 mutant (ATCC searching number 4023792); Chinese Sen Shi saccharomyces cerevisiae, epigamous (ATCC searching number 20626; 44773; 44774 and 62995); Saccharifying yeast (Saccharomycesdiastaticus Andrews et Gilliland ex van der Walt), epigamous (ATCC searching number 62987); Kluyveromyces lactis (Kluyveromyces lactis (Dombrowski) van der Walt), epigamous (ATCC searching number 76492); Angus Pichia sp. (Pichia angusta (Teunisson etc.) Kurtzman), epigamous preservation are multiple-shaped nuohan inferior yeast (Hansenula polymorpha de Morals etMaia), epigamous (ATCC searching number 26012); Aspergillus niger (Aspergillus niger van Tieghem), phorozoon (ATCC searching number 9029); Aspergillus niger (Aspergillus niger van Tieghem), phorozoon (ATCC searching number 16404); Aspergillus nidulans (Aspergillus nidulans (Eidam) Winter), phorozoon (ATCC searching number 48756); Conciliate fat Ye Shi yeast (Yarrowia lipolytica (Wickerham etc.) van der Walt et von Arx), epigamous (ATCC searching number 201847).
The suitable promoter of saccharomyces cerevisiae is comprised the gene with PGKI, GAL1 or GAL10 gene, CYCI, PHO5, TRPI, ADHI, ADH2, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvic carboxylase, phosphofructokinase, phosphotriose isomerase, glucose phosphate isomerase, glucokinase, α-mating factor pheromone, the promoter of the gene-correlation of [mating factor pheromone], the PRBI promoter, the GUT2 promoter, the GPDI promoter, with the part 5 ' control region that comprises part 5 ' control region and other promoter or with the hybrid promoter (as the promoter of EP-A-258067) of the heterozygote in upstream activation site.
The promoter of regulatable type easily that is used for schizosaccharomyces pombe (Schizosaccharomyces pomhe) is that thiamine-promoter (as Maundrell (1990) J.Biol.Chem.265,10857-10864 is described) that checks from the nmt gene and the jbp1 gene promoter of glucose repression are (as Hoffman ﹠amp; Winston (1990) Genetics 124,807-816 is described).
Transforming Pichia sp. instructs in for example with the method for expression alien gene, Cregg etc. (1993) and multinomial Phillips patent are (as No. the 4857467th, United States Patent (USP), incorporate into by reference at this), and the Pichia anomala expression test kit is from Invitrogen BV, Leek, Netherlands and Invitrogen Corp., SanDiego, California is commercially available.Suitable promoter comprises AOXI and AOX2.GIeeson etc. (1986) J.Gen.Microbiol.132,3459-3465 comprises the information about Hansenula yeast carrier and conversion, suitable promoter is MOX1 and FMD1; And how other open source literature instruction of EP 361991, Fleer etc. (1991) and Rhone-Poulenc Rorer reaches foreign protein at kluyveromyces species invading the exterior, and suitable promoter is PGKI.
Exemplary transcription stop signals comprises and is useful on 3 ' flanking sequence of the correct signal of tanscription termination and polyadenylation containing of eukaryotic gene.3 suitable ' flanking sequence can be for example natural 3 ' flanking sequence that is connected in the gene of used expression control sequenc, can be corresponding to promoter.Alternatively, 3 ' flanking sequence can be different, and termination signal can be saccharomyces cerevisiae ADHI gene in this case.
Originally the albumin fusion proteins of expectation can express with the secretion targeting sequencing, and targeting sequencing can be effective any targeting sequencing in selected yeast.Can be used for zymic targeting sequencing comprises following any:
A) MPIF-1 signal sequence (as, the amino acid/11-21 of GenBank searching number AAB51134) MKVSVAALSCLMLVTALGSQA (SEQ ID NO:6)
B) department's gland calcium protein signal sequence (MLQNSAVLLLLVISASA, SEQ ID NO:7)
C) the HSA signal sequence is preceding-former district (as, MKWVTFISLLFLFSSAYSRGVFRR, SEQID NO:8)
D) proparea of HSA signal sequence (as, MKWVTFISLLFLFSSAYS, SEQ ID NO:9) or its variant, such as for example, MKWVSFISLLFLFSSAYS, (SEQ ID NO:10)
E) the invertase signal sequence (as, MLLQAFLFLLAGFAAKISA, SEQ ID NO:11)
F) yeast mating factor alpha signal sequence (as, MRFPSIFTAVLAFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPF SNSTNNGLLFINTTIASIAAKEEGVSLEKR, SEQ ID NO:12 or MRFPSIFTAVLAFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPF SNSTNNGLLFINTTIASIAAKEEGVSLDKR, SEQ ID NO:12)
G) Kluyveromyces lactis kills and wounds the toxin targeting sequencing
H) the heterozygosis signal sequence (as, MKWVSFISLLFLFSSAYSRSLEKR, SEQ ID NO:13)
I) HSA/MF α-1 heterozygosis signal sequence (being also referred to as HSA/kex2) (as MKWVSFISLLFLFSSAYSRSLDKR, SEQ ID NO:14)
J) Kluyveromyces lactis kill and wound/MF α-1 merges targeting sequencing (as MNIFYIFLFLLSFVQGSLDKR, SEQ ID NO:15)
K) immunoglobulin Ig signal sequence (as, MGWSCIILFLVATATGVHS, SEQ ID NO:16)
L) fine protein B precursor signal sequence (as MERAAP SRRVPLPLLLLGGLALLAAGVDA, SEQ ID NO:17)
M) bunch egg collection Rhizoma Cynanchi Stauntonii body signal sequence (as MMKTLLLFVGLLLTWESGQVLG, SEQID NO:18)
N) insulin like growth factor-conjugated protein 4 signal sequence (as MLPLCLVAALLLAAGPGPSLG, SEQ ID NO:19)
O) the HSA signal sequence is preceding-and the variant in former-district is such as for example,
MKWVSFISLLFLFSSAYSRGVFRR(SEQ?ID?NO：20)，
MKWVTFISLLFLFAGVLG(SEQ?ID?NO：21)，
MKWVTFISLLFLFSGVLG(SEQ?ID?NO：22)，
MKWVTFISLLFLFGGVLG(SEQ?ID?NO：23)，
The HSA targeting sequencing HSA#64-MKWVTFISLLFLFAGVSG (SEQ IDNO:24) that modifies;
The HSA targeting sequencing HSA#66-MKWVTFISLLFLFGGVSG (SEQ IDNO:25) that modifies;
HSA (A14) targeting sequencing-MKWVTFISLLFLFAGVSG (SEQ ID NO:26) that modifies;
HSA (S14) targeting sequencing (being also referred to as the HSA#65 of the modification)-MKWVTFISLLFLFSGVSG (SEQ ID NO:27) that modifies,
HSA (G14) targeting sequencing-MKWVTFISLLFLFGGVSG (SEQ ID NO:28) that modifies, or MKWVTFISLLFLFGGVLGDLHKS (SEQ ID NO:29)
P) shared signal sequence (MPTWAWWLFLVLLLALWAPARG, SEQ ID NO:3O)
Q) acid p'tase (PH05) targeting sequencing (as, MFKSVVYSILAASLANA SEQ IDNO:31)
R) MFoz-1's preceding-sequence
S) preceding-sequence of 0 glucanase (BGL2)
T) kill and wound the toxin targeting sequencing
U) kill and wound the presequence of toxin
V) Kluyveromyces lactis kills and wounds preceding former (29 aminoacid of toxin; The aminoacid of the aminoacid of (pre) and 13 former (pro) before 16) MNIFYIFLFLLSFVQGLEHTHRRGSLDKR (SEQID NO:32)
W) saccharifying yeast glucoamylase (glucoarnylase) II secretion targeting sequencing
X) saccharomyces carlsbergensis (S.carlsbergensis) alpha-galactosidase (MEL1) secretion targeting sequencing
Y) mycocandida glucoamylase (glucoarnylase) targeting sequencing
Z) be disclosed in the heterozygosis targeting sequencing of EP-A-387319 (incorporating into by reference) at this
Aa) gp67 signal sequence (with baculovirus expression system associating) (as, the amino acid/11-19 of GenBank searching number AAA72759) or
Bb) the natural targeting sequencing of human cytokines X;
Cc) saccharomyces cerevisiae invertase (SUC2) targeting sequencing is disclosed in JP 62-096086 (grant number is 911036516, incorporates into by reference at this); Or
Dd) multiple Flos Calystegiae sepii powder enzyme-MKLAYSLLLPLAGVSASVINYKR (SEQ ID NO:33)
Ee) the TA57 propetide targeting sequencing variant #1-MKLKTVRSAVLSSLFASQVLGQPIDDTESQTTSVNLMADDTESAFATQTNSGG LDVVGLISMAKR (SEQ ID NO:34) of Xiu Shiing
Ff) the TA57 propetide targeting sequencing variant #2-MKLKTVRSAVLSSLFASQVLGQPIDDTESQTTSVNLMADDTESAFATQTNSGG LDVVGLISMAEEGEPKR (SEQ ID NO:35) of Xiu Shiing
Gg) shared signal peptide-MWWRLWWLLLLLLLLWPMVWA (SEQ ID NO:111)
Hh) the HSA/kex2 signal sequence-MKWVSFISLLFLFSSAYSGSLDKR of Xiu Shiing (SEQ ID NO:112)
Ii) shared signal peptide #2-MRPTWAWWLFLVLLLALWAPARG (SEQ ID NO:105)
Reorganization and synthetic other method that produces albumin fusion proteins
The invention still further relates to the carrier, host cell of the polynucleotide that contain code book invention albumin fusion proteins and produce albumin fusion proteins by synthetic and recombinant technique.This carrier can be for example phage, plasmid, virus or retroviral vector.Retroviral vector can be replication competent type or replication defect type.In the situation of replication defect type, virus breeding is general only to be taken place in complementary host cell.
The polynucleotide of albumin fusion proteins of the present invention of encoding can be connected to breed in the host with the carrier that contains selected marker.Usually, plasmid vector is introduced precipitation (such as calcium phosphate precipitation) or with charged lipid complex.If carrier is a virus, then can go into host cell in external use suitable package cell line packaging virus and transduction subsequently.
Polynucleotide insert son should be operably connected to suitable promoter, such as phage PL promoter, escherichia coli lac, trp, phoA and tac promoter, SV40 is early stage and the promoter of late promoter and retrovirus LTR, only gives some instances.Other suitable promoter is known to those skilled in the art.Expression construct will further contain the site that is useful on transcription initiation, termination, and contain the ribosome binding site that is useful on translation in transcriptional domain.The coded portion of the transcripton that construct is expressed can comprise translation initiation codon at the beginning of polypeptide to be translated, and comprises the termination codon (UAA, UGA or UAG) that suitably places end.
As described, expression vector can comprise at least a selected marker.Such labelling comprises dihydrofolate reductase, G418, glutamine synthase or neomycin resistance that is used for the eukaryotic cell cultivation and tetracycline, kanamycin or the ammonia benzyl mycin resistant gene that is used to cultivate in escherichia coli and other antibacterial.The representative example of suitable hosts includes but not limited to, bacterial cell is such as escherichia coli, streptomycete (Streptomyces) and Salmonella typhimurium (Salmonella typhimurium) cell; The fungal cell, such as yeast cells (as, saccharomyces cerevisiae or pichia pastoris phaff (ATCC searching number 201178)); Insect cell such as fruit bat S2 and noctuid (Spodoptera) Sf9 cell; Zooblast such as CHO, COS, NSO, 293 and Bowes melanoma cells and plant cell.To above-mentioned host cell proper culture medium and condition is known in the art.
The exemplary carrier that is used for antibacterial comprises can be from QIAGEN, pQE70, pQE60 and pQE-9 that Inc. obtains; Can be from Stratagene Cloning Systems, pBluescript carrier, Phagescript carrier, pNH8A, pNH16a, pNH18A, pNH46A that Inc. obtains; With can be from PharmaciaBiotech, ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 that Inc obtains.Exemplary eukaryotic vector comprises can be from pWLNEO, pSV2CAT, pOG44, pXT1 and the pSG of Stratagene acquisition; With pSVK3, pBPV, pMSG and the pSVL that can obtain from Pharmacia.The exemplary expression carrier that is used for Yeast system includes but not limited to that pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K and PAO815 (can be from Invitrogen, Carlbad, CA obtains).Other suitable carriers will be tangible to those skilled in the art.
In one embodiment, the polynucleotide of albumin fusion proteins of the present invention of encoding can merge with signal sequence, and this signal sequence will guide protein positioning of the present invention in protokaryon or eukaryotic specific compartment and/or guide protein of the present invention from protokaryon or eukaryotic cell secretion.For example, in escherichia coli, can expect to guide protein expression to periplasmic space.Can merge to guide expression of polypeptides to include but not limited to pelB signal sequence, maltose-binding protein (MBP) signal sequence, MBP, ompA signal sequence, the signal sequence of pericentral siphon coli heat-unsettled enterotoxin B-subunit and the signal sequence of alkali phosphatase to the signal sequence of antibacterial periplasmic space or the example of protein (or its fragment) with albumin fusion proteins of the present invention.The variety carrier that to guide protein positioning that is used for construction of fusion protein is commercially available obtainable, such as the pMAL serial carrier (especially pMAL-p series) that can obtain from New England Biolabs.In specific implementations, the polynucleotide of the albumin fusion proteins of the present invention of encoding can merge to increase expression and the purification efficiency of these polypeptide in gram negative bacteria with pelB pectin lyase signal sequence.See, United States Patent (USP) the 5th, 576,195 and 5,846, No. 818, its content is incorporated into its integral body by reference at this.
Can merge to guide its example of excretory signal peptide in mammalian cell to include but not limited to albumin fusion proteins of the present invention:
A) MPIF-1 signal sequence (as, the amino acid/11-21 of GenBank searching number AAB51134) MKVSVAALSCLMLVTALGSQA (SEQ ID NO:6)
B) department's gland calcium protein signal sequence (MLQNSAVLLLLVISASA, SEQ ID NO:7)
C) the HSA signal sequence is preceding-former district (as, MKWVTFISLLFLFSSAYSRGVFRR, SEQID NO:8)
D) proparea of HSA signal sequence (as, MKWVTFISLLFLFSSAYS, SEQ ID NO:9) or its variant, such as for example, MKWVSFISLLFLFSSAYS, (SEQ ID NO:10)
E) the invertase signal sequence (as, MLLQAFLFLLAGFAAKISA, SEQ ID NO:11)
F) yeast mating factor alpha signal sequence (as, MRFPSIFTAVLAFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPF SNSTNNGLLFINTTIASIAAKEEGVSLEKR, SEQ ID NO:12 or MRFPSIFTAVLAFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPF SNSTNNGLLFINTTIASIAAKEEGVSLDKR, SEQ ID NO:12)
G) Kluyveromyces lactis kills and wounds the toxin targeting sequencing
H) the heterozygosis signal sequence (as, MKWVSFISLLFLFSSAYSRSLEKR, SEQ ID NO:13)
I) HSA/MF α-1 heterozygosis signal sequence (being also referred to as HSA/kex2) (as MKWVSFISLLFLFSSAYSRSLDKR, SEQ ID NO:14)
J) Kluyveromyces lactis kill and wound/MF α-1 merges targeting sequencing (as MNIFYIFLFLLSFVQGSLDKR, SEQ ID NO:15)
K) immunoglobulin Ig signal sequence (as, MGWSCIILFLVATATGVHS, SEQ ID NO:16)
L) fine protein B precursor signal sequence (as
MERAAPSRRVPLPLLLLGGLALLAAGVDA，SEQ?ID?NO：17)
M) bunch egg collection Rhizoma Cynanchi Stauntonii body signal sequence (as MMKTLLLFVGLLLTWESGQVLG, SEQID NO:18)
N) insulin like growth factor-conjugated protein 4 signal sequence (as MLPLCLVAALLLAAGPGPSLG, SEQ ID NO:19)
O) the HSA signal sequence is preceding-and the variant in former-district is such as for example,
MKWVSFISLLFLFSSAYSRGVFRR(SEQ?ID?NO：20)，
MKWVTFISLLFLFAGVLG(SEQ?ID?NO：21)，
MKWVTFISLLFLFSGVLG(SEQ?ID?NO：22)，
MKWVTFISLLFLFGGVLG(SEQ?ID?NO：23)，
The HSA targeting sequencing HSA#64-MKWVTFISLLFLFAGVSG (SEQ IDNO:24) that modifies;
The HSA targeting sequencing HSA#66-MKWVTFISLLFLFGGVSG (SEQ IDNO:25) that modifies;
HSA (A14) targeting sequencing-MKWVTFISLLFLFAGVSG (SEQ ID NO:26) that modifies;
HSA (S14) targeting sequencing (being also referred to as the HSA#65 of the modification)-MKWVTFISLLFLFSGVSG (SEQ ID NO:27) that modifies,
HSA (G14) targeting sequencing-MKWVTFISLLFLFGGVSG (SEQ ID NO:28) that modifies, or MKWVTFISLLFLFGGVLGDLHKS (SEQ ID NO:29)
P) shared signal sequence (MPTWAWWLFLVLLLALWAPARG, SEQ ID NO:30)
Q) acid p'tase (PH05) targeting sequencing (as, MFKSVVYSILAASLANA SEQ IDNO:31)
R) MFoz-1's preceding-sequence
S) preceding-sequence of 0 glucanase (BGL2)
T) kill and wound the toxin targeting sequencing
U) kill and wound the presequence of toxin
V) Kluyveromyces lactis kills and wounds preceding former (29 aminoacid of toxin; The aminoacid of the aminoacid of (pre) and 13 former (pro) before 16) MNIFYIFLFLLSFVQGLEHTHRRGSLDKR (SEQID NO:32)
W) saccharifying yeast glucoamylase (glucoarnylase) II secretion targeting sequencing
X) saccharomyces carlsbergensis (S.carlsbergensis) alpha-galactosidase (MEL1) secretion targeting sequencing
Y) mycocandida glucoamylase (glucoarnylase) targeting sequencing
Z) be disclosed in the heterozygosis targeting sequencing of EP-A-387319 (incorporating into by reference) at this
Aa) gp67 signal sequence (with baculovirus expression system associating) (as, the amino acid/11-19 of GenBank searching number AAA72759) or
Bb) the natural targeting sequencing of human cytokines X;
Cc) saccharomyces cerevisiae invertase (SUC2) targeting sequencing is disclosed in JP 62-096086 (grant number is 911036516, incorporates into by reference at this); Or
Dd) multiple Flos Calystegiae sepii powder enzyme-MKLAYSLLLPLAGVSASVINYKR (SEQ ID NO:33)
Ee) the TA57 propetide targeting sequencing variant #1-MKLKTVRSAVLSSLFASQVLGQPIDDTESQTTSVNLMADDTESAFATQTNSGG LDVVGLISMAKR (SEQ ID NO:34) of Xiu Shiing
Ff) the TA57 propetide targeting sequencing variant #2-MKLKTVRSAVLSSLFASQVLGQPIDDTESQTTSVNLMADDTESAFATQTNSGG LDVVGLISMAEEGEPKR (SEQ ID NO:35) of Xiu Shiing
Gg) shared signal peptide-MWWRLWWLLLLLLLLWPMVWA (SEQ ID NO:111)
Jj) the HSA/kex2 signal sequence-MKWVSFISLLFLFSSAYSGSLDKR of Xiu Shiing (SEQID NO:112)
Kk) shared signal peptide #2-MRPTWAWWLFLVLLLALWAPARG (SEQ ID NO:105)
In one embodiment, the HSA/kex2 signal sequence of modifying (SEQ ID NO:112) merges with the amino terminal of albumin fusion proteins, and described albumin fusion proteins comprises and as herein describedly comprises the fusion rotein of albumin and human cytokines and be disclosed in WO93/15199; WO97/24445; WO03/60071; WO03/59934; And the albumin fusion proteins of PCT/US04/01369 (this incorporates into its each leisure by reference with its integral body).The HSA/kex2 signal sequence of modifying is based on and is disclosed in as Sleep etc., Biotechnology 1990, the 8 volumes, 42-46 page or leaf; With United States Patent (USP) the 5th, 302, the HSA/kex2 signal sequence of No. 697 (both incorporate into its integral body by reference at this) (SEQ ID NO:14).The HSA/kex2 targeting sequencing of modification disclosed herein contains nonconservative aminoacid replacement (Arg to Gly) at residue 19 places of parent's signal peptide.Found that the HSA/kex2 signal peptide of modifying unexpectedly obtains the better expression productive rate and/or the better lysis efficiency of albumin fusion proteins than the HSA/kex2 signal sequence of unmodified when expressing in yeast.The variant of the HSA/kex2 signal peptide of modifying is also included among the present invention.Especially the Gly residue of the position 19 of SEQ ID NO:112 can be replaced by the Pro residue.Other conservative that also comprises the HSA/kex2 signal sequence of modification replaces variant.The signal sequence of the HSA/kex2 of the modification of coding SEQ ID NO:112 with and the conservative nucleic acid that replaces variant be also included among the present invention.
Use glutamine synthase (GS) or DHFR can when having medicine methionine sulphoximine (methionine sulphoximine) or methotrexate, increase respectively as the carrier of selected marker.Based on the advantage of the carrier of glutamine synthase be the glutamine synthase feminine gender cell line (as, rat bone marrow tumour cell system NSO) is easy to obtain.The glutamine synthase expression system also can work in the cell (as Chinese hamster ovary (CHO) cell) of expressing glutamine synthase with the function that prevents endogenous gene by extra inhibitor is provided.Glutamine synthase expression system and its component are specified in PCT and announce: WO87/04462; WO86/05807; WO89/01036; WO89/10404 and WO91/06657, it is incorporated into its integral body by reference at this.In addition, the glutamine synthase expression vector can be from Lonza Biologies, and (Portsmouth NH) obtains Inc..Use the GS expression system in rat bone marrow tumour cell, to express and produce monoclonal antibody and be described in Bebbington etc., Bio/Technology 10:169 (1992) and Biblia and Robinson Biotechnol.Prog.11:1 (1995), it is incorporated into its integral body by reference at this.
The invention still further relates to the host cell that contains above-mentioned vector construction body as herein described, comprise in addition contain use techniques known in the art operationally with one or more allos control zones (as, promoter and/or enhancer) host cell of continuous nucleotide sequence of the present invention.Host cell can be higher eucaryotic cells, and such as mammalian cell (as, people's derived cell), or eukaryotic cell such as low, such as yeast cells, or host cell can be prokaryotic cell, such as bacterial cell.Can select to regulate the expression of inserting gene order, or with the ad hoc fashion modification of expectation and the host strain of processed gene product.Can improve expression when having some elicitors from some promoteres; Thereby the polypeptide expression of may command genetic modification.In addition, different host cells have translation and translation post-treatment and modification (as, phosphorylation, cracking) proteinic feature and specific mechanism.Can select of modification and the processing of suitable cell line with the expectation of the foreign protein guaranteeing to express.
Nucleic acid of the present invention and nucleic acid construct introducing host cell can be realized by the transfection of calcium phosphate transfection, the mediation of DEAE-glucosan, transfection, electroporation, transduction, infection or other method of cation lipid-mediation.These methods are described in many standard laboratory handbooks, such as Davis etc., BasicMethods In Molecular Biology (molecular biology basic skills) (1986).Having imagined polypeptide of the present invention particularly in fact can be by the host cell expression that lacks recombinant vector.
Except comprising the host cell of the vector construction body that contains this paper discussion, the present invention also comprise transformed with disappearance or replaced endogenous genetic stocks (as, can be replaced by albumin fusion proteins corresponding to the coded sequence of human cytokines corresponding to human cytokines) and/or with comprise genetic stocks (as, the heterologous polynucleotide sequence can comprise the albumin fusion proteins of the present invention corresponding to human cytokines such as for example) former generation, subculture and host cell immortalization in vertebrates source, especially mammal source.The genetic stocks that links to each other with endogenous polynucleotide endogenous polynucleotide that can activate, change and/or increase operationally.
In addition, technology known in the art can be used for by homologous recombination with heterologous polynucleotide (as, the polynucleotide of coding albumin albumen or its fragment or variant) and/or the allos control zone (as, promoter and/or enhancer) with the coding human cytokines endogenous polynucleotide sequence operationally link to each other (see as, the United States Patent (USP) the 5th of issue on June 24th, 1997,641, No. 670; No. 96/29411, international publication WO; No. 94/12650, international publication WO; Roller etc., Proc.Natl.Acad.Sci.USA 86:8932-8935 (1989); With Zijlstra etc., Nature 342:435-438 (1989), its disclosure is separately incorporated into its integral body by reference at this).
Can be by comprising that the interact well-known process of chromatography and agglutinin chromatography of ammonium sulfate or ethanol precipitation, acid extraction, anion or cation-exchange chromatography, cellulose phosphate chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, dewatering electric charge reclaims and purification albumin fusion proteins of the present invention from the reconstitution cell culture.On the other hand, adopt high performance liquid chroma-tography (" HPLC ") to come purification.
In some embodiments, albumin fusion proteins of the present invention uses the anion-exchange chromatography purification, includes but not limited to the chromatography that carries out on Q-agarose gel, DEAE agarose gel, poroid (poros) HQ, poroid DEAE, Toyopearl Q, Toyopearl QAE, Toyopearl DEAE, Resource/SourceQ and DEAE, Fractogel Q and DEAE post.
In specific implementations, albumin fusion proteins of the present invention uses the cation-exchange chromatography purification, includes but not limited to SP-agarose gel, CM agarose gel, poroid HS, poroid CM, Toyopearl SP, Toyopearl CM, Resource/Source S and CM, Fractogel S and CM post and their equivalent and suitable thing.
In specific implementations, albumin fusion proteins of the present invention uses the hydrophobic interaction chromatography purification, include but not limited to phenyl, butyl, methyl, octyl group, hexyl-agarose gel, poroid phenyl, butyl, methyl, octyl group, hexyl, Toyopearl phenyl, butyl, methyl, octyl group, hexyl, Resource/Source phenyl, butyl, methyl, octyl group, hexyl, Fractogel phenyl, butyl, methyl, octyl group, hexyl column and their equivalent and suitable thing.
In specific implementations, albumin fusion proteins of the present invention uses the size exclusion chromatography purification, includes but not limited to agarose gel S100, S200, S300, superdex resin column and their equivalent and suitable thing.
In specific implementations, albumin fusion proteins of the present invention uses affinity chromatography purification, includes but not limited to HSA or " merging target " molecule are selectively simulated dyestuff affinity post, peptide affinity post and antibody affinity post.
In some embodiments, use one or more above-mentioned chromatography method purification albumin fusion proteins of the present invention.In other embodiments, use one or more following chromatographic column purification albumin fusion proteins of the present invention: Q agarose gel FF post, SP agarose gel FF post, Q agarose gel high-efficiency column, Blue agarose gel FF post, Blue post, phenyl sepharose gel FF post, DEAE agarose gel FF or methyl post.
In addition, can use the method purification albumin fusion proteins of the present invention described in the PCT international publication WO 00/44772 (it is incorporated into its integral body by reference at this).Those skilled in the art can make amendment to be used for purification albumin fusion proteins of the present invention to the method for wherein describing simply.
Albumin fusion proteins of the present invention can be from following recovery: the product of chemosynthesis scheme; With by recombinant technique from for example comprising, antibacterial, yeast, higher plant, insecticide and mammalian cell are at the product of interior protokaryon or eucaryon host preparation.According to the host who adopts in the reorganization generation scheme, polypeptide of the present invention can be glycosylated or is nonglycosylated.In addition, albumin fusion proteins of the present invention also can be drawn together the methionine residues that contains initial modification, is results of the processing of host-mediation in some situations.Therefore well known, generally in all eukaryotic cells, remove with high efficiency from any protein after the translation by the terminal methionine of the N-of translation initiation codon coding.Although also effectively remove the terminal methionine of N-on the most protein in most of prokaryotes, to remove processing be invalid to this protokaryon concerning some protein, and this depends on the covalently bound amino acid whose character of the terminal methionine of N-.
In one embodiment, the yeast pichia pastoris phaff is used for expressing albumin fusion proteins of the present invention at eukaryotic system.Pichia pastoris phaff is methanol can be carried out metabolic methylotrophy yeast as sole carbon source.The key step of methanol metabolic pathway is to use O
2Oxidation methanol is formaldehyde.This reaction is by the catalysis of enzyme alcohol oxidase.For methanol is carried out metabolism as sole carbon source, pichia pastoris phaff must produce the high levels of alcohol oxidase, the part be since alcohol oxidase to O
2Low relatively affinity.Therefore, in depending on the growth medium that methanol is main carbon source, the promoter region of one of two alcohol oxidase genes (AOX1) is highly active.When having methanol, what the alcohol oxidase that produces from the AOX1 gene accounted for total soluble protein the pichia pastoris phaff reaches about 30%.See Ellis, S.B etc., Mol.Cell.Biol.5:1111-21 (1985); Koutz, P.J etc., Yeast 5:167-77 (1989); Tschopp, J.F. etc., Nucl.Acids Res.15:3859-76 (1987).Therefore allogeneic coding sequence, such as for example, polynucleotide of the present invention are being expressed in the Pichia sp. of growing under methanol with unusual high level under the transcriptional control of all or part AOX1 regulating and controlling sequence.
In an example, plasmid vector pPIC9K is used for expressing the DNA of coding as the listed albumin fusion proteins of the present invention of this paper at Bichi yeast system, basically as be described in " PichiaProtocols:Methods in Molecular Biology (Pichia sp. experimental program: molecular biology method); " D.R.Higgins and J.Cregg edit .The Humana Press, Totowa, NJ, 1998.By the strong AOX1 promoter that is connected in pichia pastoris phaff alkali phosphatase (PHO) secreting signal peptide (being targeting sequencing) that is positioned at the multiple clone site upstream, this expression vector allows to express and secrete polypeptide of the present invention.
As those skilled in the art with understandable, many other yeast vectors can replace pPIC9K to use, such as pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZ α, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K and PAO815, as long as proposed expression construct is provided for transcribing, translate, secreting the suitable framing signal of (if expectation) and similar behavior, comprise AUG in the required frame.
In another embodiment, by heterologous polynucleotide of the present invention being cloned into such as for example, the expression vector of pGAPZ or pGAPZ α etc., and when not having methanol the culture yeasts culture, can reach the high level expression of allogeneic coding sequence, such as for example, the polynucleotide of the albumin fusion proteins of the present invention of encoding.
In addition, can use technology chemosynthesis known in the art albumin fusion proteins of the present invention (as, see Creighton, 1983, Proteins:Structures and Molecular Principles (protein structure and molecular principle), W.H.Freeman ﹠amp; Co., N.Y. and Hunkapiller etc., Nature, 310:105-111 (1984)).For example, the polypeptide corresponding to polypeptide fragment can use peptide synthesizer synthetic.If expectation in addition can be introduced nonclassical amino acid or chemical amino acid analogue as replacing or adding to peptide sequence.Nonclassical amino acid includes but not limited to the D-isomer of common amino acid, 2,4-diamino-butanoic, the a-aminoisobutyric acid, the 4-aminobutyric acid, Abu, the 2-aminobutyric acid, g-Abu, e-Ahx, 6-aminocaprolc acid, Aib, the 2-aminoisobutyric acid, the 3-alanine, ornithine, nor-leucine, norvaline, hydroxyproline, sarcosine, citrulline, Homocitrulline, cysteic acid, tert-butyl group glycine, tert-butyl group alanine, phenylglycine, Cyclohexylalanine, the b-alanine, fluoro-aminoacid, (designer) aminoacid of design is such as the b-methylamino acid, the Ca-methylamino acid, Na-methylamino acid and general amino acid analogue.In addition, this aminoacid can be D (dextral) or L (left-handed).
Present invention resides in the translation process or afterwards through the albumin fusion proteins of the present invention of different modifying; as; by glycosylation, acetylation, phosphorylation, amidatioon, by known protection/blocking groups derivatization, proteolytic cleavage, be connected in antibody molecule or other cell ligand, etc.Can carry out number of chemical modification arbitrarily with known technology, include but not limited to by Bromine cyanide., trypsin, chymase, papain, V8 protease, NaBH
4The specificity chemical cracking; Acetylation, formylated, oxidation, reduction; Metabolism under tunicamycin exists is synthetic; Deng.
Other post translational modification that the present invention includes comprises, for example, as sugar chain, processing N-end or C-end, N-connection or the O-connection), chemical part be attached to sugar chain amino acid backbone, chemical modification N-connection or the O-connection and add or the terminal methionine residues of disappearance N-(for the result of prokaryotic host cell expression).Also can modify albumin fusion proteins, detect and protein isolate with permission such as enzyme labelling, fluorescent labeling, isotope or affinity labelling with detectable labelling.
The example of suitable enzyme comprises horseradish peroxidase, alkali phosphatase, beta galactosidase or acetylcholinesterase; The example of suitable prothetic group complex comprises Streptavidin/biotin and avidin/biotin; The example of suitable fluorescent material comprises umbelliferone, fluorescein, Fluorescein isothiocyanate, rhodamine, dichlorotriazine amine fluorescein, dansyl chloride or phycoerythrin; The example of luminescent material comprises luminol; The example of bioluminescent material comprises luciferase, luciferin and aequorin; With the example of suitable active material comprise iodine (
121I,
123I,
125I,
131I), carbon (
14C), sulfur (
35S), tritium (
3H), indium (
111In,
112In,
113MIn,
115MIn), technetium (
99Tc,
99MTc), thallium (
201Ti), gallium (
68Ga,
67Ga), palladium (
103Pd), molybdenum (
99Mo), xenon (
133Xe), fluorine (
18F),
153Sm,
177Lu,
159Gd,
149Pm,
140La,
175Yb,
166Ho,
90Y,
47Sc,
186Re,
188Re,
142Pr,
105Rh and
97Ru.
In specific implementations, albumin fusion proteins of the present invention or its fragment or variant are attached to macrocyclic chelants, described chelating agen is by including but not limited to
177Lu,
90Y,
166Ho and
153The radioactive metal ion of Sm is connected to polypeptide.In one embodiment, the radioactive metal ion that links to each other with macrocyclic chelants is
111In.In another embodiment, the radioactive metal ion that links to each other with macrocyclic chelants is
90Y.In specific implementations, this macrocyclic chelants is 1,4,7,10-tetraazacyclododecanand-N, N ', N ", N ' " tetraacethyl (DOTA).In other specific embodiment, DOTA attaches to antibody of the present invention or its fragment by linkers.Be used for example with the linkers of DOTA and conjugation of polypeptides and be this area known usually-for example see DeNardo etc., Clin Cancer Res.4 (10): 2483-90 (1998); Peterson etc., Bioconjug.Chem.10 (4): 553-7 (1999); With Zimmerman etc., Nucl.Med.Biol.26 (8): 943-50 (1999); It is incorporated into its integral body by reference at this.
As mentioned above, albumin fusion proteins of the present invention can be by natural process such as the translation post-treatment, or modifies by chemical modification technology well known in the art.The modification that should be understood that same type can exist with identical or different degree in a plurality of sites of specifying polypeptide.Polypeptide of the present invention can be ramose, for example, is the result of ubiquitinization, and polypeptide can be and has or do not have ramose ring-type.Ring-type, ramose and ramose ring type polypeptide can be from translating back natural process acquisition or can being prepared by synthetic method.Modification comprises acetylation; acidylate; the ADP-ribosylation; amidatioon; attached with the flavin covalency; attached with the heme moiety covalency; attached with nucleotide or nucleotide derivative covalency; attached with lipid or lipid derivate covalency; attached with the phosphatidylinositols covalency; crosslinked; cyclisation; form disulfide bond; demethylation; form covalent crosslink; form cysteine; form pyroglutamic acid; formylated; γ-carboxylated; glycosylation; form the GPI anchor; hydroxylating; iodate; methylate; myristylation; oxidation; Pegylation (pegylation); Proteolytic enzyme processing; phosphorylation; prenylation; racemization; selenoization (selenoylation); sulfation; what transfer-RNA mediated adds aminoacid to protein, such as arginylization and ubiquitinization.(for example see PROTEINS-STRUCTURE AND MOLECULAR PROPERTIES (albumen-structure and characterization of molecules), second edition, T.E.Creighton, W.H.Freeman and Company, NewYork (1993); POST-TRANSLATIONAL COVALENT MODIFICATION OFPROTEINS (translation back covalent modification protein), B.C.Johnson, editor, Academic Press, New York, 1-12 page or leaf (1983); Seifter etc., Meth.Enzymol.182:626-646 (1990); Rattan etc., Ann.N.Y.Acad.Sci.663:48-62 (1992)).
The antibody of albumin fusion proteins of the present invention and combined treatment albumen or fragment or variant can merge so that purification with labelled sequence such as peptide.In other embodiments, marker amino acid sequence is six-histidine peptide, such as the label that provides in the pQE carrier (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), and other, wherein many is commercially available getting.As Gentz etc., described in the Proc.Natl.Acad.Sci.USA 86:821-824 (1989), for example, six-histidine provides the purification that makes things convenient for to fusion rotein.Other peptide tag that is used for purification includes but not limited to " HA " label, and it is corresponding to epi-position (Wilson etc., Cell 37:767 (1984)) and " flag " label derived from influenza hemagglutinin protein.
Further, albumin fusion proteins of the present invention can be puted together in therapeutic part such as cytotoxin, as, cytostatics or cytocide, therapeutic agent or radioactive metal ion, as, alpha emitter such as for example, 213Bi.Cytotoxin or cytotoxic agent comprise deleterious dose of any pair cell.Example comprises Pa Litasai, Cytochalasin B, Gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracin diketone, mitoxantrone, mithramycin, actinomycin D, 1-boldenone, glucocorticoid, procaine, tetracaine, lignocaine, Propranolol and puromycin and its analog or homologue.Therapeutic agent includes but not limited to, antimetabolite (as, methotrexate, Ismipur, the 6-thioguanine, cytosine arabinoside, the 5-fluorouracil decarbazine), alkylating agent (as, chlormethine, Thiotef (thioepa) chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, mitobronitol, streptozotocin, ametycin, and (II) (DDP) cisplatin of cisplatin (cis-dichlorodiamine platinum)), anthracycline antibiotics (as, daunorubicin (daunomycin in the past) and doxorubicin), antibiotic (as, dactinomycin (D actinomycin D in the past), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotic agent (as, vincristine and vinblastine).
Conjugate of the present invention can be used for modifying specifies biological answer-reply, and described therapeutic agent or drug moiety are not interpreted as and are limited to classical chemical therapeutic agent.For example, drug moiety can be protein or the polypeptide with desired biological activity.This protein can comprise, for example, and toxin such as abrin, ricin A, Rhodopseudomonas extracellular toxin or diphtheria toxin, diphtherotoxin; Albumen such as tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, apoptosis agent, as TNF-α, TNF-β, AIM I (seeing No. 97/33899, international publication WO), AIM II (seeing No. 97/34911, international publication WO), Fas part (Takahashi etc., Int.Immunol, 6:1567-1574 (1994)), VEGI (seeing No. 99/23105, international publication WO), thrombosis agent or anti--blood vessel propellant, as angiostatin or endostatin; Or the biological answer-reply dressing agent is such as for example, lymphokine, il-1 (" IL-1 "), interleukin-2 (" IL-2 "), interleukin-6 (" IL-6 "), granulocyte macrophage colony stimulating factor (" GM-CSF "), granulocyte colony-stimulating factor (" G-CSF ") or other somatomedin.The technology that is used for this therapeutic part and protein (for example albumin fusion proteins) are puted together is known in the art.
Albumin fusion proteins also can be attached to solid support, this to immunoassay or purification by albumin fusion proteins of the present invention bonded or be incorporated into albumin fusion proteins of the present invention or be particularly useful with the associating polypeptide of albumin fusion proteins of the present invention.This solid support includes but not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polrvinyl chloride or polypropylene.
The albumin fusion proteins of puting together or do not put together therapeutic part can be united as therapeutic agent individually or with cytotoxic factor and/or cytokine and use.
Only comprise in the embodiment of VH domain of the proteic antibody of combined treatment at albumin fusion proteins of the present invention, may and/or expect that coexpression has the fusion rotein of the VL domain of the proteic same antibody of combined treatment, so that VH-albumin fusion proteins and VL albumen will associate (covalently or non-covalently) after translation.
Only comprise in the embodiment of VL domain of the proteic antibody of combined treatment at albumin fusion proteins of the present invention, may and/or expect that coexpression has the fusion rotein of the VH domain of the proteic same antibody of combined treatment, so that VL-albumin fusion proteins and VH albumen will associate (covalently or non-covalently) after translation.
Some therapeutic antibodies are the antibody of bispecific, expression combined treatment proteic antibody be have two differences heavy/light chain to the artificial hybrid antibody of two different binding sites.In order to produce albumin fusion proteins, can produce N-and the terminal segmental albumin fusion proteins of scFv that merges of C-that has with the albumin protein part corresponding to this human cytokines.More specifically, with the terminal scFv that merges of albumin N-will corresponding to weight/light (VH/VL) of the proteic original antibody of combined treatment to one of, and with the terminal scFv that merges of albuminised C-will be right corresponding to another heavy/light (VH/VL) of the proteic original antibody of combined treatment.
The present invention also provides the derivant of the chemical modification of albumin fusion proteins of the present invention, and it can provide other advantage such as the dissolubility, stability and the circulation time that increase polypeptide, or reduces immunogenicity (seeing United States Patent (USP) the 4th, 179, No. 337).Be used for deutero-chemical part and can be selected from water-soluble polymer such as Polyethylene Glycol, ethylene glycol/propylene glycol copolymers, carboxy methyl cellulose, glucosan (dextran), polyvinyl alcohol and analog.Albumin fusion proteins can be modified in intramolecular random site or the predetermined position of intramolecularly and can be comprised one, two, three or more attached chemical parts.
Polymer can be any molecular weight, and can be ramose or not ramose.For Polyethylene Glycol, molecular weight can be between about 1kDa and the about 100kDa (term " pact " is illustrated in the Polyethylene Glycol goods, the molecular weight of some molecules will than shown in molecular weight greater or lesser) so that handle and make.Depend on the desired therapeutic feature (as, the persistent period of the lasting release of expectation, to bioactively influence (if there is), be easy to handle, antigenic degree or shortage and Polyethylene Glycol be to other known effect of human cytokines or analog), can use other size.For example, the mean molecule quantity of Polyethylene Glycol can be about 200,500,1000,1500,2000,2500,3000,3500,4000,4500,5000,5500,6000,6500,7000,7500,8000,8500,9000,9500,10,000,10,500,11,000,11,500,12,000,12,500,13,000,13,500,14,000,14,500,15,000,15,500,16,000,16,500,17,000,17,500,18,000,18,500,19,000,19,500,20,000,25,000,30,000,35,000,40,000,45,000,50,000,55,000,60,000,65,000,70,000,75,000,80,000,85,000,90,000,95,000 or 100,000kDa.
As mentioned above, Polyethylene Glycol can have ramose structure.Ramose Polyethylene Glycol for example is described in, United States Patent (USP) the 5th, 643, No. 575; Morpurgo etc., Appl Biochem.Biotechnol.56:59-12 (1996); Vorobjev etc., Nucleosides Nucleotides 18:2745-2750 (1999); With Caliceti etc., Bioconjug.Chem.70:638-646 (1999), its disclosure is separately incorporated into by reference at this.
Peg molecule (or other chemical part) is should be with protein attached and consider influence to proteinic functional or antigenicity domain.Those skilled in the art there is the attachment method that can get in a large number,, is disclosed in the method (with PEG and G-CSF coupling) of EP 0401384, incorporate into by reference at this such as for example; Also see Malik etc., Exp.Hematol.20:1028-1035 (1992), report use HFC-143a sulfonic acid chloride (tresyl chloride) to come Pegylation GM-CSF.For example, Polyethylene Glycol can be by amino acid residue by reactive group such as free amine group or carboxylic group covalent bond.Reactive group is can be in conjunction with the group of activated polyglycol molecule.Amino acid residue with free amine group group can comprise lysine residue and-terminal amino acid residue; Amino acid residue with free carboxy group can comprise asparagicacid residue, glutaminic acid residue and C-terminal amino acid residue.Mercapto groups also can be used as the reactive group of attached peg molecule.For therapeutic purposes, can carry out at amino group attached, such as carrying out attached at N-end or lysine group.
As mentioned above, Polyethylene Glycol can by with a large amount of amino acid residues in arbitrary bonding and attach to protein.For example, Polyethylene Glycol can be by to the covalent bond of lysine, histidine, aspartic acid, glutamic acid or cysteine residues and be connected in protein.One or more reactive chemistries can be used for Polyethylene Glycol attach to proteinic particular amino acid residue (as, lysine, histidine, aspartic acid, glutamic acid or cysteine) or with Polyethylene Glycol attach to proteinic more than one type amino acid residue (as, lysine, histidine, aspartic acid, glutamic acid, cysteine and its combination).
People can be desirably in the protein of N-end through chemical modification especially.Using Polyethylene Glycol is the example of this compositions, can select (according to molecular weight, branch etc.) from multiple peg molecule: the type of the ratio of peg molecule and protein (polypeptide) molecule the reactant mixture, the pegylation reaction that will carry out and obtain the method for protein of selected N-end Pegylation.The method (promptly if desired, separating this part from the part of other single Pegylation) that obtains the goods of the terminal Pegylation of N-can be passed through from the material of the terminal Pegylation of the purification N-of protein molecule colony of Pegylation.The proteic modification of selectivity in the terminal chemical modification of N-can realize that it utilizes the differential responses of the dissimilar primary amino radical groups (lysine or N-end) that can be used for derivatization in specific protein by standard reductive alkylation.Under proper reaction conditions, can realize with the polymer that contains carbonyl group at the N-end proteinic roughly selective derivatizationization.
As mentioned above, Pegylation albumin fusion proteins of the present invention can be realized by any kind of mode.For example, Polyethylene Glycol can be directly or is attached by therebetween joint and albumin fusion proteins.Be used for Polyethylene Glycol and proteinic non junction system description in Delgado etc., Crit.Rev.Thera.DrugCarrier Sys.9:249-304 (1992); Francis etc., Intern.J.of Hematol.68:1-18 (1998); United States Patent (USP) the 4th, 002, No. 531; United States Patent (USP) the 5th, 349, No. 052; WO 95/06058; With WO 98/32466, its being disclosed in this and incorporating into by reference separately.
Be used for Polyethylene Glycol directly attachedly and not have the MPEG of a kind of system employing HFC-143a sulfonylation of joint therebetween with proteinic amino acid residue, it is to use HFC-143a sulfonic acid chloride (ClSO
2CH
2CF
3) modify mono methoxy (monmethoxy) Polyethylene Glycol (MPEG) and preparation.After the MPEG reaction of protein and HFC-143a sulfonylation, that Polyethylene Glycol is directly attached with proteinic amine groups.Therefore the present invention includes with protein of the present invention with have 2,2, the peg molecule reaction of 2-HFC-143a (trifluoreothane) sulfonyl group and the protein-Polyethylene Glycol conjugate of preparation.
Also can use a large amount of different joints therebetween that Polyethylene Glycol is attached to protein.For example, United States Patent (USP) the 5th, 612, No. 460, its complete disclosure is incorporated into by reference at this, discloses to be used for Polyethylene Glycol and the attached urethane joint of protein.Wherein Polyethylene Glycol attach to proteinic protein-Polyethylene Glycol conjugate by joint also can be by the reaction of protein and chemical compound is prepared, this chemical compound is such as MPEG-butanimide succinate, 1, the activatory MPEG of 1 '-carbonyl dimidazoles, MPEG-2,4,5-trichlorine amyl carbonate, MPEG-paranitrophenol carbonic ester and various MPEG-succinate derivative.Being used for Polyethylene Glycol is attached to proteinic a large amount of other polyethyleneglycol derivative and reactive chemistry is that its complete disclosure is incorporated into by reference at this described in No. 98/32466, the international publication WO.Use the Pegylation protein product of reactive chemistry preparation as herein described to comprise within the scope of the present invention.
The number (being substitution value) that attaches to the polyalkylene glycol moiety of each albumin fusion proteins of the present invention also can change.For example, the protein of Pegylation of the present invention can be on average with 1,2,3,4,5,6,7,8,9,10,12,15,17,20 or more a plurality of peg molecule attached.Similarly, average substitution degree is in the scope of every protein molecular such as 1-3,2-4,3-5,4-6,5-7,6-8,7-9,8-10,9-11,10-12,11-13,12-14,13-15,14-16,15-17,16-18, a 17-19 or 18-20 polyalkylene glycol moiety.The method of determining substitution value for example is discussed at, Delgado etc., Crit.Rev.Thera.Drug Carrier Sys.9:249-304 (1992).
Can reclaim and purification polypeptide of the present invention from chemosynthesis and reconstitution cell culture by the standard method that includes but not limited to ammonium sulfate or ethanol precipitation, acid extraction, anion or cation-exchange chromatography, cellulose phosphate chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and agglutinin chromatography.In one aspect of the invention, adopt high performance liquid chroma-tography (" HPLC ") to come purification.When polypeptide separating and/or purge process in can adopt the technology of knowing of folded protein to come the regeneration activity configuration during degeneration.
Can use the extensive known immunoassay ELISA in this area to determine the existence and the amount of albumin fusion proteins of the present invention.In an ELISA, the scheme that will be used for detected/quantified albumin fusion proteins of the present invention may further comprise the steps: with Anti-Human's serum albumin antibody sandwich elisa plate, closure plate is non-to prevent-the specificity combination, clean elisa plate, add the solution that contains albumin fusion proteins of the present invention (with one or more variable concentrations), add the second anti--human cytokines specific antibody be coupled to detectable labelling (as described herein or in addition known in the art), and detect the existence of second antibody.In the optional version of this scheme, can resist-human cytokines specific antibody bag is by elisa plate, and second reagent of labelling can be Anti-Human's albumin specific antibody.
The purposes of polynucleotide
Every kind of polynucleotide that this paper identifies can use as reagent in many ways.Below describe and to think exemplary and use known technology.
Polynucleotide of the present invention can be used for producing albumin fusion proteins of the present invention.As described in more detail below, polynucleotide of the present invention (coding albumin fusion proteins) can be used for recombinant DNA method, are used for genetic engineering with cell, cell line or the tissue of preparation expression by the albumin fusion proteins of the polynucleotide encoding of coding albumin fusion proteins of the present invention.
Polynucleotide of the present invention also can be used for gene therapy.A target of gene therapy is that normal gene is inserted into the organism with defective gene, acts on the correction genetic defect.Be disclosed in polynucleotide of the present invention means with these genetic defectes of mode targeting of pin-point accuracy are provided.Another target is to insert non-existent new gene in the host genome, thereby produces new character in host cell.Other non-limitative example of the gene therapy method that the present invention includes is described in the other places (see as, be designated as the part and the embodiment 61 and 62 of " gene therapy ") of this paper more thoroughly.
The purposes of polypeptide
Every peptide species that this paper identifies can use in many ways.Below describe and to think exemplary and the use known technology.
Albumin fusion proteins of the present invention can be used for for diversity ground appraisement organization (as, the immunohistochemistry algoscopy is such as for example, ABC immunoperoxidase (Hsu etc., J.Histochem.Cytochem.29:577-580 (1981)) or cell type (as, immunocytochemical determination method) the immunology probe is provided.
Albumin fusion proteins can be used for using to classical immunohistology method well known by persons skilled in the art measure polypeptide level in the biological sample (as, see Jalkanen etc., J.Cell.Biol.101:976-985 (1985); Jalkanen etc., J.Cell.Biol.105:3087-3096 (1987)).Other method that can be used for detecting protein gene expression comprises immunoassay, such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA).Suitable mensuration labelling is known in the art and comprises enzyme labelling, such as, glucoseoxidase; Radiosiotope, such as iodine (
131I,
125I,
123I,
121I), carbon (
14C), sulfur (
35S), tritium (
3H), indium (
115mIn,
113mIn,
112In,
111In) and technetium (
99Tc,
99mTc), thallium (
201Ti), gallium (
68Ga,
67Ga), palladium (
103Pd), molybdenum (
99Mo), xenon (
133Xe), fluorine (
18F),
153Sm,
177Lu,
159Gd,
149Pm,
140La,
175Yb,
166Ho,
90Y,
47Sc,
186Re,
188Re,
142Pr,
105Rh,
97Ru; Luminescent marking is such as luminol; With fluorescent labeling such as fluorescein and rhodamine and biotin.
Also can be by detecting albumin fusion proteins of the present invention in the imaging body.Be used for proteinic labelling of in-vivo imaging or label and comprise the labelling or the label that can (ESR) detect by X-radiography, nuclear magnetic resonance, NMR (NMR) or electron spin relaxation (electron spin relaxtion).For the X-radiography, suitable labelling comprises radiosiotope such as barium or caesium, and they launch detectable radiation but not obvious injury curee.To NMR and ESR, suitable labelling comprises the labelling with detectable feature spin (characteristic spin), such as deuterium, it can mix albumin fusion proteins by the nutrient substance that labelling offers the cell line of expressing albumin fusion proteins of the present invention.
To (for example, use suitable detectable imaging moiety such as radiosiotope
131I,
112In,
99mTc, (
131I,
125I,
123I,
121I), carbon (
14C), sulfur (
35S), tritium (
3H), indium (
115mIn,
113mIn,
112In,
111In) and technetium (
99Tc,
99mTc), thallium (
201Ti), gallium (
68Ga,
67Ga), palladium (
103Pd), molybdenum (
99Mo), xenon (
133Xe), fluorine (
18F,
153Sm,
177Lu,
159Gd,
149Pm,
140La,
175Yb,
166Ho,
90Y,
47Sc,
186Re,
188Re,
142Pr,
105Rh and
97Ru), the albumin fusion proteins of radio-opaque substance or the detectable material marking of nuclear magnetic resonance, NMR is introduced the mammal of (for example, parenteral, subcutaneous or intraperitoneal ground) examine immune system illness.Should be understood that the physique of curee in this area and the amount that used imaging system will determine to produce the required imaging moiety of diagnostic image.In the situation of radiosiotope part, for people curee, radioactive amount of injection usually will be for from about 5 to 20 millicuries
99mTc.The albumin fusion proteins of labelling subsequently will be preferentially in vivo the position that is positioned at of one or more receptors, part or substrate (corresponding to receptor, part or the substrate of the human cytokines that is used to prepare albumin fusion proteins of the present invention) (as, organ, cell, extracellular space or substrate) accumulation.Alternatively, comprise at least one fragment of therapeutic antibodies or the situation of variant at albumin fusion proteins, the position that the albumin fusion proteins of labelling subsequently will be preferentially be positioned at corresponding to the polypeptide/epi-position of the bonded polypeptide/epi-position of therapeutic antibodies (being used to prepare albumin fusion proteins of the present invention) in vivo (as, organ, cell, extracellular space or substrate) accumulation.The in-vivo tumour imaging is described in S.W.Burchiel etc., " Immunopharmacokineticsof Radiolabeled Antibodies and Their Fragments (immune labeled antibody and its segmental immune pharmacokinetics) " (the 13rd chapter of Tumor Imaging:The Radiochemical Detection of Cancer (tumor imaging: radiochemistry detects cancer), S.W.Burchiel and B.A.Rhodes edit, Masson Publishing Inc. (1982)).Scheme described herein can be easy to be used for albumin fusion proteins of the present invention by those skilled in the art's modification.
In one embodiment, the invention provides the method for a kind of specific delivery albumin fusion proteins of the present invention in the cell, by use with heterologous polypeptide or the associating albumin fusion proteins of the present invention of nucleic acid (as, by the polypeptide of the polynucleotide encoding of coding albumin fusion proteins of the present invention and/or antibody).In an example, the invention provides the method for a kind of delivery of therapeutic albumen in the cell of institute's targeting.In another example, the invention provides a kind of send single-chain nucleic acid (as, antisense or ribozyme) or double-strandednucleic acid (as, can be integrated into the genome or free the DNA that duplicates and can be transcribed of cell) method in the cell of institute's targeting.
In another embodiment, the invention provides a kind of by albumin fusion proteins of the present invention and toxin or the coupling of cytotoxicity prodrug are come specificity destroy cell (as, destroy tumor cell) method.
" toxin " mean one or more chemical compounds, radiosiotope, holotoxin, the modification of combination and activation of endogenous cytotoxic effect system toxin, toxin the catalytic subunit or be not present in any molecule or the enzyme that in the cell or on the cell surface, under qualifications, causes cell death usually.The spendable toxin of the method according to this invention includes but not limited to, radiosiotope known in the art, chemical compound be such as for example, in conjunction with the antibody of the intrinsic or inductive endogenous cell poisonous effect system complement fixation of its part (or contain), thymidine kinase, endonuclease, RNA enzyme, alpha toxin, Ricin, abrin, Rhodopseudomonas exotoxin A, diphtheria toxin, diphtherotoxin, saporin, momordin, gelonin, pokeweed antiviral protein, α-sarcina and cholera toxin." toxin " also comprises cytostatics or cytocide, therapeutic agent or radioactive metal ion, as, alpha emitter such as for example,
213Bi or other radiosiotope be such as for example,
103Pd,
133Xe,
131I,
68Ge,
57Co,
65Zn,
85Sr,
32P,
35S,
90Y,
153Sm,
153Gd,
169Yb,
51Cr,
54Mn,
75Se,
113Sn,
90Yttrium,
117Tin,
186Rhenium,
166Holmium and
188Rhenium; Luminescent marking is such as luminol; With fluorescent labeling such as fluorescein and rhodamine and biotin.In specific implementations, the invention provides a kind of passing through with polypeptide of the present invention or antibody of the present invention and radiosiotope
90Y coupling specificity destroy cell (as, destroy tumor cell) method.In another specific implementations, the invention provides a kind of passing through with polypeptide of the present invention or antibody of the present invention and radiosiotope
111In coupling specificity destroy cell (as, destroy tumor cell) method.In the further specific embodiment, the invention provides a kind of passing through with polypeptide of the present invention or antibody of the present invention and radiosiotope
131I coupling specificity destroy cell (as, destroy tumor cell) method.
Can use technology known in the art with labelling polypeptide of the present invention.This technology includes but not limited to, use difunctional put together agent (see as, United States Patent (USP) the 5th, 756,065; 5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119; 4,994,560; With 5,808, No. 003; Its content is separately incorporated into its integral body by reference at this).
Albumin fusion proteins of the present invention can be used for mammal for example diagnosis, treatment, prevention and/or the prognosis of the various illness of philtrum.These illness include but not limited to that the following title of this paper is the illness in the part of " biological activity ".
The invention provides the diagnostic method of illness, comprise that (a) uses albumin fusion proteins of the present invention to measure specific polypeptide at the cell of individuality or the expression in the body fluid; (b) institute is measured polypeptide expression level and standard polypeptide expression level relatively, thus increase that the polypeptide expression level of measuring is compared with the standard expression or minimizing expression illness.For cancer, exist the transcript of relative a large amount can indicate the susceptible body constitution that develops disease in the individual biological biopsy, maybe can be provided at the means that disease appears detecting before in actual clinical symptom.Thereby such more reliable diagnostic can allow fitness guru earlier to adopt preventive measure or initiative treatment to prevent the development of cancer or further make progress.
And, albumin fusion proteins of the present invention can be used for treatment or prevent disease or disease such as for example, neuropathy, immune system illness, muscle illness, reproduction illness, gastrointestinal tract disease, pulmonary disease, cardiovascular disease, kidney illness, propagation illness and/or Cancerous disease and disease.For example, the patient can be applied polypeptide of the present invention, work in order to replace non-existent or low-level polypeptide (as, insulin), with replenish non-existent or low-level not homopolypeptide (as, haemachrome S replaces haemachrome B, SOD, catalase, dna repair protein), with suppress polypeptide (as, oncogene or tumor inhibitor) activity, with the activity of activated polypeptides (as, by being incorporated into receptor), with by with membrane-bound receptor competition free ligand reduce membrane-bound receptor activity (as, be used to reduce the soluble TNF acceptor of inflammation), or with replying of causing expecting (as, angiogenic growth suppresses, enhancing is to the immunne response of proliferative cell or tissue).
Especially, comprise at least one fragment of therapeutic antibodies or the albumin fusion proteins of variant and also can be used for treating disease (as above described) with this paper other places.For example, use at least one fragment that comprises therapeutic antibodies or variant albumin fusion proteins can in conjunction with and/or neutralization be used to prepare the bonded polypeptide of therapeutic antibodies specificity of albumin fusion proteins and/or reduce the excessive generation of the bonded polypeptide of therapeutic antibodies specificity that is used to prepare albumin fusion proteins.Similarly, use at least one fragment that comprises therapeutic antibodies or the albumin fusion proteins of variant and can activate the bonded polypeptide of therapeutic antibodies specificity that is used to prepare albumin fusion proteins, by combining with the polypeptide that is incorporated into film (receptor).
At least, use method well known to those skilled in the art, albumin fusion proteins of the present invention can be used as the molecular weight marker on SDS-PAGE gel or the molecular sieve gel filtration column.Albumin fusion proteins of the present invention also can be used for producing antibody, this antibody can be used for measuring human cytokines of the present invention, albumin albumen and/or the albumin fusion proteins protein expression from reconstitution cell conversely, as a kind of mode of estimating host cell conversion or the conversion in biological sample.And albumin fusion proteins of the present invention can be used for testing biological activity as herein described.
The diagnostic assay method
Chemical compound of the present invention can be used for mammal for example diagnosis, treatment, prevention and/or the prognosis of the various illness of philtrum.These illness include but not limited to, in table 1 respective column to every kind of human cytokines and be " immunocompetence ", " blood related disorders ", " hyper-proliferative illness ", " kidney illness ", " cardiovascular disease ", " breathing illness ", " activity takes place anti--blood vessel ", " disease of cellular level ", " wound healing and epithelial cell proliferation ", " neural activity and neurological disorder ", " endocrine illness ", " reproductive system illness ", " infectious disease ", " regeneration " and/or " gastrointestinal tract disease with lower banner herein " part in the illness described.
For multiple illness, can from the tissue, cell or the body fluid that obtain from the individuality of suffering from this illness (as, serum, blood plasma, urine, seminal fluid, synovial fluid or spinal fluid) in detect gene expression dose with respect to " standard " gene expression dose, that is, from the material change (increase or reduce) of the tissue or the expression in the body fluid of the individuality of not suffering from this illness.Therefore the invention provides a kind of diagnostic method that can be used for diagnosing in the illness process, comprise that the expression of gene in tissue, cell or the body fluid of individuality of measuring coded polypeptide also compares measured gene expression dose and standard gene expression level, thus increase that gene expression dose is compared with standard or minimizing expression illness.These diagnostic assay methods can be in vivo or external carrying out, and such as for example, blood sample, biopsy or autopsy tissue carried out.
The present invention also can be used as prognostic indicator, thereby the patient who shows the gene expression of enhanced or reduction will obtain worse clinical effectiveness.
The expression of gene level of coded polypeptide " measure " mean directly (as, by determining or estimating absolute protein matter level or mRNA level) or relatively (as, by with second biological sample in the polypeptide level or the mRNA level relatively) qualitative or quantitative measurement or estimate the level of the specific polypeptide (as polypeptide) in first biological sample or the mRNA level of the polypeptide of the present invention of encoding corresponding to the human cytokines that is disclosed in table 1.In one embodiment, measure or estimate expression of polypeptides level or mRNA level in first biological sample, and compare with standard polypeptide level or mRNA level, this standard is determined from the average level that obtains from second biological sample of the individuality of not suffering from this illness or the colony by deriving from the individuality of not suffering from this illness.Should understand as this area, in case standard polypeptide level or mRNA level are known, then reusable it as standard in order to relatively.
" biological sample " means any biological sample that obtains from individuality, cell line, tissue culture or other source of containing polypeptide of the present invention (comprising its part) or mRNA.As directed, biological sample comprises total length or its segmental body fluid (such as serum, blood plasma, urine, synovial fluid and spinal fluid) and tissue-derived of finding express polypeptide or mRNA.The method that obtains biopsy and body fluid from mammal is well known in the art.Wherein biological sample means and comprises mRNA, and mRNA can obtain from biopsy.
Can use any suitable technique to separate total cell RNA from biological sample, such as Chomczynski and Sacchi, the guanidine-rhodanate-phenol-chloroform method of the single stage described in the Anal.Biochem.162:156-159 (1987).Use any suitable method to measure the mRNA level of coding polypeptide of the present invention subsequently.These comprise Northern engram analysis, S1 nuclease mapping, polymerase chain reaction (PCR), reverse transcription associating polymerase chain reaction (RT-PCR) and reverse transcription associating ligase chain reaction (RT-LCR).
The invention still further relates to the diagnostic assay method, such as quantitative and diagnostic assay method, be used to measure the polypeptide that is incorporated into albumin fusion proteins of the present invention, by the bonded polypeptide of albumin fusion proteins of the present invention or with the associating polypeptide of albumin fusion proteins of the present invention biological sample (as, cell and tissue) in level, comprise the normal and abnormal level of determining polypeptide.The diagnostic assay method of the unconventionality expression of therefore for example, be used to detect the polypeptide that is incorporated into albumin fusion proteins of the present invention according to the present invention, comparing by the bonded polypeptide of albumin fusion proteins of the present invention or with the associating polypeptide of albumin fusion proteins of the present invention with the normal control tissue sample can be used for detecting the existence of tumor.Can be used for determining to be incorporated into albumin fusion proteins of the present invention polypeptide, by the bonded polypeptide of albumin fusion proteins of the present invention or with the associating polypeptide of albumin fusion proteins of the present invention be to well known to those skilled in the art in determination techniques from the level in host's the sample.These assay methods comprise radioimmunoassay, competitiveness-binding assay, Western engram analysis and ELISA algoscopy.Measuring the level of polypeptide in biological sample can use any means known in the art to carry out.
Can use multiple technologies to carry out the mensuration of polypeptide level in the biological sample.For example, can be with expression of polypeptides (Jalkanen etc., the J.Cell.Biol.101:976-985 (1985) in the classical immunohistology method research organization; Jalkanen etc., J.Cell.Biol.105:3087-3096 (1987)).Other can be used for detecting the method that polypeptide gene expresses and comprises immunoassay, such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA).Suitable TPPA labelling is known in the art and comprises enzyme labelling, such as, glucoseoxidase; And radiosiotope, such as iodine (
125I,
121I), carbon (
14C), sulfur (
35S), tritium (
3H), indium (
112In) and technetium (
99mTc); With fluorescent labeling such as fluorescein and rhodamine and biotin.
Tissue to be analyzed or cell type will generally comprise the tissue of known or the gene that doubtful expression is paid close attention to or cell type (such as for example, cancer).The protein separating method that this paper adopts can be, for example, be described in Harlow and Lane (Harlow, E. and Lane, D., 1988, " Antibodies:A LaboratoryManual (laboratory manual of antibody) ", Cold Spring Harbor Laboratory Press, ColdSpring Harbor, New York) in (it is incorporated into its integral body by reference at this) those.Isolated cells can be derived from cell culture or from the patient.The cell that analysis obtains from culture can be the essential step of estimating cell, and it can be used as the part based on the gene therapy technology of cell, or alternatively, is used for the effect of test compounds to gene expression.
For example, albumin fusion proteins can be used for quantitatively or qualitative detection be incorporated into albumin fusion proteins of the present invention polypeptide, by the bonded polypeptide of albumin fusion proteins of the present invention or with the existence of the associating polypeptide of albumin fusion proteins of the present invention.This can be by for example, realizes with the immunofluorescence technique of the link coupled employing fluorescent labeling of light microscope method, flow cytometry or fluoroscopic examination albumin fusion proteins.
In one embodiment, comprise specificity in conjunction with at least one human cytokines disclosed herein (as, be disclosed in the human cytokines of table 1) or in addition at least one fragment of the antibody of human cytokines known in the art or the albumin fusion proteins of variant can be used for quantitatively or the existence of qualitative detection gene outcome or its conservative variant or fragments of peptides.This can for example pass through, and immunofluorescence technique link coupled with light microscope method, flow cytometry or fluoroscopic examination, the fluorescently-labeled antibody of employing realizes.
In addition, but the histology adopts albumin fusion proteins of the present invention in ground, as in immunofluorescence, immunoelectron microscope or non-immunologic assay method, be used for polypeptide that in situ detection is incorporated into albumin fusion proteins of the present invention, by the bonded polypeptide of albumin fusion proteins of the present invention or with the associating polypeptide of albumin fusion proteins of the present invention.Can be by removing histological specimens from the patient, and the antibody of labelling of the present invention or polypeptide be applied thereto realize in situ detection.Can cover by albumin fusion proteins and apply albumin fusion proteins on the biological sample labelling.By using this scheme, not only might determine to be incorporated into albumin fusion proteins of the present invention polypeptide, by the bonded polypeptide of albumin fusion proteins of the present invention or with the existence of the associating polypeptide of albumin fusion proteins of the present invention, also might determine its distribution in the tissue of checking.Use the present invention, those of ordinary skills are with easy to understand, and any (such as the dyeing scheme) that can revise many Histological methods is to realize this in situ detection.
Detection is incorporated into the polypeptide of albumin fusion proteins of the present invention, will be usually included in by the bonded polypeptide of albumin fusion proteins of the present invention or with the immunoassay of the associating polypeptide of albumin fusion proteins of the present invention and non-immunoassay and can have incubation sample down in conjunction with the detectable label antibody of gene outcome or its conservative variant or fragments of peptides, such as biofluid, tissue extract, the new cell of collecting or in cell culture through the lysate of the cell of incubation, and the bonded antibody of any detection by many technology well known in the art.
Can make biological sample contact and be fixed in solid support or carrier such as celluloid or can fixed cell, on other solid support of cell granulations or soluble protein.Can wash this support for suitable buffer subsequently, handle with the albumin fusion proteins of the present invention that can detect ground mark subsequently.Subsequently can this solid support of buffer secondary washing to remove unconjugated antibody or polypeptide.This antibody of labelling subsequently randomly.Can detect the amount of bonded labelling on the solid support subsequently by conventional method.
" solid support or carrier " mean can in conjunction with polypeptide (as, albumin fusion proteins or be incorporated into albumin fusion proteins of the present invention, by albumin fusion proteins of the present invention in conjunction with or with the associating polypeptide of albumin fusion proteins of the present invention) any support.Support of knowing or carrier comprise glass, polystyrene, polypropylene, polyethylene, glucosan, nylon, amylase, natural and cellulose, polyacrylamide, gabbro and the Magnet modified.For the object of the invention, the character of carrier can be to a certain degree solvable or soluble.Supporting body material can have any in fact possible node configuration, as long as link coupled molecule can be incorporated into polypeptide.Therefore the support configuration can be sphere, as pearl, or cylindrical, as the inner surface of test tube or the outer surface of rod.Alternatively, the surface can be smooth such as lamella, calibration tape etc.Exemplary support comprises polystyrene bead.One skilled in the art will know that to be used for binding antibody or antigenic many other suitable carriers, or can determine suitable carriers by using routine test.
Can determine the combination activity of the albumin fusion proteins that institute gives batch according to well-known process.By adopting routine test those skilled in the art can determine to the effective and best condition determination of each mensuration.
Except measuring the level of polypeptide the biological sample that obtains from individuality, also can detect polypeptide by imaging in vivo.For example, in an embodiment of the invention, use the ill or tumor cell of albumin fusion proteins imaging of the present invention.
Be used for the labelling of in-vivo imaging albumin fusion proteins of the present invention or label and comprise the labelling or the label that can detect by X-radiography, NMR, MRI, CAT-scanning or ESR.For the X-radiography, suitable labelling comprises radiosiotope such as barium or caesium, and they launch detectable radiation but not obvious injury curee.NMR and the suitable labelling of ESR are comprised the labelling that detectable feature spins, and such as deuterium, it can mix albumin fusion proteins by the nutrient substance of the cell line (or antibacterial or yeast strain) of labelling through transforming.
In addition, can use the albumin fusion proteins of the present invention that its existence can be detected.For example, can use with the albumin fusion proteins of the present invention of radiopacity or other suitable compound labelling and as above-mentioned the antibody of labelling is discussed, make it can be in vivo as seen.Further, such polypeptide can be used for the in-vitro diagnosis scheme.
To (for example, use suitable detectable imaging moiety such as radiosiotope
131I,
112In,
99mTc), radio-opaque substance or the mammal that can introduce (for example, parenteral, subcutaneous or intraperitoneal ground) examine illness by the polypeptide-specific antibody or the antibody fragment of the material marking of magnetic resonance detection.Should be understood that the physique of curee in this area and the amount that used imaging system will determine to produce the required imaging moiety of diagnostic image.In the situation of radiosiotope part, for people curee, radioactive amount of injection usually will be for from about 5 to 20 millicuries
99mTc.The albumin fusion proteins of labelling will preferentially contain in vivo subsequently be incorporated into albumin fusion proteins of the present invention, by albumin fusion proteins of the present invention in conjunction with or with the position accumulation of the associating polypeptide of albumin fusion proteins of the present invention or other material.The in-vivo tumour imaging is described in S.W.Burchiel etc., " Immunopharmacokinetics ofRadiolabeled Antibodies and Their Fragments (immune labeled antibody and its segmental immune pharmacokinetics) " (the 13rd chapter of Tumor Imaging:The Radiochemical Detection of Cancer (tumor imaging: radiochemistry detects cancer), S.W.Burchiel and B.A.Rhodes edit, MassonPublishing Inc. (1982)).
Wherein one of mode that can detect ground mark albumin fusion proteins of the present invention is to use this connection product (Voller in reporter enzyme and in enzyme immunoassay (EIA) by connecting, A., " TheEnzyme Linked Immunosorbent Assay (enzyme-linked immunosorbent assay) (ELISA) ", 1978, Diagnostic Horizons 2:1-7, Microbiological Associates Quarterly Publication, Walkersville, MD); Voller etc., J.Clin.Pathol.31:507-520 (1978); Butler, J.E., Meth.Enzymol.73:482-523 (1981); Maggio, E. (editor), 1980, EnzymeImmunoassay (immunoassay of enzyme), CRC Press, Boca Raton, FL; Ishikawa, E. etc., (editor), and 1981, Enzyme Immunoassay (immunoassay of enzyme), Kgaku Shoin, Tokyo).Be incorporated into antibody reporter enzyme will with for example chromogenic substrate reaction of suitable substrate, the mode of reaction is for example to produce, by spectrophotometer, fluorescence or by the detectable chemical part of visual method.The reporter enzyme that can be used for detecting ground mark antibody includes but not limited to malic dehydrogenase, staphylococcal nuclease, δ-5-steroid isomerase, Alcohol Dehydrogenase from Yeast, alpha-phosphate glycerol dehydrogenase, phosphotriose isomerase, horseradish peroxidase, alkali phosphatase, asparaginase, glucoseoxidase, beta galactosidase, ribonuclease, urase, catalase, glucose-6-phosphate dehydrogenase (G6PD), glucoamylase and acetylcholinesterase.The colorimetric analysis method of chromogenic substrate that in addition, can be by adopting reporter enzyme detects.Also can be by visually detecting with the enzymatic reaction degree of standard substance comparison substrate of preparation similarly.
Albumin fusion proteins also can and be used for any of multiple other immune detection by radio-labeled.For example, by the radioactive label albumin fusion proteins, might (for example see at radioimmunoassay (RIA), Weintraub, B., Principles of Radioimmunoassays (radioimmunoassay principle), Seventh Training Course on Radioligand Assay Techniques (the 7th training course of radioligand assay technology), The Endocrine Society, March, 1986, it is incorporated into by reference at this) in use this albumin fusion proteins.Can detect radioactive isotope with the following methods, include but not limited to gamma counter, scintillation counter or autoradiography.
In addition, chelator molecule is known in the art and can be used for the labelling albumin fusion proteins.Chelator molecule can be attached to albumin fusion proteins of the present invention so that come the described albumen of labelling with metal ion that comprises radionuclide or fluorescent labeling.For example, see Subramanian, R. and Meares, C.F., " Bifunctional Chelating Agents for Radiometal-labeled monoclonalAntibodies (bifunctional chelating agent that is used for the monoclonal antibody of radioactive metal labelling); " CancerImaging with Radiolabeled Antibodies (using radiolabeled cancer imaging) (D.M.Goldenberg edits) Kluwer Academic Publications, Boston; Saji, H., " Targeteddelivery of radiolabeled imaging and therapeutic agents:bifunctionalradiopharmaceuticals (radiolabeled imaging of targeted delivery and therapeutic agent: bifunctional radiopharmaceutical). " Crit.Rev.Ther.Drug Carrier Syst.16:209-244 (1999); Srivastava S.C. and Mease R.C., " Progress in research on ligands, nuclides and techniques forlabeling monoclonal antibodies (part of labeled monoclonal antibody, nucleic and Study on Technology progress). " Int.J.Rad.Appl.Instrum.B18:589-603 (1991); And Liu, S. and Edwards, D.S., " Bifunctional chelators for therapeutic lanthanide radiopharmaceuticals (the radiopharmaceutic bifunctional chelating agent of being used for the treatment of property group of the lanthanides). " Bioconjug.Chem.12:7-34 (2001).Any chelating agen that can be covalently bonded in described albumin fusion proteins can be used according to the invention.This chelating agen can further comprise the blank area that chelating moiety is connected with albumin fusion proteins.
In one embodiment, the chelating agen that albumin fusion proteins of the present invention is attached to acyclic is such as diethylenetriamine-N, N, N ', N ", N " pentaacetic acid (DPTA), DPTA analog and DPTA derivant.As non-limitative example, chelating agen can be 2-(to the isothiocyano benzyl)-6-methyl diethylene triamine pentacetic acid (DTPA) (1B4M-DPTA, also be called MX-DTPA), 2-methyl-6-(ρ-nitrobenzyl)-1,4,7-triazaheptane-N, N, N ', N ", N " pentaacetic acid (nitro-1B4M-DTPA or nitro-MX-DTPA); 2-(to the isothiocyano benzyl)-cyclohexyl diethylene triamine pentacetic acid (DTPA) (CHX-DTPA) or N-[2-amino-3-(ρ-nitrobenzophenone) propyl group]-trans-cyclohexane extraction-1,2-diamidogen-N, N ', N " pentaacetic acid (nitro-CHX-A-DTPA).
In another embodiment, albumin fusion proteins of the present invention is attached to the terpyridyl chelating agen of acyclic such as 6,6 " two [[N; N, N ", N " four (carboxyl methyl) amino] methyl]-4 '-(3-amino-4-methoxyphenyl)-2; 2 ': 6 ', 2 " terpyridyls (TMT-amine).
In specific implementations, the macrocyclic chelants that attaches to albumin fusion proteins of the present invention is 1,4,7,10-tetraazacyclododecanand-N, N, N ", N ' " tetraacethyl (DOTA).In other specific implementations, DOTA is attached to albumin fusion proteins of the present invention by linkers.Can be used for DOTA put together in the example of the linkers of polypeptide be this area known usually-for example see DeNardo etc., Clin.Cancer Res.4 (10): 2483-90,1998; Peterson etc., Bioconjug.Chem.70 (4): 553-7,1999; With Zimmerman etc., Nucl Med.Biol.26 (8): 943-50,1999, it is incorporated into its integral body by reference at this.In addition, United States Patent (USP) the 5th, 652,361 and 5,756, disclose for No. 065 and can put together in the chelating agen and the preparation of antibody and use their method, incorporate into by reference with its integral body at this.Although United States Patent (USP) the 5th, 652,361 and 5,756, focus on chelating agen and antibody are puted together for No. 065, those skilled in the art can be easy to revise disclosed method with chelating agen and other conjugation of polypeptides.
Can adopt bifunctional chelating agent based on macrocyclic ligand, wherein puting together is activatory arm or functional group by the carbon backbone chain that is connected in part, as described below: M.Moi etc., (2-is to nitrobenzyl-1,4,7 for J.Amer.Chem.Soc.49:2639 (1989), 10-tetraazacyclododecanand-N, N ', N ", N ' " and tetraacethyl); S.V.Deshpande etc., J.Nucl.Med.31:413 (1990); G.Ruser etc., Bioconj.Chem.1:345 (1990); C.J.Broan etc., J.C.S.Chem.Comm.23:1739 (1990); With C.J.Anderson etc., J.Nucl Med.36:850 (1995).
In one embodiment, macrocyclic chelants is attached to albumin fusion proteins of the present invention such as the optional poly-nitrogen heterocyclic ring chelating agen that contains one or more carboxyls, amino, hydroxamic acid ester, phosphonate ester or phosphate groups.In another embodiment, this chelating agen is the chelating agen that is selected from down group: DOTA, DOTA analog and DOTA derivant.
In one embodiment, the suitable chelator molecule that can attach to albumin fusion proteins of the present invention comprises DOXA (1-oxa--4,7,10-triazododecane triacetic acid), NOTA (1,4,7-7-triazacyclononane triacetic acid), TETA (1,4,8,11-tetraazacyclododecane tetradecane tetraacethyl) and THT (4 '-(3-amino-4-methoxyl group-phenyl)-6,6 " two (N ', N '-dicarboxyl methyl-N-methyl hydrazine)-2; 2 ': 6 ', 2 " terpyridyl) and its analog and derivant.See as, Ohmono etc., J.Med.Chem.35:157-162 (1992); Kung etc., J.Nucl.Med.25:326-332 (1984); Jurisson etc., Chem.Rev.93:1137-1156 (1993); With United States Patent (USP) the 5th, 367, No. 080.Other suitable chelating agen comprises and is disclosed in United States Patent (USP) the 4th, 647,447; 4,687,659; 4,885, No. 363; EP-A-71564; WO89/00557; Chelating agen with EP-A-232751.
In another embodiment, spendable suitable macro ring carboxylic acid chelating agen comprises 1,4,7 among the present invention, 10-tetraazacyclododecanand-N, N ', N " N ' "-tetraacethyl (DOTA); 1,4,8,12-tetraazacyclododecane pentadecane-N, N ', N " N ' "-tetraacethyl (15N4); 1,4,7-7-triazacyclononane-N, N ', N " triacetic acid (9N3); 1,5,9-triazododecane-N, N ', N " triacetic acid (12N3); With 6-acetyl bromide amino-benzyl-1,4,8,11-tetraazacyclododecane tetradecane-N, N ', N " N ' "-tetraacethyl (BAT).
The exemplary chelating agen that can attach to albumin fusion proteins of the present invention is α-(5-isothiocyano-2-methoxyphenyl)-1,4,7,10-tetraazacyclododecanand-1,4,7, and the 10-tetraacethyl also is called MeO-DOTA-NCS.Also can use α-(5-isothiocyano-2-methoxyphenyl)-1,4,7,10-tetraazacyclododecanand-1,4,7, the salt of 10-tetraacethyl or ester.
The albumin fusion proteins of the present invention of the attached chelating agen as described of covalency can be with the radioisotope labeling that is suitable for treating, diagnosing or treat and diagnose two kinds of purposes (through the coordination site of chelating agen).The example of suitable metal comprises Ag, At, Au, Bi, Cu, Ga, Ho, In, Lu, Pb, Pd, Pm, Pr, Rb, Re, Rh, Sc, Sr, Tc, Tl, Y and Yb.The example that is used for the radionuclide of diagnostic purpose be Fe, Gd,
111In,
67Ga or
68Ga.In another embodiment, the radionuclide that is used for diagnostic purpose is
111In or
67Ga.The example that is used for the treatment of the radionuclide of purpose is
166Ho,
165Dy,
90Y,
115mIn,
52Fe or
72Ga.In one embodiment, the radionuclide that is used for diagnostic purpose is
166Ho or
90Y.Be used for the treatment of and diagnose the example of the radionuclide of two kinds of purposes to comprise
153Sm,
177Lu,
159Gd,
175Yb or
47Sc.In one embodiment, radionuclide is
153Sm,
177Lu,
175Yb or
159Gd.
The illustrative metal radionuclide comprises
90Y,
99mTc,
111In,
47Sc,
67Ga,
51Cr,
177mSn,
67Cu,
167Tm,
97Ru,
188Re,
177Lu,
199Au,
47Sc,
67Ga,
51Cr,
177mSn,
67Cu,
167Tm,
95Ru,
188Re,
177Lu,
199Au,
203Pb and
141Ce.
In specific implementations, the albumin fusion proteins of the present invention of the attached chelating agen of covalency can be selected from down the group the metal ion labelling:
90Y,
111In,
177Lu,
166Ho,
215Bi and
225Ac.
And, γ-emission radionuclide such as
99mTc,
111In,
67Ga and
169Yb has been approved for diagnosing image or has just studied and has been used for diagnosing image, and beta emitter such as
67Cu,
111Ag,
186Re and
90Y can be used for the application of oncotherapy.Available other radionuclide also comprises gamma emitter, such as
99mTc,
111In,
67Ga and
169Yb, and beta emitter, such as
67Cu,
111Ag,
186Re,
188Re and
90Y, and other radionuclide of paying close attention to such as
211At,
212Bi,
177Lu,
86Rb,
105Rh,
153Sm,
198Au,
149Pm,
85Sr,
142Pr,
214Pb,
109Pd,
166Ho,
208Tl and
44Sc.The albumin fusion proteins of the present invention of the attached chelating agen of covalency can be used aforesaid radioisotope labeling.
In another embodiment, the albumin fusion proteins of the present invention of the attached chelating agen of covalency can be used the paramagnetic metal ion labelling, this paramagnetic metal ion comprises the ion of transition metal and lanthanide series metal, such as having 21-29,42,43,44 or the ion of metal, especially Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and the Lu of the atomic number of 57-71.Be used for paramagnetic metal that the compositions of NMR (Nuclear Magnetic Resonance)-imaging uses and comprise having 22 to 29,42,44 and the element of the atomic number of 58-70.
In another embodiment, the albumin fusion proteins of the present invention of the attached chelating agen of covalency can with the fluorescence metal ion labelling, the especially La that comprise group of the lanthanides, Ce, Pr, Nd, Pm, Sm, Eu (as,
152Eu), Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu.
In another embodiment, the albumin fusion proteins of the present invention of the attached chelating agen of covalency can be with containing the heavy metal reporter molecules labelling of (can comprise Mo, Bi, Si and W atom).
Also might be with fluorescent chemicals labelling albumin fusion proteins.When fluorescently-labeled antibody is exposed to light time of suitable wavelength, subsequently can be owing to the existence of fluoroscopic examination to it.The most frequently used fluorescent labeling chemical compound comprises Fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde(OPA) and fluorescamine.
Also can use the fluorescent emission metal such as
152Other metal of Eu or group of the lanthanides can detect the ground mark albumin fusion proteins.Use such metal-chelating group such as diethylene triamine pentacetic acid (DTPA) (DTPA) or ethylenediaminetetraacetic acid (EDTA), these metals can be attached to antibody.
Albumin fusion proteins also can be by being coupled to it chemiluminescence compound and can detecting ground mark.The existence of the albumin fusion proteins of chemiluminescence-labelling is subsequently determined by detecting the luminous existence that produces in the chemical reaction process.The example of useful especially chemiluminescent labeling chemical compound is luminol, different luminol, theromatic acridinium ester, imidazoles, acridinium salt and oxalate.
Similarly, bioluminescent compound can be used for labelling albumin fusion proteins of the present invention.Bioluminescence is the chemiluminescence that a class sees biosystem, and wherein catalytic albumen increases the efficient of chemiluminescence reaction.Determine the existence of bioluminescent protein by detecting luminous existence.With the important biomolecule luminophor that is labeled as purpose is luciferin, luciferase and aequorin.
Transgenic organism
The transgenic organism of expressing albumin fusion proteins of the present invention is also included among the present invention.Transgenic organism is to the genetically modified organism of the genetic stocks that wherein shifts reorganization, external source or clone.This genetic stocks often is called transgenic.Genetically modified nucleotide sequence can comprise one or more transcription regulating nucleotide sequences and other nucleotide sequence such as intron, and this may be for essential to optimization expression and secretion encoded protein matter.Can design transgenic to guide encoded protein matter so that express, as breast, blood, urine, ovum, hair or kind from organism from organism or the mode that from the product that organism produces, reclaims.Transgenic can be made up of following: derived from the genomic nucleotide sequence of the identical or different species of the species of target animal.Transgenic can be incorporated into common absent variable genomic locus of specific nucleic acid sequence or genetically modified normal site.
Term " germ cell line transgenic organism " refers to that wherein hereditary change or hereditary information introduced the system genitale cell, thereby gives the transgenic organism that transgenic organism is transferred to hereditary information offspring's ability.If in fact these offsprings have some or all described change or hereditary information, these offsprings also are transgenic organisms so.Described change or hereditary information can be external source to the organism species under the receiver, is external source to the particular individual receiver only, or can be the hereditary information that the receiver has had.In in the end a kind of situation, gene change or that introduce can be expressed by different way with natural gene.
Transgenic organism can be transgenic animal or transgenic plant.Transgenic animal can be by the preparation of multiple distinct methods, comprise gene targeting in transfection, electroporation, microinjection, the embryonic stem cell and recombinant virus and retroviral infection (see as, United States Patent (USP) the 4th, 736, No. 866; United States Patent (USP) the 5th, 602, No. 307; Mullins etc. (1993) Hypertension 22 (4): 630-633; Brenin etc. (1997) Surg.Oncol.6 (2) 99-110; Tuan (editor), Recombinant Gene Expression Protocols, Methods in Molecular Biology (molecular biology method, recombinant gene expression scheme) No.62, Humana Press (1997)).The method of nucleic acid fragment being introduced reorganization competence mammalian cell can be any method that helps the multiple nucleic acid molecules of cotransformation.The detailed protocol that produces transgenic animal is that those skilled in the art are easy to obtain, and comprises United States Patent (USP) the 5th, 489, disclosing in No. the 5th, 602,307, No. 743 and the United States Patent (USP).
Prepare a large amount of reorganization or transgenic mice, comprised the mice (United States Patent (USP) the 4th, 736, No. 866) of expressing activatory oncogene sequence; Express the antigenic mice of ape and monkey SV40 T-(United States Patent (USP) the 5th, 728, No. 915); Lack the mice (United States Patent (USP) the 5th, 731, No. 490) that interferon regulatory factor 1 (IRF-1) is expressed; The performance handicapped mice of dopaminergic (United States Patent (USP) the 5th, 723, No. 719); Express the mice (United States Patent (USP) the 5th, 731, No. 489) of the people's gene of at least a participation controlling of blood pressure; The mice (United States Patent (USP) the 5th, 720, No. 936) that shows very big similarity with situation about existing in the naturally occurring Alzheimer; The mice (United States Patent (USP) the 5th, 602, No. 307) that the adherent ability of mediated cell reduces; Mice (Clutter etc. (1996) Genetics 143 (4): 1753-1760) with bovine growth hormone gene; Maybe can produce mice (McCarthy (1997) the The Lancet 349 (9049): 405) that fully human antibodies is replied.
Although mice and rat are still the selected animal of most of transgenic experiments, preferred or even the alternate animal species of necessary use in certain situation.The transgenic scheme has been successfully used to multiple non-Mus animal, comprises that sheep, goat, pig, dog, cat, monkey, chimpanzee, hamster, rabbit, cattle and Cavia porcellus (see as, Kim etc. (1997) Mol.Reprod.Dev.46 (4): 515-526; Houdebine (1995) Reprod.Nutr.Dev.35 (6): 609-617; Peters (1994) Reprod.Fertil.Dev.6 (5): 643-645; Schnieke etc. (1997) Science 278 (5346): 2130-2133; And Amoah (1997) J.AnimalScience 75 (2): 578-585).
For the protein secreting of the present invention that guides transgenic-coding Ruzhong, can be placed on preferentially under the activatory promoter control of galactophore epithelial cell to transgene mammal.Can use the promoter of the gene of control coding lactoprotein, for example the promoter of casein, beta lactoglobulin, whey acidic protein or lactalbumin (sees as, DiTullio (1992) Biotechnology 10:74-77; Clark etc. (1989) Biotechnology 7:487-492; Gorton etc. (1987) Biotechnology 5:1183-1187; With (1992) FEBS Letts.297:13 such as Soulier).Selected transgene mammal will produce a large amount of breasts and have long lactation period, for example goat, cattle, camel or sheep.
Albumin fusion proteins of the present invention also can be expressed in transgenic plant, as wherein the DNA transgenic being inserted the plant of nuclear or plastom.The Plant Transformation scheme that is used for exogenous nucleic acid introduced plant cell or protoplast is known in the art.Usually see Methods in Enzymology (Enzymology method) the 153rd volume (" Recombinant DNA Part D (recombinant DNA D part) ") 1987, Wu and Grossman edit, Academic Press and European patent application EP 693554.The method that produces genetically engineered plant is further described in United States Patent (USP) the 5th, 283, No. the 5th, 482,852, No. 184, United States Patent (USP) and European patent application EP 693554, and it is all incorporated into by reference at this.
Medicine or therapeutic combination
Albumin fusion proteins of the present invention or its preparation can be used by any conventional method, comprise parenteral (as subcutaneous or intramuscular) injection or intravenous infusion.Treatment can be made up of single dose or a plurality of dosage that continue for some time.
Although may use albumin fusion proteins of the present invention separately, it also can be rendered as pharmaceutical preparation with one or more acceptable carriers.It is compatible with albumin fusion proteins and harmless to its receiver that carrier must be that " acceptable " means.Usually, carrier is aseptic and pyrogen-free water or saline.Albumin fusion proteins of the present invention is particularly suitable for the preparation in aqueous carrier such as aseptic apirogen water, saline or other isosmotic solution, because their shelf lives in solution prolong.For example, pharmaceutical composition of the present invention can be before making up a prescription for example several weeks or several months or longer time early (well in advance) be formulated as moisture form.
Can consider the shelf life that albumin fusion proteins prolongs when for example, preparing the preparation that contains albumin fusion proteins in aqueous compositions.As discussed above, many these human cytokines shelf life after being blended in HA significantly increases or prolongs.
Be fit to use in the aerocolloidal situation, can using standard scheme that albumin fusion proteins of the present invention is formulated as aerosol.Any gas that term " aerosol " comprises albumin fusion proteins of the present invention carries suspended phase, and it can be inhaled into bronchioles or nasal meatus.Particularly, the gas that aerosol comprises the microdroplet of albumin fusion proteins of the present invention carries suspension, as preparing in dose inhaler that measures or spray gun (nebulizer) or in aerosol apparatus (mist sprayer).Aerosol also comprises chemical compound of the present invention and is suspended in dry powder composite in air or other vector gas (carrier gas), can send by being blown into from for example inhaler apparatus.See Ganderton ﹠amp; Jones, Drug Delivery to the RespiratoryTract (delivering drugs is to respiratory tract), Ellis Horwood (1987); Gonda (1990) CriticalReviews in Therapeutic Drug Carrier Systems (the key summary of medicine carrier system) 6:273-313; With Raeburn etc.. (1992) Pharmacol.Toxicol.Methods 27:143-159.
Preparation of the present invention is non-immunogenic normally also, part be since the composition of the albumin fusion proteins that uses derived from suitable species.For example, use for the mankind, the human cytokines of albumin fusion proteins and albumin part be the people normally.Arbitrary therein component is in non-human-more deutero-situations, thereby can be the people's rather than external source in nature by replacing concrete epi-position that key amino acid makes this component humanization to find expression in human immune system then.
Preparation can present with unit dosage form easily and can know any method preparation by pharmaceutical field.These methods comprise the step of albumin fusion proteins with the carrier associating (association) that constitutes one or more supplementary elements.Usually by equably and closely making the product molding prepare preparation then if necessary active component and liquid-carrier or meticulous solid-state carrier or The combined.
The preparation that is fit to parenteral administration comprises moisture and non--moisture aseptic injectable solution that can contain the antioxidant, buffer agent, antibacterial and the solute that make preparation be fit to the expection receiver and the moisture and non--moisture sterile suspensions that can comprise suspending agent and thickening agent.Said preparation can the unit of being presented on-dosage or many-dose container in, for example sealed ampoule, bottle or syringe, and can be stored under lyophilization (lyophilizing) condition, only need be before being about to use interpolation sterile liquid carrier, for example water for injection.Instant injection solution and suspension can prepare from sterilized powder.Because many albumin fusion proteins of the present invention show the serum half-life that prolongs, dosage particles is compared with the standard preparation that does not merge of human cytokines and can be contained the human cytokines part than low molar concentration or than low dosage.
As an example, when albumin fusion proteins of the present invention comprises that a kind of albumen of " human cytokines: the X " row that are listed in table 1 is during as one or more human cytokines district, can calculate dosage form with respect to tiring of independent human cytokines based on tire (potency) of albumin fusion proteins, consider that simultaneously albumin fusion proteins compares serum half-life and shelf life and prolong with natural human cytokines.For example, if human cytokines usually with 0.3 to 30.0IU/kg/ week or be divided into three or seven dosage 0.9 to 12.0IU/kg/ week and used 1 year or longer.For by being blended in the albumin fusion proteins that the proteic total length HA of therapeutic (therpeutic) forms, will represent more heavy weight medicament with the DE of unit representation but can reduce dose frequency, for example reduce to twice weekly, once in a week or still less.
Preparation of the present invention or compositions can be packed with description or the packing insert of shelf life of the prolongation of pointing out the albumin fusion proteins component, or are included in together in the cover box.For example, this description or packing insert can indicate the storage requirement of recommendation, such as time, temperature and illumination, consider that the shelf life of albumin fusion proteins of the present invention extends or prolongs.This description or packing insert also can indicate the concrete advantage of albumin fusion proteins of the present invention, and such as being convenient to depot formulation, described preparation need use in the place beyond hospital, clinic or the office's condition of control.As mentioned above, preparation of the present invention can be moisture form and can be stored in and is lower than under the ecotopia and significantly do not lose therapeutic activity.
Albumin fusion proteins of the present invention also can be included in the nutritional medicine (netraceuticals).For example, albumin fusion proteins more of the present invention can be used with natural product, comprise the breast or the newborn product that obtain from the transgene mammal of expressing albumin fusion proteins.This compositions also can comprise plant or the plant product that obtains from the transgenic plant of expressing albumin fusion proteins.Albumin fusion proteins can also provide with powder or the tablet form that contains or do not contain other additives known, carrier, filler and diluent.Nutritional medicine (netraceuticals) is described in Scott Hegenhart, Food Product Design (food product design), in December, 1993.
The present invention also provides and treats and/or prevents disease or illness (such as for example, any or multiple disease disclosed herein or illness) method, by the polynucleotide (" albumin fusion polynucleotides ") of the albumin fusion proteins of the present invention that is applied in the effective dose in the pharmaceutically acceptable carrier to the curee or the albumin fusion proteins of the present invention of encoding.
Albumin fusion proteins and/or polynucleotide will be prepared and dosed administration in the mode consistent with good medical practice, consider clinical setting (the especially only side effect for the treatment of with albumin fusion proteins and/or polynucleotide), site of delivery, application process, administration schedules and the known other factors of doctor of individual patient.Therefore " effective dose " of this paper purpose is definite by these considerations.
As total suggestion, total every dosage medicine effective quantity of parenteral administration albumin fusion proteins in about 1ug/kg/ days to 10mg/kg/ days scope of weight in patients, although as mentioned above, this judgement of will receiving treatment.In one embodiment, this dosage is 0.01mg/kg/ days and in another embodiment at least, to the mankind, is about 0.01 to 1mg/kg/ day for hormone.If continue medication, use albumin fusion proteins with about 1ug/kg/ hour to about 50ug/kg/ hour close rate usually, inject 1-4 time every day or lasting h inf, for example use micropump.Also can adopt intravenous bag (bag) solution.Observe and change required treatment length and treatment back and the required interval of response takes place show the effect that depends on expectation and change.
As mentioned above, albumin fusion proteins of the present invention is compared with independent human cytokines part (or its fragment or variant) and is had higher plasma stability.When the effective dose of determining the albumin fusion proteins that every dosage is to be administered and administration administration schedules, should be taken into account this increase of plasma stability.Especially, higher plasma stability can allow with identical frequency of administration using albumin fusion proteins than low dosage, or can allow alternatively to use albumin fusion proteins with less administration.More high stability can allow to use albumin fusion proteins of the present invention more infrequently with less administration.In one embodiment, can whenever biweekly use albumin fusion proteins.In another embodiment, depend on the pharmacokinetics of albumin fusion proteins, can per three weeks once, every around once, per five weeks once or more weeks once use albumin fusion proteins.For example, as discussed above, the pharmacokinetics of IFN-α-HSA fusion rotein support every 2-4 week once or more weeks once dosage regimen and even with 4 weeks or surpass the interval administration in per 4 weeks.
Total preparation albumin fusion proteins concentration of the every dosage that provides also can be provided the treatment effective dose of the albumin fusion proteins that every dosage is to be administered.In one embodiment, every dosage is applied to total preparation albumin fusion proteins concentration of patient in the scope of about 10ug/ agent to about 2400ug/ agent.In one embodiment, to about 1000ug/ agent, or alternatively, about 1000ug/ agent is about 1200ug/ agent or the about 900ug/ agent scope of about 1800ug/ agent extremely extremely in about 100ug/ agent for total concentration.
In concrete embodiment, IFN-α of the present invention-HSA fusion rotein (as, prepare by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) with the total compound concentration administration of about 90ug/ agent to about 2400ug/ agent.In other embodiments, IFN-α of the present invention-HSA fusion rotein (as, by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296 preparations) with extremely about 2400ug/ agent of about 900ug/ agent, about 900ug/ agent is to about 2100ug/ agent, about 900ug/ agent is to about 2000ug/ agent, about 900ug/ agent is to about 1200ug/ agent, about 900ug/ agent is to total compound concentration administration of about 1800ug/ agent with in another other embodiment, with the total compound concentration administration of about 1200ug/ agent to about 1800ug/ agent.In other embodiment, IFN-α of the present invention-HSA fusion rotein (as, by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296 preparations) with total compound concentration administration of 600ug/ agent, 720ug/ agent, 800ug/ agent, 900ug/ agent, 1000ug/ agent, 1200ug/ agent, 1500ug/ agent, 1800ug/ agent, 2000ug/ agent, 2100ug/ agent or 2400ug/ agent.In other embodiment, the IFN-α of the present invention-HSA fusion rotein of total preparation dosage (as, by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296 preparations) use separately or with antiviral compound such as the ribavirin combined administration.In other embodiment, the IFN-α of the present invention-HSA fusion rotein of total preparation dosage (as, by CID2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296 preparations) with a kind of, two kinds, three kinds, four kinds or more kinds of antiviral compound such as the ribavirin combined administration.
In other embodiment, use total compound concentration IFN-α of the present invention-HSA fusion rotein (as, by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296 the preparation) with the treatment subject suffering from HCV infection.In concrete embodiment, separately or with antiviral compound such as the ribavirin combination of effective dose with about 90ug/ agent to total compound concentration of about 2400ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, prepare by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) in the untreated patient who suffers from HCV.In other embodiment, separately or with antiviral compound such as the ribavirin combination of effective dose with extremely about 2400ug/ agent of about 900ug/ agent, about 900ug/ agent is to about 2100ug/ agent, about 900ug/ agent is to about 2000ug/ agent, about 900ug/ agent is to about 1200ug/ agent, about 900ug/ agent to total compound concentration of about 1800ug/ agent and another further embodiment with about 1200ug/ agent extremely total compound concentration of about 1800ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296 preparations) in the untreated patient who suffers from HCV.In other embodiment, separately or with antiviral compound such as the ribavirin combination of effective dose with the 600ug/ agent, the 720ug/ agent, the 800ug/ agent, the 900ug/ agent, the 1000ug/ agent, the 1200ug/ agent, the 1500ug/ agent, the 1800ug/ agent, the 2000ug/ agent, total compound concentration of 2100ug/ agent or 2400ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, by CID2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296 preparations) in the untreated patient who suffers from HCV.
In other embodiment, with a kind of, two kinds, three kinds, four kinds or more kinds of antiviral compound such as the ribavirin combination of effective dose with about 90ug/ agent extremely total compound concentration of about 2400ug/ agent use the IFN-α of the present invention-HSA fusion rotein of total compound concentration in the patient of the untreated HCV of suffering from.In other embodiment, a kind of with effective dose, two kinds, three kinds, the combination of four kinds or more kinds of antiviral compound such as ribavirin is with extremely about 2400ug/ agent of about 900ug/ agent, about 900ug/ agent is to about 2100ug/ agent, about 900ug/ agent is to about 2000ug/ agent, about 900ug/ agent is to about 1200ug/ agent, about 900ug/ agent to total compound concentration of about 1800ug/ agent and further embodiment with about 1200ug/ agent extremely total compound concentration of about 1800ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296 preparations) in the untreated patient who suffers from HCV.In other embodiment, a kind of with effective dose, two kinds, three kinds, the combination of four kinds or more kinds of antiviral compound such as ribavirin is with the 600ug/ agent, the 720ug/ agent, the 800ug/ agent, the 900ug/ agent, the 1000ug/ agent, the 1200ug/ agent, the 1500ug/ agent, the 1800ug/ agent, the 2000ug/ agent, total compound concentration of 2100ug/ agent or 2400ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296 preparations) in the untreated patient who suffers from HCV.
In other embodiment, separately or with antiviral compound such as the ribavirin combination of effective dose with about 90ug/ agent to total compound concentration of about 2400ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, prepare by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) in the patient who suffers from HCV who lives through treatment.In other embodiments, separately or with antiviral compound such as the ribavirin combination of effective dose with extremely about 2400ug/ agent of about 900ug/ agent, about 900ug/ agent is to about 2100ug/ agent, about 900ug/ agent is to about 2000ug/ agent, about 900ug/ agent is to about 1200ug/ agent, about 900ug/ agent to total compound concentration of about 1800ug/ agent and other embodiment with about 1200ug/ agent extremely total compound concentration of about 1800ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296 preparations) in the patient who suffers from HCV who lives through treatment.In other embodiment, separately or with antiviral compound such as the ribavirin combination of effective dose with the 600ug/ agent, the 720ug/ agent, the 800ug/ agent, the 900ug/ agent, the 1000ug/ agent, the 1200ug/ agent, the 1500ug/ agent, the 1800ug/ agent, the 2000ug/ agent, total compound concentration of 2100ug/ agent or 2400ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296 preparations) in the patient who suffers from HCV who lives through treatment.
In other embodiment, with a kind of, two kinds, three kinds, four kinds or more kinds of antiviral compound such as the ribavirin combination of effective dose with about 90ug/ agent to total compound concentration of about 2400ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, prepare by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) in the patient who suffers from HCV who lives through treatment.In other embodiment, a kind of with effective dose, two kinds, three kinds, the combination of four kinds or more kinds of antiviral compound such as ribavirin is with extremely about 2400ug/ agent of about 900ug/ agent, the 900ug/ agent is to about 2100ug/ agent, about 900ug/ agent is to about 2000ug/ agent, about 900ug/ agent is to about 1200ug/ agent, about 900ug/ agent to total compound concentration of about 1800ug/ agent and other embodiment with about 1200ug/ agent extremely total compound concentration of about 1800ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296 preparations) in the patient who suffers from HCV who lives through treatment.In other embodiment, a kind of with effective dose, two kinds, three kinds, the combination of four kinds or more kinds of antiviral compound such as ribavirin is with the 600ug/ agent, the 720ug/ agent, the 800ug/ agent, the 900ug/ agent, the 1000ug/ agent, the 1200ug/ agent, the 1500ug/ agent, the 1800ug/ agent, the 2000ug/ agent, total compound concentration of 2100ug/ agent or 2400ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296 preparations) in the patient who suffers from HCV who lives through treatment.
Total compound concentration of albumin fusion proteins and the dosing interval of wherein using albumin fusion proteins will depend on desired effects and the particular treatment albumen used and changing.In one embodiment, weekly, every biweekly, per three weeks once, every around once or more weeks once every dosage be applied to patient's the albumin fusion proteins concentration of total preparation in about 10ug/ agent scope of about 2400ug/ agent extremely.In another embodiment, once in a week, every biweekly, per three weeks once, every around once or more weeks total concentration once in about 100ug/ agent scope of about 1000ug/ agent extremely, or alternatively, weekly, every biweekly, per three weeks once, every around once or more weeks total concentration once for about 1000ug/ agent to about 1200ug/ agent or extremely about 1800ug/ agent of about 900ug/ agent.
In concrete embodiment, every biweekly, per three weeks once, every around once or per five weeks once with about 90ug/ agent to total compound concentration of about 2400ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, prepare by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296).In other embodiment, weekly, every biweekly, per three weeks once, every around once or per five weeks once with about 900ug/ agent total compound concentration of about 2400ug/ agent extremely; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with about 900ug/ agent total compound concentration of about 2100ug/ agent extremely, once in a week, every biweekly, per three weeks once, every around once or per five weeks once with the total compound concentration of about 900ug/ agent to about 2000ug/ agent, weekly, every biweekly, per three weeks once, whenever all around once or per five weeks once with about 900ug/ agent total compound concentration of about 1200ug/ agent extremely; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with about 900ug/ agent total compound concentration of about 1800ug/ agent extremely; In another embodiment, once in a week, every biweekly, per three weeks once, every around once or per five weeks once with about 1200ug/ agent to total compound concentration administration of 1800ug/ agent IFN-α of the present invention-HSA fusion rotein (as, prepare by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296).In other embodiment, weekly, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of about 600ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 800ug/ agent, weekly, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 900ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 1000ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 1200ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 1500ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 1600ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 1800ug/ agent; Once in a week, whenever biweekly, per three weeks once, around every once or per five weeks once with total compound concentration of 2000ug/ agent, once in a week, whenever biweekly, per three weeks once, around every once or per five weeks once with total compound concentration of 2100ug/ agent, or it is weekly, whenever biweekly, per three weeks once, around every once or per five weeks once with total compound concentration of 2400ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, by CID2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296 preparations).In more embodiments, every biweekly with total compound concentration of 900ug/ agent, with further embodiment every biweekly with the total concentration of 1200ug/ agent, every around once with the total concentration of 1200ug/ agent or every around once with the total concentration of 1800ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296 preparations).In other embodiment, separately or with antiviral compound such as ribavirin combined administration always prepare dosage IFN-α of the present invention-HSA fusion rotein (as, by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296 preparations).In other embodiment, with a kind of, two kinds, three kinds, four kinds or more kinds of antiviral compound such as the ribavirin combined administration of effective dose always prepare dosage IFN-α of the present invention-HSA fusion rotein (as, by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296 preparations).
In concrete embodiment, separately or, per three weeks every biweekly with the combination of antiviral compound such as ribavirin once, every around once or per five weeks once with about 90ug/ agent to total compound concentration of about 2400ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, prepare by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) in untreated HCV patient.In other embodiment, separately or and antiviral compound, comprise, per three weeks weekly, every biweekly such as the ribavirin combination once, every around once or per five weeks once with about 900ug/ agent total compound concentration of about 2400ug/ agent extremely; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with about 900ug/ agent total compound concentration of about 2100ug/ agent extremely, once in a week, every biweekly, per three weeks once, every around once or the once about 900ug/ agent of per five weeks to total compound concentration of about 2000ug/ agent, weekly, every biweekly, per three weeks once, whenever all around once or per five weeks once with about 900ug/ agent total compound concentration of about 1200ug/ agent extremely; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with about 900ug/ agent total compound concentration of about 1800ug/ agent extremely; In other embodiments, once in a week, every biweekly, per three weeks once, every around once or per five weeks once with about 1200ug/ agent to total compound concentration of about 1800ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, prepare by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) in untreated HCV patient.In other embodiment, separately or, per three weeks weekly, every biweekly with the combination of antiviral compound such as ribavirin once, every around once or per five weeks once with total compound concentration of about 600ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 800ug/ agent, weekly, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 900ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 1000ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 1200ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 1500ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 1600ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 1800ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 2000ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 2100ug/ agent; Or weekly, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 2400ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296 preparations) in untreated HCV patient.
In concrete embodiment, with effective dose a kind of, the combination of two kinds, three kinds, four kinds or more kinds of antiviral compound such as ribavirin is every biweekly, per three weeks once, every around once or per five Mondays with about 90ug/ agent to total compound concentration of about 2400ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, prepare by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) in untreated HCV patient.In other embodiment, with one or more antiviral compounds, for example comprise, ribavirin combination and a kind of, two kinds, three kinds or more kinds of antiviral compound, for example comprise, ribavirin and optional another kind of antiviral compound combination are weekly, every biweekly, per three weeks once, every around once or per five weeks once with about 900ug/ agent total compound concentration of about 2400ug/ agent extremely; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with about 900ug/ agent total compound concentration of about 2100ug/ agent extremely; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with about 900ug/ agent total compound concentration of about 2000ug/ agent extremely; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with about 900ug/ agent total compound concentration of about 1200ug/ agent extremely; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with about 900ug/ agent total compound concentration of about 1800ug/ agent extremely; With in further embodiment, once in a week, every biweekly, per three weeks once, every around once or per five weeks once with about 1200ug/ agent to total compound concentration of about 1800ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, prepare by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) in untreated HCV patient.In other embodiment, with effective dose a kind of, the combination of two kinds, three kinds, four kinds or more kinds of antiviral compound such as ribavirin is weekly, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of about 600ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 800ug/ agent, weekly, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 900ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 1000ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 1200ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 1500ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 1600ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 1800ug/ agent; Or weekly, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 2000ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 2100ug/ agent; Or weekly, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 2400ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296 preparations) in untreated HCV patient.
In other embodiment, separately or every biweekly with total compound concentration of 900ug/ agent with the combination of antiviral compound such as ribavirin, with in other embodiment, every biweekly with the total concentration of 1200ug/ agent, around every once with the total concentration of 1200ug/ agent, or around every once with the total concentration of 1800ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296 preparations) in untreated HCV patient.In other embodiment, a kind of with effective dose, two kinds, three kinds, the combination of four kinds or more kinds of antiviral compound such as ribavirin is every biweekly with total compound concentration of 900ug/ agent, with in other embodiment, every biweekly with the total concentration of 1200ug/ agent, around every once with the total concentration of 1200ug/ agent, or around every once with the total concentration of 1800ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, by CID2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296 preparations) in untreated HCV patient.
In another embodiment, every biweekly continued at least 24 weeks with total compound concentration of 900ug/ agent, in other embodiments, every biweekly continued at least 24 weeks with the total concentration of 1200ug/ agent, around every once the total concentration with the 1200ug/ agent continued at least 24 weeks, or around every once with the total concentration of 1800ug/ agent continue to use at least 24 weeks IFN-α of the present invention-HSA fusion rotein (as, by CID2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296 preparations) in untreated HCV patient.IFN-α of the present invention-HSA fusion rotein described used to be independent or to make up such as ribavirin with a kind of, two kinds, three kinds, four kinds or more kinds of antiviral compound.In one embodiment, untreated HCV patient infection HCV genotype 2 or 3.In other embodiment, every biweekly continued at least 48 weeks with total compound concentration of 900ug/ agent, in another embodiment, every biweekly continued at least 48 weeks with the total concentration of 1200ug/ agent, around every once the total concentration with the 1200ug/ agent continued at least 48 weeks, or around every once with the total concentration of 1800ug/ agent continue to use at least 48 weeks IFN-α of the present invention-HSA fusion rotein (as, by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296 preparations) in untreated HCV patient.IFN-α of the present invention-HSA fusion rotein described used to be independent or to make up such as ribavirin with a kind of, two kinds, three kinds, four kinds or more kinds of antiviral compound.In one embodiment, untreated HCV patient infection HCV genotype 1.
Foregoing, the quick virusology response (RVR) in the 4th week of treatment has been accredited as the positive indication signal that lasting virusology is replied (SVR).And, up to the further positive indication signal that HCV RNA feminine gender has been set up as SVR of keeping in the 12nd week of treatment.In a word, reaching HCV RNA feminine gender the 4th week and may after finishing therapeutic scheme, keep the HCVRNA feminine gender up to keeping this negative patients the 12nd week.Therefore, need will with IFN-α-HSA separately or with at least a, two kinds, three kinds or four kinds of antiviral agent after such as the ribavirin combined therapy to reach HCV RNA the 4th week negative and keep the HCV patient's of this feminine gender number maximization up to the 12nd week.Therefore in other embodiment, with every biweekly and around every once combination with about 90ug/ agent to total compound concentration of about 2400ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, prepare by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) in untreated HCV patient.In other embodiments, with every biweekly and around every once combination with about 900ug/ agent total compound concentration of about 2400ug/ agent extremely; With every biweekly and around every once combination with about 900ug/ agent total compound concentration of about 2100ug/ agent extremely; With every biweekly and around every once combination with about 900ug/ agent total compound concentration of about 2000ug/ agent extremely; With every biweekly and around every once combination with about 900ug/ agent total compound concentration of about 1200ug/ agent extremely; With every biweekly and around every once combination with about 900ug/ agent total compound concentration of about 1800ug/ agent extremely; With in other embodiment, with every biweekly and around every once combination with about 1200ug/ agent to total compound concentration of about 1800ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, prepare by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) in untreated HCV patient.IFN-α of the present invention-HSA fusion rotein described used to be independent or to make up such as ribavirin with a kind of, two kinds, three kinds, four kinds or more kinds of antiviral compound.
In other embodiment, with every biweekly and around every once combination with total compound concentration of about 600ug/ agent; With every biweekly and around every once combination with total compound concentration of 800ug/ agent, with every biweekly and around every once combination with total compound concentration of 900ug/ agent; With every biweekly and around every once combination with total compound concentration of 1000ug/ agent; With every biweekly and around every once combination with total compound concentration of 1200ug/ agent; With every biweekly and around every once combination with total compound concentration of 1500ug/ agent; With every biweekly and around every once combination with total compound concentration of 1600ug/ agent; With every biweekly and around every once combination with the 1800ug/ agent; With every biweekly and around every once combination with total compound concentration of 2000ug/ agent; With every biweekly and around every once combination with total compound concentration of 2100ug/ agent; Or with every biweekly and around every once combination with total compound concentration of 2400ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296 preparations) in untreated HCV patient.IFN-α of the present invention-HSA fusion rotein described used to be independent or to make up such as ribavirin with a kind of, two kinds, three kinds or four kinds of antiviral compounds.In some embodiments, continued at least 24 weeks with a kind of, two kinds, three kinds, four kinds or more kinds of antiviral compound such as ribavirin combined administration IFN-α of the present invention-HSA fusion rotein in the HCV patient of untreated infection genotype 1, in other embodiments, continued at least 48 weeks.In other embodiment, continued for 24 weeks in the HCV patient of untreated infection genotype 2 or 3 with a kind of, two kinds, three kinds, four kinds or more kinds of antiviral compound such as ribavirin combined administration IFN-α of the present invention-HSA fusion rotein.
In one embodiment, when therapeutic scheme begins every biweekly with about 90ug/ agent extremely total compound concentration of about 2400ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, prepare by CID2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) in untreated HCV patient twice, every in the remaining time of treatment subsequently all around once with IFN-α-HSA fusant treatment.In concrete embodiment, when therapeutic scheme begins every biweekly with about 900ug/ agent extremely total compound concentration of about 2400ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, prepare by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) in untreated HCV patient twice, every in the remaining time of treatment subsequently all around once with IFN-α-HSA fusant treatment.In other embodiment, when therapeutic scheme begins every biweekly with about 900ug/ agent extremely total compound concentration of about 1800ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, prepare by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) in untreated HCV patient twice, every in the remaining time of treatment subsequently all around once with IFN-α-HSA fusant treatment.IFN-α of the present invention-HSA fusion rotein described used to be independent or to make up such as ribavirin with a kind of, two kinds, three kinds, four kinds or more kinds of antiviral compound.In some embodiments, a kind of, two kinds, three kinds, four kinds or more kinds of antiviral compound such as ribavirin combined administration IFN-α of the present invention-HSA fusion rotein continued at least 24 weeks in the HCV patient of untreated infection genotype 1, in one embodiment, continued at least 48 weeks.In other embodiment, continued for 24 weeks in the HCV patient of untreated infection genotype 2 or 3 with a kind of, two kinds, three kinds, four kinds or more kinds of antiviral compound such as ribavirin combined administration IFN-α of the present invention-HSA fusion rotein.
In other embodiment, when therapeutic scheme begins whenever biweekly with about 600ug/ agent, the 800ug/ agent, the 900ug/ agent, the 1000ug/ agent, the 1200ug/ agent, the 1500ug/ agent, the 1600ug/ agent, the 1800ug/ agent, the 2000ug/ agent, total compound concentration of 2100ug/ agent or 2400ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296 preparations) in untreated HCV patient twice, every in the remaining time of treatment subsequently all around once with IFN-α-HSA fusant treatment.IFN-α of the present invention-HSA fusion rotein described used to be independent or to make up such as ribavirin with a kind of, two kinds, three kinds, four kinds or more kinds of antiviral compound.In one embodiment, total compound concentration of IFN-α of the present invention-HSA fusion rotein is 1200ug/ agent or 1800ug/ agent.In other embodiment, continued at least 24 weeks with a kind of, two kinds, three kinds or four kinds of antiviral compound such as ribavirin combined administration IFN-α of the present invention-HSA fusion rotein in the HCV patient of untreated infection genotype 1, in another embodiment, continued at least 48 weeks.In other embodiment, continued for 24 weeks in the HCV patient of untreated infection genotype 2 or 3 with a kind of, two kinds, three kinds, four kinds or more kinds of antiviral compound such as ribavirin combined administration IFN-α of the present invention-HSA fusion rotein.
In the other specific embodiment, separately or, per three weeks every biweekly with the combination of antiviral compound such as ribavirin once, every around once or per five weeks once with about 90ug/ agent to total compound concentration of about 2000ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, prepare by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) in the HCV patient who lives through treatment.In other embodiment, separately or, per three weeks weekly, every biweekly with the combination of antiviral compound such as ribavirin once, every around once or per five weeks once with about 900ug/ agent total compound concentration of about 2400ug/ agent extremely; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with about 900ug/ agent total compound concentration of about 2100ug/ agent extremely; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with about 900ug/ agent total compound concentration of about 2000ug/ agent extremely; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with about 900ug/ agent total compound concentration of about 1200ug/ agent extremely; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with about 900ug/ agent total compound concentration of about 1800ug/ agent extremely; In other embodiments, once in a week, per three weeks once, every around once or per five weeks once or in another embodiment, every biweekly with the total compound concentration of about 1200ug/ agent to about 1800ug/ agent, use IFN-α of the present invention-HSA fusion rotein (as, by CID2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296 the preparation) in the HCV patient who lives through treatment.In other embodiment, separately or, per three weeks weekly, every biweekly with the combination of antiviral compound such as ribavirin once, every around once or per five weeks once with total compound concentration of about 600ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 800ug/ agent, weekly, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 900ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 1000ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 1200ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 1500ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 1600ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 1800ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 2000ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 2100ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 2400ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296 preparations) in the HCV patient who lives through treatment.
In embodiment more specifically, with effective dose a kind of, the combination of two kinds, three kinds, four kinds or more kinds of antiviral compound such as ribavirin is every biweekly, per three weeks once, every around once or per five weeks once with about 90ug/ agent to total compound concentration of about 2000ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, prepare by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) in the HCV patient who lives through treatment.In some embodiments, with effective dose a kind of, the combination of two kinds, three kinds, four kinds or more kinds of antiviral compound such as ribavirin is weekly, every biweekly, per three weeks once, every around once or per five weeks once with about 900ug/ agent total compound concentration of about 2400ug/ agent extremely; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with about 900ug/ agent total compound concentration of about 2100ug/ agent extremely; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with about 900ug/ agent total compound concentration of about 2000ug/ agent extremely; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with about 900ug/ agent total compound concentration of about 1200ug/ agent extremely; Once in a week, every biweekly, per three weeks once, every around once or once about 900ug/ agent of per five weeks total compound concentration of about 1800ug/ agent extremely; In other embodiments, once in a week, per three weeks once, every around once or per five weeks once or every biweekly with about 1200ug/ agent total compound concentration of about 1800ug/ agent extremely at other other embodiments, use IFN-α of the present invention-HSA fusion rotein (as, by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296 the preparation) in the HCV patient who lives through treatment.In other embodiment, with effective dose a kind of, the combination of two kinds, three kinds, four kinds or more kinds of antiviral compound such as ribavirin is weekly, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of about 600ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 800ug/ agent, weekly, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 900ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 1000ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 1200ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 1500ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 1600ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 1800ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 2000ug/ agent; Once in a week, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 2100ug/ agent; Or weekly, every biweekly, per three weeks once, every around once or per five weeks once with total compound concentration of 2400ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296 preparations) in the HCV patient who lives through treatment.
In some embodiments, separately or every biweekly with total compound concentration of 900ug/ agent with the combination of antiviral compound such as ribavirin, in one embodiment, every biweekly with the total concentration of 1200ug/ agent, around every once with the total concentration of 1200ug/ agent, or around every once with the total concentration of 1800ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296 preparations) in the HCV patient who lives through treatment.In other embodiments, a kind of with effective dose, two kinds, three kinds, the combination of four kinds or more kinds of antiviral compound such as ribavirin is every biweekly with total compound concentration of 900ug/ agent, every in another embodiment biweekly with the total concentration of 1200ug/ agent, around every once with the total concentration of 1200ug/ agent, or around every once with the total concentration of 1800ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296 preparations) in the HCV patient who lives through treatment.
In another embodiment, every biweekly continued at least 24 weeks with total compound concentration of 900ug/ agent, in some embodiments, every biweekly continued at least 24 weeks with the total concentration of 1200ug/ agent, around every once the total concentration with the 1200ug/ agent continued at least 24 weeks, or around every once with the total concentration of 1800ug/ agent continue to use at least 24 weeks IFN-α of the present invention-HSA fusion rotein (as, by CID2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296 preparations) in the HCV patient who lives through treatment.IFN-α of the present invention-HSA fusion rotein described used to be independent or to make up such as ribavirin with a kind of, two kinds, three kinds, four kinds or more kinds of antiviral compound.In one embodiment, live through treatment the HCV patient infection HCV genotype 2 or 3.In other embodiment, every biweekly continued at least 48 weeks with total compound concentration of 900ug/ agent, in another embodiment, every biweekly continued at least 48 weeks with the total concentration of 1200ug/ agent, around every once the total concentration with the 1200ug/ agent continued at least 48 weeks, or around every once with the total concentration of 1800ug/ agent continue to use at least 48 weeks IFN-α of the present invention-HSA fusion rotein (as, by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296 preparations) in the HCV patient who lives through treatment.IFN-α of the present invention-HSA fusion rotein described used to be independent or to make up such as ribavirin with a kind of, two kinds, three kinds, four kinds or more kinds of antiviral compound.In one embodiment, live through treatment the HCV patient infection HCV genotype 1.
Foregoing, the quick virusology response (RVR) in the 4th week of treatment has been accredited as the positive indication signal that lasting virusology is replied (SVR).And, up to the further positive indication signal that HCV RNA feminine gender has been set up as SVR of keeping in the 12nd week of treatment.In a word, reaching HCV RNA feminine gender the 4th week and may after finishing therapeutic scheme, keep the HCVRNA feminine gender up to keeping this negative patients the 12nd week.Therefore, need will with IFN-α-HSA separately or with at least a, two kinds, three kinds or four kinds of antiviral agent after such as the ribavirin combined therapy to reach HCV RNA the 4th week negative and keep the HCV patient's of this feminine gender number maximization up to the 12nd week.Therefore in other embodiment, with every biweekly and around every once combination with about 90ug/ agent to total compound concentration of about 2400ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, prepare by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) in the HCV patient who lives through treatment.In some embodiments, with every biweekly and around every once combination with about 900ug/ agent total compound concentration of about 2400ug/ agent extremely; With every biweekly and around every once combination with about 900ug/ agent total compound concentration of about 2100ug/ agent extremely; With every once the about 900ug/ agent of combination total compound concentration of about 2000ug/ agent extremely biweekly and around every; With every once the about 900ug/ agent of combination total compound concentration of about 1200ug/ agent extremely biweekly and around every; With every once the about 900ug/ agent of combination total compound concentration of about 1800ug/ agent extremely biweekly and around every; With in some other embodiments, with every biweekly and around every once combination with about 1200ug/ agent to total compound concentration of about 1800ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, prepare by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) in the HCV patient who lives through treatment.IFN-α of the present invention-HSA fusion rotein described used to be independent or to make up such as ribavirin with a kind of, two kinds, three kinds, four kinds or more kinds of antiviral compound.
In other embodiment, with every biweekly and around every once combination with total compound concentration of about 600ug/ agent; With every biweekly and around every once combination with total compound concentration of 800ug/ agent, with every biweekly and around every once combination with total compound concentration of 900ug/ agent; With every biweekly and around every once combination with total compound concentration of 1000ug/ agent; With every biweekly and around every once combination with total compound concentration of 1200ug/ agent; With every biweekly and around every once combination with total compound concentration of 1500ug/ agent; With every biweekly and around every once combination with total compound concentration of 1600ug/ agent; With every biweekly and around every once combination with total compound concentration of 1800ug/ agent; With every biweekly and around every once combination with total compound concentration of 2000ug/ agent; With every biweekly and around every once combination with total compound concentration of 2100ug/ agent; Or with every biweekly and around every once combination with total compound concentration of 2400ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296 preparations) in the HCV patient who lives through treatment.IFN-α of the present invention-HSA fusion rotein described used to be independent or to make up such as ribavirin with a kind of, two kinds, three kinds or four kinds of antiviral compounds.In some embodiments, continued at least 24 weeks with a kind of, two kinds, three kinds, four kinds or more kinds of antiviral compound such as ribavirin combined administration IFN-α of the present invention-HSA fusion rotein in the HCV patient of the infection genotype 1 that lives through treatment, with in other embodiment, continued at least 48 weeks.In other embodiment, continued for 24 weeks in the HCV patient of the infection genotype 2 that lives through treatment or 3 with a kind of, two kinds, three kinds, four kinds or more kinds of antiviral compound such as ribavirin combined administration IFN-α of the present invention-HSA fusion rotein.
In one embodiment, when therapeutic scheme begins every biweekly with about 90ug/ agent extremely total compound concentration of about 2400ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, prepare by CID2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) in the HCV patient twice who lives through treatment, every in the remaining time of treatment subsequently all around once with IFN-α-HSA fusant treatment.In one embodiment, when therapeutic scheme begins every biweekly with about 900ug/ agent extremely total compound concentration of about 2400ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, prepare by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) in the HCV patient twice who lives through treatment, every in the remaining time of treatment subsequently all around once with IFN-α-HSA fusant treatment.In other embodiment, when therapeutic scheme begins every biweekly with about 900ug/ agent extremely total compound concentration of about 1800ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, prepare by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) in the HCV patient twice who lives through treatment, every in the remaining time of treatment subsequently all around once with IFN-α-HSA fusant treatment.IFN-α of the present invention-HSA fusion rotein described used to be independent or to make up such as ribavirin with a kind of, two kinds, three kinds, four kinds or more kinds of antiviral compound.In other embodiments, continued at least 24 weeks with a kind of, two kinds, three kinds, four kinds or more kinds of antiviral compound such as ribavirin combined administration IFN-α of the present invention-HSA fusion rotein in the HCV patient of the infection genotype 1 that lives through treatment, with in other embodiment, continued at least 48 weeks.In other embodiment, continued for 24 weeks in the HCV patient of the infection genotype 2 that lives through treatment or 3 with a kind of, two kinds, three kinds, four kinds or more kinds of antiviral compound such as ribavirin combined administration IFN-α of the present invention-HSA fusion rotein.
In other embodiment, when therapeutic scheme begins whenever biweekly with about 600ug/ agent, the 800ug/ agent, the 900ug/ agent, the 1000ug/ agent, the 1200ug/ agent, the 1500ug/ agent, the 1600ug/ agent, the 1800ug/ agent, the 2000ug/ agent, total compound concentration of 2100ug/ agent or 2400ug/ agent use IFN-α of the present invention-HSA fusion rotein (as, by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296 preparations) in the HCV patient twice who lives through treatment, every in the remaining time of treatment subsequently all around once with IFN-α-HSA fusant treatment.IFN-α of the present invention-HSA fusion rotein described used to be independent or to make up such as ribavirin with a kind of, two kinds, three kinds, four kinds or more kinds of antiviral compound.In one embodiment, total compound concentration of IFN-α of the present invention-HSA fusion rotein is 1200ug/ agent or 1800ug/ agent.In other embodiment, continued at least 24 weeks with a kind of, two kinds, three kinds or four kinds of antiviral compound such as ribavirin combined administration IFN-α of the present invention-HSA fusion rotein in the HCV patient of the infection genotype 1 that lives through treatment, in another embodiment, continued at least 48 weeks.In other embodiment, continued for 24 weeks in the HCV patient of the infection genotype 2 that lives through treatment or 3 with a kind of, two kinds, three kinds, four kinds or more kinds of antiviral compound such as ribavirin combined administration IFN-α of the present invention-HSA fusion rotein.
Albumin fusion proteins and/or polynucleotide can be oral, in the rectum, parenteral, brain pond, intravaginal, intraperitoneal, (as with powder, ointment, gel, drop or transdermal patch), buccal or use partly with mouth or nasal spray." pharmaceutically acceptable carrier " refers to that any non-toxicity is solid-state, semisolid or liquid filler, diluent, encapsulating material or formulation aid.Term used herein " parenteral " refers to comprise intravenous, intramuscular, intraperitoneal, breastbone is interior, subcutaneous and the method for application of intra-articular injection and infusion.
Albumin fusion proteins of the present invention and/or polynucleotide also are fit to use with slow-released system.The albumin fusion proteins of slow release and/or the example of polynucleotide with in oral, rectum, parenteral, the brain pond, intravaginal, intraperitoneal, (as with powder, ointment, gel, drop or transdermal patch), buccal or use partly with mouth or nasal spray." pharmaceutically acceptable carrier " refers to that the non-toxicity of any kind is solid-state, semisolid or liquid filler, diluent, encapsulating material or formulation aid.Term used herein " parenteral " refers to comprise intravenous, intramuscular, intraperitoneal, breastbone is interior, subcutaneous and the method for application of intra-articular injection and infusion.Other example of the albumin fusion proteins of slow release and/or polynucleotide comprises suitable polymeric material (such as for example, with the semipermeable polymers substrate of formed article form as, film or microcapsule), suitable hydrophobic material (emulsion in for example acceptable oil) or ion exchange resin and sl. sol. derivant (such as for example, sl. sol. salt).
The substrate of slow release comprises polyactide (United States Patent (USP) the 3rd, 773, No. 919, EP 58,481), the copolymer (Sidman etc. of L-glutamic acid and γ-ethyl-L-glutamate, Biopolymers 22:547-556 (1983)), poly-(2-hydroxyethyl methacrylate) (Langer etc., J.Biomed.Mater.Res.15:167-277 (1981) and Langer, Chem.Tech.12:98-105 (1982)), ethylene vinyl acetate (ethylenevinyl acetate) (Langer etc., the same) or poly--D-(-)-3-hydroxybutyric acid (EP 133,988).
Albumin fusion proteins of the present invention and/or polynucleotide that the albumin fusion proteins of slow release and/or polynucleotide also comprise liposome (see usually Langer, Science 249:1527-1533 (1990); Treat etc., Liposomes in the Therapy of Infectious Disease and Cancer (liposome in treatment infectious disease and the cancer), Lopez-Berestein, Lopez-Berestein and Fidler (volume), Liss, New York, pp.317-327 and 353-365 (1989)).The liposome that is contained albumin fusion proteins and/or polynucleotide by known method preparation itself: DE 3,218, and 121; Epstein etc., Proc.Natl.Acad.Sci. (USA) 82:3688-3692 (1985); Hwang etc., Proc.Natl.Acad.Sci (USA) 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese patent application 83-118008 number; United States Patent (USP) the 4th, 485,045 and 4,544, No. 545; With EP 102,324.Usually, liposome is little (about 200-800 dust) single-layer type, and wherein lipid content is greater than about 30 molar percentage cholesterol, for optimal treatment is adjusted selected ratio.
In another other embodiment, send albumin fusion proteins of the present invention and/or polynucleotide (see above-mentioned Langer with pump; Sefton, CRC Crit.Ref.Biomed.Eng.14:201 (1987); Buchwald etc., Surgery 88:507 (1980); Saudek etc., N.Engl.J.Med.321:574 (1989)).
Other controlled release system is discussed at the summary (Science 249:1527-1533 (1990)) of Langer.
For parenteral administration, in one embodiment, albumin fusion proteins and/or polynucleotide are generally prepared by mixing with pharmaceutically acceptable carrier with its purity with expected degree, with unit dose injection form (solution, suspension or emulsion), pharmaceutically acceptable carrier promptly the dosage that adopts and concentration to the non-toxicity of receiver and with the compatible carrier of other composition of preparation.For example, preparation does not comprise known to treating deleterious oxidant and other chemical compound.
Usually by evenly and closely being contacted, albumin fusion proteins and/or polynucleotide and liquid-carrier or meticulous solid-state carrier or both prepare preparation.If necessary subsequently, make product be shaped to the preparation of expectation.Exemplary carrier be the parenteral carrier or with the isoosmotic solution of receiver's blood.The example of this carrier inert matter comprises water, saline, Ringer's mixture and dextrose solution.Also can use non-moisture inert matter such as fixing oil and ethyl oleate and liposome.
Carrier contains minor amounts of additives aptly, such as the material that strengthens isotonicity and chemical stability.This material is toxic to receiver's right and wrong with the dosage and the concentration that are adopted, and comprises buffer agent such as phosphate, citrate, succinate, acetic acid and other organic acid or its salt; Antioxidant is such as ascorbic acid; Low-molecular-weight (being less than about ten residues) polypeptide, as, poly arginine or tripeptides; Albumen is such as serum albumin, gelatin or immunoglobulin; Hydrophilic polymer is such as polyvinylpyrrolidone; Aminoacid is such as glycine, glutamic acid, aspartic acid or arginine; Monosaccharide, disaccharide and other saccharide comprise cellulose or derivatives thereof, glucose, mannose (manose) or dextrin; Chelating agen is such as EDTA; Sugar alcohol such as mannitol or sorbitol; Counter ion counterionsl gegenions are such as sodium; And/or non-ionic surface active agent such as polysorbate (for example comprises, Tween-20), poloxamer or PEG.
Albumin fusion proteins usually in this inert matter with about 0.1mg/ml to 100mg/ml for example 1-10mg/ml concentration, with about 3 to 8 pH preparation.Should understand and use more aforementioned excipient, carrier or stabilizing agent will cause forming polypeptide salt.
Being used for the treatment of any medicine of using can be aseptic.Filter and to be easy to realize aseptic by aseptic filter membrane (as, 0.2 micron membranes).Albumin fusion proteins and/or polynucleotide generally place the container with aseptic inlet port, and for example, having can be by the intravenous solution bag or the bottle of the saturating plug of hypodermic injection needle thorn.
Albumin fusion proteins and/or polynucleotide are the unit of being stored in or many-dose container for example in sealed ampoule or the bottle, as aqueous solution or as the reconstitutable lyophilizate preparation usually.As the example of lyophilized formulations, to 1% (w/v) albumin fusion proteins and/or the polynucleotide aqueous solution of 10-ml bottle filling 5ml aseptic filtration, and the mixture of lyophilizing gained.By using freeze dried albumin fusion proteins of water for injection,bacteriostatic reconstruct and/or polynucleotide to prepare infusion solution.
In concrete embodiment, the albumin fusion proteins preparation comprises 0.01M sodium phosphate, 0.15mM sodium chloride, 0.16 micromole sodium caprylate/milligram fusion rotein, 15 mcg/ml polysorbate80s, and pH 7.2.In another embodiment, the albumin fusion proteins preparation is by 0.01M sodium phosphate, 0.15mM sodium chloride, 0.16 micromole sodium caprylate/milligram fusion rotein, 15 mcg/ml polysorbate80s, and pH 7.2 forms.Select pH and buffer with the coupling physiological condition, and add salt as tension regulator (tonicifier).Select sodium caprylate, can increase the heat stability of albumen in solution owing to report it.The final polysorbate that adds is as general surfactant, and it reduces the surface tension of solution and reduces the non--specific adsorption of albumin fusion proteins to container closure systems.
The present invention also provides the pharmaceutical pack or the cover box of the one or more containers that comprise the composition that is filled with one or more albumin fusion proteins of the present invention and/or polynucleotide.This container can attach the notice with the form of government organs' regulation of manufacturing, use or the sale of management medicine or biological product, and the mechanism that this notice reflection management is made, used or sells is to the approval of human administration.In addition, albumin fusion proteins and/or polynucleotide can be used in combination with other therapeutic compound.
Albumin fusion proteins of the present invention and/or polynucleotide can be separately or with the adjuvant combined administration.Can include but not limited to the adjuvant that albumin fusion proteins of the present invention and/or polynucleotide are used, Alumen, Alumen add dexycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21 (Genentech, Inc.), BCG (as,
), the deactivation goods of MPL and coryne bacterium parvum (Corynebacterium parvum).In concrete embodiment, albumin fusion proteins of the present invention and/or polynucleotide and Alumen combined administration.In another embodiment, albumin fusion proteins of the present invention and/or polynucleotide and QS-21 combined administration.Can include but not limited to monophosphoryl lipid matter immunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005, aluminum salt, MF-59 and virion adjuvant technology with the further adjuvant that albumin fusion proteins of the present invention and/or polynucleotide are used.Can include but not limited to the vaccine that albumin fusion proteins of the present invention and/or polynucleotide are used, be used to prevent the vaccine of MMR (measles,mumps,rubella), poliomyelitis (polio), chickenpox, tetanus/diphtheria (diphtheria), hepatitis A, hepatitis B, Type B hemophilus influenza, pertussis (whooping cough), pneumonia, influenza, Lyme disease, rotavirus, cholera, yellow fever, Japanese encephalitis, poliomyelitis (poliomyelitis), rabies, typhoid fever and pertussis (pertussis).Can be together, as, as mixture, respectively but (simultaneously) or (concurrently) simultaneously concurrently; Or sequentially use combination.The scheme that this medicament that comprises wherein combination is used together as the treatment mixture, the medicament that also comprises wherein combination respectively but use simultaneously, as, the scheme that enters same individuality via different intravenous pipes." combination " used further to comprise and used one of the chemical compound given or medicament at first separately, uses second kind subsequently.
Albumin fusion proteins of the present invention and/or polynucleotide can be separately or with other therapeutic agent combined administration.Can include but not limited to chemotherapeutics, antibiotic, steroidal and non-steroidal anti-inflammatory agent, routine immunization therapeutic agent and/or following therapeutic treatment with the albumin fusion proteins and/or the polynucleotide medicament of albumin fusion proteins of the present invention and/or polynucleotide combined administration.Can be together, as, as mixture, respectively but simultaneously or concurrently (concurrently); Or sequentially use combination.The scheme that this medicament that comprises wherein combination is used together as the treatment mixture, the medicament that also comprises wherein combination respectively but use simultaneously, as, the scheme that enters same individuality via different intravenous pipes." combination " used further to comprise and used one of the chemical compound given or medicament at first separately, uses second kind subsequently.
In one embodiment, albumin fusion proteins of the present invention and/or polynucleotide and anticoagulant combined administration.Can include but not limited to the anticoagulant that compositions of the present invention is used, heparin, low molecular weight heparin, warfarin sodium (as,
), dicoumarol, 4 hydroxy coumarin, anisindione be (as, MIRADON
TM), the acenocoumarol class (as, acenocoumarol, SINTHROME
TM), dihydroindene-1,3-diketone, phenprocoumon are (as, MARCUMAR
TM), ethylbisoumacetate is (as, TROMEXAN
TM) and aspirin.In concrete embodiment, compositions of the present invention and heparin and/or warfarin combined administration.In another embodiment, compositions of the present invention and warfarin combined administration.In another embodiment, compositions of the present invention and warfarin and aspirin combined administration.In another embodiment, compositions of the present invention and heparin combined administration.In another embodiment, compositions of the present invention and heparin and aspirin combined administration.
In another embodiment, albumin fusion proteins of the present invention and/or polynucleotide and thrombolytic drug combined administration.Can include but not limited to the thrombolytic drug that compositions of the present invention is used, plasminogen, lys-plasminogen, α 2-antiplasmin, streptokinase (streptokinae) (as, KABIKINASE
TM), antiresplace is (as, EMESfASE
TM), tissue plasminogen activator (t-PA, altevase, ACTIVASE
TM), urokinase is (as, ABBOKINASE
TM), Saruplase (sauruplase), (prourokinase, single chain urokinase type plasminogen activator) and aminocaproic acid be (as, AMICAR
TM).In concrete embodiment, compositions of the present invention and tissue plasminogen activator and aspirin combined administration.
In another embodiment, albumin fusion proteins of the present invention and/or polynucleotide and antiplatelet drug combined administration.Can include but not limited to the antiplatelet drug that compositions of the present invention is used, aspirin, dipyridamole (as, PERSANTINE
TM) and ticlopidine (as, TICLID
TM).
In concrete embodiment, expection is used in combination anticoagulant, thromboembolism and/or antiplatelet drug with prevention, diagnosis and/or treatment thrombosis, artery thrombosis, venous thrombosis, thromboembolism, pulmonary infarction, arteriosclerosis, myocardial infarction, transient ischemic attack, unstable angina with albumin fusion proteins of the present invention and/or polynucleotide.In concrete embodiment, expection is used in combination anticoagulant, thrombolytic drug and/or antiplatelet drug with albumin fusion proteins of the present invention and/or polynucleotide and blocks (occulsion) with the prevention saphenous vein graft, with reduce angioplasty may with the thrombotic risk of peri-operation period (periprocedural), suffers from the risk of the atrial fibrillation patients apoplexy that comprises non-rheumatic atrial fibrillation with reduction, with reduction and artificial heart valve and or the relevant thromboembolism risk of mitral valve disease.Therapeutic agent of the present invention includes but not limited to separately or with other therapeutic use of anti-platelet agents, anticoagulant and/or thrombolytic drug combination, prevent health install outward (as, the vascular access diverter of blood vessel interpolation pipe, hemodialysis patients, haemodialysis control unit and cardiovascular shunt machine) obstruction.
In some embodiments, albumin fusion proteins of the present invention and/or polynucleotide and antiretroviral agent, nucleoside/nucleotide reverse transcriptase inhibitors (NRTI), non-nucleoside reverse transcriptase inhibitors (NNRTI) and/or protease inhibitor (PI) combined administration.Can include but not limited to RETROVIR with the NRTI of albumin fusion proteins of the present invention and/or polynucleotide combined administration
TM(zidovudine/AZT), VIDEX
TM(Didanosine/ddI), HIVID
TM(zalcitabine/ddC), ZERTT
TM(stavudine/d4T), EPIVIR
TM(lamivudine/3TC) and COMBIVTR
TM(zidovudine/lamivudine).Can include but not limited to VIRAMUNE with the NNRTI of albumin fusion proteins of the present invention and/or polynucleotide combined administration
TM(nevirapine), RESCRIPTOR
TM(delavirdine) and SUSTIVA
TM(efavirenz).Can include but not limited to CRIXIVAN with the protease inhibitor of albumin fusion proteins of the present invention and/or polynucleotide combined administration
TM(indinavir), NORVIR
TM(ritonavir), INVIRASE
TM(Saquinavir) and VTRACEPT
TM(nelfinavir).In concrete embodiment, antiretroviral agent, nucleoside reverse transcriptase inhibitor, non-nucleoside reverse transcriptase inhibitors and/or protease inhibitor can use with treatment AIDS and/or prevention or treatment HIV infection with albumin fusion proteins of the present invention and/or polynucleotide in any combination.
Other NRTI comprises lodenosine (LODENOSINE
TM) (F-ddA; The adenosine NRTI that a kind of acid is stable; Triangle/Abbott; COVIRACIL
TM(emtricitabine/FTC; Relevant with lamivudine (3TC) but have 3 to 10 times of big external activities on the structure; Triangle/Abbott); DOTC (BCH-10652, also relevant on the structure with lamivudine, but the lamivudine resistance isolates of general proportions is kept active; Biochem Pharma); (approval of FDA refusal is used for the anti-HIV treatment to adefovirdipivoxil; Gilead Sciences);
(adefovir dipivoxil, the active prodrug of adefovirdipivoxil; Its activity form is PMEA-pp); Tenofovir (TENOFOVIR
TM) (two-POC PMPA, the PMPA prodrug; Gilead); DAPD/DXG (the active metabolite of DAPD; Triangle/Abbott); D-D4FC (relevant with 3TC, that AZT/3TC-resistance virus is had activity); GW420867X (Glaxo Wellcome); ZIAGEN
TM(Abacavir/159U89; Glaxo Wellcome Inc.); CS-87 (3 ' azido-2 ', 3 '-two BrdUs; WO 99/66936); And the prodrug forms (WO 98/17281) that has S-acyl group-2-sulfur ethyl (SATE) of β-L-FD4C and β-L-FddC.
Other NNRTI comprises COACTINON
TM(emivirine/MKC-442, the potent NNRTI of HEPT class; Triangle/Abbott); Capravirine (CAPRAVIRINE
TM) (AG-1549/S-1153, NNRTI of future generation have activity to the virus that contains the K103N sudden change; Agouron); (activity is 20 to 50 times of its predecessor delavirdine and the K103N mutant is had activity PNU-142721; Pharmacia﹠amp; Upjohn); DPC-961 and DPC-963 (second filial generation derivant of efavirenz, being designed to has activity to the virus with K103N sudden change; DuPont); (specific activity HBY097 is big 25 times and the K103N mutant had activity for GW-420867X; Glaxo Wellcome); CALANOLIDE A is (from the medicament of the natural generation of rubber tree; To containing Y181C sudden change or K103N sudden change or both virus activity is arranged); And propolis (WO 99/49830).
Other protease inhibitor comprises LOPINAVTR
TM(ABT378/r; Abbott Laboratories); BMS-232632 (azepine peptide; Bristol-Myres Squibb); Tipranavir (TIPRANAVIR
TM) (PNU-140690, non-peptide class dihydro pyrone; Pharmacia ﹠amp; Upjohn); PD-178390 (non-peptide class dihydro pyrone; Parke-Davis); BMS 232632 (azepine peptide; Bristol-Myers Squibb); L-756,423 (indinavir analog; Merck); DMP-450 (ring-type carbamide compound; Avid ﹠amp; DuPont); AG-1776 (intends peptide, protease inhibitor resistance virus is had external activity; Agouron); VX-175/GW-433908 (the phosphate ester prodrug of amprenavir; Vertex ﹠amp; Glaxo Welcome); CGP61755 (Ciba) and AGENERASE
TM(amprenavir; Glaxo Wellcome Inc.).
Other antiretroviral agent comprises fusion inhibitor/gp41 bonding agent.Fusion inhibitor/gp41 bonding agent comprises T-20, and (from the peptide of the residue 643-678 in HIV gp41 transmembrane protein external structure territory, it is in conjunction with the gp41 of resting state and prevent to be converted into the fusion attitude; Trimeris) and T-1249 (second filial generation fusion inhibitor; Trimeris).
Other antiretroviral agent comprises fusion inhibitor/chemokine receptor anagonists.Fusion inhibitor/chemokine receptor anagonists comprises the CXCR4 antagonist, such as AMD 3100 (a kind of bicyclam), SDF-1 and its analog, and ALX40-4C (a kind of cationic peptide), T22 (18 amino acid whose peptides; Trimeris) and T22 analog T134 and T140; The CCR5 antagonist is such as RANTES (9-68), AOP-RANTES, NNY-RANTES and TAK-779; With the CCR5/CXCR4 antagonist, such as NSC 651016 (distamycin analog).Also comprise CCR2B, CCR3 and CCR6 antagonist.Chemokine receptor agonists such as RANTES, SDF-1, MIP-1 α, MIP-1 β etc. also can suppress to merge.
Other antiretroviral agent comprises integrase inhibitor.Integrase inhibitor comprises two caffeoyl quinine (DFQA) acid; L-chicoric acid (a kind of two caffeoyl winestone (DCTA) acid); Alizarin bordeaux (QLC) and relevant anthraquinone; ZINTEVIR
TM(AR 177, act on a kind of oligonucleotide of cell surface rather than real integrase inhibitor probably; Arondex); With aphthols such as being disclosed in those of WO 98/50347.
Other antiretroviral agent comprises that hydroxyurea sample chemical compound is such as BCX-34 (a kind of purine nucleoside phosphorylase inhibitor; Biocryst); The ribonucleotide reductase inhibitor is such as DIDOX
TM(Molecules for Health); Inosine list phosphate dehydrogenase (IMPDH) inhibitor is such as VX-497 (Vertex); And mycophenolic acid (mycopholic acid) is such as CellCept (mycophenolic acid acid ethyl ester; Roche).
Other antiretroviral agent comprises that viral integrase enzyme inhibitor, viral genome nuclear transposition inhibitor are such as two (methyl ketone) chemical compounds of arlydene; The solvable complex and the AMD-3100 of the inhibitor that HIV enters such as AOP-RANTES, NNY-RANTES, RANTES-IgG fusion rotein, RANTES and glycosaminoglycans (GAG); Nucleocapsid zinc refers to that inhibitor is such as the dithiane chemical compound; The target of HIV Tat and Rev; With medicine reinforcing agent (pharmacoenhancer) such as ABT-378.
Other antiretroviral therapy and complementary therapy comprise cytokine and lymphokine such as MIP-1 α, MIP-1 β, SDF-1 α, IL-2, PROLEUKIN
TM(aldesleukin/L2-7001; Chiron), IL-4, IL-10, IL-12 and IL-13; Interferon is such as IFN-α 2a, IFN-α 2b or IFN-β; The antagonist of TNF, NFKB, GM-CSF, M-CSF and IL-10; Regulate medicament such as the ciclosporin and the prednisone of immune activation; Vaccine is such as Remune
TM(HIV Immunogen), APL 400-003 (Apollon), reorganization gp120 and fragment, bivalence (B/E) recombinant envelope glycoprotein, rgp120CM235, MN rgp120, SF-2rgp120, the solvable CD4 complex of gp120/, Δ JR-FL albumen, derived from the branch of discontinuous gp120C3/C4 domain synthetic peptide, the immunogen that can merge and Gag, Pol, Nef and Tat vaccine; Based on the therapy of gene such as hereditary straining element (GSE; WO 98/54366) and cell in chemotactic factor (intrakine) (targeting ER is with CC chemotactic factor (Yang etc., the PNAS 94:11567-72 (1997) of the genetic modification of the surface expression that hinders new synthetic CCR5; Chen etc., Nat.Med.3:1110-16 (1997)); Antibody such as anti--CXCR4 antibody 12G5, anti--CCR5 antibody 2D7,5C7, PA8, PA9, PA10, PA11, PA12 and PA14, anti-CD 4 antibodies Q4120 and RPA-T4, anti--CCR3 antibody 7B11, anti--gp120 antibody 17b, 48d, 447-52D, 257-D, 268-D and 50.1, anti--Tat antibody, anti-TNF-Alpha antibodies and monoclonal antibody 33A; Aromatic hydrocarbons (AH) receptor stimulating agent and antagonist such as TCDD, 3,3 ', 4,4 ', 5-pentachlorodiphenyl, 3,3 ', 4,4 '-tetrachloro biphenyl and α-naphthoflavene (WO 98/30213); With antioxidant such as γ-L-glutamyl-L-ethycysteine (γ-GCE; WO 99/56764).
In other embodiment, albumin fusion proteins of the present invention and/or polynucleotide and one or more antiviral agent combined administrations.In other embodiment, albumin fusion proteins of the present invention and/or polynucleotide and two or more antiviral agent combined administrations.In other embodiment, albumin fusion proteins of the present invention and/or polynucleotide and three kinds or more kinds of antiviral agent combined administration.
In one embodiment, can include but not limited to the micromolecular inhibitor of viral enzyme, the micromolecular inhibitor of RNA polymerase, antiviral agent, antisense oligonucleotide inhibitor, thiazolide, antiviral antibody, novel immunomodulator, cyclophilin inhibitor, liver protectant and interferon enhancer with the antiviral agent that albumin fusion proteins of the present invention and/or polynucleotide are used based on nucleic acid.In concrete embodiment, can include but not limited to acyclovir, ribavirin, ribavirin analog, amantadine, remantidine, Maxamine or thymalfasin with the antiviral agent that albumin fusion proteins of the present invention and/or polynucleotide are used.Particularly, the interferon albumin fusion proteins can with any of these medicament combined administration.And the interferon-ALPHA albumin fusion proteins also can be used with any of these medicament, and in one embodiment, interferon-ALPHA 2a or 2b albumin fusion proteins can be used with any of these medicament.In addition, the interferon beta albumin fusion proteins also can be used with any of these medicament.In addition, any IFN heterozygosis albumin fusion proteins can with any of these medicament combined administration.
In one embodiment, interferon albumin fusion proteins of the present invention and ribavirin or ribavirin analog combined administration.In another embodiment, can include but not limited to the ribavirin or the ribavirin analog of interferon albumin fusion proteins combined administration
(Hoffman-LaRoche, Nutley, N.J.),
(Schering Corp., Kenilworth, NJ.), virazole
(Valeant, Costa Mesa, CA), RIBAVIN
TM(Lupin, Baltimore, MD), RIBAZID
TM(EpIa, Kirachi, Pakistan), Tribavirin, VIRAMIDINE
TM(Valeant, Costa Mesa, CA) and RIBASPHERE
TM(Three Rivers Pharmaceuticals, Cranberry Township, PA).In other embodiment, interferon-ALPHA albumin fusion proteins and ribavirin or ribavirin analog combined administration.In other embodiment, interferon-ALPHA 2a albumin fusion proteins and ribavirin or ribavirin analog combined administration.In other embodiment, interferon alpha 2 b albumin fusion proteins and ribavirin or ribavirin analog combined administration.In other embodiment, interferon beta albumin fusion proteins and ribavirin or ribavirin analog combined administration.In other embodiment, hybrid interferon albumin fusion proteins and ribavirin or ribavirin analog combined administration.In other embodiment, interferon albumin fusion proteins of the present invention and ribavirin or ribavirin analog and one or more other antiviral agent combined administrations.In other embodiment, include but not limited to the micromolecular inhibitor of viral enzyme, the micromolecular inhibitor of RNA polymerase, antiviral agent, antisense oligonucleotide inhibitor, thiazolide, antiviral antibody, novel immunomodulator, cyclophilin inhibitor, liver protectant and interferon enhancer with other antiviral agent of interferon albumin fusion proteins of the present invention and ribavirin or ribavirin analog combined administration based on nucleic acid.
In other embodiment, can be separately or with one or more antiviral agent combined administrations albumin fusion proteins of the present invention and/or polynucleotide with the treatment viral infection.In one embodiment, interferon-albumin fusion proteins of the present invention can with one or more antiviral agent combined administrations.In other embodiment, this viral infection is from hepatites virus infections.In another embodiment, hepatitis virus is hepatitis C virus (HCV).Can include but not limited to the micromolecular inhibitor of viral enzyme, the micromolecular inhibitor of RNA polymerase, antiviral agent, antisense oligonucleotide inhibitor, thiazolide, antiviral antibody, novel immunomodulator, cyclophilin inhibitor, liver protectant and interferon enhancer with the antiviral agent that albumin fusion proteins of the present invention and/or polynucleotide are used based on nucleic acid.
Can include but not limited to the antiviral enzyme inhibitor of albumin fusion proteins of the present invention and/or polynucleotide combined administration, VX-950 (protease inhibitor, Vertex, Cambridge, MA), VX-497 (U.S. mooring basin cloth (merimepodib), oral IMPDH inhibitor, Vertex, Cambridge, MA), BILB1941 (protease inhibitor, Boehringer Ingelheim, Germany), SCH7 (protease inhibitor, Schering Corp., Kenilworth, N.J.), MX-3253 (celgosivir, alpha-glucosidase inhibitor, Migenix, Vancouver, BC), IDN-6556 (caspase inhibitor, Pfizer, New York, NY), UT231B (glucosidase inhibitor, United Therapeutics, Silver Spring, MD), R1626 (hiv protease inhibitor, F.Hoffman-La Roche, Switzerland), ITMN-B (ITMN-191, protease inhibitor, InterMune, Brisbane, CA), SCH 503034 (protease inhibitor, Schering Corp., Kenilworth, NJ.), ACH 806 (GS9132, the oral protein enzyme inhibitor, Achillion, New Haven, CT/Gilead Sciences, Foster City, CA).
Can be nucleoside analog or non-nucleoside inhibitor (NNI) with the antiviral AG14361 of albumin fusion proteins of the present invention and/or polynucleotide combined administration.In one embodiment, the antiviral AG14361 suppresses the HCV RNA polymerase.In one embodiment, the antiviral AG14361 can be nucleoside analog, includes but not limited to, NM283 (23 '-oral prodrugs of C-methyl-cytidine, Idenix, Cambride, MA), R1626 (F.Hoffman-La Roche, Switzerland) and 2 '-the C-methyl nucleoside.In another embodiment, the antiviral AG14361 can be the non-nucleoside inhibitor, include but not limited to, JTK-103, JTK-003 and JTK-109 (Japan Tabacco, Tokyo, Japan), R803 (Rigel, South San Francisco, CA), HCV-371, HCV-086 and HCV-796 (ViroPharm, Exton, PA/Wyeth, Madison, NJ), XTL-2125 (BC2125, XTLbio, New York, NY), A-831 (Arrow Therapeutics, London, United Kingdom).Can include but not limited to MK-0608 (Merck, Whitehouse Station with other antiviral AG14361 of albumin fusion proteins of the present invention and/or polynucleotide combined administration; NJ), NS5a (oral non-nucleoside AG14361, Genelabs, Redwood City, CA), NS5b (oral nucleoside polymerase inhibitor, Genelabs, Redwood City, CA) and ANA025-1 (AG14361, AnadysPharmaceuticals, San Diego, CA).
Can include but not limited to antisense oligonucleotide, ribozyme and siRNA or short hairpin RNA (shRNA) with the medicament based on antiviral nucleic acid of albumin fusion proteins of the present invention and/or polynucleotide combined administration.Can comprise with the antiviral antisense oligonucleotide inhibitor of albumin fusion proteins of the present invention and/or polynucleotide combined administration but be not limited to,
AVI-4065 (AVIBiopharma, Portland, OR).
In another embodiment, can with albumin fusion proteins of the present invention and/or polynucleotide combined administration thiazolide.In one embodiment, can include but not limited to the thiazolide of albumin fusion proteins of the present invention and/or polynucleotide combined administration
(nitazoxanide, RomarkLaboratories, L.C., Tampa, FL).
Can include but not limited to the antiviral immunity regulator of albumin fusion proteins of the present invention and/or polynucleotide combined administration,
(thymosin, thymalfasin, SciClonePharmaceuticals Int ' l, Hong Kong) and toll-sample receptor (TLR) agonist, include but not limited to, ANA245 (TLR-7 agonist, Anadys Pharmaceuticals, San Diego, CA), the ANA975 (oral prodrugs of ANA245, Anadys Pharmaceuticals, San Diego, CA) and CPG-10101 (ACTILON
TM, the TLR-9 agonist, Coley Pharmaceutical Group, Wellesley, MA).Can comprise with the cyclophilin inhibitor of albumin fusion proteins of the present invention and/or polynucleotide combined administration but be not limited to, and Debio-025 (Debiopharm Group, Lausanne, Switzerland).Can include but not limited to the liver protectant of albumin fusion proteins of the present invention and/or polynucleotide combined administration, and NOV-205 (Novelos Therapeutics, Newton, MA).
Can include but not limited to the interferon enhancer of albumin fusion proteins of the present invention and/or polynucleotide combined administration EMZ702 (Transition Therapeutics, Toronto, Ontario).And, can include but not limited to Tarvacin (Ba Wei former times monoclonal antibody (bavituximab) with the antiviral antibody of albumin fusion proteins of the present invention and/or polynucleotide combined administration, a kind of Humanized monoclonal antibodies of Phosphatidylserine of target tumor endothelial cell surface, Peregrine Pharmaceuticals, Inc., Tustin, CA).
In one embodiment, can be separately or with the albumin fusion proteins of one or more antiviral agent combined administrations that the present invention includes are interferon-albumin fusion proteins.In other embodiment, the interferon of interferon-albumin fusion proteins partly is an interferon-ALPHA.The non-limitative example of the interferon-ALPHA that the present invention includes includes but not limited to, is disclosed in the interferon-ALPHA albumen of the treatment albumen row of table 1.In concrete embodiment, interferon-ALPHA part is formed or comprised alternatively by following: Intederon Alpha-2a, Interferon Alpha-2b, interferon c, Interferon Alfacon-1, interferon alfacon-1, interferon alfa-n1, Alferon N, interferon-ALPHA any commercial obtains form such as for example
A (Schering Corp., Kenil worth, NJ.),
A (Hoffman-La Roche, Nutley, N.J.), the Berofor interferon-ALPHA (Boehringer Ingelheim Pharmaceutical, Inc., Ridgefied, Conn.), OMNIFERON
TM(Viragen, Inc., Plantation, FL), MULTIFERON
TM(Viragen, Inc., Plantation, FL)
(GlaxoSmithKline, London, Great Britian),
(Amgen, Inc., Thousands Oaks, CA),
(Sumitomo, Japan),
(Nautilus Biotech, France), MAXY-ALPHA
TM(Maxygen, Redwood City, CA/Hoffman-La Roche, Nutley, N.J.) or the interferon-ALPHA product of any purification or its fragment.In other embodiment, the interferon-ALPHA of IFN-α-HSA fusion rotein part is formed or is comprised alternatively by following: for prolonging the interferon-ALPHA that release or controlled release are modified or prepared.For example, interferon-ALPHA part is formed or is comprised alternatively by following: commercially available prolongation release or controlled release interferon-ALPHA, include but not limited to interferon-' alpha '-XL (Flamel Technologies, France) and LOCTERON
TM(BioLexTherapeutics/OctoPlus, Pittsboro, NC).In other embodiment, the interferon-ALPHA of IFN-α-HSA fusion rotein part can be modified by connecting chemical part.For example, the interferon-ALPHA part can be modified by Pegylation.Therefore in other embodiment, the interferon-ALPHA of IFN-α-HSA fusion rotein part is formed or is comprised alternatively by following: the Pegylation form of Intederon Alpha-2a, 2b or Interferon Alfacon-1 and include but not limited to commercially available pegylated interferon alfa, such as for example
(Schering Corp., Kenilworth, N.J.),
(Hoffman-La Roche, Nutley, N.J.), PEG-OMNIFERON
TM(Viragen, Inc., Plantation, FL) or its fragment.In other embodiment, the interferon of albumin fusion proteins partly is interferon-ALPHA 2a or 2b interferon, the interferon albumin fusion proteins can with any of these medicament combined administration.And in another embodiment, the interferon of interferon-albumin fusion proteins partly is interferon beta or interferon heterozygote.In other embodiment, the interferon that does not the merge part of interferon-albumin fusion proteins can be used in combination separately or with one or more antiviral agent that the present invention includes.
In other embodiments, albumin fusion proteins of the present invention and/or polynucleotide can with anti--opportunistic infection agent combined administration.Can include but not limited to TRIMETHOPRIM-SULFAMETHOXAZOLE with the anti--opportunistic agent of albumin fusion proteins of the present invention and/or polynucleotide combined administration
TM, DAPSONE
TM, PENTAMIDINE
TM, ATOVAQUONE
TM, ISONIAZID
TM, RIFAMPIN
TM, PYRAZINAMIDE
TM, ETHAMBUTOL
TM, RIFABUTIN
TM, CLARITHROMYCIN
TM, AZITHROMYCIN
TM, GANCICLOVIR
TM, FOSCARNET
TM, CIDOFOVIR
TM, FLUCONAZOLE
TM, ITRACONAZOLE
TM, KETOCONAZOLE
TM, ACYCLOVIR
TM, FAMCICOLVIR
TM, PYRIMETHAMINE
TM, LEUCOVORIN
TM, NEUPOGEN
TM(filgrastim/G-CSF) and LEUKINE
TM(Sargramostim/GM-CSF).In concrete embodiment, albumin fusion proteins of the present invention and/or polynucleotide with TRIMETHOPRIM-SULFAMETHOXAZOLE
TM, DAPSONE
TM, PENTAMIDINE
TMAnd/or ATOVAQUONE
TMAnyly be used in combination with the treatment of pre-defense sector or prevent opportunistic Pneumocystis carinii (Pneumocystis carinii) pneumonia.In another embodiment, albumin fusion proteins of the present invention and/or polynucleotide with ISONIAZID
TM, RIFAMPIN
TM, PYRAZINAMIDE
TMAnd/or ETHAMBUTOL
TMAnyly be used in combination with the treatment of pre-defense sector or prevent opportunistic Mycobacterium avium (Mycobacterium avium) MOI.In another embodiment, albumin fusion proteins of the present invention and/or polynucleotide with RIFABUTIN
TM, CLARITHROMYCIN
TMAnd/or AZITHROMYCIN
TMAnyly be used in combination with the treatment of pre-defense sector or prevent that opportunistic mycobacterium tuberculosis (Mycobacterium tuberculosis) from infecting.In another embodiment, albumin fusion proteins of the present invention and/or polynucleotide with GANCICLOVIR
TM, FOSCARNET
TMAnd/or CIDOFOVIR
TMAnyly be used in combination with the treatment of pre-defense sector or prevent the opportunistic cytomegalovirus infection.In another embodiment, albumin fusion proteins of the present invention and/or polynucleotide with FLUCONAZOLE
TM, ITRACONAZOLE
TMAnd/or KETOCONAZOLE
TMAnyly be used in combination with the treatment of pre-defense sector or prevent opportunistic fungal infection.In another embodiment, albumin fusion proteins of the present invention and/or polynucleotide with ACYCLOVIR
TMAnd/or FAMCICOLVIR
TMAnyly be used in combination with the treatment of pre-defense sector or prevent opportunistic I type and/or the II herpes simplex virus type infects.In another embodiment, albumin fusion proteins of the present invention and/or polynucleotide with PYRIMETHAMINE
TMAnd/or LEUCOVORIN
TMAnyly be used in combination with the treatment of pre-defense sector or prevent that opportunistic toxoplasma gondii (Toxoplasma gondii) from infecting.In another embodiment, albumin fusion proteins of the present invention and/or polynucleotide with LEUCOVORIN
TMAnd/or NEUPOGEN
TMAnyly be used in combination with the treatment of pre-defense sector or prevent the opportunistic bacterial infection.
In other embodiment, albumin fusion proteins of the present invention and/or polynucleotide and antibiotic combined administration.Can include but not limited to amoxicillin, beta-lactam enzyme, aminoglycoside, beta-lactam (glycopeptide), beta-lactamase, clindamycin, chloromycetin, cephalosporins, ciprofloxacin, erythromycin, fluoroquinolones, Macrolide, metronidazole, penicillins, quinolones, rapamycin, rifampicin, streptomycin, sulphanilamide, Tetracyclines, trimethoprim, trimethoprim-sulfamethoxazole and vancomycin with the antibiotic that albumin fusion proteins of the present invention and/or polynucleotide are used.
In other embodiments, albumin fusion proteins of the present invention and/or polynucleotide and immunostimulant combined administration.Can include but not limited to the immunostimulant of albumin fusion proteins of the present invention and/or polynucleotide combined administration, levamisole (as, ERGAMISOL
TM), inosine pranobex is (as INOSIPLEX
TM), interferon (as interferon-ALPHA) and interleukin (as, IL-2).
In other embodiments, albumin fusion proteins of the present invention and/or polynucleotide and immunosuppressant combined administration.Can include but not limited to steroid, ciclosporin, ciclosporin analog, cyclophosphamide Methyllprednisolone, prednisone, azathioprine, FK-506,15-deoxyspergualin and other immunosuppressant that works by the function that suppresses response T cell with the immunosuppressant of albumin fusion proteins of the present invention and/or polynucleotide combined administration.Can include but not limited to prednisolone, methotrexate, Thalidomide, methoxsalen, rapamycin, leflunomide, mizoribine (BREDININ with other immunosuppressant of albumin fusion proteins of the present invention and/or polynucleotide combined administration
TM), brequinar, deoxyspergualin and aza spiro alkane (azaspirane) (SKF 105685), ORTHOCLONE
3 (muromonab-CD3s), SANDIMMUNE
TM, NEORAL
TM, SANGDYA
TM(ciclosporin),
(FK506, tacrolimus),
(mycophenolic acid ethyl ester (motefil), its active metabolite is a mycophenolic acid), IMURAN
TM(azathioprine), glucocorticoid, adrenocortical steroid are such as DELTASONE
TM(prednisone) and HYDELTRASOL
TM(prednisolone), FOLEX
TMAnd MEXATE
TM(methotrexate), OXSORALEN-ULTRA
TM(methoxsalen) and RAPAMUNE
TM(sirolimus).In concrete embodiment, immunosuppressant can be used for preventing organ or marrow graft rejection.
In other embodiment, albumin fusion proteins of the present invention and/or polynucleotide separately or with one or more intravenous immunoglobulin product combined administrations.Can include but not limited to GAMMAR with the intravenous immunoglobulin product that albumin fusion proteins of the present invention and/or polynucleotide are used
TM, IVEEGAM
TM, SANDOGLOBULIN
TM, GAMMAGARD S/D
TM, ATGAM
TM(antithymocyte globulin (glubulin)) and GAMIMUNE
TMIn concrete embodiment, albumin fusion proteins of the present invention and/or polynucleotide in transplantation treatment (as, bone marrow transplantation) with intravenous immunoglobulin product combined administration.
In another embodiment, albumin fusion proteins of the present invention and/or polynucleotide are separately or be applied to the patient in the part body as combined therapy or externally be applied to cell with the treatment cancer.In concrete embodiment, albumin fusion proteins is IL-2-albumin fusant especially, in the passive immunotherapy cancer such as (Science Express such as Dudley, on JIUYUE 19th, 2002, at www.scienceexpress.org, incorporate into by reference with its integral body at this) described in inheritance cell transfer treatment metastatic melanoma during repetitive administration.
In some embodiments, albumin fusion proteins of the present invention and/or polynucleotide separately or with the antiinflammatory combined administration.Can include but not limited to the antiinflammatory that albumin fusion proteins of the present invention and/or polynucleotide are used, corticosteroid is (as betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, prednisone and triamcinolone), nonsteroidal antiinflammatory drug (as, diclofenac, diflunisal, etodolac, fenoprofen, floctafenine, esflurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenamic acid, mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin, Phenylbutazone, piroxicam, sulindac, tenoxicam, tiaprofenic acid and tolmetin), and hydryllin, the aminoaryl carboxylic acid derivates, the Arylacetic acids derivant, arylbutyric acid derivatives, aryl carboxylic acid, aryl propionic acid derivatives, pyrazoles, pyrazolone, salicyclic acid derivatives, the thiazine carboxylic acid amides, e-acetylamino caproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide, ditazole, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole and tenidap.
In other embodiment, compositions of the present invention separately or with anti--angiogenic agent combined administration.Can include but not limited to anti--angiogenic agent that compositions of the present invention is used, his spit of fland (Entremed of blood vessel, Rockville, MD), troponin-1 (Boston Life Sciences, Boston, MA), the various forms of tissue depressant, VEGI, plasminogen activator inhibitor-1, plasminogen activator inhibitor-2 and light " d group " transition metal of the tissue depressant of the anti-invasion factor, tretinoin and its derivant, paclitaxel (Taxol), suramin, metalloproteases-1, metalloproteases-2.
Light " d group " transition metal comprises, for example, and vanadium, molybdenum, tungsten, titanium, niobium and tantalum material.This transition metal material can form transition metal complex.The suitable complex of above-mentioned transition metal material comprises the oxo transition metal complex.
The representation example of vanudium complex comprises oxo vanudium complex such as vanadate and vanadyl complex.Suitable vanadate complex comprises metavanadate and positive vanadate complex such as for example, ammonium metavanadate, sodium metavanadate and sodium vanadate.Suitable vanadyl complex comprises, and for example, acetopyruvic acid vanadyl and vanadium oxysulfate comprise hydration vanadium oxysulfate such as single hydration vanadium oxysulfate and three hydration vanadium oxysulfates.
The representation example of tungsten and molybdenum complex also comprises the oxo complex.Suitable oxo tungsten complex comprises tungstates and tungsten oxide complex.Suitable tungstates complex comprises ammonium tungstate, artificial schellite, Disodium tungstate (Na2WO4) dihydrate and wolframic acid.Suitable tungsten oxide comprises tungsten oxide (IV) and tungsten oxide (VI).Suitable oxo molybdenum complex comprises molybdate, molybdenum oxide and molybdenyl complex.Suitable molybdate complex comprises ammonium molybdate and its hydrate, sodium molybdate and its hydrate and potassium molybdate and its hydrate.Suitable molybdenum oxide comprises molybdenum oxide (VI), molybdenum oxide (VI) and molybdic acid.Suitable molybdenyl complex comprises, for example, and acetopyruvic acid oxygen molybdenum.Tungsten that other is suitable and molybdenum complex comprise derived from for example, the hydroxyl derivant of glycerol, tartaric acid and sugar.
A large amount of other anti--angiogenesis factors also can be used in the context of the present invention.Representative example includes but not limited to, platelet factor 4; Protamine sulfate; Sulphation chitin derivatives (from the preparation of snowflake Carapax Eriocheir sinensis), (Murata etc., Cancer Res.51:22-26, (1991)); Sulfated polysaccharides Peptidoglycan complex (SP-PG) (this compound functions can strengthen by having steroid such as estrogen and citric acid tamoxifen); Staurosporine; The matrix metabolism instrumentality comprises for example proline analogs, cis hydroxyl groups proline, d, L-3,4-dehydroproline, thioproline, α, α-Lian Biding, aminopropionitrile fumarate; 4-propyl group-5-(4-pyridine radicals)-2 (3H)-azolactones; Methotrexate; Mitoxantrone; Heparin; Interferon; 2 macroglobulin-serum; ChIMP-3 (Pavloff etc., J.Bio.Chem.267:17321-17326, (1992)); Chymostatin (Tomkinson etc., Biochem J.286:475-480, (1992)); Cyclodextrin 14 sulfuric esters; Eponemycin; Camptothecine; Amebacilin (Ingber etc., Nature 348:555-557, (1990)); Kidon (Ono) (" GST "; Matsubara and Ziff, J.Clin.Invest.79:1440-1446, (1987)); Anticollagenase-serum; α 2-antiplasmin (Holmes etc., J.Biol.Chem.262 (4): 1659-1664, (1987)); Bisantrene (National Cancer Institute); Lobenzarit Disodium (N-(2)-carboxyl phenyl-4-chloro ortho-aminobenzoic acid (chloroanthronilic acid) disodium or " CCA "; (Takeuchi etc., Agents Actions 36:312-316, (1992)); With inhibitors of metalloproteinase such as BB94.
Other the anti--angiogenesis factor that also can be used for the context of the invention comprise Thalidomide (Celgene, Warren, NJ); Suppress the angiogenesis steroid; AGM-1470 (H.Brem and J.Folkman JPediatr.Surg.28:445-51 (1993)); Beta 2 integrin alpha v β 3 antagonisies (C.Storgard etc., JClin.Invest.103:47-54 (1999)); Carboxynaminolmidazole; Carboxylamide triazole (CAI) (National Cancer Institute, Bethesda, MD); Conbretastatin A-4 (CA4P) (OXiGENE, Boston, MA); Squalamine (Magainin Pharmaceuticals, PlymouthMeeting, PA); TNP-470 (Tap Pharmaceuticals, Deerfield, IL); ZD-0101AstraZeneca (London, UK); APRA (CT2584); Benfluralin, Byrostatin-1 (SC339555); CGP-41251 (PKC 412); CM101; Dexrazoxane (ICRF 187); DMXAA; His spit of fland of endothelium; Flavopridiol; Genestein; GTE; ImmTher; Iressa (ZD1839); Octreotide (somatostatin); Panretin; Penacillamine; Photopoint; PI-88; Prinomastat (AG-3340) Purlytin; Suradista (FCE26644); Tamoxifen (Nolvadex); Tazarotene; Tetrathiomolybdate; Xeloda (capecitabine); And 5-fluorouracil.
Can number of mechanisms work with the anti--angiogenic agent of chemical compound combined administration of the present invention, include but not limited to, the proteolysis that suppresses extracellular matrix, hinder the function of endotheliocyte-extracellular matrix adhesion molecule, the integrin receptor of expressing in the function of antagonizing vessel generation inducer such as somatomedin and the inhibition propagation endotheliocyte.The example of interference cell epimatrix proteolysis and the anti--angiogenesis inhibitor that can use with combination of compositions of the present invention includes but not limited to, AG-3340 (Agouron, La Jolla, CA), BAY-12-9566 (Bayer, West Haven, CT), BMS-275291 (BristolMyers Squibb, Princeton, NJ), CGS-27032A (Novartis, East Hanover, NJ), Marimastat (British Biotech, Oxford, UK) and Metastat (Aeterna, St-Foy, Quebec).Work and the example of the anti--angiogenesis inhibitor that can use with combination of compositions of the present invention includes but not limited to by the function that hinders endotheliocyte-extracellular matrix adhesion molecule, EMD-121974 (MerckKcgaA Darmstadt, Germany) and Vitaxin (Ixsys, La Jolla, CA/Medimmune, Gaithersburg, MD).Direct antagonism or suppress blood vessel generation inducer and work and the example of the anti--angiogenic agent that can use with combination of compositions of the present invention includes but not limited to, Angiozyme (Ribozyme, Boulder, CO), anti-VEGF antibodies (Genentech, S.San Francisco, CA), PTK-787/ZK-225846 (Novartis, Basel, Switzerland), SU-101 (Sugen, S.SanFrancisco, CA), SU-5416 (Sugen/Pharmacia Upjohn, Bridgewater, NJ) and SU-6668 (Sugen).Other anti--angiogenic agent works and suppresses the blood vessel generation indirectly.The example of the indirect inhibitor that can take place with the blood vessel that combination of compositions of the present invention is used includes but not limited to, IM-862 (Cytran, Kirkland, WA), interferon-' alpha ', IL-12 (Roche, Nutley, NJ) and poly-sulphuric acid pentosan (Georgetown University, Washington, DC).
In concrete embodiment, expect that compositions of the present invention and anti--angiogenic agent are used in combination with treatment, prevention and/or alleviate autoimmune disease, such as for example, autoimmune disease as herein described.
In concrete embodiment, expect that compositions of the present invention and anti--angiogenic agent are used in combination with treatment, prevention and/or releasing arthritis.In specific implementations more, expect combination of compositions of the present invention anti--angiogenic agent uses with treatment, prevention and/or rheumatoid arthritis.
In another embodiment, the encode polynucleotide combined administration of the polynucleotide of polypeptide of the present invention and angiogenic proteins or coding angiogenic proteins.The example of the angiogenic proteins that can use with compositions of the present invention includes but not limited to, acid and basic fibroblast growth factor, VEGF-1, VEGF-2, VEGF-3, epidermal growth factor α and β, the platelet-endothelial cell growth factor (ECGF) of deriving, platelet-derivative growth factor, tumor necrosis factor, hepatocyte growth factor, insulin like growth factor, colony stimulating factor, M-CSF, granulocyte/M-CSF and nitricoxide synthase.
In other embodiment, compositions of the present invention and chemotherapeutics combined administration.Can include but not limited to that with the chemotherapeutics that albumin fusion proteins of the present invention and/or polynucleotide are used alkylating agent such as nitrogen mustards (for example, chlormethine, cyclophosphamide, cyclophosphamide, ifosfamide, melphalan (L-Sarcolysin) and chlorambucil), aziridine and methyl melamine class are (for example, hexamethyl tripolycyanamide and plug are for group), alkyl sulfonate esters (for example, busulfan), nitrourea (for example, carmustine (BCNU), lomustine (CCNU), semustine (Semustine) and streptozocin (streptozocin)), triazenes (for example, dacarbazine (DTIC; Dimethyl triazenes imidazole carboxamide)), folacin (for example, methotrexate (9-methylpteroylglutamic acid)), pyrimidine analogue (for example, fluorouracil (Fluorouacil) (5-fluorouracil; 5-FU), floxuridine (fluorodeoxyuridine; FudR) with cytosine arabinoside (cytosine arabinoside (cytosinearabinoside))), purine analogue and relevant inhibitor (for example, mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG) and spray Si Tating (2 '-deoxycoformycin)), vinca alkaloids (for example, vinblastine (VLB, vincaleucoblastine (vinblastine sulfate))) and vincristine (vincristine sulfate)), etoposide (for example, etoposide and teniposide), antibiotic (for example, dactinomycin (actinomycin D), daunorubicin (daunorubicin; Daunorubicin), doxorubicin, bleomycin A5, plicamycin (mithramycin) and mitomycin (ametycin), enzyme are (for example, the altheine enzyme), the biological response instrumentality (for example, interferon-' alpha ' and interferon-' alpha '-2b), iridium-platinum complex are (for example, cisplatin (cis-DDP) and carboplatin), anthraquinone (mitoxantrone), the urea that replaces are (for example, hydroxyurea), methylhydrazine derivant (for example, procarbazine (N-methylhydrazine; MIH), adrenocortical steroid (for example, prednisone), Progesterone (for example, hydroxyprogesterone caproate, medroxyprogesterone, Medroxyprogesterone Acetate and acetic acid megestrol), estrogen (for example, diethylstilbestrol (DES), stilphostrol, estradiol and ethinylestradiol), antiestrogen (for example, tamoxifen), androgen (Testosterone Propionate and fluoxymesterone), antiandrogen (for example, flutamide), gonadotropin releasing hormone analogues (for example, Acetate), other hormone and hormone analogs are (for example, methyltestosterone, estramustine, estramustine phosphate sodium, chlorotrianisene and testolactone), and other (for example, dacarbazine (dicarbazine), glutamic acid and mitotane).
In one embodiment, compositions of the present invention and one or more following drug regimens are used: infliximab (is also referred to as Remicade
TMCentocor, Inc.), Trocade (Roche, RO-32-3555), leflunomide (is also referred to as Arava
TM, from Hoechst Marion Roussel), Kineret
TM(a kind of IL-1 receptor antagonist also is called Antril (Synergen), from Amgen, Inc.).
In concrete embodiment, compositions of the present invention and CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) combination or with one or more composition combined administrations of CHOP.In one embodiment, compositions of the present invention and anti-CD 20 antibodies, human monoclonal anti-CD 20 antibodies combined administration.In another embodiment, compositions of the present invention and anti-CD 20 antibodies and CHOP combination or with the combination in any of one or more compositions of anti-CD 20 antibodies and CHOP especially cyclophosphamide and/or prednisone combined administration.In concrete embodiment, compositions Mabthera combined administration of the present invention.In other embodiment, compositions of the present invention is used with combination in any of one or more compositions of Mabthera and CHOP or Mabthera and CHOP especially cyclophosphamide and/or prednisone.In concrete embodiment, compositions of the present invention and tositumomab combined administration.In other embodiment, compositions of the present invention is used with combination in any of one or more compositions of tositumomab and CHOP or tositumomab and CHOP especially cyclophosphamide and/or prednisone.Anti-CD 20 antibodies can randomly be united with radiosiotope, toxin or cytotoxicity prodrug.
In another embodiment, compositions of the present invention and Zevalin
TMCombined administration.In other embodiment, compositions of the present invention and Zevalin
TMWith CHOP or Zevalin
TMUse together with combination in any of one or more compositions of CHOP especially cyclophosphamide and/or prednisone.Zevalin
TMCan unite with one or more radiosiotope (radisotope).Exemplary isotope is
90Y and
111In.
In other embodiment, albumin fusion proteins of the present invention and/or polynucleotide and combination of cytokines are used.Can include but not limited to IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15, anti-CD 40, CD40L, IFN-γ and TNF-α with the cytokine that albumin fusion proteins of the present invention and/or polynucleotide are used.In another embodiment, albumin fusion proteins of the present invention and/or polynucleotide can be used with any interleukin, include but not limited to IL-1 α, IL-1 β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20 and IL-21.
In one embodiment, member's combined administration of albumin fusion proteins of the present invention and/or polynucleotide and TNF family.The TNF that can use with albumin fusion proteins of the present invention and/or polynucleotide, TNF is correlated with or TNF-sample molecule includes but not limited to, the soluble form of TNF-α, lymphotoxin-α (LT-α also is called TNF-β), LT-β (seeing compound heterotrimer LT-α 2-β), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-γ (No. 96/14328, international publication WO), AIM-I (No. 97/33899, international publication WO), endokine-α (No. 98/07880, international publication WO), OPG, with neutrokine-α (No. 98/18921, international publication WO, OX40, and nerve growth factor (NGF), soluble form with Fas, CD30, CD27, CD40 and 4-IBB, TR2 (No. 96/34095, international publication WO), DR3 (international publication WO97/33904 number), DR4 (No. 98/32856, international publication WO), TR5 (international publication WO98/30693 number), TRANK, TR9 (No. 98/56892, international publication WO), TR10 (No. 98/54202, international publication WO), 312C2 (No. 98/06842, international publication WO), and TR12, with soluble form CD154, CD70 and CD153.
In other embodiment, albumin fusion proteins of the present invention and/or polynucleotide and angiogenic proteins combined administration.Can include but not limited to the angiogenic proteins that albumin fusion proteins of the present invention and/or polynucleotide are used, glioma derivative growth factor (GDGF), as be disclosed in European patent EP-399816 number; Platelet derived growth factor-A (PDGF-A), as be disclosed in European patent EP-682110 number; Platelet derived growth factor-B (PDGF-B), as be disclosed in European patent EP-282317 number; Placental growth factor (PlGF), as be disclosed in international publication WO92/06194 number; Placental growth factor-2 (PlGF-2), as be disclosed in Hauser etc., Growth Factors, 4:259-268 (1993); VEGF (VEGF), as be disclosed in international publication WO90/13649 number; VEGF-A (VEGF-A), as be disclosed in European patent EP-506477 number; VEGF-2 (VEGF-2), as be disclosed in international publication WO96/39515 number; Vascular endothelial growth factor B (VEGF-3); Vascular endothelial growth factor B-186 (VEGF-B 186), as be disclosed in No. 96/26736, international publication WO; VEGF-D (VEGF-D), as be disclosed in No. 98/02543, international publication WO; VEGF-D (VEGF-D), as be disclosed in No. 98/07832, international publication WO; And VEGF-E (VEGF-E), as be disclosed in No. 19639601, Deutsche Bundespatent DE.Above-mentioned list of references is incorporated into its integral body by reference at this.
In other embodiment, albumin fusion proteins of the present invention and/or polynucleotide and fibroblast growth factor combined administration.Can include but not limited to FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14 and FGF-15 with the fibroblast growth factor that albumin fusion proteins of the present invention and/or polynucleotide are used.
In other embodiment, albumin fusion proteins of the present invention and/or polynucleotide and hemopoietic growth factor combined administration.Can include but not limited to granulocyte macrophage colony stimulating factor (GM-CSF) (Sargramostim, LEUKINE with the hemopoietic growth factor that albumin fusion proteins of the present invention and/or polynucleotide are used
TM, PROKINE
TM), granulocyte colony-stimulating factor (G-CSF) (filgrastim, NEUPOGEN
TM), M-CSF (M-CSF, CSF-1) erythropoietin (Epoetin Alfa, EPOGEN
TM, PROCRIT
TM), any or multiple, interferon-or the thrombopoietin of stem cell factor (SCF, c-kit part, steel factor), megakaryocyte colony stimulating factor, PIXY321 (a kind of GMCSF/IL-3 fusion rotein), interleukin, especially IL-1 to IL-12.
In some embodiments, albumin fusion proteins of the present invention and/or polynucleotide and adrenergic blocking drug combined administration, described adrenergic blocking drug is such as for example, acebutolol, atenolol, betaxolol, bisoprolol, carteolol, labetalol, metoprolol, nadolol, oxprenolol, penbutolol, pindolol, Propranolol, sotalol and timolol.
In another embodiment, albumin fusion proteins of the present invention and/or polynucleotide and arrhythmia medicine (as, adenosine, acylamino-arone (amidoarone), bretylium tosylate, Folium Digitalis Purpureae, digoxin, Digitoxin, diltiazem (diliazem), disopyramide, esmolol, flecainide, lignocaine, mexiletine, moracizine, phenytoin, procainamide, N-Acetylprocainamide, Propafenone, Propranolol, quinidine, sotalol, tocainide and verapamil) combined administration.
In another embodiment, albumin fusion proteins of the present invention and/or polynucleotide and diuretic combined administration, such as carbonic anhydrase-inhibitor (as, acetazolamide, daranide and Mei She azoles rice), the osmotic diuresis agent (as, glycerol, isosorbide, mannitol and urea), suppress Na
+-K
+-2Cl
-The diuretic of common transportation (as, furosemide, bumetanide, azosemide, piretanide, tripamide, etacrynic acid, muzolimine and torsemide), thiazide and thiazide-sample diuretic (as, bendroflumethiazide, benzthiazide, chlorothiazide, hydrochlorothiazide, hydroflumethiazide, methyclothiazide, polythiazide, trichlormethiazide, chlortalidone, indapamide, metolazone and quinethazone), Potassium-sparing diuretic (as, amiloride and triamterene) and mineralocorticoid receptor antagonists (as, spironolactone, canrenone and canrenoate potassium).
Use albumin fusion proteins of the present invention and/or polynucleotide in one embodiment, with to the therapeutic combination of endocrine and/or hormonal imbalance illness.Treatment to endocrine and/or hormonal imbalance illness includes but not limited to,
127The radioactive isotope of I, iodine such as
131I and
123I; Recombinant human growth hormone is such as HUMATROPE
TM(reorganization somatropin); Growth hormone analogs is such as PROTROPIN
TM(somatrem); Dopamine agonist is such as PARLODEL
TM(bromocriptine); Somatostatin analogue is such as SANDOSTATIN
TM(octreotide); Alasin is such as PREGNYL
TM, A.P.L.
TMAnd PROFASI
TM(chorionic-gonadotropin hormone (CG)), PERGONAL
TM(menotropin) and METRODIN
TM(Urofollitropin (uFSH)); Synthetic human gonadotropin releasing hormone preparation is such as FACTREL
TMAnd LUTREPULSE
TM(hydrochloric acid promoting sexual gland hormone); Synthetic promoting sexual gland hormone agonist is such as LUPRON
TM(acetic acid Acetate), SUPPRELIN
TM(acetic acid histrelin), SYNAREL
TM(acetic acid nafarelin) and ZOLADEX
TM(acetic acid goserelin); The synthesising preparation of thyrotropin-releasing hormone is such as RELEFACT TRH
TMAnd THYPINONE
TM(Protirelin); Recombinant human TSH is such as THYROGEN
TMThe synthesising preparation of thyroxin natural isomer sodium salt is such as L-T
4 TM, SYNTHROID
TMAnd LEVOTHROID
TM(levothyroxine sodium), L-T3
TM, CYTOMEL
TMAnd TRIOSTAT
TM(Cynomel (liothyroine sodium)) and THYROLAR
TM(liotrix); The antithyroid chemical compound is such as 6-n-pro-pyl deracil (propylthio uracil), 1-methyl-2-mercaptoimidazole and TAPAZOLE
TM(U.S. match azoles), NEO-MERCAZOLE
TM(carbimazole); B-adrenergic receptor antagonist such as Propranolol and esmolol; Ca
2+Channel blocker; Dexamethasone and iodate radiopaque contrast medium are such as TELEPAQUE
TM(iopanoic acid) and ORAGRAFIN
TM(sodium iopodate).
Other treatment to endocrine and/or hormonal imbalance illness includes but not limited to, estrogen or (congugated) estrogen of puting together are such as ESTRACE
TM(estradiol), ESTINYL
TM(ethinylestradiol), PREMARIN
TM, ESTRATAB
TM, ORTHO-EST
TM, OGEN
TMAnd piperazine estrone (estrone), ESTROVIS
TM(quinestrol), ESTRADERM
TM(estradiol), DELESTROGEN
TMAnd VALERGEN
TM(estradiol valerate), DEPO-ESTRADIOL CYPIONATE
TMWith ESTROJECT LA
TM(estradiol cypionate); Antiestrogen is such as NOLVADEX
TM(tamoxifen), SEROPHENE
TMAnd CLOMID
TM(clomifene); Progesterone is such as DURALUTIN
TM(hydroxycaproic acid Progesterone), MPA
TMAnd DEPO-PROVERA
TM(Medroxyprogesterone Acetate), PROVERA
TMAnd CYCRIN
TM(MPA), MEGACE
TM(acetic acid megestrol), NORLUTIN
TM(norethindrone) and NORLUTATE
TMAnd AYGESTIN
TM(SH 420); The Progesterone implant is such as NORPLANT SYSTEM
TM(norgestrel subcutaneous implant); Antiprogestin such as RU 486
TM(mifepristone); Mestranol is such as ENOVID
TM(Norethynodrel+mestranol), PROGESTASERT
TM(discharging the intrauterine device of Progesterone), LOESTRIN
TM, BREVICON
TM, MODICON
TM, GENORA
TM, NELONA
TM, NORINYL
TM, OVACON-35
TMAnd OVACON-50
TM(ethinylestradiol/norethindrone), LEVLEN
TM, NORDETTE
TM, TRI-LEVLEN
TMAnd TRIPHASIL-21
TM(ethinylestradiol/levonorgestrel) LO/OVRAL
TMAnd OVRAL
TM(ethinylestradiol/norgestrel), DEMULEN
TM(ethinylestradiol/oxalic acid etynodiol), NORINYL
TM, ORTHO-NOVUM
TM, NORETHIN
TM, GENORA
TMAnd NELOVA
TM(norethindrone/mestranol), DESOGEN
TMAnd ORTHO-CEPT
TM(ethinylestradiol/desogestrel), ORTHO-CYCLEN
TMAnd ORTHO-TRICYCLEN
TM(ethinylestradiol/norgestimate), MICRONOR
TMAnd NOR-QD
TM(norethindrone) and OVRETTE
TM(norgestrel).
Other treatment to endocrine and/or hormonal imbalance illness includes but not limited to that the testosterone ester is such as acetic acid metenolone and testosterone undecanoate; Parenteral and oral androgenic are such as TESTOJECT-50
TM(testosterone), TESTEX
TM(Testosterone Propionate), DELATESTRYL
TM(testosterone enanthatas), DEPO-TESTOSTERONE
TM(depo-testosterone), DANOCRINE
TM(danazol), HALOTESTIN
TM(fluoxymesterone), ORETON METHYL
TM, TESTRED
TMAnd VIRILON
TM(methyltestosterone) and OXANDRIN
TM(oxandrolone); The testosterone intradermic system is such as TESTODERM
TMAndrogen receptor antagonists and 5-alpha-reductase inhibitors are such as ANDROCUR
TM(acetic acid cyproterone), EULEXIN
TM(flutamide) and PROSCAR
TM(finasteride); Adrenocorticotropic hormone preparation is such as CORTROSYN
TM(thyroliberin); Adrenocortical steroid and its synthetic analogues are such as ACLOVATE
TM(alclometasone diproionate), CYCLOCORT
TM(amcinonide), BECLOVENT
TMAnd VANCERIL
TM(beclomethasone), CELESTONE
TM(betamethasone), BENISONE
TMAnd UTICORT
TM(betamethasone benzoate), DIPROSONE
TM(betamethasone dipropionate), CELESTONE PHOSPHATE
TM(betamethasone sodium phosphate), CELESTONE SOLUSPAN
TM(betamethasone sodium phosphate and betamethasone sodium acetate), BETA-VAL
TMAnd VALISONE
TM(betamethasone valerate), TEMOVATE
TM(dipropionic acid clobetasol), CLODERM
TM(pivalic acid clocortolone), CORTEF
TMAnd HYDROCORTONE
TM(hydrocortisone (hydrocortisone)), HYDROCORTONEACETATE
TM(acetic acid hydrocortisone (hydrocortisone)), LOCOID
TM(butanoic acid hydrocortisone (hydrocortisone)), HYDROCORTONE PHOSPHATE
TM(hydrocortisone (hydrocortisone) sodium phosphate), A-HYDROCORT
TMWith SOLU CORTEF
TM(hydrocortisone (hydrocortisone) sodium succinate), WESTCORT
TM(valeric acid hydrocortisone (hydrocortisone)), CORTISONE ACETATE
TM(acetic acid cortisone), DESOWEN
TMAnd TRIDESILON
TM(desonide), TOPICORT
TM(desoximetasone), DECADRON
TM(dexamethasone), DECADRON LA
TM(acetic acid dexamethasone), DECADRON PHOSPHATE
TMWith HEXADROL PHOSPHATE
TM(dexamethasone sodium phosphate), FLORONE
TMAnd MAXIFLOR
TM(oxalic acid diflorasone), FLORINEFACETATE
TM(acetic acid fludrocortisone), AEROBID
TMAnd NASALIDE
TM(flunisolide), FLUONID
TMAnd SYNALAR
TM(fluocinolone acetonide), LIDEX
TM(fluocinonide), FLUOR-OP
TMAnd FML
TM(fluorometholone), CORDRAN
TM(flurandrenolide), HALOG
TM(halcinonide), HMS LIZUIFILM
TM(medrysone), MEDROL
TM(methylprednisolone), DEPO-MEDROL
TMWith MEDROL ACETATE
TM(methyl acetic acid prednisone), A-METHAPRED
TMAnd SOLUMEDROL
TM(methylprednisolone sodium succinate), ELOCON
TM(momestasone furoate), HALDRONE
TM(acetic acid 6.alpha.-fluoro-16.alpha.-methylprednisolone), DELTA-CORTEF
TM(prednisolone), ECONOPRED
TM(prednisone acetate dragon), HYDELTRASOL
TM(Inflamase), HYDELTRA-T.B.A
TM(uncle's Hydeltra T. B. A), DELTASONE
TM(prednisone), ARISTOCORT
TMAnd KENACORT
TM(triamcinolone), KENALOG
TM(triamcinolone acetonide), ARISTOCORT
TMWith KENACORT DIACETATE
TM(oxalic acid triamcinolone) and ARISTOSPAN
TM(triamcinolone hexacetonide); The inhibitor of adrenocortical steroid biosynthesis and effect is such as CYTADREN
TM(aminoglutethimide), NIZORAL
TM(ketoconazole), MODRASTANE
TM(trilostane) and METOPIRONE
TM(metyrapone); Cattle, pig or human insulin or its mixture; Insulin analog; The recombinant human insulin is such as HUMULIN
TMAnd NOVOLIN
TMOral hypoglycemic is such as ORAMIDE
TMAnd ORINASE
TM(tolbutamide), DIABINESE
TM(chlorpropamide), TOLAMIDE
TMAnd TOLINASE
TM(tolazamide), DYMELOR
TM(acetohexamide), glibenclamide, MICRONASE
TM, DIBETA
TMAnd GLYNASE
TM(glyburide), GLUCOTROL
TM(glipizide) and DIAMICRON
TM(gliclazide), GLUCOPHAGE
TM(metformin), ciglitazone, pioglitazone and alpha-glucosidase inhibitor; Cattle or pig glucagon; Somatostatin is such as SANDOSTATIN
TM(octreotide); With the diazoxide class such as PROGLYCEM
TM(diazoxide).
In one embodiment, albumin fusion proteins of the present invention and/or polynucleotide and the therapeutic combination of uterus activeness illness used.Treatment to uterus activeness illness includes but not limited to, estrogenic such as the estrogen of puting together (as,
With
), estradiol (as,
With
), estropipate (estropipate) and chlorotrianisene; The Progesterone medicine (as,
(medroxyprogesterone),
(SH 420 (norethidrone acetate)),
Progesterone and acetic acid megestrol); And estrogen/Progesterone combination treatment is such as for example, estrogen/medroxyprogesterone of puting together (as, PREMPRO
TMWith
) and SH 420/ethinylestradiol (ethinyl estsradiol) (as, FEMHRT
TM).
In other embodiment, the drug regimen of albumin fusion proteins of the present invention and/or polynucleotide and effective treatment iron deficiency anemia and low hematochrome anemia is used, described medicine includes but not limited to, ferrous sulfate (ferrous sulfate (iron sulfate), FEOSOL
TM), ferrous fumarate is (as, FEOSTAT
TM), Ferrous gluconate is (as, FERGON
TM), polysaccharide-ferrum complex is (as, NIFEREX
TM), the dextran iron injection is (as, INFED
TM), copper sulfate, pyridoxol, riboflavin, vitamin B
12, the cobalamin injection is (as, REDISOL
TM, RUBRAMIN PC
TM), hydroxocobalamin, folic acid be (as, FOLVITE
TM), folinic acid (folinic acid, 5-CHOH4PteGlu, citrovorum factor) or WELLCOVORIN (calcium salt of folinic acid), transferrins or ferritin.
In some embodiments, albumin fusion proteins of the present invention and/or polynucleotide and the drug regimen that is used for the treatment of the psychosis illness are used.Can include but not limited to the psychosis medicine that albumin fusion proteins of the present invention and/or polynucleotide are used, the ataraxy medicine (as, chlorpromazine, chlorprothixene, clozapine, fluphenazine, haloperidol, loxapine, mesoridazine, molindone, olanzapine, perphenazine, pimozide, Quetiapine, risperidone, thioridazine, tiotixene, trifluoperazine and triflupromazine), antimaniacal drugs (as, carbamazepine, divalproex sodium, lithium carbonate and Lithium Citrate de), antidepressants (as, amitriptyline, amoxapine, BUP, citalopram, clomipramine, desipramine, doxepin, fluvoxamine, fluoxetine, imipramine, isocarboxazid, maprotiline, mirtazapine, nefazodone, nortriptyline, paroxetine, phenelzine, protriptyline, Sertraline, tranylcypromine; Trazodone, trimeprimine and venlafaxine), antianxiety drugs (as, alprazolam, buspirone, chlordiazepoxide, the tall and erect acid of chlorine, diazepam, halazepam, lorazepam, oxazepam and prazepam) and analeptic (as, dexamphetamine (d-amphetamine), methylphenidate and pemoline).
In other embodiments, albumin fusion proteins of the present invention and/or polynucleotide and the drug regimen that is used for the treatment of neurological disorder are used.Can include but not limited to the neurologic agent that albumin fusion proteins of the present invention and/or polynucleotide are used, antuepileptic (as, carbamazepine, clonazepam, ethosuximide, phenobarbital, phenytoin, primidone, valproic acid, divalproex sodium, non-ammonia ester, gabapentin, lamotrigine, levetiracetam, oxcarbazepine, Tiagabine, topiramate, zonisamide, diazepam, lorazepam and clonazepam), antiparkinsonism drug (as, levodopa/carbidopa, selegiline, amantadine, bromocriptine, pergolide, ropinirole, pramipexole, benzatropine; Biperiden; Profenamine; Procyclidine; Benzhexol, tolcapone) and ALS therapeutic agent (as riluzole).
In another embodiment, albumin fusion proteins of the present invention and/or polynucleotide and vasodilation and/or calcium channel blocker combined administration.Can include but not limited to the vasodilation that albumin fusion proteins of the present invention and/or polynucleotide are used, Angiotensin-Converting (ACE) inhibitor (as, papaverine, isoxsuprine, benazepril, captopril, cilazapril, enalapril, enalaprilat, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, spirapril, trandolapril and buphenine) and nitrate (as, isosorbide dinitrate, Ismo 20 and glycerin trinitrate).Can include but not limited to amlodipine, bepridil, diltiazem, felodipine, flunarizine, Isradipine, nicardipine, nifedipine, nimodipine and verapamil with the example of the calcium channel blocker of albumin fusion proteins of the present invention and/or polynucleotide combined administration.
In some embodiments, albumin fusion proteins of the present invention and/or polynucleotide and the therapeutic combination of gastrointestinal tract disease used.Can include but not limited to H with the treatment that albumin fusion proteins of the present invention and/or polynucleotide are used to gastrointestinal tract disease
2Histamine receptor antagonists (as, TAGAMET
TM(cimetidine), ZANTAC
TM(ranitidine), PEPCID
TM(famotidine) and AXID
TM(nizatidine)); H
+, K
+Atpase inhibitor (as, PREVACID
TM(lansoprazole) and PRILOSEC
TM(omeprazole)); Bismuth compound (as, PEPTO-BISMOL
TM(bismuth subsalicylate) and DE-NOL
TM(bismuth subcitrate)); Various antacid; Sucralfate; Prostaglandin analogue is (as CYTOTEC
TM(misoprostol)); The muscarine cholinergic antagonist; Laxative (as, surfactant laxative, analeptic laxative, saline and infiltration laxative); Diarrhea (as, LOMOTIL
TM(diphenoxylate), MOTOFEN
TM(difenoxin (diphenoxin)) and IMODIUM
TM(loperamide hydrochloride)), the synthetic analogues of somatostatin is such as SANDOSTATIN
TM(octreotide), Bendectin (as, ZOFRAN
TM(ondansetron), KYTRIL
TM(Granisetron Hydrochloride), tropisetron, dolasetron, metoclopramide, chlorpromazine, perphenazine, prochlorperazine, promethazine, thiethylperazine, triflupromazine, domperidone, haloperidol, droperidol, trimethobenzamide, dexamethasone, methylprednisolone, dronabinol and nabilone); The D2 antagonist (as, metoclopramide, trimethobenzamide and chlorpromazine); Cholate; Chenodeoxycholic acid; Ursodesoxycholic acid; With pancreas enzyme preparation such as pancreatin and pancreatic lipase.
In other embodiment, albumin fusion proteins of the present invention and/or polynucleotide and other therapeutic or preventative therapy are such as for example X-ray therapy combined administration.
The present invention also provides the pharmaceutical pack or the cover box of the one or more containers that comprise one or more compositions that are filled with the pharmaceutical composition that comprises albumin fusion proteins of the present invention.Randomly these containers can be with the notice with the form of administrative organization's regulation of manufacturing, use or the sale of management medicine or biological product, and the administrative organization that this notice reflection management is made, used or sells is to the approval of human administration.
Gene therapy
The construct of albumin fusion proteins of the present invention of encoding can be used as the albumin fusion proteins of the part of gene therapy scheme with the delivery treatments effective dose.Introduce nucleic acid is to use the nucleic acid that contains the albumin fusion proteins of the present invention of encoding to the illustrative methods of cell viral vector in the body.With the advantage of viral vector infection cell is that the targeted cells of larger proportion can obtain nucleic acid.In addition, the molecule effective expression in the cell that obtains viral vector nucleic acid as encoding in the viral vector by the cDNA that contains in the viral vector.
Retroviral vector and adeno-associated virus vector can be used as the recombination delivery system to shift the exogenous nucleic acid molecule of coding albumin fusion proteins in the body.These carriers provide to the effective nucleic acid delivery of cell, and the nucleic acid stability that shifts be incorporated in the host chromosome DNA.Exploitation only produces and duplicates-and the retroviral specialized cell line of defective (being called " incasing cells ") increased the effectiveness that retrovirus is used for gene therapy, and the deficiency retrovirus is characterized as being the gene transfer (summary sees Miller, A.D. (1990) Blood 76:271) that is used for the gene therapy purpose.The replication defect type retrovirus can be packaged in the virion, and virion can be used for infecting target cell with standard technique by using helper virus.Be used to produce recombinant retrovirus and with this viroid in the external or body rules of infection cell be found in Current Protocols in Molecular Biology, Ausubel, F.M. etc., (volume) GreenePublishing Associates, (1989), 9.10-9.14 part and other standard laboratory handbook.
Available another kind of viral gene delivery system uses adenovirus-derivative vector among the present invention.The genome that can handle adenovirus is so that its coding and express interested gene outcome, but the ability of duplicating in normal lytic virus life cycle is by inactivation.For example see Berkner etc., Biotechniques 6:616 (1988); Rosenfeld etc., Science 252:431-434 (1991); With Rosenfeld etc., Cell 68:143-155 (1992).Derived from adenovirus strains A d type 5d1324 or other adenovirus bacterial strain (as, Ad2, Ad3, Ad7 etc.) suitable adenovirus vector be well known by persons skilled in the art.Recombinant adenovirus can be favourable in certain situation, because they can not infect nondividing cell and can be used for infecting a variety of cell types, comprise epithelial cell (Rosenfeld etc., (1992) are above-mentioned).In addition, virion is metastable and is suitable for purification and concentrates, and as mentioned above, influenced pattern of infection thereby can modify.In addition, the adenovirus DNA of being introduced (foreign DNA that is comprised with this paper) is not integrated into the host cell gene group and remains episome, thereby the DNA that avoids being introduced is integrated in the host genome situation of (as, retrovirus DNA) owing to inserting the potential problems that mutation takes place.And, with respect to other gene delivery vector, the adenoviral gene group to the carrying capacity of foreign DNA big (as many as 8 kilobase) (Berkner etc., above-mentioned; Haj-Ahmand etc., J.Virol.57:267 (1986)).
In another embodiment, of the present invention non--the viral gene delivery system depend on the endocytosis approach so that described nucleic acid molecule by the cellular uptake of targeting.The exemplary genes delivery system of this class comprises liposome flavor, poly--lysine conjugate and artificial viral envelope.In representative embodiments, the nucleic acid molecules of albumin fusion proteins of the present invention of encoding can be captured to carry on the surface positive charge also (choose wantonly) with in the liposome at the antibody labeling of the cell surface antigen of target tissue (as, lipofectin) (Mizuno etc. (1992) No Shinkei Geka 20:547-551; PCT announces WO91/06309; No. the 1047381st, Japanese patent application; With European patent publication EP-A-43075 number).
The genes delivery system of gene of albumin fusion proteins of the present invention of encoding can be introduced the patient by any big metering method.For example, can be systematically as introduce the pharmaceutical preparation of genes delivery system by intravenous injection, and the transfection specificity that specific proteins transduction mainly provides according to gene delivery vector in the target cell, because cell-type that the transcription regulating nucleotide sequence that the control acceptor gene is expressed causes or tissue-type are expressed or its combination and taking place.In other embodiments, initially send recombination more to be confined to introduce animal be suitable localization (localized).For example, gene delivery vector can be introduced by conduit (seeing United States Patent (USP) the 5th, 328, No. 470) or by stereotactic injection (as (1994) PNAS 91:3054-3057 such as Chen).The pharmaceutical preparation of gene therapy construct can mainly be made up of the genes delivery system in acceptable diluent, maybe can comprise the sustained-release matrix that wherein embeds gene delivery vector.Can be at albumin fusion proteins from the situation of reconstitution cell such as the complete preparation of retroviral vector, pharmaceutical preparation can comprise one or more cells that produces albumin fusion proteins.
Other gene therapy method
The present invention also comprises the gene therapy method of treatment or prevention illness, disease and disease.Gene therapy method relates to introduces animal to realize the expression of albumin fusion proteins of the present invention with nucleic acid (DNA, RNA and antisense DNA or RNA) sequence.This method requires the polynucleotide of coding albumin fusion proteins of the present invention to be operably connected to promoter and essential any other genetic elements of target tissue expressed fusion protein.This genoid treatment and delivery technique are known in the art, for example see, and WO90/11092, it is incorporated into by reference at this.
Therefore, for example, patient's cell can in vitro be transformed with the polynucleotide (DNA or RNA) of the promoter that comprises the polynucleotide that are operably connected to coding albumin fusion proteins of the present invention, and institute's engineered cells is provided in subsequently the patient of stand-by fusion rotein treatment of the present invention.These class methods are well known in the art.For example, see Belldegrun, A. etc., J.Natl.Cancer Inst.85:207-216 (1993); Ferrantini, M. etc., Cancer Research 53:1107-1112 (1993); Ferrantini, M. etc., J.Immunology 153:4604-4615 (1994); Kaido, T. etc., Int.J.Cancer 60:221-229 (1995); Ogura, H. etc., Cancer Research 50:5102-5106 (1990); Santodonato, L. etc., Human Gene Therapy 7:1-10 (1996); Santodonato, L. etc., Gene Therapy 4:1246-1255 (1997); And Zhang, J.-F. etc., Cancer Gene Therapy 3:31-38 (1996)), it is incorporated into by reference at this.In one embodiment, the cell of being transformed is an arterial cell.Arterial cell can be via being injected directly into tremulous pulse, introducing the patient again to the tremulous pulse surrounding tissue or via tube injection.
As discussed in detail below, polynucleotide constructs can be sent to any method of zooblast by sending injectable materials, such as, be expelled to the interstitial space of tissue (heart, muscle, skin, lung, liver and allied organization).Polynucleotide constructs can pharmaceutically acceptable liquid state or aqueous vehicle send.
In one embodiment, the encode polynucleotide of albumin fusion proteins of the present invention are sent with naked polynucleotide.Term " naked " polynucleotide, DNA or RNA refer to not contain the sequence that act as auxiliary, as to promote or be convenient to enter cell any delivery vector (comprising virus sequence, virion, Liposomal formulation, lipofectin or precipitant and analog).Yet the polynucleotide of the albumin fusion proteins of the present invention of encoding can also be sent by Liposomal formulation, and lipofectin preparation and analog can be by method preparations well known to those skilled in the art.These class methods for example are described in, United States Patent (USP) the 5th, 593, and 972,5,589,466 and 5,580, No. 859, it is incorporated into by reference at this.
The polynucleotide carrier construct that is used for gene therapy method can be unconformity and goes into host genome and also do not contain the construct that allows the sequence of duplicating.Suitable carriers comprises can be from pWLNEO, pSV2CAT, pOG44, pXT1 and the pSG of Stratagene acquisition; Can be from pSVK3, pBPV, pMSG and the pSVL of Pharmacia acquisition; With pEF1/V5, pcDNA3.1 and the pRc/CMV2 that can obtain from Invitrogen.Other suitable carriers is easily significantly to those skilled in the art.
Any strong promoter well known by persons skilled in the art can be used for driving the expression of polynucleotide sequence.Suitable promoter comprises adenovirus promoter, such as adenovirus major late promoter; Or allogeneic promoter, such as cytomegalovirus (CMV) promoter; Breathe syncytial virus (RSV) promoter; Inducible promoter is such as MMT promoter, metallothionein promoter; Heat-inducible promoter; The albumin promoter; The ApoAI promoter; Human globin promoter; Viral thymidine kinase promoter is such as herpes simplex thymidine kinase promoter; Retrovirus LTR; The b-actin promoter; With human growth hormone's promoter.Promoter also can be the natural promoter corresponding to the gene of the treatment protein part of albumin fusion proteins of the present invention.
Be different from other gene therapy technology, a main advantage the naked nucleic acid sequence being introduced target cell is that polynucleotide are synthetic temporary in cell.Research shows, non--replicability DNA sequence can be introduced cell and continue nearly six months period so that the polypeptide that produces expectation to be provided.
Polynucleotide constructs can be delivered to the interstitial space of animal tissue, described tissue comprises muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, intestinal, testis, ovary, uterus, rectum, nervous system, eye, body of gland and connective tissue.The interstitial space of tissue comprises in the collagen fiber of elastic fiber in mucopolysaccharide matrix, blood vessel wall or the chamber wall between iuntercellular, fluid, organ-tissue reticular fiber, fibrous tissue or the connective tissue sheath's muscle cell or the same matrix in the lacuna osseous.Be the space that the lymph fluid of circulating plasma and lymph-space occupies similarly.Because reason discussed below, can be delivered to the interstitial space of muscular tissue.Can send polynucleotide constructs easily by being expelled to the tissue that comprises these cells.Polynucleotide constructs can be delivered to the forever non--somatoblast that is differentiation and express therein, although send and express and can realize in cell non--differentiation or not exclusively differentiation (such as for example, blood stem cell or skin flbroblast).Muscle cell obtains and to express the ability of polynucleotide especially strong in the body.
For the injection of naked nucleic acid sequence, the DNA of effective dose or the amount of RNA will be the scope of about 0.05mg/kg body weight to about 50mg/kg body weight.In one embodiment, dosage will be about 5mg/kg extremely from about 0.005mg/kg to about 20mg/kg and about in another embodiment 0.05mg/kg.Certainly as one of ordinary skill in the art will appreciate, this dosage will change according to the tissue site of injection.Suitable and nucleotide sequence effective dose can be determined by those of ordinary skills easily, and can be depending on the disease and the route of administration of being treated.
In one embodiment, route of administration is the interstitial space that is expelled to tissue by parenteral route.Yet also can use other parenteral route,, be particularly useful for being delivered to lung or bronchial tissue, throat or nasal mucosa such as the inhalation aerosol preparation.In addition, the naked DNA construct can be during angioplasty by catheter delivery used in this method to tremulous pulse.
Naked polynucleotide are sent by any method known in the art, include but not limited to, in the direct needle injection of site of delivery, intravenous injection, local application, conduit infusion and what is called " particle gun ".These delivering methods are known in the art.
Construct also can be sent with delivery vector such as virus sequence, virion, Liposomal formulation, lipofectin, precipitant etc.This class delivering method is known in the art.
In some embodiments, polynucleotide constructs is compounded in the Liposomal formulation.Be used for Liposomal formulation of the present invention and comprise cation (positively charged), anion (electronegative) and neutral preparation.Yet can use cationic-liposome, because between cationic-liposome and polyanionic nucleic acid, can form tight charge recombination body.The plasmid DNA (it is incorporated into by reference at this for Feigner etc., Proc.Natl.Acad.Sci USA (1987) 84:7413-7416) that has shown delivery functions form in the cationic-liposome-mediated cell; MRNA (it is incorporated into by reference at this for Malone etc., Proc.Natl.Acad.Sci USA (1989) 86:6077-6081); With purification transcription factor (it is incorporated into by reference at this for Debs etc., J.Biol.Chem. (1990) 265:10189-10192).
Cationic-liposome is easy to obtain.For example, N[1-2,3-two oil base oxygen bases) propyl group]-N, N, N-triethyl ammonium (DOTMA) liposome is particularly useful and can be from GIBCO BRL with trade mark Lipofectin, and GrandIsland, N.Y. obtain (also to see Feigner etc., Proc.Natl Acad.Sci USA (1987) 84:7413-7416, it is incorporated into by reference at this).Other commercially available liposome comprises transfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).
Other cationic-liposome can use the material preparation of well known technology from being easy to obtain.See as PCT and announce No. 90/11092, WO (it is incorporated into by reference at this), described synthetic DOTAP (1, two (oleoyl oxygen base)-3-(trimethyl ammonium) propane of 2-) liposome.Preparation DOTMA liposome is described in document, sees as, P.Feigner etc., and Proc.Natl.Acad.Sci USA 84:7413-7417, it is incorporated into by reference at this.Similarity method can be used for from other cation lipid material preparation liposome.
Similarly, anion and neutral fat plastid are easy to such as (Birmingham Ala.) obtains, and maybe can use the material that is easy to obtain to be easy to preparation from Avanti Polar Lipids.This class material comprises phosphatidyl, choline, cholesterol, PHOSPHATIDYL ETHANOLAMINE, dioleyl phosphatidyl choline (DOPC), dioleoyl phosphatidyl glycerol (DOPG), dioleoyl phosphatidyl (phoshatidyl) ethanolamine (DOPE) and other.These materials can also be suitable ratio mix with DOTMA and DOTAP parent material.The method of using these material preparation liposomees is well known in the art.
For example, can variously be used in combination commercially available dioleyl phosphatidyl choline (DOPC), dioleoyl phosphatidyl glycerol (DOPG) and dioleoyl PHOSPHATIDYL ETHANOLAMINE (DOPE) and add or do not add cholesterol to prepare conventional liposome.Therefore for example, can prepare DOPG/DOPC vesicle (vesicle) to the supersound process bottle by DOPG and the DOPC of dry each 50mg under nitrogen current.With sample place spend the night under the vacuum pump and next day with the deionized water hydration.Use Heat Systems 350 type supersound process instrument that inverted cup (bath type) probe is housed to be provided with sample supersound process 2 hours in adding a cover bottle subsequently with maximum, simultaneously with water-bath 15 degrees centigrade of circulations.Alternatively, can prepare electronegative vesicle to produce the multilamellar vesicle or to be pressed through nucleopore membranes without supersound process to produce the monolayer vesicle of different size.Other method is known and is obtainable to those skilled in the art.
Liposome can comprise multilamellar vesicle (MLV), little monolayer vesicle (SUV) or big monolayer vesicle (LUV), and wherein SUV is a specific implementations.Use well known method to prepare various liposome-nucleic acid complexes.See as, Straubinger etc., Methods of Immunology (1983), 101:512-527, it is incorporated into by reference at this.For example, can be by phospholipid membrane being deposited on the glass tube walls and preparing the MLV that contains nucleic acid with the solution hydration of the material waiting to be coated subsequently.Prepare SUV by the supersound process MLV that prolongs with the unilamellar liposome that produces homogeneous group.To treat that captive material adds the suspension of preformed MLV, supersound process subsequently.When use contained the liposome of cation lipid, exsiccant lipid film was resuspended in suitable solution such as sterilized water or isotonic buffer solution such as among the 10mM Tris/NaCl, supersound process, and subsequently preformed liposome is directly mixed with DNA.Because positively charged liposome is incorporated into cation DNA, liposome and DNA form stabilized complex.SUV is used for the small nucleic acids fragment.LUV is by well known big metering method preparation.Common method comprises Ca
2+-EDTA chelating (Papahadjopoulos etc., Biochim.Biophys.Acta (1975) 394:483; Wilson etc., Cell 17:77 (1979)); Ether injection (Deamer, D. and Bangham, A., Biochim.Biophys.Acta 443:629 (1976); Ostro etc., Biochem.Biophys.Res.Commun.76:836 (1977); Fraley etc., Proc.Natl.Acad.Sci USA 76:3348 (1979)); Detergent dialysis (Enoch, H. and Strittmatter, P., Proc.Natl.Acad.Sci USA 76:145 (1979)); With anti-phase evaporation (REV) (Fraley etc., J.Biol.Chem.255:10431 (1980); Szoka, F. and Papahadjopoulos, D., Proc.Natl.Acad.Sci USA 75:145 (1978); Schaefer-Ridder etc., Science 215:166 (1982)), above-mentioned file is incorporated into by reference at this.
Usually, the ratio of DNA and liposome is from about 10: 1 to about 1: 10.In one embodiment, ratio is from about 5: 1 to about 1: 5.In another embodiment, ratio is about 3: 1 to about 1: 3.In another embodiment, ratio is about 1: 1.
United States Patent (USP) the 5th, 676, No. 954 (it is incorporated into by reference at this) reported and will be expelled to mice with the compound genetic stocks of cationic-liposome carrier.United States Patent (USP) the 4th, 897,355,4,946,787,5,049,386,5,459,127,5,589,466,5,693,622,5,580,859,5,703, No. 055 and international publication WO No. 94/9469 (it is incorporated into by reference at this) are provided for the DNA transfection to cell and mammiferous cation lipid.United States Patent (USP) the 5th, 589,466,5,693,622,5,580,859,5,703, No. 055 and international publication WO provide for No. 94/9469 DNA-cation lipid complex are delivered to mammiferous method.
In some embodiments, the retroviral particle that uses the RNA contain the sequence that comprises the albumin fusion proteins of the present invention of encode in vitro or the interior engineered cells of body.The retrovirus of retroviral plasmid vector of can therefrom deriving includes but not limited to, Moloney murine leukemia virus, SNV, rous sarcoma virus, Harvey sarcoma virus, birds leukosis virus, gibbon ape leukemia virus, HIV (human immunodeficiency virus), myeloproliferative sarcoma virus and mammary tumor virus.
Adopt retroviral plasmid vector to transduce package cell line to form production cell line.The example of incasing cells that can be transfected includes but not limited to, PE501, PA317, R-2, R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12 and DAN cell line, as Miller, described in the Human Gene Therapy 1:5-14 (1990), it is incorporated into its integral body by reference at this.Carrier can be via any way transduction incasing cells known in the art.This mode includes but not limited to, electroporation, use liposome and CaPO
4Precipitation.A selection scheme, retroviral plasmid vector can be coated liposome or is coupled to lipid, is applied to the host subsequently.
Production cell line produces infectious retroviral vector particle, and it comprises the polynucleotide of the albumin fusion proteins of the present invention of encoding.Can adopt the external or body of this retroviral vector particle eukaryotic cell of transduceing subsequently interiorly.The eukaryotic cell of transduction will be expressed fusion rotein of the present invention (protin).
In some other embodiments, cell in vitro or in the body is transformed with the polynucleotide that contain in the adenovirus vector.Can handle adenovirus so that its coding and express fusion rotein of the present invention, and simultaneously its ability of in normal lytic virus life cycle, duplicating by inactivation.Realize gland virus expression and viral DNA is not incorporated in the host cell chromosome, thereby alleviated about inserting the problem of mutation.In addition, adenovirus has been used as intestinal vaccine alive for many years, has excellent security (Am.Rev.Respir.Dis.109:233-238 (1974) such as Schwartz).Finally, adenovirus mediated gene transfer is proved to be in a large amount of situations, comprises α-1-antitrypsin and CFTR are transferred to (Rosenfeld, M.A. etc. (1991) Science 252:431-434 in the cotton mouse lung; Rosenfeld etc., (1992) Cell 68:143-155).In addition, attempt to determine that adenovirus is that the broad research of the virulence factor of human cancer is (negative) (Green, M. etc. (1979) the Proc.Natl.Acad.Sci.USA 76:6606) that negates without exception.
Can be used for suitable adenovirus vector of the present invention and for example be described in, Kozarsky and Wilson, Curr.Opin.Genet.Devel.3:499-503 (1993); Rosenfeld etc., Cell 68:143-155 (1992); Engelhardt etc., Human Genet.Ther.4:759-769 (1993); Yang etc., Nature Genet.7:362-369 (1994); Wilson etc., Nature 365:691-692 (1993); With United States Patent (USP) the 5th, 652, No. 224, it is incorporated into by reference at this.For example, adenovirus vector Ad2 can be with also growing in human 293 cells.These cells contain the E1 district of adenovirus and E1a and E1b are expressed in composing type ground, this by the product that this gene that lacks from carrier is provided with the defective adenoviral complementation.Except Ad2, the adenovirus of other kind (as, Ad3, Ad5 and Ad7) also can be used for the present invention.
In one embodiment, being used for adenovirus of the present invention is replication defective.The adenovirus require helper virus of replication defective and/or the help of package cell line form infectious particles.The virus of gained can infection cell and can be expressed the interested polynucleotide that are operably connected to promoter, but in most cells reproducible not.The adenovirus of replication defective can lack the following gene of one or more all or parts: E1a, E1b, E3, E4, E2a or L1 to L5.
In some other embodiments, use adeno associated virus (AAV) ground engineered cells in vitro or in the body.AAV is naturally occurring defective virus, needs helper virus to produce infectious particles (Muzyczka, N., Curr.Topics in Microbiol.Immunol.158:97 (1992)).It still can be incorporated into its DNA one of minority virus in non--somatoblast.Containing the carrier of few AAV to 300 base pairs can be packaged and can integrate, but the space of foreign DNA is limited to about 4.5kb.It is known in the art producing and using the method for this AAV.For example see United States Patent (USP) the 5th, 139,941,5,173,414,5,354,678,5,436,146,5,474,935,5,478,745 and 5,589, No. 377.
For example, be used for proper A AV carrier of the present invention and will comprise all sequences that dna replication dna, encapsidate and host-cellular integration are essential.Use standard cloning process, such as Sambrook etc., MolecularCloning:A Laboratory Manual, those described in the Cold Spring Harbor Press (1989) are inserted into polynucleotide constructs in the AAV carrier.Use any standard technique subsequently, comprise that the AAV carrier transfections of will recombinating such as lipofection, electroporation, calcium phosphate precipitation are to the incasing cells that has infected helper virus.Suitable helper virus comprises adenovirus; Cytomegalovirus, vaccinia virus or herpesvirus.Behind transfection and the infection incasing cells, incasing cells contains generation the infectious AAV virion of polynucleotide constructs.These virions are used in vitro subsequently or body is transduceed interiorly eukaryotic cell.The cell of transduction will contain and be integrated into its genomic polynucleotide constructs, and will express fusion rotein of the present invention.
Another kind of gene therapy method comprises via be operably connected allos control zone and endogenous polynucleotide sequence (as the polypeptide of the present invention of encoding) of homologous recombination and (seeing as, No. the 5th, 641,670, the United States Patent (USP) of authorizing on June 24th, 1997; No. 96/29411, the international publication WO of JIUYUE in 1996 announcement on the 26th; No. 94/12650, the international publication WO that announced on August 4th, 1994; Koller etc., Proc.Natl.Acad.Sci.USA 86:8932-8935 (1989); With Zijlstra etc., Nature 342:435-438 (1989), it is incorporated into by reference at this.This method comprises that activation is present in the target cell but does not express in this cell usually or with the gene of the horizontal expression that is lower than expectation.
Use standard technique known in the art to prepare polynucleotide constructs, it contains the targeting sequence of promoter and promoter flank.Suitable promoter is as herein described.Targeting sequence and endogenous sequence are enough complementary to allow homologous recombination promoter-targeting sequence and endogenous sequence.The targeting sequence is with 5 ' end of the endogenous polynucleotide sequence of close enough expectation, thereby promoter will be operably connected to endogenous sequence behind homologous recombination.
Can use pcr amplification promoter and targeting sequence.In one embodiment, the promoter of amplification 5 ' contain different restriction enzyme sites with 3 ' end.In another embodiment, 3 of the first targeting sequence ' end contains identical restriction enzyme site with 5 ' end of the promoter of amplification, and 5 ' end of the second targeting sequence contains identical restriction enzyme site with 3 ' end of the promoter of amplification.The promoter of amplification and targeting sequence are digested and are linked together.
Promoter-targeting sequence construct body is delivered to cell as naked polynucleotide or with transfection-promoter such as liposome, virus sequence, virion, totivirus, lipofection thing, precipitant etc., as above describes in more detail.Can send P promoter-targeting sequence by any method, comprise direct needle injection, intravenous injection, local application, conduit infusion, particle accelerator etc.These methods are described in more detail following.
Promoter-targeting sequence construct body is obtained by cell.Between construct and the endogenous sequence homologous recombination takes place, so that endogenous sequence is placed under the promoter control.Promoter is with the expression of rear drive endogenous sequence.
The polynucleotide of albumin fusion proteins of the present invention of encoding can contain the secretory signal sequence of being convenient to secretory protein.Usually, signal sequence is positioned at the coding region of waiting the polynucleotide of being expressed or in 5 of this coding region ' end.Signal sequence can with interested polynucleotide homology or allos and can with treat transfected cell homology or allos.In addition, can use means known in the art chemosynthesis signal sequence.
Can make and use any above-mentioned polynucleotide constructs in any way, as long as this mode causes one or more molecules to be enough to the providing scale of therapeutical effect to reach.This comprises direct needle injection, system's injection, conduit infusion, biological projectile syringe (biolistic injector), particle accelerator (i.e. " particle gun "), gel foam sponge storage (gelfoam sponge depots), other commercially obtains storage material, osmotic pumps (as, Alza micropump), oral or suppository is solid-state (sheet or ball) pharmaceutical preparation and in intra-operative decant (decanting) or local application.For example, naked calcium phosphate-sedimentary plasmid is injected directly into rat liver and Rats Spleen or the plasmid of albumen-Bao quilt is injected directly into portal vein caused the gene expression (Kaneda etc., Science 243:375 (1989)) of alien gene in rat liver.
In one embodiment, methods of use for topical application is to pass through direct injection.In another embodiment, with the compound albumin fusion proteins of the present invention of delivery vector by be injected directly into tremulous pulse and use or local application in arteriosomes.In arteriosomes, refer to the compositions local application in tremulous pulse is counted cm range and at embodiment injectable composition in tremulous pulse number millimeter scope.
Another kind of methods of use for topical application is in surgical wound or contacts polynucleotide constructs of the present invention on every side.For example, the patient can experience operation and polynucleotide constructs can be applied to wound inner tissue surface or construct can be injected in the wound inner tissue zone.
The therapeutic combination that can be used for systemic application comprises and the compound fusion rotein of the present invention of the delivery vector of targeting of the present invention.The suitable delivery vector that is used for systemic application comprise the liposome that comprises part so that the carrier targeting in specific part.In concrete embodiment, the suitable delivery vector that is used for systemic application comprise the liposome that comprises albumin fusion proteins of the present invention so that the carrier targeting in specific part.
In one embodiment, the systemic application method comprises that intravenous injection, aerosol, oral and percutaneous (part) send.Intravenous injection can use in this area standard method to carry out.Aerosol is sent and also can be used this area standard method to carry out (for example see, Stribling etc., Proc.Natl.Acad.Sci.USA189:11277-11281,1992, it is incorporated into by reference at this).Oral delivery can be by making polynucleotide constructs of the present invention and compound the carrying out of carrier that can stand the degraded of digestive enzyme in the animal intestinal.The example of this carrier comprises plastic packaging capsule or tablet, such as known in the art those.Local delivery can by mix polynucleotide constructs of the present invention and the lipophilic reagent that can pass skin (as, DMSO) carry out.
Determine that the effective dose for the treatment of delivered substance can be depending on a large amount of factors, for example comprise, the chemical constitution of material and biological activity, animal age and weight, accurate disease and its severity and the route of administration that need treat.Therapeutic frequency depends on a large amount of factors, uses the amount of polynucleotide constructs and curee's health status and medical history such as every dosage.To determine accurately amount, dosage number and time of administration arrangement by attending doctor or veterinary.
Albumin fusion proteins of the present invention can be applied to any animal, for example, and mammal and birds.Exemplary mammal comprises people, dog, cat, mice, rat, rabbit, sheep, cattle, horse and pig, artificial specific implementations.
Biological activity
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be tested one or more biological activitys in mensuration.If albumin fusion proteins and/or polynucleotide show activity in particular assay, the treatment albumen corresponding to fusion rotein (portein) can be involved in the disease relevant with this biological activity probably.Therefore fusion rotein can be used for treating relevant disease.
In some embodiments, the present invention is contained treatment and is listed in the disease of table 1 " indication Y " row or the method for illness, comprise that the patient to expectation this treatment, prevention or alleviation uses albumin fusion proteins of the present invention with the amount of effective treatment, prevention or alleviation disease or illness, this fusion rotein comprises corresponding to the proteic treatment protein part of treatment that is disclosed in table 1 " treatment albumin X " row (with the disease to be treated or the same delegation of illness that are listed in table 1 " indication Y " row).
In other embodiment, the present invention contain treatment to particular treatment albumen be listed in table 1 " indication: Y " row disease or the method for illness, comprise that the patient to expectation this treatment, prevention or alleviation uses albumin fusion proteins of the present invention with the amount of effective treatment, prevention or alleviation disease or illness, this fusion rotein comprises corresponding to the proteic treatment protein part of the treatment relevant with indication among the embodiment.
The present invention comprises the albumin fusion proteins that the coded cell of polynucleotide of coding SEQ ID NO:Y produces particularly.When these polynucleotide were used for from the cellular expression encoded protein, the natural secretion of cell and procedure of processing produced the albumen of the signal sequence that lacks the row 4 that are listed in table 2 clearly and/or 11.The concrete aminoacid sequence of listed signal sequence shows in this manual or is well known in the art.Therefore in some embodiments, the present invention comprises the albumin fusion proteins (row 4 and/or 11 targeting sequencings that show that lack table 2) that is produced by cell.Also in other embodiments, polypeptide comprises SEQ IDNO:Y and is not listed in the row 4 of table 2 and/or 11 concrete targeting sequencing.The compositions that comprises these two kinds of embodiments (comprising pharmaceutical composition) also can be provided.Comprise disease or illness that these albumin fusion proteins are listed as proteic " indication: Y " that is listed in table 1 of particular treatment with treatment, prevention or alleviation specifically.
In some embodiments, fusion rotein of the present invention can be used for diagnosis, prognosis evaluation, prevent and/or treat and relate to hormonal system and (for example see, below " endocrine illness " part), nervous system (for example sees, below " neuropathy " part), immune system (for example sees, below " immunocompetence " part), respiratory system (for example sees, below " breathe illness " part), cardiovascular system (for example sees, below " cardiovascular disease " part), reproductive system (for example sees, below " reproductive system illness " part), digestive system (for example sees, below " gastrointestinal tract disease " part) disease and the disease and/or the illness of illness, the disease and/or the illness that relate to cell proliferation (for example see, below " hyper-proliferative illness " part), and/or relate to the disease of blood or illness (for example see, below " blood-related disorders " part).
In some embodiments, albumin fusion proteins of the present invention can be used for diagnosing and/or prognosis evaluation and relevant disease and/or the illness of wherein expressing corresponding to the gene of the treatment protein part of fusion rotein of the present invention of tissue.
Therefore the polynucleotide of fusion rotein of the present invention and coding albumin fusion proteins of the present invention are used to diagnose, measure and/or treat and active relevant disease and/or illness, described activity includes but not limited to, prohormone activation, neurotransmitter activity, cellular signal transduction, cell proliferation, cell differentiation and cell migration.
More generally, fusion rotein of the present invention can be used for diagnosing, prognosis evaluation, prevents and/or treats disease and/or the illness relevant with following system with the polynucleotide of coding albumin fusion proteins of the present invention.
Immunocompetence
The polynucleotide of albumin fusion proteins of the present invention and coding albumin fusion proteins of the present invention can be used for the immune disease of treatment, prevention, diagnosis and/or prognosis evaluation, illness and/or disease, by for example, activate or suppress propagation, the differentiation of immunocyte or move (mobility) (chemotaxis).Immunocyte produces via the process that is called hemopoietic, produces myelocyte (platelet, erythrocyte, neutrophil cell and macrophage) and lymphoid cell (B and T lymphocyte) from pluripotent stem cell.The etiology of these immunological diseases, illness and/or disease can be (such as cancer and some autoimmune diseases) heredity, somatic, acquired (as, by chemotherapy or toxin) or infective.And the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used as the labelling of specific disease of immune system or illness or detect thing.
In another embodiment, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins can be used for treating immune disease and illness and/or inhibition or strengthen the immunne response of the cell generation relevant with the tissue of wherein expressing polypeptide of the present invention.
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for treatment, prevention, diagnosis and/or prognosis evaluation immunodeficiency, comprise inborn and acquired immunodeficiency.Wherein the example of the B cellular immunity deficiency of immunoglobulin level B cell function and/or B cell number minimizing comprises: the chain agammaglobulinemia of X-(Bu Ludunshi disease); the chain infant agammaglobulinemia of X-; the chain immunodeficiency of X-is accompanied high IgM; the chain immunodeficiency of non-X-is accompanied high IgM; the chain lymphoproliferative syndrome of X-(XLP); agammaglobulinemia comprises inborn and acquired agammaglobulinemia disease; the adult is the property sent out agammaglobulinemia just; tardy property agammaglobulinemia; dysgammaglobulinemia; hypogammag lobulinemia; non-specific hypogammag lobulinemia; recessive agammaglobulinemia (Swiss type); selective IgM deficiency; selective IgA deficiency; selectivity IgG subclass defective; IgG subclass defective (being with or without the IgA defective); Ig defective companion IgM increases; IgG and IgA defective companion IgM increase; antibody defective with normal or rising Ig; the Ig heavy chain deletion; kappa chain deficiency; B cell lymphocytic hyperplasia illness (BLPD); common anomaly immunodeficiency (CVID); common anomaly immunodeficiency (CVI) (acquired); with the temporary hypogammag lobulinemia of infant.
In concrete embodiment, use polynucleotide treatment, prevention, diagnosis and/or prognosis evaluation asynergy-capillary dilation or the disease relevant of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding with asynergy-capillary dilation.
Wherein the example of the innate immunity defective of T cell and/or B cell function and/or decreased number includes but not limited to: DiGeorge deformity (DiGeorge anomaly), serious symptom combination immunodeficiency (SCID) (includes but not limited to the chain SCID of X-, autosomal recessive SCID, adenosine deaminase deficiency, purine nucleoside phosphorylase (PNP) defective, II class MHC defective (bare lymphocyte symdrome), WAS and ataxia telangiectasia), thymic dysplasia, third and fourth pharyngeal pouch syndrome, 22q11.2 disappearance, chronic mucocutaneous candidiasis, natural killer cell defective (NK), congenital CD4+T-lymphopenia, main T cell defect (non-specific) companion immunodeficiency, nonspecific immunity defective with cell-mediated immunity.
In concrete embodiment, use polynucleotide treatment, prevention, diagnosis and/or prognosis evaluation DiGeorge deformity or the disease lopsided relevant of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding with DiGeorge.
Use the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding to treat, prevention, other immunodeficiency of diagnosis and/or prognosis evaluation includes but not limited to, chronic granulo matosis, CH, myeloperoxidase deficiency, leukocyte glucose-6-phosphate dehydrogenase (G6PD) defective, the chain lymphoproliferative syndrome of X-(XLP), leukocyte adhesion deficiency, the complement component defective (comprises C1, C2, C3, C4, C5, C6, C7, C8 and/or C9 deficiency), reticular dysgenesis, thymic alymphoplasia-aplasia, immunodeficiency companion thymoma, the congenital leukopenia of serious symptom, abnormal development companion immunodeficiency, newborn neutrophil cell reduces, short-limb dwarfism disease, and Nezelof syndrome-combination Ig immunodeficiency (Nezelof syndrome-combined immunodeficiency withIgs).
In one embodiment, use polynucleotide treatment, prevention, diagnosis and/or the prognosis evaluation immunodeficiency of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding and/or the disease relevant with above-mentioned immunodeficiency.
Polynucleotide at an embodiment fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used as the medicament that booster immunization is replied in the middle of the immunodeficiency individuality.In concrete embodiment, the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used as the medicament that booster immunization is replied in the middle of B cell and/or T cellular immunity deficiency individuality.
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for treatment, prevention, diagnosis and/or prognosis evaluation autoimmune illness.Many autoimmune illness are because immunocyte is identified as exogenous material with self irrelevantly.This inappropriate identification causes immunne response, damages host tissue.Therefore, use and can suppress immunne response, especially the polynucleotide of T cell proliferation, differentiation or the chemotactic fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can be the effective treatment that prevents the autoimmune illness.
The polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can be treated, prevention, the diagnosis and/or the autoimmune disease of prognosis evaluation or illness include but not limited to following one or more: systemic lupus erythematosus (sle), rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis, autoimmune thyroiditis, struma lymphomatosa, autoimmune hemolytic anemia, hemolytic anemia, thrombocytopenia, idiopathic thrombocytopenic purpura, the newborn thrombocytopenia of autoimmunity, idiopathic thrombocytopenic purpura, purpura (as, anaphylactoid purpura (Henloch-Scoenleinpurpura)), the autoimmunity cytopenia, goodpasture syndrome, pemphigus vulgaris, myasthenia gravis, Graves disease (hyperthyroidism) and insulin resistance diabetes (diabetesmellitus).
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be treated, other illness that may have the autoimmune composition of prevention and/or diagnosis includes but not limited to, the arthritis of II Collagen Type VI-cause, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, the neuritis, uveitis (uveitisophthalmia), the multiple endocrine glands disease, Lay Te Shi disease, stiff-man syndrome, autoimmune pulmonary inflammation disease, autism, guillain-Barre syndrome, insulin-dependent diabetes and autoimmunity inflammatory ophthalmic are suffered from.
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be treated, prevention, other illness that may have the autoimmune composition of diagnosis and/or prognosis evaluation includes but not limited to, have anti--collagen antibodies scleroderma (normal by as, kernel and other antinuclear antibodies characterize), MCTD (normal by as, antibody (as ribonucleoprotein) to extractible nuclear antigen characterizes), polymyositis (normal by as, nonhistones ANA characterizes), pernicious anemia (normal by as, anti-parietal cell, microsome and intrinsic factor antibody characterize), congenital Addison's disease (normal by as, body fluid and cell-mediated adrenal cells toxicity characterize, infertility (normal by as, antisperm antibody characterizes), glomerulonephritis (normal by as, antiglomerular basement membrane antibody or immunocomplex characterize), bullous pemphigoid (normal by as, IgG in the basement membrane and complement characterize), siogren's syndrome (normal by as, organize the nonhistones ANA of antibody and/or specificity (SS-B) to characterize) more, diabetes (normal by as, cell-mediated characterizes with the body fluid insular cellular antibody), with adrenergic resistance (comprising adrenergic resistance companion asthma or cystic fibrosis) (normal by as, B-adrenergic receptor antibody characterizes).
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be treated, prevention, other illness that may have the autoimmune composition of diagnosis and/or prognosis evaluation includes but not limited to, chronic active hepatitis (normal by as, smooth muscle antibody (SMA) characterizes), primary biliary cirrhosis (normal by as, mitochondrial antibody characterizes), other endocrine gland asthenia exhaust (some situations often by as, specific tissue antibody characterizes), vitiligo (normal by as, melanocyte antibody characterizes), vasculitis (normal by as, complement and/or low SC characterize in Ig and the blood vessel wall), (post-MI) behind the myocardial infarction (normal by as, heart antibody characterizes), cardiotomy syndrome (normal by as, heart antibody characterizes), urticaria (normal by as, IgG and IgM antibody characterize to IgE), atopic dermatitis (normal by as, IgG and IgM antibody characterize to IgE), asthma (normal by as, IgG and IgM antibody characterize to IgE), with many other inflammatories, granulomatous, degeneration and atrophic illness.
In one embodiment, use polynucleotide treatment, prevention, diagnosis and/or prognosis evaluation autoimmune disease and illness and/or the disease relevant with illness of the fusion rotein for example of the present invention and/or the albumin fusion proteins of the present invention of encoding with above-mentioned disease.In concrete embodiment, use polynucleotide treatment, prevention and/or the diagnostics classes rheumatic arthritis of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding.
In another embodiment, use polynucleotide treatment, prevention and/or the diagnostic system lupus erythematosus of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding.In another embodiment, use polynucleotide treatment, prevention and/or the diagnosis idiopathic thrombocytopenic purpura of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding.
Use polynucleotide treatment, prevention and/or the diagnosis of iga nephropathy of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding in another specific implementations.
In one embodiment, use polynucleotide treatment, prevention, diagnosis and/or prognosis evaluation autoimmune disease and illness and/or the disease relevant with illness of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding with above-mentioned disease.
In some embodiments, the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding are as immunosuppressant.
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for treating, disease, illness and/or the disease of prevention, prognosis evaluation and/or diagnosis hematopoietic cell.The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for increasing differentiation and the propagation that hematopoietic cell comprises pluripotent stem cell, reduce relevant disease, illness and/or disease to be devoted to treatment or prevention with some (perhaps many) type hematopoietic cells, include but not limited to that leukopenia, neutrophil cell reduce disease, anemia and thrombocytopenia.Alternatively, the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for increasing differentiation and the propagation that hematopoietic cell comprises pluripotent stem cell, increase relevant disease, illness and/or disease with treatment or prevention with some (perhaps many) type hematopoietic cells, include but not limited to histiocytosis.
Use the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding also can treat, prevent, diagnosis and/or prognosis evaluation anaphylaxis and disease, such as asthma (especially allergic asthma) or other breathing problem.And these molecules can be used for treatment, prevention, prognosis evaluation and/or diagnosis of allergy, incompatible to antigen molecule allergy or blood group.
In addition, the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for treating, prevent, the anaphylaxis of diagnosis and/or prognosis evaluation IgE-mediation.This anaphylaxis includes but not limited to asthma, rhinitis and eczema.In concrete embodiment, the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for external or the interior IgE of adjusting of body concentration.
And the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for diagnosis, prognosis evaluation, prevent and/or treat inflammatory disease.For example, because activation, propagation and/or the differentiation of the cell that the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can suppress to relate in the inflammatory reaction, so these molecules can be used for preventing and/or treating chronic and the acute inflammation disease.This inflammatory disease includes but not limited to, for example, the inflammation relevant with infection (as, septic shock, septicemia or system's inflammatory reaction syndrome), ischemia reperfusion injury, endotoxin cause death, the inductive injury of lung of hyperacute rejection, nephritis, cytokine or chemotactic factor of complement-mediation, inflammatory bowel, clone disease, cytokine (as, TNF or IL-1) excessively produce, breathe illness (as, asthma and anaphylaxis); Gastrointestinal tract disease (as, inflammatory bowel); Cancer (as, stomach, ovary, lung, bladder, liver and mammary gland); The CNS illness (as, multiple sclerosis; Ischemic brain injury and/or apoplexy, traumatic brain injury, nervus retrogression illness (as, parkinson disease and Alzheimer); The dementia that AIDS-is relevant; And prion disease); Cardiovascular disease (as, arteriosclerosis, myocarditis, cardiovascular disease and cardiopulmonary bypass complication); And many other disease, disease and illness that are characterized as inflammation (as, hepatitis, rheumatoid arthritis, gout, wound, pancreatitis, sarcoidosis, dermatitis, renal ischaemia-reperfusion injury, Graves disease, systemic lupus erythematosus (sle), diabetes and allograft rejection).
Because inflammation is basic defense mechanism, the inflammatory illness can influence almost any tissue of health.Therefore, the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for treated tissue-specificity inflammatory illness, include but not limited to adrenalitis, alveolitis, angiocholecystitis, appendicitis, balanitis, blepharitis, bronchitis, bursitis, carditis (carditis), cellulitis, cervicitis, cholecystitis, chorditis, cochlitis, colitis, conjunctivitis, cystitis, dermatitis, diverticulitis, encephalitis, endocarditis, esophagitis, salpingitis, fibrositis, folliculitis, gastritis, gastroenteritis, gingivitis, glossitis, hepatosplenitis, keratitis, labyrinthitis, laryngitis, lymphangitis, mastitis, otitis media, meningitis, metritis, mucositis, myocarditis, myositis (myosititis), myringitis, nephritis, the neuritis, orchitis, osteochondritis, otitis, pericarditis, tenosynovitis (peritendonitis), peritonitis, pharyngitis, phlebitis, poliomyelitis, prostatitis, pulpitis, retinitis, rhinitis, salpingitis, scleritis, sclerochoroiditis, scrotitis, sinusitis, spondylitis, pimelitis, stomatitis, synovitis, salpingitis, tendinitis, tonsillitis, urethritis and vaginitis.
In concrete embodiment, the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for diagnosis, prognosis evaluation, prevent and/or treat organ-graft refection and graft versus host disease.The organ rejection destroys the tissue of transplanting by the host immune cell via immunne response and takes place.Similarly, immunne response also relates to GVHD, but in this situation, the immune cell destruction host tissue that external source is transplanted.The activation of inhibition immunne response of the present invention, especially T-cell, propagation, differentiation or chemotactic polypeptide, antibody or polynucleotide and/or its agonist or antagonist can be effective treatment of prevention organ rejection or GVHD.In concrete embodiment, the fusion rotein of activation, propagation, differentiation or the chemotactic of inhibition immunne response of the present invention, especially T-cell and/or the polynucleotide of the albumin fusion proteins of the present invention of encoding can be effective treatment of prevention experimental allergic and super acute heteroplastic transplantation repulsion.
In other embodiments, the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for diagnosis, prognosis evaluation, prevent and/or treat immune complex disease, include but not limited to the vasculitis that serum sickness, post-streptococcal glomerulonephritis, polyarteritis nodosa and immune complex cause.
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for treatment, mensuration and/or prevention infection former (infectious agent).For example,, especially increase propagation, activation and/or the differentiation of B and/or T cell, can treat, mensuration and/or prophylaxis against infection diseases by increasing immunne response.Can be by strengthening existing immunne response or increasing immunne response by beginning new immunne response.Alternatively, the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding also can directly suppress infector (REFERENCE TO RELATED is listed part of infector or the like), and needn't cause immunne response.
In another embodiment, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding are as the vaccine adjuvant that strengthens antigenic immune responsiveness.In specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding are as the adjuvant that strengthens tumor-specific immune response.
In another specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding are as strengthening the adjuvant that antiviral immunity is replied.Can use compositions of the present invention as adjuvant and enhanced antiviral immunity reply and comprise virus as herein described or otherwise known in the art and viral relevant disease or symptom.In specific implementations, compositions of the present invention strengthens being selected from the immunne response by virus, disease or the symptom of the following group of forming as adjuvant: AIDS, meningitis, dengue fever, EBV and hepatitis (as, hepatitis B).In another specific implementations, compositions of the present invention strengthens being selected from the immunne response by virus, disease or the symptom of the following group of forming: HIV/AIDS, breathing syncytial virus, dengue fever, rotavirus, Japanese B encephalitis, first type and influenza B, parainfluenza, measles, cytomegalovirus, rabies, junin fever, chikungunya disease, Rift valley fever, herpes simplex and yellow fever as adjuvant.
In another specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding are as the adjuvant that strengthens anti--antibacterial or anti--fungus immunne response.Use compositions of the present invention as adjuvant can enhancedly resist-antibacterial or anti--fungus immunne response comprise antibacterial as herein described or otherwise known in the art or fungus and antibacterial or fungus relevant disease or symptom.In specific implementations, compositions of the present invention strengthens at being selected from by the antibacterial of the following group of forming or the immunne response of fungus, disease or symptom as adjuvant: tetanus, diphtheria, botulism and epidemic cerebrospinal meningitis B.
In another specific implementations, compositions of the present invention strengthens being selected from antibacterial or the fungus by the following group of forming as adjuvant, the immunne response of disease or symptom: vibrio cholera (Vibrio cholerae), Mycobacterium leprae (Mycobacterium leprae), salmonella typhi (Salmonella typhi), salmonella paratyphi (Salmonella paratyphi), meningitis naphthalene plucked instrument Salmonella (Meisseria meningitidis), streptococcus pneumoniae (Streptococcus pneumoniae), the B group B streptococcus, some kind of Shigella (Shigella spp.), enterotoxigenic E.Coli, enterohemorrhagic Escherichia coli and borrelia burgdorferi (Borrelia burgdorferi).
In another specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding strengthen the anti-parasitic immunne response as adjuvant.Use compositions of the present invention can enhanced anti-parasitic immunne response comprise parasite as herein described or otherwise known in the art and parasite relevant disease or symptom as adjuvant.In specific implementations, compositions of the present invention strengthens parasitic immunne response as adjuvant.In another specific implementations, compositions of the present invention strengthens plasmodium (malaria) or leishmanial immunne response as adjuvant.
In another specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding also can be used for treating infectious disease, comprise silicosis, sarcoidosis and idiopathic pulmonary fibrosis, for example by preventing raising and activating of mononuclear phagocyte.
In another specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding produce antibody to suppress or to strengthen immune-mediated response at polypeptide of the present invention as antigen.
In one embodiment, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding be applied to animal (as, mice, rat, rabbit, hamster, Cavia porcellus, pig, miniature pig (micro-pig), chicken, camel, goat, horse, cattle, sheep, dog, cat, the non-human primates and the mankind, in specific implementations for human) come the booster immunization system with one or more antibody of producing recruitment (as, IgG, IgA, IgM and IgE) with the generation of inducing high-affinity antibody more and immunoglobulins conversion (as, IgG, IgA, IgM and IgE) and/or to increase immunne response.
In another specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding are as the stimulus object of B cell to the pathogen response.
In another specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding are as the activator of T cell.
In another specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding are as the medicament that promoted its immune state before individuality is accepted immunosuppressive therapy.
In another specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding are as inducing the more medicament of the antibody of high-affinity.
In another specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding are as the medicament that increases serum immune globulin concentration.
In another specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding are as the medicament of the recovery of accelerating immunocompromised individuality.
In another specific implementations, the medicament that the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding are replied with oppose old colony and/or neonate booster immunization.
In another specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding bone marrow transplantation and/or other transplanting (as, of the same race or xenotransplant) before, during or afterwards as immune-system enhancers.For transplanting, can be before transplanting, simultaneously and/or use compositions of the present invention afterwards.In specific implementations, using compositions of the present invention after the transplanting, before the recovery of T cell colony begins.In another specific implementations, after transplanting, after the T cell colony begins to recover but before the B cell colony recovers fully, use compositions of the present invention first.
In another specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding suffer from the medicament of the individual booster immunization response of acquired B cell function forfeiture with opposing.Cause and to alleviate or the situation of the acquired B cell function forfeiture for the treatment of includes but not limited to HIV infection, AIDS, bone marrow transplantation and B cell chronic lymphocytic leukemia (CLL) by the polynucleotide of using the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding.
In another specific implementations, the medicament that the individual booster immunization that the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding suffer from temporary immunodeficiency with opposing is replied.Cause and to alleviate or the situation of the temporary immunodeficiency for the treatment of includes but not limited to by the polynucleotide of using the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding, from the viral infection recovery of (as, influenza), the disease relevant, from the recovery of infectious mononucleosis or with the disease that stress be correlated with, from the recovery of measles, from the recovery of blood transfusion with from the recovery of operation with malnutrition.
In another specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding are as the regulator of the antigen presentation of mononuclear cell, dendritic cell and/or B-cell.In one embodiment, enhancement antigen is presented or the antagonism antigen presentation in the external or body of the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding.And in related embodiment, the enhancing of this antigen presentation or antagonism can be used as Anti-tumor treatment or in order to regulate immune system.
In another specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding are as the medicament that guides individual immune system towards the development humoral response (be TH2) opposite with the TH1 cell response.
In another specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding are as induced tumor propagation, and make it to the antitumor agent means of susceptible more thus.For example, multiple myeloma is slow fissility disease, is that nearly all Anti-tumor therapy all is difficult to treat it therefore.If force these cells to be bred quickly, may change their susceptibility feature so.
In another specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding are used as the stimulus object that the B cell produces in such as AIDS, chronic lymphocytic illness and/or common anomaly immunodeficiency pathology such as (Common Variable Immunodificiency).
In another specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding are as producing and/or the adenoid therapy of regenerating after operation, wound or genetic defect.In another specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding are used for pretreatment bone marrow sample before transplanting.
In another specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding cause the heredity of immunologic inadequacy/immunodeficiency (such as observed in SCID patient) to bear the therapy based on gene of the illness of (genetically inherited) as treatment.
In another specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding influence monocytic management of parasitic diseases such as leishmania as the means of activated monocyte/macrophage with defence.
In another specific implementations, the polynucleotide that utilize the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding as means regulate by polypeptide of the present invention cause produce by excretory cytokine.
In another embodiment, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding are used for one or more application as herein described, because they can be applicable to veterinary.
In another specific implementations, the polynucleotide that utilize the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding stop various aspects at foreign substance or the immunne response of self as means.Expectation stops the disease or the examples of disorders of some aspect of immunne response to comprise autoimmune illness such as lupus and arthritis, and to the immune responsiveness of skin allergy, inflammation, enteropathy, damage and the disease/illness relevant with pathogen.
In another specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding are as prevention B cell proliferation and the Ig excretory therapy relevant with autoimmune disease such as idiopathic thrombocytopenic purpura, systemic lupus erythematosus (sle) and multiple sclerosis.
In another specific implementations, the polynucleotide of the agonist of polypeptide of the present invention, antibody, polynucleotide and/or fusion rotein of the present invention or antagonist and/or the albumin fusion proteins of the present invention of encoding are as B and/or the T cell inhibitor at endothelial cell migration.This activity disorganize structure or natural replying, and can be used for for example interrupting immunne response and stop septicemia.
In another specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding are used for the treatment of as tangible chronic hypergammaglobulinemia in the diseases such as the uncertain MG of meaning (MGUS), waldenstrom's disease (Waldenstrom ' s disease), relevant spy's property sent out MG and plasmocytoma.
In another specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for for example suppressing the activation and the polypeptide chemotaxis of macrophage and precursor and neutrophil cell, basophilic granulocyte, bone-marrow-derived lymphocyte and some T-cell subsets such as activating T cell and cd8 cell toxicity T cell and natural killer cell in some autoimmune and chronic inflammatory and infectious disease.The example of autoimmune disease is on the books in this article, comprises multiple sclerosis and insulin-dependent diabetes.
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding also can be used for the too much syndrome of the treatment special property sent out eosinophilic granulocyte, for example generation and the migration by preventing the eosinophilic granulocyte.
In another specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding are used to strengthen or suppress the lysis of complement-mediated.
In another specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding are used for strengthening or suppressing the antibody dependent cellular cytotoxicity.
In another specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding also can be used for treating arteriosclerosis, for example by preventing the infiltration of mononuclear cell in arterial wall.
In another specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for treating adult respiratory distress syndrome (ARDS).
In another specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for stimulating wound and tissue repair, stimulation blood vessel that the reparation of blood vessel or lymphatic disease or illness takes place and/or stimulates.In addition, the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for the stimulating mucosal surface regeneration.
In specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding are used to diagnose, prognosis evaluation, treat and/or prevent that to produce defective, recurrent infection and/or function of immune system obstacle with constitutional or acquired immunodeficiency, serum immune globulin be the illness of feature.And, the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for treatment or prevention joint, bone, skin, and/or the infection of the parotid gland, the infection of blood dissemination (as, septicemia, meningitis, septic arthritis, and/or osteomyelitis), autoimmune disease (as autoimmune disease disclosed herein), the inflammatory illness, and cancerate, and/or with these infection, disease, illness and/or cancerate relevant any disease or illness or disease, include but not limited to, CVID, other primary immunodeficiency, the HIV disease, CLL, the recurrent bronchitis, sinusitis, otitis media, conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster (as, serious herpes zoster), and/or Pneumocystis carinii (Pneumocystis carnii).Can utilize the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding to prevent, other disease and the illness of diagnosis, prognosis evaluation and/or treatment include but not limited to HIV infection, HTLV-BLV infection, lymphopenia, phagocyte bactericidal dysfunction anemia, thrombocytopenia and hemoglobinuria.
In another embodiment, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding are used for the treatment of and/or diagnose and suffer from common variable immunodeficiency (" CVID "; Also be called " acquired agammaglobulinemia disease " and " acquired hypogammaglobulinemia ") or the individuality of subclass that should disease.
In specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for diagnosis, prognosis evaluation, prevent and/or treat cancer or neoplasm, comprise immunocyte or immuning tissue-associated cancer or neoplasm.Cancer that the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can prevent, diagnose or treat or neoplastic example include but not limited to, disease and the illness described in the part that the disease that acute myeloid leukemia, chronic granulocytic leukemia, Hokdkin disease, non_hodgkin lymphoma, acute lymphocytic anemia (ALL) chronic lymphocytic leukemia, plasmocytoma, multiple myeloma, Burkitt lymphoma, EBV-transform and/or this paper other places title are " excess proliferative illness ".
In another specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding are as the therapy of the cell proliferation that reduces large B cell lymphoid tumor.
In another specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding are as the means that reduce the B cell responsibility relevant with chronic granulocytic leukemia with Ig.
In specific implementations, compositions of the present invention is used as the medicament that booster immunization is replied in B cellular immunity deficiency individuality (for example having experienced the individuality of partial or complete splenectomy).
The blood related disorders
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for regulating hemostasis (stopping hemorrhage) or thromboembolism (dissolved blood clot) activity.For example, by increasing hemostasis or thrombolysis activity, the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for treating or preclude blood is condensed disease, illness and/or disease (as, afibrinogenemia, coagulation factor deficiency, hemophilia), the wound that causes of blood blood platelet disorder, illness and/or disease (as, thrombocytopenia) or wound, operation or other reason.Perhaps, can reduce that the hemostasis or the polynucleotide of the fusion rotein of the present invention of thrombolysis activity and/or the albumin fusion proteins of the present invention of encoding can be used for suppressing or the dissolving blood clotting.These molecules can be significant in treatment or prevention of cardiac outbreak (infarction), apoplexy or cicatrization.
In specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for prevention, diagnosis, prediction and/or treatment thrombosis, artery thrombosis, venous thrombosis, thromboembolism, pulmonary infarction, arteriosclerosis, myocardial infarction, transient ischemic attack, unstable angina.In specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for preventing the obstruction (occulsion) of saphenous vein graft, with reduce angioplasty may with the risk of peri-operation period thrombosis (periprocedural thromosis), suffers from the risk of patient's apoplexy of atrial fibrillation (comprising non-rheumatic atrial fibrillation) with reduction, with reduction and artificial heart valve and or the relevant thromboembolism risk of mitral valve disease.Other purposes of the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding includes but not limited to, prevent health install outward (Extrcorporeal device) (as, the vascular access diverter of blood vessel interpolation pipe, hemodialysis patients, haemodialysis control unit and cardiovascular shunt machine) obstruction.
In another embodiment, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for preventing, diagnose, predict and/or treat the disease and the illness of blood and/or the blood formation organ relevant with the tissue of expressing polypeptide of the present invention.
The polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for regulating hemopoietic activity (formation blood cell).For example, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for increasing the amount of whole blood cell or its subgroup, such as for example, erythrocyte, lymphocyte (B or T cell), medullary cell (as, basophilic granulocyte, eosinophilic granulocyte, neutrophil cell, mastocyte, macrophage) and platelet.The ability that reduces the amount of blood cell or blood cell subgroup may be useful for preventing, detect, diagnose and/or treat following anemia and leukopenia.Perhaps, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for reducing the amount of whole blood cell or its subgroup, such as for example, erythrocyte, lymphocyte (B or T cell), medullary cell (as, basophilic granulocyte, eosinophilic granulocyte, neutrophil cell, mastocyte, macrophage) and platelet.The ability that reduces the amount of blood cell or blood cell subgroup can be useful such as for example eosinophilia for prevention, detection, diagnosis and/or treatment leukocytosis (leukocytose).
The polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for prevention, treatment or diagnosis blood dyscrasia.
Anemia be wherein in erythrocyte number or the erythrocyte amount of haemachrome (carrying the albumen of oxygen) be lower than the disease of normal value.Anemia can be caused by excessive minimizing or erythrophthoric increase (haemolysis) hemorrhage, that erythrocyte produces.The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for treatment, prevention and/or diagnosis anemia.The anemia that can use the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding to treat, prevent or diagnose comprises iron deficiency anemia, low hematochrome anemia, microcytic anemia, chlorosis (chlorosis), hereditary sideroblastic anemia (siderob; Astic anemia), specially send out that property acquired sideroblastic anemia, red blood cell development are complete, megaloblastic anemia (as, pernicious anemia (vitamin B12 deficiency) and folic acid deficiency anemia), aplastic anemia, hemolytic anemia (as, autoimmune hemolytic anemia (helolytic anemia), microangiopathic hemolytic anemia and sudden nocturnal hemoglobinuria).The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for treating, prevent and/or the anemia of diagnosis and disease association, include but not limited to the anemia relevant with systemic lupus erythematosus (sle), cancer, lymphoma, chronic nephropathy and splenomegaly.The anemia that the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for treating, prevention and/or diagnostic medicine treatment produce is such as the anemia relevant with methyldopa, dapsone and/or sulfonamide.In addition, the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for treating, preventing and/or diagnosis and the unusual relevant anemia of red blood cell structure, including but not limited to hereditary spherocytosis, hereditary elliptocytosis, glucose-6-pbosphate dehydrogenase deficiency and sicklemia.
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for treatment, prevention and/or diagnosis haemachrome unusual (as, unusual with the haemachrome of sicklemia, haemachrome C disease, haemachrome S-C disease and haemachrome E disease association).In addition, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for diagnosis, prognosis evaluation, prevent and/or treat thalassemia, include but not limited to α-Di Zhonghaipinxue and β-thalassemic main and less important form.
In another embodiment, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for diagnosis, prognosis evaluation, prevent and/or treat bleeding disorder, include but not limited to thrombocytopenia (as, idiopathic thrombocytopenic purpura and thrombocytopenic purpura,thrombotic), Von WillebrandShi disease, heritability platelet illness is (as storage pool disease (storagepool disease) such as CH and Hai-Pu two Cotards, the thromboxane A2 dysfunction, thrombasthenia and Bai-Suo two Cotards), hemolytic uremic syndrome, hemophilia (hemophelia) such as hemophilia (hemophelia) A or proconvertin lack and christmas disease (Christmas disease) or plasma thromboplastin component shortage, hereditary hemorrhagic telangiectasia (Hemorhhagic Telangiectsia) claim rendu-Osler-Weber syndrome again, anaphylactoid purpura (HenochSchonlein purpura) and disseminated inravascular coagulation.
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can use any thrombotest known in the art to monitor to the influence of blood coagulation time, include but not limited to whole blood partial thromboplastin time (PTT), activated partial Thromboplastin time (aPTT), activated clotting time (ACT), multiple calcium activated clotting time or lee-White clotting time.
Several conditions and multiple medicine can cause dysfunction of platelet.Therefore in specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for diagnosis, prognosis evaluation, prevent and/or treat acquired dysfunction of platelet, such as following renal failure, leukemia, multiple myeloma, liver cirrhosis, with the dysfunction of platelet and the dysfunction of platelet relevant with Drug therapy of systemic lupus erythematosus (sle), described Drug therapy comprises the aspirin of high dose, ticlopidine, nonsteroidal antiinflammatory drug (is used for arthritis, pain and sprain) and penicillin treat.
In another embodiment, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for diagnosing, prognosis evaluation, prevent and/or treat and be characterized as the leukocyte number and increase or reduce or increase or reduce with the leukocyte number relevant disease and illness.Leukopenia takes place when being lower than normal value when the leukocyte number reduces to.Leukopenia includes but not limited to that neutrophil cell reduces and lymphopenia.The leukocyte number is called leukocytosis than normal value increase.Health produces the leukocyte that number increases between infection period.Therefore leukocytosis may only be the normal physiological mathematic(al) parameter that reflection is infected.Perhaps, leukocytosis may be indicated damage or other diseases such as cancer.Leukocytosis (leukocytose) includes but not limited to eosinophilia and macrophage gathering.In specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for diagnosis, prognosis evaluation, prevent and/or treat leukopenia.In other specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for diagnosis, prognosis evaluation, prevent and/or treat leukocytosis.
Leukopenia can be that all types leukocyte generally reduces, and perhaps can be the specific leukocytic exhaustion of some type.Therefore in specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for diagnosing, prognosis evaluation, prevent and/or treat and be called the neutrophil cell decreased number that neutrophil cell reduces.Can use the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding to diagnose, prognosis evaluation, the neutrophil cell minimizing that prevents and/or treats includes but not limited to child's heritability agranulocytosis, the familial neutrophil cell reduces, the circulation neutrophil cell reduces, the diet shortage (as, vitamin B12 deficiency or folic acid deficiency) neutrophil cell minimizing that cause or associated, Drug therapy (as, antibiotherapy is treated such as penicillin, sulfoamide (sulfonamide) treatment, the anticoagulant treatment, anticonvulsant drug, antithyroid drug and cancer chemotherapy) neutrophil cell minimizing that cause or associated, destroy the neutrophil cell minimizing that increase causes with neutrophil cell, wherein neutrophil cell destroys to increase and can follow in some antibacterial or viral infection, irritated illness, autoimmune disease, individuality suffers from the disease of splenomegaly (as Felty syndrome, malaria and sarcoidosis) and some drugs therapeutic scheme and taking place.
The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for diagnosis, prognosis evaluation, prevent and/or treat lymphopenia (B and/or T lymphocyte number reduce), include but not limited to by following that cause or associated lymphopenia: stress, Drug therapy (is treated as corticosteroid medication, cancer chemotherapy and/or X-ray therapy), AIDS infect and/or other disease such as for example, cancer, rheumatoid arthritis, systemic lupus erythematosus (sle), chronic infection, some viral infection and/or heritability illness (as, the DiGeorge syndrome, WAS, severe combined immunodeficiency, blood capillary mutual aid sexual maladjustment (ataxia telangiectsia)).
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for diagnosing, prognosis evaluation, prevent and/or treat disease and the illness relevant with macrophage number and/or macrophage function, include but not limited to familial splenic anemia, niemann-Pick disease, letterer-Siwe disease and Hand Sch.
In another embodiment, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for diagnosing, prognosis evaluation, prevent and/or treat disease and the illness relevant with eosinophilic granulocyte's number and/or eosinophilic granulocyte's function, include but not limited to the special too much syndrome of property eosinophilic granulocyte, eosinophilia-muscle pain syndrome and the Hand Sch sent out.
In another embodiment, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for diagnosis, prognosis evaluation, prevent and/or treat leukemia and lymphoma, include but not limited to acute lymphocytic (lymphoblast property (lymphpblastic)) leukemia (ALL), acute bone marrow (myeloid, bone marrow, myeloblast property or bone marrow mononuclear cell) leukemia, chronic lymphocytic leukemia (as, B cell leukemia, the T chronic myeloid leukemia, Sezary syndrome, and hairy cell leukemia (leukenia)), chronic myeloid (bone marrow, bone marrow or granulocytic) leukemia, hodgkin's lymphoma, non Hodgkin lymphoma, Burkitt lymphoma and mycosis fungoides.
In other embodiments, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for diagnosis, prognosis evaluation, prevent and/or treat plasmacytic disease and illness, include but not limited to plasma cell dyscrasias, MG (gammaopathies), uncertain MG, multiple myeloma, macroglobulinemia, waldenstrom's macroglobulinemia, cryoglobulinemia and the Raynaud's phenomenon of meaning.
In other embodiments, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for treatment, prevention and/or diagnosis myelosis illness, include but not limited to polycythemia vera, relative erythrocytosis, secondary polycythemia, myelofibrosis, acute myelofibrosis, agnogenic myeloid metaplasia (myelod metaplasia), thrombocytosis (comprising constitutional and Secondary cases thrombocytosis) and chronic granulocytic leukemia.
In other embodiments, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used as preoperative treatment to increase the blood cell generation.
In other embodiments, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used as that the migration that strengthens neutrophil cell, eosinophilic granulocyte and macrophage, phagocytosis, superoxides produce, the Cytotoxic medicament of antibody dependent cellular.
In other embodiments, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used as the medicament that increased stem cell number in the circulation before the stem cell extraction method.In another specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used as the medicament that increased stem cell number in the circulation before thrombapheresis.
In other embodiments, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used as increases the medicament that cytokine produces.
In other embodiments, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for prevention, diagnosis and/or treatment constitutional hemopoietic illness.
The hyper-proliferative illness
In some embodiments, the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for treatment or detect the hyper-proliferative illness, comprise neoplasm.The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can suppress the propagation of illness via direct or indirect interaction.Perhaps, the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can be bred the cell that other can suppress the hyper-proliferative illness.
For example,, especially increase the antigenic property of hyper-proliferative illness, or, can treat the hyper-proliferative illness by propagation, differentiation or mobilization T cell by increasing the immunne response of hyper-proliferative illness.Can be by strengthening existing immunne response or increasing this immunne response by beginning new immunne response.Perhaps, reduce the method that immunne response also can be used as treatment hyper-proliferative illness, such as chemotherapeutics.
Can use the example of the hyper-proliferative illness that the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding treat or detect to include but not limited to be positioned at the neoplasm at following position: colon, abdominal part, bone, mammary gland, digestive system, liver, pancreas, peritoneum, endocrine body of gland (adrenal gland, parathyroid gland, hypophysis, testis, ovary, thymus, thyroid), eye, head and neck, neural (maincenter and periphery), lymphsystem, pelvis, skin, soft tissue, spleen, chest and urogenital tract.
Similarly, other hyper-proliferative illness can be treated or detect to the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding also.The example of such hyper-proliferative illness includes but not limited to: acute child's lymphoblastic leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia, adrenocortical carcinoma, adult's (constitutional) hepatocarcinoma, adult's (constitutional) hepatocarcinoma, adult's acute lymphoblastic leukemia, adult's acute myelogenous leukemia, adult's Hodgkin, adult's hodgkin's lymphoma, adult lymphoid cellularity leukemia, adult's non Hodgkin lymphoma, become the human primary liver cancer, adult's soft tissue sarcoma, the AIDS-lymphoma of being correlated with, AIDS-is relevant to cancerate, anus cancer, astrocytoma, cancer of biliary duct, bladder cancer, osteocarcinoma, the brain stem glioma, cerebroma, breast carcinoma, renal pelvis and carcinoma of ureter, central nervous system's (constitutional) lymphoma, central nervous system lymphoma, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, child's (constitutional) hepatocarcinoma, child's (constitutional) hepatocarcinoma, the children acute lymphoblastic leukemia, the children acute myelomatosis, child's brain stem glioma, child's cerebellar astrocytoma, child's cerebral astrocytoma, child's extracranial germ cell tumor, child's Hodgkin, child's hodgkin's lymphoma, child's hypothalamus and visual pathway glioma, child's lymphoblastic leukemia, child's medulloblastoma, child's non Hodgkin lymphoma, child's pineal gland and curtain are gone up original neuroectodermal tumor, child's primary hepatocarcinoma, child's rhabdomyosarcoma, child's soft tissue sarcoma, child's visual pathway and hypothalamus glioma, chronic lymphocytic leukemia, chronic granulocytic leukemia, colon cancer, cutaneous T cell lymphoma, the endocrine islet-cell carcinoma, carcinoma of endometrium, ependymoma, epithelial cancer, the esophageal carcinoma, Ewing sarcoma and related neoplasms, the exocrinosity cancer of pancreas, the extracranial germ cell tumor, the outer sexual cell tumor of gonad, cholangiocarcinoma, cancer eye, female breast carcinoma, familial splenic anemia, carcinoma of gallbladder, gastric cancer, the gastrointestinal carcinoid tumor, the gastrointestinal tumor, germinoma, trimester of pregnancy trophoblastic tumor, hairy cell, head and neck cancer, hepatocarcinoma, Hodgkin, hodgkin's lymphoma, hypergammaglobulinemia, hypopharyngeal carcinoma, intestinal cancer, intraocular melanoma, islet-cell carcinoma, the islet cells cancer of pancreas, Kaposi sarcoma, renal carcinoma, laryngeal carcinoma, lip and oral cancer, hepatocarcinoma, pulmonary carcinoma, the lymphocytic hyperplasia illness, macroglobulinemia, male breast carcinoma, malignant mesothe, malignant thymoma, medulloblastoma, melanoma, mesothelioma, the recessive constitutional squamous epithelial cancer of transitivity neck cancer, transitivity constitutional squamous epithelial cancer neck cancer, transitivity squamous epithelial cancer neck cancer, multiple myeloma, multiple myeloma/plasma cell neoplasm, myelodysplastic syndrome, myelocytic leukemia, myeloid leukemia, the myelosis illness, nasal cavity and paranasal sinuses, nasopharyngeal carcinoma, neuroblastoma, trimester of pregnancy non Hodgkin lymphoma, non-melanoma skin cancer, nonsmall-cell lung cancer, recessive constitutional transitivity squamous epithelial cancer neck cancer, the oropharynx cancer, bone-/the malignant fibrous sarcoma, osteosarcoma/malignant fibrohistiocytoma, the malignant fibrohistiocytoma of osteosarcoma/bone, epithelial ovarian cancer, the ovarian germ cell tumor, ovary hangs down pernicious potential tumor, cancer of pancreas; paraprotein mass formed by blood stasis; purpura; parathyroid gland cancer; carcinoma of penis; pheochromocytoma; pituitary tumor; plasma cell neoplasm/multiple myeloma; primary central nervous system lymphoma; primary hepatocarcinoma; carcinoma of prostate; rectal cancer; renal cell carcinoma; renal pelvis and carcinoma of ureter; retinoblastoma; rhabdomyosarcoma; salivary-gland carcinoma; sarcoid sarcoma; Sezary syndrome; skin carcinoma; small cell lung cancer; carcinoma of small intestine; soft tissue sarcoma; squamous epithelial cancer neck cancer; gastric cancer; original neuroderm and strobile adenoma on the curtain; T-cell lymphoma; carcinoma of testis; thymoma; thyroid carcinoma; renal pelvis and transitional cell carcinoma of ureter; divide a word with a hyphen at the end of a line renal pelvis and carcinoma of ureter; trophoblastic tumor; ureter and renal pelvis cell carcinoma; carcinoma of urethra; uterus carcinoma; sarcoma of uterus; cancer of vagina; visual pathway and hypothalamus glioma; carcinoma vulvae; waldenstrom's macroglobulinemia; Wilms' tumor; with any other hyperproliferation disease that is arranged in above-mentioned tract, except neoplasia.
In another embodiment, use the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding to diagnose, prognosis evaluation, prevent and/or treat cancerate before disease and prevent progress to neoplasm or malignant state (including but not limited to aforesaid illness).Such use is necessary in the following cases: situation in advance to neoplasm or malignant state (including but not limited to aforesaid illness) progress occurs when (comprising known or suspicious situation in advance), hypertrophy has particularly appearred comprising, change give birth to, (summary to this misgrowth disease sees Robbins and Angell during the situation of particularly non-newborn sexual cells growth such as abnormal development, 1976, Basic Pathology (basic pathology), the 2nd edition, W.B.Saunders Co., Philadelphia, the 68-79 page or leaf).
Hypertrophy is a kind of form of the controlled propagation of cell, comprises in tissue or the organ that cell number increases and significantly do not change structure or function.Can use the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding to diagnose, prognosis evaluation, the hypertrophy illness that prevents and/or treats includes but not limited to, blood vessel folliculus mediastinal lymph nodes hypertrophy, ALH is with the eosinophilia, the atypia melanocytic hyperplasia, basal cell hyperplasia, optimum giant lymph node hyperplasia, hypercementosis, inborn adrenal hyperplasia, inborn sebaceous hyperplasia, cystic hyperplasia, cystic hyperplasia of breast, denture hypertrophy, ductal hyperplasia, endometrial hyperplasia, fibromuscular hyperplasia, focal epithelial hyperplasia, gum hypertrophy, fibrous inflammatory hyperplasia, the inflammatory papillary hyperplasia, intravascular papillary endothelial hyperplasia, nodular hyperplasia of prostate gland, nodular regenerative hyperplasia, pseudoepitheliomatous hyperplasia, senile sebaceous gland hyperplasia and verrucous hyperplasia.
Changing life is a kind of form of cell controlled growth, and wherein the cell of one type adult or differentiation fully replaces the one-tenth somatic cell of another type.Can use the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding to diagnose, prognosis evaluation, the change natural disposition illness that prevents and/or treats includes but not limited to that agnogenic medullization is living, Apocrine metaplasia, atypiaization is given birth to, give birth to from realizing (autoparenchymatous metaplasia), connective tissueization is given birth to, epithelial metaplasia, intestinalization is given birth to, metaplastic anemia, change the natural disposition skeletonization, metaplastic polyp, medullization is given birth to, the constitutional medullization is given birth to, the Secondary cases medullization is given birth to, squamous metaplasia, amniotic membrane squamous metaplasia and symptomatic medullization are given birth to.
Abnormal development is the omen of cancer normally, and is mainly seen in epithelium; It is the most unordered non-tumprigenicity cell growth forms, comprises the forfeiture of conforming forfeiture of cell individual and cellularity sexual orientation.The abnormal development cell often has unusual huge deep dyed color nuclear, and the performance pleomorphism.Abnormal development typically takes place when having chronic stimulation or inflammation.Can use the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding to diagnose, prognosis evaluation, the abnormal development illness that prevents and/or treats includes but not limited to the hidroschesis ectodermal dysplasia, foreface (anterofacial) abnormal development, asphyxiating breast abnormal development, atrium-finger abnormal development, bronchopulmonary dysplasia, encephalodysplasia, cervical atypical hyperplasia (cervical dysplasia), chondroectodermal dysplasia, clavicle skull abnormal development, inborn ectodermal dysplasia, skull backbone (craniodiaphysial) abnormal development, cranium wrist metatarsal abnormal development, skull epiphysis (craniometaphysial) abnormal development, dentinal dysplasia, diaphyseal sclerosis, ectodermal dysplasia, enamel development is unusual, the brain eyeball development is unusual, epiphysis half limb abnormal development, dysplasia epiphysealis multiplex, chondrodystrophia congenita punctata, epithelial development is unusual, face-refer to-genital development is unusual, familial jaw fiber abnormal development, familial white gauffer sexual abnormality, fiber flesh sexual abnormality, bone fibres abnormal development, red bone sexual abnormality, heritability kidney-retinal development is unusual, the perspiration ectodermal dysplasia, the hypohidrosis ectodermal dysplasia, the lymphopenia thymus development is unusual, development of breast is unusual, mandibular bone face abnormal development, metaphysis abnormal development, Mondini abnormal development (Mondini dysplasia), single fibrous dysplasia of bone, mucous epithelium abnormal development, dysplasia epiphysealis multiplex, eye ear vertebra abnormal development, the eye tooth refers to abnormal development, eye vertebra abnormal development, odontogenic abnormal development, eye ramus of mandible abnormal development, periapical cementum abnormal development, multiple fibroid abnormal development, pseudoachondroplasia vertebra epiphysis (pseudoachondroplasticspondyloepiphysial) abnormal development, retinal development is unusual, in every-dysplasia of eye, spinal column epiphysis (spondyloepiphysial) abnormal development and chamber be abnormal development (ventriculoradialdysplasia) radially.
Other can use that the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding are diagnosed, prognosis evaluation, the tumor that prevents and/or treats take place before illness include but not limited to optimum abnormality proliferation illness (as, benign tumor, Fibrocystic disease disease, tissue hypertrophy, polyp intestinal, polyp of colon and esophagus abnormal development), leukoplakia, keratosis, bowen's disease, farmersskin (Farmer ' s skin), solar cheilitis and solar keratosis.
In another embodiment, the illness that the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for diagnosing and/or prognosis evaluation is relevant with the tissue of expression polypeptide of the present invention.
In another embodiment, as described herein puting together in the albumin fusion proteins of the present invention of toxin or radioactive isotope and/or the polynucleotide of the albumin fusion proteins of the present invention of encoding can be used for treating cancer and neoplasm, includes but not limited to cancer as herein described and neoplasm.In further embodiment, as described herein puting together in the albumin fusion proteins of the present invention of toxin or radioactive isotope and/or the polynucleotide of the albumin fusion proteins of the present invention of encoding can be used for treating acute myeloid leukemia.
In addition, the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can influence apoptosis, therefore can be used for treating disease multiple and that cell survival increases or the apoptosis inhibition is relevant.For example, can utilize polynucleotide of the present invention, polypeptide, and/or agonist or antagonist are diagnosed, prognosis evaluation, the disease with cell survival increases or the apoptosis inhibition is relevant that prevents and/or treats comprises that cancer is (such as follicular lymphoma, cancer with p53 sudden change, and hormone-dependent tumor, include but not limited to colon cancer, cardiac tumor, cancer of pancreas, melanoma, retinoblastoma, glioblastoma, pulmonary carcinoma, intestinal cancer, carcinoma of testis, gastric cancer, neuroblastoma, myxoma, muscular tumor, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast carcinoma, carcinoma of prostate, Kaposi sarcoma and ovarian cancer); The autoimmune illness such as, multiple sclerosis, siogren's syndrome, struma lymphomatosa, biliary cirrhosis, Behcet, Crohn disease, polymyositis, systemic lupus erythematosus (sle) and immune-related glomerulonephritis and rheumatic arthritis) and viral infection (such as herpesvirus, poxvirus and adenovirus), inflammation, graft versus host disease, acute grafing repel and chronic transplanting rejection.
In other embodiments, the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding are used to suppress growth, progress and/or the transfer of the especially above listed cancer of cancer.
Can use the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding to diagnose, prognosis evaluation, prevention, and/or other of treatment increases relevant disease or disease include but not limited to cancerate progress and/or transfer with related disorders with cell survival, described cancerate and related disorders such as leukemia (comprise acute leukemia (as, acute lymphoblastic leukemia, acute myeloid leukemia (comprises myeloblast, promyelocyte, bone marrow mononuclear cell, monocarpotic cellularity and erythroleukemia)) and chronic leukemia (as, chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (as, Hodgkin and non-hodgkin's disease), multiple myeloma, waldenstrom's macroglobulinemia, heavy chain disease and solid tumor include but not limited to that sarcoma and cancer are such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovioma, mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, cancer of pancreas, breast carcinoma, ovarian cancer, carcinoma of prostate, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, syringocarcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchus source property cancer, renal cell carcinoma, hepatoma, cancer of biliary duct, choriocarcinoma, spermocytoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, pulmonary carcinoma, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma (emangioblastoma), acoustic neuroma, oligodendroglioma, meningioma (menangioma), melanoma, neuroblastoma and retinoblastoma.
Can use the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding to diagnose, prognosis evaluation, prevent and/or treat increase relevant disease with apoptosis and comprise AIDS; Nervus retrogression illness (such as Alzheimer, parkinson disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration and cerebroma or relevant disease before); Autoimmune illness (such as multiple sclerosis, siogren's syndrome, struma lymphomatosa, biliary cirrhosis, Behcet, Crohn disease, polymyositis, systemic lupus erythematosus (sle) and relevant glomerulonephritis of immunity and rheumatoid arthritis) myelodysplastic syndrome (such as aplastic anemia), graft versus host disease, ischemia injury (causing), hepar damnification (as, hepatitis be correlated with hepar damnification, ischemia/reperfusion injury, cholestasis (bile duct injury) and hepatocarcinoma) such as myocardial infarction, apoplexy and reperfusion injury; The hepatic disease that toxin causes (hepatic disease that causes such as ethanol), septic shock, cachexia and apositia.
Can use the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding to diagnose, prognosis evaluation, hyperproliferation disease that prevents and/or treats and/or illness include but not limited to be positioned at the neoplasm at following position: liver, abdominal part, bone, mammary gland, digestive system, pancreas, peritoneum, endocrine body of gland (adrenal gland, parathyroid gland, hypophysis, testis, ovary, thymus, thyroid), eye, head and neck, neural (maincenter and periphery), lymphsystem, pelvis, skin, soft tissue, spleen, chest and urogenital tract.
Similarly, the polynucleotide diagnosis of the also available fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding, prognosis evaluation, prevent and/or treat other hyper-proliferative illness.The example of such hyper-proliferative illness includes but not limited to: hypergammaglobulinemia, lymphocytic hyperplasia illness, paraprotein mass formed by blood stasis, purpura, sarcoidosis, Sezary syndrome, Wal Dan Sitelun (Waldenstron) macroglobulinemia, Gaucher disease, histiocytosis and be arranged in any other hyperproliferation disease of above-mentioned tract except neoplasia.
Another embodiment uses the polynucleotide of coding albumin fusion proteins of the present invention to suppress abnormal division, the application of the invention and/or its albumen fusant or segmental gene therapy.
Therefore the invention provides a kind of method for the treatment of the cell proliferation illness, this method system is by inserting the polynucleotide of coding albumin fusion proteins of the present invention to the abnormality proliferation cell, and wherein said polynucleotide suppress described expression.
Another embodiment of the present invention provides a kind of method for the treatment of cell proliferation illness in the individuality, comprises using one or more cells that one or more active gene of the present invention copies abnormality proliferation to.In one embodiment, polynucleotide of the present invention are to include the encode DNA construct of recombinant expression carrier of DNA sequence of described polynucleotide of efficient expression.In another embodiment of the present invention, utilize retrovirus, or utilize adenovirus vector in another embodiment, the DNA construct of coding fusion rotein of the present invention is inserted cell to be treated (see G J.Nabel etc., PNAS 199996:324-326, it is incorporated into by reference at this).In one embodiment, viral vector is a deficiency, and can not transform non-proliferative cell, only can transform the cell in the propagation.And, in another embodiment, polynucleotide of the present invention insert proliferative cell or individually with the combination of other polynucleotide or merge after the back inserts proliferative cell, can be regulated by external irritant (being magnetic, specificity micromolecule, chemicals or medicament administration etc.), these external irritants act on the promoter of described polynucleotide upstream and induce the expression of encoded protein product.Like this, can regulate (promptly increase, reduce or suppress expression of the present invention) useful therapeutical effect of the present invention significantly based on described external irritant.
Polynucleotide of the present invention can be used for suppressing oncogene or antigenic expression." suppress oncogene express " mean that repressor gene is transcribed, degrading genes transcript (premessenger RNA), suppress montage, destroy messenger RNA, stop proteic post translational modification, destroy the normal function of albumen or Profilin.
In order to be locally applied to the abnormality proliferation cell, can use polynucleotide of the present invention by the known any method of those skilled in the art, include but not limited to transfection, electroporation, cell microinjection or in carrier such as liposome, lipofection reagent (lipofectin), or with the form of naked polynucleotide, or any other method of addressing in full by description.Polynucleotide of the present invention can be sent by the known delivery system, such as, but not limited to, retroviral vector (Gilboa, J.Virology 44:845 (1982); Hocke, Nature 320:275 (1986); Wilson etc., Proc.Natl.Acad.Sci.U.S.A.85:3014), vaccinia virus system (Chakrabarty etc., Mol.Cell Biol.5:3403 (1985) or other effective dna delivery system well known by persons skilled in the art (Yates etc., Nature 313:812 (1985)).These lists of references are only for exemplary and incorporate into by reference at this.In order to send specifically or the cell of transfection abnormality proliferation and do not relate to cell in the non-division, can use retrovirus well known by persons skilled in the art or adenovirus (as on the books described in this area) delivery system with this paper other places.Require host DNA to duplicate owing to integrate retrovirus DNA, and owing to lack the required reverse transcription virus gene of retrovirus life cycle, retrovirus can not self replication.Use this retrovirus delivery system will make described gene and construct targeting not relate to normal cell in the non-division in the abnormality proliferation cell to polynucleotide of the present invention.
Be used for injection needle directly is directed to the imaging device of disease location by use, polynucleotide of the present invention directly can be delivered to the cell proliferation illness/disease location in internal, body cavity and the similar position.Also can when surgical intervention, polynucleotide of the present invention be applied to disease location.
" cell breeding disease " mean influence any one organ, body cavity or body part or their any combination, any single place or many places local anomaly propagation (no matter optimum or pernicious) with cell, cell mass or tissue is the mankind or the Animal diseases or the illness of feature.
Can use the polynucleotide of the present invention of any amount, as long as it has biologically inhibitory action to the propagation of treatment cell.And it is possible using more than a kind of polynucleotide of the present invention simultaneously to same position.Propagation or rate of growth that " inhibition biology " means growth inhibited partially or completely and reduce cell.Suppressing dosage biology can be by estimating polynucleotide of the present invention to the effect of the cell growth of the target malignant cell in tissue culture growth or abnormality proliferation, effect or the known any other method of those of ordinary skills of tumor growth in animal and the cell culture are determined.
And, describe elsewhere as this paper, the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can use separately, use with the form of albumen fusant, or with other polypeptide coupling, the blood vessel that suppresses proliferative cell or tissue directly or indirectly takes place.In one embodiment, described angiogenesis inhibitor effect can realize indirectly, for example by suppressing hemopoietic, tomour specific sexual cell, (see J Natl Cancer Inst such as Joseph IB such as tumor-associated macrophages, 90 (21): 1648-53 (1998), it is incorporated into by reference at this).
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for by apoptosis-induced proliferative cell or the tissue of suppressing.These fusion rotein and/or polynucleotide can work directly or indirectly and induce the apoptosis of proliferative cell and tissue, for example in the activation of activation death domain receptor, albumen (TRAMP) apoptosis induction ligand (TRAIL) receptor-1 and-2 relevant with TNF of the apoptosis mediation that described death domain receptor such as tumor necrosis factor (TNF) receptor-1, CD95 (Fas/APO-1), TNF receptor are correlated with (sees Schulze-Osthoff K etc., Eur J Biochem254 (3): 439-59 (1998), it is incorporated into by reference at this).And, in another embodiment of the present invention, these fusion rotein and/or polynucleotide---individually or with other small-molecule drug or adjuvant such as apoptonin, Galectins, thioredoxin, anti-inflammatory protein combination---can be apoptosis-induced via other mechanism, for example can activate in the proteic activation of apoptosis at other, perhaps by stimulating these proteic expression (referring to for example, Mutat Res 400 (1-2): 447-55 (1998), Med Hypotheses.50 (5): 423-33 (1998), Chem Biol Interact.Apr 24; 111-112:23-34 (1998), J MolMed.76 (6): 402-12 (1998), Int J Tissue React; 20 (1): 3-15 (1998), it is all incorporated into by reference at this).
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for suppressing the transfer of proliferative cell or tissue.Inhibition can be used as the direct result of using these albumin fusion proteins and/or polynucleotide and takes place, or take place indirectly, for example expression of alpha-4 integrin (sees as Curr Top Microbiol Immunol 1998 such as the activation transferable albumen of known inhibition; 231:125-41, it is incorporated into by reference at this).This therapeutical effect of the present invention can separately or unite small-molecule drug or adjuvant is realized.
In another embodiment, the invention provides a kind of to expression be incorporated into the polypeptide of albumin fusion proteins of the present invention, by the bonded polypeptide of albumin fusion proteins of the present invention or the method for compositions of sending the polynucleotide that contain the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding with the target cell of the associating polypeptide of albumin fusion proteins of the present invention.Albumin fusion proteins of the present invention can associate via hydrophobic, hydrophilic, ion and/or covalent interaction and heterologous polypeptide, heterologous nucleic acids, toxin or prodrug.
Albumin fusion proteins of the present invention can be used for strengthening directly or indirectly the immunogenicity and/or the antigenicity of proliferative cell or tissue, for example when albumin fusion proteins of the present invention " inoculation " (vaccinate) immunne response directly enhancing will take place with in response to propagation antigen and immunogen the time; Be presented as and for example activate the known expression that strengthens the albumen (as chemotactic factor) of described antigen and immunogenic immunne response and indirectly strengthen.
The kidney illness
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for treating, prevent, the illness of diagnosis and/or prognosis evaluation kidney system.The kidney illness that can use compositions diagnosis of the present invention, prognosis evaluation, prevents and/or treats includes but not limited to urinary system illness, autoimmune illness, sclerosis and necrosis, electrolyte imbalance and the renal carcinoma of renal failure, nephritis, renal blood vessels illness, metabolic and congenital kidney disease, kidney.
Can use compositions diagnosis of the present invention, prognosis evaluation, the kidney disease that prevents and/or treats includes but not limited to, acute renal failure, chronic renal failure, arterial thrombosis renal failure, end-stage renal disease, the kidney inflammatory diseases (as, acute glomerulonephritis, infect the back glomerulonephritis, the glomerulonephritis of rapid progress, nephrotic syndrome, membranous glomerulonephritis, the familial nephrotic syndrome, film proliferative glomerulonephritis I and II, mesangium hypertrophy glomerulonephritis, chronic glomerulonephritis, the acute tubular interstitial nephritis, the chronic renal tubulointerstitial nephritis, glomerulonephritis behind the acute meningitis from Streptococcus pneumoniae (PSGN), pyelonephritis, lupus nephritis, chronic nephritis, interstitial nephritis, with glomerulonephritis after the streptococcal infection), the renal blood vessels illness (as, the kidney infarction, the arterial thrombosis nephropathy, cortical necrosis, malignant nephrosclerosis, renal venous thrombosis, renal perfusion is low, renal retinopathy, renal ischaemia-perfusion again, thrombosis of renal artery and renal artery stenosis), the kidney disease that causes with urethral disease (as, pyelonephritis, hydronephrosis, urolithiasis (renal calculus, nephrolithiasis), reflux nephropathy, urinary tract infection, urine retention, with acute or chronic one-sided obstructive uropathy).
In addition, compositions of the present invention can be used for diagnosis, prognosis evaluation, prevent and/or treat the metabolic of kidney and inborn illness (as, uremia, renal amyloidosis, renal osteodystrophy, renal tubular acidosis, renal glucosuria, nephrogenic diabetes insipidus, cystinuria, fanconi's syndrome, kidney fiber cryptomere osteosis (kidney osteomalacia), how Hart pounces on disease, Bartter, the Li Deer Cotard, multicystic kidney disease, MCD, marrow sample medullary sponge kidney, Alport's syndrome, nail patella syndrome, congenital nephrotic syndrome, the CRUSH syndrome, horseshoe kidney, diabetic nephropathy, nephrogenic diabetes insipidus, analgesic nephropathy, renal calculus and membranous nephropathy), with kidney autoimmune illness (as, systemic lupus erythematosus (sle) (SLE), Goodpasture, IgA nephropathy, with IgM mesangium hypertrophy glomerulonephritis).
Compositions of the present invention also can be used for diagnosis, prognosis evaluation, prevent and/or treat the hardening of kidney or gangrenosum acne illness (as, glomerulosclerosis, diabetic nephropathy, FSG (FSGS), gangrenosum acne glomerulonephritis and the necrosis of renal papillae shape), cancer kidney (as, nephroncus, hypernephroma, nephroblastoma, renal cell carcinoma, transitional cell carcinoma, renal adenocarcinoma, squamous cell carcinoma and Wilms' tumor), and electrolyte imbalance (as, nephrocalcinosis, pyuria, edema, hydronephrosis (hydronephritis), albuminuria, hyponatremia, hypernatremia, hypokalemia, hyperpotassemia, hypocalcemia, hypercalcemia, hypophosphatemia and hyperphosphatemia).
Can use any method known in the art to use compositions of the present invention, include but not limited to, at the direct needle injection of site of delivery, intravenous injection, local application, conduit infusion, biological projectile syringe, particle accelerator, gel foam sponge storage, other commercially available storage material, osmotic pumps, oral or suppository solid-state drug preparation, send at intra-operative decant (decanting) or local application, aerosol.Such method is known in the art.Can use the part of compositions of the present invention, as described in more detail below as treatment.Sending the method for polynucleotide of the present invention describes in more detail at this paper.
Cardiovascular disease
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for treatment, prevention, diagnosis and/or prognosis evaluation cardiovascular disease, include but not limited to that peripheral arterial disease is such as the limb ischemia.
Cardiovascular disease includes but not limited to, Cardiovascular abnormality such as tremulous pulse-fistula of artery, arteriovenous fistula, arteriovenous malformation of brain, congenital heart defect, pulmonary atresia and scimitar syndrome.Congenital heart defect includes but not limited to, coarctation of aorta, cor triatriatum, malformation of coronary artery, spider heart, dextrocardia, patent ductus arteriosus, Ai Busitanshi deformity, Eisenmenger complex, hypoplastic left heart syndrome, position, a left side, tetralogy of Fallot, trunk transposition, double outlet of right ventricle, tricuspid atresia, persistent truncus arteriosus and cardiac septum defective are risen Bach's Cotard, trilogy of Fallot, ventricle cardiac septum defective such as aorticopulmonary septum defective, endocardial cushion defective, Shandong.
Cardiovascular disease includes but not limited to that also heart disease is such as arrhythmia, carcinoid heart disease, the cardiac output height, cardiac output is low, cardiac tamponade, endocarditis (comprising bacteroidal), arterio-cardiac aneurysm, asystole, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, cardiac hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, rupture of heart after the infarction, ventricular septal rupture, lular heart disease, cardiomyopathy, myocardial ischemia, pericardial effusion, pericarditis (comprising constriction and tuberculosis), pneumopericardium, postpericardiotomy syndrome, lung heart disease, rheumatic heart disease, ventricular dysfunction, congested, the cardiovascular pregnancy complications, scimitar syndrome, cardiovascular syphilis and cardiovascular tuberculosis disease.
Arrhythmia includes but not limited to, syndrome, Wolff Parkinson White syndrome, sick sinus syndrome, tachycardia and ventricular fibrillation before arrhythmia, atrial fibrillation, atrial flutter, bradycardia, additional shrinkage, adams-Stokes syndrome, bundle branch block, sinoatrial block, long QT syndrome, parasystole, lown-Ganong-Levine syndrome, the excitement of Mahaim-type.Tachycardia comprises paroxysmal tachycardia, supraventricular tachycardia, accelerated idioventricular rhythm, atrioventricular nodal reentrant tachycardia, atopy atrial tachycardia, atopy junctional tachycardia, sino-atrial node reentry tachycardia, sinus tachycardia, torsive ventricular tachycardia and ventricular tachycardia.
Lular heart disease includes but not limited to, aortic incompetence, aortic stenosis, heart (hear) noise, aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitral incompetence, mitral stenosis, pulmonary atresia, pulmonic insufficiency, pulmonary stenosis, tricuspid atresia, tricuspid insufficiency and tricuspid stenosis.
Cardiomyopathy includes but not limited to, narrow under alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, the aorta artery lobe, subvalvular pulmonary stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis, myocardium endocardium fibrosis, Kearns syndrome (KearnsSyndrome), reperfusion injury of cardiac muscle and myocarditis.
Myocardial ischemia includes but not limited to coronary artery disease, such as angina pectoris, coronary aneurysm, coronary atherosclerosis, crown thrombosis, coronary vasospasm, myocardial infarction and myocardial stunning.
Cardiovascular disease comprises that also angiopathy is such as aneurysm, angiodysplasia, angiomatosis, BA, hippel-Lindau disease, io-osteohypertrophy's syndrome, Sturge-Weber syndrome, vasodilation, aortopathy, high iS-One arteritis, aortitis, leriche syndrome, arterial occlusive disease, arteritis, endarteritis (enarteritis), polyarteritis nodosa, cerebrovascular disease, diabetic angiopathy, diabetic retinopathy, thromboembolism, thrombosis, erythromelalgia, hemorrhoid, hepatic veno-occulusive disease, hypertension, hypotension, ischemia, peripheral blood vessel, phlebitis, pulmonary veno-occlusive disease, Raynaud disease, the CREST syndrome, the retinal vein occlusion, scimitar syndrome, superior vena cava syndrome, telangiectasis, ataxia telangiectasia (atacia telangiectasia), hereditary hemorrhagic telangiectasia, varicocele, cirso-, varicose ulcer, vasculitis and venous insufficiency.
Aneurysm includes but not limited to dissecting aneurysm, false aneurysm, infectious aneurysm, ruptured aneurysm, aortic aneurysm, cerebral aneurysm, coronary aneurysm, cardiac aneurysm and iliac artery tumor.
Arterial occlusive disease includes but not limited to, arteriosclerosis, intermittent claudication, carotid artery stenosis, fiber flesh sexual abnormality, mesenteric vascular occlusion, moyamoya, renal artery block, retinal artery occlusion and thromboangiitis obliterans.
Cerebrovascular disease includes but not limited to, carotid disease, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, arteriovenous malformation of brain, cerebral arterial disease, cerebral embolism and thrombosis, carotid artery thrombosis, hole thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma, subarachnoid hemorrhage (subaraxhnoid hemorrhage), cerebral infarction, cerebral ischemia (comprising temporary transient), subclavian steal syndrome, periventricular leukomalacia, vascular headache, cluster headache, migraine and VBI.
Thromboembolism includes but not limited to, air embolism, amniotic embolism, cholesterol thromboembolism, blue toe syndrome, fat embolism, pulmonary infarction and thromboembolism (thromoboembolism).Thrombosis includes but not limited to, coronary artery thrombosis, hepatic vein thrombosis formation, the retinal vein occlusion, carotid artery thrombosis, hole thrombosis, Wallenberg's syndrome and thrombophlebitis.
The ischemic illness includes but not limited to, cerebral ischemia, ischemic colitis, compartment syndrome, anterior compartment syndrome, myocardial ischemia, reperfusion injury and periphery limb ischemia.Vasculitis includes but not limited to, aortitis, arteritis, Behcet, churg-Strauss syndrome, mucocutaneous lymph syndrome, thromboangiitis obliterans, hypersensitivity angiitis, Xu Lan-Heng Nuoshi purpura, allergic skin vasculitis and Wei Genashi granulomatosis.
Can use any method known in the art to use the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding, include but not limited to, at the direct needle injection of site of delivery, intravenous injection, local application, conduit infusion, biological projectile syringe, particle accelerator, gel foam sponge storage, other commercially available storage material, osmotic pumps, oral or suppository solid-state drug preparation, send at intra-operative decant (decanting) or local application, aerosol.These methods are known in the art.The method of sending polynucleotide has in more detail in this article to be described.
Breathe illness
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for treatment, prevention, diagnosis and/or prognosis evaluation respiratory system disease and/or illness.
Respiratory system disease and illness include but not limited to, nasal vestibulitis, the anallergic rhinitis (as, acute rhinitis, chronic rhinitis, the atrophic rhinitis, vasomotor rhinitis), nasal polyp, and sinusitis, juvenile angiofibroma, rhinocarcinoma and property childhood papilloma, polyp of vocal cord, lesser tubercle (singer's nodule joint), contact ulcer of larynx, vocal cord paralysis, the larynx bulging, pharyngitis (as, viral and bacillary), tonsillitis, the tonsil cellulitis, parapharyngeal abscess, laryngitis, larynx bulging and throat cancer (as, nasopharyngeal carcinoma, carcinoma of tonsil, laryngeal carcinoma), pulmonary carcinoma (as, squamous cell carcinoma, minicell (oat cell) cancer, large cell carcinoma and adenocarcinoma), allergia illness (eosinophilic granulocyte's pneumonia, the allergy pneumonia (as, external allergic pulmonary alveolitis, allergic interstitial pneumonitis, the organic dust pneumoconiosis, allergic bronchopulmonary aspergillosis, asthma, Wei Genashi granulomatosis (granuloma vasculitis), Goodpasture)), pneumonia (as, bacterial pneumonia (as, streptococcus pneumoniae (Streptococcus pneumoniae) (pneumococcal pneumonia), staphylococcus aureus (Staphylococcus aureus) (staphylococcus pneumonia), the gram negative bacteria pneumonia (as, Klebsiella (Klebsiella) and some kind of Rhodopseudomonas (Pseudomas) cause), mycoplasma pneumoniae (Mycoplasma pneumoniae) pneumonia, hemophilus influenza (Hemophilusinfluenzae) pneumonia, legionella pneumophila (Legionella pneumophila) (legionnaires disease), and chlamydia psittaci (Chlamydia psittaci) (psittacosis)) and viral pneumonia (as, influenza, fowl pox (chickenpox).
Other respiratory system disease and illness include but not limited to bronchiolitis, poliomyelitis (poliomyelitis), croup, respiratory syncytial virus infection, parotitis, erythema infectiosum (erythema infectiosum), roseola infantum, progressive rubella panencephalitis, German measles and subacute sclerosing panencephalitis), fungal pneumonia (as, fungal infection among the crowd of histoplasmosis, coccidioidomycosis, blastomycosis, immune system severe inhibition (as, the cryptococcosis that Cryptococcus histolyticus (Cryptococcus neoformans) causes; The aspergillosis that some kind of aspergillus (Aspergillus) causes; The candidiasis that Candida (Candida) causes; And mucormycosis)), Pneumocystis carinii (Pneumocystis carinii) (pneumocystis carinii pneumonia), atypical pneumonia (as, mycoplasma (Mycoplasma) and chlamydia (Chlamydia) belong to some kind), the opportunistic infection pneumonia, nosocomial pneumonia, chemical pneumonitis, and aspiration pneumonitis, the pleura illness (as, pleuritis, hydrothorax, and pneumothorax is (as simple spontaneous pneumothorax, concurrent spontaneous pneumothorax, tension pneumothorax)), obstructive airway diseases is (as asthma, chronic obstructive pulmonary disease (COPD), emphysema, chronic or acute bronchitis), occupational pneumonopathy is (as silicosis, black lung (coal worker's pneumoconiosis disease), asbestosis, berylliosis, occupational asthma (asthsma), byssinosis and benign pneumoconiosis disease (pneumoconioses)), the infiltrative type lung disease (as, pulmonary fibrosis is (as FA, common interstitial pneumonia), idiopathic pulmonary fibrosis, desquamative interstitial pneumonia, LIP, histiocytosis X is (as Letterer-Siwe disease, Hand Sch, eosinophilic granulocyte's granuloma), idiopathic pulmonary hemosiderosis, sarcoidosis and pulmonary alveolar proteinosis), adult respiratory distress syndrome (being also referred to as for example adult respiratory distress syndrome), edema, pulmonary infarction, bronchitis is (as viral, bacillary), bronchiectasis, pulmonary atelectasis, pulmonary abscess (causing) and cystic fibrosis as staphylococcus aureus (Staphylococcus aureus) or legionella pneumophila (Legionella pneumophila).
Anti--blood vessel takes place active
Endogenous stimulus object and the natural balance between the mortifier that blood vessel takes place are that inhibition influences dominant balance.Rastinejad etc., Cell 56:345-355 (1989)., normal physiologic conditions rare at those issues in the angiopoietic situation of tissue regeneration promoting (such as wound healing, neomorph, fetal development and female reproduction process), and blood vessel takes place strictly to be regulated and spatially goes up with the time and defined.In the situation that pathologic vessels takes place, such as being grown to solid tumor in the situation of feature, these regulation and control are malfunctioning.The blood vessel of imbalance changes pathologic into, and keeps the progress of many tumors and non--tumor disease.Have multiple serious disease to be arranged by unusual neovascularization, these diseases comprise that the ophthalmic of solid tumor growth and transfer, arthritis, some types suffers from and psoriasis.Referring to as Moses etc., Biotech.9:630-634 (1991); Folkman etc., N.Engl.J.Med., 333:1757-1763 (1995); Auerbach etc., J.Microvasc.Res.29:401-411 (1985); Folkman, Advances in Cancer Research (cancer research progress) .Klein and Weinhouse edit Academic Press, New York, 175-203 page or leaf (1985); Patz, Am.J.Opthalmol.94:715-743 (1982); With Folkman etc., the summary of Science221:719-725 (1983).Under multiple pathological condition, the blood vessel generating process plays the contribution effect to morbid state.For example, existing mass data shows that the solid tumor growth depends on blood vessel and takes place.Folkman and Klagsbrun, Science 235:442-447 (1987).
The present invention provides possibility for treat disease relevant with neovascularization or illness by the polynucleotide of using the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding.Pernicious and the transitivity disease that can use polynucleotide of the present invention and polypeptide or agonist or antagonist to treat includes but not limited to as herein described and otherwise known in the art cancerate, (summary to these illness sees Fishman etc. for solid tumor and cancer, Medicine (medical science), the 2nd edition, J.B.LippincottCo., Philadelphia (1985)).Therefore the invention provides a kind of method for the treatment of blood vessel generation relevant disease and/or illness, comprise to the albumin fusion proteins of the present invention of the individual administering therapeutic effective dose that needs are arranged and/or the polynucleotide of the albumin fusion proteins of the present invention of encoding.For example, the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used in multiple other method with therapeutic ground managing cancer or tumor.Can include but not limited to solid tumor with the cancer that the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding are treated, comprise prostate, lung, mammary gland, ovary, stomach, pancreas, larynx, esophagus, testis, liver, the parotid gland, bile duct, colon, rectum, cervix uteri, uterus, endometrium, kidney, bladder, thyroid carcinoma; Primary tumor and metastasis; Melanoma; Glioblastoma; Kaposi sarcoma; Leiomyosarcoma; Nonsmall-cell lung cancer; Colorectal carcinoma; Cancerate late period; With the blood dissemination tumor such as leukemia.For example, the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can be sent partly with treatment cancer such as skin cancer, head and neck tumor, breast tumor and Kaposi sarcoma.
In others, the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for treating shallow phenotype bladder cancer, for example use by intravesical.The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can directly be sent near tumor or the tumor locus via injection or conduit.Certainly, it will be understood by those skilled in the art that suitable method of application will be according to cancer to be treated and difference.Other sends mode discussion in this article.
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for treating except cancer other and involve the illness that blood vessel takes place.These illness include but not limited to: benign tumor, for example hemangioma, acoustic neuroma, neurofibroma, trachoma and purulent granuloma; Atheromatous plaque (artheroscleric plaques); Eye angiogenesis disease, for example, diabetic retinopathy, retinopathy of prematurity, degeneration of macula, corneal graft rejection, neovascular glaucoma, Terry's sign disease, rubescent, retinoblastoma, uveitis (uvietis) and eye pterygium (abnormal vascular growth); Rheumatoid arthritis; Psoriasis; Wound healing postpones; Endometriosis; Blood vessel takes place; Granulation forms; Hypertrophic cicatrix (keloid); Associativity is not fractured; Scleroderma; Trachoma; Blood vessel adheres to; Myocardial vascular takes place; The coronary artery side is propped up; The brain side is propped up; Arteriovenous malformotion; Ischemic limb blood vessel takes place; Ao-Wei Er Shi (Osler-Webber) syndrome; Plaque neovessels forms; Telangiectasis; Bleeder's joint; Fibrohemangioma; Fiber flesh sexual abnormality; The wound granulation; Crohn disease; And arteriosclerosis.
For example, provide the method for treatment hypertrophic cicatrix and keloid among the present invention in one aspect, comprise the polynucleotide of using the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding step to hypertrophic cicatrix or keloid.
In an embodiment of the invention, the polynucleotide with the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding are injected directly in hypertrophic cicatrix or the keloid to stop the progress of these infringements.This treatment has been notified aspect the situation (as burn) that causes the morbidity of hypertrophic cicatrix and keloid especially valuable in preventative processing, and can be at existing time progress of propagation phase (after theproliferative phase has had time to progress) (after the initial damage about 14 days) begin treatment afterwards but before hypertrophic cicatrix or keloid morbidity.As mentioned above, the present invention also provides the method for the neovascular diseases of treatment eye, and described disease for example comprises, cornea neovascularization, neovascular glaucoma, propagation diabetic retinopathy, Terry's sign disease and degeneration of macula.
And the ophthalmic relevant with neovascularization that can use the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding to treat suffered from and being included but not limited to: neovascular glaucoma, diabetic retinopathy, retinoblastoma, Terry's sign disease, uveitis, retinopathy of prematurity, degeneration of macula, corneal transplantation neovascularization and other ophthalmia disease, eye neoplasms and with choroid or the relevant disease of iris neovascularization.Referring to for example Waltman etc., Am.J.Ophthal.85:704-710 (1978) and Gartner etc., the summary of Surv.Ophthal.22:291-312 (1978).
Therefore the method for treatment ocular neovascular disease such as cornea neovascularization (comprising the corneal transplantation neovascularization) is provided in one aspect of the invention, the step that comprises the chemical compound (as the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding) to patient's cornea administering therapeutic effective dose is so that vascularization is suppressed.In brief, cornea is the tissue that does not have blood vessel usually.Yet in some pathology situations, blood capillary can extend into cornea from the cornea week vascular plexus of limbus of corneae.When vascularization of cornea, shadowed takes place in simultaneously, causes patient's visual acuity to descend.If cornea is muddy fully, may completely lose vision.A large amount of illness can cause the cornea neovascularization, for example comprise, corneal infection (as, trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis), the immunology process (as, transplant rejection and ectodermosis pluriorificialis), alkali burn, wound, inflammation (any reason), toxicity and malnutrition state and wear the complication of adherent lens.
In some embodiments of the present invention, can be prepared as the normal saline solution that is used for local application (cooperation is generally used for any antiseptic and the antibacterial of ophthalmic preparation), and use with the form of eye dropping liquid.Solution or suspension can be prepared as its pure form and use repeatedly every day.Perhaps, the anti--angiogenesis compositions of preparation also can directly be applied to cornea as mentioned above.In some embodiments, prepare this anti--angiogenesis compositions with the mucoadhesive polymer that can be incorporated into cornea.In further embodiment, this anti-angiogenesis or angiogenesis inhibitor compositions can be used as the supplementary means of conventional steroid therapy.Topical therapeutic also is used for to preventability the known corneal injury (such as chemical burn) that has induction of vascular to generate the high likelihood of response.Under these situations, begin treatment (probably with the steroid coupling) is to help prevention complication subsequently immediately.
In other embodiments, aforesaid chemical compound can be injected directly into corneal stroma by ophthalmologists under microscope guides.Injection site can be different along with the form of individual pathological changes, but the target of using is that compositions is placed vascular system forward position (promptly between blood vessel and normal cornea).Under most of situations, this need carry out (perilimbic) the cornea injection of edge week is not subjected to the blood vessel progress with " protection " cornea influence.This method also can be used behind corneal wound prophylactically to stop the cornea neovascularization at once.In this case, can inject material into edge week between keratopathy and its possible limbus of corneae blood source in the cornea.This method also can be used to prevent the blood capillary of corneal transplant to invade in a similar manner.The slow release form may only need 2-3 time injection every year.Also can add steroid in the injection solution to reduce the inflammation that injection itself causes.
In another aspect of this invention, the method of treatment neovascular glaucoma is provided, comprise step, so that vascularization is suppressed to the polynucleotide of the albumin fusion proteins of the present invention of patient's eye administering therapeutic effective dose and/or the albumin fusion proteins of the present invention of encoding.In one embodiment, chemical compound can be applied to the neovascular glaucoma of eye with the treatment old model partly.In other embodiments, can chemical compound be implanted to the angle of anterior chamber zone by injection.In other embodiments, chemical compound also can be placed in any position so that chemical compound is released into aqueous humor continuously.In another aspect of this invention, the method of treatment proliferating diabetic retinopathy is provided, comprise step, so that vascularization is suppressed to the polynucleotide of the albumin fusion proteins of the present invention of patient's eye administering therapeutic effective dose and/or the albumin fusion proteins of the present invention of encoding.
In some embodiments of the present invention,, can treat proliferating diabetic retinopathy by being injected into aqueous humor or vitreous body to increase polynucleotide, polypeptide, antagonist and/or the local concentration of agonist in retina.In some embodiments, should to need to obtain to begin this treatment before the serious disease of photocoagulation.
In another aspect of the present invention, the method of treatment Terry's sign disease is provided, comprise step, so that vascularization is suppressed to the polynucleotide of the albumin fusion proteins of the present invention of patient's eye administering therapeutic effective dose and/or the albumin fusion proteins of the present invention of encoding.Chemical compound can be implanted and use partly via intravitreal injection and/or via ophthalmic.
In addition, the medicable illness of the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding includes but not limited to that hemangioma, arthritis, psoriasis, fibrohemangioma, atheromatous plaque, wound healing delay, granulation formation, bleeder's joint, hypertrophic cicatrix, non-associativity fracture, Ao-Wei two Cotards, purulent granuloma, scleroderma, trachoma and blood vessel adhere to.
And, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be treated, prevention, the illness and/or the state of diagnosis and/or prognosis evaluation include but not limited to, solid tumor, the blood dissemination tumor is such as leukemia, neoplasm metastasis, Kaposi sarcoma, benign tumor, hemangioma for example, acoustic neuroma, neurofibroma, trachoma, and purulent granuloma, rheumatoid arthritis, psoriasis, eye angiogenesis disease, for example diabetic retinopathy, retinopathy of prematurity, degeneration of macula, corneal graft rejection, neovascular glaucoma, Terry's sign disease, rubescent, retinoblastoma, and uveitis (uvietis), wound healing postpones, endometriosis, (vascluogenesis) takes place in blood vessel, granulation forms, hypertrophic cicatrix (keloid), associativity is not fractured, scleroderma, trachoma, blood vessel adheres to, myocardial vascular takes place, the coronary artery side is propped up, the brain side is propped up, arteriovenous malformotion, ischemic limb blood vessel takes place, Ao-Wei Er Shi (Osler-Webber) syndrome, plaque neovessels forms, telangiectasis, bleeder's joint, fibrohemangioma, fiber flesh sexual abnormality, the wound granulation, Crohn disease, arteriosclerosis, by preventing the required angiopoietic control pregnancy agent of embryo nidation control menstruation, occur as disease such as the cat scratch disease (Rochele minaliaquintosa) of pathological consequences with blood vessel, ulcer (helicobacter pylori (Helicobacter pylori)), bartonellosis and BA.
In aspect of described control fertility method, use the chemical compound of the amount that is enough to stop embryo nidation before sexual intercourse and fertilization take place or after having taken place, thereby the effective ways that provide control to give birth to may be " afterwards " methods.The polynucleotide that also can use the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding are with the control menstruation, or implant with peritoneal lavage fluid or peritoneum and to use with the treatment endometriosis.
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can mix in the operation suture thread with the prevention property sewed up granuloma.
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for multiple operation plan.For example, in one aspect of the invention, compositions (with for example spraying or the form of thin film) be used in remove before the tumor bag by or spray certain zone, so that malignant tissue is kept apart with normal structure on every side, and/or prevent disease is diffused into surrounding tissue.In others of the present invention, compositions (as with Sprayable) can be sent with bag via endoscope operation and be taken place by the blood vessel of tumor or inhibition desired locations.The present invention other other aspect in, can be used for any scheme that may use operation reticulated (surgical mesh) with the operation reticulated of angiogenesis inhibitor compositions bag quilt of the present invention.For example in an embodiment of the invention, the operation reticulated of filling the angiogenesis inhibitor compositions can be used on during the abdominal part carcinectomy operation (after colectomy) structure is provided support and discharge a certain amount of anti-angiogenesis.
Of the present invention further aspect in, the method of handling the tumor resection position is provided, has been included in behind the tumor resection polynucleotide from the albumin fusion proteins of the present invention of encoding to margins of excision that use albumin fusion proteins of the present invention and/or and forms neovascularity with the local recurrence that suppresses cancer with at this position.In an embodiment of the invention, anti-angiogenic compounds directly be applied to the tumor resection position (as by smear, brush or alternate manner with anti--angiogenesis chemical compound bag by the tumor resection edge).Perhaps, anti-angiogenic compounds can be incorporated known surgical thickener (surgical paste) into before using.In some embodiments of the present invention, anti-angiogenic compounds is used after hepatectomy cancerates He behind the operative procedure on nerve.
In one aspect of the invention, the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can be applied to the margins of excision of kinds of tumors, and described tumor comprises for example mammary gland, colon, brain and liver tumor.For example in an embodiment of the invention, anti-angiogenic compounds can be applied to the neural tumor position after excision, so that the neovascularization at this position is suppressed.
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding also can be used with other anti-angiogenesis.The representation example of other anti-angiogenesis comprises: the various forms of the tissue depressant of the anti-invasion factor, tretinoin and its derivant, paclitaxel, suramin, metalloproteases-1, the tissue depressant of metalloproteases-2, plasminogen activator inhibitor-1, plasminogen activator inhibitor-2 and light " d family " transition metal.
Light " d family " transition metal comprises for example vanadium, molybdenum, tungsten, titanium, niobium and tantalum kind.This transition metal material can form transition metal complex.The suitable complex of above-mentioned transition metal material comprises oxo transition metal complex (oxo transition metal complexes).
The representation example of vanudium complex comprises oxo vanudium complex such as vanadate and vanadyl complex.Suitable vanadate complex comprises metavanadate and positive vanadate complex such as for example ammonium metavanadate, sodium metavanadate and sodium vanadate.Suitable vanadyl complex comprises, for example VAAC and comprise the vanadium oxysulfate of hydration vanadium oxysulfate such as single hydration vanadium oxysulfate and three hydration vanadium oxysulfates.
The representation example of tungsten and molybdenum complex also comprises the oxo complex.Suitable oxo tungsten complex comprises tungstates and tungsten oxide complex.Suitable tungstates complex comprises ammonium tungstate, artificial schellite, Disodium tungstate (Na2WO4) dihydrate and wolframic acid.Suitable tungsten oxide comprises tungsten oxide (IV) and tungsten oxide (VI).Suitable oxo molybdenum complex comprises molybdate, molybdenum oxide and molybdenyl complex.Suitable molybdate complex comprises ammonium molybdate and its hydrate, sodium molybdate and its hydrate and potassium molybdate and its hydrate.Suitable molybdenum oxide comprises molybdenum oxide (VI), molybdenum oxide (VI) and molybdic acid.Suitable molybdenyl complex comprises, for example molybdenyl levulinic ketone ester.Tungsten that other is suitable and molybdenum complex comprise derived from for example hydroxyl derivant of glycerol, tartaric acid and sugar.
A lot of other anti-angiogenesis also can be used for the present invention.Representative example comprises platelet factor 4; Protamine sulfate; Sulphation chitin derivatives (from the preparation of snowflake Carapax Eriocheir sinensis), (Murata etc., CancerRes.51:22-26, (1991)); Sulfated polysaccharides Peptidoglycan complex (SP-PG) (this compound functions can strengthen by having steroid such as estrogen and citric acid tamoxifen); Staurosporine; The matrix metabolism instrumentality comprises for example proline analogs, cis hydroxyl groups proline, d, L-3,4-dehydroproline, thioproline, α, α-Lian Biding, aminopropionitrile fumarate; 4-propyl group-5-(4-pyridine radicals)-2 (3H)-azolactones; Methotrexate; Mitoxantrone; Heparin; Interferon; 2 macroglobulin-serum; ChIMP-3 (Pavloff etc., J.Bio.Chem.267:17321-17326, (1992)); Chymostatin (Tomkinson etc., Biochem J.286:475-480, (1992)); Cyclodextrin 14 sulfuric esters; Eponemycin; Camptothecine; Amebacilin (Ingber etc., Nature 348:555-557, (1990)); Kidon (Ono) (" GST "; Matsubara and Ziff, J.Clin.Invest.79:1440-1446, (1987)); Anticollagenase-serum; α 2-antiplasmin (Holmes etc., J.Biol.Chem.262 (4): 1659-1664, (1987)); Bisantrene (National Cancer Institute); Lobenzarit Disodium (N-(2)-carboxyl phenyl-4-chloro-o-amino benzoic acid (anthronilic acid) disodium or " CCA "; Takeuchi etc., Agents Actions36:312-316, (1992)); Thalidomide; Blood vessel is stablized steroid (angostatic steroid); AGM-1470; Carboxyamino imidazoles (carboxynaminolmidazole); With inhibitors of metalloproteinase such as BB94.
The disease of cellular level
Use the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding to treat, prevention, the disease with cell survival increases or the apoptosis inhibition is relevant of diagnosis and/or prognosis evaluation comprises that cancer is (such as follicular lymphoma, cancer with p53 sudden change, tumor with depending on hormone includes but not limited to colon cancer, cardiac tumor, cancer of pancreas, melanoma, retinoblastoma, glioblastoma, pulmonary carcinoma, intestinal cancer, carcinoma of testis, gastric cancer, neuroblastoma, myxoma, muscular tumor, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast carcinoma, carcinoma of prostate, Kaposi sarcoma and ovarian cancer); Autoimmune illness such as multiple sclerosis, siogren's syndrome, struma lymphomatosa, biliary cirrhosis, Behcet, Crohn disease, polymyositis, systemic lupus erythematosus (sle) and relevant glomerulonephritis of immunity and rheumatoid arthritis) and viral infection (such as herpesvirus, poxvirus and adenovirus), inflammation, graft versus host disease, acute grafing repulsion and chronic transplanting rejection.
In some embodiments, the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding are used to suppress growth, progress and/or the transfer (metasis) of the especially above listed cancer of cancer.
Other disease or the disease relevant with the cell survival increase that the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can treat or detect include but not limited to, cancerate and the progress and/or the transfer of related disorders, (comprise that acute leukemia is (as acute lymphoblastic leukemia such as leukemia, acute myeloid leukemia (comprises myeloblast, promyelocyte, bone marrow mononuclear cell, monocarpotic cellularity and erythroleukemia)) and chronic leukemia (as chronic myeloid (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (how outstanding king's evil and Fei Hejiejinshi disease), multiple myeloma, waldenstrom's macroglobulinemia, heavy chain disease and solid tumor include but not limited to that sarcoma and cancer are such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovioma, mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, cancer of pancreas, breast carcinoma, ovarian cancer, carcinoma of prostate, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, syringocarcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchus source property cancer, renal cell carcinoma, hepatoma, cancer of biliary duct, choriocarcinoma, spermocytoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, pulmonary carcinoma, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma (hemangioblastoma), acoustic neuroma, oligodendroglioma, meningioma (menangioma), melanoma, neuroblastoma and retinoblastoma.
Use the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding to treat, prevent, the increasing relevant disease with apoptosis and include but not limited to AIDS of diagnosis and/or prognosis evaluation (prognesed); Neurodegenerative illness (such as Alzheimer, parkinson disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration and cerebroma or relevant disease before); The autoimmune illness is (such as multiple sclerosis, siogren's syndrome, struma lymphomatosa, biliary cirrhosis, Behcet, Crohn disease, polymyositis, systemic lupus erythematosus (sle) and relevant glomerulonephritis of immunity and rheumatoid arthritis) myelodysplastic syndrome (such as aplastic anemia), graft versus host disease, ischemia injury is (such as myocardial infarction, apoplexy and reperfusion injury cause), hepar damnification is (as the relevant hepar damnification of hepatitis, ischemia/reperfusion injury, cholestasis (bile duct injury) and hepatocarcinoma); The hepatic disease that toxin causes (causing), septic shock, cachexia and apositia such as ethanol.
Wound healing and epithelial cell proliferation
According to further aspect of the present invention, the method of using the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding for therapeutic purposes is provided, for example the purpose for wound healing stimulates epithelial cell proliferation and substrate keratinocyte and hair follicle stimulating to produce and the corium wound healing.The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for stimulating wound healing clinically, described wound comprises operation wound, the excision wound, the diacope that comprises the breaking-up of corium and epidermis, ocular tissue's wound, the dental tissue wound, oral wounds, diabetic ulcer, corium ulcer, elbow ulcer, ulcer of artery, venous stasis ulcers, be exposed to the burn that heat or chemicals cause, with other unusual wound healing disease, such as uremia, malnutrition, vitamin deficiency and with steroid, the complication that radiotherapy is relevant with the antimetabolite whole body therapeutic with antitumor drug.The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding are used in corium forfeiture back and promote corium to rebuild.
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for increasing skin graft to the adhesion of wound bed and be used to stimulate the epithelium from wound bed to form again.The polynucleotide that below are the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for increasing its adherent graft type to wound bed: autograft, artificial skin, allograft, the self-skin transplant thing, from body surface skin (autoepdermic) graft, no vascular (avacular) graft, Bu-Bu Er Shi graft, bone graft, brephoplastic graft, the skin grafting dermepenthesis thing, postpone graft (delayed graft), the dermal transplantation thing, epidermic graft, fascia graft, full pachydermia (full thickness) graft, the allograft thing, xenograft, syngenesiograft, hypertrophy skin grafting dermepenthesis thing, the corneal lamellar graft, mesh skin (mesh) graft, mucosal graft, ollier-Thiersch graft, nethike embrane (omenpal) graft, patch graft, the pedicle graft thing, the holostrome corneal graft, the layering skin graft, thick layering skin grafting dermepenthesis thing.The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for promoting skin intensity and improve the apparent of old and feeble skin.
Think that the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding also will produce the variation of epithelial cell proliferation in hepatocyte growth and lung, mammary gland, pancreas, stomach, small intestinal and the large intestine.The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can promote epithelial cell proliferation, described epithelial cell such as sebaceous gland cell, hair follicle, hepatocyte, II type pneumonocyte, produce other epithelial cell and the ancestors thereof that comprise in mucinous goblet cell and skin, lung, liver and the gastrointestinal tract.The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can promote endotheliocyte, keratinocyte and substrate keratinocyte propagation.
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding also can be used for alleviating the intestinal toxic side effects that radiotherapy, chemotherapy or viral infection cause.The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can have cytoprotection to mucous membrane of small intestine.Mucositis (canker sore) healing that the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding also can stimulate chemotherapy and viral infection to cause.
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be further used for the skin defect (comprising burn) of through thickness and segment thickness skin holomorphosis (being the regeneration of hair follicle, sweat gland and sebaceous gland), treat other skin defect such as psoriasis.The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for treating epidermolysis bullosa, epidermolysis bullosa is the adherent defective of epidermis and its below corium, cause frequent bulla that occur, open, that pain is arranged, described fusion rotein and/or polynucleotide are treated them by the reepithelialization (reepithelializatoin) that quickens these damages.The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding also can be used for treating harmonization of the stomach duodenum (doudenal) ulcer and form cicatrix quickly and regeneration gland mucosa and duodenal mucosa liner help healing by mucosal lining.Inflammatory bowel such as Crohn disease and ulcerative colitis are respectively to cause small intestinal or the destructive disease of large intestine mucomembranous surface.Therefore the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for promoting redress (resurfacing) of mucomembranous surface to make progress to help faster healing and prophylaxis of inflammatory bowel disease.Utilize the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding to treat; generation has remarkable effect to the mucus in the whole gastrointestinal tract; and can be used for protecting intestinal mucosa not to be subjected to the influence of the harmful substance of intake body, perhaps be used for after operation, protecting intestinal mucosa.The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for treating and the relevant disease of low expression.
And the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for preventing and cure owing to the infringement of various pathologic state to lung.The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can stimulate proliferation and break up and the reparation that promotes alveolar and bronchioles (brochiolar) epithelium with prevention or treat acute or the chronic pulmonary infringement.For example, use polynucleotide of the present invention or polypeptide, agonist or antagonist effectively to treat to cause the emphysema of carrying out property alveolar (aveoli) forfeiture and cause the inhalation injury (promptly suck smog and burn cause) of bronchioles epithelium and alveolar necrosis.The polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding also can be used for stimulating II type pneumonocyte propagation and differentiation, this can help treatment or prevent disease such as the hyaline membrane disease, such as child's respiratory distress syndrome and premature infant's bronchopulmonary dysplasia (displasia).
But therefore the polynucleotide cell cultured supernatant of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding propagation and differentiation can be used for alleviating or treat the liver injury that fulminant type liver failure, viral hepatitis and toxicant (being acetaminophen, carbon tetrachloride (carbon tetraholoride) and other hepatotoxin known in the art) that hepatic disease and condition of illness such as liver cirrhosis cause cause.
In addition, the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for treatment or prevent diabetes morbidity.Be diagnosed as recently I and type ii diabetes, still have some islet cellss to keep among the patient of functions, alleviate, delay or prophylactic permanent sign thereby the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for keeping islet function.And the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for auxiliary islet cell transplantation to improve or to promote islet cell function.
Neural activity and neurological disorder
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for diagnosis and/or treatment brain and/or neural disease, illness, infringement or damage.The medicable nervous system disease of compositions of the present invention (as the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding) includes but not limited to: nervous system injury, and the disease or the illness that cause aixs cylinder disconnection, neuron minimizing or degeneration or demyelination.Nervous system damage among the medicable patient of the method according to this invention (comprising human and non-human mammal patient) includes but not limited to the following infringement of maincenter (comprising spinal cord, brain) or peripheral nervous system: (1) ischemic lesions, anoxia causes neuronal damage or death in the wherein neural part, comprises cerebral infarction or ischemia or spinal infarction or ischemia; (2) traumatic lesion comprises infringement that physical damnification causes or the infringement relevant with operation, for example cuts off the infringement or the pressurized damage of a neural part; (3) malignant lesions, wherein neural part is destroyed or is damaged by malignant tissue, and to be that nervous system is relevant cancerate or cancerating derived from non-neural system tissue in this malignant tissue; (4) infectious infringement, a wherein neural part be owing to infect destroyed or damage, for example by abscess or relevant with HIV (human immunodeficiency virus), herpes zoster or herpes simplex infections or Lyme disease, tuberculosis or prunus mume (sieb.) sieb.et zucc. poison; (5) degeneration infringement, wherein neural part is owing to include but not limited to the destroyed or damage of the degeneration process of the degeneration relevant with parkinson disease, Alzheimer, Huntington Chorea or amyotrophic lateral sclerosis (ALS); (6) with nutrition disease or the relevant infringement of illness, wherein neural part is by the nutrition illness or metabolic disorders destroys or damage, and described nutrition illness or metabolic disorders include but not limited to vitamin B12 deficiency, folic acid deficiency, acute hemorrhagic polioencephalitis, Nicotiana tabacum L.-alcohol amblyopia, marchiafava-Bignami disease (primary degeneration of corpus callosum) and alcoholic cerebellar degeneration; (7) with the relevant neurological damage of systemic disease (including but not limited to diabetes (diabetic neuropathy, bell's palsy), systemic lupus erythematosus (sle), cancer or sarcoidosis); (8) toxicant comprises the infringement that ethanol, lead or specific neurotoxin cause; (9) demyelination infringement, wherein neural part is destroyed or is damaged by demyelination, and described demyelination includes but not limited to multiple sclerosis, HIV (human immunodeficiency virus) relevant myelopathy, transverse myelopathy or the various cause of disease, progressive multifocal leukoencephalopathy and central pontine myelinolysis.
In one embodiment, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding are used for the neuroprotective cell and are not subjected to anoxybiotic destruction.In further embodiment, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding are used for the destruction that the neuroprotective cell is not subjected to cerebral anoxia.According to this embodiment, the neural cell injury that compositions of the present invention is used for the treatment of or prevention is relevant with cerebral anoxia.In aspect a non-exclusionism of this embodiment, the neural cell injury that polynucleotide are used for the treatment of or prevention is relevant with cerebral ischemia of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding.In aspect another non-exclusionism of this embodiment, the neural cell injury that polynucleotide are used for the treatment of or prevention is relevant with cerebral infarction of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding.
In another embodiment, the neural cell injury that polynucleotide are used for the treatment of or prevention is relevant with apoplexy of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding.In specific implementations, the damage of the cranial nerve cell that polynucleotide are used for the treatment of or prevention is relevant with apoplexy of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding.
In another embodiment, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding are used for the treatment of or prevent and the relevant neural cell injury of having a heart attack.In specific implementations, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding are used for the treatment of or prevent the cranial nerve cell relevant with heart attack to damage.
Can be used for treating or prevent the compositions of the present invention of nervous system disease to promote the biological activity of neuronic survival or differentiation to select by detecting it.For example but, do not cause that the present composition of any following effect can be used according to the invention as restriction: (1) exist or do not exist increase under anoxia or the anoxia condition cultivate in the neuronic time-to-live; (2) increase in the cultivation or neuronic germination in the body; (3) increase to cultivate in or the generation of neuron correlation molecule (as for motor neuron, choline acetyltransterase or acetylcholinesterase) in the body; Or (4) reduce the symptom of neuron dysfunction in the body.These effects can be measured by any method known in the art.In some nonrestrictive embodiments, the increase of neuronal survival can use this paper listed or method otherwise known in the art come to be measured routinely, such as Zhang etc., Proc Natl Acad Sci USA 97:3637-42 (2000) or Arakawa etc., J.Neurosci., 10:3507-15 (1990); The available methods known in the art of increase that neuron germinates detect, such as Pestronk etc., and Exp.Neurol., 70:65-82 (1980), or Brown etc., Ann.Rev.Neurosci., listed method among the 4:17-42 (1981); The increase that the neuron correlation molecule produces can be used measurements such as biological detection, enzyme detection, antibodies, the detection of Northern trace, and said method all uses technology known in the art and depends on molecule to be measured; And the motor neuron dysfunction can be measured by the physical manifestations (as weakness, motor neuron conduction velocity or function anergy) of estimating the motor neuron illness.
In specific implementations, medicable motor neuron illness includes but not limited to according to the present invention, the illness that can influence motor neuron and other ingredient of nervous system is such as infarction, infect, the contact toxin, wound, the operation infringement, degenerative disease or cancerate, and the affect the nerves illness of illness such as amyotrophic lateral sclerosis of unit of selectivity, include but not limited to progressive spinal muscular atrophy, PBP, primary lateral sclerosis, child and juvenile muscular atrophy, child's progressive bulbar palsy (Fazio Londe syndrome), poliomyelitis and post poliomyelitis syndrome, and hereditary motor and sensory neuropathy (charcot-Marie-Tooth disease).
Further, the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can work in neuronal survival, synapse formation, conduction, Neural Differentiation etc.Therefore compositions of the present invention (polynucleotide that comprise the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding) can be used for diagnosing and/or treats or prevent disease or the illness relevant with these effects, includes but not limited to learn and/or cognitive illness.Compositions of the present invention also can be used for treatment or prevention neurodegenerative disease state and/or behavior illness.These neurodegenerative disease states and/or behavior illness include but not limited to, Alzheimer, parkinson disease, Huntington Chorea, tourette syndrome, schizophrenia, mania, dementia, paranoia, mandatory illness, terrified property illness, deficiency of learning ability, ALS, psychosis, autism and behavior change, comprise ingest, sleep pattern, balance and consciousness illness.In addition, compositions of the present invention also can work in treatment, prevention and/or detection growth illness relevant with developmental embryo or property related disorders.
In addition; the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for the neuroprotective cell and avoid the disease relevant with cerebrovascular disease; infringement; illness or damage include but not limited to that carotid disease is (as carotid artery thrombosis; carotid artery stenosis; or moyamoya); cerebral amyloid angiopathy; cerebral aneurysm; cerebral anoxia; cerebral arteriosclerosis; arteriovenous malformation of brain; cerebral arterial disease; cerebral embolism and thrombosis are (as carotid artery thrombosis; hole thrombosis; or Wallenberg's syndrome); cerebral hemorrhage is (as epidural or subdural hematoma; or subarachnoid hemorrhage); cerebral infarction; cerebral ischemia is (as transient cerebral ischemia; subclavian steal syndrome; or VBI); vascular dementia (as multiple infarct); periventricular white matter malacosis and vascular headache (as cluster headache or migraine).
According to further aspect again of the present invention, provide therapeutic purposes for the polynucleotide that utilize the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding for example to excite nerve and learn cell proliferation and/or differentiation method.Therefore, the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for treatment and/or detect neurological disease.And the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used as the labelling or the detection of specific nervous system disease or illness.
The example of the neurological disease that the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can treat or detect comprises disease of brain, such as the metabolic disease of brain, comprise that phenylketonuria is such as maternal phenylketonuria disease, pyruvate carboxylase deficiency, pyruvate dehydrogenase complex deficiency, Wernicke encephalopathy, cerebral edema, brain neoplasm is such as little brain neoplasm, comprise an act following neoplasm, ventricles of the brain neoplasm is such as the choroid plexus neoplasm, the hypothalamus neoplasm, neoplasm on the curtain, canavan's disease, little disease of brain comprise that such as cerebellar ataxia spinocerebellar degeneration is such as ataxia telangiectasia, the cerebellum dyssynergia, Freed relies uncommon (Friederich ' s) family name's ataxia, Ma-Yue disease, olivopontocerebellar atrophy, little brain neoplasm is such as neoplasm under the curtain, DCS is such as schilder's encephalitis, globoid cell leukodystrophy, metachromatic leukodystrophy and subacute sclerosing panencephalitis.
Other neurological disease that the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can treat or detect comprises that cerebrovascular disease is (such as comprising carotid artery thrombosis, the carotid disease of carotid artery stenosis and moyamoya), cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, arteriovenous malformation of brain, cerebral arterial disease, cerebral embolism and thrombosis are such as carotid artery thrombosis, hole thrombosis and Wallenberg's syndrome, cerebral hemorrhage is such as epidural hematoma, subdural hematoma and subarachnoid hemorrhage, cerebral infarction, cerebral ischemia is such as transient cerebral ischemia, subclavian steal syndrome and VBI, vascular dementia is such as multi infarct dementia, the periventricular white matter malacosis, vascular headache such as cluster headache and migraine.
Other neurological disease that the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can treat or detect comprises dull-witted such as the AIDS chronic brain syndrome, presenile dementia such as Alzheimer and Ke-Ya syndrome, alzheimer disease such as Alzheimer and progressive supranuclear plasy, vascular dementia is such as multi infarct dementia, encephalitis, comprise schilder's encephalitis, viral encephalitis is such as epidemic encephalitis, Japanese encephalitis, St. Louis encephalitis, tick encephalitis and west Nile fever, acute disseminated encephalomyelitis, meningoencephalitis is such as the uveomeningoencephalitis syndrome, parkinson disease and subacute sclerosing panencephalitis after the encephalitis, cerebral malacia is such as periventricular leukomalacia, epilepsy is such as generalized epilepsy, comprise child's spasm, inattentive epilepsy, myoclonic epilepsy comprises the MERRF syndrome, clonic spasm type epilepsy, the localized epilepsy of localized epilepsy such as complexity, frontal epilepsy and temporal-lobe epilepsy, post-traumatic epilepsy, status epilepticus such as EPC and hallerman-Streiff syndrome.
Other neurological disease that the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can treat or detect comprises hydrocephalus such as dandy-Walker syndrome and normal pressure hydrocephalus, the hypothalamus disease is such as the hypothalamus neoplasm, encephalic malaria, sleeping sickness comprises and dampinging off, bulbar poliomyelitis, the false tumor of brain, the Rett syndrome, Reye syndrome, the thalamus disease, brain toxoplasmosis, intracranial tuberculoma and Ze Weige syndrome, central nervous system infection is such as the AIDS chronic brain syndrome, brain abscess, subdural empyema, encephalomyelitis, such as equine encephalomyelitis, Venezuelan equine encephalomyelitis, necrotizing hemorrhagic encephalomyelitis, visna (Visna), and encephalic malaria.
Other neurological disease that the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can treat or detect comprises that meningitis is such as arachnoiditis, aseptic meningitis (meningtitis) is such as viral meningitis (meningtitis), comprise lymphocytic choriomeningitis, bacterial meningitis (meningtitis), comprise Haemophilus meningitis (meningtitis), listeria meningitis (meningtitis), meningococcal meningitis (meningtitis) is such as WF, pneumococcal meningitis (meningtitis) and meningeal tuberculosis disease, fungal meningitis such as cryptococcal meningitis (meningtitis), subdural effusion, meningoencephalitis such as uveomeningoencephalitis (uvemeningoencephalitic) syndrome, myelitis is such as transverse myelitis, neurosyphilis is such as tabes dorsalis, poliomyelitis comprises bulbar poliomyelitis and post poliomyelitis syndrome, prion disease is (such as Ke-Ya syndrome, bovine spongiform encephalopathy, lucky this syndrome, Kuru disease, scrapie), and brain toxoplasmosis.
Other neurological disease that the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can treat or detect comprises that central nervous system's neoplasm is such as brain neoplasm, comprise that little brain neoplasm is such as neoplasm under the curtain, ventricles of the brain neoplasm is such as the choroid plexus neoplasm, hypothalamus neoplasm and curtain are gone up neoplasm, the meninges neoplasm, the spinal cord neoplasm, comprise the epidural neoplasm, demyelination is such as canavan's disease, DCS (sceloris), comprise adrenoleukodystrophy, schilder's encephalitis, globoid cell leukodystrophy, DCS is such as metachromatic leukodystrophy, the allergic effect encephalomyelitis, necrotizing hemorrhagic encephalomyelitis, progressive multifocal leukoencephalopathy, multiple sclerosis, central pontine myelinolysis, transverse myelitis, optic neuromyelitis, scrapie, swayback (Swayback), chronic fatigue syndrome, visna, high pressure nervous syndrome, meningism, diseases of spinal cord is such as spy's property sent out myatonia, amyotrophic lateral sclerosis, Duchenne-Arandisease is such as familial spinal muscular atrophy, compression of spinal cord, the spinal cord neoplasm is such as the epidural neoplasm, syringomyelia, tabes dorsalis, stiff-man syndrome, mental retardation is such as Angelman syndrome, cri du chat syndrome, Cornelia De Lange Syndrome, mongolism, the sick G (M1) of ganglioside disease (Gangliosidoses) such as ganglioside, sandhoff disease, Tay Sachs disease, how Hart pounces on disease, homocystinuria, Laurence-Moon-Biedl syndrome, L-N, maple sugar urine disease, sticking fat is stored up disease such as fucosidosis, the neuron ceroid-lipofuscinosis, OCR, phenylketonuria is such as maternal phenylketonuria disease, PW, the Rett syndrome, rubinstein-Taybi syndrome, tuberous sclerosis, the WAGR syndrome, nervous system abnormality is such as holoprosencephaly, neural tube defect comprises hydranencephaly (hydrangencephaly) such as anencephaly, anold-Chiari deformity, the brain bulging, the meninges bulging, the bulging of spinal cord spinal meninges, spinal dysraphism such as spina bifida cystica and spina bifida occulta.
Other neurological disease that the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can treat or detect comprises heritability motion and esthesioneurosis, comprise Xia-Ma Er Shi disease, hereditary optic atrophy, Refsum's disease, hereditary spastic paraplegia, familial spinal muscular atrophy, heritability sensation and autonomic neuropathy are such as spy's property sent out analgesia and dysautonomia, neurological's performance is (such as the agnosia that comprises gerstmann's syndrome, amnesia is such as retrograde amnesia, apraxia, neurogenic bladder, damping off, communication disorders comprises deafness such as dysacousis, the part hearing disability, loudness recruitment and tinnitus; Aphasis comprises logagraphia, anomia, Broca's aphasia and Wernicke's aphasia such as aphasia; Reading disorder such as acquired dyslexia, development of speech illness, speech disorder comprise anomia, Broca's aphasia and Wernicke's aphasia such as aphasia; Dysphonia; Communication disorders comprises dysarthria, echolalia, mutism and stutter such as speech disorder; Pararthria is such as crying out and hoarseness, decerebrate state, delirium, fasciculation, hallucination, meningism, the motion illness is such as the angelman syndrome, ataxia, athetosis, chorea, dystonia, hypokinesia, muscle tone is low excessively, myoclonus, twitch, torticollis and trembling, muscle tone too high such as muscle rigidity such as stiff-man syndrome, muscular spasticity, paralysis is such as facial paralysis, comprise herpes auris, gastroparesis, hemiplegia, ophthalmoplegia is such as diplopia, duane's syndrome, Horner syndrome, chronic progressive external ophthalmoplegia is such as the Keams syndrome, bulbar paralysis, tropical spastic paresis, paraplegia is such as Brown-Se﹠1﹠quard syndrome, quadriplegia, respiratory paralysis and vocal cord paralysis, paresis, phantom limb, sense of taste illness such as ageusia and dysgeusia, the vision illness is such as amblyopia, blind, color defect, diplopia, hemianopsia, dim spot and subnormal vision, the sleep illness is such as hypersomnia, comprise Kleine-Levin syndrome, the insomnia, and somnambulism, spasm is such as gnathospasma, loss of consciousness is such as stupor, persistent vegetable state and faint and dizzy, neuromuscular disease is such as spy's property sent out myatonia, amyotrophic lateral sclerosis, the Lambert-Eaton myasthenic syndrome, motor neuron disease, amyotrophy is such as Duchenne-Arandisease, charcot-Marie atrophy disease and familial spinal muscular atrophy, post poliomyelitis syndrome, muscular dystrophy, myasthenia gravis, myotonia atrophica, the special property sent out paramyotonia (Myotonia Confenita), nemaline myopathy, familial periodic paralysis, multiple light myoclonus (Multiplex Paramyloclonus), spastic lower extremity paralysis in the torrid zone and stiff-man syndrome, diseases in peripheral nerve system is such as dactylalgia, amyloid neuropathy, autonomic nervous system diseases is such as Adie's syndrome, barre-Lieou syndrome, dysautonomia, bernard's syndrome, reflex sympathetic dystrophy and orthostatic hypotension syndrome, the cranial nerve disease such as the acoustic nerve disease such as acoustic neuroma, comprise 2 type neurofibromatosiss, the nervus facialis disease is such as opsialgia, Mai-Luo two Cotards, the ocular motility illness comprises amblyopia, nystagmus, oculomotor paralysis, ophthalmoplegia is such as duane's syndrome, bernard's syndrome, chronic progressive external ophthalmoplegia, comprise the Kearns syndrome, stravismus such as esotropia and exotropia, oculomotor paralysis, optic nerve disease comprises hereditary optic atrophy such as optic atrophy, drusen of optic disc, optic neuritis is such as optic neuromyelitis, papilloedema, trigeminal neuralgia, vocal cord paralysis, demyelination such as optic neuromyelitis and swayback, with diabetic neuropathy such as diabetic foot.
Other neurological disease that the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can treat or detect comprises that the nerve compression syndrome is such as carpal tunnel syndrome, tarsal tunnel syndrome, thoracic outlet syndrome is such as cervical rib syndrome, ulnar nerve pressurized syndrome, neuralgia is such as causalgia, cervico-brachial neuralgia, opsialgia and trigeminal neuralgia, the neuritis is such as experimental allergic effect neuritis, ophthalmoneuritis, polyneuritis, polyradiculoneuritis and radiculitis (radiculities) are such as polyradiculitis, heritability motion and esthesioneurosis are such as the charcot-Marie atrophy disease, hereditary optic atrophy, Refsum's disease, hereditary spastic paraplegia and familial spinal muscular atrophy, heritability sensation and autonomic neuropathy comprise the special property sent out analgesia and dysautonomia, the POEMS syndrome, sciatica, gustatory sweating and tetany).
The endocrine illness
The albumin fusion proteins of the present invention and/or the albumin fusion proteins polynucleotide of the present invention of encoding can be used for treating, prevent, diagnosis and/or prognosis evaluation relate to hormone imbalances illness and/or disease and/or hormonal system illness or disease.
Hormone control physical growth, sexual function, metabolism and other function of hormonal system glandular secretion.Illness can be classified in two ways: upsetting hormone generation and tissue can not be in response to hormone.The cause of disease of these hormone imbalances or endocrine system disease, illness or disease can be heredity, somatocyte such as cancer and some autoimmune disease, acquired (causing) or infective as chemotherapy, damage or toxin.And the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used as specified disease or the illness labelling that relates to hormonal system and/or hormone imbalances or survey thing.
Hormonal system and/or hormone imbalances and/or disease comprise the illness of uterine activity, include but not limited to: pregnant and childbirth complication (the slow or childbirth termination as preterm labor, postterm pregnancy, spontaneous abortion and childbirth); With menstrual cycle illness and/or disease (as dysmenorrhea and endometriosis).
Hormonal system and/or hormone imbalances illness and/or disease comprise pancreas illness and/or disease, such as for example diabetes, diabetes insipidus, congenital pancreas hypoplasia, pheochromocytoma-islet cell tumor syndrome; Adrenal gland's illness and/or disease are such as for example Addison's disease, corticosteroid shortage, manlike disease, hirsutism, cushing's syndrome, aldosteronism, pheochromocytoma; Hypophysis body of gland illness and/or disease are such as for example hyperpituitarism, hypopituitarism, growth hormone deficiency dwarfism, pituitary adenoma, panhypopituitarism, acromegaly, gigantism; Thyroid illness and/or disease, include but not limited to hyperthyroidism, hypothyroidism, Plummer disease, Graves disease (graves), toxic nodular goiter, thyroiditis (struma lymphomatosa, subacute granulomatous thyroiditis and reticent lymphocytic thyroiditis), Pan De Randt Cotard, myxedema, cretinism, thyrotoxicosis, thyroid hormone coupling defect, thymic dysplasia, thyroid oxyphil cell tumor, thyroid cancer, thyroid carcinoma, medullary thyroid carcinoma; Parathyroid gland illness and/or disease are such as for example hyperparathyroidism, hypoparathyroidism; Hypothalamus illness and/or disease.
In addition, hormonal system and/or hormone imbalances illness and/or disease also can comprise testis or ovary illness and/or disease, comprise cancer.Other illness and/or the disease of testis or ovary comprise that further for example ovarian cancer, polycystic ovarian syndrome, reifenstenin-Albright syndrome, vanishing testes syndrome (bilateral anorchia), congenital deficiency interstitial cell, cryptorchidism, Noonan syndrome, myotonic dystrophy, testis blood capillary hemangioma (optimum), testicular tumor form and new testis.
And hormonal system and/or hormone imbalances illness and/or disease also can comprise as multiple endocrine glands shortage syndrome, pheochromocytoma, neuroblastoma, multiple endocrine neoplasia and illness and/or diseases such as endocrine tissue's illness and/or cancer.
In another embodiment, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for diagnosing, prognosis evaluation, prevent and/or treat and wherein express relevant endocrinopathy and/or the illness of tissue corresponding to the human cytokines of albumin human cytokines part of the present invention.
The reproductive system illness
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for diagnosis, treatment or prevention reproductive system disease and/or illness.The medicable reproductive system illness of compositions of the present invention includes but not limited to, the reproductive system damage, infect, new natural disposition illness, birth defect and cause the disease of infertility or illness, gestation, childbirth or production complication and puerperal problem.
Reproductive system illness and/or disease comprise testis disease and/or illness, comprise atrophy of testis, testicular feminization, cryptorchidism (one-sided and bilateral), anorchia, ectopic testis, epididymitis and orchitis (normally infect such as for example gonorrhea, parotitis, tuberculosis and syphilis cause), testicular torsion, the nodositas deferentitis, germ cell tumor is (as spermocytoma, embryo's sexual cell cancer, malignant teratoma, choriocarcinoma, yolk sac tumor and teratoma), stromal tumor (as leydig cell tumor of testis), hydrocele, hematoma of scrotum, varicocele, seminal cyst, inguinal hernia, the illness that produces with sperm is (as immotile-cilia syndrome, azoospermia, azoospermia, azoospermia, oligospermia and teratozoospermia).
The reproductive system illness also comprises prostate diseases, such as acute non--bacterial prostatitis, chronic non--bacterial prostatitis, acute bacterial prostatitis, chronic bacterial prostatitis, prostate dystonia (prostatodystonia), prostatosis, granulomatous prostatitis, malakoplakia, benign prostatauxe or hypertrophy and the new natural disposition illness of prostate, comprise adenocarcinoma, transitional cell carcinoma, duct carcinoma and squamous cell carcinoma.
In addition, compositions of the present invention can be used for diagnosis, treats and/or prevents penis and urethra illness or disease, comprise the inflammatory illness, such as balanoposthitis, balanitis xerotica obliterans, phimosis, paraphimosis, syphilis, herpes simplex virus, gonorrhea, nongonococcai urethritis, chlamydia, mycoplasma, trichomonacide, HIV, AIDS, the special Cotard of Lay, condyloma acuminatum, flat condyloma and pearly penile papules; Urethra is unusual, such as hypospadias, epispadias and phimosis; The preceding pathological changes that cancerates comprises erythroplasia of Queyrat, bowen's disease, bowenoid papulosis (paplosis), Buscke-Lowenstein giant condyloma acuminatum and excipuliform (varrucous) cancer; The penis cancer comprises squamous cell carcinoma, cancer in situ, verrucous carcinoma and dissemination carcinoma of penis; The new natural disposition illness of urethra comprises penis carcinoma of urethra, ball film carcinoma of urethra (bulbomembranousurethral carcinoma) and prostate-urethra cancer; With the erection illness, such as priapism, Perun Nie Shi disease, erection disturbance and sexual impotence.
And disease of the vas deferens and/or illness comprise vasculitis (vasculititis) and CBAVD (congenital bilateral does not have deferent duct); In addition, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for diagnosis, treat and/or prevent seminal vesicle disease and/or illness, comprise cysticercosis, congenital chloride diarrhea and POLYCYSTIC KIDNEY DISEASE.
Other illness and/or the disease of male reproductive system comprise, for example reifenstenin-Albright syndrome, young's syndrome, premature ejaculation, diabetes, cystic fibrosis, Ka Tagena Cotard, hyperpyrexia (high fever), multiple sclerosis and gynecomastia.
Further, polynucleotide of the present invention, the polynucleotide of the fusion rotein and/or the albumin fusion proteins of the present invention of encoding can be used for diagnosis, treat and/or prevent vagina and vulva disease and/or illness, comprise bacterial vaginosis, the candida mycoderma vaginitis, herpes simplex virus, chancroid, granuloma inguinale, lymphogranuloma venereum, scabies, the human papillomavirus, the vagina wound, the pudendum wound, adenopathy, the chlamydia vaginitis, gonorrhea, trichomonal vaginitis, condyloma acuminatum, syphilis, molluscum contagiosum, atrophic vaginitis, Paget, lichen sclerosus, lichen planus, vulvodynia, toxic shock syndrome, vulvismus, vulvovaginitis, vulvar vestibulitis, with new natural disposition illness, such as the squamous cell hypertrophy, clear cell carcinoma, basal cell carcinoma, melanoma, Intradermal neoplasia on bartholin gland carcinoma and the pudendum.
Uterus disease and/or disease comprise dysmenorrhea, retroversion, endometriosis, fibroma, endometriosis, anovulatory bleeding, amenorrhea, cushing's syndrome, hydatidiform mole, Asherman's syndrome, premature menopause, sexual precosity, metropolypus, anovulatory dysfunctional uterine hemorrhage (as because unusual hormone signal) and new natural disposition illness, such as adenocarcinoma, leiomyosarcoma (keiomyosarcomas) and sarcoma.In addition, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used as the labelling of congenital abnormal uterus or survey thing, and be used for diagnosis, treat and/or prevent congenital abnormal uterus, such as uterus bicornis, uterus septus, simple unicornuate uterus, have non-cavity property rudimentary horn unicornuate uterus, have unicornuate uterus, unicornuate uterus, uterus arcuatus, double uterus (uterine didelfus) and the T shape uterus of non--cavity property rudimentary horn of being communicated with cavity property angle of connection.
Ovarian disease and/or illness comprise do not ovulate, polycystic ovarian syndrome (Stein-Leventhal), ovarian cyst, hypo ovaria, ovary are insensitive to promoting sexual gland hormone, the excessive production androgen of ovary, right ovarian vein syndrome, amenorrhea, hirsutism (hirutism) and ovary cancer (including but not limited to nascent and time natural disposition cancerous growths, Sertoli-Leydig glucagonoma, ovary inner membrance sample adenocarcinoma, ovary papillary serous adenocarcinoma, ovary mucinous adenocarcinoma and ovary krukenberg's tumor).
Cervix diseases and/or illness comprise cervicitis, chronic cervicitis, mucus purulence cervicitis, cervical atypical hyperplasia, cervical polyp, naboths cysts, cervical erosion, incompetence,cervical and cervix uteri neoplasm (comprising that for example cervical cancer, squamous metaplasia, squamous cell carcinoma, glandular scale glucagonoma form and the cylindrical cell neoplasia).
In addition, reproductive system disease and/or illness comprise pregnant illness and/or disease, comprise miscarriage and stillbirth, such as early abortion, late abortion, spontaneous abortion, induced labor, TA, threatened abortion, missed abortion, incomplete abortion, artificial abortion, habitual abortion, missed abortion and septic abortion; Ectopic pregnancy, anemia, Rh are incompatible, trimester of pregnancy vaginal hemorrhage, gestational diabetes, intrauterine growth retardation, polyhydramnios, HELLP syndrome, placental abruption, placenta previa, hypermesis, preeclampsia, eclamposia, herpes gestationis and gestational urticaria.In addition, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for diagnosis, treat and/or prevent the disease that can make gestation complicated, comprise heart disease, heart failure, rheumatic heart disease, congenital heart disease, mitral valve prolapse, hypertension, anemia, nephropathy, infectious disease is (as rubella, cytomegalovirus, toxoplasmosis, infectious hepatitis, chlamydia, HIV, AIDS and genital herpes), diabetes, Graves disease, thyroiditis, hypothyroidism, struma lymphomatosa, chronic active hepatitis, liver cirrhosis, primary biliary cirrhosis, asthma, systemic lupus erythematosus (sle) (systemic lupuseryematosis), rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, appendicitis, ovarian cyst, gallbladder disease, and intestinal obstruction.
With childbirth with produce that relevant complication comprises that premature rupture of fetal membrane, preterm labor, postterm pregnancy, postmature infant, production made progress slowly, fetal distress (as heart rate (fetus or mother), breathing problem and malposition of fetus unusually), shoulder dystocia, prolapse of cord, amniotic embolism and abnormal uterine bleeding.
Further, the disease in stage in puerperal and/or illness comprise endometritis, myometritis, parametritis, peritonitis, pelvic thrombophlebitis, pulmonary infarction, endotoxemia, pyelonephritis, thrombophlebitis of saphenous vein, mastitis, cystitis, postpartum hemorrhage and inversion of uterus.
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding other female reproductive system illness and/or disease diagnosable, that treat and/or prevent comprise for example Turner syndrome, pseudohermaphroditism, premenstrual syndrome, pelvic inflammatory disease, pelvic congestion (congestion of blood vessel), frigidity, ahedonia, dyspareunia, salpingorrhexis and middle pain.
Infectious disease
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for treatment or detect infector.For example, especially increase the propagation and the differentiation of B and/or T cell, can treat infectious disease by increasing immunne response.Can be by strengthening existing immunne response or increasing immunne response by beginning new immunne response.Perhaps, the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding also can directly suppress infector and needn't cause immunne response.
Virus is an example that can cause the infector of disease that the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can treat or detect or symptom.The example of virus includes but not limited to following DNA and RNA viruses and Viraceae: arbovirus, Adenoviridae, Arenaviridae, arteritis virus, double-core ribonucleic acid virus section, Bunyaviridae, Caliciviridae, Circoviridae, coronaviridae, dengue virus, EBV, HIV, flaviviridae, Hepadnaviridae (hepatitis virus), herpetoviridae is (such as cytomegalovirus, herpes simplex, herpes zoster), the unimolecule minus-stranded rna virus is (as Paramyxoviridae, Morbillivirus, Rhabdoviridae), orthomyxoviridae family is (as influenza A, influenza B and parainfluenza), Papillomavirus, papovaviridae, Parvoviridae, Picornaviridae, Poxviridae (such as variola or cowpox), Reoviridae (as rotavirus), Retroviridae (HTLV-I, HTLV-II, slow virus) and Togaviridae (as rubella virus genus).The virus of these sections can cause multiple disease or symptom, includes but not limited to: arthritis, bronchiolitis, breathe syncytial virus, encephalitis, eye infections is (as conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (first type, B-mode, third type, penta type, chronic active, the fourth type), Japanese B encephalitis, junin fever, chikungunya disease, Rift Valley fever, yellow fever, meningitis, opportunistic infection (as AIDS), pneumonia, Burkitt lymphoma, chickenpox, hemorrhagic fever, measles, parotitis, parainfluenza, rabies, flu, poliomyelitis, leukemia, rubella, sexually transmitted disease (STD), dermatosis is (as Ka Boxi, wart) and viremia.The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for treatment or detect any of these symptom or disease.In specific implementations, the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding are used for the treatment of: meningitis, dengue fever, EBV and/or hepatitis (as hepatitis B).In other specific implementations, the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding are used for the treatment of not the patient in response to one or more other commercially available hepatitis vaccine.In the further specific embodiment, the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding are used for the treatment of AIDS or HIV or hepatitis (heptatitis) and infect the infection of (as chronic hepatitis C infection) combination HIV.
Similarly, can cause that antibacterial that the polynucleotide of disease or symptom and albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can treat or detect and fungus are former to include but not limited to following Gram-negative and gram-positive bacterium, antibacterial section and fungus: actinomyces (as Nocard's bacillus (Norcardia)), acinetobacter (Acinetobacter), Cryptococcus histolyticus (Cryptococcusneoformans), aspergillus (Aspergillus), Bacillaceae (as Bacillus anthracis (Bacillusanthrasis)), Bacteroides (as bacteroides fragilis (Bacteroides fragilis)), blastomycosis, Bordetella, Borrelia (as borrelia burgdorferi (Borrelia burgdorferi)), Brucella, candidiasis, campylobacter, chlamydia, clostridium (Clostridium) is as bacillus botulinus (Clostridiumbotulinum), clostridium difficile (Clostridium dificile), bacillus perfringens (Clostridiumperfringens), clostridium tetani (Clostridium tetani)), Coccidioides (Coccidioides), Corynebacterium (as corynebacterium diphtheriae (Corynebacterium diptheriae)), Cryptococcus (Cryptococcus), dermatomycosis (Dermatocycoses), escherichia coli (as enterotoxigenic E.Coli and enterohemorrhagic Escherichia coli), Enterobacter (Enterobacter) (as clostridium perfringen (Enterobacteraerogenes)), enterobacteriaceae (Enterobacteriaceae) (Klebsiella (Klebsiella), Salmonella (Salmonella) is (as salmonella typhi (Salmonella typhi), Salmonella enteritidis (Salmonella enteritidis), salmonella typhi (Salmonella typhi)), Serratia (Serratia), Yersinia (Yersinia), Shigella (Shigella)), erysipelothrix (Erysipelothrix), haemophilus (as influenza B haemophilus (Haemophilus influenza type B)), Helicobacterium (Helicobacter), Legionella (Legionella) (as having a liking for lung Legionnella (Legionellapneumophila)), Leptospira (Leptospira), listeria (Listeria) (as listerisa monocytogenes in mjme (Listeria monocytogenes)), mycoplasma, mycobacterium (Mycobacterium) (as Mycobacterium leprae (Mycobacterium leprae) and Mycobacterium tuberculosis (Mycobacterium tuberculosis)), vibrio (Vibrio) (as vibrio cholera (Vibrio cholerae)), naphthalene plucked instrument Salmonella section (Neisseriaceae) is (as gonorrhea naphthalene plucked instrument Salmonella (Neisseria gonorrhea), meningitis naphthalene plucked instrument Salmonella (Neisseria meningitidis)), Pasteurellaceae (Pasteurellacea), Bacillus proteus (Proteus), Rhodopseudomonas (Pseudomonas) (as Pseudomonas aeruginosa (Pseudomonasaeruginosa)), Rickettsiaceae (Rickettsiaceae), Spirochaetes (Spirochetes) is (as some kind of treponema, some kind of Leptospira, some kind of Borrelia), Shigella, staphylococcus (Staphylococcus) (as staphylococcus aureus (Staphylococcus aureus)), meningococcus, Pn and Streptococcus are (as streptococcus pneumoniae (Streptococcus pneumoniae) and A, B and C group B streptococcus) and Ureaplasma urealyticum (Ureaplasmas).These antibacterials, parasitism and the inducible disease of fungus family or symptom include but not limited to: antibiotic resistance infects, bacteremia, endocarditis, septicemia, eye infections (as conjunctivitis), uveitis, tuberculosis, gingivitis, bacterial diarrhea, opportunistic infection (as the AIDS infections relating), paronychia, prosthese-infections relating, dental caries, Lay Te Shi disease, respiratory tract infection is such as pertussis or empyema, septicemia, Lyme disease, cat scratch disease, dysentery, paratyphoid fever, alimentary toxicosis, legionellosis, chronic and acute inflammation, erythema, yeast infection, typhoid fever, pneumonia, gonorrhea, meningitis (as first type and epidemic cerebrospinal meningitis B (mengitis)), chlamydia, syphilis (syphillis), diphtheria, leprosy, brucellosis, peptic ulcer, anthrax, spontaneous abortion, birth defect, pneumonia, pulmonary infection, ear infection, deaf, blind, drowsiness, uncomfortable, vomiting, chronic diarrhea, Crohn disease, colitis, vaginosis, sterile, pelvic inflammatory disease, candidiasis, paratuberculosis, tuberculosis, lupus, botulism, gangrene, tetanus, impetigo, rheumatic fever, scarlet fever, sexually transmitted disease (STD), dermatosis is (as cellulitis, dermatomycosis), toxemia, urinary tract infection, wound infection, (noscomial) infects in the hospital.The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for treatment or detect any of these symptom or disease.In specific implementations, the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding are used for the treatment of: tetanus, diphtheria (diptheria), botulism and/or epidemic cerebrospinal meningitis B.
And, cause the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding to treat, the disease of prevention and/or diagnosis or the parasitic former following family or the monoid of including but not limited to of symptom: amebiasis, babesiasis, coccidiosis, cryptosporidiosis, double-core amebiasis (Dientamoebiasis), covering disease, ectoparsite, giardiasis, anthelmintic, leishmaniasis, schistosomicide (Schistisoma), theileriasis, toxoplasmosis, african trypanosomiasis, with trichomonacide and sporozoon (as Plasmodium vivax (Plasmodium virax), plasmodium falciparum (Plasmodium falciparium), malariae (Plasmodium malariae) and Plasmodium ovale (Plasmodium ovale)).These parasite can cause multiple disease or symptom, include but not limited to: scabies, chigger disease, eye infections, intestinal diseases (as dysentery, giardiasis), hepatic disease, lung disease, opportunistic infection (relevant as AIDS), malaria, pregnancy complications and toxoplasmosis.The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for treatment, prevention and/or diagnosis any of these symptom or disease.In specific implementations, the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding are used for the treatment of, prevent and/or diagnose malaria.
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be by using effective dose albumin fusion proteins of the present invention in the patient, or by from patient's emigrated cells, supply polynucleotide of the present invention and send the cell of transforming back to patient (ex vivo treatment) to this cell.And the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used as antigen in the vaccine to cause the immunne response at infectious disease.
Regeneration
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for differentiation, propagation and attract cell, cause tissue regeneration.(seeing Science 276:59-87 (1997)).Tissue regeneration can be used for repairing, replaces or protects the impaired tissue owing to birth defect, wound (wound, burn, cutting or ulcer), aging, disease (as osteoporosis, osteoarthritis (osteocarthritis), periodontal, liver failure), operation (comprising plastic aesthetic surgery), fibrosis, reperfusion injury or the infringement of general cytokine.
Can use the regenerated tissue of the present invention to comprise organ (as pancreas, liver, intestinal, kidney, skin, endothelium), muscle (smooth muscle, skeletal muscle or cardiac muscle), vascular system (comprising blood vessel and lymph), nerve, hemopoietic and skeleton (bone, cartilage, tendon and ligament) tissue.In one embodiment, regeneration does not have cicatrization or cicatrization to reduce.Survive again and can comprise that blood vessel takes place.
And the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can increase the regeneration of the tissue that is difficult to heal.For example increase the recovery time after the regeneration of tendon/ligament can be accelerated to damage.The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding also can be used for prophylactically avoiding damage.Medicable disease specific comprises tendinitis, carpal tunnel syndrome and other tendon or ligament defective.The further example of the tissue regeneration of non-healing of wound comprises pressure ulcer, the ulcer relevant with vascular insufficiency, operation and trauma wounds.
Similarly, use the also renewable nerve of polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding and cerebral tissue with propagation and differentiation neurocyte.Use the medicable disease of this method to comprise maincenter and diseases in peripheral nerve system, neuropathy or machinery and traumatic illness (as spinal cord illness, head trauma, cerebrovascular disease and apoplexy (stoke)).Particularly, relevant with peripheral nerve injury disease, peripheral neurophaty (causing as chemotherapy or other medical therapy), localized neuropathy and central nervous system disease (as Alzheimer, parkinson disease, Huntington Chorea, amyotrophic lateral sclerosis and orthostatic hypotension syndrome) all can use the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding to treat.
Gastrointestinal tract disease
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for treatment, prevention, diagnosis and/or prognosis evaluation gastrointestinal tract disease, comprise inflammatory diseases and/or disease, infection, cancer (as intestinal neoplasm (enteric carcinoid tumor, small intestinal non_hodgkin lymphoma, small intestinal (smallbowl) lymphoma)) and ulcer, such as peptic ulcer.
Gastrointestinal tract disease comprises dysphagia, odynophagia, the esophagus inflammation, peptic esophagitis, gastric reflux, submucosal fibrosis and narrow, Ma-Wei Er Shi infringement, leiomyoma, lipoma, the epidermis cancer, adenoma (adeoncarcinomas), the gastric retention illness, gastroenteritis, gastratrophia, stomach/gastric cancer, polyp of stomach, the autoimmune illness is such as pernicious anemia, pyloric stenosis, gastritis is (bacillary, viral, eosinophilic granulocyte, pressure causes, chronic corrosivity, atrophic, plasma cell and giant hypertrophy gastritis), and disease of peritoneum is (as chyloperitoneum (chyloperioneum), hemoperitoneum, mesenteric cyst, adenomesenteritis, mesenteric vascular occlusion, panniculitis, neoplasm, peritonitis, pneumoperitoneum, subphrenic abscess (bubphrenic abscess)).
Gastrointestinal tract disease also comprises the illness relevant with small intestinal, such as malabsorption syndrome, expand, irritable bowel syndrome, sugar does not tolerate, celiac disease, duodenal ulcer, duodenitis, intertropica stomatitis, Whipple, intestinal lymphangiectasia, Crohn disease, appendicitis, ileocleisis, Meckel's diverticulum, many diverticulums, small intestinal and large intestine can not full rotations, lymphoma, with antibacterial and parasitic disease (such as traveler's diarrhea, injury and paratyphoid fever, cholera, ascarid (ascariasis (Ascariasis lumbricoides)), ancylostome (Ancylostoma duodenale (Ancylostoma duodenale)), pinworm (pinworm (Enterobius vermicularis)), cestode (taeniasis bovis (Taenia saginata), Echinococcus granulosus (Echinococcus granulosus), Bothriocephalus and taeniasis suis (T.solium)) infect.
Hepatic disease and/or illness comprise intrahepatic cholestasis (alagille syndrome, biliary cirrhosis), fatty liver (alcoholic fatty liver, the Lay syndrome), hepatic vein thrombosis forms, hepatolenticular degeneration (hepatolentricular degeneration), hepatomegaly, hepatopulmonary syndrome, hepatorenal syndrome, portal hypertension (esophagus and gastric varices), liver abscess (amebic liver abscess), liver cirrhosis (ethanol, bile and experimental), dirty disease (the fatty liver of alcoholic liver, hepatitis, liver cirrhosis), parasitic (hepatic echinococcosis, fascioliasis, amebic liver abscess), jaundice (hemolytic, hepatocyte property and cholestasis), cholestasis, portal hypertension, liver is expanded, ascites, hepatitis (alcoholic hepatitis, animal hepatitis, chronic hepatitis (autoimmune, hepatitis B, hepatitis C, hepatitis D, drug-induced), toxic hepatitis, viral human hepatitis (hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E), Wilson's disease, granulomatous hepatitis, the Secondary cases biliary cirrhosis, hepatic encephalopathy, portal hypertension, varicosis, hepatic encephalopathy, primary biliary cirrhosis, primary sclerosing cholangitis, adenoma, hemangioma, cholelithiasis, liver failure (hepatic encephalopathy, acute hepatic depletion), with liver neoplasm (angiomyoliopma, calcification liver metastasis, cyst liver metastasis, epithelioma, fibrous layer hepatocarcinoma, focal nodular hyperplasia, adenoma of liver, hepatobiliary cystadenoma, hepatoblastoma, hepatocarcinoma, hepatoma, hepatocarcinoma, the liver vessel endothelioma, mesenchymal hamartoma, the liver mesenchymoma, nodular regenerative hyperplasia, optimum liver neoplasm (hepatic cyst [simple cyst, the dirty disease of polycystic liver, hepatobiliary cystadenoma, choledochal cyst], mesenchymoma [mesenchymal hamartoma, child's hemangioendothelioma, hemangioma, peliosis hepatis, lipoma, inflammatory pseudotumor, miscellany], epithelioma [epithelial duct (bile duct hamartoma, cholangioadenoma), hepatocyte (adenoma, focal nodular hyperplasia, nodular regenerative hyperplasia)], pernicious liver neoplasm is [hepatocellular, hepatoblastoma, hepatocarcinoma, bile duct cell, cancer of biliary duct, cystadenocarcinoma, vascular tumor, angiosarcoma, Kaposi sarcoma, hemangioendothelioma, other tumor, embryonic sarcoma, fibrosarcoma, leiomyosarcoma, rhabdomyosarcoma, carcinosarcoma, teratoma, carcinoid, squamous cell carcinoma, the constitutional lymphoma]), peliosis hepatis, the erythrocyte hepatic porphyria, hepatic porphyria (acute intemittent porphyria, porphyria cutanea tarda), the Ze Weige syndrome).
Pancreatic diseases and/or illness comprise acute pancreatitis, chronic pancreatitis (acute necrotizing pancreatitis, alcoholic-toxic pancreatitis), neoplasm (pancreas adenocarcinoma, cystadenocarcinoma, insulinoma, gastrinoma and glucagonoma of pancreas, capsule neoplasm, islet cells tumor, Pancreatoblastoma (pancreoblastoma)) and other pancreatic diseases (as cystic fibrosis, cyst (pancreatic pseudocyst, pancreatic fistula, functional defect)).
Gallbladder disease comprises cholelithiasis (cholelithiasis and choledocholithiasis), post-cholecystectomy syndrome, diverticulum of gallbladder disease, acute cholecystitis, chronic cholecystitis, tumor of bile duct and mucous cyst.
Large intestine disease and/or illness comprise antibiotic-relevant colitis, diverticulitis, ulcerative colitis, acquired megacolon, abscess, fungus and bacterial infection, anal orifice and rectal intestine illness (as crack, hemorrhoid), colonic diseases (colitis, colon neoplasm [colon cancer, adenoma polyp of colon (as villous adenoma), colon cancer, colorectal carcinoma], diverticulitis of colon, diverticulosis of colon, megacolon [Hirschsprung disease, toxic megacolon]; Sigmoid diseases [proctocolitis, the sigmoid colon neoplasm]), constipation, Crohn disease, diarrhoea (infantile diarrhea, dysentery), dudenal disease (duodenum neoplasm, duodenal obstruction, duodenal ulcer, duodenitis), enteritis (enterocolitis), the HIV enteropathy, ileum disease (ileum neoplasm, ileitis), immunoproliferative small intestine disease, inflammatory bowel (ulcerative colitis, Crohn disease), intestinal atresia, parasitosis (anisakiasis, balantidiasis, yeast infection, cryptosporidiosis, double-core amebiasis (dientamoebiasis), amebic dysentery, giardiasis), intestinal fistula (rectal fistula), intestinal neoplasm (cecal neoplasm, the colon neoplasm, the duodenum neoplasm, the ileum neoplasm, polyp intestinal, the jejunum neoplasm, the rectum neoplasm), intestinal obstruction (afferent loop syndrome, duodenal obstruction, impacted feces, intestinal pseudo-obstruction [volvulus of cecum], intussusception), intestinal perforation, polyp intestinal (polyp of colon, Gardner syndrome, Peutz Jeghers syndrome), jejunum disease (jejunum neoplasm), malabsorption syndrome (blind loop syndrome, celiac disease, lactose intolerance, short intestinal (short bowl) syndrome, intertropica stomatitis, Whipple), mesenteric vascular occlusion, pneumatosis cystoides intestinalis, protein loss enteropathy (intestinal lymphangiectasia), recial disease (anus, fecal incontinence, hemorrhoid, proctitis, rectal fistula, proctoptosis, proctoptosis), peptic ulcer (duodenal ulcer, peptic esophagitis, hemorrhage, perforation, gastric ulcer, Zollinger Ellison syndrome), dumping syndrome (dumping syndrome), gastric disease is (as achlorhydria, duodenogastric reflux (bile reflux), gastric antrum vasodilation disease, gastric fistula, gastric outlet obstruction, gastritis (atrophic or plumpness), gastroparesis, flatulence, gastric diverticulum, stomach neoplasm (gastric cancer, polyp of stomach, adenocarcinoma of stomach, the hypertrophy polyp of stomach), gastric rupture, gastric ulcer, gastric volvulus), tuberculosis, visceroptosis, vomiting is (as hematemesis, the hyperemesis gravidarum, operation is nausea and vomiting afterwards) and hemorrhagic colitis.
The further disease and/or the illness of gastronintestinal system comprise bile duct disease, split such as abdomen, fistula is (as leak, esophageal fistula, gastric fistula, intestinal fistula, pancreatic fistula), neoplasm is (as the bile duct neoplasm, the esophagus neoplasm, such as esophageal adenocarcinoma, esophageal squamous cell carcinoma, the gastrointestinal neoplasm, the pancreas neoplasm is such as pancreas adenocarcinoma, pancreas bursa neoplasm, pancreas capsule neoplasm, Pancreatoblastoma and peritoneum neoplasm), esophagel disease is (as the bulla disease, candidiasis, the glycogen acanthosis, ulcer, the Barrett esophageal varix, locking, cyst, diverticulum (as divicine), fistula (as tracheo esophageal fistula), mobility's illness is (as the CREST syndrome, swallow illness, relaxing can not, spasm, gastroesophageal reflux), neoplasm, perforation is (as boerhaave syndrome, the Mallory Weiss syndrome), narrow, esophagitis, diaphragmatocele (as hiatal hernia); Gastrointestinal disease is such as gastroenteritis (infecting as eholera morbus, norwalk virus), hemorrhage (as hematemesis, tarry stool, digestive ulcerative bleeding), stomach neoplasm (gastric cancer, polyp of stomach, adenocarcinoma of stomach, gastric cancer)), hernia (as congenital diaphragmatic hernia, femoral hernia, inguinal hernia, obturator hernia, umbilical hernia, abdominal hernia) and intestinal diseases (as caecum disease (appendicitis, cecal neoplasm)).
Chemotaxis
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can have chemotactic activity.Chemotaxis molecular line or mobilization cell (as mononuclear cell, fibroblast, neutrophil cell, T-cell, mastocyte, eosinocyte, epithelium and/or endotheliocyte) are to the interior specific part of body, such as inflammation, infection or hyper-proliferative position.By the cell of the being mobilized specific wound or unusual of to beat back subsequently and/or to heal.
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can increase the chemotactic activity of specific cells.These chemotaxis molecules can be used for the treatment of inflammation, infection, hyper-proliferative illness or any immune system illness by increasing targeting cell number of ad-hoc location in body subsequently.For example by attracting immunocyte to damaged location, the chemotaxis molecule can be used for wound and other wound of treated tissue.Chemotaxis molecule of the present invention also can attract fibroblast, and this can be used for treating wound.
Expect that also the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can suppress chemotactic activity.These molecules also can be used for treating illness.Therefore the polynucleotide of the fusion rotein of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used as chemotactic inhibitor.
In conjunction with active
Albumin fusion proteins of the present invention can be used for screening can be in conjunction with the human cytokines molecule partly or the bonded molecule of human cytokines part energy of fusion rotein of fusion rotein.Fusion rotein can activate (as agonist, partial agonist etc.) with combining of molecule, increase, suppresses the activity of (as antagonist, partial antagonist etc.) or minimizing fusion rotein or institute's binding molecule.The example of this quasi-molecule comprises antibody, oligonucleotide, albumen (as receptor) or micromolecule.
In one embodiment, the native ligand (as part fragment or natural substrate, part, structure or functional simulation thing) of the human cytokines of this molecule and fusion rotein of the present invention part is closely related.(see Coligan etc., Current Protocols in Immunology 1 (2): the 5th chapter (1991)).Similarly, this molecule can be closely related by bonded natural receptor with the human cytokines of albumin fusion proteins of the present invention part, or at least with can being closely related of this receptor by the bonded fragment of human cytokines part (as avtive spot) of albumin fusion proteins of the present invention.Under any situation, can use known technology reasonably to design molecule.
In one embodiment, screen these molecules and comprise the suitable cell that produces expression albumin fusion proteins of the present invention.Exemplary cells comprises from mammal, yeast, fruit bat or colibacillary cell.
Combining of candidate compound and albumin fusion proteins of the present invention can be only tested in detection, wherein in conjunction with being detected by label, or detects in the mensuration system that comprises with competitor's competition of labelling.Further, mensuration can be tested candidate compound and whether caused the signal that produces by being incorporated into fusion rotein.
Perhaps, can use acellular goods, the fusion rotein/molecule that invests solid support, chemical library or natural prodcuts mixture to detect.Detect also can only comprise the steps: to mix candidate compound with the solution that contains albumin fusion proteins, measurement fusion albumen/molecular activity or combine, also comparison fusion rotein/molecular activity or with the combining of standard substance.
In one embodiment, use monoclonal or polyclonal antibody, but ELISA detects fusion rotein level or activity in the measuring samples (as biological sample).Antibody can be by being incorporated into albumin fusion proteins or by measurement fusion protein level or activity with albumin fusion proteins competition substrate directly or indirectly.
In addition, human cytokines receptor partly in conjunction with albumin fusion proteins of the present invention can be identified by several different methods well known by persons skilled in the art, for example part elutriation and FACS sorting (Coligan etc., Current Protocols in Immun. (the up-to-date experimental program of immunology), 1 (2), the 5th chapter, (1991)).For example in the situation of the human cytokines of fusion rotein part corresponding to FGF, can adopt expression cloning, wherein from response to the cell of albumin fusion proteins (for example known NIH3T3 cell that contains multiple receptor at the FGF family protein, and SC-3 cell) RNA of preparation polyadenylation, to be divided into a plurality of ponds (pools) by the cDNA library that this RNA produces, with their transfections not in response to COS cell or other cell of albumin fusion proteins.Behind labelling albumin fusion proteins of the present invention, the transfectional cell that is grown in microscope slide is exposed to albumin fusion proteins of the present invention.Albumin fusion proteins is labelling in many ways, the recognition site that comprises iodate or mix site-specificity protein kinase.
Fixing and cultivate after, microscope slide is carried out radioautographic analysis.Identify positive pond, prepare subpool (sub-pool) and carry out transfection again that final generation coding is inferred the monoclonal of receptor by the process of dividing subpool (sub-pooling) and screening more repeatedly.
As a kind of alternative approach of identifying receptor, can or extract prepared product with the cell membrane of the affine acceptor molecule that is connected in therapeutic (Therapeutoc) protein part of expressing albumin fusion proteins of the present invention of the albumin fusion proteins light of labelling, the material after the connection can be analyzed by PAGE and resolve and x-ray film is exposed.The complex of labelling that contains the receptor of fusion rotein can be downcut, resolve to fragments of peptides, and carry out albumen micrometering preface.Can use the aminoacid sequence that obtains from the micrometering preface to design one group of degenerate oligonucleotide probe and screen receptor is inferred in the cDNA library with identification code gene.
And, the technology of gene reorganization, motif reorganization, exon reorganization and/or codon reorganization (being generically and collectively referred to as " DNA reorganization ") can be used for regulating the fusion rotein of albumin fusion proteins of the present invention and/or human cytokines partly or the activity of albumin part, thereby produces the agonist and the antagonist of albumin fusion proteins of the present invention effectively.Generally referring to United States Patent (USP) the 5th, 605,793,5,811,238,5,830,721,5,834,252 and 5,837, No. 458 and Patten, P.A. etc., Curr.Opinion Biotechnol.8:724-33 (1997); Harayama, S.Trends Biotechnol.16 (2): 76-82 (1998); Hansson, L.O. etc., J.Mol.Biol.287:265-76 (1999); And Lorenzo, M.M. and Blasco, R.Biotechniques 24 (2): 308-13 (1998); These patents and publication are all incorporated into by reference at this).In one embodiment, DNA reorganization can realize changing the polynucleotide of coding albumin fusion proteins of the present invention and the albumin fusion proteins that therefore changes coding.DNA reorganization comprises by homologous recombination or locus specificity reorganization, two or more DNA sections is assembled into the molecule of expectation.In another embodiment,, can change the polynucleotide of coding albumin fusion proteins of the present invention, and therefore change the albumin fusion proteins of coding by before reorganization, it being carried out fallibility PCR, adds the random mutagenesis of random nucleotide or other method.In another embodiment, one or more parts of albumin fusion proteins of the present invention, motif, section, partly, domain, fragment etc. can be with one or more components of one or more heterologous molecule, motif, section, partly, reorganization such as domain, fragment.In some embodiments, this heterologous molecule is the family member.In further embodiment, this heterologous molecule is a somatomedin, such as for example platelet derived growth factor (PDGF), insulin like growth factor (IGF-I), transforming growth factor (TGF)-α, epidermal growth factor (EGF), fibroblast growth factor (FGF), TGF-β, bone morphogenetic protein (BMP)-2, BMP-4, BMP-5, BMP-6, BMP-7, activin (activin) A and B, decapentaplegic (dpp), 6OA, OP-2, procaine benzylpenicillin, growth and differentiation factor (GDF), nodal, MIS, inhibin-α, TGF-β 1, TGF-β 2, TGF-β 3, TGF-β 5 and neuroglia derived neurotrophic factor (GDNF).
The human cytokines part that other exemplary fragment is an albumin fusion proteins of the present invention and/or the bioactive fragment of albumin part.Bioactive fragment is performance and the human cytokines part of albumin fusion proteins of the present invention and/or the active similar but not necessarily identical active fragment of albumin part.Segmental biological activity can comprise that improved expectation is active or reduce do not expect activity.
In addition, the invention provides a kind of SCREENED COMPOUND to identify the method for the chemical compound of regulating albumin fusion proteins effect of the present invention.An example of this class algoscopy be included under the normal proliferating cells condition of culture of fibroblast with mammal fibroblast, albumin fusion proteins of the present invention and chemical compound to be screened with
3[H] thymidine mixes.Can carry out blank determination under the chemical compound be screened not existing, and exist the amount of fibroblast proliferation down to compare with by determining to absorb under every kind of situation with chemical compound
3[H] thymidine and determine whether chemical compound stimulates proliferation.The amount of fibroblast proliferation is by measurement
3The liquid phase flicker chromatograph that [H] thymidine mixes is measured.Agonist and agonist compounds be scheme evaluation thus all.
In another approach, cultivate in the presence of chemical compound expressing the mammalian cell of receptor of human cytokines part of fusion rotein of the present invention or the fusion rotein of the present invention of film preparation thing and tape label thing.Can measure this chemical compound enhancing subsequently or stop this interactional ability.Perhaps, known second messenger system is being treated that the response behind SCREENED COMPOUND and the acceptor interaction measures, and measuring the ability that this chemical compound is incorporated into receptor and causes second message,second messenger's response, to determine whether this chemical compound is potential fusion rotein.This second messenger system includes but not limited to cAMP guanylate cyclase, ion channel or phosphoinositide hydrolysis.
Above-mentioned all these mensuration all can be used as diagnosis or prognostic markers.The molecule that uses these algoscopys to find can or suppress fusion rotein/molecule by activation and be used for the treatment of disease or cause particular result (as angiogenic growth) in the patient.And these algoscopys can be found such agent, and the cell or tissue that they can suppress or strengthen through proper handling produces albumin fusion proteins of the present invention.
Therefore, the present invention includes the method for identifying the chemical compound be incorporated into albumin fusion proteins of the present invention, may further comprise the steps: (a) with candidate's binding compounds with albumin fusion proteins incubation of the present invention; (b) determine whether to have taken place combination.And, the present invention includes the method for identifying agonist/antagonist, may further comprise the steps: (a) with candidate compound with albumin fusion proteins incubation of the present invention, (b) measure biological activity and determine (b) whether the biological activity of fusion rotein is changed.
Targeted delivery
In another embodiment, the invention provides a kind of method to the target cell delivering compositions, wherein target cell is expressed the receptor of the component of albumin fusion proteins of the present invention.
As described herein, fusion rotein of the present invention (as albumin fusion proteins) can associate via hydrophobic, hydrophilic, ion and/or covalent interaction and heterologous polypeptide, heterologous nucleic acids, toxin or prodrug.In one embodiment, the invention provides the method for a kind of specific delivery compositions of the present invention to cell, this method system is by using and heterologous polypeptide or the associating fusion rotein of the present invention of nucleic acid (comprising antibody).In an example, the invention provides the method for a kind of delivery of therapeutic albumen to the cell of institute's targeting.In another example, the invention provides a kind of method of sending single-chain nucleic acid (as antisensenucleic acids or ribozyme) or double-strandednucleic acid (as the genome that can be integrated into cell or free the DNA that duplicates and can be transcribed) to the cell of institute's targeting.
In another embodiment, the invention provides a species specificity and destroy the cell method of (as destroying tumor cell), this method system passes through albumin fusion proteins of the present invention (as polypeptide of the present invention or antibody of the present invention) and toxin or cytotoxicity prodrug combined administration.
" toxin " means chemical compound, radiosiotope, the holotoxin of combination and activation of endogenous cytotoxic effect device system, the toxin of modification, catalytic subunit or any molecule or enzyme that causes cell death in the cell or on the cell surface, under qualifications that be not present in usually of toxin.The spendable toxin of the method according to this invention includes but not limited to, radiosiotope known in the art, chemical compound are such as for example in conjunction with antibody (or it contains the part of complement fixation part), thymidine kinase, endonuclease, RNA enzyme, alpha toxin, ricin, Agglutinin, Rhodopseudomonas exotoxin A, diphtheria toxin, diphtherotoxin, Saponin, momordin, gelonin, pokeweed antiviral protein, α-Zhou Qujunsu and cholera toxin inherent or that induce the endogenous cell poisonous effect system of generation." cytotoxicity prodrug " refers to so non-toxic chemical, and its enzymatic conversion that is present in the cell usually is a cytotoxic compound.The spendable cytotoxicity prodrug of the method according to this invention includes but not limited to glutamyl derivative, etoposide or ametycin, cytosine arabinoside, the phosphoric acid derivatives of daunorubicin (daunorubisin) and the phenoxy-acetamide derivant of doxorubicin of benzoic acid mustard alkylating agent.
Drug screening
The polynucleotide screening that further contemplates that albumin fusion proteins of the present invention or these fusion rotein of encoding changes albumin fusion proteins of the present invention or corresponding to the purposes of the proteic active molecule of the human cytokines part of albumin fusion proteins.This method will comprise fusion rotein will be contacted with the chemical compound that selected suspection has antagonist or agonist activity, and in the activity of measuring fusion rotein in conjunction with the back.
The present invention is particularly useful for by using albumin fusion proteins of the present invention or its binding fragment to screen therapeutic compound in multiple drug screening technology any.The albumin fusion proteins that adopts in this class testing can attached on the solid support, be expressed on the cell surface, be free in the solution or be positioned at cell.A kind of drug screening method uses eucaryon or the prokaryotic host cell with the recombinant nucleic acid stable conversion of expressing albumin fusion proteins.In competitive combination detects, at this cell transformed or from cultivating the supernatant screening of medicaments that this cell obtains.For example can measure the formation of the complex between tested agent and the albumin fusion proteins of the present invention.
Therefore the invention provides the active medicine of influence albumin fusion proteins mediation of the present invention or the screening technique of any other agent.These methods comprise the existence that such agent is contacted albumin fusion proteins of the present invention or its fragment and detect complex between described agent and albumin fusion proteins or its fragment by well known method.This competitive in conjunction with in detecting, agent to be screened normally is labeled.After the cultivation, the agent that free agent and combining form are existed separates, and the free or amount of compound label the measuring in conjunction with the ability of albumin fusion proteins of the present invention that be the specific function agent not.
Another kind of drug screening technology provides high flux screening and albumin fusion proteins of the present invention to have the chemical compound of suitable binding affinity, this method is described in detail in the european patent application 84/03564 of JIUYUE in 1984 announcement on the 13rd, and it is incorporated into by reference at this.Briefly, go up the different little peptide test compounds of synthesizing big figure at solid state substrate such as plastics pin (plastic pin) or some other surfaces.Make reaction of peptide test compounds and albumin fusion proteins of the present invention and washing.Detect bonded peptide by well known method subsequently.Can be directly the albumin fusion proteins of purification be coated on the plate to be used for aforementioned drug screening technology.In addition, can utilize non-neutral antibody capture peptide and being fixed on the solid support.
The present invention also comprises use competitive drug screening assay algoscopy, wherein can compete albumin fusion proteins or its segmental combination with test compounds specifically in conjunction with the neutralizing antibody of albumin fusion proteins of the present invention.The existence of the peptide of one or more identical epitopes is arranged with antibody test albumin fusion proteins any and of the present invention by this way.
Binding peptide and other molecule
The present invention comprises that also evaluation is in conjunction with the screening technique of the polypeptide of albumin fusion proteins of the present invention and non-polypeptide with by its binding molecule of identifying.These binding molecules can be used as for example agonist and the antagonist of albumin fusion proteins of the present invention.Such agonist and antagonist can use in the treatment embodiment of following detailed description according to the present invention.
This method comprises the steps: to make albumin fusion proteins of the present invention to contact a plurality of molecules; With the molecule of identifying in conjunction with albumin fusion proteins.
The step that makes albumin fusion proteins of the present invention contact a plurality of molecules can be carried out in many ways.For example can consider albumin fusion proteins is fixed on the solid support and the solution of a plurality of molecules is contacted with immobilized polypeptide.This scheme is similar to the affinity chromatography method, and affinity substrate comprises immobilized albumin fusion proteins of the present invention.Can select purification albumin fusion proteins to be had the molecule of selectivity affinity by affinity subsequently.The character of solid support, method, affinity that albumin fusion proteins is connected in solid support are separated or solvent and the condition selected are to be conventional substantially, and are that those of ordinary skills know.
Perhaps, also a plurality of polypeptide can be divided into isolating basically fraction, such fraction comprises the subgroup of polypeptide or independent polypeptide.For example, can separate a plurality of polypeptide by gel electrophoresis, column chromatography or similar polypeptide separation method well known by persons skilled in the art.Independent polypeptide also can prepare by transformed host cells, and on its outer surface or on every side (as recombinant phage) expresses.Then can be with the independent separator of albumin fusion proteins of the present invention " detection ", randomly, inducer is then surveyed in the presence of inducer if desired, to determine that whether any selectivity affinity takes place between albumin fusion proteins and the single clone interacts.Making before albumin fusion proteins contact comprises each fraction of independent polypeptide, can earlier polypeptide be transferred to solid support for more convenient.This solid support can only be a slice filter membrane, the filter membrane of making such as celluloid or nylon.By this way, can transform from expression library, comprise that coding has albumin fusion proteins of the present invention a group host cell of DNA construct of polypeptide of selectivity affinity and identify positive colony.In addition, the amino acid sequence of polypeptide that albumin fusion proteins of the present invention is had the selectivity affinity can be directly definite by conventional method, and perhaps, the coded sequence of the DNA of coded polypeptide often can be determined more easily.Then can be from corresponding DNA sequence derivation primary sequence.If determine aminoacid sequence, can use micrometering preface technology from polypeptide itself.Sequencing technologies can comprise mass spectral analysis.
In some cases, before attempting definite or detecting the interactional existence of selectivity affinity, may be preferably from any unconjugated polypeptide of the mixture flush away of albumin fusion proteins of the present invention and a plurality of polypeptide.When albumin fusion proteins of the present invention or a plurality of polypeptide were incorporated into solid support, such washing step can be special expectation.
The a plurality of molecules that provide according to this method can provide by the mode of diverse libraries, such as can be used for screening specificity in conjunction with the molecule of albumin fusion proteins of the present invention at random or the peptide or the non-peptide library of combination.Many libraries are known in the art and spendable, as the library of chemosynthesis, recombinant (as phage display library) with based in vitro translated library.The example in the library of chemosynthesis is described in Fodor etc., Science 251:767-773 (1991); Houghten etc., Nature 354:84-86 (1991); Lam etc., Nature 354:82-84 (1991); Medynski, Bio/Technology 12:709-710 (1994); Gallop etc., J.Medicinal Chemistry 37 (9): 1233-1251 (1994); Ohlmeyer etc., Proc.Natl.Acad.Sci.USA 90:10922-10926 (1993); Erb etc., Proc.Natl.Acad.Sci.USA 91:11422-11426 (1994); Houghten etc., Biotechniques 13:412 (1992); Jayawickreme etc., Proc.Natl.Acad.Sci.USA 91:1614-1618 (1994); Salmon etc., Proc.Natl.Acad.Sci.USA 90:11708-11712 (1993); PCT announces WO93/20242 number; With Brenner and Lerner, Proc.Natl.Acad.Sci.USA 89:5381-5383 (1992).
The example of phage display library is described in Scott etc., Science 249:386-390 (1990); Devlin etc., Science, 249:404-406 (1990); Christian etc., 1992, J.Mol.Biol.227:711-7181992); Lenstra, J.Immunol.Meth.152:149-157 (1992); Kay etc., Gene128:59-65 (1993); Announce WO No. 94/18318 with the PCT on August 18th, 1994.
Include but not limited to as described below based in vitro translated library: the PCT on April 18th, 1991 announces WO No. 91/05058; With Mattheakis etc., Proc.Natl.Acad.Sci.USA 91:9022-9026 (1994).
As the example of non-peptide library, the benzodiazepine library (see as Bunin etc., Proc.Natl.Acad.Sci.USA 91:4708-4712 (1994)) can be suitable for using.Also can use class peptide library (Simon etc., Proc.Natl.Acad.Sci.USA 89:9367-9371 (1992)).Another example in spendable library is described in (Proc.Natl.Acad.Sci.USA 91:11138-11142 (1994)) such as Ostresh, and wherein the amide functional group of peptide is by permethylated (permethylated) combinatorial library with the generation chemical conversion.
The kind that can be used for non-peptide library of the present invention is a lot.For example (Bio/Technology 13:351-360 (1995) has listed benzodiazepine in the chemical substance that constitutes basis, various library, hydantoin, NSC-135758, biphenyl, sugar analogue, β-sulfydryl ketone, Arylacetic acids, acylpiperidine .alpha.-5:6-benzopyran, cubane, xanthine, aminimide with azolactone for Ecker and Crooke.
Non-peptide library can be divided into two types widely: (decorated) monomer and the oligomer of modification.Simple relatively supporting structure is adopted in the monomer library of modifying, and adds multiple functional group on it.Mostly support is to have the molecule of known useful pharmacological activity.For example support can be benzodiazepine.
The monomer of big figure is used in non--peptide oligomer library, and they fit together in some way, forms the shape that depends on monomer sequence.Already used monomeric unit comprises carbamate, pyrrolinone and morpholino.Class peptide---class wherein side chain is connected in the peptide sample oligomer of α amino group rather than alpha-carbon atom---is the basis in another kind of non-peptide oligomer library.The monomer of single type is used in the first non-peptide oligomer library, therefore contains multiple main chain (repeating backbone).Has used more than a kind of monomer in recent library, makes the library have more motilities.
Can realize library screening by any method in the multiple known method commonly used.Referring to for example disclose the peptide library screening below with reference to document: Parmley etc., Adv.Exp.Med.Biol.251:215-218 (1989); Scott etc., Science 249:386-390 (1990); Fowlkes etc., Biotechnologys13:422-427 (1992); Oldenburg etc., Proc.Natl.Acad.Sci.USA 89:5393-5397 (1992); Yu etc., Cell 76:933-945 (1994); Staudt etc., Science 241:577-580 (1988); Bock etc., Nature 355:564-566 (1992); Tuerk etc., Proc.Natl.Acad.Sci.USA 89:6988-6992 (1992); Ellington etc., Nature 355:850-852 (1992); Authorize No. the 5th, 223,409, No. the 5th, 096,815, United States Patent (USP), the United States Patent (USP) of Ladner etc. and United States Patent (USP) the 5th, 198, No. 346; Rebar etc., Science 263:671-673 (1993); Announce WO94/18318 number with PCT.
In specific implementations, can be fixed in albumin fusion proteins of the present invention on the solid phase by making library member contact, and collect and be incorporated into the library member of albumin fusion proteins and screen to identify molecule in conjunction with albumin fusion proteins of the present invention.This being called,, the example of screening technique of " elutriation " technology was described in for example Parmley etc., Gene 73:305-318 (1988); Fowlkes etc., Biotechniques13:422-427 (1992); PCT announces WO No. 94/18318; The list of references of being quoted with this paper.
In another embodiment, in yeast, select two-hybrid system (Fields etc., the Nature 340:245-246 (1989) of interaction protein; Chien etc., Proc.Natl.Acad.Sci.USA 88:9578-9582 (1991) can be used for identifying that specificity is incorporated into the molecule of polypeptide of the present invention.
When binding molecule was polypeptide, this polypeptide can be selected from any peptide library easily, comprised (biased) peptide library of random peptide library, combined peptide library or bias.Term used herein " bias " is meant handles the library production method, thereby multifarious one or more parameters that the molecule (being peptide under this situation) of domination gained is gathered limit.
Therefore the peptide library of the true random set that will produce peptide, wherein the probability in the assigned address discovery specific amino acids of peptide is identical to all 20 seed amino acids.Mode below but for example also available is introduced the library with bias: stipulate that a lysine appears in every five amino acid, or the position 4,8 and 9 in regulation decapeptide library is fixed as and only comprises arginine.Obviously, can expect the bias of many types, and the invention is not restricted to any specific bias.In addition, the present invention has considered the peptide library of particular type, such as the phage display peptide library with utilize the peptide library of DNA construct (comprise have DNA insert segmental bacteriophage lambda) carrier.
As mentioned above, be the situation of polypeptide at binding molecule, this polypeptide can have about 6 to being less than about 60 amino acid residues, for example about 6 to about 10 amino acid residues, and further, about 6 to about 22 aminoacid.In another embodiment, the aminoacid that has 15-100 aminoacid or 20-50 aminoacid scope in conjunction with polypeptide.
Selected can be in conjunction with polypeptide by chemosynthesis or recombinant expressed acquisition.
Other activity
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for stimulating in treatment because the blood vessel of the tissue of various diseases situation (such as thrombosis, arteriosclerosis and other cardiovascular disorder) and ischemia forms again.The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding also can be used for stimulating blood vessel to take place and limb regeneration, as discussed above.
Also can be used for of the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding, treat because the wound due to damage, burn, operation back tissue repair and the ulcer, this is because they have mitogenetic effect to the cell (such as fibroblast and Skeletal Muscle Cell) of various separate sources, therefore can promote the reparation or the replacement of impaired or sick tissue.
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding also can be used for the neuron infringement that stimulating neuronal growth and treatment and prevention take place in some neuron illness or neurodegenerative disease such as Alzheimer, parkinson disease and AIDS AIDS-related complex AIDS.The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can have the ability that stimulates the chondrocyte growth, therefore can be used for strengthening bone and periodontal regenerative and help tissue transplantation or the bone transplanting.
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding also can be used for by stimulating the keratinocyte growth to prevent the skin aging that Exposure to Sunlight causes.
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding also can be used for preventing alopecia.Similarly, when using with other combination of cytokines, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for hemopoietic cell and medullary cell growth and differentiation.
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding also are used in the cell culture of keeping organ before the transplanting or supporting former generation tissue.The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding also can be used for inducing being organized in the body early embryo of mesoderm origin to break up.
Except hematopoietic lineage as discussed above, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding also can increase or reduce the differentiation or the propagation of embryonic stem cell.
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding also can be used for regulating the mammal feature, such as height, body weight, hair color, wink, skin, fatty tissue percentage ratio, pigmentation, size and profile (as cosmetic surgery).Similarly, the polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for regulating the mammal metabolism, influence catabolism, anabolism, processing, utilization and the storage of energy.
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding can be used for changing the mammiferous mental status or condition, by influencing biorhythm, cardiac rhythm (caricadic rhythms), depressed (comprising depressed illness), violence trend, tolerance, reproductive performance (for example by activin or inhibin sample activity), hormone or endocrine level, appetite, libido, memory, pressure or other awareness to pain.
The polynucleotide of the albumin fusion proteins of the present invention and/or the albumin fusion proteins of the present invention of encoding also can be used as food additive or antiseptic, such as in order to increase or reduce storage capacity, fat content, lipid, protein, sugar, vitamin, mineral, cofactor or other nutritional labeling.
Above-mentioned application can be used for host widely.This host includes but not limited to, the mankind, muroid, rabbit, goat, Cavia porcellus, camel, horse, mice, rat, hamster, pig, miniature pig, chicken, goat, cattle, sheep, dog, cat, the non--human primates and mankind.In specific implementations, this host is mice, rabbit, goat, Cavia porcellus, chicken, rat, hamster, pig, sheep, dog or cat.In some embodiments, this host is a mammal.In other embodiments, this host is human.
As above described the present invention prevailingly, will be more readily understood the present invention with reference to following examples, embodiment provides as example explanation not mean restriction.
Need not to further describe, believe the change that those of ordinary skills use above description and exemplary embodiment hereinafter can make and utilize among the present invention to be found, and implement method required for protection.Therefore following work embodiment points out in the embodiments of the present invention particularly, and is not interpreted as and limits remainder of the present disclosure by any way.
Embodiment
The generation of embodiment 1:pScNHSA and pScCHSA
Carrier pScNHSA (ATCC preserving number PTA-3279) and carrier pScCHSA (ATCC preserving number PTA-3276) are the derivants of pPPC0005 (ATCC preserving number PTA-3278), and as cloning vehicle, the polynucleotide of coding human cytokines or its fragment or variant are inserted described two kinds of carriers, make the polynucleotide of its contiguous human serum albumin of coding " HSA ", and be in the same translation reading frame with these polynucleotide.PScCHSA can be used for producing human cytokines-HSA fusant, and pScNHSA can be used for producing HSA-human cytokines fusant.
The structure of pScCHSA: the albumin that albumin part is positioned at therapeutic partial C end merges
Body
Introduce the restriction site of Xho I and Cla I by changing nucleotide sequence that pPPC0005 goes up the chimeric HSA signal peptide of coding, preparation helps encoding the dna clone of human cytokines to the carrier of the N-terminal of the albuminised DNA of encoding mature.
At first, remove the intrinsic Xho I of pPPC0005 and Cla I site (being positioned at 3 ' end of ADH1 terminator sequence) with Xho I and Cla I digestion pPPC0005, then flat sticking terminal with T4DNA polymerase benefit, reconnect flat end at last, obtain pPPC0006.
Secondly, adopt the two-wheeled PCR artificially restriction site of introducing Xho I and ClaI of on pPPC0006, encode in the nucleotide sequence of HSA signal peptide (HSA targeting sequencing and from the chimera in the kex2 site of mating factor α " MAF ").First round PCR uses the primer that is shown as SEQ ID NO:36 and SEQID NO:37 to increase.The primer that its sequence is shown as SEQ ID NO:36 comprises coded portion HSA signal peptide sequence, from the kex2 site of mating factor α targeting sequencing, and the aminoterminal nucleotide sequence of section H SA mature form.This section sequence is introduced 4 point mutation, is created in Xho I and Cla I site that chimeric signal peptide and HSA mature form junction are found.Underline in the sequence shown in these 4 sudden changes below.In pPPC0005, it is T, G, T and G that the nucleotide of these 4 positions is held 3 ' end from 5 '.
5 '-GC
CT
CGA
GAAAAGAGATGCACACAAGAGTGAGGTTGCTCATCG
ATTTAAAGATTTGGG-3 ' (SEQ ID NO:36) and
5’-AATCGATGAGCAACCTCACTCTTGTGTGCATCTCTTTTCTCGAGGCTCCTGGAATAAGC-3’(SEQ?ID?NO：37)。Second take turns PCR and increase then with upstream flank primer 5 '-TACAAACTTAAGAGTCCAATTAGC-3 ' (SEQ ID NO:38) and downstream flank primer 5 '-CACTTCTCTAGAGTGGTTTCATATGTCTT (SEQ ID NO:39).With the PCR product purification that obtains,, connect into the same loci of pPPC0006 then, obtain pScCHSA with Afl ∏ and Xba I digestion.The site of manually having introduced Xho I and Cla I in its signal sequence of the plasmid of Xing Chenging like this.The existence in this Xho I site causes the change of the terminal single amino acids of signal sequence, has become LEKR from LDKR.To have 5 ' end Sal I site (it is compatible with Xho I site) and nucleotide sequence 3 ' end Cla I site, that comprise the albumin fusion proteins therapeutic polynucleotide partly of encoding and connect into Xho I and Cla I site among the pScCHSA, not have the change of D in the final albumin fusion proteins expression plasmid to E.Sal I with recovered the initial aminoacid sequence of signal peptide sequence being connected of Xho I.The DNA of coding albumin fusion proteins therapeutic part can be inserted in Kex2 site (point of contact of Kex2 is positioned at after two basic amino acid sequence KR of signal peptide end) afterwards, before the Cla I site.
The structure of pScNHSA: the albumin that albumin part is positioned at the N-terminal of human cytokines merges
Body
By increasing the restriction site of 38 base pairs on pScCHSA, preparation helps encoding the dna clone of human cytokines to the carrier of the C-terminal of the albuminised DNA of encoding mature.Between the Bsu36 I of the proteic nucleotide sequence end of encoding mature HSA and Hind III site, add Asc I, Fse I and Pme I restriction site.This complementary synthetic primer (SEQ ID NO:40 and SEQ ID NO:41) by using two to contain Asc I, Fse I and Pme I restriction site (underlining), 5 '-AAGCTGCCTTAGGCTTATAATAA
GGCGCGCCGGCCGGCCGTTTAAACTAAGCTTAATTCT-3 ' (SEQ ID NO:40) and 5 '-AGAATTAAGCTTA
GTTTAAACGGCCGGCCGGCGCGCCTTATTATAAGCCTAAGGCAGCTT-3 ' (SEQ ID NO:41) and realizing.Behind these primer annealings,, connect into the same loci of pScCHSA, obtain pScNHSA with Bsu36 I and Hind III digestion.
Embodiment 2: be used for the general construct preparation of yeast conversion
Carrier pScNHSA and pScCHSA can be used as cloning vehicle, and the polynucleotide of coding human cytokines or its fragment or variant are inserted described two kinds of carriers, make the polynucleotide of its contiguous human serum albumins of encoding mature " HSA ".PScCHSA is used to produce human cytokines-HSA fusant, and pScNHSA is used to produce HSA-human cytokines fusant.
Comprise the preparation of the albumin fusion constructs of HSA-human cytokines fusion product
Use (for example helps primer that fusion constructs produces, by increasing restriction site, the seamless fusion rotein of encoding, coding joint sequence etc.) carry out the DNA (as be shown as SEQ ID NO:X sequence or sequence known in the art) of pcr amplification coding human cytokines.For example, those skilled in the art can design 5 ' end primer and 3 ' end primer, and will the encode polynucleotide of last 4 aminoacid (with containing the Bsu36I site) of HSA mature form of 5 ' end primer are added in the 5 ' end of the DNA of the human cytokines of encoding; 3 ' end primer is at 3 ' terminal termination codon and the suitable cloning site of adding of human cytokines coded sequence.Such as, the forward primer that is used for the DNA of amplification coding human cytokines can have sequence 5 '-aagctG
CCTTAGGCTTA-(N)
15-3 ' (SEQ ID NO:42), wherein underlined sequence is the Bsu36I site, ripe proteic last 4 aminoacid of HSA (ALGL) of uppercase nucleotide coding, (N)
15Identical with preceding 15 nucleotide of the interested human cytokines of coding.Similarly, the reverse primer that is used for the DNA of amplification coding human cytokines can have sequence 5 '-GCGCGCGTTTAAAC
GGCGCGCC (N)
15-3 ' (SEQ IDNO:43), wherein the sequence of italic is Pme I site, and the sequence that adds double underline is Fse I site, and the sequence that adds single underscore is Asc I site, and the nucleotide that adds frame is the reverse complementary sequence of two series connection termination codoies, (N)
15Identical with the reverse complementary sequence of last 15 nucleotide of the interested human cytokines of coding.The PCR product is once amplification, promptly cuts with Bsu 36I and Asc I, Fse I or Pme I thrin, connects into pScNHSA then.
It is the change of the terminal single amino acids of HSA-kex2 signal sequence that the existence in the interior Xho I site of the chimeric targeting sequencing of HSA causes chimeric signal sequence, has become LEKR (SEQ ID NO:45) from LDKR (SEQ ID NO:44).
Comprise the preparation of the albumin fusion constructs of gene-HSA fusion product
Similar with said method, can use polynucleotide that DNA:5 ' the end primer of following primer pcr amplification coding human cytokines will contain a Sal I site and last 3 the aminoacid DKR of coding HSA targeting sequencing to be added in the 5 ' end of the DNA of coding human cytokines; 3 ' end primer is several amino acid and the polynucleotide that contain Cla I site before 3 ' the terminal adding encoding mature HSA of the DNA of coding human cytokines.For example, the forward primer that is used for the DNA of amplification coding human cytokines can have sequence 5 ' aggagc
GtcGACAAAAGA (N)
15-3 ' (SEQ ID NO:46), wherein underlined sequence is Sal I site, last 3 aminoacid (DKR) of uppercase nucleotide coding HSA targeting sequencing, (N)
15Identical with preceding 15 nucleotide of the interested human cytokines of coding.Similarly, the reverse primer that is used for the DNA of amplification coding human cytokines can have sequence 5 '-CTTTAAATCG
ATGAGCAACCTCACTCTTGTGTGCATC(N)
15-3 ' (SEQ IDNO:47), wherein the sequence of italic is Cla I site, underlined nucleotide is the reverse complementary sequence of DNA of preceding 9 aminoacid (DAHKSEVAH, SEQ ID NO:48) of coding HSA mature form, (N)
15Identical with the reverse complementary sequence of last 15 nucleotide of the interested human cytokines of coding.The PCR product promptly with Sal I and Cla I digestion, connects into the pScCHSA through Xho I and Cla I digestion once amplification.May want different signals or targeting sequencing, for example, invertase " INV " (Swiss-Prot searching number P00724), mating factor α " MAF " (Genbank searching number AAA18405), MPIF (Geneseq AAF82936), fine protein B (Swiss-Prot searching number P23142), bunch egg collection white (Swiss-Prot searching number P10909), IGFBP (insulin-like growth factor binding protein) 4 (Swiss-Prot searching number P22692), the available standard method sub-clone known in the art of the variation of HSA targeting sequencing (permutations) is on appropriate carriers.
Be adapted at the preparation of the albumin fusion constructs of expression in the saccharomyces cerevisiae (S.cerevisiae)
Will be in the Not I site of the pSAC35 with LEU2 selected marker from the Not I fragment cloning of the DNA that contains coding N-terminal or C-terminal albumin fusion proteins of pScNHSA or pScCHSA.The carrier that obtains like this is used for the conversion of saccharomyces cerevisiae expression system.
Embodiment 3: the general expression in saccharomyces cerevisiae
Transform by lithium acetate, electroporation, or additive method known in the art, or as Sambrook, Fritsch and Maniatis, 1989, " Molecular Cloning:A Laboratory Manual (molecular cloning laboratory manual); second edition ", 1-3 volume and Ausubel etc., 2000, Massachusetts GeneralHospital and Harvard Medical School " Current Protocols in MolecularBiology " (Massachusetts general hospital and Harvard Medical School " modern molecular biology general scheme "), the described method of part in the 1-4 volume can transform the expression vector that is fit to yeast expression into saccharomyces cerevisiae.By transforming, expression vector is introduced Wine brewing yeast strain DXY1, D88 or BXP10,10 milliliters of YEPD (yeast extracts of 1% mass/volume, the peptone of 2% mass/volume, the dextrose of 2% mass/volume) the transformant individuality in can be 30 ℃ of growths 3 days, grow after 60 hours, collect the cell that is in stable phase.With cell 3000g clarification in centrifugal 10 minutes cell, collect supernatant.
Except the LEU2 selected marker, pSAC35 (Sleep etc., 1990, Biotechnology 8:42 and referring to Fig. 3) comprise 2 microns plasmids of complete yeast, PRB1 promoter and ADH1 termination signal that copy function is provided.
Embodiment 4: the albumin fusion proteins of expressing from saccharomyces cerevisiae albumin fusant generally pure
Change
In some embodiment, albumin fusion proteins of the present invention comprises the mature form of the HSA of the N-terminal that is fused to human cytokines or its part mature form (as the mature form of human cytokines listed in the table 1, or being shown as the mature form of the human cytokines of SEQ ID NO:Z in the table 2) or C-terminal.In one embodiment of the invention, albumin fusion proteins of the present invention also comprises one section signal sequence that guides newborn fused polypeptide at the host's secretory pathway that is used for expressing.In one embodiment, the signal peptide of signal sequence encoding is removed, and sophisticated albumin fusion proteins direct secretion is in culture medium.Albumin fusion proteins of the present invention (for example can comprise the allos signal sequence, the proteic non-natural signal sequence of particular treatment), include but not limited to that MAF, INV, immunoglobulin, fine protein B, bunch egg collection are white, the variant (including but not limited to chimeric HSA/MAF targeting sequencing) of IGFBP (insulin-like growth factor binding protein) 4, HSA targeting sequencing, or other allos signal sequence known in the art.Example comprises those listed in the table 2 signal sequences and/or description aforementioned " Expression of Fusion Protein " and/or " additive method of reorganization and synthetic preparation albumin fusion proteins " listed signal sequence of part.In some embodiment, fusion rotein of the present invention also comprises the methionine residues of a N-terminal.The polynucleotide (comprising fragment and/or variant) of these polypeptide of encoding are also included among the present invention.
The albumin fusion proteins of expressing in the aforementioned yeast can be as follows carries out small scale purification with the peptide affinity column of Dyax.Express the phosphate buffer (pH 6.2) of the yeast supernatant of albumin fusion proteins with 3mM, the NaCl of 20mM and 0.01% polysorbas20 diafiltration are to reduce volume and to remove pigment.Solution filters with the device of 0.22 μ m then.Sample is to the peptide affinity column of Dyax on the filtrate.Pillar Tris/HCl buffer (pH 8.2) eluting of 100mM.Collection contains proteic peak fraction, analyzes with SDS-PAGE after concentrating 5 times.
Available following method is carried out large scale purification.Surpass supernatant diafiltration in 20mM Tris/HCl (pH8.0) of 2 liters, be concentrated to 500 milliliters.Sample is to 50 milliliters of quick posts of DEAE-Sepharose of pre-equilibration on the spissated protein solution, after the pillar rinsing, with 20mM Tris/HCl (pH 8.0) eluted protein with NaCl linear gradient of 0 to 0.4M NaCl.To contain proteic those fraction and compile, with the sodium phosphate (NaH of 0.5M
2PO
4) adjust to pH 6.8.(the NH that in protein solution, adds final concentration 0.9M
4)
2SO
4, sample is to 50 milliliters of Butyl650S posts of pre-equilibration on the complete soln.(0.9 arrives 0M (NH to albumen with the ammonium sulfate linear gradient elution
4)
2SO
4).Those fraction that will contain the albumin fusant once more compile, with the Na of 10mM
2HPO
4/ citrate buffer solution (pH5.75) diafiltration is gone up on the quick post of SP-Sepharose of sample to 50 milliliter pre-equilibration then.The NaCl linear gradient elution of albumen from 0 to 0.5M.Mix the fraction that contains proteins of interest, buffer is converted to the Na of 10mM with the Amicon concentrator
2HPO
4/ citric acid (pH 6.25), electrical conductivity is less than 2.5mS/cm.On the Q-Sepharose high-efficiency column with sample to 15 milliliter pre-equilibration on this protein solution, after the pillar rinsing, albumen is used from the NaCl linear gradient elution of 0 to 0.15M NaCl.Then, the albumen of purification can be formulated in the specific buffer constituent by buffer-exchanged.
Embodiment 5: be used for the general construct preparation of mammalian cell transfection
The preparation of the albumin fusion constructs that is adapted at expressing in the mammal cell line
Can be used in the expression vector that uses in the mammalian cell culture system and make up the albumin fusion constructs.By standard method known in the art (for example, pcr amplification, restrictive diges-tion and be connected), can be with N-terminal or the C-terminal of the dna clone of coding human cytokines HSA in the mammalian expression vector.Build after the expression vector, just can carry out the transfection of mammalian expression systems.Suitable carriers known in the art includes but not limited to, for example, and carrier pC4 and/or can be, Inc. (Portsmouth, carrier NH) available from LonzaBiologics.
The dna clone of the human serum albumin of coding to the pC4 carrier that is fit to the mammal culture systems, is formed plasmid pC4:HSA (ATCC preserving number PTA-3277).Contain dihydrofolate reductase " DHFR " gene on this carrier, this gene is used in when methotrexate exists and selects.
The pC4:HSA carrier is used in and expresses albumin fusion proteins in the Chinese hamster ovary celI.For the expression in other mammalian cell culture system, may want with comprise or by the fragment sub-clone formed of DNA of coding albumin fusion proteins in alternate expression vector.For example, can with the fragment sub-clone that comprises or form by the DNA of encoding mature albumin fusion proteins in another expression vector, include but not limited to any mammalian expression vector described herein.
In one embodiment, by methods known in the art, to by Lonza Biologics, (Portsmouth on the carrier that NH) provides, is used for the expression at the NS0 cell to Inc. with the DNA sub-clone of coding albumin fusion constructs.
Comprise the preparation of the albumin fusion constructs of HSA-human cytokines fusion product
The albumin fusion constructs that can obtaining medical treatment property protein part be positioned at ripe albumin sequence C end with pC4:HSA (ATCC preserving number PTA-3277).For example, can be with the dna clone of coding human cytokines or its fragment or variant between the Bsu 36I and Asc I restriction site of carrier.Be cloned into after Bsu 36I and the Asc I site, can use the same design of primers (SEQ ID NO:42 and 43 is referring to embodiment 2) that is used for the clone of yeast vector system.
Comprise the preparation of the albumin fusion constructs of gene-HSA fusion product
The albumin fusion constructs of can obtaining medical treatment property protein part being cloned into ripe albumin sequence N-terminal with pC4:HSA (ATCC preserving number PTA-3277).For example, coding can be contained the dna clone of human cytokines of himself signal sequence between the Bam HI (or Hind III) and Cla I site of pC4:HSA.Be cloned into after Bam HI or the Hind III site, can before the translation initiation codon of DNA of coding human cytokines, add one section Kozak sequence (CCGCCACC
ATG, SEQID NO:49).If human cytokines does not contain signal sequence, the DNA of coding human cytokines can be cloned between the Xho I and Cla I site of pC4:HSA.When using Xho I site, can use following 5 ' (SEQ ID NO:50) and 3 ' (SEQ ID NO:51) terminal exemplary PCR primer: 5 '-CCGCCG
CTCGAGGGGTGTGTTTCGTCGA (N)
18-3 ' (SEQ ID NO:50) and 5 '-AGTCCC
ATCGATGAGCAACCTCACTCTTGTGTGCATC (N)
18-3 ' (SEQ IDNO:51).
In the described 5 ' primer (SEQ ID NO:50), underlined sequence is Xho I site; Last 7 aminoacid of the natural human serum albumin targeting sequencing of dna encoding after Xho I site and the Xho I site.In SEQ ID NO:50, " (N)
18" be with the coding interested human cytokines preceding 18 DNA that nucleotide is identical.In the described 3 ' primer (SEQ ID NO:51), underlined sequence is Cla I site; Cla I site and the DNA after it are the reverse complementary sequences of preceding 10 amino acid whose DNA of encoding mature HSA albumen (SEQ IDNO:1).In SEQ ID NO:51, " (N)
18" be the reverse complementary sequence of DNA of last 18 nucleotide of the interested human cytokines of coding.Use this two primers, can the interested human cytokines of pcr amplification, purified pcr product with Xho I and the digestion of Cla I restriction endonuclease, is cloned into it in Xho I and Cla I site of pC4:HSA carrier then then.
If want alternate targeting sequencing, can be by standard method known in the art with chimeric albumin leading (being the HSA-kex2 signal peptide) or alternate leading alternative native albumin targeting sequencing.(for example, those skilled in the art can substitute leading by conventional pcr amplification, and to the albumin fusion constructs, it is leading to substitute albumin, has kept the reading frame simultaneously with PCR product sub-clone).
Embodiment 6: the general expression in mammal cell line
Pass through calcium phosphate precipitation, lipofection amine (lipofectamine), electroporation or other transfection method known in the art and/or as Sambrook, Fritsch and Maniatis, 1989, " Molecular Cloning:ALaboratory Manual (molecular cloning laboratory manual); second edition " and Ausubel etc., 2000, Massachusetts General Hospital and Harvard Medical School " Current Protocolsin Molecular Biology (Massachusetts general hospital and Harvard Medical School " modern molecular biology general scheme ") ", other method described in the 1-4 volume, but the albumin fusion constructs transfection that the expression vector that is adapted at expressing in the mammal cell line produces is in suitable cell line.Then, under the condition that selective agent exists, select transfectional cell by the selected marker on the expression vector.
PC4 expression vector (ATCC searching number 209646) is the derivant of plasmid pSV2-DHFR (ATCC searching number 37146).PC4 comprises long terminal repetition " the LTR " (Cullen etc. of rous sarcoma virus strong promoter, in March, 1985, Molecular and Cellular Biology, 438-447) and the fragment (Boshart etc. of cytomegalovirus " CMV " enhancer, 1985, Cell 41:521-530).This carrier also comprises the poly-adenosine of 3 ' intron, rat proinsulin protogene and termination signal and by the mice DHFR gene of SV40 early promoter control.Chinese hamster ovary " CHO " cell or other cell line that lacks active DHFR gene can be used for transfection.By means known in the art with the albumin fusion constructs transfection on the pC4 in Chinese hamster ovary celI, albumin fusion proteins is expressed in Chinese hamster ovary celI, cut away targeting sequencing then, be secreted in the supernatant.Can from supernatant, obtain albumin fusion proteins by purification subsequently.
By Lonza Biologics, (Portsmouth, the pEE12.1 expression vector that NH) provides are pEE6 (Stephens and Cockett, 1989, Nucl.Acids Res. to Inc.
17: derivant 7110).This carrier comprises promoter, enhancer and the complete 5 '-untranslated region of the main immediate early gene of human cytomegalovirus " hCMV-MIE " (international publication WO89/01036 number) that is positioned at sequence interested upstream, with the glutamine synthetase gene (Murphy etc. that are used on the selection culture medium that contains methionine sulphoximine (methionine sulphoximine), selecting transfectional cell, 1991, Biochem J.227:277-279; Bebbington etc., 1992, Bio/Technology 10:169-175; United States Patent (USP) the 5th, 122, No. 464).By means known in the art the albumin fusion constructs transfection on the pEE12.1 is arrived in the NS0 cell (international publication WO86/05807 number), albumin fusion proteins is expressed in the NS0 cell, cut away targeting sequencing then, be secreted in the supernatant.Can use technology described herein or other technology known in the art purification from supernatant to obtain albumin fusion proteins subsequently.
For example, can analyze the expression of albumin fusion proteins by SDS-PAGE and Western trace, reversed-phase HPLC analysis or other method known in the art.
Can obtain the stable CHO and the NS0 cell line of transfection albumin fusion constructs by means known in the art (as the transfection of lipofection amine), for example for containing the carrier of dihydrofolate reductase " DHFR " gene as selected marker, the methotrexate of available 100nM is selected, and perhaps selects by growing under the condition that lacks glutamine.Mainly anti-or anti-ly carry out immunoblotting and check expression as one with containing posteriorly as one then at the serum of the antibody of specific albumin fusion proteins human cytokines part with anti-HSA serum.
Anti-carry out immunoblotting and detect expression as one with anti-HSA serum.Measure specific production rate by enzyme-linked immunosorbent assay (ELISA), the capture antibodies of ELISA can be the monoclonal antibody at albumin fusant human cytokines part, detecting antibody can be the biotinylated antibody of monoclonal anti HSA (or vice versa), carry out the combination of horseradish peroxidase/strepto-affinity element then, analyze with reference to the scheme that manufacturer provides.
Embodiment 7: the expression of albumin fusion proteins in mammalian cell
Albumin fusion proteins of the present invention can be expressed in mammalian cell.Typical mammalian expression vector comprises that promoter element, albumen coded sequence and the termination of mediation mRNA transcription initiation transcribe the signal required with the polyadenylic acid transcript.Other element comprises that enhancer, Kozak sequence and flank are the intervening sequence of RNA donor splicing site and acceptor site.From the early stage and late promoter of SV40, can realize transcribing efficiently from the length terminal repetition (LTR) of retrovirus retrovirus (for example RSV, HTLVI, HIVI) and the early promoter of cytomegalovirus (CMV).Yet, also can use cell element (for example, the promoter of people's actin-like protein).
The suitable expression vector of using in the present invention's practice comprises, for example, such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), carrier such as pRSVcat (ATCC 37152), pSV2dhfr (ATCC37146), pBC12MI (ATCC 67109), pCMVSport 2.0 and pCMVSport 3.0.Spendable mammalian host cell includes but not limited to human Hela, 293, H9 and Jurkat cell, mouse NIH 3T3 and C127 cell, Cos 1, Cos 7 and CV1, Carnis Coturnicis japonicae QC1-3 cell, mouse Lcell and Chinese hamster ovary (CHO) cell.
Perhaps, albumin fusion proteins also can be expressed in the stable cell lines of the polynucleotide that contain the coding albumin fusion proteins that is incorporated on the chromosome.With make the evaluation of transfectional cell and be separated into possibility such as selected marker cotransfections such as DHFR, gpt, neomycin or hygromycin.
The polynucleotide of the encoding fusion protein of transfection also can be expressed the fusion rotein of a large amount of codings by amplification.DHFR (dihydrofolate reductase) labelling can be used for developing carry hundreds of or even the cell line of several thousand gene of interest copies (referring to for example Alt etc., J.Biol.Chem.253:1357-1370 (1978); Hamlin etc., Biochem.et Biophys.Acta, 1097:107-143 (1990); Page etc., Biotechnology 9:64-68 (1991)).Another useful selected marker is that (Murphy etc., Biochem are (1991) J.227:277-279 for glutamine synthase (GS); Bebbington etc., Bio/Technology10:169-175 (1992)).Use these labellings, mammalian cell can be grown on the culture medium selecting, and selects to have the cell of high resistance then.These cell lines contain the gene that is incorporated into the amplification on the chromosome.Chinese hamster ovary (CHO) cell and NSO cell are usually used in proteic preparation.
The derivant of plasmid pSV2-dhfr (ATCC searching number 37146): expression vector pC4 (ATCC searching number 209646) and pC6 (ATCC searching number 209647) contain rous sarcoma virus strong promoter (LTR) (Cullen etc., Molecular and Cellular Biology, 438-447 (in March, 1985)) adds the fragment (Boshart etc., Cell 41:521-530 (1985)) of CMV-enhancer.Have the multiple clone site of restriction endonuclease Bam HI, Xba I and Asp 718 cleavage sites for example and help the clone of gene of interest.These carriers also contain the poly-adenosine signal of 3 ' intron, rat proinsulin protogene and termination signal and by the mice DHFR gene of SV40 early promoter control.
Particularly, for example, after plasmid pC6 digests with suitable restriction endonuclease, by means known in the art calf small intestinal phosphatase dephosphorylation.From 1% agarose gel, separate then and obtain carrier.
The polynucleotide of albumin fusion proteins can produce by technology known in the art in the code book invention, and these polynucleotide can increase by round pcr known in the art.If use naturally occurring signal sequence to produce fusion rotein among the present invention, then carrier does not need other signal peptide.Perhaps, if do not use naturally occurring signal sequence, can modify carrier and introduce allogenic signal sequence (referring to No. 96/34891, international publication WO for example).
But the amplified fragments of encoding fusion protein can use the test kit that commercial sources obtains (La Jolla Ca.) separates from 1% agarose gel and obtains for " Geneclean ", BIO 101 Inc. among the present invention.The fragment that obtains is with the digestion of suitable restriction endonuclease, and then with 1% agarose gel purification.
Then, the amplified fragments of albumin fusion proteins can digest with same restriction endonuclease in the code book invention, and with 1% agarose gel purification.With the T4DNA ligase fragment that separation obtains is connected with dephosphorylized carrier.Transformed into escherichia coli (E.coli) HB101 or XL-1 Blue cell use for example restriction enzyme analysis evaluation to contain the segmental antibacterial of insertion plasmid pC6 then.
The Chinese hamster ovary cell that lacks active DHFR gene is used for transfection.With lipofectin reagent (lipofectin) (above-mentioned Felgner etc.) with 5 μ g expression plasmid pC6 or pC4 and 0.5 μ g pSVneo plasmid co-transfection.Plasmid pSV2-neo contains dominant selectable marker: from the neo gene of Tn5, the enzyme of coding is given comprising one group of antibiotic resistance of G418.Cell inoculation is being added on the α of 1mg/mlG418-MEM culture medium.After 2 days, cell is through trypsinization, be seeded in added 10,25 or the hybridoma clone plate of α-MEM culture medium of 50ng/ml methotrexate and 1mg/ml G418 (Greiner, Germany) on.After about 10-14 days, monoclonal is through trypsinization, is seeded in the have the variable concentrations methotrexate 6 hole culture dishs or 10 milliliters of culture bottles of (50nM, 100nM, 200nM, 400nM, 800nM).The clone that will be grown in the maximum concentration methotrexate then transfer to new contain in addition 6 orifice plates of higher concentration methotrexate (1 μ M, 2 μ M, 5 μ M, 10mM, 20mM) on.Repeat same method, until the clone who obtains on 100-200 μ M concentration, to grow.The required Expression of Fusion Protein of methods analyst by for example SDS-PAGE and Western trace or reversed-phase HPLC analysis.
Embodiment 8: the albumin of expressing in the albumin fusion constructs in mammal cell line merges
Proteic general purification
In some embodiment, albumin fusion proteins of the present invention comprises the N-terminal that is fused to human cytokines or its a part of mature form (as the mature form of listed human cytokines in the table 1, or being shown as the mature form of the human cytokines of SEQ ID NO:Z in the table 2) or the HSA mature form of C-terminal.In certain embodiment of the present invention, albumin fusion proteins of the present invention also comprises one section signal sequence that guides newborn fused polypeptide at the host's secretory pathway that is used for expressing.In one embodiment, the signal peptide of signal sequence encoding is removed, and sophisticated albumin fusion proteins direct secretion is in culture medium.Albumin fusion proteins of the present invention (for example can comprise the allos signal sequence, the proteic non-natural signal sequence of particular treatment), include but not limited to that MAF, INV, immunoglobulin, fine protein B, bunch egg collection are white, the variant (including but not limited to chimeric HSA/MAF targeting sequencing) of IGFBP (insulin-like growth factor binding protein) 4, HSA targeting sequencing, or other allos signal sequence known in the art.Exemplary signal sequence comprises signal sequence listed in the table 2 and/or description aforementioned " Expression of Fusion Protein " and/or " additive method of reorganization and synthetic preparation albumin fusion proteins " listed signal sequence of part.In some embodiment, fusion rotein of the present invention also comprises the methionine residues of a N-terminal.The polynucleotide (comprising fragment and/or variant) of these polypeptide of encoding are also included among the present invention.
Because of the difference of the expression system that uses, can use different schemes to come albumin fusion proteins in the purification mammal cell line supernatant.
Purification from CHO and 293T cell line
Comprise from the Chinese hamster ovary celI supernatant or from the purification step of the albumin fusion proteins of the 293T cell conditioned medium liquid of transient transfection, the initial acquisition of the anion HQ resin of use sodium phosphate buffer and phosphate gradient elution and use salt gradient subsequently are eluted in affinity chromatograph on the Blue Sepharose FF post.BlueSepharose FF removes main BSA/ myosin pollutant.Can remove and reduce endotoxic pollution with the phosphate gradient to being further purified of Poros PI 50 resins, and concentrated albumin fusion proteins.
Purification from NSO cell line
Can comprise Q-Sepharose anion-exchange chromatography, SP-Sepharose purification, the Phenyl-650M purification of one-step elution and the last diafiltration subsequently of one-step elution subsequently from the purification step of the albumin fusion proteins of NS0 cell conditioned medium liquid.
The albumen of purification can be prepared by buffer-exchanged.
Embodiment 9: the expression of albumin fusion proteins in antibacterial
Use comprises the polynucleotide of albumin fusion proteins in the code book invention of antibacterial signal sequence corresponding to DNA sequence 5 ' and 3 ' terminal PCR oligonucleotide primers amplification, come the synthetic fragment of inserting.The primer that is used for the polynucleotide of amplification coding insert can contain the restriction site such as Bam HI and Xba I at 5 ' end of primer, so as with extension amplification outcome to expression vector.For example, and Bam HI and Xba I and bacterial expression vector pQE-9 (Qiagen, Inc., Chatsworth, CA) restriction enzyme sites on is corresponding.This plasmid vector coding antibiotic resistance (Amp
r), antibacterial origin of replication (ori), IPTG promoter/operator (P/O), ribosome binding site (RBS), 6 histidine-tagged (6-His) and the restriction endonuclease cloning sites regulated.
The pQE-9 carrier connects into this pQE-9 carrier with amplified fragments after Bam HI and Xba I digestion, be retained in the initial reading frame of antibacterial RBS simultaneously.Connect mixture be used for transformed into escherichia coli bacterial strain M15/rep4 (Qiagen, Inc.), this bacterial strain contains the pREP4 plasmid of multicopy, this plasmid expression lac I repressor is also given kalamycin resistance (Kan
r).Identify transformant by the ability that can on the LB flat board, grow, select to have the bacterium colony of ampicillin/kalamycin resistance.Separation obtains plasmid DNA and confirms by restriction analysis.
Contain being cloned in of required construct and added grow overnight in the liquid LB culture medium of ampicillin (100 μ g/ml) and kanamycin (25 μ g/ml).Use overnight culture to inoculate big culture with 1: 100 to 1: 250 ratio.Cell grows into optical density 600 (O.D.
600) between 0.4 to 0.6.Adding IPTG (isopropyl-β-D-sulfo-galactopyranose glycosides) makes its final concentration reach 1mM.IPTG by inactivation lac I repressor, induce startup (clearing) P/O, cause gene expression to increase.
Cell continued growth 3 to 4 hours.Then by centrifugal (6000g, 20 minutes) collecting cell.Cell precipitation is dissolved in the guanidine hydrochloride of 6 moles of chaotropic agents or for example in the carbamide and 2 mercapto ethanol that concentration is higher than 0.14M of 8M, 4 ℃ stir 3-4 hour (referring to, for example, Burton etc., Eur.J.Biochem.179:379-387 (1989)).Centrifugal removal cell debris, will contain on the supernatant of polypeptide sample to the affine resin column of nickel-nitrilo--triacetic acid (" Ni-NTA ") (from above-mentioned QIAGEN, Inc.).The albumen that has the 6-His label is attached on the Ni-NTA resin with high-affinity, and can (see above-mentioned QIAexpressionist (1995) QIAGEN for details, Inc.) by a simple step program purification.
Briefly, on the supernatant in 6M guanidine hydrochloride (pH 8) sample to pillar.With 6M guanidine hydrochloride (pH 8) the rinsing pillar of 10 times of volumes, use the 6M guanidine hydrochloride (pH6) of 10 times of volumes to continue rinsing then earlier, use 6M guanidine hydrochloride (pH 5) eluting polypeptide at last.
By adding that with phosphate buffered saline (PBS) (PBS) or 50mM sodium acetate buffer (pH 6) 200mM NaCl dialyses the albumen of renaturation purification.Perhaps, can be successfully folding again by proteopexyization is made albumen on the Ni-NTA post.Exemplary condition is as follows: carry out renaturation with having 6M to the linear gradient of 1M carbamide and the 500mM NaCl, 20% glycerol, the 20mMTris/HCl (pH 7.4) that contain protease inhibitor.Renaturation should or be carried out at 1.5 hours for more time.After the renaturation, add 250mM imidazoles eluted protein.At last by adding that with PBS or 50mM sodium acetate buffer (pH6) the 200mM NaCl step of dialysing removes imidazoles.The albumen of purification is kept at 4 ℃ or frozen in-80 ℃.
Except above-mentioned expression vector, the present invention also comprises and is called pHE4a (ATCC searching number 209645, be preserved on February 25th, 1998) expression vector, this carrier contains the phage operator and the promoter element of polynucleotide that is operably connected to coding albumin fusion proteins of the present invention.This carrier comprises: 1) neomycin phosphotransferase gene is a selected marker, 2) escherichia coli origin of replication, the 3) promoter sequence of T5 phage, 4) two lac operon sequence, 5) Shine-Delgarno sequence and 6) lacoperator repressor gene (lacIq).Origin of replication (oriC) derived from pUC19 (LTI, Gaithersburg, MD).Promoter and operator sequence are synthetic.
By usefulness Nde I and Xba I, Bam HI, Xho I or ASP 718 restriction enzyme digestion pHE4a carriers, and, separate bigger fragment (stuffer should be about 310 base pairs), DNA can be inserted pHE4a restriction enzyme digestion product race glue.According to PCR scheme described herein or other method known in the art, use PCR primer to obtain the DNA insert with restriction site Nde I (5 ' primer) and Xba I, Bam HI, Xho I or ASP 718 (3 ' primer).The PCR insert is through the restriction enzyme digestion of gel-purified and suitable enzymes.Scheme according to standard is connected insert with carrier.
Alternative artificial constructed carrier in the such scheme, expressing protein in bacterial system.
Embodiment 10: separate selected cDNA clone from the preservation sample
Many albumin fusion constructs of the present invention are preserved in ATCC (as shown in table 3).These albumin fusion constructs can comprise any in the following expression vector: saccharomyces cerevisiae expression vector pSAC35, mammalian expression vector pC4 or mammalian expression vector pEE12.1.
PSAC35 (Sleep etc., 1990, Biotechnology 8:42), pC4 (ATCC searching number 209646; Cullen etc., Molecular and Cellular Biology, 438-447 (1985); Boshart etc., Cell 41:521-530 (1985)) and pEE12.1 (Lonza Biologics, Inc.; Stephens and Cockett, Nucl.Acids Res.17:7110 (1989); International publication WO89/01036 number; Murphy etc., Biochem are (1991) J.227:277-279; Bebbington etc., Bio/Technology10:169-175 (1992); United States Patent (USP) the 5th, 122, No. 464; International publication WO86/05807 number) carrier comprises and is used for the ampicillin resistance gene of growing at bacterial cell.Use the described technology in this area (as Hanahan), but these carriers and/or comprise the albumin fusion constructs transformed into escherichia coli bacterial strain of these carriers, XL-1 Blue (Stratagene Cloning Systems as Stratagene, Inc., 11011N.Torrey Pines Road, La Jolla, CA, 92037), be layered on the Luria-Broth agar plate that contains 100 μ g/ml ampicillin 37 ℃ of grow overnight then.
To any given albumin fusion constructs, the preserved material in the sample of quoting in the table 3 with ATCC preserving number also can contain one or more extra albumin fusion constructs, the albumin fusion proteins that each coding is different.Therefore, the preservation thing with same ATCC preserving number contains the albumin fusion constructs of identifying at least one table 3 corresponding line.
Separate specific albumin fusion constructs in the preservation sample of the plasmid DNA of the albumin fusion constructs that can from table 3, quote by two kinds of methods.
Method 1: screening
At first, use means known in the art, use plasmid DNA sample, directly separate the albumin fusion constructs corresponding to the polynucleotide probes screening preservation of individual construct ID in the table 1 number SEQ IDNO:X.For example, according to the sequence of having reported, can use Applied Biosystems dna synthesizer to synthesize to have one section specific polynucleotide of 30-40 nucleotide.Can use the T4 polynucleotide kinase for example to use
32P-γ-ATP labeled oligonucleotide and purification is (for example according to conventional methods, Maniatis etc., Molecular Cloning:A Laboratory Manual (molecular cloning laboratory manual), Cold SpringHarbor Press, Cold Spring, NY (1982)).Use technology well known by persons skilled in the art, the technology of mentioning in the technology that provides as carrier supplying manufacturer or above-mentioned relevant publication of quoting or the patent will be transformed into from the albumin fusion constructs of given ATCC preservation thing as described above in the suitable hosts (as XL-1 Blue (Stratagene)).Transformant is layered on 1.5% the agar plate (containing suitable selective agent, as ampicillin), and density is each dull and stereotyped about 150 transformant (bacterium colony) of.(for example be used for the bacterial clump method for screening according to routine, Sambrook etc., Molecular Cloning:A LaboratoryManual (molecular cloning laboratory manual), the 2nd edition, (1989), Cold Spring Harbor LaboratoryPress, 1.93 to 1.104 pages) or known other method of those skilled in the art, screen these flat boards with nylon membrane.
Method 2:PCR
Perhaps, for example by using two 5 ' and 3 ' ends can hybridize to the primer of 17-20 nucleotide of preservation albumin fusion constructs at the DNA of the given albumin fusion proteins of coding, can be from preservation albumin fusion constructs sample with SEQ ID NO:X the DNA of the given albumin fusion proteins of amplification coding.Under normal condition, move the polymerase chain reaction, for example, in the reactant mixture of 25 μ l, contain the above-mentioned cDNA template of 0.5 μ g.Suitable reactant mixture comprises 1.5-5mM MgCl
2, 0.01% (mass/volume) gelatin, each 20 μ M of dATP, dCTP, dGTP, dTTP, the Taq polymerase of every primer 2 5pmol and 0.25 unit.With 35 circulation PCR of the automatic thermal cycler of Perkin-Elmer Cetus operation (94 ℃ of degeneration 1 minute; Annealed 1 minute for 55 ℃; 72 ℃ were extended 1 minute).Amplified production is analyzed with agarose gel electrophoresis, downcuts the DNA band with expection molecular weight, carries out purification then.Verify that by sub-clone and sequenced dna product the PCR product that obtains is selected sequence.
There is several method to can be used for identifying not to be present in 5 ' or 3 ' the non-coded portion of the gene among the preservation clone.Clone's concentration method that these methods include but not limited to the filter membrane probe technique, use specific probe with the similar or the same scheme of known in the art 5 ' and 3 ' " RACE " scheme.For example, a kind of method that is similar to 5 ' RACE can be used for producing disappearance 5 ' terminal (Fromont-Racine etc., Nucleic Acids Res., 21 (7): 1683-1684 (1993)) of required total length transcript.
Briefly, special RNA oligonucleotide is connected to the 5 ' end that supposition contains the RNA colony of full-length gene rna transcription thing.With the 5 ' part of the primer that contains the RNA oligonucleotide that is specific to connection with the required full-length gene of primer collection pcr amplification of the primer that is specific to the gene of interest known array.This amplified production can be checked order, and can be used for producing full-length gene.
Although can use poly-A+RNA, said method isolating total RNA from required source begins.If necessary, the RNA prepared product phosphoric acid enzyme that obtains is handled with degraded of removing possibility interfere with subsequent RNA ligase step or the 5 ' phosphate groups of damage RNA.Answer the inactivation phosphatase then, handle RNA to remove the cap that is present in messenger RNA 5 ' end with tobacco acid pyrophosphatase.This reaction makes one 5 ' phosphate groups of 5 ' terminal reservation of the RNA that cuts medicated cap, uses the T4RNA ligase this RNA can be connected on the RNA oligonucleotide.
RNA prepared product with above-mentioned modification carries out the synthetic of the first chain cDNA as template, and it uses the gene specific oligonucleotide.First chain synthesis reaction is as the template of the required 5 ' end of pcr amplification, and this pcr amplification uses the primer of the RNA oligonucleotide that is specific to connection and is specific to the primer of gene of interest known array.Then, with the order-checking of the product that obtains and analyze to confirm that 5 ' end sequence belongs to required gene.
Embodiment 11: the fusants that merge more
In addition, albumin fusion proteins (for example, containing the human cytokines (or its fragment or variant) that is fused to albumin (or its fragment or variant)) also can be fused on other albumen, produces " many fusion rotein ".These many fusion rotein have many application.For example, albumin fusion proteins of the present invention can be fused to and be beneficial to purification on His label, HA label, protein A, IgG domain and the maltose-binding protein (referring to for example EP A 394,827; Traunecker etc., Nature 331:84-86 (1988)).Be fused to the Subcellular Localization that nuclear localization signal on the polypeptide of the present invention can be specific with targeting proteins, and the heterodimer of covalency or homodimer can improve or reduce the activity of albumin fusion proteins.In addition, extra protein sequence is fused to the dissolubility and/or the stability that can further improve fusion rotein on the albumin fusion proteins of the present invention.Above-mentioned fusion rotein can obtain by using technology known in the art or the customary technology of revising known in the art and/or revising following proposal, and following proposal has been summarized the fusion of polypeptide and IgG molecule.
Briefly, can use the human Fc part of the primer PCR amplification IgG molecule of striding following sequence 5 ' and 3 ' end.These primers also should have suitable restriction enzyme sites, are beneficial to it is cloned into (as mammal or Yeast expression carrier) in the expression vector.
For example, if use pC4 (ATCC searching number 209646), the human Fc part can be connected in the Bam HI cloning site.Note, should destroy the Bam HI site of 3 ' end.Then, the carrier that contains human Fc part is used Bam HI restriction enzyme digestion again, after the carrier linearisation, the polynucleotide of code book invention albumin fusion proteins (use technology known in the art to produce and separate) is connected in this BamHI site.Notice that the polynucleotide of clone's code book invention fusion rotein do not have termination codon, otherwise can not produce the fusion rotein that contains Fc.
If use naturally occurring signal sequence to produce albumin fusion proteins among the present invention, pC4 does not need other signal peptide.Perhaps, if do not use naturally occurring signal sequence, can modify carrier and introduce allogenic signal sequence (referring to international publication for example WO96/34891 number).
The Fc district of IgG:
GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCAC
CTGAATTCGAGGGTGCACCGTCAGTCTTCC
TCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACAT
GCGTGGTGGTGGACGTAAGCCACGAAGACC
CTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAA
AGCCGCGGGAGGAGCAGTACAACAGCACG
TACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAG
TACAAGTG?CAAGGTCTCCAACAAAGCCCTC
CCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAG
GTGTACACCCTGCCCCCATCCCGGGATGA
GCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGA
CATCGCCGTGGAGTGGGAGAGCAATGGGC
AGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCT
TCCTCTACAGCAAGCTCACCGTGGACAAGA
GCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACA
ACCACTACACGCAGAAGAGCCTCTCCCTGT
CTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT (SEQ?ID?NO：52)
Embodiment 12: produce antibody from albumin fusion proteins
Hybridoma technology
Many methods in conjunction with albumin fusion proteins of the present invention and an albumin fusion proteins part of the present invention (for example can prepare, the human cytokines of fusion rotein part or albumin part) antibody (referring to Current Protocols (general scheme), the 2nd chapter).An example is in these methods, and the prepared product of preparation and purification albumin fusion proteins of the present invention or an albumin fusion proteins part of the present invention makes it be substantially free of natural pollutant.Subsequently the prepared product that obtains is introduced and produced the have bigger specific activity polyclonal antiserum of (specific activity) in the animal.
Be specific to monoclonal antibody (Kohler etc., the Nature 256:495 (1975) of an albumin fusion proteins of the present invention or an albumin fusion proteins part of the present invention by the hybridoma technology preparation; Kohler etc., Eur.J.Immunol.6:511 (1976); Kohler etc., Eur.J.Immunol.6:292 (1976); Hammerling etc. are at Monoclonal Antibodies and T-Cell Hybridomas (monoclonal antibody and T quadroma), Elsevier, N.Y., 563-681 (1981)).Usually, with albumin fusion proteins of the present invention or albumin fusion proteins of the present invention part immune animal (as mice).Extract the splenocyte of described mice, and merge with suitable myeloma cell line.Can use any suitable myeloma cell line according to the present invention; Yet can use the parent myeloma cell lines (SP2O) that can obtain from ATCC.After the fusion, the hybridoma that obtains is optionally kept on the HAT culture medium, and subsequently by cloning as (Gastroenterology 80:225-232 (1981)) described limiting dilutions such as Wands.Detect the hybridoma that obtains by this selection subsequently and be used to identify that secretion can be in conjunction with the clone of the antibody of an albumin fusion proteins of the present invention or an albumin fusion proteins part of the present invention.
Perhaps, in the scheme in one two step, using anti-idiotype antibody to produce can be in conjunction with other antibody of an albumin fusion proteins of the present invention or an albumin fusion proteins part of the present invention.It also is the antigenic fact that this method has been utilized antibody itself, and therefore might obtain can be in conjunction with the antibody of second antibody.According to this method, with the special antibody mediated immunity animal (as mice) of albumen.The splenocyte of this animal is used to produce hybridoma subsequently, identify the clone who produces a kind of antibody by screening this hybridoma, the ability of this antibodies albumin fusion proteins of the present invention (or albumin fusion proteins part of the present invention)-specific antibody can be albumin fusion proteins of the present invention or an albumin fusion proteins of the present invention part is blocked.This antibody comprises the anti-idiotype antibody at fusion rotein of the present invention (or albumin fusion proteins part of the present invention)-specific antibody, is used for the formation that immune animal is induced more fusion rotein of the present invention (or albumin fusion proteins part of the present invention)-specific antibody.
In order in human body, to use antibody, with antibody " humanization ".Can use genetic constructs to produce humanized antibody derived from the hybridoma of above-mentioned generation monoclonal antibody.Produce chimeric known in the art with method humanized antibody, and discuss herein (referring to Morrison, Science229:1202 (1985); Oi etc., BioTechniques 4:214 (1986); Cabilly etc., United States Patent (USP) the 4th, 816, No. 567; Taniguchi etc., EP 171496; Morrison etc., EP 173494; Neuberger etc., WO 8601533; Robinson etc., No. 8702671, international publication WO; Boulianne etc., Nature 312:643 (1984); Neuberger etc., Nature 314:268 (1985)).
From the scFv library, separate antibody fragment at an albumin fusion proteins of the present invention or an albumin fusion proteins part of the present invention.Will be from human PBL isolating naturally occurring V gene constructed in the library that has at reactive antibody fragment of an albumin fusion proteins of the present invention or an albumin fusion proteins part of the present invention, its donor can be to have contacted an albumin fusion proteins of the present invention or an albumin fusion proteins part of the present invention, also can be do not contact (referring to for example United States Patent (USP) the 5th, 885, No. 793, complete by reference income this paper).
The rescue in library.The scFv library is to make up as the RNA from human PBL as described in No. 92/01047, the international publication WO.In order to save phage displaying antibody fragment, with containing general 10 of phasmid
950 milliliters of individual escherichia coli inoculations contain 1% glucose and 100 μ g/ml ampicillin 2 * TY (2 * TY-AMP-GLU), reach 0.8 by oscillating growth to O.D..Get 50 milliliters of 2 * TY-AMP-GLU of 5 milliliters of this culture inoculations then, and add 2 * 10
8Δ gene 3 helper phages of TU No. 92/01047, international publication WO (M13 Δ gene III, referring to), culture are in 37 ℃ of dead-beat incubations 45 minutes, and then in 37 ℃ of vibration incubations 45 minutes.Centrifugal 10 minutes of the culture 4000r.p.m. that obtains is resuspended in 2 liters of 2 * TY that contain 100 μ g/ml ampicillin and 50 μ g/ml kanamycin with precipitation, and grow overnight.At last as preparation phage as described in No. 92/01047, the international publication WO.
Prepare not encoding gene III albumen of M13 Δ gene III:M13 Δ gene III helper phage as follows, therefore show that the phage (phasmid) of antibody fragment has the affinity of bigger conjugated antigen.Build up in the process in the morphology of phages,, obtain infectious M13 Δ gene III granule by in containing the cell that the proteic pUC19 derivant of wild type gene III can be provided, cultivating helper phage.Culture is in 37 ℃ of dead-beat incubations 1 hour, and then in 37 ℃ of vibration incubations 1 hour.(centrifugal 10 minutes of IEC-Centra 8400r.p.m.) is resuspended in that (2 * TY-AMP-KAN), 37 ℃ of shaken cultivation are spent the night in 300 milliliters of 2 * TY broth bouillons that contain 100 μ g ampicillin/ml and 25 μ g kanamycin/ml behind the cell centrifugation.By two step PEG precipitation (Sambrook etc., 1990) purification and concentrated phage particles from culture medium, then phage particle is resuspended in 2 milliliters of PBS, filter 0.45 μ m filter membrane (Minisart NML; Sartorius), the final concentration that obtains at last is about 10
13Transduced unit/ml (amicillin resistance clone).
The elutriation in library.PBS bag with 4 milliliter of 100 μ g/ml or 10 a μ g/ml albumin fusion proteins of the present invention or an albumin fusion proteins part of the present invention is spent the night by immunity pipe (Nunc).Use 2% Marvel-PBS in 37 ℃ of sealed tubes 2 hours, PBS rinsing 3 times then.Add about 10
13The phage of TU places to spin upside down on the platform and rotates in pipe, and room temperature incubation 30 minutes left standstill other 1.5 hours then.Respectively wash pipe 10 times with PBS that contains 0.1% tween 20 and PBS.Add the triethylamine of 1 milliliter of 100mM, and place and spin upside down on the platform 15 minutes wash-out bacteriophages of rotation, use Tris-HCl (pH 7.4) neutralization solution of 0.5 milliliter of 1.0M then immediately.Arise from 37 ℃ of incubations 30 minutes by phage and antibacterial one with eluting, phage is used to infect the e. coli tg1 of 10 milliliters of mid-log phases.Then escherichia coli are layered on the TYE flat board that contains 1% glucose and 100 μ g/ml ampicillin.The above is saved with Δ gene 3 helper phages for another example in the antibacterial library that obtains, and is used for the phage that next round is selected with preparation.Repeat said process then and carry out totally 4 affinity purifications of taking turns, the 3rd and 4 take turns with the PBS and the PBS rinsing that contain 0.1% tween 20 and respectively are increased to 20 times.
The table of conjugate exists.Be used for ehec infection HB 2151 from the 3rd and the 4th wash-out bacteriophage of taking turns selection, from single bacterium colony, produce soluble scFv (Marks etc., 1991) to detect.Albumin fusion proteins of the present invention or albumin fusion proteins of the present invention part bag with the 10pg/ml that is dissolved in 50mM bicarbonate (pH 9.6) are carried out ELISA by microtitration plate.Positive colony among the ELISA can pass through PCR fingerprinting (referring to No. 92/01047, international publication WO for example) and order-checking subsequently further characterizes.These ELISA positive colonies also can further be identified by technology known in the art, such as for example, epitope mapping, binding affinity, receptor signal transduction, blocking-up or the bonded ability of competitive inhibition antibody/antigen and competitive excitement or antagonistic activity.
Embodiment 13:[
3
H]-picked-up of 2-deoxyglucose experiment
Fat, skeletal muscle regulating liver-QI are the insulin sensitivity tissues.Insulin can stimulate the picked-up/transhipment of glucose to go into these tissues.For fat and skeletal muscle, the transduction of insulin initial signal finally causes glucose transporter 4 molecules (GLUT4) from specific transporte to cells surface, intracellular region chamber.In case be positioned at cell surface, GLUT4 just allows the picked-up/transhipment of glucose.
[
3
H]-picked-up of 2-deoxyglucose
Any combination that one or more list the medicine that is used for treating diabetes lack or the condition that exists under, many fat and muscle relevant cell system can be used for the mensuration of glucose uptake/transport activity.Especially, 3T3-L1 mouse fibroblast cell and L6 Os Mus bone myocyte can be divided into 3T3-L1 adipose cell and myotube respectively, as [
3H]-suitable external model (Urso etc., J Biol Chem, 274 (43): 30864-73 (1999) of 2-deoxyglucose picked-up experiment; Wang etc., J Mol Endocrinol, 19 (3): 241-8 (1997); Haspel etc., J Membr Biol, 169 (1): 45-53 (1999); Tsakiridis etc., Endocrinology, 136 (10): 4315-22 (1995)).Briefly, 2 * 10
5The adipose cell of individual cell/100 μ L or the L6 cell of differentiation are transferred in the culture medium after differentiation with the poly-l-lysine of 50 μ g/mL and are handled in the 96 hole tissue culturing plates (" TC ") of (i.e. bag by), then at 5% CO
2In be incubated overnight in 37 ℃.Cell at first with the LG DMEM culture medium rinsing that does not contain serum once, use the same culture medium in 100 μ L/ holes then, any or several combinations of listing the medicine that is used for treating diabetes of the buffer in reuse 100 μ L/ holes (for example, 1nM, 10nM and 100nM increase the therapeutic agent of the present invention (as be disclosed as SEQ ID NO:Y specific fusant and its fragment and variant) of concentration) lacking or existing under the condition of 1nM insulin, place 37 ℃ 16 hours, make it be in starvation.With the HEPES buffer saline rinsing culture plate in 100 μ L/ holes 3 times.10 μ M labellings [
3H]-the 2-deoxyglucose (Amersham, #TRK672) and the unlabelled 2-deoxyglucose of 10 μ M (SIGMA D-3179) under the condition of Cun Zaiing, is added in the insulin of 1nM in the HEPES buffer saline, place 37 ℃ 30 minutes.In contrast, under similarity condition, carry out same experiment, just do not add insulin.(SIGMA C6762) measures non-specific uptake to add the Cytochalasin B that final concentration is 10 μ M with 100 μ L/ holes in each hole.Cell HEPES buffer saline rinsing 3 times.10 μ M of adding labelling [
3H]-the 2-deoxyglucose of 2-deoxyglucose and unlabelled 10 μ M, room temperature 10 minutes.Cell is with cold phosphate buffered saline (PBS) " PBS " rinsing 3 times.The NaOH that adds the 0.2N in 150 μ L/ holes, the incubation of room temperature vibration subsequently 20 minutes, cell is cleaved.Then with sample transfer in the scintillation vial that is added with 5 milliliters of scintillation solutions.With the β scintillation counter scintillation vial is counted.In difference for lacking or existing the intake under two conditions of insulin to determine with following equation: [(insulin of per minute is counted the special cpm/ of " cpm "-non-(the non-special cpm of no insulin cpm-)].The average response of adipose cell and myotube drops on about 5 times and 3 times respectively in the limit value of contrast.
The differentiation of cell
At T-75cm
2Bottle in allow a cell to converge fully.Discard culture medium, replaced to before 25 milliliters the differentiation culture medium culturing 48 hours.Cell in 37 ℃ at 5%CO
2With incubation under 85% humidity.After 48 hours, discard the preceding culture medium of differentiation, replace to 25 milliliters of division culture mediums and cultivated 48 hours.Cell again in 37 ℃ at 5%CO
2With incubation under 85% humidity.After 48 hours, discard culture medium, replace to culture medium after 30 milliliters of differentiation.Cell continued to keep on culture medium after the differentiation 14-20 days, or until realizing differentiation fully.Changed a subculture in every 2-3 days.Human adipose cell can be from Zen-Bio, and INC (#SA-1096) buys.
Embodiment 14:[
3
H]-thymidine mixes the in vitro tests of pancreatic cell system
Recent studies show that, GLP-1 induces the differentiation of rat ductus pancreaticus epithelial cell line ARIP with the pattern that depends on time and dosage, relevant (the Hui etc. of rising of this inducing action and islets of langerhans duodenum homeobox-1 (IDX-1) and Insulin mRNA level, 2001, Diabetes, 50 (4): 785-96).IDX-1 improves GLP-1 receptor mRNA level then.
The cell type of test
The RIN-M cell: these cells can (ATCC cell line numbering CRL-2057) obtain from U.S. typical organization culture collection center.RIN-M cell line is derived from radiation-induced portable Islet cells tumor.This cell line is set up from the nude mice tumor xenogeneic graft.This cell produces and secretion islets of langerhans polypeptide hormone, and produces L-DOPA decarboxylase (sign with amine precursor picked-up and decarboxylation or the active cell of APUD).
The ARIP cell: these cells are the exocrine pancreas cells with epithelium form, can (ATCC cell line numbering CRL-1674) obtain from U.S. typical organization culture collection center.Also referring to list of references: Jessop, N.W. and Hay, R.J., " Characteristics of two rat pancreatic exocrinecell lines derived from transplantable tumors (derived from the feature of two pancreas in rat exocrine cells system of transplantable tumor) ", In Vitro 16:212, (1980); Cockell, M. etc., " Identification of a cell-specific DNA-binding activity that interacts with atranscriptional activator of genes expressed in the acinar pancreas (with the evaluation of the interactional cell-specific dna binding activity of gene transcription activator of in pancreatic acini, expressing) ", Mol.Cell.Biol.9:2464-2476, (1989); Roux, E. etc., " The cell-specifictranscription factor PTF1 contains two different subunits that interact with theDNA (and cell-specific transcription factor PTF1 contain two different can with the interactional subunit of DNA) " Genes Dev.3:1613-1624, (1989); And Hui, H. etc., " Glucagon-like peptide1induces differentiation of islet duodenal homeobox-1-positive pancreatic ductalcells into insulin-secreting cells (glucagon-like peptide 1 induces the male pancreatic ductal cell of islets of langerhans duodenum homeobox to be divided into insulin secretory cell) " Diebetes 50:785-796, (2001).
The preparation of cell
(HyClone, (Hyclone, #SH300027.01), per 6 to 8 days with 1: 3 to 1: 6 ratio successive transfer culture in RPMI1640 culture medium #SH30088.03) containing 10% hyclone for the RIN-M cell line growth.Changed a subculture in per 3 to 4 days.
ARIP cell line (ATCC numbers CRL-1674) be grown in the L-glutaminate, 1.5g/L sodium bicarbonate and 10% hyclone that contain 2mM HamShi F12K culture medium (ATCC, #30-2004) in.Twice with 1: 3 to 1: 6 ratio successive transfer culture ARIP cell line weekly.Changed a subculture in per 3 to 4 days.
Testing program
Cell is inoculated in 96 orifice plates with 4000 cells/well, cultivates to reach 50% converge in 48 to 72 hours.Then cell is transferred to the culture medium that does not contain serum with 100 μ L/ holes.After incubation 48-72 hour, serum and/or therapeutic agent of the present invention (as albumin fusion proteins of the present invention and its fragment or variant) are added to the hole.Incubation is 36 hours again.Will [
3H]-(AmershamPharmacia #TRK120) is diluted to 1 micromicrocurie/5 microlitres to thymidine (5-20Ci/mmol).Every hole adds 5 microlitres behind 36 hours incubations, continues incubation 24 hours.With the soft cessation reaction of rinsing cell of cold phosphate buffered saline (PBS) " PBS ".The ice-cold TCA that uses 100 microlitres 10% then was in 4 ℃ of fixed cells 15 minutes.Discard PBS, add the NaOH of 200 microlitre 0.2N.Plate was in room temperature vibration incubation 1 hour.Solution is transferred in the scintillation vial, added 5 milliliters of scintillation solutions compatible, the violent mixing with aqueous solution.Scintillation vial is counted with the β scintillation counter.Only use buffer as negative control.Hyclone is as positive control.
Embodiment 15: the glycosuria test
Glycosuria (i.e. excessive sugar in the urine) is measured easily, can be used as the index of diabetes morbid state.Compare the symptom that urine excessive in patient's sample is IDDM and NIDDM with the normal patient sample.The reduction of excessive glucose content in the urine due to this type of IDDM and NIDDM patient's therapeutic efficiency is shown as thus.In the embodiment of a monitoring IDDM and NIDDM, use technology known in the art to measure the existence of glucose in patient's urine sample.The concentration that human glycosuria is defined as glucose in the urine surpasses 100mg/100ml.Even the mensuration that can carry out serum glucose by the blood sample that obtains the patient is measured the excessive sugar level of glycosuria patient more accurately.
Embodiment 16: detect the stimulation of B cell proliferation and differentiation or the test of inhibition
The generation of functional humoral immunoresponse(HI) requires two class signals conduction solubility and related (cognate) between B-pedigree cell and its microenvironment.Signal can just produce to stimulate, and allows B-pedigree cell to continue its procedural growth, or negative the stimulation, and indicator cells interrupts its present development pathway.Up to now, find that many zests and inhibition signal can influence the response of B cell, comprise IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-13, IL-14 and IL-15.What is interesting is that these signals itself are the weak effect things, but with various altogether stimulatory protein(SP) combinations, can induce the B cell colony activation, propagation, break up, go back to the nest, tolerate and dead.
Study the most clearly one of B cell co-stimulatory albumen classification be the TNF superfamily.In this family, found that CD40, CD27 and CD30 and they part CD154, CD70 and CD153 separately regulates many immunne response.The test that is used to detect and/or observe these B-cell colonys and precursor thereof propagation and differentiation is to determine the useful instrument of various albumen to the influence of the propagation of these B-cell colonys and differentiation.Hereinafter listed the test that two designs are used for detecting differentiation, propagation or the inhibition of B-cell colony and precursor thereof.
In vitro tests-can evaluate albumin fusion proteins of the present invention (comprising the fragment that contains human cytokines or the fusion rotein of variant and/or albumin or albuminised fragment or variant) is induced B-cell colony and precursor activation, propagation, differentiation or inhibition and/or dead ability.Albumin fusion proteins of the present invention to the human tonsil B cell activity of purification qualitatively from 0.1 to 10, measure in the dosage range of 000ng/mL, B-lymphocyte irritant test evaluation altogether with standard, in this test, the tonsil B cell of purification is cultivated under the condition that staphylococcus aureus (Staphylococcus aureus) CowanI (SAC) of formalin fixed or immobilized anti-human IgM antibody exist as initiator.Such as secondary signal and SAC and the collaborative crosslinked B cell proliferation that causes of IgM of IL-2 and IL-15, measure by mixing of tritium-labeled thymidine.Use this test can easily identify new synergist.This test comprises that removing the CD3 positive cell by magnetic bead (MACS) separates human tonsil B cell.The cell colony that obtains contains and surpasses 95% B cell, evaluates by the expression of CD45R (B220).
The different dilutions of each sample are added in each hole of 96 orifice plates, and the usefulness culture medium that is added with cumulative volume 150 μ l in the hole (contains 10% FBS, 5 * 10
-5The streptomycin and 10 of the 2ME of M, the penicillin of 100U/ml, 10 μ g/ml
-5RPMI 1640 culture medium of the SAC of dilution doubly) suspend 10
5The B cell.Added after the factor 72 hours, and began to use
3Propagation or inhibition that the pulse (1uCi/ hole) that H-thymidine (6.7Ci/mM) carried out 20 hours comes quantitative cell.Positive and negative control is respectively IL2 and culture medium.
Twice usefulness in vivo test-every day buffer or contain buffer injection (i.p.) BALB/c mouse of the albumin fusion proteins of the present invention (comprising the fragment that contains human cytokines or the fusion rotein of variant and/or albumin or albumin fragment or variant) of 2mg/Kg only.Mice was accepted this processing in continuous 4 days, put to death mice then, collected various tissues and serum and was used for analyzing.The H﹠amp of the spleen of handling by more normal spleen with albumin fusion proteins of the present invention; The active result of fusion rotein to splenocyte identified in the E section, such as, the phenomenal growth of the diffusion of periarterial lymphatic sheath and/or red pulp district band nucleus, this can indicate the activation of differentiation of B cell colony and propagation.The immunohistochemistry research of carrying out with the anti-CD45R of B cell marking (B220) is used for determining that whether any physiological change (as the spleen structural damage) of splenocyte is to build up in the loose B cellular regions that limits of T cellular regions B cell expressivity by infiltration to increase and cause.
The spleen of the mice of handling with albumin fusion proteins with flow cytometry is used to indicate albumin fusion proteins whether to compare the ratio that increases the dim B cell of ThB+/CD45R (B220) specifically with control mice.
Similarly, the result of the mature B cell expressivity that increases in the body of prediction is the relative increase of Ig serum titer.Correspondingly, IgM and the IgA serum levels between the mice of comparison buffer and fusion rotein processing.
Embodiment 17:T cell proliferation test
PBMC is carried out the inductive proliferation test of CD3, by
3The picked-up of H-thymidine is measured.Test is carried out as follows: with the mAb (HIT3a of the CD3 in 100 μ l/ holes, Pharmingen) or the contrast mAb (B33.1) of isotype coupling bag spent the night by 4 ℃ of 96 orifice plates and (be dissolved in the bicarbonate buffer of 0.05M, pH 9.5,1 μ g/ml), use the PBS rinsing then 3 times.From human peripheral blood, separate PBMC with the F/H gradient centrifugation, in RPMI (albumin fusion proteins of the present invention (comprising the fragment that contains human cytokines or the fusion rotein of variant and/or albumin or albuminised fragment or variant) that contains 10% FCS, P/S and variable concentrations), be added to the mAb bag by four repeating holes of plate in (5 * 10
4/ hole, cumulative volume 200 μ l).Only associated protein buffer and culture medium are in contrast.Cultivate after 48 hours for 37 ℃,,, then take out 100 μ l supernatant, be saved in-20 ℃ of mensuration that are used for IL-2 (or other cytokine) if observe the influence of on cell proliferation with centrifugal 2 minutes of plate 1000rpm.In the hole, add 100 μ l then and contain 0.5 μ Ci's
3The culture medium of H-thymidine was cultivated 18-24 hour in 37 ℃.Collect each hole, mix
3The mensuration that the H-thymidine is used to breed.Independent anti-CD3 is as the positive control of propagation.IL-2 (100U/ml) is also as the contrast that strengthens propagation.The control antibodies of inducing T cell propagation is not as the negative control of fusion rotein effect of the present invention.
Embodiment 18: fusion rotein of the present invention to MHC ∏ class, altogether stimulate and the expression of adhesion molecule and
Influence to the human dendritic cell differentiation of mononuclear cell and monocyte derived
Dendritic cell are to be produced by the propagation precursor expansion (expansion) that is present in peripheral blood: (elutriated) monocyte fraction of the PBMC of adhesion or elutriation was cultivated 7-10 days with GM-CSF (50ng/ml) and IL-4 (20ng/ml).These dendritic cell have the feature phenotype (CD1, CD80, CD86, CD40 and the antigenic expression of MHC ∏ class) of immature cell.Use such as the activity factor of TNF-α and handle the rapid change (rise of the following mediation CD83 of MHC I class and ∏ class, the enhancing that is total to stimulation and adhesion molecule expression, FC γ R ∏) that causes surperficial phenotype.These changes are relevant with the functional maturation of the enhancing of antigen presentation ability and dendritic cell.
The facs analysis of surface antigen can be undertaken by following scheme.Cell was handled 1-3 days with albumin fusion proteins of the present invention that increases concentration or LPS (positive control), with the PBS rinsing that contains 1%BSA and 0.02mM Hydrazoic acid,sodium salt, then with the suitable FITC-of dilution in 1: 20 or the monoclonal antibody of PE-labelling jointly in 4 ℃ of incubations 30 minutes.Post rinse once after, in the flow cytometry of the enterprising row labels cell of FACScan (Becton Dickinson).
The influence that the pair cell factor producesThe cytokine (particularly IL-12) that dendritic cell produce plays a significant role in startup depends on the immunne response of T cell.IL-12 influences the generation of Thl helper T cell immunne response strongly, and inducing cytotoxic T and NK cell play a role.The scheme of measuring IL-12 release with ELISA is as described below.Handle dendritic cell (10 with the albumin fusion proteins of the present invention that increases concentration
6/ ml) 24 hours.The LPS (100ng/ml) that is added to cell culture is as positive control.The supernatant of collecting cell culture, with business-like ELISA test kit (as R﹠amp; D Systems (Minneapolis, MN)) analyzes the content of IL-12.The standard scheme that uses this test kit to provide.
Influence to MHC ∏ class, common stimulation and adhesion molecule expressionCan identify the cell surface antigen of 3 extended familys on the mononuclear cell: the molecule and the Fc receptor of adhesion molecule, participation antigen presentation.By regulating the expression of MHC ∏ class antigen and other costimulatory molecules (as B7 and ICAM-1), can cause the change of mononuclear cell antigen presentation ability and the change of inducing T cell activation capability.The enhancing of Fc expression of receptor may be relevant with the release and the phagocytosis of improved mononuclear cell cytotoxicity, cytokine.
The facs analysis that is used to detect surface antigen can be undertaken by following scheme.Mononuclear cell was handled 1-5 days with albumin fusion proteins of the present invention that increases concentration or LPS (positive control), with the PBS rinsing that contains 1%BSA and 0.02mM Hydrazoic acid,sodium salt, then with the suitable FITC-of dilution in 1: 20 or the monoclonal antibody of PE-labelling jointly in 4 ℃ of incubations 30 minutes.Float Xian Yici again, at last in the flow cytometry of the enterprising row labels cell of FACScan (Becton Dickinson).
The survival of monocyte activation and/or increaseIt is known in the art studying the test that activates (or inactivation) mononuclear cell and/or increase the molecule of monocytic survival (or reducing monocytic survival), can be used for usually determining whether molecule of the present invention can be used as monocytic inhibitor or activator plays a role.Albumin fusion proteins of the present invention can screen by following 3 tests.For each of these experiments, PERIPHERAL BLOOD MONONUCLEAR CELL (PBMC) all be by Histopaque gradient (Sigma) centrifugal from single donor leukocyte bag (American Red Cross, Baltimore, MD) in purification.Then, by the adverse current centrifugal elutriation, from PBMC, separate obtaining mononuclear cell.
The monocyte survival testWhen cultivating in the culture medium that is lacking serum or other stimulation, human peripheral blood mononuclear cells can little by little be lost viability.Its death is derived from inherent regulation process (apoptosis).In culture, add the survival that can significantly improve cell such as the activity factor of TNF-α, stop the fragmentation of DNA.Iodate third ingot (PI) dyeing is used for the mensuration of apoptosis, and its scheme is as follows.Mononuclear cell was cultivated in following culture medium 48 hours in polypropylene tube: do not contain serum culture medium (positive control), contain the culture medium (negative control) of 100ng/ml TNF-α and contain the culture medium of fusion rotein that variable concentrations is surveyed.With containing final concentration is that the PBS of 5 μ g/ml PI is with 2 * 10
6The concentration suspension cell of/ml, room temperature incubation carry out FACScan and analyze after 5 minutes.In this experiment example, it is that fragmentation with DNA is relevant that the picked-up of PI shows.
The influence that the pair cell factor dischargesA critical function of monocyte/macrophage is that they discharge cytokine by stimulating the back, to the adjusting activity of immune other cell colony.Measure following the carrying out of ELISA of release of cytokines.The human monocyte is with 5 * 10
5The density of individual cell/ml and the common incubation of albumin fusion proteins of the present invention that increases concentration, and lacking incubation under the same incubation conditions of fusion rotein.In order to produce IL-12, under the condition that fusion rotein exists, cause (prime) cell with IFN (100U/ml) and spend the night.Add LPS (10ng/ml) then.Collection condition culture medium after 24 hours, and make it keep freezing state until use.Use business-like ELISA test kit (as R﹠amp then; D Systems (Minneapolis, MN)) and the standard scheme mensuration TNF-α, IL-10, MCP-1 and the IL-8 that provide with test kit.
Oxidative burstThe mononuclear cell of purification is with 2-1 * 10
5Individual cells/well is taped against in 96 orifice plates.In the culture medium of cumulative volume 0.2ml (RPMI 1640+10%FCS, glutamine and antibiotic), the albumin fusion proteins of the present invention that increases concentration is joined in the hole.Behind the incubation 3 days, centrifugal flat board discards the culture medium in the hole.To the macrophage monolayer, add phenol red solution (140mMNaCl, 10mM kaliumphosphate buffer (pH 7.0), 5.5mM dextrose, 0.56mM is phenol red and 19U/ml HRPO) and the stimulus object (200nM PMA) of 0.2ml in every hole.Dull and stereotyped in 37 ℃ of incubations 2 hours, every hole adds the NaOH cessation reaction of 20 μ l 1N.Measure the absorbance of 610nm.For calculating the H that macrophage produces
2O
2Amount, the H of known molar concentration is drawn in each experiment
2O
2The standard curve of solution.
Embodiment 19: albumin fusion proteins of the present invention is to the influence of vascular endothelial cell growth
The 1st day, with human umbilical vein endothelial cell (HUVEC) with 2-5 * 10
4The density of individual cell/35mm dish is inoculated into the M199 culture medium, this culture medium contain 4% hyclone (FBS), 16 units/ml heparin and 50 units/ml endothelial cell growth additive (ECGS, Biotechnique, Inc.).The 2nd day, culture medium is replaced with the M199 culture medium that contains 10%FBS and 8 units/ml heparin.Add the albumin fusion proteins of the present invention of variable concentrations and such as the positive control of VEGF and basic FGF (bFGF).The 4th day and the 6th day, change culture medium.The 8th day, measure cell number with the Coulter enumerator.
The increase explanation fusion rotein of HUVEC cell number can be bred vascular endothelial cell, and the minimizing of HUVEC cell number explanation fusion rotein suppresses vascular endothelial cell.
Embodiment 20: rat corneal wound healing model
This animal model has shown the influence of albumin fusion proteins of the present invention to neovascularization.Experimental program comprises:
From the CC to the hypothallus, make the otch of a 1-1.5 millimeters long.
Below described notching edge, insert a spatula (spatula) towards the tail of the eye.
Make a pocket (its bottom is apart from the edge 1-1.5 millimeter (its base is 1-1.5mm formthe edge of the eye) of eyes).
The piller that will contain 50ng-5ug albumin fusion proteins of the present invention is put into pocket.
The processing of albumin fusion proteins of the present invention also can be added in the corneal wound place partly, and dosage range is 20mg-500mg (handle every day, handled 5 days).
Embodiment 21: the wound healing model that diabetic mice and glucocorticoid are impaired
Diabetes db+/db+ mouse model
But prove the process of albumin fusion proteins healing acceleration of the present invention with the mice wound healing model of hereditary diabetes.Holostrome skin wound healing model in the db+/db+ mice be study more clearlyly, clinical relevant and repeatably impaired wound healing model.The healing of diabetic wounds depends on the formation of granulation tissue and the formation again of epithelium, rather than shrinks (Gartner, M.H. etc., J.Surg.Res.52:389, (1992); Greenhalgh, D.G. etc., Am.J.Pathol.136:1235, (1990)).
Diabetic animal has observed many typical characteristics in the ∏ type diabetes.With littermate normal heterozygosis mice (db+ /+m) compare (db+/db+) mice obesity of isozygotying.Saltant diabetic mice (db+/db+) has single autosomal recessive sudden change (db+) (Coleman etc., Proc.Natl.Acad.Sci.USA 77:283-293,1982) on No. 4 chromosomes.Animal shows as polyphagia, polydipsia and polyuria.Saltant diabetic mice (db+/db+) blood sugar level raises, insulin level is normal or rising, and cell-mediated immunity is suppressed (Mandel etc., J.Immunol.120:1375, (1978); Debray-Sachs, M. etc., Clin.Exp.Immunol.51 (1): 1-7, (1983); Leiter etc., Am.J.of Pathol.114:46-55, (1985)).These animals also show as peripheral neurophaty, myocardium complication, microvascular lesions, basement membrane thickening and glomerular filtration unusual (Norido, F. etc., Exp.Neurol.83 (2): 221-232, (1984); Robertson etc., Diabetes 29 (1): 60-67, (1980); Giacomelli etc., Lab Invest.40 (4): 460-473, (1979); Coleman, D.L., Diabetes 31 (supplementary issue): 1-6, (1982)).The hyperglycemia to insulin resistant (Mandel etc., J.Immunol.120:1375-1377, (1978)) of human type ii diabetes has taken place to be similar in these diabetic mices that isozygoty.
Observed feature description in these animals, observed agglutination similar (Greenhalgh etc., Am.J.of Pathol.136:1235-1246, (1990)) in the agglutination of this model and the human diabetes.
This research is used be the female C57BL/KsJ mice (db+/db+) of hereditary diabetes and non-diabetic heterozygosis mice littermate with it (db+ /+m) (Jackson laboratory).6 is all big when these animals were bought, and 8 weeks are big when beginning one's study.Animal is raised separately, ad libitum access and drinking water.All operations all is to use aseptic technique to carry out.Experiment is according to Human Genome Sciences's management of laboratory animal and uses the regulation of committee (Human Genome Sciences, Inc.Institutional Animal Care andUse Committee) and the criterion (Guidelines forthe Care and Use of Laboratory Animals) of criterion and management of laboratory animal and use to carry out.
The scheme that produces wound is (Tsuboi, R. and Rifkin, D.B., J.Exp.Med.172:245-251, (1990)) of carrying out according to the method for having delivered.Briefly, producing wound same day, first peritoneal injection be dissolved in deionized water A Fuding (Avertin, 0.01mg/ml), tribromo-ethanol and 2-methyl-2-butanols anesthetized animal.Hair is scraped in the dorsal part zone of animal, cleaned skin with 70% alcoholic solution and iodine.Before producing wound, operative region is dried with sterile gauze.Then, organize perforator to produce one 8 millimeters holostrome skin wound with Keyes.After wound produced, the peripheral skin that stretches carefully prevented that wound from enlarging at once.The opening that always keeps wound in the experimentation.Produce the same day from wound, carried out surface treatment in continuous 5 days.Before the processing, gently clear up wound with aseptic saline and gauze sponge earlier.
Operation the same day and per afterwards two days is with the naked eye checked wound and is taken a picture at the fixed range place.At 1-5 days with measure the closure that wound is determined wound the 8th day every day.Measure wound horizontally and vertically with calibrated Jameson slide calliper rule.If granulation tissue no longer as seen, and wound covered by successive epithelium, thinks that then wound heals.
Employed albumin fusion proteins of the present invention is in the scope of various dose, and each wound is from 4mg to 500mg every day in the media, handles 8 days.The media matched group media solution-treated of 50mL.
The 8th day, animal imposed euthanasia by peritoneal injection pentobarbital sodium (300mg/kg).Collect wound then and peripheral skin is used for histology and immunohistochemical analysis.Tissue sample is placed in the tissue cassette of the formalin that contains 10% neutral buffered, between the vivisection sponge, be used for further processing.
Estimate 3 groups of samples of every group of 10 animals (5 diabetes samples and 5 non-diabetic contrasts): 1) media placebo, 2) not treatment group, 3) the treatment group.
Wound by measuring vertical and horizontal axis and the whole square area that obtains wound are analyzed the closure of wound.Estimate the contraction of wound then by the difference of establishing initial wound area (the 0th day) and processing back wound area (the 8th day).The 1st day wound area is 64mm
2, big or small corresponding with punch skin.Use following formula to calculate:
The 8th day aperture area of a.[]-[the 1st day aperture area]/[the 1st day aperture area]
Sample is cut into slices wax embedding block with the Reichert-Jung microtome with 10% buffered formalin fixed perpendicular to wound surface (5mm).Conventional hematoxylin-eosin staining (H﹠amp is carried out in the wound cross section of section; E).The histology of wound is used to assess whether the morphology of skin of agglutination and reparation is apparent changes because of the processing of albumin fusion proteins of the present invention.This assessment comprises checking cell accumulation, inflammatory cell, blood capillary, fibroblast, epithelium forms again and the existing of epidermis Maturity (Greenhalgh, D.G. etc., Am.J.Pathol.136:1235,1990).Calibrated lens micrometer is used by blind method observer.
Tissue slice also by the keratin antibody dyeing of immunohistochemical method with the anti-people of multi-clone rabbit, detects with ABC Elite detection system.Human skin is as positive tissue contrast; But not the IgG of immunity is as negative control.The degree that forms again by the assess wound epithelium with calibrated lens micrometer is determined the growth of keratinocyte.
Prove the existence of proliferating cell nuclear antigen/cyclin in the skin sample (PCNA) with ABC Elite detection system, use anti-PCNA antibody (1: 50).Human colon's cancer is as the contrast of positive tissue, and human cerebral tissue is as negative tissue contrast.Each specimen comprises with what non-immune mouse IgG replaced and does not contain an anti-section.Degree (by the grade of 0-8) according to propagation is carried out classification to these sections, and lower grade has reflected slight propagation degree, and higher grade has reflected intensive propagation degree.
Analyze experimental data with non-paired t test.P value＜0.05 is considered as significantly.
The rat model that steroid is impaired
Prove fully that all steroid can suppress healing (Wahl, the Glucocorticoids and Wound healing (glucocorticoid and wound healing) of wound in the system in the various external and bodies.In Anti-Inflammatory Steroid Action:Basic and Clinical Aspects (anti-inflammatory steroid effect: basis and clinicing aspect) .280-302, (1989); Wahl etc., J.Immunol.115:476-481,1975; Werb etc., J.Exp.Med.147:1684-1694,1978).Vascular permeability (Ebert etc., An.Intern.Med.37:701-705,1952), fibroblast proliferation, collagen protein synthesis (Beck etc., Growth Factors.5:295-304,1991 take place, reduce by suppressing blood vessel; Haynes etc., J.Clin.Invest.61:703-797,1978) and make the instantaneous minimizing of circulation mononuclear cell (Haynes etc., J.Clin.Invest.61:703-797,1978; Wahl, " Glucocorticoids and Wound healing (glucocorticoid and wound healing) ", at Anti-Inflammatory Steroid Action:Basic and ClinicalAspects (anti-inflammatory steroid effect: basis and clinicing aspect), Academic Press, New York, the 280-302 page or leaf, 1989), glucocorticoid can postpone the healing of wound.To impaired wound healing systematically the administration steroid be phenomenon (Beck etc., Growth Factors.5:295-304,1991 of relatively determining in the rat; Haynes etc., J.Clin.Invest.61:703-797,1978; Wahl, " Glucocorticoids andWound healing (glucocorticoid and wound healing) ", at Anti-Inflammatory SteroidAction:Basic and Clinical Aspects (anti-inflammatory steroid effect: basis and clinicing aspect), Academic Press, New York, the 280-302 page or leaf, 1989; Pierce etc., Proc.Natl.Acad.Sci.USA 86:2229-2233,1989).
But, use the influence of holostrome skin being excised skin wound to make the impaired repeatedly surperficial fusion rotein of rat assessment of healing owing to the methylprednisolone that is administered systemically for proving albumin fusion proteins healing acceleration process of the present invention.
What use in the present embodiment is the male Sprague Dawley of the adult rat (Charles River laboratory) of the youth of heavy 250-300g.8 is all big when these animals were bought, and 9 weeks are big when beginning one's study.When wound forms, be administered systemically by methylprednisolone (intramuscular injection 17mg/kg/ rat), make the healing reaction of rat impaired.Animal is raised separately, ad libitum access and drinking water.All operations all is to use aseptic technique to carry out.This research is according to Human Genome Sciences's management of laboratory animal and uses the regulation of committee (Human Genome Sciences, Inc.Institutional Animal Care and UseCommittee) and the criterion (Guidelines for theCare and Use of Laboratory Animals) of criterion and management of laboratory animal and use to carry out.
The scheme of generation wound as previously mentioned.Producing the wound same day, intramuscular injection ketamine (50mg/kg) and xylazine (5mg/kg) anesthetized animal.Hair is scraped in the dorsal part zone of animal, cleaned skin with 70% ethanol and iodine solution.Before producing wound, operative region is dried with sterile gauze.Organize perforator to produce one 8 millimeters holostrome skin wound with Keyes.The opening that always keeps wound in the experimentation.Produce the same day from wound, after the methylprednisolone administration, continuous 7 days, carry out the surface once a day and apply test material.Before the processing, clean a wound carefully with Sterile Saline and gauze sponge.
Produce and with the naked eye to check wound when the wound same day and processing finish and take a picture at the fixed range place.At 1-5 days with measure the closure that wound is determined wound the 8th day every day.Measure wound horizontally and vertically with calibrated Jameson slide calliper rule.If granulation tissue no longer as seen, and wound covered by successive epithelium, thinks that then wound heals.
Employed albumin fusion proteins of the present invention is in the scope of various dose, and each wound is from 4mg to 500mg every day in the media, handles 8 days.The media matched group media solution-treated of 50mL.
The 8th day, animal imposed euthanasia by peritoneal injection pentobarbital sodium (300mg/kg).Collect wound then and peripheral skin is used for histologic analysis.Tissue specimen is placed in the tissue cassette of the formalin that contains 10% neutral buffered, between the vivisection sponge, be used for further processing.
Estimate 3 groups of samples of every group of 10 animals (5 methylprednisolones are handled sample and 5 samples that do not contain glucocorticoid): 1) not treatment group, 2) the media placebo, 3) the treatment group.
Wound by measuring vertical and horizontal axis and the whole area that obtains wound are analyzed the closure of wound.Come the closure of assess wound then by the difference of establishing initial wound area (the 0th day) and processing back wound area (the 8th day).The 1st day wound area is 64mm
2, big or small corresponding with punch skin.Use following formula to calculate:
The 8th day aperture area of b.[]-[the 1st day aperture area]/[the 1st day aperture area]
Specimen is cut into slices wax embedding block with the Olympus microtome with 10% buffered formalin fixed perpendicular to wound surface (5mm).Conventional hematoxylin-eosin staining (H﹠amp is carried out in the wound cross section of section; E).The histology of wound is allowed whether the morphology of skin of assessment agglutination and reparation is apparent improves because of the processing of albumin fusion proteins of the present invention.Blind method observer uses calibrated lens micrometer to determine the distance of wound breach.
Analyze experimental data with non-paired t test.P value＜0.05 is considered as significantly.
Embodiment 22: the lymphedema animal model
The purpose of this experimental technique is to produce suitable with consistent lymphedema model, is used for measuring the therapeutic effect of albumin fusion proteins of the present invention at generation of rat hindlimb lymphatic vessel and lymph circulation system reconstructing.Expanding volume by influenced limb, the amount of lymph vasculature quantitatively, total plasma protein and histopathological analysis, measure its effectiveness.The acute lymphoblastic edema was observed 7-10 days.May the more important thing is that the chronic development of edema continues to observe 3-4 week.
Before beginning operation, taking a blood sample is used for the analysis of protein concentration.The male rat of heavily about 350g is handled with pentobarbital.Subsequently, right lower limb is scraped hair from the knee to the hip.Scraping the zone of hair wipes away totally with being soaked with 70% alcoholic acid gauze cotton.Take a blood sample and be used for total serum protein mensuration.Before the injection dyestuff, define 2 measuring heights (metapedes is caught up with 0.5cm, is positioned at the middle part of back of the body foot) and afterwards, carry out girth and stereometry in the foot.The EvanShi indigo plant of injecting 0.05ml 1% at the Intradermal back side of right side foot and left side foot.Behind the injection dyestuff, carry out girth and stereometry in the foot.
Mark as the position with knee joint, ring cutting obtains the otch at midleg groin place, locatees Femur blood vessel.Dissect and separate flap with tweezers and mosquito forceps.After having located Femur blood vessel, just can locate along blood vessel side and the lymphatic vessel below blood vessel.The main lymphatic vessel in this zone is with electric coagulation or use the stitching thread ligation then.
At microscopically, flush end is dissected the muscle (closing on semitendinosus m. and adductor muscle) of lower limb back.Ding the Wei lymphonodi poplitei.Prick 2 near-ends and 2 the far-end lymphatic vessels and the far-end blood supply of lymphonodi poplitei by sewing up toe-in then.Again by cutting connective tissue Chu Qu lymphonodi poplitei and any fatty tissue that accompanies.
Handled is controlled because that this operation causes is any slight hemorrhage.After the lymphsystem closure, with liquid skin (Vetbond, AJ Buck) sealing flap.Skin edge separately stays the breach of an about 0.5cm with underlying muscle tissue sealing, the while around lower limb.If necessary, also can be by skin closure is fixed skin on underlying muscle.
For avoiding infection, animal is with netting independent stable breeding (not having the pad grass).By the animal in best edema peak (typically appearing at 5-7 days) the detection every day recovery.Observation platform edema peak then.Be the intensity of assessment lymphedema, totally 7 days every day before operation and afterwards, measure girth and volume of last 2 assigned addresses of each foot.Whether measure the influence of plasma protein to lymphedema, also studying analysis of protein is a useful assay method (perimeter).Weight at 2 position finding contrasts and edema limb.Analysis is carried out with blind sexual norm.
Girth is measured: prevent moving of limb by brief gas anesthesia, measure the girth of limb with cloth.Measure average 2 readings by 2 different people at anklebone and back of the body foot place.Reading is from contrast and edema limb.
Stereometry: perform the operation the same day, and used the pentobarbital anesthetized animal, and before operation, measure.For carrying out the stereometry of every day, use halothane (fast braking and fast quick-recovery) anesthetized animal tout court, both legs are all scraped hair, do same labelling on the lower limb with being marked at of waterproof.Earlier lower limb is immersed in the water, immerse then in the instrument, measure with Buxco edema software (Chen/Victor) to the height of each labelling.One personal record data, and another person is dipped into marked region with the animal limb.
The mensuration of plasma protein: take a blood sample before the operation, when finishing, the rotation separation of serum is used for comparison total protein and Ca subsequently
2+
The limb weight ratio is: after taking a blood sample, for the tissue of collecting animal ready.With quillitine excision animal limb, the lower limb that will test and contrast downcuts at the ligature place then, weighs.When disconnecting, carry out weighing the second time, claim heavy sensation in the foot in the tibiocalcaneal part joint.
Histology's preparation: dissect and be positioned knee (popliteal) transversus of back, district places in the metal pattern, fills with frozen rubber, immerses cold methybutane, puts into the sample sack of-80 ℃ of labellings, until beginning section.After the section, with the lymphsystem of fluorescence microscope muscle.
Embodiment 23: albumin fusion proteins of the present invention is to pressing down that the inductive adhesion molecule of TNF-α is expressed
System
LR is comprised the specific receptor-ligand interaction between the cell surface adhesion molecule (CAM) on lymphocyte and the blood vessel endothelium to inflammation and blood vessel generation area.The process of sticking together under the normal and pathological state is followed a cascade that multistep is rapid, and this cascade involves cell-cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and endothelial leucocyte adhesion molecule-1 (E-the selects albumen) expression on endotheliocyte (EC).These molecules and other molecules are in the expression on the blood vessel endothelium has determined generating process in inflammatory reaction, and leukocyte adheres to the local vascular structure and extravasates into the efficient of local organization.The Local enrichment of cytokine and somatomedin has participated in regulation and control that these content-addressable memorys are reached.
Tumor necrosis factor (TNF-α) (a kind of effective proinflammatory cytokine) is the stimulus object of all 3 kinds of CAM on the endotheliocyte, may participate in many inflammatory reactions, can cause pathologic result usually.
Can detect the potentiality that albumin fusion proteins of the present invention mediates the inhibition that the inductive content-addressable memory of TNF-α is reached.When stimulating altogether, detect the expression of the last CAM of EC that measures TNF-α processing as the improved ELISA of solid phase absorbent with EC with FGF protein family member.
For carrying out this experiment, from merge the band gleanings, obtain human umbilical vein endothelial cell (HUVEC) culture, be kept at (EGM-2 in the growth medium that is added with 10%FCS and 1% penicillin/streptomycin; Clonetics, San Diego CA), places to contain 5%CO
237 ℃ of humidified incubator in.In the EGM culture medium with HUVEC with 1 * 10
4The density of individual cells/well is inoculated in 96 orifice plates, in 37 ℃ of cultivations 18-24 hour, or until converging.Monolayer was handled 24 hours in 37 ℃ with given cytokine and/or somatomedin with the RPMI-1640 solution rinsing that does not contain serum that is added with 100U/ml penicillin and 100mg/ml streptomycin 3 times subsequently.Behind the incubation, the expression of CAM in the evaluation cell.
96 orifice plates that human umbilical vein endothelial cell (HUVEC) are grown in standard are interior until converging.From cell, discard growth medium, replace to 199 culture medium (10%FBS) of 90 μ l.Specimen and positive or negative contrast are added in the plate, and three repeat (with the volume of 10 μ l).With flat board in 37 ℃ of incubations 5 hours (selecting albumen and integrin expression) or 24 hours (having only integrin expression).Culture medium is removed in the flat board suction, and paraformaldehyde-PBS that every hole adds 100 μ l 0.1% (contains Ca
++And Mg
++).With flat board place 4 ℃ 30 minutes.
Remove the fixative in the hole,, discharge with PBS (+Ca and Mg)+0.5%BSA rinsing hole 1 time.Do not make orifice drying.Anti-arriving in test and the control wells that adds 10 μ l dilution.It is 10 μ g/ml (the antibody mother solutions of 1: 10 dilution 0.1mg/ml) that anti-ICAM-1-biotin, anti-VCAM-1-biotin and anti-E-select the working concentration of albumen-biotin.Cell is under moist environment, in 37 ℃ of incubations 30 minutes.The hole is with PBS (+Ca and Mg)+0.5%BSA rinsing 3 times.
The ExtrAvidin-alkali phosphatase (1: 5,000 dilution) that adds every hole 20 μ l dilution then was in 37 ℃ of incubations 30 minutes.The hole is with PBS (+Ca and Mg)+0.5%BSA rinsing 3 times.1 paranitrophenol phosphoric acid pNPP is dissolved in (pH 10.4) in the 5ml glycine buffer.Each instrument connection adds the pNPP substrate of the 100 μ l that are dissolved in glycine buffer.Prepare three multiple gauge orifices, it uses the work dilution of the ExtrAvidin-alkali phosphatase in the glycine buffer: 1: 5, and 000 (10
0)＞10
-0.5＞10
-1＞10
-1.5Every kind of diluent of 5 μ l is added in three repeating holes, and the AP content in the every hole that obtains like this is 5.50ng, 1.74ng, 0.55ng, 0.18ng.The pNPP reagent that must add 100 μ l then in each gauge orifice.Flat board must be in 37 ℃ of incubations 4 hours.Add the NaOH of 50 μ l 3M in porose.The result carries out quantitative analysis at plate reader 405nm place.The background deduction option blank well of only filling glycine buffer.Template is set at the concentration (5.50ng, 1.74ng, 0.55ng, 0.18ng) of AP-conjugate in each gauge orifice of indication.The result is shown as the amount of bonded AP-conjugate in each sample.
The structure of embodiment 24:GAS report construct
A signal transduction pathway that participates in cell differentiation and propagation is called the Jak-STAT approach.Activated albumen activates site " GAS " element with γ on being positioned at many gene promoters or interferon-sensitive response element (" ISRE ") combines in the Jak-STAT approach.Albumen changes Expression of Related Genes with combining of these elements.
GAS and ISRE element are discerned by the transcription factor that a class is called signal transducer and transcriptional activator or " STAT ".There are 6 members in STAT family.The same with Stat2, Stat1 and Stat3 are present in many cell types (is general because of the response to IFN-α).Stat4 is more restricted, although it is present in t helper cell I class, in the cell after IL-12 handles, is not present in many cell types.Stat5 is called the mammoplasia factor at first, but its concentration in comprising myelocytic many other cells is higher.It can be activated by many cytokines in tissue culture cells.
The tyrosine of STAT by one group of tyrosine phosphorylation that is called Janus kinases (" Jak ") family after, be activated, examine from the Cytoplasm transporte to cells.Jak represents unique solubility family tyrosine kinase, comprises Tyk2, Jak1, Jak2 and Jak3.These kinases show as the obvious sequence similarity, do not have catalytic activity in resting cell usually.
Jak is activated (selecting from summary Schidler and Darnell, Ann.Rev.Biochem.64:621-51,1995) by many receptors that are summarized in following table.The cytokine receptor family that can activate Jak is divided into two groups: (a) the 1st class comprises the receptor of IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-11, IL-12, IL-15, Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF and thrombopoietin; (b) the 2nd class comprises IFN-a, IFN-g and IL-10.The 1st receptoroid has one section conservative cysteine motif (one group of 4 conservative cysteine and 1 tryptophan) and WSXWS motif (membrane-proximal region, coding Trp-Ser-Xaa-Trp-Ser (SEQ ID NO:53)).
Therefore, after part was attached on the receptor, Jak was activated, and then activated STAT, then STAT transported and with the GAS combination of elements.Whole this process all is included in the Jak-STAT signal transduction pathway.Therefore, the activation by the Jak-STAT approach that reflects in conjunction with GAS or ISRE element can be used for indicating the albumen that participates in cell proliferation and differentiation.For example, known somatomedin and cytokine can activate Jak-STAT approach (seeing the following form 4).Therefore, by using the GAS element that is connected with reporter molecules, can identify the activator of Jak-STAT approach.
Table 4
JAK
STAT
GAS (element) or ISRE
Part
Tyk2
Jak1
Jak2
Jak3
IFN family
IFN-a/B + + - - 1，2，3 ISRE
IFN-g + + - 1 GAS(IRF1＞Lys6＞IFP)
IL-10 + ？ ？ - 1，3
Gp130 family
IL-6 (multi-purpose)+++? 1,3 GAS (IRF1＞Ly6＞IFP)
IL-11 (multi-purpose)? +? 1,3
OnM (multi-purpose)? ++? 1,3
LIF (multi-purpose)? ++? 1,3
CNTF (multi-purpose)-/+++? 1,3
G-CSF (multi-purpose)? +? 1,3
IL-12 (multi-purpose)+-++ 1,3
G-C family
IL-2 (lymphocyte)-+-+1,3,5 GAS
IL-4 (lymph/marrow sample)-+-+6 GAS (IRF1=IFP＞＞Ly6) (IgH)
IL-7 (lymphocyte)-+-+5 GAS
IL-9 (lymphocyte)-+-+5 GAS
IL-13 (lymphocyte)-+? 6 GAS
IL-15 ？ + ？ + 5 GAS
Gp140 family
IL-3 (marrow sample)--+-5 GAS (IRF1＞IFP＞＞Ly6)
IL-5 (marrow sample)--+-5 GAS
GM-CSF (marrow sample)--+-5 GAS
Somatotropin family
GH ？ - + - 5
PRL ？ +/- + - 1，3，5
EPO ？ - + - 5 GAS(B-CAS＞IRF1＝IFP＞＞Ly6)
Receptor tyrosine kinase
EGF ？ + + - 1，3 GAS(IRF1)
PDGF ？ + + - 1，3
CSF-1? 1,3 GAS of++-(non-IRF1)
For structure is used for promoter element as the synthetic GAS of containing of biologic test as described in the embodiment 27-29, make up one section GAS-SV40 promoter sequence with the strategy of PCR-based.5 ' primer contains the GAS binding site of 4 tandem copies, and this site is present in the IRF1 promoter, before proves behind many cytokine inductions, can combine (Rothman etc. with STAT, Immunity 1:457-468,1994), can certainly use other GAS or ISRE element.5 ' primer also comprises one section sequence with the complementary 18bp of SV40 early promoter sequence, and flank has an Xho I site.5 ' primer sequence is: 5 '-GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAATGA TTTCCCCGAAATATCTGCCATCTCAATTAG-3 ' (SEQ IDNO:54).
Downstream primer and the complementation of SV40 promoter, flank have Hind III site: 5 '-GCGGCAAGCTTTTTGCAAAGCCTAGGC-3 ' (SEQ ID NO:55).
Use is available from β-gal of Clontech: the SV40 promoter templates that exists in the promoter plasmid carries out pcr amplification.The PCR fragment that obtains digests with Xho I/Hind III, and sub-clone is to BLSK2-(Stratagene).Use forward and reverse primer to check order and verify that insert contains following sequence: 5:
CTCGAGATTFCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAATGATTTCCCCGAA ATATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATC CCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTT TTTFATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGT GAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAA
AAGCTT: 3 ' (SEQ ID NO:56)
With this GAS promoter element that links to each other with the SV40 promoter, report carrier that just can artificial constructed GAS:SEAP2.The reporter molecules here is excretory alkali phosphatase or " SEAP ".Yet, significantly, in present embodiment or any other embodiment, all replaceable SEAP of any reporter molecules.Can be used for replacing the albumen that reporter molecules comprises that chloramphenicol acetyltransferase (CAT), luciferase, alkali phosphatase, beta galactosidase, green fluorescent protein (GFP) or any available antibodies detect of knowing of SEAP.
Above-mentioned sequence proves, synthetic GAS-SV40 promoter element uses Hind III and Xho I sub-clone to the pSEAP-promoter vector available from Clontech, replace the SV40 promoter with the GAS:SV40 promoter element of amplification effectively, obtained the GAS-SEAP carrier.Yet this carrier does not contain neomycin resistance gene, therefore, is not suitable for mammalian expression systems.
Therefore, in order to produce the stable cell line of mammal of expressing the GAS-SEAP reporter molecules, downcut the GAS-SEAP box with Sal I and Not I from the GAS-SEAP carrier, insert the skeleton carrier that contains neomycin resistance gene with the restriction site in the multiple clone site, as pGFP-1 (Clontech), obtain the GAS-SEAP/Neo carrier.This carrier transfection promptly can be used as the bonded reporter molecules of GAS described in embodiment 27-29 behind mammalian cell.
Use said method and replace GAS, can obtain other carrier with different promoter sequences.For example, the structure that contains the reporter molecules of EGR and NF-κ B promoter sequence is described in embodiment 27-31.Yet, use scheme described in these embodiment can replace many other promoteres.For example, can replace SRE, IL-2, NFAT or osteocalcin promoter (as: GAS/NF-κ B/EGR, GAS/NF-κ B, IL-2/NFAT or NF-κ B/GAS) alone or in combination.Similarly, other cell line also can be used to measure the activity of report construct, as HELA (epithelium), HUVEC (endothelium), Reh (B-cell), Saos-2 (osteoblast), HUVAC (aorta) or myocardial cell.
Embodiment 25:SEAP activity test
As the reporter molecules of the test described in this paper disclosed embodiment, the activity of SEAP can use Tropix Phospho-light test kit (catalog number (Cat.No.) BP-400) to measure according to following general approach.The TropixPhospho-light test kit provides dilution, test and the reaction buffer of using below.
2.5 times dilution buffer liquid are injected allotter, to containing the dilution buffer liquid that Optiplate that 35 μ l contain albumin fusion proteins solution of the present invention distributes 2.5 times of 15 μ l.Dull and stereotyped with plastic-enclosed device sealing, 65 ℃ of incubations 30 minutes.Separately each Optiplate is to avoid heating uneven.
Sample was cooled to room temperature 15 minutes.The turned letter allotter, the injection testing buffer.Add 50ml test buffer, room temperature incubation 5 minutes.The turned letter allotter injects reaction buffer (seeing the following form).Add 50 μ l reaction buffers, room temperature incubation 20 minutes.Because the intensity of chemiluminescence signal depends on the time, be with 5 flat boards that read on the luminometer in about 10 minutes, so answer 5 flat boards of single treatment, beginning is second group after 10 minutes.
Read the relative light unit on the luminometer.It is blank setting H12, print result.The activity of chemiluminescent increase order report molecule.
Table 5
| Dull and stereotyped number | Reaction buffer diluent (ml) | CSPD(ml) | Dull and stereotyped number | Reaction buffer diluent (ml) | CSPD(ml) |
| 10 | 60 | 3 | 31 | 165 | 8.25 |
| 11 | 65 | 3.25 | 32 | 170 | 8.5 |
| 12 | 70 | 3.5 | 33 | 175 | 8.75 |
| 13 | 75 | 3.75 | 34 | 180 | 9 |
| 14 | 80 | 4 | 35 | 185 | 9.25 |
| 15 | 85 | 4.25 | 36 | 190 | 9.5 |
| 16 | 90 | 4.5 | 37 | 195 | 9.75 |
| 17 | 95 | 4.75 | 38 | 200 | 10 |
| 18 | 100 | 5 | 39 | 205 | 10.25 |
| 19 | 105 | 5.25 | 40 | 210 | 10.5 |
| 20 | 110 | 5.5 | 41 | 215 | 10.75 |
| 21 | 115 | 5.75 | 42 | 220 | 11 |
| 22 | 120 | 6 | 43 | 225 | 11.25 |
| 23 | 125 | 6.25 | 44 | 230 | 11.5 |
| 24 | 130 | 6.5 | 45 | 235 | 11.75 |
| 25 | 135 | 6.75 | 46 | 240 | 12 |
| 26 | 140 | 7 | 47 | 245 | 12.25 |
| 27 | 145 | 7.25 | 48 | 250 | 12.5 |
| 28 | 150 | 7.5 | 49 | 255 | 12.75 |
| 29 | 155 | 7.75 | 50 | 260 | 13 |
| 30 | 160 | 8 |
Embodiment 26: the neuronal activity qualification test
When cell broke up and breed, one group of gene can be activated by many different signal transduction pathways.One of them gene EGR1 (early growth responsive genes 1) can be induced in various tissues and cell type through activating.The promoter of EGR1 is responsible for this inducing.Use the EGR1 promoter that is connected with reporter molecules, can assess the ability of fusion rotein active cell of the present invention.
Especially, following proposal can be used for assessing the neuronal activity in the PC12 cell line.Known PC12 cell (rat pheochromocyte oncocyte) can breed and/or break up after being activated by many mitogens (such as TPA (myristoyl phorbol acetas), NGF (nerve growth factor) and EGF (epidermal growth factor)).This processing activates the EGR1 expression of gene.Therefore, with the PC12 cell of the construct stable transfection that contains the EGR1 promoter that is connected with the SEAP reporter molecules, can assess the activation of albumin fusion proteins of the present invention to the PC12 cell.
EGR/SEAP report construct can be assembled by following proposal.EGR-1 promoter sequence (633 to+1) (Sakamoto K etc., Oncogene 6:867-871,1991) can use following primer to obtain through pcr amplification from the human genome DNA:
Article one, primer: 5 ' GCGCTCGAGGGATGACAGCGATAGAACCCCGG-3 ' (SEQ ID NO:57)
Second primer: 5 ' GCGAAGCTTCGCGACTCCCCGGATCCGCCTC-3 ' (SEQ ID NO:58)
Use the GAS:SEAP/Neo carrier that produces among the embodiment 24, the EGR1 amplified production can be inserted this carrier.With restriction endonuclease Xho I/Hind III linearisation GAS:SEAP/Neo carrier, cut away the GAS/SV40 implant.With same these enzyme restriction enzyme digestions EGR1 amplified production.Connection carrier and EGR1 promoter then.
96 orifice plates that are used for cell culture for preparation, the dull and stereotyped 2 milliliters of coating buffers (dilution in 1: 30 is dissolved in 30% alcoholic acid type i collagen (Upstate Biotech Inc. catalog number (Cat.No.) 08-115) (filter sterilised)) that add of each 10cm, or the every hole 50ml of 96 orifice plates, air drying 2 hours.
Grow on the 10cm tissue culture dish that wraps quilt in advance that contains RPMI-1640 culture medium (Bio Whittaker) the PC12 cell routine, this culture medium contains 10% horse serum (JRH BIOSCIENCES, catalog number (Cat.No.) 12449-78P) and 5% heat inactivation hyclone (FBS), be added with the penicillin of 100 unit/ml and the streptomycin of 100 μ g/ml simultaneously.Finished 1-4 partition in every 3-4 days.Scrape off and remove cell from flat board, by drawing above 15 re-suspended cells up and down.
Use technology known in the art with the transfection of EGR/SEAP/Neo construct in the PC12 cell.By cell is grown among the G418 of 300 μ g/ml, obtain the stable cell of EGR/SEAP/PC12.The culture medium that does not contain G418 is used for conventional growth, but every 1-2 month, cell should regrow and go down to posterity several times in the G418 of 300 μ g/ml.
For carrying out neuronal activity test, screen and contain about 70% to the 80% 10cm flat board that converges cell by removing old culture medium.With PBS (phosphate buffered saline (PBS)) rinsing cell once.Making cell be in starvation on low blood serum medium (containing 1% horse serum, 0.5%FBS and antibiotic RPMI-1640) then spends the night.
In morning next day, remove culture medium, with PBS rinsing cell.Scrape off cell from flat board, with 2 milliliters low blood serum medium suspension cells.The counting cells number adds how low blood serum medium so that final cell density reaches 5 * 10
5Individual cells/ml.
Add 200 μ l cell suspending liquids and in each hole of 96 orifice plates, (be equal to 1 * 10
5Individual cells/well).The albumin fusion proteins of the present invention that adds a series of variable concentrations, 37 ℃ of incubations 48 to 72 hours.The available known somatomedin that can activate the PC12 cell by EGR is as positive control, such as the neure growth factor (NGF) of 50ng/ μ l.Usually observing the SEAP that surpasses 50 times in the positive control hole induces.SEAP test can be used technology known in the art routinely and/or carry out as technology as described in the embodiment 25.
The test of embodiment 27:T cytoactive
Following proposal is used to assess the T cytoactive, is tested and appraised the factor and determines the T cell can be bred and/or break up to albumin fusion proteins of the present invention whether.Can use the GAS/SEAP/Neo construct that produces among the embodiment 24 to assess the T cytoactive.Therefore, improve the active factor table of SEAP and understand that it activates the ability of Jak-STAT signal transduction pathway.This tests employed T cell is JurkatT cell (ATCC searching number TIB-152), although also can use Molt-3 cell (ATCC searching number CRL-1552) and Molt-4 cell (ATCC searching number CRL-1582).
Jurkat T cell is a lymphoblast CD4+Th1 accessory cell.In order to produce stable cell line, use DMRIE-C (Life Technologies), general 200 ten thousand Jurkat cells are with the transfection of GAS-SEAP/Neo carrier (the transfection scheme is as described below).Transfectional cell selects the 1mg/ml gentamycin is had the transfectant of resistance with the density inoculation of 20,000 cells in about every hole.With the amplification of resistance bacterium colony, measure it to increasing the response of concentration interferon gamma.The dose response of having showed a selected clone.
Especially, following proposal will produce enough cells, be used for every hole, 75 holes 200 μ l cells.Therefore, perhaps scale is enlarged, perhaps repeatedly repeat to produce the cell that enough is used for a plurality of 96 orifice plates.The Jurkat cell is maintained in the RPMI+10% serum that contains 1% penicillin-streptomycin.In the T25 bottle, mix the plasmid DNA of 2.5 milliliters of OPTI-MEM (Life Technologies) and 10 μ g.Add 2.5 milliliters of OPTI-MEM that contain 50 μ l DMRIE-C, room temperature incubation 15-45 minute.
Between incubation period, counting cells concentration centrifugally obtains (the each transfection 10 of requisite number purpose cell
7Individual cell), be resuspended to final concentration 10 with OPTI-MEM
7Individual cells/ml.Add 1 * 10 among 1 milliliter of OPTI-MEM then
7Individual cell in the T25 bottle, 37 ℃ of incubations 6 hours.Behind the incubation, add 10 milliliters of RPMI+15% serum.
The report cell line that Jurkat:GAS-SEAP is stable maintains in the RPMI+10% serum that contains 1mg/ml gentamycin and 1% penicillin-streptomycin.These cells are handled with one or more fusion rotein of the present invention of variable concentrations.
Fusion rotein is handled the same day, and cell is used fresh RPMI+10% serum rinsing and is resuspended to the density of every milliliter of 500,000 cells.The exact number of required cell will depend on the number of the variable concentrations fusion rotein of the number of fusion rotein and screening.Concerning 1 96 orifice plate, approximately need 1,000 ten thousand cells (10 plates, 100,000,000 cells).
The porose disc that contains the Jurkat cell of handling with fusion rotein places 48 hours (note: this time is variable, between 48-72 hour) in the incubation case.Use 12 passage pipettors with every hole 35 μ l sample transfer to opaque 96 orifice plate then.Opaque flat board should be covered (using the sellophene covering), is stored in-20 ℃, until carrying out the 25 described SEAP tests as embodiment.The flat board that contains residue processing cell places 4 ℃, is used as the material source of particular bore repeated trials when needing.
The interferon gamma of available known 100 units per ml that activate Jurkat T cell is as positive control.Generally observedly in the positive control hole surpass 30 times induce.
It is apparent to those skilled in the art that such scheme can be used for making up instantaneous and stable transfectional cell.
The test of embodiment 28:T cytoactive
NF-κ B (nuclear factor κ B) be by be exposed to LPS or thrombin, by the expression of some viral gene product with by the activated transcription factor of many stimulus object, these stimulus object comprise inflammatory cytokine IL-1 and TNF, CD30 and CD40, lymphotoxin-α and lymphotoxin-β.As transcription factor, NF-κ B regulates many expression of gene, and these genes participate in immunocyte activation, control apoptosis (NF-κ B can protect cell to avoid apoptosis), B and T cell development, antiviral and antimicrobial and reply and multiple stress.
Under the non-incentive condition, NF-κ B and I-κ B (inhibitor κ B) remain in the Cytoplasm.Yet after the stimulation, I-κ B is caused NF-κ B to shuttle back and forth in nucleus by phosphorylation and degraded, has therefore activated transcribing of target gene.The activated target gene of NF-κ B comprises IL-2, IL-6, GM-CSF, ICAM-1 and 1 class MHC.
Because central role of NF-κ B and in response to the ability of multiple stimulation, we use the report construct with NF-κ B promoter element structure to screen fusion rotein.The activator of NF-κ B or inhibitor can be used to treatment, prevent and/or diagnose the illness.For example, the inhibitor of NF-κ B can be used for treating and the relevant disease of acute or chronic NF-κ B activation, as rheumatoid arthritis.
In order to make up the carrier that contains NF-κ B promoter element, we use the strategy of PCR-based.Forward primer contain 4 tandem copies NF-κ B binding site (GGGGACTTTCCC) (SEQ IDNO:59), with the sequence of the terminal complementary one section 18bp of SV40 early promoter sequence 5 ', flank has Xho I site:
5’：GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTTCCATCCTGCCATCTCAATTAG：3’(SEQ?ID?NO：60)
Downstream primer and SV40 promoter 3 ' are terminal complementary, and flank has a Hind III site:
5’：GCGGCAAGCTTTTTGCAAAGCCTAGGC：3’(SEQ?ID?NO：55)
With being present in the pB-gal that obtains from Clontech: the SV40 promoter templates of promoter plasmid carries out pcr amplification.The PCR fragment that obtains digests with Xho I and Hind III, and sub-clone is to BLSK2-(Stratagene) then.Order-checking with T7 and T3 primer confirms that insert contains following sequence: 5 ': CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTTCCATCTGCC ATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTA ACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTA TGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCT TTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTT:3 ' (SEQ ID NO:61)
Next, use Xho I and Hind III, replace the SV40 minimal promoter element that is present in pSEAP2-promoter plasmid (Clontech) with this NF-κ B/SV40 fragment.Yet this carrier does not contain neomycin resistance gene, therefore, is not suitable for mammalian expression systems.
In order to produce stable mammal cell line, cut away NF-κ B/SV40/SEAP box with restriction endonuclease Sal I and Not I from above-mentioned NF-κ B/SEAP carrier, be inserted into the carrier that contains neomycin resistance.Especially, behind Sal I and Not I restriction enzyme digestion pGFP-1, NF-κ B/SV40/SEAP box is inserted pGFP-1 (Clontech), replace the GFP gene.
After having produced NF-κ B/SV40/SEAP/Neo carrier, just can produce and keep stable Jurkat T cell according to the scheme described in the embodiment 25.Similarly, the method that is used to detect fusion rotein and these stable Jurkat T cells is also as described in the embodiment 25.As positive control, external source TNF α (0.1,1,10ng) is added hand-hole H9, H10 and H11, generally observed 5-10 activation doubly.
Embodiment 29: identify the activity test of marrow sample
By determining fusion rotein and whether breed and/or breaking up myeloid cell, assess the marrow sample activity of albumin fusion proteins of the present invention with following proposal.Use the GAS/SEAP/Neo construct that produces among the embodiment 24 to assess the myeloid cell activity.Therefore, improve the active factor table of SEAP and understand that it activates the ability of Jak-STAT signal transduction pathway.The myeloid cell that uses in this test is U937 (a kind of premonocyte system), although also can use TF-1, HL60 or KG1.
For GAS/SEAP/Neo construct transient transfection U937 cell, use DEAE-dextran method (Kharbanda etc., 1994, Cell Growth ﹠amp to produce among the embodiment 24; Differentiation, 5:259-265).At first, collect 2 * 10
7Individual U937 cell, and use the PBS rinsing.The U937 cell grows in usually and contains 10% heat inactivation hyclone (FBS) and be added with in the RPMI-1640 culture medium of 100 units/ml penicillin and 100mg/ml streptomycin.
Then, with Tris-HCl (pH 7.4) the buffer suspension cell of 1ml 20mM, contain DEAE-dextran, 8 μ g GAS-SEAP2 plasmid DNA, 140mMNaCl, 5mM KCl, the 375 μ M Na of 0.5mg/ml in this buffer
2HPO
47H
2O, 1mM MgCl
2With 675 μ M CaCl
2In 37 ℃ of incubations 45 minutes.
RPMI-1640 culture medium rinsing cell with containing 10%FBS is resuspended in cell 10 milliliters of complete mediums then, 37 ℃ of incubations 36 hours.
By cell is grown among the G418 of 400 μ g/ml, can obtain the stable cell of GAS-SEAP/U937.The culture medium that does not contain G418 is used for conventional growth, but every 1-2 month, cell should regrow and go down to posterity several times in the G418 of 400 μ g/ml.
Collect 1 * 10
8Individual cell (these cells enough are used for the test of 10 96 orifice plates) is used for measuring, and uses the PBS rinsing.In 200 milliliters of above-mentioned growth mediums, making whole density is 5 * 10 with cell suspension
5Individual cells/ml.The every hole of 96 orifice plates adds 200 μ l cells (or 1 * 10
5Individual cells/well).
The fusion rotein that adds variable concentrations.37 ℃ of incubations 48 to 72 hours.The interferon gamma of 100 units per ml of available known activation U937 cell is as positive control.Generally can be observed in the positive control hole and surpass 30 times induce.Carry out the SEAP test of supernatant according to the scheme described in means known in the art and/or the embodiment 25.
Embodiment 30: identify the test that micromolecule concentration and membrane permeability change
Known ligand and receptors bind can change level and the pH and the membrane potential of micromolecule (as calcium, potassium, sodium) in the born of the same parents.These changes can be tested and appraised with the test of the fusion rotein of specific cells receptors bind and measure.Although following proposal has been described the test that is used for calcium, be used to detect potassium, sodium, pH, membrane potential or any micromolecular change that other can detect by fluorescent probe can easily change this scheme.
Following test is used fluorescence imaging to read plate instrument (" FLIPR ") and is measured change with the bonded fluorescence molecule of micromolecule (Molecular Probes).Significantly, except calcium fluorescent molecule fluo-4 (MolecularProbes, Inc.; Catalog number (Cat.No.) F-14202) outside, can use any micromolecular fluorescence molecule that detects.
For sticking together cell, with cell with 10,000-20,000 cells/well is inoculated on Co-star black 96 orifice plates with clear bottom.Dull and stereotyped at CO
2Incubation is 20 hours in the incubation case.Wash in the plate instrument at Biotek and to stick together cell 2 times, keep 100 μ l buffer after the last rinsing with 200 μ l HBSS (HankShi balanced salt solution) rinsings.
The fluo-4 mother solution for preparing 1mg/ml with 10%pluronic acid DMSO.For making cell be loaded with fluo-4, every hole adds the fluo-4 of 50 μ l, 12 μ g/ml.With flat board in 37 ℃ at CO
2Incubation is 60 minutes in the incubation case.Wash in the plate instrument with dull and stereotyped 4 times of HBSS rinsing at Biotek, keep 100 μ l buffer.
For the non-cell that sticks together, get off cell is centrifugal from culture medium.In 50 milliliters of conical pipes, use the HBSS re-suspended cell to 2-5 * 10
6Individual cells/ml.Every ml cells float adds the fluo-4 solution that 4 μ l are dissolved in the 1mg/ml of 10%pluronic acid DMSO.Then pipe was placed 37 ℃ of water-bath 30-60 minutes.Cell is resuspended to 1 * 10 with HBSS rinsing 2 times
6Individual cells/ml installs to microplate with 100 μ l/ hole branches.With plate centrifugal 5 minutes with 1000rpm.Use 200 μ l Denley cell washing liquid rinsing plates 1 time then, with after a step be pumped to final volume 100 μ l.
To based on acellular test, every hole contains a kind of fluorescence molecule, as fluo-4.Fusion rotein of the present invention is added in the hole, detects the change of fluorescence.
For measuring the fluorescence of cellular calcium, FLIPR is set at following parameter: (1) system gain is 300-800mW; (2) time of exposure is 0.4 second; (3) camera F/ terminates as F/2; (4) excite and be 488nm; (5) be emitted as 530nm; (6) sample is incorporated as 50 μ l.The enhanced emission of 530nm shows by albumin fusion proteins of the present invention or by the caused extracellular signal conduction of the inductive molecule of albumin fusion proteins of the present invention incident, this has caused Ca in the born of the same parents
++The increase of concentration.
Embodiment 31: identify the test of tyrosine kinase activity
Protein tyrosine kinase (PTK) is represented one group of various film and kytoplasm kinases of striding.The receptor of the many mitogenesis and the metabolism growth factor is arranged in receptor protein tyrosine kinase (RPTK) group, comprise PDGF, FGF, EGF, NGF, HGF and Insulin receptor INSR subfamily.In addition, the big RPTK family that also has a corresponding part the unknown.The part of RPTK mainly comprises the secreted small protein, also comprises film combination and extracellular matrix protein.
The process that part activates RPTK comprises ligand-mediated receptor dimerizationization, causes the activation of the transphosphorylation and the cytoplasmic tyrosine kinase of receptor subunit.Cytoplasmic tyrosine kinase comprises the relevant tyrosine kinase (as src, yes, lck, lyn, fyn) of the receptor of src family and non-receptor coupling and cytosol protein tyrosine kinase, as Jak family, the signal transduction that Jak family member mediation is triggered by cytokine receptor superfamily (as interleukin, interferon, GM-CSF and leptin (Leptin)).
Because many known factors can stimulate the activity of tyrosine kinase, identify that whether albumin fusion proteins of the present invention or the inductive molecule of fusion rotein of the present invention can activate tyrosine kinase signal transduction pathway is interested.Therefore, the design following proposal is used to identify this quasi-molecule that can activate tyrosine kinase signal transduction pathway.
With the density of 25,000 cells in every approximately hole target cell (as former generation keratinocyte) is inoculated in purchase from Nalge Nunc (Naperville, 96 hole Loprodyne Silent screen plates IL).Dull and stereotyped with 100% ethanol rinsing 2 times, each 30 minutes and sterilize, use water rinse then, dried overnight.(mentioned reagent is all available from Sigma Chemicals (St.Louis for some dull and stereotyped type i collagen (50mg/ml), gelatin (2%) or polylysines (50mg/ml) of cultivating grade with 100 ml cells, MO)) or 10% Matrigel (available from Becton Dickinson, Bedford, MA) or the calf serum bag by 2 hours, use the PBS rinsing, be stored in 4 ℃.By with every hole 5,000 cell to growth medium, after 48 hours, uses these dull and stereotyped cell inoculations of going up growth as supplier Alamar Biosciences, Inc. (Sacramento, CA) described alamarBlue pair cell number carries out indirect quantitative analysis.Use (Bedford, the dull and stereotyped covering of Falcon MA) (catalog number (Cat.No.) 3071) covering Loprodyne Silent screen plate available from Becton Dickinson.Also can use Falcon trace detection III type Tissue Culture Plate in some proliferation experiment.
Be the preparation extract, with A431 cell inoculation (20,000 cell/200 milliliter/hole) to the nylon membrane of Loprodyne flat board, overnight incubation in complete medium.Cell incubation on the basal medium that does not contain serum was made the cell dormancy in 24 hours.After handling 5-20 minute with EGF (60ng/ml) or variable concentrations albumin fusion proteins of the present invention, discard culture medium, every hole adds 100 milliliters and extracts buffer (20mM HEPES pH7.5,0.15M NaCl, 1%Triton X-100,0.1%SDS, 2mMNa
3VO
4, 2mM Na
4P
2O
7With available from Boeheringer Mannheim (Indianapolis, protease inhibitor cocktail IN) (catalog number (Cat.No.) 1836170)), flat board places on the gyratory shaker 4 ℃ to shake 5 minutes.Then flat board is placed in the vacuum transfer pipe, make extract pass through the bottom of the membrane filtration of 0.45mm to each hole with the domestic depurator.Collect the extract in vacuum tube bottom 96 hole accepter/bread boards, place on ice rapidly.Be to obtain the extract of centrifugal clarification, the detergent dissolving was taken out the inclusions in every hole after 5 minutes, centrifugal 15 minutes of 4 ℃ of 16,000 * g.
Measure the tyrosine kinase activity level of filtering the back extract.Although the method for known many detection tyrosine kinase activities, this paper describes a kind of method.
Usually, the tyrosine kinase activity of albumin fusion proteins of the present invention is to estimate by the ability that definite its phosphorylation specific substrates (biotinylated peptide) goes up tyrosine residue.The biotinylated peptide that can be used as this purpose comprises PSK1 (corresponding to the aminoacid 6-20 of cell division kinases cdc2-p34) and PSK2 (corresponding to the amino acid/11-17 of gastrin).These two kinds of substrates that peptide all is many tyrosine kinase can have been bought from Boeheringer Mannheim.
The tyrosine-kinase enzyme reaction adds following component by order and sets up.At first, add the biotinylated peptide of 10 μ l, 5 μ M, add 10 μ l ATP/Mg then
2+(5mM ATP/50mM MgCl
2), add 10 μ l 5 * test buffer (40mM salt imidazole acid, pH 7.3,40mM β-phosphoglycerol, 1mM EGTA, 100mM MgCl then
2, 5mM MnCl
2, 0.5mg/ml BSA), add 5 μ l vanadic acid sodiums (1mM) then, add 5 μ l water again.Gently mix each component, with reactant mixture in 30 ℃ of precincubation 2 minutes.Add 10 μ l control enzyme or filter after the supernatant initial action.
Add 10 μ l 120mM EDTA and stop tyrosine-kinase enzyme test reaction, then reaction is placed on ice.
The mensuration of tyrosine kinase activity: 50 μ l sample aliquot of reactant mixture are transferred on microtitration plate (MTP) module 37 ℃ of incubations 20 minutes.This makes 96 orifice plates of the plain bag of strepto-affinity quilt combine with biotinylated peptide.With the PBS rinsing MTP module in 300 μ l/ holes 4 times.Every then hole adds the anti-phosphotyrosine antibody (anti-P-Tyr-POD (0.5 units per ml)) of 75 μ l and horseradish peroxidase, 37 ℃ of incubations 1 hour.Rinsing hole as stated above.
Add 100 μ l peroxidase substrate solution (Boeheringer Mannheim) then, room temperature incubation at least 5 minutes (maximum 30 minutes).Read the absorbance that the plate instrument is measured 405nm place sample with ELISA.It is quantitative that bonded Peroxidase activity level is read the plate instrument with ELISA, and this level has reflected the activity level of tyrosine kinase.
Embodiment 32: identify the phosphorylation activity test
As substituting and/or replenish as the potential of embodiment 31 described protein tyrosine kinase activity tests, the main intracellular signal transduction intermediate of also available detection activates the test of (phosphorylation).For example, a specific test as described below can detect Erk-1 and the kinase whose tyrosine phosphorylation of Erk-2.Yet, other molecule, as Raf, JNK, the kinase whose kinases of p38MAP, Map (MEK), MEK kinases, Src, muscle specific kinases (MuSK), IRAK, Tec and Janus and any other phosphorylation of phosphoserine, phosphotyrosine or phosphothreonine molecule, also can detect by replace Erk-1 or Erk-2 in the following test with these molecules.
Particularly, by being prepared test panel by the hole of 96 hole elisa plates with 2 hours bags of 0.1 milliliter of Protein G (1ug/ml) room temperature.Use PBS rinsing flat board then, the BSA/PBS room temperature sealing with 3% 1 hour.Use 2 kinds of business-like monoclonal antibodies (100ng/ hole) (Santa Cruz Biotechnology) to handle Protein G flat board (room temperature 1 hour) then at Erk-1 and Erk-2.(for detecting other molecule, this step can easily change by replacing to the monoclonal antibody that can detect any above-mentioned molecule.) with after PBS rinsing 3-5 time, with flat board be stored in 4 ℃ stand-by.
The A431 cell is inoculated on the 96 hole Loprodyne screen plates with 20,000 cells/well, incubated overnight on growth medium.Then cell is placed basal medium (DMEM) to make its hungry 48 hours, the fusion rotein of the present invention of reuse EGF (6ng/ hole) or variable concentrations was handled 5-20 minute.Dissolved cell then, with the extracting solution Direct Filtration in test panel.
With extract room temperature incubation after 1 hour, rinsing hole once more.As positive control, substitute the A431 extract with business-like map kinase preparation (10ng/ hole).Dull and stereotyped then commercialization polyclone (rabbit) antibody (1 μ g/ml) with specific recognition Erk-1 and Erk-2 tyrosine phosphorylation epi-position is handled dull and stereotyped (room temperature 1 hour).This antibody has been used the standard scheme biotinylation.Incubation in Wallac DELFIA instrument comes quantitative bonded polyclonal antibody (time-resolved fluorescence) by continuous and europium-strepto-affinity element and europium fluorescence-enhancing agent then.The enhanced fluorescence signal that is higher than background shows the phosphorylation that fusion rotein of the present invention or the inductive molecule of albumin fusion proteins of the present invention cause.
Embodiment 33: the phosphorylation test
For detecting the phosphorylation activity of albumin fusion proteins of the present invention, use as United States Patent (USP) the 5th, 958 No. 405 described phosphorylation tests (taking in this paper by reference).Briefly, can use gamma marker
32P-ATP phosphorylated protein substrate, the radioactivity of mixing with the quantitative analysis of gamma activity isotope enumerator is measured phosphorylation activity then.With fusion rotein of the present invention and protein substrate,
32P-ATP and the common incubation of kinase buffer liquid.Then by electrophoresis with free
32P-ATP with mix substrate
32P separates, and counting mixes
32P, and with negative control relatively.The radiocounting that is higher than negative control has shown the phosphorylation activity of fusion rotein.
Embodiment 34: the phosphorylation that detects albumin fusion proteins of the present invention when polypeptide ligand exists is lived
Property (activation)
Means known in the art or methods described herein can be used for determining the phosphorylation activity of albumin fusion proteins of the present invention.In one embodiment, the method for determining phosphorylation activity is by using as United States Patent (USP) the 5th, 817 No. 471 described tyrosine phosphorylation tests (taking in this paper by reference).
Embodiment 35: stimulate bone marrow CD34+ cell proliferation test
The multiplication capacity of human CD34+ when this test is based on hemopoietic growth factor and exists is estimated the ability that fusion rotein of the present invention stimulates the CD34+ cell proliferation.
Previous studies show that most ripe precursors only respond single signal.The response of how jejune precursor needs at least 2 kinds of signals.Therefore, for measuring fusion rotein of the present invention to the active influence of many CFU-GM hemopoietic, when hemopoietic growth factor existed or do not exist, this test comprised a kind of given fusion rotein of the present invention.At stem cell factor (SCF) when existing, with isolated cells and specimen co-cultivation 5 days.Independent SCF is very limited to the influence of bone marrow (BM) cell proliferation, works in following conduct of this condition " survival " factor.Yet in case combined with any factor (as IL-3) that these cells is shown as stimulating effect, SCF will cause cooperative effect.Therefore, if the fusion rotein of test has stimulus effects to hemopoietic progenitor cell, this activity can easily detect.Because normal BM cell contains low-level circulating cells, therefore, may not detect any depression effect of given fusion rotein.Correspondingly, the test of CFU-GM depression effect can be formerly detected this inductive inhibition of proliferation in the cell that external SCF+IL+3 stimulates then with the chemical compound of being estimated contacts.
Briefly, with means known in the art separation of C D34+ cell.After melting cell, be resuspended in (QBSF 60 does not contain the culture medium of serum, contains 1% L-glutaminate (500 milliliters) QualityBiological, Inc., Gaithersburg, MD, catalog number (Cat.No.) 160-204-101) in the culture medium.After the soft centrifugation step of 200 * g several times, cell was had a rest 1 hour.Cell counting is adjusted into 2.5 * 10
5Individual cells/ml.During this time, add 100 μ l sterilized water in 96 orifice plates hole on every side.The cytokine that can measure with albumin fusion proteins of the present invention in this test is the rhSCF (R﹠amp of independent 50ng/ml; D Systems, Minneapolis, MN, catalog number (Cat.No.) 255-SC) and rhSCF and the rhIL-3 (R﹠amp of 30ng/ml; DSystems, Minneapolis, MN, catalog number (Cat.No.) 203-ML) combination.After 1 hour, cytokine, the albumin fusion proteins of the present invention of variable concentrations and the cell of 20 μ l dilution that 10 μ l are prepared are added in the culture medium that is present in the hole, make final cumulative volume reach 100 μ l.Then flat board is placed 37 ℃/5%CO
2Incubation case 5 days.
Test preceding 18 hours collecting cells, in 10 μ l volumes, every hole adds [3H] thymidine in 0.5 μ Ci/ hole and determines multiplication rate.Stop experiment from each 96 orifice plate collecting cell to bag filter (filtermat) with Tomtec Harvester 96.After the collection,, and place that dull and stereotyped and OmniFilter coils in the OmniFilter assembly of forming by OmniFilter with bag filter drying, pruning (trim).Every hole adds the Microscint of 60 μ l, presses pressure seal mouth membrane closure flat board with TopSeal-A.With bar code 15 labelings be attached to be used on first flat board the counting.The flat board that will seal then loads, and determines radioactive level and print to analyze the data of collecting by Packard Top enumerator.Radioactive level has reflected the proliferative amount of cell.
The activity that given fusion rotein stimulates bone marrow CD34+ cell proliferation has been measured in the described research of present embodiment.The activity of fusion rotein of the present invention and polynucleotide (as gene therapy) and agonist and antagonist is measured in the research that those skilled in the art can easily change institute's example.Albumin fusion proteins of the present invention stimulates the ability of bone marrow CD34+ cell proliferation to show albumin fusion proteins and/or can be used for influencing the diagnosis and the treatment of the disease that immune system and hemocyte take place corresponding to the polynucleotide of fusion rotein.Typical application as above-mentioned " immunocompetence " and " infectious disease " part and other place of this paper as described in.
Embodiment 36: extracellular matrix strengthens cellular response (EMECR) test
The purpose that extracellular matrix strengthens cellular response (EMECR) test is to estimate in the background of the inductive signal of extracellular matrix (ECM), and fusion rotein of the present invention acts on the ability of hematopoietic stem cell.
Cell is made response to regulatory factor in the background of the signal that receives from peripheral microenvironment.For example, fibroblast and endothelium and epithelial stem cell reproducible not when the signal that lacks from ECM.Hematopoietic stem cell can carry out self renewal in bone marrow, but can not in external suspension culture.Stem cell depends on the interaction of they and stromal cell and ECM albumen fibronectin (fn) in external ability of carrying out self renewal.Cell is to be subjected to α to sticking together of fn
5. β
1And α
4. β
1The integrin receptor mediation, these receptors are expressed by human and mouse hematopoietic stem cell.Integrate and be responsible for to stimulate the factor of stem cell self renewal also not identify with the ECM environment.The discovery of this factor should be of great use in the application of gene therapy and bone marrow transplantation.
Briefly, 96 orifice plates of polystyrene, the processing of non-tissue culture are with 0.2 μ g/cm
2Bag by concentration with fn fragment bag quilt.Bone marrow cells in mice is layered on 0.2 milliliter of culture medium that does not contain serum (1,000 cells/well).Cultured cells is as positive control under the condition that IL-3 (5ng/ml)+SCF (50ng/ml) exists, and under this condition, estimates seldom self renewal but tangible differentiation is arranged of stem cell.When existing and lack SCF (5.0ng/ml), measure albumin fusion proteins of the present invention with suitable negative control, the administration volume that contains the compositions of albumin fusion proteins of the present invention has herein been represented 10% of overall test volume.With the cell incubation on the flat board at low-oxygen environment (5%CO
2, 7%O
2And 88%N
2) tissue culture's incubation case in its growth of 7 angels.The number of hole internal breeding cell then comes quantitatively by the thymidine that mensuration is mixed cell DNA.Need the phenotype of cell to characterize to the positive checking of hitting of this test, this can realize by the scale and the suitable antibody reagent and the FACScan at cell surface antigen of use of amplification culture system.
If find that a kind of specific fusion rotein of the present invention is the stimulus object of hemopoietic progenitor cell, fusion rotein and can be used for such as the diagnosis and the treatment that influence the disease that immune system and hemocyte take place so corresponding to the polynucleotide of fusion rotein.Typical application as above-mentioned " immunocompetence " and " infectious disease " part and other place of this paper as described in.Fusion rotein also can be used for the propagation of stem cell and various blood lineage committed CFU-GM and the differentiation and/or the propagation of various cell types.
In addition, the polynucleotide of albumin fusion proteins of the present invention and code book invention albumin fusion proteins also can be used for suppressing the propagation and the differentiation of hematopoietic cell, therefore can be used for protecting bone marrow stem cell to avoid the injury of chemotherapeutics in the chemotherapy process.This anti-proliferative effect can make more that the chemotherapeutics administration of high dose becomes possibility, therefore can more effectively carry out chemotherapeutic treatment.
In addition, because stromal cell plays a significant role in the generation of hematopoietic lineage cell, the polynucleotide of fusion rotein of the present invention and code book invention albumin fusion proteins also can be used for the treatment and the diagnosis of hemopoietic associated conditions (as anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia).Its application comprises the earlier external back of medullary cell culturing in vivo, bone marrow transplantation, marrow-reconstitution, neoplastic radiotherapy or chemotherapy.
Embodiment 37: the propagation of human dermal fibroblast and aortic smooth muscle cell
Albumin fusion proteins of the present invention is added in normal human subject dermal fibroblast (NHDF) and human aortic smooth muscle cell (AoSMC) culture, and each sample carries out 2 tests simultaneously.First test detects the influence of fusion rotein to the propagation of normal human subject dermal fibroblast (NHDF) or aortic smooth muscle cell (AoSMC).The misgrowth of fibroblast or smooth muscle cell is several pathological processes parts of (comprising fibrosis and restenosis).Second test detects the generation of the IL6 that NHDF and SMC cause.The generation presentation function of IL6 activates.The many cytokines of activated cell and the generation of other factor increase, and can cause short inflammation or immunoregulatory result.For detecting common stimulation or suppressing active, test is carried out under the condition that has and do not have TNF α to stimulate altogether.
Briefly, the 1st day, in 100 μ l culture medium, set up 96 hole black plates with 1000 cells/well (NHDF) or 2000 cells/well (AoSMC).The NHDF culture medium contains: the FB basal medium of Clonetics, 1mg/ml hFGF, 5mg/ml insulin, 50mg/ml gentamycin, 2%FBS; And the AoSMC culture medium contains: the SM basal medium of Clonetics, 0.5 μ g/mlhEGF, 5mg/ml insulin, 1 μ g/ml hFGF, 50mg/ml gentamycin, 50 μ g/ml amphotericin Bs, 5%FBS.37 ℃ of incubations after 4-5 hour, with the culture medium suction, replace to the growth retardation culture medium at least.The growth retardation culture medium of NHDF contains: fibroblast basal medium, 50mg/ml gentamycin, 2%FBS; And the growth retardation culture medium of AoSMC contains SM basal medium, 50mg/ml gentamycin, 50 μ g/ml amphotericin Bs, 0.4%FBS.37 ℃ of incubations to the 2 days.
The 2nd day, the serial dilution thing and the template of design albumin fusion proteins of the present invention made it always comprise culture medium contrast and known protein contrast.To stimulating and suppressing two kinds of experiments, with growth retardation culture medium diluted protein.To suppressing experiment, it is 2ng/ml (NHDF) or 5ng/ml (AoSMC) that TNF α is added to final concentration.The culture medium that contains contrast or albumin fusion proteins of the present invention that adds 1/3 volume, 37 ℃/5%CO
2Incubation to the 5 days.
The 5th day, wrap the ELISA that is carried out IL6 by 96 orifice plates with the anti-human IL6 monoclonal antibody in 50-100 μ l/ hole (with the PBS dilution of pH 7.4), room temperature is incubated overnight.
The 6th day, with the flat board turned letter, trace was to napkin in tank.Preparation contains the test buffer of PBS and 4%BSA.The Pierce Super Block sealing buffer that is dissolved in PBS with 200 μ l/ holes sealed dull and stereotyped 1-2 hour, used rinsing buffer (PBS, 0.05% tween 20) rinsing flat board then.To napkin, the biotin labeled antibody of monoclonal that adds the anti-human IL-6 that dilutes in 50 μ l/ holes then is to 0.50mg/ml with dull and stereotyped trace.The diluent of preparation IL-6 mother solution in culture medium (30,10,3,1,0.3,0ng/ml).First row at flat board adds two multiple samples.Cover flat board, incubation is 2 hours on the room temperature shaking table.
With rinsing buffer rinsing flat board, trace is to napkin.With the strepto-affinity element of test buffer, add 100 μ l/ holes with 1: 1000 dilution EU labelling.Cover flat board, room temperature incubation 1 hour.Reuse rinsing buffer rinsing flat board, trace is to napkin.
The enhancing solution that adds 100 μ l/ holes.Shook 5 minutes.Flat board is placed reading in the Wallac DELFIA exometer.The reading tabulation of each test from triple duplicate sample product shown, and average.
The positive findings of this test has illustrated the propagation of AoSMC cell, has illustrated that also albumin fusion proteins can participate in the propagation of dermal fibroblast and/or the propagation of smooth muscle cell.Positive findings also illustrates the many potential application of the polynucleotide of fusion rotein and coding albumin fusion proteins.For example, take place as inflammation and immunoreation, the healing of wound, the blood vessel that is described in detail in this description.Especially, fusion rotein can be used for the healing and the corium to regenerate of wound and promotes blood vessel and the generation of lymphoid blood vessel.Vessel growth can be used for for example treatment of cardiovascular disease.In addition, the fusion rotein that shows antagonistic activity in this test can be used as anti-angiogenic dose (as angiogenesis inhibitor) and is used for the treatment of and involves disease, disease and/or the illness that blood vessel takes place.These diseases, disease and/or illness are known in the art and/or described herein, such as for example malignant tumor, solid tumor, benign tumor, as hemangioma, acoustic neuroma, neurofibroma, trachoma and purulent granuloma; Atherosclerosis (artheroscleric) speckle; Eye angiogenesis disease is as the uveitis and the pterygium (abnormal vascular growth) of diabetic renal papillary necrosis, retinopathy of prematurity, degeneration of macula, corneal graft rejection, neovascular glaucoma, Terry's sign disease, rubescent, retinoblastoma, eye; Rheumatoid arthritis; Psoriasis; The wound healing that postpones; Endometritis; Angiogenesis; Granulation forms; Loose bud trace (keloid); Associativity is not fractured; Scleroderma; Trachoma; Blood vessel sticks together; Myocardial vascular takes place; Crown pleurapophysis; The brain pleurapophysis; Arteriovenous malformotion; Ischemic limb angiogenesis takes place; Ao-Wei Er Shi (Osler-Webber) syndrome; Plaque neovessels forms; Telangiectasis; Bleeder's joint; Fibrohemangioma; Fibrillar muscle abnormal development; The wound granulation forms; Crohn disease and atherosclerosis.In addition, the albumin fusion proteins as antagonist can be used for treating anti-hyperproliferation disease known in the art and/or as herein described and/or anti-inflammatory disease in this test.
Embodiment 38: the expression of cell adhesion molecule (CAM) on endotheliocyte
Raise lymphocyte and comprise the interaction of the special receptor-ligand between the cell surface adhesion molecule (CAM) on lymphocyte and the blood vessel endothelium to inflammation and blood vessel generation area.Under the normal and pathological state, this process of sticking together is followed a cascade that multistep is rapid, comprises cell-cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and endothelial leucocyte adhesion molecule-1 (E-the selects albumen) expression on endotheliocyte (EC).These molecules and other molecule are in the expression on the blood vessel endothelium has determined generating process in inflammatory reaction, and leukocyte adheres to the local vascular structure and extravasates into the efficient of local organization.The Local enrichment of cytokine and somatomedin has participated in regulation and control that these content-addressable memorys are reached.
Briefly, endotheliocyte (as human umbilical vein endothelial cell (HUVEC)) is grown on 96 orifice plates of standard it is converged, and removes the growth medium of cell, replaces to 199 culture medium (10% hyclone (FBS)) of 100 μ l.Sample that will be used to test (containing albumin fusion proteins of the present invention) and positive or negative contrast three repeatedly are added to dull and stereotyped interior (10 μ l volume).Flat board was placed 37 ℃ of incubations then 5 hours (select albumen and integrin expression) or 24 hours (having only integrin expression).Culture medium is removed in the flat board suction, and paraformaldehyde-PBS that every hole adds 100 μ l 0.1% (contains Ca
++And Mg
++).With flat board place 4 ℃ 30 minutes.Remove the fixative in the hole,, discharge with PBS (+Ca and Mg)+0.5%BSA rinsing 1 time.Anti-arriving in test and the control wells that adds 10 μ l dilution.It is 10 μ g/ml (the antibody mother solutions of 1: 10 dilution 0.1mg/ml) that anti-ICAM-1-biotin, anti-VCAM-1-biotin and anti-E-select the working concentration of albumen-biotin.Cell is under moist environment, in 37 ℃ of incubations 30 minutes.The hole is with PBS (+Ca and Mg)+0.5%BSA rinsing 3 times.Every then hole added the ExtrAvidin-alkali phosphatase (1: 5,000 dilution is called the work diluent herein) of 20 μ l dilution, in 37 ℃ of incubations 30 minutes.The hole is with PBS (+Ca and Mg)+0.5%BSA rinsing 3 times.1 paranitrophenol phosphoric acid pNPP is dissolved in (pH 10.4) in the 5ml glycine buffer.Each instrument connection adds the pNPP substrate of the 100 μ l that are dissolved in glycine buffer.Preparation standard three repeating holes, its use are dissolved in the work diluent of the ExtrAvidin-alkali phosphatase of glycine buffer: 1: 5, and 000 (10
0)＞10
-0.5＞10
-1＞10
-1.5Every kind of diluent of 5 μ l is added in three repeating holes, and the AP content in the every hole that obtains like this is 5.50ng, 1.74ng, 0.55ng, 0.18ng.The pNPP reagent that adds 100 μ l then in each gauge orifice.With flat board in 37 ℃ of incubations 4 hours.Add the NaOH of 50 μ l 3M in porose.Read flat board with plate reader at the 405nm place, the background deduction option blank well that only contains glycine buffer.In addition, template is set at the concentration (5.50ng, 1.74ng, 0.55ng, 0.18ng) of AP-conjugate in each gauge orifice of indication.The result is shown as the amount of bonded AP-conjugate in each sample.
The test of embodiment 39:Alamar Blue endothelial cell proliferation
This test can be used for quantitatively determining protein mediated to the inductive cattle lymph of bFGF endotheliocyte (LEC), bovine aortic endothelial cells (BAEC) or the effect of human blood capillary myometrium cell (UTMEC) inhibition of proliferation.This test comprises the fluorescence growth indicator that detects based on metabolic activity.The Alamar Blue proliferation test of standard is prepared in EGM-2MV, wherein is added with 10ng/ml bFGF as the endotheliocyte stimulus.This test also can be used for many kinds of endotheliocytes by slight change growth medium and cell concentration.The albumen that will test batch is diluted to suitable diluent.The culture medium that does not contain serum (GIBCO SFM) that does not have bFGF is as non-stimulation contrast, and angiostatin or TSP-1 are included as known inhibition contrast.
Briefly, LEC, BAEC or UTMEC are inoculated into 96 orifice plates that contain growth medium with 5000 to 2000 cells/well, place 37 ℃ to spend the night.After the cell incubated overnight, remove growth medium, replace to GIBCO EC-SFM.In three repeating holes, cell is handled with the suitable diluent of albumin fusion proteins of the present invention or reference protein sample (preparation is in SFM), add simultaneously bFGF to concentration be 10ng/ml.Cell was put back to 37 ℃ of incubation casees 3 days with flat board after sample treatment.After 3 days, every hole adds 10 milliliters of Alamar Blue mother solutions (Biosource, catalog number (Cat.No.) DAL1100), and flat board was put back to 37 ℃ of incubation casees 4 hours.Flat board excites and 590nm emission place reading at 530nm with CytoFluor fluorescence reader then.Directly the output result is with the relative fluorescence unit record.
Alamar Blue is a kind of oxidation-reduction indicator, can be by fluorescing and changing color to because response is made in the electronation of the growth medium that cell growth causes.When cell was grown on culture medium, intrinsic metabolic activity caused the electronation of instant surrounding enviroment.The reduction relevant with growth makes indicator become reduction (fluorescein) form from oxidation (non-fluorescent blue) form and (promptly stimulates proliferation and will produce stronger signal, suppress propagation and will produce more weak signal, resultant signal is proportional with total cellular score and their metabolic activity).The background activity level is observed in independent hungry culture medium.This with compare from positive control sample (growth medium that contains bFGF) and the observed result of diluted protein solution.
Embodiment 40: detect the inhibitory action to mixed lymphocyte reaction
This test can be used to detect and estimates the inhibitory action of fusion rotein of the present invention to mixed lymphocyte reaction (MLR).To the inhibition of MLR may be because the adjusting that the direct influence of on cell proliferation and viability, costimulatory molecules produce to the adjusting of interaction cell, to the cytokine of the adjusting of sticking together effect between lymphocyte and accessory cell or accessory cell.Many cells may be the target cells that suppresses the albumin fusion proteins of MLR, and single nuclear fraction comprises T, B and NK cell lymphocyte because this tests employed peripheral blood, and mononuclear cell and dendritic cell.
The albumin fusion proteins of the present invention that discovery can suppress MLR can be applicable to lymphocyte and monocyte activation or the relevant disease of propagation.These diseases include but not limited to asthma, arthritis, diabetes, inflammatory dermatopathy, psoriasis, eczema, systemic lupus erythematosus (sle), multiple sclerosis, glomerulonephritis, inflammatory bowel, Crohn disease, ulcerative colitis, arteriosclerosis, liver cirrhosis, graft versus host disease, host versus graft disease, hepatitis, leukemia and lymphoma.
Briefly, from the PBMC of human donor by density gradient centrifugation with LSM (
Density 1.0770g/ml, Organon Teknika Corporation, West Chester PA) comes purification.(Life Technologies, Grand Island adjust to 2 * 10 in NY) at the RPMI-1640 that has added 10%FCS and 2mM glutamine from the PBMC of two donors
6Individual cells/ml.PBMC from the 3rd donor adjusts to 2 * 10
5Individual cells/ml.50 microlitres are added in the hole of microtitration plate at the bottom of 96 hole circles from the PBMC of each donor.The fusion rotein diluent of test material (50 μ l) three repeatedly is added in the microtiter well.Add (proteins of interest) specimen with final 1: 4 dilution; Add rhuIL-2 (R﹠amp; D Systems, Minneapolis, MN, catalog number (Cat.No.) 202-IL) to make its final concentration be 1 μ g/ml; Add anti-CD4mAb (R﹠amp; D Systems, clone 34930.11, catalog number (Cat.No.) MAB379) to make its final concentration be 10 μ g/ml.Cell is at 5%CO
2In cultivated 7-8 days in 37 ℃, add 1 μ C[in the last 16 hours culture hole
3H] thymidine.Collecting cell is determined the thymidine that mixes with Packard Topcount.Data are expressed as the average and the standard deviation of triple repetition measurement amounts.
The sample of fusion rotein interested is independently screening in the experiment, and with can suppress lymphopoietic negative control and handle (anti-CD4mAb) and can strengthen lymphopoietic positive control processing (IL-2 can be recombined material or supernatant) and compare.
Embodiment 41: the proteinase activity test
Following test can be used to assess the proteinase activity of albumin fusion proteins of the present invention.
Gelatin and casease spectrometry are substantially as described carrying out (Heusen etc., Anal.Biochem.102:196-202,1980 such as Heusen etc. and Wilson; Wilson etc., Journal of Urology, 149:653-658,1993).Sample is containing electrophoresis on 1% gelatin or caseic 10% polyacrylamide/0.1%SDS gel, and soaking at room temperature is in 2.5%Triton 1 hour, in 0.1M glycine (pH 8.3) in 37 ℃ of electrophoresis 5 to 16 hours.After amido black dyeing, the Proteolytic enzyme zone is compared with the background of blue-black and is revealed as bright zone.Trypsin Sigma T8642) as positive control.
Proteinase activity also can be determined by monitoring n-a-benzoyl-L-arginine ethyl ester (BAEE) cracking (SigmaB-4500).Be reflected at (25mM NaPO
4, 1mM EDTA and 1mM BAEE, pH 7.5) in set up.Add sample, the change of the mode monitoring 260nm place absorbance that on Beckman DU-6 spectrophotometer, drives with the time.Trypsin is as positive control.
Other is based on carrying out (Bergmeyer etc. from the absorbance at the test determination 280nm place of the release of the acid-soluble peptide of casein or hemoglobin or the colorimetric analysis test of using the Folin method as method as described in the Bergmeyer etc., Methods of Enzymatic Analysis, 5,1984).Other test comprises the dissolving (Ward, Applied Science, 251-317,1983) of chromogenic substrate.
Embodiment 42: the substrate specificity of identifying serine protease
Means known in the art or methods described herein can be used to determine have the substrate specificity of the albumin fusion proteins of the present invention of serine protease.In one embodiment, the method for determining substrate specificity is by using as the described location scanning synthetic combinatorial libraries of GB 2324529 (the whole this paper of income).
Embodiment 43: part is in conjunction with test
Following test can be used to assess the ligand-binding activity of albumin fusion proteins of the present invention.
A kind of direct method that part provides definite receptor agents to learn in conjunction with test, and applicable to high throughput format.Part at the purification of albumin fusion proteins of the present invention is used in conjunction with research to high specific acitivity (50-2000Ci/mmol) through radioactive label.Evidence, radiolabeled process do not reduce the activity of part to fusion rotein.The experimental condition of optimizing buffer, ion, pH and other regulon (as nucleotide) is set up for film and the full cell polypeptide feasible signal to noise ratio for the two of originating.In these tests, specific polypeptide deducts the radioactivity of being surveyed in conjunction with being defined as total binding radioactivity when the competitive part of excessive non-marked exists.If possible, can use more than one competitive parts to define residual non-specific binding.
Embodiment 44: the function test in the xenopus leavis oocytes (Xenopus oocyte)
From the linearization plasmid template of code book invention albumin fusion proteins to add medicated cap rna transcription thing external synthetic according to standard scheme with RNA polymerase.External, making its final concentration with the aqueous suspension transcript is 0.2mg/ml.Take out the to grow up ovary leaf of female Bufo siccus obtains the V phase to remove follicular oocyte, uses the microinjection device that rna transcription thing (10ng/ oocyte) is injected with 50nl.Measure the electric current that single xenopus leavis oocytes response is exposed to fusion rotein and polypeptide agonist with 2 electrode voltage pincers.Under the room temperature condition, do not containing Ca
2+The BarthShi culture medium in record.The Xenopus laevis system can be used to screen known ligand and the tissue/cell extract obtains the activity part.
Embodiment 45: little physiological measurement test
Activate many second messenger systems and cause that small amount of acid discharges from cell.The acid that forms mainly is the required metabolic activity enhanced results of supply intracellular signal conductive process.The change of pericellular pH in culture medium is very little, but the little physiological measurement instrument of available CYTOSENSOR (Molecular DevicesLtd., Menlo Park Calif.) detects.Therefore, CYTOSENSOR can detect albumin fusion proteins of the present invention and activate the ability of utilizing the link coupled second message,second messenger of intracellular signal pathway with energy.
Embodiment 46: the screening of extract/cell conditioned medium liquid
There are many mammalian receptors still not have relevant activation part (agonist).Therefore, the active ligand of these receptors may be not included in the part storehouse of having identified at present.Correspondingly, also can be by functionally identifying the human cytokines part of albumin fusion proteins of the present invention and/or the native ligand of albumin part at tissue extract screening (using functional screenings such as calcium, cAMP, little physiological measurement instrument, oocyte electricity physiology) albumin fusion proteins of the present invention.The extract that produces the response of positive function is subfragrnentization sequentially, until separating and identifying the activity part.
Embodiment 47:ATP is in conjunction with test
Following test can be used to assess the ATP of fusion rotein of the present invention in conjunction with activity.
The ATP of albumin fusion proteins of the present invention is available as United States Patent (USP) the 5th, 858 in conjunction with activity, and No. 719 (whole by reference income this paper) described ATP detects in conjunction with testing.Briefly, pass through in competitive trials, to measure with the bonded ATP of albumin fusion proteins of the present invention with the photoaffinity labeling that 8-nitrine-ATP carries out.Contain the ATP of proteic reactant mixture of 1mg/ml abc transport and variable concentrations or non-water-disintegrable ATP analog adenosine-5 '-imidodiphosphoric acid jointly in 4 ℃ of incubations 10 minutes.Add 8-nitrine-ATP (Sigma Chem.Corp., St.Louis, MO) and 8-nitrine-ATP (
32P-ATP) mixture of (5mCi/ μ mol, ICN, Irvine CA), making final concentration is 100 μ M, the 0.5ml sample aliquot is put into the hole of porcelain point plate, and place on ice.Shining dull and stereotyped 2 times from the distance of dull and stereotyped 2.5cm with shortwave 254nm uviol lamp, each 1 minute, midfeather cooling in 1 minute.Add dithiothreitol, DTT and come cessation reaction to final concentration 2mM.The incubation thing is carried out SDS-PAGE electrophoresis, drying and autoradiography.Cutting-out is corresponding to the protein band of albumin fusion proteins of the present invention, its radioactivity of quantitative analysis.Radioactivity reduces along with the increase of ATP or adenosine-5 '-imidodiphosphoric acid, and measuring of the affinity of ATP to fusion rotein is provided.
Embodiment 48: identify and the interactional signal transducer of albumin fusion proteins of the present invention
Albumin fusion proteins of the present invention can be used as the research tool of evaluation, sign and the purification of signal transduction pathway albumen or receptor protein.Briefly, the fusion rotein of the present invention of labelling can be used as the reagent of purification and its interacting molecule.In an embodiment of affinity purification, albumin fusion proteins of the present invention and chromatographic column covalent coupling.Cross post derived from the not celliferous extract of inferring target cell (as cancerous tissue), the molecule with suitable affinity combines with albumin fusion proteins.Reclaim protein complexes and dissociate from pillar, the molecule that reclaims is carried out the N-terminal protein sequencing.The aminoacid sequence that obtains is used for subsequently identifying that capture molecules or design degenerate oligonucleotide probe are from the suitable relevant gene of cDNA library clone.
The bioassay of embodiment 49:IL-6
Many tests known in the art can be used for measuring the multiplication effect of albumin fusion proteins of the present invention.For example, such test is the bioassay (95:3251-56 1998, takes in this paper by reference for Proc.Natl.Acad.Sci., U.S.A.) as described IL-6 such as Marz.37 ℃ of incubations are after 68 hours, by adding tetrazolium tetrazolium bromide (MTT) and in 37 ℃ of incubations 4 hours again, measuring the living cells number.With SDS cracking B9 cell, measure optical density at the 570nm place.The contrast that contains IL-6 is a positive control, and the contrast of factor-containing is not a negative control.Briefly, depend on the B9 Mus cell 3 times of IL-6,, add 50 μ l fusion rotein of the present invention and utilize with the concentration bed board of 50 μ l with 5,000 cells in every hole with the culture medium rinsing that does not contain IL-6.Specimen (containing albumin fusion proteins of the present invention) has shown fusion protein mediated multiplication effect with respect to the enhanced propagation of negative control.
Embodiment 50: to the support of Embryo Gallus domesticus neuronal survival
Whether can support the viability of sympathetic neuron cell for measuring albumin fusion proteins of the present invention, and the Embryo Gallus domesticus neuronal survival test of use Senaldi etc. (Proc.Natl.Acad.Sci.U.S.A., 96:11458-63,1998, take in this paper by reference).Briefly, isolated movement neuron and sympathetic neuron from Embryo Gallus domesticus are resuspended in the L15 culture medium respectively and (contain 10%FCS, glucose, sodium selenite, progesterone, conalbumin, putrescine and insulin; Life Technologies, Rockville, MD) the Eagles culture medium with the Dulbecco improvement [contains 10%FCS, glutamine, penicillin and 25mMHepes buffer (pH 7.2); Life Technologies, Rockville, MD], under the condition that the fusion rotein of the present invention of variable concentrations purification exists, at 5%CO
2In in 37 ℃ of incubations, negative control is without any cytokine.After 3 days, by estimating cellular morphology and using the colorimetric test of Mosmann to determine neuronic survival (Mosmann, T., J.Immunol.Methods, 65:55-63,1983).Compare with the contrast that lacks cytokine, enhanced neuronal cell viability shows that albumin fusion proteins strengthens the ability of neuronal cell survival.
Embodiment 51: the phosphatase activity test
Following test can be used to assess serine/threonine phosphatase (PTPase) activity of albumin fusion proteins of the present invention.
For measuring serine/threonine phosphatase (PTPase) activity, can use extensively to be test well known by persons skilled in the art.For example, serine/threonine phosphatase (PSPase) activity of albumin fusion proteins of the present invention can be used New England Biolabs, and the PSPase test kit of Inc. is measured.[
32P] there is down the protein kinase phosphorylation that the serine of sphingomyelins basic protein (MyBP) (a kind of substrate of PSPase) and threonine residues are depended on cAMP in ATP.Then, can by measure from
32The inorganic phosphate radical that the MyBP of P labelling discharges is determined proteic serine/threonine phosphatase activity.
Embodiment 52: serine/threonine phosphatase and other proteic interaction
For example, the fusion rotein of the present invention (determining as embodiment 51) with serine/threonine phosphatase activity can be used as the research tool of other interaction protein or receptor protein or the proteic evaluation of other signal transduction pathways, sign and purification.Briefly, the fusion rotein of the present invention of labelling can be used as the reagent of purification molecule interactional with it.In an embodiment of affinity purification, albumin fusion proteins of the present invention and chromatographic column covalent coupling.Cross post derived from the not celliferous extract of inferring target cell (as neurocyte or hepatocyte), the molecule with suitable affinity combines with fusion rotein.Reclaim the fusion rotein complex and dissociate from pillar, the molecule that reclaims is carried out the N-terminal protein sequencing.The aminoacid sequence that obtains is used for subsequently identifying that capture molecules or design degenerate oligonucleotide probe are from the suitable relevant gene of cDNA library clone.
Embodiment 53: the heparanase activity test
Many tests known in the art can be used to measure the heparanase activity of albumin fusion proteins of the present invention.In an example, the heparanase activity of albumin fusion proteins of the present invention is as mensuration (Nat.Med. such as Vlodavsky, 5:793-802,1999) as described in Vlodavsky etc.Briefly, with cell lysate, conditioning culture medium, intact cell (every 35-mm dish 1 * 10
6Individual cell), the fusion rotein of cell culture supernatant liquid or purification with
35The ECM of S labelling or the deutero-peak of soluble E CM I Dan Baijutang are jointly in 37 ℃ of incubations 18 hours (pH 6.2-6.6).The incubation culture medium is centrifugal, by gel filtration at Sepharose CL-6B post (clear liquid analytically on 0.9 * 30cm).With each fraction of PBS eluting, and measure its radioactivity.The degradation fragment of Heparan sulfate side chain from Sepharose 6B post at 0.5＜K
Av＜0.8 (peak ∏) locates eluting.Each experiment is done 3 times at least.As show the activity of albumin fusion proteins cracking Heparan sulfate of the present invention as described in the Vlodavsky corresponding to the degradation fragment of peak H.
Embodiment 54: the immobilization of biomolecule
Present embodiment provides the method for stablizing albumin fusion proteins of the present invention in non-host cell lipid bilayer construction (referring to for example Bieri etc., Nature Biotech 17:1105-1108,1999, whole by reference income this paper), this method is fit to the fusion rotein of the present invention in the various above-mentioned functions tests of research.Briefly, be used for the special chemical reaction of biotinylated carbohydrate and be used for biotin label is limited in albumin fusion proteins of the present invention, so have consistent direction after making immobilization.Wash albumin fusion proteins solution of the present invention and the 20mM NaIO of 50 μ M on the film
4And 1.5mg/ml (4mM) BACH or 2mg/ml (7.5mM) biotin-hydrazides room temperature incubation 1 hour (reaction volume, 150 μ l).Sample was in 4 ℃ of dialysis (Pierce Slidealizer box, 10kDa holds back, Pierce Chemical Co., Rockford IL) earlier 5 hours then, and exchange buffering liquid after per 1 hour is used 500ml buffer R (0.15M NaCl, 1mM MgCl at last
2, the 10mM sodium phosphate, pH 7) dialysis 12 hours.Before in being added to cup, with ROG50 (adding the buffer R of 50mM Octyl glucoside) buffer with 1: 5 dilute sample.
Embodiment 55: the metal proteinase activity test
Metalloproteases is (as Zn with metal ion
2+) as the peptidohydrolase of catalytic mechanism.The metal proteinase activity of albumin fusion proteins of the present invention can be measured according to means known in the art.Exemplary method hereinafter is provided:
The Proteolytic enzyme of α-2-macroglobulin
Be checking proteinase activity, the fusion rotein of the present invention of purification and substrate α-2-macroglobulin (0.2 units per ml; Boehringer Mannheim Germany) is mixed in 1 * test buffer (50mMHEPES, pH 7.5,0.2M NaCl, 10mM CaCl
2, 25 μ M ZnCl
2And 0.05%Brij-35), 37 ℃ incubation 1-5 days.Trypsin is as positive control.Negative control only contains the α-2-macroglobulin that is dissolved in the test buffer.Collect sample, in containing the SDS-PAGE sample-loading buffer of 5%2-mercaptoethanol, boiled 5 minutes, go up then on the SDS-polyacrylamide gel of sample to 8%.Dye by silver behind the electrophoresis and observe albumen.The Proteolytic enzyme of having compared more low-molecular-weight carrying means of appearance with negative control.
Inhibitors of metalloproteinase is to α-proteoclastic inhibitory action of 2-macroglobulin
Known inhibitors of metalloproteinase (metal-chelator (EDTA, EGTA and HgCl
2), peptide inhibitors of metalloproteinase (TIMP-1 and TIMP-2) and commercialization micromolecule MMP inhibitor) also can be used to characterize the proteolytic activity of albumin fusion proteins of the present invention.Spendable 3 kinds of synthetic MMP inhibitor are MMP inhibitor I, [to the IC of MMP-1 and MMP-8
50=1.0 μ M; IC to MMP-9
50=30 μ M; IC to MMP-3
50=150 μ M]; MMP-3 (extracellular matrix degrading enzyme 1) inhibitor I[is to the IC of MMP-3
50=5 μ M] and MMP-3 inhibitor ∏ [to the K of MMP-3
i=130nM]; These inhibitor can buy (catalog number (Cat.No.) is respectively 444250,444218 and 444225) by Calbiochem.Briefly, the micromolecule MMP inhibitor of variable concentrations and the fusion rotein of the present invention of purification (50 μ g/ml) (50mM HEPES, pH 7.5,0.2M NaCl, 10mM CaCl in 22.9 μ l, 1 * HEPES buffer
2, 25 μ M ZnCl
2And 0.05%Brij-35) mix, room temperature (24 ℃) incubation 2 hours adds 7.1 μ l substrate α-2-macroglobulin (0.2 units per ml) then, 37 ℃ of incubations 20 hours.After adding 4 * sample-loading buffer cessation reaction, boiled rapidly 5 minutes.Behind the SDS-PAGE, dye visible protein band by silver.
Synthetic fluorescence peptide substrates breaking test
The substrate specificity that has confirmed to have the fusion rotein of the present invention of metal proteinase activity can use technology known in the art to determine, as uses synthetic fluorescence peptide substrates (available from BACHEM BioscienceInc.).The test substrate comprises: M-1985, M-2225, M-2105, M-2110 and M-2255.Preceding 4 is the MMP substrate, and last 1 is the substrate of tumor necrosis factor (TNF-α) invertase (TACE).These substrates can prepare in 1: 1 dimethyl sulfoxine (DMSO) and water.Mother solution is 50-500 μ M.Fluorescent test is to use the Perkin Elmer LS 50B luminescent spectrum instrument of being furnished with water bath with thermostatic control to carry out.Excitation wavelength is 328nm, and emission wavelength is 393nm.Briefly, test is undertaken by following scheme: with 176 μ l, 1 * HEPES buffer (0.2M NaCl, 10mM CaCl
2, 0.05%Brij-35 and 50mM HEPES, pH 7.5) and 4 μ l substrate solutions (50 μ M) in 25 ℃ of incubations 15 minutes, the fusion rotein of the present invention that adds 20 μ l purification then is in test glass.The final concentration of substrate is 1 μ M.Monitored initial hydrolysis rate 30 minutes.
The morbidity of diabetes in the embodiment 56:NOD mice
The feature of female NOD (non-obese diabetes) mice is to show as IDDM (that finds among its process and the mankind is similar), although disease is female more obvious in than male NOD mice.Unless hereinafter specialize, term " NOD mice " refers to female NOD mice.The NOD mice has the destruction gradually of the β cell that is caused by chronic autoimmune disease.Therefore, NOD mice when birth blood glucose is normal or blood glucose levels is normal.Yet when big, the NOD mice begins the hyperglycemia that becomes, and illustrate that its most of pancreatic beta cells are damaged, and correspondingly pancreas can not produce enough insulins to general 15-16 week.Therefore, the reason of disease and progress are all similar with human IDDM patient.
Can in female NOD/LtJ mice (can be, Bar Harbor, Me. buys), carry out the in vivo test that immunotherapy is renderd a service from The Jackson Laboratory.Bibliographical information is arranged, and diabetes take place when can be in 24 weeks big in 80% female mice, and the morbidity of insulitis starts from 6-8 between week.The NOD mice is inbred, can regulate tactful high response to panimmunity.The average weight of adult NOD mice (6-8 week is big) is the 20-25 gram.
These mices or for untreated (contrast) or for therapeutic agent of the present invention (as albumin fusion proteins of the present invention and fragment and variant) separately or with other above-mentioned therapeutic compound of mentioning mice of processing jointly.These different processing are measured the available following method of the influence of diabetes progress:
14 weeks can be carried out phenotype to female NOD mice according to glucose tolerance and determine when big.Glucose tolerance can be measured with intraperitoneal glucose tolerance test (IPGTT).Briefly, at 0 minute and peritoneal injection glucose (1 gram/kg body weight) after 60 minutes, from the other vascular plexus extraction of socket of the eye blood sample.Normal tolerance is defined as that plasma glucose is less than 144mg% in the time of 0 minute, or is less than 160mg% in the time of 60 minutes.Blood glucose levels is determined with Glucometer Elite instrument.
Based on this phenotype analytical, animal is assigned to different experimental grouies.Especially, have the animal that higher blood glucose levels raises and to be assigned to the impaired glucose tolerance group.Mice can arbitrarily be fed, and acidifying water (pH 2.3) is provided.
Glucose tolerates and does not tolerate mice and can further be divided into matched group, albumin fusion proteins group of the present invention and albumin fusion proteins/therapeutic compound combination group again.But control group mice accepts to inject between peritoneum media, 6 times weekly every day.But albumin fusion proteins group mice is accepted the therapeutic agent of the present invention (as albumin fusion proteins of the present invention and fragment and variant) that injection between peritoneum is dissolved in media, 6 times weekly every day.Albumin fusion proteins/therapeutic compound combination group mice can be accepted the combination of above-mentioned albumin fusion proteins and therapeutic compound.
Glucose level in the NOD mouse retention can be used Labstix, and (Bayer Diagnostics, Hampshire England) measure weekly twice.Body weight and fluidic picked-up also can be measured weekly twice.The morbidity of diabetes is defined as the appearance of glycosuria in double mensuration.After the processing in 10 weeks, can carry out one time IPGTT again, then next day animal just can be condemned to death.
In 10 all processes of handling, glucose tolerance and glucose do not tolerate control animal in the group respectively with 60% and 86% ratio generation diabetes (referring to United States Patent (USP) the 5th, 866, No. 546, Gross etc.).Therefore, if do not intervene, even in the NOD mice of glucose tolerance originally, also diabetes can appear in high proportion.
Can verify the result by the level of blood-glucose in measuring the NOD mice before and after treatment.The glucose tolerance is measured as stated above with the level that does not tolerate the blood-glucose of mice in described all groups.
In an alternative embodiment, therapeutic agent of the present invention (as be published as SEQ ID NO:Y specific fusions and fragment and variant) can carry out quantitative assay by spectrum analysis, and suitable protein content can be resuspended in 50.mu.l phosphate buffered saline (PBS) (PBS)/agent before the injection.2 injections (being separated by for 1 week) can be carried out subcutaneous administration under the skin of back of each mice.Can monitor in different at two before the immunity, also can monitor weekly once in the entire process process, after this continue.Glucose (Keto-Diastix.RTM. during test is urinated weekly; Miles Inc., Kankakee, Ill), the glycosuria mice can detect its serum glucose (ExacTech.RTM., MediSense, Inc., Waltham, Mass.).When surpassing 2.5 grams per liters, fasting glucose is diagnosed as diabetes.
The histologic analysis of embodiment 57:NOD mice
The combination of the provable present composition of histologic analysis of NOD mouse tissue sample and/or the present composition and other Remedies for diabetes improves the ability of β cell relative concentration in the pancreas.Experimental technique is as described below:
Mice from embodiment 56 is condemned to death when the processing phase finishes, and extracts tissue sample from pancreas.Then sample is fixed in 0.9% brinish 10% formalin, is embedded in the wax.Slice spacings that can 150.mu.m is cut 2 groups 5 continuous 5.mu.m sections and is used for immune labeled.Can carry out section immune labeled: insulin (Cavia porcellus glucagon antiserum diluent 1: 1000, ICN Thames U.K.) and glucagon (the anti-pancreas glucagon of rabbit antiserum diluent 1: 2000), with with the link coupled anti-Cavia porcellus (Dako of peroxidase, High Wycombe, U.K.) or with the link coupled anti-rabbit anti-serum of peroxidase (diluent 1: 50 Dako) detects.
Compositions of the present invention to the influence of the visible energy of β cell may be or may not be to resemble it not tolerate in the animal strong the influence of the clinical manifestation of diabetes at glucose tolerance and glucose.
Embodiment 58: NIDDM mouse model in the body
(this mice is with conventional food or is rich in fat (35.5% w/w for Bar Harbor, ME) available 3 all big male C 57 BL/6 J mouses from Jackson Laboratory; Bioserv.Frenchtown, NJ) or fructose (60% w/w; Harlan Teklad, Madison, food WI) is fed.Conventional food is fibrous by the fructose of the starch of the protein of the fat of 4.5% w/w, 23% w/w, 31.9% w/w, 3.7% w/w and 5.3% w/w.Higher fatty acid (Adeps Sus domestica) food is fibrous by the fructose of the starch of the protein of the fat of 35.5% w/w, 20% w/w, 36.4% w/w, 0.0% w/w and 0.1% w/w.High fructose food is fibrous by the fructose of the starch of the protein of the fat of 5% w/w, 20% w/w, 0.0% w/w, 60% w/w and 9.4% w/w.Mice is in the control of 22 ℃ ± 3 ℃ temperature and 50% ± 20% humidity and to have a room inner lane of 12 hours illumination (6am is to 6pm)/dark cycles foster, every cage is no more than 5 (Luo etc., 1998, Metabolism47 (6): 663-8, " Nongenetic mouse models of non-insulin-dependent diabetesmellitus " (the nongenetic mouse model of noninsulindependent diabetes); Larsen etc., Diabetes50 (11): 2530-9,2001, " Systemic administration of the long-acting GLP-1derivative NN2211induces lasting and reversible weight loss in both normal andobese rats " (systemic administration of long-acting GLP-1 derivant NN2211 can be induced lasting and reversible weight saving in normal and the obese rat)).At separately food of contact after 3 weeks, (MO) or media (0.05mol/L citric acid, pH 4.5) peritoneal injection mice, ensuing 4 weeks are kept same food for Sigma, St.Louis with the streptozotocin " STZ " of 100mg/kg body weight.Under non-fasted conditions, obtain the blood sample in STZ injection 1,2 and 4 weeks of back by the end of cutting tail.Sample is used to measure non-fasting plasma glucose and concentration of insulin.Write down the picked-up of body weight and food weekly.
For directly determining the influence of food rich in fat, after finishing above-mentioned 7 periods in week, begin 3 groups of mices are experimentized: fatty nursing group, with the conventional food nursing group of media injection with the fat nursing group of STZ injection to insulin stimulating glucose disposal ability.Experiment is preceding with mice fasting 4 hours.In first group of experiment, (Pitman-Moor, Mundelein IL) make mouse anesthesia by sucking methoxiflurane.By injection ([IV] 0.1U/kg body weight) insulin regular (Sigma) in the tail cava vein, blood is collected from a different tail vein in injection back 3,6,9,12 and 15 minutes.Measure the plasma glucose concentration of these samples, the half-life (t that glucose disappears from blood plasma
1/2) (NC) (a kind of pharmacokinetics/pharmacodynamics software program) calculates available WinNonlin for ScientificConsulting, Apex.
In second group of experiment, by peritoneal injection pentobarbital sodium (Sigma) anesthetized mice.Open the abdominal cavity, expose main abdomen vein, insert No. 24 IV conduits (Johnson-Johnson Medical, Arlington, TX).Conduit is fixed to contiguous abdomen venous muscular tissue, makes otch in syringe junction bottom, and be suspended on the PE50 plastic tube that is pre-charged with, plastic tube is connected to contains the syringe that injects solution again.By stitching the abdominal cavity is closed then.In this way, the obstruction that flows backwards and cause from the body lower because of blood will do not had.Inject mice with glucose (24.1mg/kg/min) and insulin (10mU/kg/min) continuously with the injection volume of 10 μ l/min.Blood sample (each 70 μ l) is used for the mensuration of plasma glucose and insulin concentration after beginning to inject the back can extract socket of the eye in 90,105,120 and 135 minutes.The meansigma methods of these 4 samples is used to assess the concentration of every animal steady state blood plasma glucose (SSPG) and insulin (SSPI).
At last, the therapeutic composition (separately or with any or multiple treatment treatment of diabetes drug regimen that is enumerated as) of estimating albumin fusion proteins, the application in " NIDDM " mouse model of following 2 groups of STZ injection reduces the experiment of the ability of plasma glucose: the C57BL/6J that C57BL/6J that (1) fat is fed and (2) fructose are fed.The plasma glucose concentration scope of mice that is used for these researchs from 255 to 555mg/dL.Mice is handled by random assortment: with media handle, with albumin fusions therapeutic agent individual processing of the present invention or with any or multiple combined treatment for the treatment of the treatment of diabetes medicine that is enumerated as.Administration is 3 doses altogether.Extracted the mensuration that the tail vein sample is used for plasma glucose concentration before the 1st dose of administration and after the last potion administration in 3 hours.
Plasma glucose concentration can be measured by colorimetric enzymatic assay with the glucose diagnostic kit (Sigma catalog number (Cat.No.) 315) of Sigma.Plasma insulin level can be used the rat insulin RIA test kit (RI-13K of Linco Research; St.Charles MO) measures.
Embodiment 59: the participation that insulin action is set up in external H4IIe-SEAP reporter gene test
Various H4IIe reporter genes
H4IIe/rMEP-SEAP: isolating malate dehydrogenase promoter (rMEP) contains the PPAR-γ element of an insulin approach from rat.But this reporter gene construct stable transfection liver H4IIe cell line.
H4IIe/SREBP-SEAP: sterin regulating element conjugated protein (SREBP-1c) is to act on the promoter of many insulin replies genes (as fatty acid synthetase FAS) and be adjusted to the transcription factor that the fatty acid metabolism key gene is expressed in fibrocyte, adipose cell and the hepatocyte.SREBP-1c (be also referred to as adipose cell decision and differentiation factor 1 (ADD-1)) is considered in the adipose cell insulin to the main mediators of the effect of gene expression.Its activity is subjected to the adjusting of insulin, sterin and glucose level.But this report gene construct stable transfection liver H4IIe cell line.
H4IIe/FAS-SEAP: fatty acid synthetase reporter gene construct contains the SREBP response FAS promoter of a minimum.But this report gene construct stable transfection liver H4IIe cell line.
H4IIe/PEPCK-SEAP: phosphoenolpy ruvate carboxy kinase (PEPCK) promoter is the main site of hormonal regulation PEPCK genetic transcription, the activity of scalable PEPCK.Therefore a directed rate-limiting step of PEPCK catalysis glycogen heteroplasia approach needs careful control to keep blood glucose levels in normal range.This report gene construct stable transfection liver H4IIe cell line.
But these reporter gene constructs are stable transfection 3T3-L1 fibroblast and L6 sarcoplast also.Then, as described in embodiment 13, these stable cell lines can be divided into 3T3-L1 adipose cell and L6 myotube.Next, the cell line of differentiation can be used in the following SEAP test.
Growth and test medium
Growth medium comprises 10% hyclone (FBS), 10% calf serum, 1%NEAA, 1 * penicillin/streptomycin and 0.75mg/ml G418 (being used for H4IIe/rFAS-SEAP and H4IIe/SREBP-SEAP) or 0.50mg/ml G418 (being used for H4IIe/rMEP-SEAP).The growth medium that is used for H4IIe/PEPCK-SEAP is made up of 10%FBS, 1% penicillin/streptomycin, 15mMHEPES buffer saline and 0.50mg/ml G418.
The test medium that is used for H4IIe/rFAS-SEAP, H4IIe/SREBP-SEAP and H4IIe/rMEP-SEAP reporter gene is made up of LG DMEM culture medium (Life Technologies), 1%NEAA, 1 * penicillin/streptomycin.The test medium that is used for the H4IIe/PEPCK-SEAP reporter gene is made up of 0.1%FBS, 1% penicillin/streptomycin and 15mM HEPES buffer saline.
Method
Inoculate 96 orifice plates with 75,000 cells/well with 100 μ l/ hole growth mediums, begin to adhere to until the cell that is in exponential phase.With 200 μ l/ holes growth medium being replaced to test medium (test medium that contains 0.5 μ M dexamethasone that is used for the H4IIe/PEPCK-SEAP cell is 100 μ l/ holes, about 20 hours of incubation) makes cell be in hungry 48 hours.Replace test medium with the fresh test medium in 100 μ l/ holes then, 50 μ l sample aliquot of the cell conditioned medium liquid that will obtain from the transfectional cell series of expressing therapeutic agent of the present invention (as albumin fusion proteins of the present invention and fragment and variant) are added in the hole.Supernatant from the empty carrier transfectional cell series is used as negative control.Add 10nM and/or 100nM insulin and in the hole, be used as positive control.Behind 48 hours incubations, the collection condition culture medium is measured SEAP activity (Phospha-Light System protocol, Tropix BP2500).Briefly, sample diluted 30 minutes SEAP with the endogenous non-placentation of inactivation of 65 ℃ of incubations with dilution buffer liquid with 1: 4.50 μ l sample aliquot of dilute sample are mixed with 50 μ l SEAP test buffer (containing the activated inhibitor mixed thing of non-Placenta Hominis SEAP isozyme), and incubation is 5 minutes again.50 μ l sample aliquot of CSPD chemical luminous substrate (diluting with 1: 20 with the luminous enhancer of Emerald) join in the mixture incubation 15-20 minute.Flat board reading on the flat-plate luminous meter of Dynex.
Embodiment 60: transgenic animal
Albumin fusion proteins of the present invention also can be expressed in transgenic animal.The animal of any species can be used to produce transgenic animal, includes but not limited to mice, rat, rabbit, hamster, Cavia porcellus, pig, miniature pig, goat, sheep, cattle and inhuman primates (as baboon, monkey and chimpanzee).In a specific embodiment, technology described herein or technology known in the art are used for expressing fusion rotein of the present invention the mankind, as the part of gene therapy scheme.
Any technology known in the art can be used to the polynucleotide of code book invention albumin fusion proteins are introduced animal to produce the original system (founder line) of transgenic animal.These technology include but not limited to pronucleus microinjection (Paterson etc., Appl.Microbiol.Biotechnol.40:691-698,1994; Carver etc., Biotechnology (NY) 11:1263-1270,1993; Wright etc., Biotechnology (NY) 9:830-834,1991 and Hoppe etc., United States Patent (USP) the 4th, 873, No. 191,1989); The gene transfer of retrovirus retrovirus mediation is to kind of system (Van der Putten etc., Proc.Natl.Acad.Sci., USA 82:6148-6152,1985), blastocyst or an embryo; The gene targeting of embryonic stem cell (Thompson etc., Cell56:313-321,1989); Cell or embryo's electroporation (Lo etc., 1983, Mol Cell.Biol., 3:1803-1814,1983), introduce polynucleotide of the present invention (referring to Ulmer etc., Science 259:1745,1993) with particle gun; Nucleic acid construct is introduced embryo's multipotent stem cells and stem cell is shifted back blastocyst; With the gene transfer (Lavitrano etc., Cell57:717-723,1989) of sperm mediation etc.The summary of these technology is referring to Gordon, " Transgenic Animals, " (" transgenic animal ") Intl.Rev.Cytol.115:171-229,1989, whole by reference income this paper.
Any technology known in the art can be used for producing the transgene clone of the polynucleotide that contain code book invention albumin fusion proteins, for example, in the future self-induction is to cultivation embryo, the fetus of resting state or become somatic consideration convey to move on to the interior (Campell etc. of enucleation oocyte, Nature 380:64-66,1996; Wilmut etc., Nature 385:810-813,1997).
The invention provides the transgenic animal of the polynucleotide that all carry code book invention albumin fusion proteins in all cells, and only at some but the animal of carrying these polynucleotide in the not all cell, i.e. mosaic animal or chimera.Transgenic can be used as single transgenic or a plurality of copy (as concatermer, as a series connection or a series connection to tail head to head) is integrated.Transgenic also can optionally be introduced and be activated (Lasko etc., Proc.Natl.Acad.Sci.USA 89:6232-6236,1992) in particular cell types by following instruction as Lasko etc.This cell type specificity activates required adjusting sequence and depends on interested particular cell types, is tangible to those skilled in the art.When the chromosomal foci that the polynucleotide of code book invention fusion rotein need be incorporated into corresponding to the endogenous gene of the human cytokines of fusion rotein of the present invention part or albumin part, can use gene targeting.Briefly, when using this technology, design contains the carrier of some and the homologous nucleotide sequence of endogenous gene, is used for being integrated into and destroying by the homologous recombination with chromosome sequence the purpose of the function of endogenous gene nucleotide sequence.Transgenic also can optionally be introduced specific cell type by following instruction as Gu etc., therefore inactivation endogenous gene (Gu etc., Science 265:103-106,1994) in this cell type only.The required adjusting sequence of this cell type specificity inactivation depends on interested particular cell types, is tangible to those skilled in the art.
After having produced transgenic animal, can use standard technique to measure the expression of recombination.Initial screening can be verified the realizing of integration of the polynucleotide of code book invention fusion rotein by Southern engram analysis or round pcr analyze animal tissue.The mRNA expression of the polynucleotide of code book invention fusion rotein also can use following technology to assess in the transgenic animal tissue, and these technology include but not limited to tissue sample, in situ hybridization analysis and the reverse transcriptional PCR (rt-PCR) that the Northern engram analysis obtains from animal.The special antibody of the also available fusion rotein of the sample of the tissue of expressed fusion protein is analyzed by immunocytochemistry or immunohistochemical method.
After having produced original animal, they can breeding, inbreeding, outbreeding or hybridization produce specific animal population.The example of these breeding strategies includes but not limited to: original animal and an above integration site outbreeding are to set up independent system; The independent system inbreeding to produce because each genetically modified expression effect and of adding up with the compound transgenic body of higher level express transgenic; Thereby the animal enhancing expression that the hybridization of heterozygosis transgenic animal is isozygotied to produce given integration site, and save the needs that screen animal by DNA analysis; The hybridization that is of independently isozygotying produces compound heterozygosis or isozygotys and is; With the different background that transgenic (being the polynucleotide of code book invention albumin fusion proteins) is placed on suitable interested experimental model by breeding.The application of transgenic animal of the present invention includes but not limited to: the animal model system that can be used for describing in detail the biological function of the human cytokines of fusion rotein of the present invention and fusion rotein of the present invention and/or albumin component, study the chemical compound that illness relevant with unconventionality expression and/or disease and screening can effectively improve this illness and/or disease.
Embodiment 61: use in Therapeutic Method-elder generation external back body of gene therapy
A kind of method of gene therapy is to transplant the fibroblast that can express albumin fusion proteins of the present invention to arrive the patient.Usually, fibroblast obtains from the curee by skin biopsy.The tissue that obtains is placed on the culture medium for tissue culture, is separated into small pieces.Little block organization is placed on the moist surface of tissue culture flasks, and each bottle is put about 10.With culture bottle inversion, jam-pack, place ambient temperature overnight.After the room temperature 24 hours, culture bottle is put upside down, piece of tissue remains fixed in the bottom of culture bottle, adds the fresh culture HamShi F12 culture medium of 10%FBS, penicillin and streptomycin (as contain).Culture bottle is 37 ℃ of about 1 weeks of incubation then.
At this moment, add fresh culture, changed in every then several days.After cultivating for 2 weeks again, the monolayer fibroblast appears.Use the trypsinization monolayer, and the expansion scale is to bigger culture bottle.
Flank digests with EcoRI and HindIII for the long terminal repetition pMV-7 (Kirschmeier, P.T. etc., DNA, 7:219-25 (1988)) of Moloney murine sarcoma virus, handles with calf small intestinal phosphatase then.The bead purification is used in the classification on agarose gel of linearizing carrier.
The polynucleotide of code book invention albumin fusion proteins can use technology known in the art to generate, and use corresponding to 5 ' and 3 ' end sequence and randomly have suitable restriction site and the PCR primer amplification of initial/termination codon (if necessary).In one embodiment, 5 ' primer contains an EcoRI site, and 3 ' primer comprises a HindIII site.When the T4DNA ligase exists, the Moloney murine sarcoma virus linearity skeleton of equivalent and the EcoRI and the HindIII fragment of amplification are added to together.The mixture that obtains maintains under the condition that is suitable for two kinds of fragments connections.Use then to connect mixture transform bacteria HB101, antibacterial is layered on the agar plate that contains kanamycin then, and purpose is in order to confirm that carrier has the interested gene of correct insertion.
Amphicheirality pA317 or GP+am12 incasing cells are grown on the tissue culture of Eagles culture medium (DMEM) of the Dulbecco improvement that contains 10% calf serum (CS), penicillin and streptomycin to converging density.The MSV carrier that will contain gene then is added to culture medium, with the carrier transduction incasing cells.Incasing cells produces the infectious viral particle (incasing cells is called the production cell now) that contains gene now.
Fresh culture is added to the production cell of transduction, subsequently from converging of 10cm flat board producing the cell harvesting culture medium.The exhausted culture medium that contains infectious viral particle removes by filter the production cell of disengaging by the Millipore filter, uses this culture medium to infect fibroblast then.Converge flat board from the fibroblast Asia and remove culture medium, replace to rapidly from the culture medium of producing cell.Remove this culture medium, and replace to fresh culture.If the virus titer height, so in fact all fibroblasts do not need to select with infected.If titre is very low, be necessary to use retrovirus vector so with selected marker (as neo or his).After fibroblast is infected effectively, be parsed into fibrocyte and determine whether to have produced albumin fusion proteins.
Then engineered fibroblast is transplanted to the host after converging separately or growing on cytodex 3 microcarrier beads.
Embodiment 62: use in the Therapeutic Method-body of gene therapy
Another aspect of the present invention is to use the treatment of vivo gene Therapeutic Method disease, disease and illness.Gene therapy method relates to introduces animal with the naked nucleic acid of code book invention albumin fusion proteins (DNA, RNA and antisense DNA or RNA) sequence.The polynucleotide of code book invention albumin fusion proteins can be operatively attached to (i.e. combination with it) promoter or polypeptide is expressed required any other genetic elements at target tissue.This gene therapy and delivery technique and method are known in the art, for example, and WO90/11092, WO98/11779; No. the 5693622nd, 5705151,5580859, United States Patent (USP); Tabata etc., Cardiovasc.Res.35 (3): 470-479 (1997); Chao etc., Pharmacol.Res.35 (6): 517-522 (1997); Wolff, Neuromuscul.Disord.7 (5): 314-318 (1997); Schwartz etc., Gene Ther.3 (5): 405-411 (1996); Tsurumi etc., Circulation94 (12): 3281-3290 (1996) (taking in this paper by reference).
Polynucleotide constructs can be sent to the method for zooblast with any injectable materials of sending, such as, be expelled to the interstitial space of tissue (heart, muscle, skin, lung, liver, intestinal or the like).Polynucleotide constructs can be sent in pharmaceutically acceptable liquid or water carrier.
Term " naked " polynucleotide, DNA or RNA refer to not contain the sequence with helping, promote or be beneficial to any delivery vehicle that enters cell, comprise virus sequence, virion, liposome formulation agent, lipofectin reagent or precipitant or the like.Yet, the polynucleotide of code book invention albumin fusion proteins can send in the liposome formulation agent also that (such as those as (1995) Biol.Cell 85 (1) such as (1995) Ann.NY Acad.Sci.772:126-139 such as Felgner P.L. and Abdallah B.: the instruction of 1-7), it can prepare by method well-known to those skilled in the art.
Be used in polynucleotide carrier construct in the gene therapy method and can comprise that unconformity advances host genome, also do not contain the construct that allows the sequence of duplicating.Any strong promoter well known by persons skilled in the art can be used for driving the expression of DNA.Different with other gene therapy technology, introducing naked nucleic acid sequence is that polynucleotide are synthetic temporary in the cell to the main advantage of target cell.Studies show that non-repetition DNA sequence can introduce cell, provide the generation of required polypeptide to reach 6 months time.
Polynucleotide constructs can be delivered to the interstitial space of animal tissue, and these tissues comprise muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, intestinal, testis, ovary, uterus, rectum, nervous system, eye, body of gland and connective tissue.Organize interstitial space comprise tissue fluid, mucopolysaccharide substrate, blood vessel or chamber wall between the organ-tissue reticular fiber elastic fiber, fibrous tissue collagen fiber or in the connective tissue of clad muscle cell or the same substrate in the skeleton lacuna.The spatial class that it and circulation blood plasma and vasculolymphatic lymph fluid occupy seemingly.The interstitial space that is delivered to muscular tissue can carry out owing to following reason.They can comprise that organizing easily of these cells sent by being expelled to.They can be delivered to and express in persistent, the Unseparated Cell of differentiation, can be in undifferentiated or obtain in the noble cells fully although send and express, and such as for example blood stem cell or skin flbroblast.In the body muscle cell obtain and to express the ability of polynucleotide strong especially.
To naked polynucleotide injection, the effective dose of DNA or RNA is in the scope from about 0.05g/kg body weight to about 50mg/kg body weight.In one embodiment, dosage will be to about 20mg/kg from about 0.005mg/kg; In another embodiment, be to about 5mg/kg from about 0.05mg/kg.Certainly, those of ordinary skills will recognize that this dosage will change according to the tissue site of injection.Suitable and the effective dosage of nucleotide sequence can easily be determined by those of ordinary skills, may depend on the illness and the route of administration of being treated.An exemplary route of administration is the interstitial space that is expelled to tissue by parenteral route.Yet, also can use other parenteral route, such as the suction aerosol preparaton that is used in particular for being delivered to lung or bronchial tissue, throat or nasal mucosa.In addition, the catheter delivery that can be in the angioplasty process uses in by scheme of naked polynucleotide constructs is to tremulous pulse.
The injection polynucleotide can be determined as follows to the dose response effect of muscle in the body.Preparation is used to produce the suitable template DNA of the mRNA of code book invention polypeptide according to the recombinant DNA method of standard.Template DNA can be ring-type or linearity, can be used as naked DNA or forms complex with liposome.Inject the mice musculus quadriceps with the template DNA of various amounts then.
By the A Fuding (Avertin) of peritoneal injection 0.3ml 2.5%, anaesthetize big female and male Balb/C mouse of 5-6 week.On foreleg, make the otch of a 1.5cm, directly visible musculus quadriceps.The template DNA that is dissolved in the 0.1ml carrier was injected in 1 minute by No. 27 pins with the 1cc syringe, inserted the position from the distally of muscle and entered the about 0.5cm of knee, and about 0.2cm is dark.On the injection site, insert stitching thread so that skin is closed with the rustless steel icarceration in following location.
Suitable incubation is (as 7 days) after the time, prepare the muscle extract by prescinding whole musculus quadriceps.The transverse section of per the 5th the 15 μ m of single musculus quadriceps is observed protein expression with histochemical stain.The time course of expressing fusion protein can be similar pattern carry out, except collect the musculus quadriceps of different mices at different time.The persistency of injection back DNA in muscle can be determined by the Southern engram analysis behind preparation total cell dna and HIRT supernatant from injection and control mice.Suitable dose and other treatment parameter that the result of above-mentioned mouse experiment can be used to extrapolate and use naked DNA in human and other animals.
Embodiment 62: the biological action of fusion rotein of the present invention
Astrocyte and neuron test
Can test that albumin fusion proteins of the present invention promotes existence, neurite outwards grows or the phenotypic differentiation of cortical neuron cell, and to induce the glial fibrillary acidic protein immuning positive cell be the activity of astrocyte propagation.The cortical cell that selection is used for biologic test is based on FGF-1 and FGF-2 generally expressing and the existence of FGF-2 processing enhancing cortical neuron of report before at cortex construction.For example, thymidine mixes test and can be used to illustrate that albumin fusion proteins of the present invention is to these cell activity.
In addition, the previous FGF-2 (basic FGF) that describes has shown the growth (Walicke etc. that neuron existence and neurite outwards grow at the report of external biological action to cortex or hippocampal neuron, " Fibroblast growth factor promotes survival of dissociated hippocampal neuronsand enhances neurite extension " (fibroblast growth factor promotes the existence of dissociated hippocampal neuron and strengthened the extension of neurite) Proc.Natl.Acad.Sci.USA 83:3012-3016. (1986), the whole by reference income this paper of test).Yet the report of the experiment of doing shows on the PC-12 cell, and these two kinds respond synonym not necessarily, may not only depend on tested FGF, also depend on those receptors of expressing on target cell.Use former generation cortical neuron to cultivate example, can compare the response that albumin fusion proteins of the present invention induces ability that neurite outwards grows and FGF-2 to obtain, for example use thymidine to mix test.
Fibroblast and endotheliocyte test
(San Diego CA) obtains human lung fibroblast, maintains on the growth medium from Clonetics from Clonetics.(SanDiego CA) obtains microvascular dermal endothelial cell from Cell Applications.To proliferation test, human lung fibroblast and microvascular dermal endothelial cell can 5,000 cells/well cultivate 96 orifice plates that contain growth medium 1 day.Cell incubation 1 day in the basal medium of 0.1%BSA then.After fresh 0.1%BSA culture medium replacement culture medium, the proteic tester fusion protein incubation of cell and the present invention 3 days.Every hole adds Alamar Blue, and (AlamarBiosciences, Sacramento is CA) to final concentration 10%.Cell incubation 4 hours.Measure the viability of cell by reading on CytoFluor fluorescence reader.To PGE
2Test, human lung fibroblast was cultivated 1 day in 96 orifice plates with 5,000 cells/well.After culture medium was replaced by the 0.1%BSA basal medium, cell was under the condition that is with or without IL-1 α, with FGF-2 or fusion rotein incubation of the present invention 24 hours.Collect supernatant, (Cayman, Ann Arbor MI) carry out PGE with the EIA test kit
2Test.To the IL-6 test, human lung fibroblast was cultivated 1 day in 96 orifice plates with 5,000 cells/well.After culture medium is replaced by the 0.1%BSA basal medium, cell and FGF-2 or be with or without albumin fusion proteins of the present invention and/or IL-1 α incubation 24 hours.Collect supernatant, (Endogen, Cambridge MA) carry out the IL-6 test with the ELISA test kit.
Before adding Alamar Blue, human lung fibroblast and FGF-2 or albumin fusion proteins of the present invention were cultivated on basal medium 3 days, assessed the effect to fibroblastic growth.FGF-2 should demonstrate stimulation at 10-2500ng/ml, and this can be used to compare with the stimulation of fusion rotein of the present invention.
The cell proliferation that mixes based on [3H] thymidine
Following [3H] thymidine mixes test and can be used to measure human cytokines (as growth factor protein) to the effect such as the cell proliferation of fibroblast, epithelial cell or immature muscle cell.
By incubation on the culture medium that does not contain serum 18 hours, make the Asia converge culture and be stuck in the G1 phase.Added human cytokines then 24 hours, during in the end 4 hours, with [3H] thymidine labelling culture, [3H] thymidine final concentration be 0.33 μ M (25Ci/mmol, Amersham, Arlington Heights, IL).[3H] thymidine that mixes was with 10% ice-cold trichloroacetic acid precipitation 24 hours.Then, cell is sequentially used 10% ice-cold trichloroacetic acid and icy water rinsing then.In 0.5M NaOH, after the cracking, compile lysate and PBS rinsing thing (500ml), measure radioactive amount.
The parkinson model
The forfeiture of motor function is because because the defective of the striatum dopamine that the degeneration of nigrostriatum dopaminergic projection neuron causes in the parkinson disease.A parkinson animal model of extensively identifying comprises systemic administration 1-methyl-4- phenyl 1,2,3,6-tetrahydropyridine (MPTP).In CNS, MPTP is absorbed by astrocyte, becomes 1-methyl-4-phenylpyridinium (MPP by monoamine oxidase-B catabolism
+) and discharge.Then, by the affine dopamine reuptake transport protein of height, MPP
+Accumulate in dopaminergic neuron on one's own initiative.Then by electrochemical gradient MPP
+Concentrate at mitochondrion, selectivity suppresses the nicotinamide adenine diphosphonic acid: ubiquinone oxide-reductase enzyme (complex I), therefore disturbed electron transport, and finally produce oxygen-derived free radicals.
Tissue culture's example show FGF-2 (basic FGF) have nutritional activities to substantia nigra dopaminergic neuron (Ferrari etc., Dev.Biol.1989).Recently; doctor's Unsicker seminar shows, uses FGF-2 with the gel foam graft and causes substantia nigra dopaminergic neuron protection almost completely to striatum, makes it avoid exposing relevant toxicity (Otto and Unsicker with MPTP; J.Neuroscience, 1990).
Data based on FGF-2; can estimate albumin fusion proteins of the present invention and determine whether it has the effect of similar to FGF-2 external enhancing dopaminergic neuron existence, but also in its protection striatum of body build-in test dopaminergic neuron avoid handling the effect of relevant damage with MPTP.At first in dopaminergic neuron cell culture example, the latent effect of vitro detection albumin fusion proteins of the present invention.Prepare culture by from the 14th day Wistar rat embryo of gestation, dissecting the midbrain base plate.Use trypsin disintegrated tissue, and with 200,000 cell/cm
2Density be inoculated on the glass cover slide of poly-ornithine (polyorthinine)-laminin bag quilt.Cell maintains on the EagleShi culture medium of the F12 culture medium that contains hormonal supplementation (N1) and Dulbecco improvement.After the external cultivation in 8 days, culture is fixed with paraformaldehyde, handles with tyrosine hydroxylase (specific marker of dopaminergic neuron), and carries out immunohistochemical staining.The dissociated cell culture of preparation from fetal rat.Changed culture medium, and added the factor at that time in per 3 days.
Because dopaminergic neuron is isolating from the 14th day animal of gestation, this developmental stage has passed through the period of dopaminergic precursor propagation, and on behalf of the number of the dopaminergic neuron of external existence, the increase of tyrosine hydroxylase immuning positive neuron number increase.Therefore, if human cytokines of the present invention shows as the existence that prolongs dopaminergic neuron, illustrate that then fusion rotein may participate in parkinson disease.
Embodiment 64: pancreatic beta cell is transplanted combined therapy
Transplanting is the common type of treatment autoimmune disease, particularly when target autologous tissue has been badly damaged.For example, restriction never in any form, pancreas transplantation and islet cell transplantation be the treatment commonly used of IDDM select (referring to for example, Stewart etc., Journal of Clinical Endocrinology ﹠amp; Metabolism86 (3): 984-988 (2001); Brunicardi, Transplant.Proc.28:2138-40 (1996); Kendall﹠amp; Robertson, Diabetes Metab.22:157-163 (1996); Hamano etc., Kove J.Med.Sci.42:93-104 (1996); Larsen ﹠amp; Stratta, Diabetes Metab.22:139-146 (1996) and Kinkhabwala etc., Am.J.Surg.171:516-520 (1996)).As any implantation method, transplantation treatment autoimmune patient comprises that the host who reduces transplanted tissue repels the processing of risk.Yet autoimmune disease comprises additionally and independently risk: the host's autoimmune response that is pre-existing in that destroys original autologous tissue will produce same damaging influence to transplanted tissue.Correspondingly, present invention resides in the method and composition that uses albumin fusion proteins combined immunization regulator of the present invention/immunosuppressant treatment autoimmune pancreas disease in the individuality that stands the autoimmune disease transplantation treatment.
According to the present invention, use above-mentioned compositions and preparaton based on the albumin fusions, prevent and treat by originally at host's intrasubject immune response of original autologous tissue cause to transplant organ, tissue or cells injury.Use and before transplanting or after transplanting, to carry out, use the 2-4 agent, often be separated by a week.
Following immunomodulator/immunosuppressant can be used with albumin fusions therapeutic agent of the present invention in islet cells or pancreas transplantation jointly, and these immunomodulator/immunosuppressant include but not limited to AI-401, CDP-571 (monoclonal antibody of anti-TNF), CG-1088, Diamyd (vaccine for diabetes), ICM3 (monoclonal antibody of anti-ICAM-3), linomide (Roquinimex), NBI-6024 (the peptide part of change), TM-27, VX-740 (HMR-3480), Caspase 8 protease inhibitor, Thalidomide, hOKT3 γ 1 (Ala-ala) (monoclonal antibody of anti-CD3), oraferon α, orally administering lactobacillus and LymphoStat-B
TM
The evaluation of embodiment 65:VH and VL domain and clone
A kind of method of identifying and cloning VH and VL domain from the cell line of expressing specific antibodies is with VH and the specific primer of VL the cDNA that obtains from the cell line of expressing antibodies to be carried out PCR.Briefly, isolation of RNA from cell line, and as the increase template of RT-PCR of the VH of antibody of EBV expression of cell lines and VL domain of design.Cell can
(LifeTechnologies, Rockville.MD) middle cracking is with the chloroform extraction of 1/5 volume for reagent.After adding chloroform, make solution room temperature incubation 10 minutes, on desk centrifuge 4 ℃ 14, centrifugal 15 minutes of 000rpm.Collect supernatant, with isopyknic isopropanol precipitating RNA.On desk centrifuge 4 ℃ 14,000rpm made sedimentary RNA agglomerating in centrifugal 15 minutes.After centrifugal, discard supernatant, with 75% ethanol rinsing.After the rinsing, RNA is once more centrifugal 5 minutes of 4 ℃ of 800rpm.Discard supernatant, make little group at air drying.RNA is dissolved in the DEPC water, be heated to 60 ℃ 10 minutes.Determine the amount of RNA by spectrodensitometry.
According to means known in the art, can use reverse transcription and hexamer primer synthetic cDNA from 1.5-2.5 microgram RNA at random.Use cDNA as template pcr amplification VH and VL domain then.The primer of VH and VL gene of being used for increasing is presented in the table 6.Wall scroll 5 ' primer and wall scroll 3 ' primer are used in general PCR reaction.Sometimes, prescribe a time limit when the amount that can utilize the RNA template has, or, can use 5 ' and/or 3 ' primer sets for higher efficient.For example, in single PCR reaction, use all 5 VH-5 ' primers and all JH3 ' primers sometimes.In the 50 microlitre volumes of the high-fidelity Taq polymerase that contains 1 * PCR buffer, every kind of dNTP of 2mM, 0.7 unit, 5 ' primer mixture, 3 ' primer mixture and 7.5 microlitre cDNA, carry out the PCR reaction.The two 5 ' and 3 ' primer mixture of VH and VL can be by with 22 picomoles and the 28 picomoles acquisition that pools together respectively of each wall scroll primer.The PCR condition is: 96 ℃ 5 minutes; It then is 94 ℃ 1 minute, 50 ℃ of 25 circulation 1 minute and 72 ℃ 1 minute; Then be one and extended 72 ℃ of circulations 10 minutes.After reaction was finished, sample cell was stored in 4 ℃.
Table 6: the primer sequence of be used for increasing VH and VL domain
The primer title
SEQ ID NO
Primer sequence (5 '-3 ')
The VH primer
Hu?VH1-5’ 62 CAGGTGCAGCTGGTGCAGTCTGG
Hu?VH2-5’ 63 CAGGTCAACTTAAGGGAGTCTGG
Hu?VH3-5’ 64 GAGGTGCAGCTGGTGGAGTCTGG
Hu?VH4-5’ 65 CAGGTGCAGCTGCAGGAGTCGGG
Hu?VH5-5’ 66 GAGGTGCAGCTGTTGCAGTCTGC
Hu?VH6-5’ 67 CAGGTACAGCTGCAGCAGTCAGG
Hu?JH1，2-5’68 TGAGGAGACGGTGACCAGGGTGCC
Hu?JH3-5’ 69 TGAAGAGACGGTGACCATTGTCCC
Hu?JH4，5-5’70 TGAGGAGACGGTGACCAGGGTTCC
Hu?JH6-5’ 71 TGAGGAGACGGTGACCGTGGTCCC
The VL primer
Hu?Vκ1-5’ 72 GACATCCAGATGACCCAGTCTCC
Hu?Vκ2a-5’?73 GATGTTGTGATGACTCAGTCTCC
Hu?Vκ2b-5’?74 GATATTGTGATGACTCAGTCTCC
Hu?Vκ3-5’ 75 GAAATTGTGTTGACGCAGTCTCC
Hu?Vκ4-5’ 76 GACATCGTGATGACCCAGTCTCC
Hu?Vκ5-5’ 77 GAAACGACACTCACGCAGTCTCC
Hu?Vκ6-5’ 78 GAAATTGTGCTGACTCAGTCTCC
Hu?Vλ1-5’ 79 CAGTCTGTGTTGACGCAGCCGCC
Hu?Vλ2-5’ 80 CAGTCTGCCCTGACTCAGCCTGC
Hu?Vλ3-5’ 81 TCCTATGTGCTGACTCAGCCACC
Hu?Vλ3b-5’?82 TCTTCTGAGCTGACTCAGGACCC
Hu?Vλ4-5’ 83 CACGTTATACTGACTCAACCGCC
Hu?Vλ5-5’ 84 CAGGCTGTGCTCACTCAGCCGTC
Hu?Vλ6-5’ 85 AATTTTATGCTGACTCAGCCCCA
Hu?Jκ1-3’ 86 ACGTTTGATTTCCACCTTGGTCCC
Hu?Jκ2-3’ 87 ACGTTTGATCTCCAGCTTGGTCCC
Hu?Jκ3-3’ 88 ACGTTTGATATCCACTTTGGTCCC
Hu?Jκ4-3’ 89 ACGTTTGATCTCCACCTTGGTCCC
Hu?Jκ5-3’ 90 ACGTTTAATCTCCAGTCGTGTCCC
Hu?Jλ1-3’ 91 CAGTCTGTGTTGACGCAGCCGCC
Hu?Jλ2-3’ 92 CAGTCTGCCCTGACTCAGCCTGC
Hu?Jλ3-3’ 93 TCCTATGTGCTGACTCAGCCACC
Hu?Jλ3b-3’?94 TCTTCTGAGCTGACTCAGGACCC
Hu?Jλ4-3’ 95 CACGTTATACTGACTCAACCGCC
Hu?Jλ5-3’ 96 CAGGCTGTGCTCACTCAGCCGTC
Hu?Jλ6-3’ 97 AATTTTATGCTGACTCAGCCCCA
PCR sample electrophoresis on 1.3% agarose gel then.DNA band (the VH domain :～506 base pairs of expection size; VL domain: 344 base pairs) downcut, use the means known in the art purification from glue.The PCR product of purification can connect into the PCR cloning vehicle (the TA carrier, from InvitrogenInc., Carlsbad, CA).Can be behind the transfection Escherichia coli through the single clone's of indigo plant/white screening and separating PCR product.Ke Long PCR product can use the method order-checking that is generally known in the art then.
The PCR band that contains VH domain and VL domain also can be used to produce total length Ig expression vector.VH and VL domain can be cloned the carrier that into contains heavy chain (as IgG 1 or IgG 4) or light chain (human κ or human λ) constant region nucleotide sequence, when proper host cell was advanced in transfection, complete heavy chain or light chain molecule can be expressed from these carriers like this.In addition, when clone's heavy chain and light chain when all (from one or two carrier) expresses in a cell line, they can be assembled into the complete functional antibodies molecule that is secreted in the cell culture medium.The method that the polynucleotide in use coding VH and VL antibody structure territory produce the expression vector of coding complete antibody molecule is known in the art.
Embodiment 66:HA-cytokine or HA-growth factor fusion protein (such as NGF, BDNFa,
BDNFb and BDNFc) preparation
The cDNA of the cells of interest factor or somatomedin (as NGF) can separate by many methods, and these methods comprise uses the PCR of standard method from the cDNA library, by RT-PCR and the synthetic oligonucleotide primer thing by using a series of overlappings.All these proteic nucleotide sequences are all known and can obtain.Can cut out cDNA at 5 ' and 3 ' end and produce restriction site, so that can use oligonucleotide joint cDNA to be cloned the carrier that into contains HA cDNA.Can use or not use intervening sequence to be cloned into N or C-terminal.The cDNA of NGF (or other cytokine) is cloned into such as carriers such as pPPC0005 (Fig. 2), pScCHSA, pScNHSA or pC4:HSA, downcut the expressed intact box and be inserted into plasmid pSAC35 from these carriers then, albumin fusion proteins can be expressed in yeast.Can collect excretory albumin fusion proteins from yeast then, and from culture medium purification, test its biologic activity.To the expression in mammal cell line, adopt similar scheme, except the expression cassette that uses utilizes mammiferous promoter, targeting sequencing and terminator (referring to embodiment 1).Downcut this expression cassette then, insert the plasmid that is fit to transfection mammalian cell system.
The preparation of embodiment 67:HA-IFN fusion rotein (as IFN α)
The cDNA of interferon interested (as IFN α) can separate by many methods, and these methods comprise but do not get rid of and use the PCR of standard method from the cDNA library, by RT-PCR and the synthetic oligonucleotide primer thing by using a series of overlappings.The nucleotide sequence of interferon (as IFN α) is all known and can obtain, for example at United States Patent (USP) the 5th, 326, and 859 and 4,588, No. 585, in EP32134 and as the public database of GenBank.Can cut out cDNA at 5 ' and 3 ' end and produce restriction site, so that can use oligonucleotide joint cDNA to be cloned the carrier that into contains HA cDNA.Can use or not use intervening sequence to be cloned into HA sequence of N or C-terminal.The cDNA of IFN α (or other interferon) is cloned into such as carriers such as pPPC0005 (Fig. 2), pScCHSA, pScNHSA or pC4:HSA, downcut the expressed intact box and be inserted into plasmid pSAC35 from these carriers then, albumin fusion proteins can be expressed in yeast.Collect excretory albumin fusion proteins from yeast then, and from culture medium purification, test its biologic activity.To the expression in mammal cell line, adopt similar scheme, except the expression cassette that uses utilizes mammiferous promoter, targeting sequencing and terminator (referring to embodiment 1).Downcut this expression cassette then, insert the plasmid that is fit to transfection mammalian cell system.
From bottle, reclaim albumen to greatest extent
Even when with low concentration when packaged, albumin fusion proteins of the present invention has high stability.In addition, although protein concentration is low, even add when reducing with bonded other albumen of bottle wall when aqueous solution does not comprise, the fusion rotein of still observing reclaims.The recovery of the HA-IFN solution that bottle stores and mother solution are relatively.6 or 30 μ g/ml HA-IFN solution are put in the bottle, are stored in 4 ℃.After 48 or 72 hours, take out the volume that originally equals the 10ng sample, in IFN sandwich ELISA, measure.The estimated value of estimated value and high concentration mother solution relatively.As shown, do not have the loss of sample in these bottles substantially, be illustrated as and stop sample loss to the bottle wall and to add such as albuminised exogenous material be unnecessary.
The body internal stability and the bioavailability of HA-α-IFN fusant
Be to determine the body internal stability and the bioavailability of HA-α-IFN fusant molecule, the fusant molecule (from yeast) of purification is administered to monkey.Serum half-life and bioavailability from the soluble prolongation of pharmaceutical composition of HA-α-IFN fusant preparation.Correspondingly, can prepare to compare and contain the more pharmaceutical composition of low dosage alpha-interferon activity with natural interferon-alpha molecule.
The pharmaceutical composition that contains HA-α-IFN fusant can be used to treat or prevent to suffer from any can be by using the disease that α-IFN regulates or the disease of patient of morbid state.These diseases include but not limited to hairy cell, Kaposi sarcoma, genital wart and anus wart, chronic hepatitis B, chronic non-A non-B hepatitis, hepatitis C particularly, hepatitis D, chronic granulocytic leukemia, renal cell carcinoma, bladder cancer, ovarian cancer and cervical cancer, skin carcinoma, recurrence type respiratory papillomatosis, Fei Hejiejinshi and cutaneous T cell lymphoma, melanoma, multiple myeloma, AIDS, multiple sclerosis, glioblastomas etc. are (referring to Interferon Alpha (interferon-ALPHA),: AHFS Drug Information (AHFS drug information), 1997).
Correspondingly, the present invention includes the pharmaceutical composition of preparing with the dosage that is fit to human administration that contains HA-α-IFN fusion rotein, polypeptide or peptide.The present invention also comprises the patient's of this treatment of treatment needs method, and this treatment comprises the step of using the pharmaceutical composition that contains at least a HA-α-IFN fusion rotein, polypeptide or peptide at least.
Difunctional HA-α-IFN fusant
Can modify HA-α-IFN expression vector makes it comprise the insertion that is used for difunctional HA-α-IFN expressing fusion protein.For example, remove or two termination codoies in transfer code sequence downstream after, the cDNA of second kind of proteins of interest can insert the downstream of " rHA-IFN " sequence, and is in same reading frame with it.
In a kind of difunctional HA-α-IFN fusion rotein of form, an end that can be fused to fusion molecule HA component at the antibody or the fragment of bone-marrow-derived lymphocyte stimulatory protein(SP) (GenBank searching number 4455139) or polypeptide.This bifunctional protein can be used for regulating any immune response by α-IFN component generation of fusant.
The preparation of embodiment 68:HA scfv fusion protein
Several method can produce single-chain antibody, these methods include but not limited to: from phage library select, cDNA by clonal antibody comes the variable region of clone-specific antibody and uses the flank constant region to clone the variable region as primer, or by synthetic oligonucleotide corresponding to any specific antibody variable region.Can cut out cDNA to produce restriction site, so that can use oligonucleotide joint cDNA to be cloned the carrier that into contains HA cDNA at 5 ' and 3 ' end.Can use or not use intervening sequence to be cloned into N or the C-terminal of HA.Cell cDNA is cloned into such as carriers such as pPPC0005 (Fig. 2), pScCHSA, pScNHSA or pC4:HSA, downcut expressed intact boxes and be inserted into plasmid pSAC35 from these carriers then, albumin fusion proteins can be expressed in yeast.
In fusion molecule of the present invention, V
HAnd V
LCan be connected by one of following method or its: at V
HC-terminal and V
LPeptide linker between the N-terminal; V
HAnd V
LBetween Kex2p protease cutting site so that after the secretion with both cuttings separately, self-then combination; And can make V
HAnd V
LBetween form the localized cystine residue of mode that disulfide bond connects together them.One is optionally selected is with V
HPlace HA or the segmental N-terminal of HA domain, V
LPlace HA or the segmental C-terminal of HA domain.
Collect excretory albumin fusion proteins from yeast then, and from culture medium purification, test its activity.To the expression in mammal cell line, adopt similar scheme, except the expression cassette that uses utilizes mammiferous promoter, targeting sequencing and terminator (referring to embodiment 1).Downcut this expression cassette then, insert the plasmid that is fit to transfection mammalian cell system.The antibody of Chan Shenging can use immuno-chemical method purification from culture medium of standard by this way, and tests itself and its antigenic combination.
Embodiment 69: as the inhibition factor of HA fusion rotein and peptide (such as the HA-antiviral,
HA-is antibiotic, HA-enzyme inhibitor and HA-antiallergic albumen) preparation
The cDNA of peptide interested (as antibacterial peptide) can separate by many methods, and these methods include but not limited to use the PCR of standard method from the cDNA library, by RT-PCR and the synthetic oligonucleotide primer thing by using a series of overlappings.Can cut out cDNA to produce restriction site, so that can use oligonucleotide joint cDNA to be cloned the carrier that into contains HA cDNA at 5 ' and 3 ' end.Can use or not use intervening sequence to be cloned into N or C-terminal.The cDNA of peptide is cloned into such as carriers such as pPPC0005 (Fig. 2), pScCHSA, pScNHSA or pC4:HSA, downcut expressed intact boxes and insert plasmid pSAC35 from these carriers then, albumin fusion proteins can be expressed in yeast.Can collect excretory albumin fusion proteins from yeast then, and from culture medium purification, test its biologic activity.To the expression in mammal cell line, adopt similar scheme, except the expression cassette that uses utilizes mammiferous promoter, targeting sequencing and terminator (referring to embodiment 1).Downcut this expression cassette then, insert the plasmid that is fit to transfection mammalian cell system.
Embodiment 70: the preparation of targeting HA fusion rotein
The cDNA of proteins of interest can use the standard molecular biology method to separate or can use synthetic acquisition of oligonucleotide of several overlappings from the cDNA library.Can transform nucleotide suitable among the cDNA and form restriction site easily, also allow albumen cDNA to be connected to albumin cDNA.Can guide albumen to enter the targeting proteins of cell or the cDNA of peptide (as single-chain antibody or peptide, as nuclear localization signal) also can be fused to albuminised another end.Protein of interest and targeting peptide are cloned into such as carriers such as pPPC0005 (Fig. 2), pScCHSA, pScNHSA or pC4:HSA, and these carriers allow to merge with albumin cDNA.Albuminised by this way N and C-terminal all are fused on other albumen.Downcut the cDNA that merges from pPPC0005 then, insert, albumin fusion proteins can be expressed in yeast such as plasmids such as pSAC35.All such schemes can use the standard molecular biology method to carry out.Can collect excretory albumin fusion proteins from yeast, and from culture medium purification, use suitable biochemistry and test its biologic activity biology and the targeting activity.
Embodiment 71: the generation of construct ID2249 (IFN α 2-HSA)
Construct ID2249 (pSAC35:IFN α 2.HSA) comprises the DNA of coding IFN α 2 albumin fusion proteins in saccharomyces cerevisiae expression vector pSAC35, this albumin fusion proteins has the chimeric targeting sequencing of HSA, and the back is the aminoterminal IFN α 2 proteic mature forms (being C1-E165) that are fused to the HSA mature form.
The clone of IFN α 2cDNA
Use the polynucleotide of following primer IFN α 2-1 and IFN α 2-2PCR amplification coding IFN α 2.The pcr amplification thing cuts with Sal I/Cla I, and connects to advance the pScCHSA of Xho I/Cla I cutting.Construct ID 2249 coding contains the chimeric targeting sequencing of HSA, IFN α 2 mature forms, then is the proteic albumin fusion proteins of ripe HSA.
2 oligonucleotide of polynucleotide synthetic pcr amplification coding IFN α 2 mature forms that are fit to) (IFN α 2-1 and IFN α 2-2:
IFNα2-1：
5’-CGCGCGC
GTCGACAAAAGATGTGATCTGCCTCAAACCCACA-3’(SEQ?ID?NO：348)
IFNα2-2：
5’-GCGCGC
ATCGATGAGCAACCTCACTCTTGTGTGCATCTTCCTTACTTCTTAAACTTTCT-3’(SEQ?ID?NO：349)
IFN α 2-1 primer contains the nucleotide of a Sal I cloning site (underscore demonstration), last 3 amino acid residues of the chimeric HSA targeting sequencing of coding and 22 nucleotide (black matrix demonstration) of preceding 7 amino acid residues of coding IFN α 2 mature forms.In IFN α 2-2, Cla I site (underscore demonstration) and DNA thereafter are the reverse complementary sequences of preceding 10 the amino acid whose DNA of encoding mature HSA albumen, and last 22 nucleotide (black matrix demonstration) are the reverse complementary sequences (referring to embodiment 2) of the DNA of coding IFN α 2 last 7 amino acid residues.The pcr amplification thing of IFN α 2-HSA uses these primer generations, purification, digests with Sal I and Cla I restriction endonuclease, and the clone enters the Xho I and the Cla I site of pScCHSA carrier.After sequence was proved, the expression cassette of these IFN α 2 albumin fusion proteins of encoding was advanced the pSAC35 of Not I digestion by sub-clone.
In addition, the existing of N-terminal susceptible of proof expection IFN α 2 sequences of the albumin fusion proteins of expressing by the amino acid sequencing analysis (vide infra).
Use other IFN α 2 albumin fusion proteins of different targeting sequencings to make up (referring to embodiment 2) by means known in the art.The example of various targeting sequencings includes but not limited to invertase " INV " (construct 2343 and 2410) and mating factor α " MAF " (construct 2366).These IFN α 2 albumin fusion proteins can be advanced such as mammalian expression vectors (referring to embodiment 5) such as foregoing pC4 (construct 2382) and pEE12.1 by sub-clone.Also can make up IFN α 2 albumin fusion proteins (construct 2381) of therapeutic partial fusion to the HSA C-terminal.
IFN α 2 albumin fusion proteins of the present invention can comprise the mature form (being that Asp-25 is to Leu-609) of the HSA that is fused to IFN α 2 mature forms (being that Cys-1 is to Glu-165) N-or C-end.In an embodiment of the invention, IFN α 2 albumin fusion proteins of the present invention further comprise the signal sequence that guides newborn fused polypeptide to enter the host's secretory pathway that is used to express.In a further embodiment, the signal peptide of signal sequence encoding is removed, and ripe IFN α 2 albumin fusion proteins direct secretion are in culture medium.IFN α 2 albumin fusion proteins of the present invention can comprise the allos signal sequence, include but not limited to that MAF, INV, Ig, fine albumen (fibulin) B, bunch egg collection are white, the variant (including but not limited to chimeric HSA/MAF targeting sequencing) of IGFBP (insulin-like growth factor binding protein) 4, HSA targeting sequencing, or other allos signal sequence known in the art.In one embodiment, IFN α 2 albumin fusion proteins of the present invention comprise natural IFN α 2.In a further embodiment, IFN α 2 albumin fusion proteins of the present invention further comprise the methionine residues of N-terminal.The polynucleotide (comprising fragment and/or variant) of these polypeptide of encoding are also included among the present invention.
The expression of construct ID 2249 and purification
In saccharomyces cerevisiae, express
Construct 2249 is transformed into Wine brewing yeast strain BXP10 and carries out (referring to embodiment 3) by means known in the art.Growing can be at the stable phase collecting cell after 72 hours.By collecting supernatant at 3000g clarification in 10 minutes cell.Analyze expression by detecting with anti-HSA serum (Kent Laboratories) or as an anti-immunoblotting.Can obtain IFN α 2 albumin fusion proteins of the about 88.5kDa of molecular weight.
Purification from the brewing yeast cell supernatant
The cell conditioned medium liquid that contains IFN α 2 albumin fusion proteins of expressing from construct ID 2249 brewing yeast cell can be used Dyax peptide affinity column small scale purification (referring to embodiment 4), or by following 5 step large scale purifications: diafiltration, use the quick post of DEAE-Sepharose anion-exchange chromatography, use butyl 650S post hydrophobic interaction chromatography (HIC), use the cation-exchange chromatography of quick post of SP-Sepharose or Blue-Sepharose chromatography and use the efficient chromatography (referring to embodiment 4) of Q-Sepharose high-efficiency column chromatography.IFN α 2 albumin fusion proteins can with 100-250mM NaCl from the quick post of DEAE-Sepharose eluting, with 150-250mM NaCl eluting and with 5-7.5mS/cm eluting from the Q-Sepharose high-efficiency column from the quick post of SP-Sepharose.The N-terminal order-checking should produce the sequence C DLPQ (SEQ ID NO:98) corresponding to IFN α 2 mature forms.
Use external ISRE-SEAP test can measure the activity of IFN α 2
Method
The result
The purification prepared product explanation of IFN α 2-HSA is in the ISRE-SEAP test, and concentration range is from 10
-1To 10
1Ng/mL (referring to Fig. 4) or 10
-10To 10
-8The linear relatively increase of ng/mL (referring to Fig. 5).
Induce OAS in the interferon-ALPHA fusant body of construct ID 2249 codings
Method
OAS enzyme (2 '-5 '-oligoadenylate synthetase) activates at the disturbed element of transcriptional level in response to viral infection resisting.The effect of interferon construct can be by measuring from monkey acquisition blood sample of handling and the transcriptional activation of analyzing 2 kinds of OAS mRNA (p41 and p69) these samples.Merely hit from every group of 4 animal pers in 7 different time points (the 0th day, the 1st day, the 2nd day, the 4th day, the 8th day, the 10th day and the 14th day) and to obtain the whole blood of volume 0.5mL.Each group comprise media contrast, intravenous injection in the 1st day 30 μ g/kg HSA-IFN, the 1st day subcutaneous injection 30 μ g/kg HSA-IFN, the 1st day subcutaneous injection 300 μ g/kg HSA-IFN and at the 1st, 3 and 5 day subcutaneous injection 40 μ g/kg interferon-ALPHA (Schering-Plough) as positive control.Use p41-OAS and p69-OAS specific probe to determine the level of p41 and p69mRNA transcript by real-time quantitative PCR (Taqman).The OASmRNA level is next quantitatively with respect to the endogenous contrast of 18S ribosomal RNA.The albumin fusant of construct 2249 codings can carry out similar experiment.
The result
Opposite with the monkey that IFN α handles, in the monkey that the HSA-interferon is handled, observe the remarkable increase (data of p41 are referring to Fig. 6) of the mRNA transcript level of p41 and p69OAS.Effect has continued nearly 10 days.
Embodiment 72:IFN α albumin fusion proteins indication
IFN α albumin fusion proteins (include but not limited to construct 2249,2343,2410,2366,2382 and 2381 codings those) can be used to treatment, prevention, improves and/or detects multiple sclerosis.Other indication includes but not limited to viral infection, comprises severe acute respiratory syndrome (SARS) and other coronavirus infection; Filovirus (filoviruses) includes but not limited to Ebola virus (Ebolavirus) and Marburg virus (Marburg virus); Arenavirus (arenavirus) includes but not limited to pichinde virus (Pichende virus), Lassa virus (Lassa virus), Junin virus (Junin virus), MAC (Machupo virus), guanarito virus (Guanarito virus); And lymphocytic choriomeningitis virus (LCMV); Bunyavirus (bunyavirus) includes but not limited to Punta Toro virus (Punta toro virus), crimean-Congo hemorrhagic fever virus (Crimean-Congohemorrhagic fever virus), pappataci fever virus (sandfly fever virus), Rift valley fever virus (Rift Valley fever virus), La Crosse virus (La Crosse virus) and Hantaan virus (hantavirus); Banzi virus includes but not limited to yellow heat, BAN (Banzi virus), west nile virus, dengue virus, Japanese encephalitis virus, tick borne encephalitis, Omsk hemorrhagic fever (Omsk HemorrhagicFever) and Kyasanur Forest disease virus (Kyasanur Forest Disease virus); Togavirus includes but not limited to Venezuela, east, Western equine encephalitis virus, ross river virus (Ross River virus) and rubella virus (Rubella virus); Vaccinia subgroup virus (orthopox virus) includes but not limited to vaccine, cowpox, variola and monkeypox; Herpesvirus; Influenza A/B; Respiratory syncytial virus (RSV); Parainfluenza; Measles; Rhinovirus; Adenovirus; Semliki Forest virus (Semliki Forest virus); Viral hemorrhagic fever; Baculovirus (Rhabdovirus); Paramyxovirus includes but not limited to Nipah virus (Nipahvirus) and Heng Dela virus (Hendra virus); Other virokine that is accredited as the high priority disease factor with U.S. CDC (is A, B and the C class factor; Referring to for example Moran, Emerg.Med.Clin.North.Am.2002; 20 (2): 311-30 and Darling etc., Emerg.Med.Clin.North.Am.2002; 20 (2): 273-309).In one embodiment, IFN α-albumin fusion proteins or IFN heterozygosis fusion rotein and CCR5 antagonist combination are used, further with only ribavirin, IL-2, IL-12, pentafuside in one of at least or associating inverase treatment (as HAART), prepare adult and HIV-1 infection, HCV or the HIV-1 of pediatric patients and the medicine of HCV coinfection that treatment was treated and lived through in treatment for the first time.
Embodiment 73:HSA-IFN α 2b associating ribavirin is slow at genotype 1, first interferon therapy
Antiviral activity among property hepatitis C (HCV) patient
Background:
Conventional therapy at genotype 1, first interferon therapy (first IFN treatment) HCV patient uses interferon-ALPHA associating 48 weeks of ribavirin (RBV).Yet this treatment has significant application limitation.Because the known side effect of current interferon therapy, use interferon at every turn after, patient's quality of life has reduced significantly.Current scheme requires administration at least weekly, and prolong the period that causes reducing quality of life.Therefore a large amount of patient's therapy discontinued have the research report, and interruption rate surpasses 50%.In addition, current interferon therapy also has the remarkable hematology of significant proportion to reduce, and this need reduce the dosage of RBV, or the more important thing is, temporarily stops the interferon therapy method until blood values normalization.Therefore, the therapeutic scheme that needs to improve the first interferon therapy patient's genotype 1HCV of treatment is obviously arranged.
Principle:
Produce HSA-IFN α 2b by merging ripe albuminised C-terminal hereditarily to the N-terminal of ripe Interferon Alpha-2b.Safety and efficient with HSA-IFN α 2b associating RBV treatment are estimated in the HCV human patients of genotype 1, first interferon therapy in the clinical research that active control (actively controlled) arranged, and compare with the conventional therapy with PEG-IFN α-2a (PEG-IFN) associating RBV as active control.
Method:
The human HCV patient of genotype 1, first interferon therapy treats with HSA-IFN α 2b or active control PEG-IFN associating ribavirin (RBV).More specifically, 458 human subject are divided into 4 subcutaneous (sc) treatment groups at random: (a) PEG-IFN (Q1w) of weekly 180 μ g dosage; (b) the HSA-IFN α 2B (Q2w) of per 2 all one time 900 μ g dosage; (c) the HSA-IFN α 2b Q2w of 1200 μ g dosage; Or (d) per 4 the week one time 1200 μ g dosage HSA-IFN α 2b (Q4w).
Each curee of each treatment group also accepts 1000-1200mg/ days RBV according to body weight.Stratified basis comprises body weight index (BMI) (＜25kg/m
2Or 〉=25kg/m
2) and HCV RNA titre (＜800,000IU/ml or 〉=800,000IU/ml).
The treatment persistent period of this research was 48 weeks, with following up a case by regular visits in 24 weeks.The main effectiveness terminal point of this research is that the virusology that continues is replied (SVR), is defined as treatment and finishes back 24 week detections less than virus load (HCV RNA＜10IU/ml).
HCV RNA titre is to use PCR in real time test Quantasure
TM(Labcorp) measure, its range of sensitivity (quantitatively level (LOQ)) is 43IU to 6,900 ten thousand IU/mL, and detection level (LOD) is 10IU/mL.Use standard technique known in the art to measure alanine transaminase (ALT) and blood effect, comprise neutrophil cell absolute counting (ANC), hemoglobin and platelet count.
The patient of purpose treatment (ITT) is defined as the experimenter that all randomizations and treatment are organized in each treatment, and no matter whether the patient has any missing data point or withdraw from this research.The patient of the purpose treatment (MITT) of improvement is defined as the patient that can conceivablely have the prescription on individual diagnosis of the 24th week based on their record date in this research.
Result and discussion:
Experimenter's demography, antiviral response and hematology's minimizing are summarised in table 7 (preliminary interim analysis) and table 11 (final interim analysis).Generally speaking, all 4 kinds of therapeutic schemes tolerances fine, between the treatment group in 3-4 level lab index value or because the interruption of side effect does not have significant difference.
Table 7.
The preliminary interim analysis of demography, antiviral response and blood effect
*P value＜0.05 HSA-IFN α 2b, 1200 μ g Q2w are to Q4w;
Value 0.068HSA-IFN α 2b 1200 μ g Q2w are right
PEG-IFN;
#P value＜0.05 HSA-IFN α 2B, 1200 μ g Q4w are to PEG-IFN;
*P value＜0.05 HSA-IFN α 2b, 1200 μ g Q4w are to all other groups
Table 8.
The final interim analysis of demography, antiviral response and blood effect
*P value＜0.05 HSA-IFN α 2b, 1200 μ g Q2w are to Q4w;
It is right to be worth 0.15 HSA-IFN α 2b, 1200 μ g Q2w
PEG-IFN;
#P value＜0.05 HSA-IFN α 2B, 1200 μ g Q4w are to PEG-IFN;
*P value＜0.05 HSA-IFN α 2b, 1200 μ gQ4w are to all other groups
The antiviral response indication signal of SVR
The antiviral response of SVR indication signal definition (has HCV RNA titre＜LOQ), with second stage slope＞0.6log/wk (second slope) for the negative HCV RNA titre of treatment during the 12nd week.Two kinds of activity of each stage indication of the antiviral response slope of curve.Phase I is represented the direct antiviral activity that responds.Second stage predicted treatment chemical compound is to the destruction of HCV infection cell.Therefore the second stage slope is the good indication signal of SVR (positive predictive value (PPV)＞90%) in the value of＞0.6log/wk.
The SVR antiviral response indication signal that the 12nd all ITT are organized in HSA-IFN α 2b 1200 μ g Q2w treatments is the highest, compare with 49% with 75/114 experimenter or 65.8% ((final interim analysis) (70/112 or 62.5% (preliminary interim analysis))) of PEG-IFN contrast treatment, among the HSA-IFN α 2b 1200 μ g Q2w, 82/110 experimenter or 74.5% ((final interim analysis) (77/104 or 74.0% (preliminary analysis))) having HCV RNA negative levels (be that level is lower than LOQ (＜43IU/mL)), and 58% shows second stage slope＞0.6log/wk.These data show antiviral activity that HSA-IFN α 2b 1200 μ g Q2w therapeutic schemes have at least with the conventional PEG-IFN treatment in the 12nd week quite.Because compare with PEG-IFN contrast treatment, in HSA-IFN α 2b 1200 μ g Q2w therapeutic schemes, RNA feminine gender (having patient's number that HCV RNA titre level is lower than LOQ) during the 12nd week is about 9% (final interim analysis) and 12% higher (preliminary interim analysis), and the second stage slope be about 9% higher (to the preliminary interim analysis of final sum the two), possible HSA-IFN α 2b 1200 μ g Q2w therapeutic schemes can cause than the higher effect of conventional PEG-IFN treatment.In HSA-IFN α 2b 900 μ g Q2w and HSA-IFN α 2b 1200 μ g Q4w treatment groups, have HCV RNA negative patients number and treat similar to conventional PEG-IFN.
At the SVR antiviral response indication signal indication in the 20th and 24 whens week is that HCV RNA titre is lower than detection level (LOD) (＜10IU/mL) experimenter.SVR antiviral response indication signal when HSA-IFN α 2b 1200 μ g Q2w treatments organized for the 20th week is the highest, compare with 77/114 or 67.5% (final interim analysis) of PEG-IFN contrast treatment, in the HSA-IFN α 2b 1200 μ g Q2w treatment groups, 82/110 or 74.5% (final interim analysis) have detection less than the HCV rna level (promptly＜10IU/mL).Similarly, SVR antiviral response indication signal when IFN α 2b 1200 μ g Q2w treatments organized for the 24th week is the highest, wherein 64/91 experimenter or 70.3% (final interim analysis) have detection less than the HCV rna level, and PEG-IFN contrast treatment have 57/90 or 63.3% have detection less than the HCV rna level.The data in the 20th and 24 whens week show that all HSA-IFN α 2b 1200 μ g Q2w therapeutic schemes have at least and suitable safety and the antiviral activity of conventional PEG-IFN treatment, and have improved administration arrangement.
The horizontal normalization of ALT
Measuring of common patient's liver function is the level of alanine transaminase (ALT).A feature of HCV infected patient is the impaired high Serum ALT levels of indication liver.Therefore, the horizontal normalization of ALT is represented the improvement of liver function, to have favourable prognosis in response to treatment.Although all therapeutic schemes have the ability of some normalization ALT levels; in HSA-IFN α 2b 1200 μ g Q4w therapeutic schemes, observe remarkable influence; compare with conventional PEG-IFN treatment; in HSA-IFN α 2b 1200 μ g Q4w therapeutic schemes, surpass the ALT level that the many patient'ss (preliminary interim analysis of final sum) of twice have obtained normalization.Therefore, on the normalization liver function, the HSA-IFN α 2b of 1200 μ g Q4w dosage is unexpectedly more effective than the HCV patient of conventional PEG-IFN therapeutic gene type 1, first interferon therapy.
The blood effect
Be maximization SVR ratio, in combined treatment, guarantee to comply with and the IFN and the RBV that are exposed to full dosage is crucial.
In with IFN and RBV therapeutic alliance process, it is common that the hematology reduces.Because RBV induces the minimizing of the hemoglobin (Hb) of haemolysis to require to reduce the dosage of RBV.Especially, Hb＜12g/dL required to reduce RBV from 1000-1200mg/ days to 800mg/ days.RBV dosage is crucial to stoping the HCV recurrence.HSA-IFN α 2b 1200 μ g Q4w therapeutic schemes unexpectedly have minimizing (52% 65% (the final interim analysis) than PEG-IFN of Hb＜12g/dL significantly still less; 49.1% 64% (preliminary interim analysis)) than PEG-IFN.This may be interpreted as with HSA-IFN α 2b 1200 μ gQ4w therapeutic schemes and has realized lower relapse rate, allows improved SVR.
ANC＜750/mm
3Minimizing require to reduce the dosage of IFN component in the therapeutic alliance.Astoundingly, HSA-IFN α 2b 1200 μ g Q4w therapeutic schemes have remarkable ANC＜750/mm still less than PEG-IFN
3(be respectively 6% to 20.2% (final interim analysis); 4.3% to 17.5% (preliminary interim analysis)).This can be interpreted as higher SVE ratio once more, considers that the required dosage still less of HSA-IFN α 2b 1200 μ g Q4w therapeutic schemes reduces.
The 12nd all HSA-IFN α 2b Q2w with PEG-IFN treatment group similar hematology have been taken place to be reduced.Yet astoundingly, observed hematology reduces than observed low by about 75% in PEG-IFN treatment group in the 12nd all HSA-IFN α 2b 1200 μ g Q4w treatment groups.These results show with conventional PEG-IFN treatment and compare that HSA-IFN α 2b Q4w can provide better safety and improved relapse rate.
The antiviral response in the 12nd week after treatment is finished
The lasting virus in the 12nd week was replied (SVR12) and is compared with 54.4% of PEG-IFN after the treatment that ITT analyzes was finished, 900Q2w is 59.3%, 1200Q2w be 55.5% and 1200Q4w be 52.6%.Adhere among the patient of IFN and RBV treatment 〉=80%, compare the SVR12 ratio of 900Q2w and 1200Q2w the highest (being respectively 73.8% and 72.0%) with 63.0% of PEG-IFN with 67.5% of 1200Q4w.Attention: heavier patient (〉=75kg) in, when the SVR12 of PEG-IFN ratio reduced, the SVR12 ratio of alb-IFN remained unchanged.Similarly, in 〉=80% patient who adheres to treating, the relapse rate of all alb-IFN group reduces, and to compare 900Q2w (13.0%) the most remarkable with PEG-IFN (29.7%), p=0.0261.Generally speaking, the measurable SVR12 of antiviral response feature in treatment 12 week, and the response characteristic of 1200Q4w group has the highest positive predictive value.Because the interruption of side effect is 6.1% in the PEG-IFN group, by contrast, 900Q2w is 9.3%, and 1200Q4w is 12.1% and the 1200Q2w group is 19.1%.The hematology of alb-IFN 1200Q4w group reduces minimum, and is suitable between other group.The quality of life of measuring by SF-36 is that the 900Q2w group is best.
The antiviral response in the 24th week after treatment is finished
As shown in table 9, analyze based on ITT, the treatment back reached in the 24th week continue virus reply (SVR) (patient's percentage ratio of HCV RNA＜10IU/ml) is compared with 57.9% of PEG-IFN, 900Q2w is 58.5%, 1200Q2w be 55.5% and 1200Q4w be 50.9%.Generally speaking, after treatment the 24th the week all patients that adhere to treating in, compare with 66.7% of PEG-IFN, the SVR ratio be 900Q2w 72.3%, 1200Q2w be 70.6% and 1200Q4w be 62%.Astoundingly, in the body weight patient that 75kg adheres to treating at least, the SVR ratio of alb-IFN group remains unchanged, and the decline of PEG-IFN.In this subgroup, compare with 53.3% of PEG-IFN, the SVR ratio be 900Q2w 74.2%, 1200Q2w be 67.9% and 1200Q4w be 61.0%.Therefore, in body weight at least among the patient of 75kg, the HSA-IFN α 2b administration of 900Q2w, 1200Q2w and 1200Q4w is compared with conventional PEG-IFN treatment and is caused higher effect, although even accept the patient of PEG-IFN conventional therapy because 2 to 3 doses of administration arrangement average every month of extra administrations.
Demography, antiviral efficacy and the blood effect analysis in the 24th week after table 9. treatment is finished
| Alb-IFN 900μgQ2w (n＝118) | Alb-IFN 1200μgQ2w (n＝110) | Alb-IFN 1200μgQ4w (n＝116) | Peg-IFNα-2a 180μg?Qw (n＝114) | |
| Demography male BMI average (kg/m 2) body weight 〉=75kg HCV RNA intermediate value (log IU/ml) | 66(55.9％) 25.4 59(50.0％) 6.1 | 64(58.2％) 25.6 59(53.6％) 6.0 | 78(67.2％) 26.1 62(53.4％) 6.1 | 66(57.9％) 25.1 46(40.4％) 6.1 |
| The SVR ratio is overall 〉=80% adhere to body weight (＜75kg, 〉=75kg) | 69(58.5％) 47/65(72.3％) (70.6％，74.2％) | 61(55.5％) 36/51(70.6％) (73.9％，67.9％) | 59(50.9％) 49/79(62.0％) (63.2％，61.0％) | 66(57.9％) 48/72(66.7％) (76.2％，53.3％) |
| Lab A NC＜750/mm 3 Hb＜10g/dL | 28(23.7％) 25(21.2％) | 25(22.7％) 31(28.2％) | 12(10.3％) 14(12.1％) | 23(20.2％) 22(19.3％) |
Quality of life is analyzed
Based on the SF-36 health survey, organize all time points in whole 48 all treatments and gather mensuration by physiology part and spiritual part SP-36, compare with the patient of PEG-IFN treatment group, report the destruction of less health-related quality of life the patient of albumin interferon (Albuferon) 900Q2w treatment group.In addition, during that month before going to a doctor in the 12nd and 24 weeks, with PEG-IFN 12 be respectively 19.2% 24 weeks and compare with 22.4%, have only 3.0% and 5.8% work patient to report absence 7 days or longer work in albumin interferon 900Q2w treatment group.In the 12nd and 24 week treatments, aspect all 10 SF-36 in, with respect to PEG-IFN, 900Q2w treatment group performance better.
Conclusion
In the 12nd week, observe the antiviral activity of genotype 1, first IFN being treated the maximum of HCV in HSA-IFN α 2b 1200 μ g Q2w treatment groups.Also observe similar effect at HSA-IFN α 2b 1200 μ g Q2w with PEG-IFN treatment group to hematology's minimizing.In addition, in the 20th and 24 weeks, also observe maximum antiviral activity in HSA-IFN α 2b 1200 μ g Q2w treatment groups.In the 20th and 24 weeks, continue to observe suitable antiviral activity in HSA-IFN α 2b 900 μ g Q2w treatment groups.In the 24th week after treatment is finished, observe the maximum effect that genotype 1, first IFN are treated HCV in HSA-IFN α 2b 900 μ g Q2w treatment groups.Therefore, HSA-IFN α 2b 900 μ g Q2w can provide effect and the safety suitable at least with Current Standard therapy PEG-IFN, and improved administration arrangement is arranged, and is interpreted as the convenience bigger to the patient.The required injection of 900 μ g Q2w administrations arrangement is half of PEG-IFN, has better quality of life influence and the SVR ratio suitable with PEG-IFN.In addition, 1200 μ g Q2w can provide the suitable at least safety with Current Standard therapy PEG-IFN, and suitable effect and improved administration arrangement are arranged, and are interpreted as the convenience bigger to the patient.
Compare suitable effect with HSA-IFN α 2b 1200 μ g Q4w schemes treatments showing astoundingly in the 12nd week, although even accept the experimenter of PEG-IFN conventional therapy because the administration arrangement has additionally been accepted 3 doses with conventional PEG-IFN treatment.HSA-IFN α 2b 1200 μ g Q4w compare suitable effect with conventional PEG-IFN treatment and lasted till for the 20th and 24 weeks.Also observe improved stable liver function and the remarkable ability that reduces the blood factor minimizing in the 12nd all HSA-IFN α 2b 1200 μ gQ4w treatment groups, illustrate that the improvement of hepatic injury among these patients of treatment back and the incidence rate of dosage minimizing or temporary transient termination and possible recurrence reduce.With HSA-IFN α 2b 1200 μ g Q4w treatment the 24th week of back, the patient who surpasses half reaches lasting virusology and replys.Therefore; these results show with HSA-IFN α 2b 1200 μ g Q4w treatment can provide and use the suitable effect of PEG-IFN therapeutic alliance; and have the ability of improved administration arrangement, bigger normalization liver function and the advantage that lower hematology reduces, cause bigger the complying with and the better result in convenient and treatment back of patient.In a word, consider the recent progress in HCV treatment field, HSA-IFN α 2b Q4w therapeutic scheme (for example will have Ideal Characteristics, suitable effect, better toleration, because the bigger bigger convenience that causes of complying with), to become the interferon skeleton that the interferon anti-reflecting virus therapeutic alliance is selected.
Advantageously, in 900Q2w and 1200Q2w HSA-IFN α 2b treatment group, at least 80% adheres to treating and body weight patient's the 24th week after treatment is finished of 75kg at least, when comparing with PEG-IFN, reaches better SVR ratio.In the whole therapeutic process of heavier patient, reach the ability of better SVR ratio, and the ability of keeping effect is in some country (as the U.S.) particular importance, because surpass 75kg in the weight in patients of those national vast scales.
In sum, these results show the HCV patient who treats with HSA-IFN α 2b and RBV therapeutic alliance genotype 1, first IFN at least with the same with the RBV therapeutic alliance effective with conventional PEG-IFN, and have improved administration arrangement advantage.Especially, these results show that uniting the RBV treatment with HSA-IFN α 2b associating RBV treatment with conventional PEG-IFN compares, can have or similar safety and better effect or better safety with similar effect or existing better effect better safety is arranged again, but have improved and highly favourable administration arrangement.
Embodiment 74: chronic hepatitis C (HCV) patient of genotype 1, first interferon therapy
In per 2 weeks once or once use HSA-IFN α 2b associating ribavirin per 4 weeks in the 12nd week of treatment
Confirm suitable antiviral response
Background
As top discussion, the current interferon therapy that is used for the treatment of chronic hepatitis C (CHC) includes but not limited to symptom, the fatigue and depressed of similar influenza with pronounced side effects.Therefore, patient's quality of life has significantly reduced in the interferon therapy process.Because current standard treatment requires administration at least weekly, be significant the period of reducing quality of life.In addition, the requirement of administration also is counted as inconvenience to the patient weekly.The combination that frequent administration arrangement and patients ' life quality significantly consume causes patient's therapy discontinued of significant number.Therefore, significant need improves the first IFN therapeutic gene type 1HCV patient's of treatment therapeutic scheme, and this will cause the growth of patient's convenience and toleration.The main purpose of administration therapeutic method is to reach the HCV rna level that is lower than quantitative level and keep this level in the interferon therapy process, replys (SVR) or recovery from illness so that reach lasting virusology.Yet owing to compare with per 2 all administrations, administration causes being administered to 2 times of patients' medication amount weekly, and compares with per 4 all administrations, and administration causes being administered to 4 times of patients' medication amount weekly, and can this administration arrangement reach this target is with suspicion.
Principle
Quick virus when treating for the 4th week is replied and is continued the strong positive indication signal that virusology is replied (SVR) among the HCV patient that (RVR) is interferon (PEG-IFN) treatment with Pegylation.Produce HSA-IFN α 2b by merging ripe albuminised C-terminal hereditarily to the N-terminal of ripe Interferon Alpha-2b.Safety and effect with HSA-IFN α 2b associating ribavirin (RBV) treatment are estimated in the HCV human patients of genotype 1, first IFN treatment in the clinical research that active control is arranged, and compare with the conventional therapy with PEG-IFN α-2a (PEG-IFN) associating RBV as active control.The early stage antiviral response that the HSA-IFN α 2b of per 2 weeks (Q2w) or (Q4w) administration of per 4 weeks causes is compared with PEG-IFN, determines to treat whether can keep the RNA feminine gender with frequency administration arrangement still less with HSA-IFN α 2b associating RBV, as HCV RNA＜LOQ.
Method
The human HCV patient of genotype 1, first IFN treatment is as treatment as described in the embodiment 73.Reaching RVR the 12nd week in treatment (treats and analyzes early stage virus among the patient of the 4th all HCV RNA＜LOQ) and reply that (EVR is defined as 〉=2log HCV RNA reduces, or the RNA feminine gender is defined as HCVRNA＜LOQ), be presented at table 10.
Table 10.
Treating the early stage virus that has RVR patient the 12nd week replys
| ITT analyzes | Peg-IFNα2a 180μg?Q1w (n＝114) | HSA-IFNα2b 1200μgQ2w (n＝110) | HSA-IFNα2b 900μg?Q2w (n＝118) | HSA-IFNα2b 1200μg?Q4w (n＝116) |
| Patient with RVR is (among the 4th when week RVR patient of HCV RNA＜LOQ): the 12nd week: do not reach the 12nd week of EVR: HCVRNA＜LOQ | 26％(30/114) 0％(0/30) 97％(29/30) | 34％(37/110) 0％(0/37) 100％(37/37) | 25％(29/118) 3％(1/29) 97％(28/29) | 18％(21/116) 0％(0/21) 100％(21/21) |
| Have among these patients of patient of 2log decline during the 2nd week: the 12nd week: do not reach the 12nd week of EVR: HCVRNA＜LOQ | 32％(36/114) 6％(2/36) 92％(33/36) | 47％(52/110) 6％(3/52) 92％(48/52) | 36％(43/118) 2％(1/43) 95％(41/43) | 41％(47/116) 2％(1/47) 91％(43/47) |
Result and discussion
The 2nd week research is gone to a doctor to occur in and is accepted before the 2nd dose of HSA-IFN α 2b or the 3rd dose of PEG-IFN.Accept 900 μ g HSA-IFN α 2b Q2w and reach the time in 2 weeks 〉=patient's ratio of 2log HCV RNA with accept being in similar proportion of PEG-IFN patient.Two treatment groups of 1200 μ g reach the time in the 2nd week 〉=and patient's ratio that 2log HCV RNA reduces all is higher than PEG-IFN, and no matter the patient is per 2 week or per 4 all administrations (P=0.03).The patient that great majority have HCV RNA 〉=2log minimizing reaches the RNA feminine gender in 12 weeks.In addition, in these patients' that reach the RNA feminine gender the 12nd week ratio at the similar (92%PEG-IFN of all treatment groups; 95%HSA-IFN α 2b 900Q2w; 92%HSA-IFN α 2b 1200Q2w and 91%HSA-IFN α 2b 1200Q4w).Therefore, the antiviral response in the 2nd when week has well been predicted the response result when Q2w and Q4w HSA-IFN α 2b treatment organized for the 12nd week.Having the RNA negative patients during the 4th and 12 weeks has the chance greater than 90% to reach SVR.In addition, these data show even by the 2nd week treating (after using single agent HSA-IFN α 2b), 1200 μ g Q2w or Q4wHSA-IFN α 2b treatment are effectively same with the PEG-IFN treatment at least, and possibility even more effective than the PEG-IFN treatment.In addition, because in genotype 1 colony that is difficult to treat, 1200 μ g dosage HSA-IFN α 2b provide than 900 μ g dosage HSA-IFN α 2b or the bigger antiviral activities of PEG-IFN, it will be more effective may increasing dosage (as to 1800 μ g).
Although the time reached in the 2nd week 〉=patient's ratio that 2log HCV RNA reduces is higher in 1200 μ g Q4w treatment groups, and the patient's ratio HSA-IFN α 2b 1200 μ g Q2w treatment groups or the PEG-IFN treatment group that reach RVR in the HSA-IFN α 2b 1200 μ g Q4w treatment groups are low.Reach patient's ratio EPG-IFN treatment group height of RVR in the HSA-IFN α 2b 1200 μ g Q2w treatment groups.In addition, the 12nd when week RVR and EVR and RNA feminine gender be positively related (P＜0.001).During the 12nd week, in all treatment groups, the patient with RVR has EVR or the HCV negative rate of similar 97-100%.Therefore, although it is lower than PEG-IFN treatment group in HSA-IFN α 2b 1200 μ g Q4w treatment groups to reach RVR patient's ratio, these patients of 100% keep the RNA feminine gender during the 12nd week, illustrate that this therapeutic scheme is a kind of feasible therapeutic scheme.In addition, the HSA-IFN α 2b 1200 μ g treatment groups ratio that reach RVR patient higher than PEG-IFN treatment group shows, increases the 2nd dose in Q4w treatment group during the 2nd week and may increase RVR.
In sum, data show when the patient of the first treatment of therapeutic gene type 1, the more not frequent administration arrangement of HSA-IFN α 2b associating RBV may with PEG-IFN equally effectively or more effective.In addition, cumulative data shows, comprise with use HSA-IFN α 2b in total concentration 1200 μ g or bigger (as 1800 μ g) pro-4 all therapeutic schemes per 2 weeks, using the Therapeutic Method in 24-48 week per 4 weeks then may be the same with the Current Standard therapy good or even better.In addition, these data show that this combinational therapeutic methods will provide the convenience and the toleration of Q4w therapy for the patient, and effect and Q2w therapy are quite or better.
Embodiment 75: chronic hepatitis C (HCV) no response patient is sharp to HSA-IFN α 2b associating
The response of Ba Weilin
Background
The U.S. surpasses 4 million peoples and infects hepatitis C virus (HCV), makes this virus become the modal hepatopathy cause of the U.S..Treatment was counted as the most effective treatment to the patient when interferon-ALPHA (IFN α) was with or without antiviral molecule (ribavirin (RBV)) in history.More in recent years, the Pegylation form of interferon-ALPHA goes through and RBV unites the treatment that is used for HCV.More effective when the interferon of these Pegylations is presented at treatment HCV than standard interferon or interferon associating ribavirin therapy, become conventional treatment to HCV.
Yet this treatment has significant application limitation.Except the known side effect that in therapeutic process, significantly reduces patients ' life quality and conventional therapy with the significant hematology of significant proportion reduce, conventional therapy is inoperative to the patient who stands current HCV treatment of vast scale.Patient to the beginning conventional therapy of the clinical studies show about 45% of the HCV patient's that before do not accept IFN α-RBV treatment treatment can not remove HCV, and keeps chronic infection (for example, nonresponder).In this colony, patient's ratio that treatment is responded significantly still less.
Clinical research person in response to not uniting the needs that RBV treats the non-responder colony that responds to previous I FN α treatment or IFN α among the HCV patient, controls these patients by IFN α and RBV conventional therapy with Pegylation again.Referring to Shiffman etc., " Peginterferon alfa-2a andribavirin in patients with chronic hepatitis C who have failed prior treatment, " (suffering from chronic hepatitis C and previous glycol interferon alpha-2a and ribavirin for the treatment of among the patient who fails) Gastroenterology 126 (4): 1015-23 (2004).Although the patient of 35% this test of registration does not have HCV RNA sign after controlling for 20 weeks again with conventional therapy, many these patients recurrence of having no progeny in treatment.Therefore, have only 18% patient in fact to reach lasting virus and reply (SVR), and cured HCV.Similarly, when before the no response patient of IFN α treatment failure of the conventional Pegylation of usefulness when controlling again with the IFN α of another kind of Pegylation, based on have only among these patients of anecdotal evidence (anecdotalevidence)～5-10% can reach SVR.Therefore, the patient colony of not only a kind of interferon therapy failure but also all current interferon therapy failures continues significantly to increase.Correspondingly, obviously be necessary to improve therapeutic scheme, it not only is general HCV treatment, and before used interferon therapy (as live through IFN α treatment) for treatment and treated and unresponsive patient, those the most refractory no response patients' (as control or before treated the patient of failure again with current traditional remedies) alternative treatment particularly.
Principle
Produce HSA-IFN α 2b by merging ripe albuminised C-terminal hereditarily to the N-terminal of ripe Interferon Alpha-2b.Living through in the clinical research of randomization and open-label with safety, toleration and the effect of HSA-IFN α 2b associating RBV treatment in the unresponsive HCV human patients of IFN α treatment and estimating.Purpose for this research, the nonresponder is defined as those owing to can not reach the HCV patient that HCV rna level 2-log reduced (as early stage virus response of the 12nd week or EVR12) and stop previous treatment when the 12nd week, or therapeutic scheme fails to reach the patient of SVR after finishing.The patient of recurring that has no progeny in the treatment gets rid of outside this research.In addition, at least 50% patient before failed with the treatment of Pegylation IFN α therapeutic scheme in this research.
Method
The no response HCV patient that the mankind of previous at least one IFN α therapeutic scheme failure live through IFN α treatment changes into 3 subcutaneous (sc) HSA-IFN α 2b treatment groups at random: (a) per 2 weeks of 900 μ g once (Q2w); (b) 1200 μ g Q2w and (c) per 4 weeks of 1200 μ g once (Q4w).Each experimenter in each treatment group also accepts 1000-1200mg/ days RBV.After the data of safety of assessment from these initial 3 crowds of people, the other 2 crowds of people that accept HSA-IFN α 2b with 1500 μ gQ2w or 1800 μ g Q2w that add 1000-1200mg/ days RBV of associating by order progressively increase the dosage of HSA-IFN α 2b.
The treatment persistent period of this research was 48 weeks, with following up a case by regular visits in 24 weeks.The main effectiveness terminal point of this research is that the virusology that continues is replied (SVR).
HCV RNA titre is to use PCR in real time test Quantasure
TM(Labcorp) measure, its range of sensitivity is 10IU to 1 hundred million IU/mL.Use standard technique known in the art to measure alanine transaminase (ALT) and blood effect, comprise neutrophil cell absolute counting (ANC), hemoglobin and platelet count.
Result and discussion
Demography
Identified and can be used as the many Demographics that have the patient's of treatment no response tendency (prevalence) individual index.The indication signal comprises (1) genotype 1 before these crucial no response treatments, (2) high baseline HCV rna level intermediate value, and the no response that had before treated PEG+RBV (3), (4) non-descendants American, the fibrosis level in late period of (5) F3-F4 (is used
Classify) and (6) high BMI (as 〉=25mg/kg).May best overall no response index be previous PEG-RBV treatment failure and previous in this respect number based on the failure of IFN therapy.
Experimenter's demography is summarised in table 11.Generally speaking, all experimenters' demography is similar in all treatment groups.Most of experimenters are exposed to and surpass a kind of therapy that contains interferon, and before with PEG+RBV treatment failure.In addition, although the baseline genius morbi is suitable between 5 treatment groups, 1800 μ g Q2w treatment groups have preceding HCV RNA of significantly higher treatment and the highest previous PEG+RBV failure ratio.Therefore, the most refractory patient of the representative of the experimenter in 1800 μ g Q2w treatment groups colony.
Table 11.
Demography and baseline characteristic
| 900μgQ2w N＝23 | 1200μgQ2w N＝24 | 1200μgQ4w N＝24 | 1500μgQ2w N＝22 | 1800μgQ2w N＝22 | The P value | |
| Median age | 53 | 50 | 49 | 51 | 47 | 0.0687 |
| Male % | 60.9％ | 58.3％ | 62.5％ | 86.4％ | 59.1％ | 0.2095 |
| |
20(87.0％) | 24(100％) | 22(91.7％) | 21(95.5％) | 21(95.5％) | 0.4531 |
| HCVRNA intermediate value (log10IU/ml) | 7.1 | 6.6 | 6.2 | 7.0 | 7.6 | ＜0.0001 |
| PEG+RBV | 14(60.9％) | 16(66.7％) | 15(62.5％) | 16(72.2％) | 20(90.9％) | 0.1370 |
| Non-descendants American | 2(8.7％) | 3(12.5％) | 1(4.2％) | 7(31.8％) | 2(9.1％) | 0.0940 |
| F3-F4 | 7(30.4％) | 7(29.2％) | 4(16.7％) | 9(40.9％) | 7(31.8％) | 0.5012 |
| BMI≥25kg/m 2 | 20(87.0％) | 21(87.5％) | 18(75.0％) | 18(81.8％) | 17(77.3％) | 0.7637 |
| Have 〉=a kind of previous experimenter % that treats | 47.8％ | 66.7％ | 66.6％ | 72.7％ | 54.5％ | 0.4087 |
Effect and biologic activity
As shown in Figure 7, in genotype 1, PEG+RBV nonresponder, the slope that the HCV RNA of 900-1500 μ g treatment group reduces during the 12nd week is suitable.Surprisingly, the HCV RNA reduction maximum of 1800 μ g treatment groups.Among the patient of the HCV RNA minimizing of 1500 μ g and 1800 μ g treatment groups this subgroup when the 24th week is suitable.
During the 24th week, experimenter's ratio of HCV RNA feminine gender is suitable between 900-1500 μ g treatment group.Similar with the 12nd week, genotype 1, PEG+RBV nonresponder's subgroup the analysis showed that the responsiveness the highest (data not shown) of 1800 μ g treatment groups during the 24th week.
Based on the judgement of researcher with consider from cumulative data and show the high negative indication signal that lacks EVR12 and the SVR RNA feminine gender when the 24th week, allow the curee during the 24th week because the shortage effect is interrupted based on interferon therapy.
Great majority had the RNA negative patients and keep negative when 48 weeks during the 24th week.The overall treatment response in latter stage (ETR, the HCV feminine gender during the 48th week) of 900-1200 μ g treatment group is 31% (Fig. 8 A).Therefore, in the experimenter of the 12nd week (as EVR12) or the HCV RNA feminine gender that became in the 24th week, a high proportion of curee reaches ETR.In genotype 1PEG+RBV nonresponder, the scope that ETR leads leads the highest (Fig. 8 B) from the ETR of about 15% to 27%, 1500 μ g and 1800 μ g treatment groups.
In addition, during following up a case by regular visits in the 12nd week after the 48 week treatments, the experimenter of significant number continues HCV RNA feminine gender, for example reaches SVR.It is 21% (Fig. 8 C) that the overall SVR of 900-1200 μ g treatment group leads.In genotype 1PEG+RBV nonresponder, the scope that SVR leads from 10% to 15.4% (Fig. 8 D).
In a word, these data show with 900-1200 μ g HSA-IFN α 2b associating RBV treats the no response HCV patient who lives through IFN α treatment of the failure of the PEG+RBV with high percentage ratio, causes strong and suitable antiviral activity.Treat at this refractory and also to observe low virus in the no response colony and break through (breakthrough) (as detecting, but positive at 2 or more time point subsequently) and low relapse rate less than HCV RNA.In addition, 12 week of treatment are also observed significantly higher minimizing in 1800 μ g treatment groups, show that the patient with this dosage HSA-IFN α 2b associating ribavirin therapy can have the remarkable increase that SVR leads.
Table 12.
GT1 and PEG+RBV nonresponder's antiviral response
| 900μg?Q2w N＝12 | 1200μg?Q2w N＝16 | 1200μg?Q4w N＝13 | 1500μg?Q2w N＝15 | 1800μg?Q2w N＝19 | |
| The 12nd all EVR12 a 95％C.I. b | 5(41.7％) (15.1％，72.3％) | 4(25％) (7.3％，52.4％) | 3(23.1％) (7.2％，52.4％) | 5(33.3％) (11.8％，61.6％) | 12(63.2％) (38.4％，83.7％) |
| The 24th week was detected less than HCVRNA 95%C.I. b | 2(16.7％) (2.1％，48.4％) | 3(18.8％) (4.0％，45，6％) | 2(15.4％) (1.9％，45.4％) | 4(26.7％) (7.8％，55.1％) | 6(31.6％) (12.6％，56.6％) |
| The 48th all ETR a 95％C.I. b | 3(25％) (5.5％，57.2％) | 3(18.8％) (4.0％，45.6％) | 2(15.4％) (1.9％，45.4％) |
A be defined as detection less than HCV RNA or 〉=the HCV RNA of 2log reduces
Strict 95% confidence interval of b
The blood effect
In the combined treatment, guarantee compliance and be exposed to the IFN of all dosage and RBV maximization SVR is led is crucial.
Hematology's minimizing is common during IFN and RBV therapeutic alliance.Recurrence is crucial to RBV dosage to prevention HCV.Yet, because the minimizing of hemoglobin (Hb) that the inductive haemolysis of RBV causes and platelet (PLT) counting requires to reduce RBV dosage.Neutrophil cell absolute counting (ANC)＜750/mm
3Minimizing require to reduce the dosage of IFN component in the therapeutic alliance.
Although observe the minimizing of some ANC and PLT, these minimizings are suitable between all Q2w treatment groups, and reach a platform about 4 to 8 weeks.Similarly, Hb is suitable between all Q2w treatment groups (comprising 1800 μ g treatment groups) with respect to the minimizing of baseline up to the 12nd week and afterwards.The blood values of Q4w treatment group reduces still less.Generally, there is 12/115 experimenter to reduce dosage for the control side effect.The minimizing of most of HSA-IFN α 2b dosage is because the ANC minimizing of summarizing as therapeutic scheme causes.Do not observe dose response between Q2w treatment group.Therefore, in high-dose therapy group more, do not observe the needs of the increase that dosage reduces.
In a word, although observe some minimizings of blood values in the HSA-IFN α 2b associating RBV treatment, these minimizings are suitable between the treatment group, and showing to live through in the safety between the no response HCV patient of IFN α treatment with 900-1800 μ g HSA-IFN α 2b associating RBV treatment does not have significant difference.
Conclusion
In sum, these results show in the no response HCV patient who lives through IFN α treatment of remarkable ratio (patient who comprises previous PEG+RBV therapeutic scheme failure), can effectively reach SVR with HSA-IFN α 2b and RBV treatment, therefore eradicate HCV.Especially, these results show that causing this to study 21% among all patients with HSA-IFN α 2b900-1200 μ g associating RBV treatment reaches SVR.The patient of previous PEG+RBV therapy failure even can reach SVR has these height refractory of 10% to 15.4% to treat patients and reaches SVR.In addition, treat in the patient colony at refractory, 1800 μ g treatment groups show the highest negative ratio of the 24th all HCV RNA, show that this treatment can cause these patients even higher SVR to lead.In addition, safety is similar between all HSA-IFN α 2b treatment groups.In addition, these results show that HSA-IFN α 2b uses around can per effectively two thoughtful, provides improved administration arrangement.Therefore, these results show the patient of HSA-IFN α 2b associating RBV treatment for failing based on the treatment of interferon, particularly the patient of the interferon of conventional Pegylation-RBV treatment failure provides the effectively also treatment of safety to select, have highly favourable and improved administration arrangement, this is that current this area lacks.
Embodiment 76:HSA-IFN α 2b associating ribavirin is controlled at genotype 2 or 3, first interferon
Antiviral activity among chronic hepatitis C (HCV) patient who treats
Background
World wide has 1.7 hundred million people of surpassing to infect hepatitis C virus (HCV), and this virus becomes significant publilc health worry, and becomes the modal hepatopathy cause of world wide rapidly.Acute HCV infection is normally asymptomatic, and early diagnosis is a problem.In fact, HCV infects that to tend to be a kind of chronic disease, and wherein about 70% actute infection becomes persistence.Therefore, although new incidence of infection is descending, the propagation that prediction HCV infects can remain unchanged in the near future.
Treatment was counted as suffering from the most effective treatment of chronic hepatitis C (CHC) patient when interferon-ALPHA (IFN α) was with or without antiviral molecule (ribavirin (RBV)) in history.More recent, the Pegylation form of interferon-ALPHA goes through and RBV unites the treatment that is used for HCV.More effective when the interferon of these Pegylations is presented at treatment HCV than standard interferon or interferon associating ribavirin therapy, become conventional treatment to HCV.
The overall antiviral response (SVR) that continues to current recommended therapy alters a great deal in CHC patient, depends on virus and host's characteristic, particularly virogene type.For example, the SVR with patient of the most common genotype 1 leads the about 42-46% of scope.On the other hand, the SVR with patient of more uncommon genotype 2 or 3 leads at 76-80%.In addition, genotype 2 or 3 patient treat not as patient's refractory of genotype 1 significantly, can by the shorter time treatment and more the ribavirin of low dosage treat.
Come therapeutic gene type 2 or 3 to cause that the patient of remarkable ratio reaches SVR although follow 24 all follow-up period interferon associating RBV 24 weeks of treatment with Pegylation of current recommendation, it is significant based on the common application limitation of interferon therapy that this therapeutic scheme still keeps.Especially, the treatment of current recommendation still locks into and uses the side effect that the back obviously reduces patients ' life quality at every turn.Therefore, need to continue new treatment effective and better tolerance to infect genotype 2 or 3HCV patient's Therapeutic Method.
Principle
Produce HSA-IFN α 2b by merging ripe albuminised C-terminal hereditarily to the N-terminal of ripe Interferon Alpha-2b.Safety and effect with HSA-IFN α 2b associating RBV treatment are estimated in the CHC human patients of genotype 2 or 3, first interferon therapy in the clinical research of randomized, multicenter, open-label.
Method:
The human HCV patient of 43 genotype 2 or 3, first interferon therapy is randomized into 2 subcutaneous (sc) HSA-IFN α 2b treatment groups: (a) per 2 weeks of 1500 μ g dosage once (Q2w) or per 4 weeks of (2) 1500 μ g dosage once (Q4w).Each experimenter of each treatment group also accepts 800mg/ days RBV.Stratified main basis comprise genotype (2 or 3) and HCV RNA (＜800,000IU/ml or 〉=800,000IU/ml).
The treatment persistent period of this research was 24 weeks, with following up a case by regular visits in 24 weeks.Main effect terminal point is that the virusology that continues is replied (SVR).
HCV RNA titre is to use PCR in real time Quantasure
TM(Labcorp) measure, its range of sensitivity (quantitative limit (LOQ)) is 43IU to 6,900 ten thousand IU/mL.Use stable state estimation model (HOMA) to estimate insulin resistance.
The patient of purpose treatment (ITT) is defined as the experimenter that all randomizations and treatment are organized in each treatment, and no matter whether the patient has any missing data point or withdraw from this research.The patient of the purpose treatment (MITT) of improvement is defined as the patient that can conceivablely have the prescription on individual diagnosis of the 12nd week based on their record date in this research.
Result and discussion
During the 4th and 12 weeks, experimenter's demography and antiviral response are summarised in table 13.Generally, HSA-IFN α 2b all tolerances well in two treatment groups.
Table 13.
Demography and antiviral response
The treatment of ITT=purpose; The purpose treatment of MITT=improvement (qualified experimenter went to a doctor in the 12nd week)
HCV RNA reduction and have the genotype 2 of HCV RNA＜LOQ or patient's ratio of 3 is suitable between HSA-IFN α 2b Q2w and HSA-IFN α 2b Q4w treatment group.In the 4th when week, have 68.2% of the genotype 2 of HCV RNA＜LOQ or 76.2% and 1500 μ g Q4w treatment groups that patient's ratio of 3 is 1500 μ g Q2w.In the 12nd when week, a high proportion of genotype 2 or 3 patients have HCV RNA＜LOQ (82.4% and the 1500Q4w of 1500Q2w 88.9%) in two treatment groups.
Therefore, these results show with 1500 μ g HSA-IFN α 2b and cause strong antiviral response rate with the CHC patient of Q2w or Q4w week therapeutic gene type 2 or 3.In addition, in the patient of genotype 2 or 3, show and use the suitable effect of HSA-IFN α 2b 1500 μ g Q2w schemes treatment with the treatment of HSA-IFN α 2b 1500 μ g Q4w schemes.Therefore, these results show with HSA-IFN α 2b 1500 μ g effective equally with the treatment to HCV infection genotype 2 or 3 patients of current recommendation at least with Q2w or Q4w treatment, and have the advantage that very big improved administration is arranged, may cause better toleration of patient and convenience.
Embodiment 77: control with HSA-IFN α 2b associating ribavirin therapy genotype 1, first interferon
Chronic hepatitis C (HCV) patient's who treats quality of life (QOL)
Background
Mention as the front, the HCV patient that routine is united ribavirin (RBV) therapeutic gene type 1, first interferon therapy (first IFN treatment) with pegylated interferon alfa had significant application limitation in 48 weeks.Because the known side effect of interferon therapy of current recommendation, use interferon at every turn after, patient's quality of life has reduced significantly.Current scheme requires administration at least weekly, period and because the increase that loses the work capacity natural law that treatment causes of the reduction quality of life that causes prolonging.Therefore a large amount of patient's therapy discontinued have some research reports, and interruption rate surpasses 50%.Therefore, significant need improves the therapeutic scheme of the first interferon therapy patient's genotype 1HCV of treatment, compares with current standard treatment to have improved influence to quality of life.
Principle:
Safety and effect with HSA-IFN α 2b associating RBV treatment are estimated in the HCV human patients of genotype 1, first interferon therapy in the clinical research that active control is arranged, and with as the described conventional therapies of uniting RBV with PEG-IFN α 2a (PEG-IFN) of embodiment 73 as active control compare.During 12 all treatments, the treatment for the treatment of with HSA-IFN α 2b associating RBV compares with the influence that loses work capacity natural law (as not removing the natural law of work) and with PEG-IFN associating RBV QOL.
Method
458 human gene's type 1HCV patients are carried out randomization and treatment as described in embodiment 73.Before treatment, when treating the 4th week and the 12nd week, pass through SF-
(QualityMetric, Lincoln RI) determine that QOL and assessment lose the natural law of work capacity to rating model.Especially, 8 SF-36v2 aspects of assessment: body function (PF), health role (RP), health misery (BP), general health (GH), vitality (VT), social function (SF), emotion role (RE) and mental health (MH).Preceding 4 aspects (PF, RP, BP and GH) are corresponding to healthy part, and remaining 4 aspects (VT, SF, RE and MH) are corresponding to the mental health part of QOL model.
Treat in whole the 12nd week, the conversion (original) of estimating 8 SF-36v2 aspects divides, and summarizes (PCS) branch based on normal body part and summarize (MCS) branch with the psychology part.
Result and discussion
Table 14.
Quality of life is measured
*P value＜0.05 couple Peg-IFN;
#Surpass (MCID) minimum clinical significant differences
In the 12nd when week, the QOL that accepts 900 μ g HSA-IFN α 2b Q2w patients when at every turn measuring improves with respect to PEG-IFN, at MCS and PCS, and reaches significance,statistical among in 8 independent aspects 5.In 1200 μ g Q2w and 1200 μ g Q4w HSA-IFN α 2b treatment groups, in the each mensuration of reality, the deterioration of QOL has reduced with respect to PEG-IFN, and with observed clinical significant difference in and the mental health painful at health.
Generally, in any HSA-IFN α 2b treatment group, genotype 1HCV patient since the natural law that their HCV infects and treatment is not subsequently gone work than the genotype 1HCV patient in the PEG-IFN treatment group still less.Especially, the patient who accepts 900 μ g HSA-IFN α 2b Q2w has and lacks 75% MDW than the patient who accepts PEG-IFN, and the patient who accepts 1200 μ g HSA-IFN α 2b Q2w or 1200 μ g HSA-IFN α 2b Q4w has and lacks 25% MDW than the patient who accepts PEG-IFN.
The antiviral activity of HSA-IFN α 2b shown in the comprehensive embodiment 73, these results show HCV patient with HSA-IFN α 2b and RBV therapeutic alliance genotype 1, first interferon therapy at least with effective equally with the treatment of conventional PEG-IFN and RBV therapeutic alliance, and have the advantage of improving administration arrangement and improvement QOL.Especially, these results show with HSA-IFN α 2b associating RBV treatment can be provided than the improved and highly favourable therapeutic scheme of conventional PEG-IFN associating RBV treatment, i.e. the reduction that provides the natural law that does not go work still less that can provide than PEG-IFN associating RBV treatment and QOL index to worsen to the patient.
Embodiment 78: chronic hepatitis C (HCV) patient of genotype 1, first interferon therapy
In, per 4 weeks are used HSA-IFN α 2b, and additionally use potion when the 2nd week, unite every day simultaneously
The ribavirin of administration carries out open-label, randomization, multicenter preliminary study
Background
The dynamics of virus modeling of Alb-IFN has proved that nonlinear pair of stage of HCV RNA reduces.Behind the initial treatment, virus load has the decline of dose dependent rapidly (" phase I "), be subsequently during the treatment next weeks relatively slowly but " second stage " of the decay of chain index formula.Phase I HCVRNA minimizing shows dose dependent.Similarly, the minimizing of the second stage of HCV RNA has dose dependent.These look like as standard and PEG-IFN α treats the strong indication signal that observed lasting virus reduces (SVR).Neumann etc., Hepatology 36 (4 pt 2): 357a (2002); Perelson etc., Hepatology 42:749-754 (2005).
Principle
Per 4 weeks are used HSA-IFN α 2b, and additionally use 1 dose the time in the 2nd week, unite the safety and the effect of two agent therapies of the ribavirin of administration every day simultaneously and estimate in open-label, randomization, multicenter preliminary study.In the patient who suffers from genotype 1 chronic hepatitis C and first interferon therapy, comparison two agent therapies and the HSA-IFN α 2b that uses the ribavirin of HSA-IFN α 2b associating administration every day per 4 weeks and per 2 weeks use a half-value dose unite the ribavirin of administration every day.
Method
Qualified patient advances one of 3 treatment groups with 1: 1: 1 ratio randomization as shown in Table 15.900Q2w treatment group is as the internal reference of this research, because should the treatment group show that having acceptable safety reduced with the HCV RNA similar to PEG-IFN α-2a.1800Q2/4 treatment group will be accepted extra 1 dose of alb-IFN, 1800 μ g the time in the 2nd week, and the 4th quick virusology when all is replied (RVR4) rate and possible SVR leads thereby the HCV RNA reduction when improving for the 4th week improves.The dosage minimizing is also included within this research design.900Q2w is organized, at the observed remarkable antiviral activity of similar dosage, select alb-IFN dosage is reduced to lowest dosage levels 600 μ g in studying mutually based on another 2a first interferon therapy, genotype 1 chronic hepatitis C patient.To 1800Q4w and 1800Q2/4w group, like per 2 phases, be exposed to 600 μ g alb-IFN dosage, select dosage is reduced to per 4 all 1200 μ g.
About 225 patients are randomized (being 75 patients of each treatment group).According to when screening HCVRNA (〉=400,000IU/mL or＜400,000IU/mL) and according to the race randomization is carried out layering.The treatment total duration was 48 weeks.All patients that finish the treatment of 48 weeks will return to go to a doctor in the 52nd week and the 60th week.The patient who has HCV RNA＜100IU/mL during the 60th week will return to carry out the SVR evaluation when the 72nd week.
The time do not reach in the 12nd week EVR (EVR12, be defined as 〉=2-log10 reduces or HCV RNA＜LOQ[43IU/mL]) or the 24th week or the patient that has HCV RNA 〉=100IU/mL afterwards will interrupt research and treat.These patients will be considered to the SVR failure, will not need to carry out further HDV RNA evaluation.
Prescription on individual diagnosis was followed up a case by regular visits in safety after the patient of all early stage therapy discontinued must finish the treatment in 4 and 12 weeks.In addition, follow up a case by regular visits to the patient who has HCV RNA＜100IU/mL when going to a doctor in the treatment back safety of 12 weeks and will after treatment, return to carry out the SVR evaluation 24 weeks.
Safety is followed up a case by regular visits to the prescription on individual diagnosis after the treatment in 4 and 12 weeks, and the patient who has positive alb-IFN or the response of HSA antibody during the research any time also must finish the treatment back safety of 24 weeks and follow up a case by regular visits to prescription on individual diagnosis, no matter its HCV RNA replys.
Estimate HCV RNA by effective real-time polymerase chain reaction (PCR) test, its dynamic range be 43IU/mL (LOQ) to 6,900 ten thousand IU/mL, lower detectability (LOD) is 15IU/mL.
Table 15. treatment group
| The treatment group | The dosing interval of subcutaneous administration Alb-IFN (total number of using) | Orally administered ribavirin ＊ | |
| | Per | 2 all 900 μ g alb-IFN (24 doses) | 1000 or 1200mg/ |
| 1800Q4w | Per | ||
| 4 all 1800 μ g alb-IFN (12 doses) | 1000 or 1200mg/ days | ||
| 1800Q2/4w | Per 4 all 1800 μ g alb-IFN, extra during the 2nd week | 1000 or 1200mg/ days |
| 1 dose (13 doses) |
*The ribavirin administration will be based on the body weight (kg) of patient in the time of the 1st day, and every day, dosage was separately used for 2 times, took with food, totally 48 weeks:
The patient 1000mg/ of body weight＜75kg days (the 400mg morning and 600mg afternoon)
The patient 1200mg/ of body weight 〉=75kg days (the 600mg morning and 600mg afternoon)
Efficacy assessment
Collect HCV RNA sample at following consultation time: the 1st day, the 4th day, the 1st week, the 2nd week, the 4th week, the 32nd day and the 6th, 8,12,16,20,24,32,40,48,52,60 and 72 weeks, except those patients who does not reach the 24th week of EVR12 or have HCV RNA 〉=100IU/mL afterwards.
Safety evaluation
Whole research process detects patient's side effect (AE), serious side effects (SAE), laboratory abnormalities and immunogenicity state.The severity of side effect carries out classification according to the microorganism and infectious disease subregion (DMID) the toxicity table of improvement.
Other assessment
Collect pharmacokinetics (PK) sample in the 1st day, the 4th day, the 8th day (the 1st week), the 15th day (the 2nd week), the 29th day (the 4th week), the 32nd day, the 57th day (the 8th week) and the 85th day (the 12nd week).Once collect questionnaire as a result, the SF36 health survey of acute version and the work productivity and energy damage questionnaire (WPAI-SHP) that the patient reports the 52nd, 60 and 72 weeks of whole 48 all treatment phases and follow-up period when going to a doctor per 2 weeks.
Data analysis
Purpose treatment (ITT) colony forms patient randomized by all, that accept at least 1 dose of research medicine.Follow the ITT principle, analyze the patient according to the treatment that they distribute when the randomization.Safety colony will be made up of all patients who accepts 1 dose of research medicine at least, and analyze according to the treatment of accepting.
The safety of alb-IFN and toleration will be estimated by the overall security of checking each treatment group, comprise AE/SAE, will cause treating the AE of interruption, AE and the laboratory abnormalities that causes dosage to reduce or omit.The treatment group presents death, other serious side effects, causes side effect that alb-IFN interrupts and the AE that causes dosage to reduce or omit.The ratio of these incidents will be estimated in all 3 treatment groups with 95% confidence interval (CI).In addition, if expection cell counting (cell count) is lower than 5, use Pearson X 2 test or FisherShi Precision Test to assess the comparison of matched group.
The responsiveness of classification (categorical) effect terminal point will be with their corresponding 95%CI report.Similarly, if the expection cell counting is lower than 5, use Pearson X 2 test or FisherShi Precision Test to assess the comparison of matched group.Use Kaplan-Meier method and logrank to check each matched group relatively to estimate to reach the time of the HCV RNA that is lower than detection.
Each file of quoting (comprise patent, patent application, patent announcement, journal of writings, summary, laboratory manual, book or other open) complete open, and the whole by reference this paper of going into of unique identifier available information that mentions among the application such as the data base of GenBank, GeneSeq or CAS registration.
In addition, the whole by reference income this paper of the description of following each international application and U. S. application and sequence table: international application PCT/US02/40891 number, December was submitted on the 23rd in 2002; International application PCT/US2004/001369 number, on January 20th, 2004 submitted to; International application PCT/US2005/004041 number, on February 9th, 2005 submitted to; International application PCT/US06/29391 number, on July 31st, 2006 submitted to; No. the 10/775th, 204, U. S. application, on February 11st, 2004 submitted to; No. the 11/175th, 690, U. S. application, on July 7th, 2005 submitted to; No. the 11/429th, 373, U. S. application, on May 8th, 2006 submitted to; No. the 11/429th, 276, U. S. application, on May 8th, 2006 submitted to; No. the 11/429th, 374, U. S. application, on May 8th, 2006 submitted to; No. the 11/495th, 624, U. S. application, on July 31st, 2006 submitted to; With No. the 60/707th, 521, U.S. Provisional Application, on August 12nd, 2005 submitted to; On August 31st, 60/712,386,2005 submitted to; On November 3rd, 60/732,724,2005 submitted to; On February 28th, 60/776,914,2006 submitted to; On March 13rd, 60/781,361,2006 submitted to; On June 2nd, 60/810,182,2006 submitted to; On June 15th, 60/813,682,2006 submitted to; JIUYUE was submitted on the 14th in 60/844,349,2006; On November 13rd, 60/858,410,2006 submitted to; On February 20th, 60/902,039,2007 submitted to; With 60/942,647, on June 7th, 2007 submitted to.
Relate to the explanation of the biomaterial of preservation
(PCT detailed rules for the implementation 13 two)
A. the indication of doing below relates to the biomaterial of the preservation that description the 30th page table 3 mentions.
B.
Preservation proves: further preservation proves at another page or leaf:
Depositary institution's title: American type culture collection (ATCC)
Depositary institution address: 10801 University Boulevard
Manassas，Virginia?20110-2209
United States of America (U.S.)
| Preserving | Preservation day | ||
| 1. | PTA-3763 | 2001-10-04 | |
| 2. | PTA-3940 | 2001-12-19 | |
| 3. | PTA-3942 | 2001-12-19 | |
| 4. | PTA-3939 | 2001-12-19 | |
| 5. | PTA-4670 | 2002-09-16 |
Canada
Applicant's requirement, be authorized to or apply for out of court up to Canadian Patent on the basis of application, or abandoned and no longer recovery, or be withdrawn, patent chief executive only authorizes the biological material specimens of the preservation that the application's indication is provided to the specified independently expert of chief executive, correspondingly, the applicant must be by written statement notice international office before the technology that is used for international application published is ready to complete.
Norway
The applicant is requirement hereby, is arranged to be disclosed in public examination (by Norw P office) until application, or finally is not disclosed examination by the decision of Norw P office, and sample supply has only the expert of this area effective.According to Norw P bill clause 22 and 33 (3), the requirement of this effectiveness should be submitted to Norw P office being not later than application by the applicant the disclosed time.If the applicant has submitted this requirement to, any third party requires the requirement of sampling should indicate the expert that will use.This expert can be anyone on the generally acknowledged list of expert worked out of Norw P office, or the applicant in case, ratify anyone.
Australia
The applicant proposes notice hereby, microbiological specimens only supply with before the patent authorizing or application lost efficacy, reject or recall before to skilled and to the profitless addressee of the present invention effectively (Australian Patent regulations 3.25 (3) bars).
Finland
The applicant is requirement hereby, is disclosed public examination (by national patent and article committee) up to application, or finally is not arranged to be disclosed in public examination by national patent and the decision of article committee, and sample supply is only effective to the expert of this area.
Britain
The applicant is requirement hereby, and microbiological specimens is supplied with only effective to the expert.The requirement of this effectiveness must be submitted to international office by the applicant before the technology of the international publication that is used to apply for is ready to complete.
Denmark
The applicant is requirement hereby, is disclosed in public examination (by Danish Patent office) up to application, or finally underground in public examination by the decision of Danish Patent office, and sample supply is only effective to the expert of this area.According to Danish Patent bill clause 22 and 33 (3), the requirement of this effectiveness should be submitted to Danish Patent office being not later than application by the applicant the disclosed time.If the applicant has submitted this requirement to, any third party requires the requirement of sampling should indicate the expert that will use.This expert can be anyone on the generally acknowledged list of expert worked out of Danish Patent office, or the applicant in case, stipulate anyone.
Sweden
The applicant is requirement hereby, is disclosed in public examination (by Swedish patent office) up to application, or finally underground in public examination by the decision of Swedish patent office, and sample supply is only effective to the expert of this area.The requirement of this effectiveness should be submitted to the international office form of the PCT/RO/134 table that is replicated in PCT applicant's guide volume I appendix Z (preferably with) before expired by the applicant 16 months distance priority dates.If the applicant has submitted this requirement to, any third party requires the requirement of sampling should indicate the expert that will use.This expert can be anyone on the generally acknowledged list of expert worked out of Swedish patent office, or the applicant in case, ratify anyone.
Holland
The applicant is requirement hereby, up to the HOII P date of grant or up to applying for out of court or recalling or the Expiration Date, with reference to patent statute 31F (1), can only supply with microorganism by sample is distributed to the expert.According to Kingdom of the Netherlands patents act clause 22C or 25, the requirement of this effectiveness must offer Dutch industrial property office by the applicant before application is open, no matter be which generation more early in two dates.
Sequence table
＜110〉Human Genome Sciences Inc. (HUMAN GEN0ME SCIENCES, INC.)
＜120〉albumin fusion proteins
<130>PF619PP5
<160>189
<170>Patentin?Ver.2.0
<210>1
<211>585
<212>PRT
＜213〉human (Homo sapiens)
<400>1
Asp?Ala?His?Lys?Ser?Glu?Val?Ala?His?Arg?Phe?Lys?Asp?Leu?Gly?Glu
1 5 10 15
Glu?Asn?Phe?Lys?Ala?Leu?Val?Leu?Ile?Ala?Phe?Ala?Gln?Tyr?Leu?Gln
20 25 30
Gln?Cys?Pro?Phe?Glu?Asp?His?Val?Lys?Leu?Val?Asn?Glu?Val?Thr?Glu
35 40 45
Phe?Ala?Lys?Thr?Cys?Val?Ala?Asp?Glu?Ser?Ala?Glu?Asn?Cys?Asp?Lys
50 55 60
Ser?Leu?His?Thr?Leu?Phe?Gly?Asp?Lys?Leu?Cys?Thr?Val?Ala?Thr?Leu
65 70 75 80
Arg?Glu?Thr?Tyr?Gly?Glu?Met?Ala?Asp?Cys?Cys?Ala?Lys?Gln?Glu?Pro
85 90 95
Glu?Arg?Asn?Glu?Cys?Phe?Leu?Gln?His?Lys?Asp?Asp?Asn?Pro?Asn?Leu
100 105 110
Pro?Arg?Leu?Val?Arg?Pro?Glu?Val?Asp?Val?Met?Cys?Thr?Ala?Phe?His
115 120 125
Asp?Asn?Glu?Glu?Thr?Phe?Leu?Lys?Lys?Tyr?Leu?Tyr?Glu?Ile?Ala?Arg
130 135 140
Arg?His?Pro?Tyr?Phe?Tyr?Ala?Pro?Glu?Leu?Leu?Phe?Phe?Ala?Lys?Arg
145 150 155 160
Tyr?Lys?Ala?Ala?Phe?Thr?Glu?Cys?Cys?Gln?Ala?Ala?Asp?Lys?Ala?Ala
165 170 175
Cys?Leu?Leu?Pro?Lys?Leu?Asp?Glu?Leu?Arg?Asp?Glu?Gly?Lys?Ala?Ser
180 185 190
Ser?Ala?Lys?Gln?Arg?Leu?Lys?Cys?Ala?Ser?Leu?Gln?Lys?Phe?Gly?Glu
195 200 205
Arg?Ala?Phe?Lys?Ala?Trp?Ala?Val?Ala?Arg?Leu?Ser?Gln?Arg?Phe?Pro
210 215 220
Lys?Ala?Glu?Phe?Ala?Glu?Val?Ser?Lys?Leu?Val?Thr?Asp?Leu?Thr?Lys
225 230 235 240
Val?His?Thr?Glu?Cys?Cys?His?Gly?Asp?Leu?Leu?Glu?Cys?Ala?Asp?Asp
245 250 255
Arg?Ala?Asp?Leu?Ala?Lys?Tyr?Ile?Cys?Glu?Asn?Gln?Asp?Ser?Ile?Ser
260 265 270
Ser?Lys?Leu?Lys?Glu?Cys?Cys?Glu?Lys?Pro?Leu?Leu?Glu?Lys?Ser?His
275 280 285
Cys?Ile?Ala?Glu?Val?Glu?Asn?Asp?Glu?Met?Pro?Ala?Asp?Leu?Pro?Ser
290 295 300
Leu?Ala?Ala?Asp?Phe?Val?Glu?Ser?Lys?Asp?Val?Cys?Lys?Asn?Tyr?Ala
305 310 315 320
Glu?Ala?Lys?Asp?Val?Phe?Leu?Gly?Met?Phe?Leu?Tyr?Glu?Tyr?Ala?Arg
325 330 335
Arg?His?Pro?Asp?Tyr?Ser?Val?Val?Leu?Leu?Leu?Arg?Leu?Ala?Lys?Thr
340 345 350
Tyr?Glu?Thr?Thr?Leu?Glu?Lys?Cys?Cys?Ala?Ala?Ala?Asp?Pro?His?Glu
355 360 365
Cys?Tyr?Ala?Lys?Val?Phe?Asp?Glu?Phe?Lys?Pro?Leu?Val?Glu?Glu?Pro
370 375 380
Gln?Asn?Leu?Ile?Lys?Gln?Asn?Cys?Glu?Leu?Phe?Glu?Gln?Leu?Gly?Glu
385 390 395 400
Tyr?Lys?Phe?Gln?Asn?Ala?Leu?Leu?Val?Arg?Tyr?Thr?Lys?Lys?Val?Pro
405 410 415
Gln?Val?Ser?Thr?Pro?Thr?Leu?Val?Glu?Val?Ser?Arg?Asn?Leu?Gly?Lys
420 425 430
Val?Gly?Ser?Lys?Cys?Cys?Lys?His?Pro?Glu?Ala?Lys?Arg?Met?Pro?Cys
435 440 445
Ala?Glu?Asp?Tyr?Leu?Ser?Val?Val?Leu?Asn?Gln?Leu?Cys?Val?Leu?His
450 455 460
Glu?Lys?Thr?Pro?Val?Ser?Asp?Arg?Val?Thr?Lys?Cys?Cys?Thr?Glu?Ser
465 470 475 480
Leu?Val?Asn?Arg?Arg?Pro?Cys?Phe?Ser?Ala?Leu?Glu?Val?Asp?Glu?Thr
485 490 495
Tyr?Val?Pro?Lys?Glu?Phe?Asn?Ala?Glu?Thr?Phe?Thr?Phe?His?Ala?Asp
500 505 510
Ile?Cys?Thr?Leu?Ser?Glu?Lys?Glu?Arg?Gln?Ile?Lys?Lys?Gln?Thr?Ala
515 520 525
Leu?Val?Glu?Leu?Val?Lys?His?Lys?Pro?Lys?Ala?Thr?Lys?Glu?Gln?Leu
530 535 540
Lys?Ala?Val?Met?Asp?Asp?Phe?Ala?Ala?Phe?Val?Glu?Lys?Cys?Cys?Lys
545 550 555 560
Ala?Asp?Asp?Lys?Glu?Thr?Cys?Phe?Ala?Glu?Glu?Gly?Lys?Lys?Leu?Val
565 570 575
Ala?Ala?Ser?Gln?Ala?Ala?Leu?Gly?Leu
580 585
<210>2
<211>1782
<212>DNA
＜213〉human (Homo sapiens)
<400>2
gatgcacaca?agagtgaggt?tgctcatcgg?tttaaagatt?tgggagaaga?aaatttcaaa 60
gccttggtgt?tgattgcctt?tgctcagtat?cttcagcagt?gtccatttga?agatcatgta 120
aaattagtga?atgaagtaac?tgaatttgca?aaaacatgtg?ttgctgatga?gtcagctgaa 180
aattgtgaca?aatcacttca?tacccttttt?ggagacaaat?tatgcacagt?tgcaactctt 240
cgtgaaacct?atggtgaaat?ggctgactgc?tgtgcaaaac?aagaacctga?gagaaatgaa 300
tgcttcttgc?aacacaaaga?tgacaaccca?aacctccccc?gattggtgag?accagaggtt 360
gatgtgatgt?gcactgcttt?tcatgacaat?gaagagacat?ttttgaaaaa?atacttatat 420
gaaattgcca?gaagacatcc?ttacttttat?gccccggaac?tccttttctt?tgctaaaagg 480
tataaagctg?cttttacaga?atgttgccaa?gctgctgata?aagctgcctg?cctgttgcca 540
aagctcgatg?aacttcggga?tgaagggaag?gcttcgtctg?ccaaacagag?actcaaatgt 600
gccagtctcc?aaaaatttgg?agaaagagct?ttcaaagcat?gggcagtggc?tcgcctgagc 660
cagagatttc?ccaaagctga?gtttgcagaa?gtttccaagt?tagtgacaga?tcttaccaaa 720
gtccacacgg?aatgctgcca?tggagatctg?cttgaatgtg?ctgatgacag?ggcggacctt 780
gccaagtata?tctgtgaaaa?tcaggattcg?atctccagta?aactgaagga?atgctgtgaa 840
aaacctctgt?tggaaaaatc?ccactgcatt?gccgaagtgg?aaaatgatga?gatgcctgct 900
gacttgcctt?cattagctgc?tgattttgtt?gaaagtaagg?atgtttgcaa?aaactatgct 960
gaggcaaagg?atgtcttcct?gggcatgttt?ttgtatgaat?atgcaagaag?gcatcctgat 1020
tactctgtcg?tgctgctgct?gagacttgcc?aagacatatg?aaaccactct?agagaagtgc 1080
tgtgccgctg?cagatcctca?tgaatgctat?gccaaagtgt?tcgatgaatt?taaacctctt 1140
gtggaagagc?ctcagaattt?aatcaaacaa?aactgtgagc?tttttgagca?gcttggagag 1200
tacaaattcc?agaatgcgct?attagttcgt?tacaccaaga?aagtacccca?agtgtcaact 1260
ccaactcttg?tagaggtctc?aagaaaccta?ggaaaagtgg?gcagcaaatg?ttgtaaacat 1320
cctgaagcaa?aaagaatgcc?ctgtgcagaa?gactatctat?ccgtggtcct?gaaccagtta 1380
tgtgtgttgc?atgagaaaac?gccagtaagt?gacagagtca?caaaatgctg?cacagagtcc 1440
ttggtgaaca?ggcgaccatg?cttttcagct?ctggaagtcg?atgaaacata?cgttcccaaa 1500
gagtttaatg?ctgaaacatt?caccttccat?gcagatatat?gcacactttc?tgagaaggag 1560
agacaaatca?agaaacaaac?tgcacttgtt?gagcttgtga?aacacaagcc?caaggcaaca 1620
aaagagcaac?tgaaagctgt?tatggatgat?ttcgcagctt?ttgtagagaa?gtgctgcaag 1680
gctgacgata?aggagacctg?ctttgccgag?gagggtaaaa?aacttgttgc?tgcaagtcaa 1740
gctgccttag?gcttataaca?tctacattta?aaagcatctc?ag 1782
<210>3
<211>609
<212>PRT
＜213〉human (Homo sapiens)
<400>3
Met?Lys?Trp?Val?Thr?Phe?Ile?Ser?Leu?Leu?Phe?Leu?Phe?Ser?Ser?Ala
1 5 10 15
Tyr?Ser?Arg?Gly?Val?Phe?Arg?Arg?Asp?Ala?His?Lys?Ser?Glu?Val?Ala
20 25 30
His?Arg?Phe?Lys?Asp?Leu?Gly?Glu?Glu?Asn?Phe?Lys?Ala?Leu?Val?Leu
35 40 45
Ile?Ala?Phe?Ala?Gln?Tyr?Leu?Gln?Gln?Cys?Pro?Phe?Glu?Asp?His?Val
50 55 60
Lys?Leu?Val?Asn?Glu?Val?Thr?Glu?Phe?Ala?Lys?Thr?Cys?Val?Ala?Asp
65 70 75 80
Glu?Ser?Ala?Glu?Asn?Cys?Asp?Lys?Ser?Leu?His?Thr?Leu?Phe?Gly?Asp
85 90 95
Lys?Leu?Cys?Thr?Val?Ala?Thr?Leu?Arg?Glu?Thr?Tyr?Gly?Glu?Met?Ala
100 105 110
Asp?Cys?Cys?Ala?Lys?Gln?Glu?Pro?Glu?Arg?Asn?Glu?Cys?Phe?Leu?Gln
115 120 125
His?Lys?Asp?Asp?Asn?Pro?Asn?Leu?Pro?Arg?Leu?Val?Arg?Pro?Glu?Val
130 135 140
Asp?Val?Met?Cys?Thr?Ala?Phe?His?Asp?Asn?Glu?Glu?Thr?Phe?Leu?Lys
145 150 155 160
Lys?Tyr?Leu?Tyr?Glu?Ile?Ala?Arg?Arg?His?Pro?Tyr?Phe?Tyr?Ala?Pro
165 170 175
Glu?Leu?Leu?Phe?Phe?Ala?Lys?Arg?Tyr?Lys?Ala?Ala?Phe?Thr?Glu?Cys
180 185 190
Cys?Gln?Ala?Ala?Asp?Lys?Ala?Ala?Cys?Leu?Leu?Pro?Lys?Leu?Asp?Glu
195 200 205
Leu?Arg?Asp?Glu?Gly?Lys?Ala?Ser?Ser?Ala?Lys?Gln?Arg?Leu?Lys?Cys
210 215 220
Ala?Ser?Leu?Gln?Lys?Phe?Gly?Glu?Arg?Ala?Phe?Lys?Ala?Trp?Ala?Val
225 230 235 240
Ala?Arg?Leu?Ser?Gln?Arg?Phe?Pro?Lys?Ala?Glu?Phe?Ala?Glu?Val?Ser
245 250 255
Lys?Leu?Val?Thr?Asp?Leu?Thr?Lys?Val?His?Thr?Glu?Cys?Cys?His?Gly
260 265 270
Asp?Leu?Leu?Glu?Cys?Ala?Asp?Asp?Arg?Ala?Asp?Leu?Ala?Lys?Tyr?Ile
275 280 285
Cys?Glu?Asn?Gln?Asp?Ser?Ile?Ser?Ser?Lys?Leu?Lys?Glu?Cys?Cys?Glu
290 295 300
Lys?Pro?Leu?Leu?Glu?Lys?Ser?His?Cys?Ile?Ala?Glu?Val?Glu?Asn?Asp
305 310 315 320
Glu?Met?Pro?Ala?Asp?Leu?Pro?Ser?Leu?Ala?Ala?Asp?Phe?Val?Glu?Ser
325 330 335
Lys?Asp?Val?Cys?Lys?Asn?Tyr?Ala?Glu?Ala?Lys?Asp?Val?Phe?Leu?Gly
340 345 350
Met?Phe?Leu?Tyr?Glu?Tyr?Ala?Arg?Arg?His?Pro?Asp?Tyr?Ser?Val?Val
355 360 365
Leu?Leu?Leu?Arg?Leu?Ala?Lys?Thr?Tyr?Glu?Thr?Thr?Leu?Glu?Lys?Cys
370 375 380
Cys?Ala?Ala?Ala?Asp?Pro?His?Glu?Cys?Tyr?Ala?Lys?Val?Phe?Asp?Glu
385 390 395 400
Phe?Lys?Pro?Leu?Val?Glu?Glu?Pro?Gln?Asn?Leu?Ile?Lys?Gln?Asn?Cys
405 410 415
Glu?Leu?Phe?Glu?Gln?Leu?Gly?Glu?Tyr?Lys?Phe?Gln?Asn?Ala?Leu?Leu
420 425 430
Val?Arg?Tyr?Thr?Lys?Lys?Val?Pro?Gln?Val?Ser?Thr?Pro?Thr?Leu?Val
435 440 445
Glu?Val?Ser?Arg?Asn?Leu?Gly?Lys?Val?Gly?Ser?Lys?Cys?Cys?Lys?His
450 455 460
Pro?Glu?Ala?Lys?Arg?Met?Pro?Cys?Ala?Glu?Asp?Tyr?Leu?Ser?Val?Val
465 470 475 480
Leu?Asn?Gln?Leu?Cys?Val?Leu?His?Glu?Lys?Thr?Pro?Val?Ser?Asp?Arg
485 490 495
Val?Thr?Lys?Cys?Cys?Thr?Glu?Ser?Leu?Val?Asn?Arg?Arg?Pro?Cys?Phe
500 505 510
Ser?Ala?Leu?Glu?Val?Asp?Glu?Thr?Tyr?Val?Pro?Lys?Glu?Phe?Asn?Ala
515 520 525
Glu?Thr?Phe?Thr?Phe?His?Ala?Asp?Ile?Cys?Thr?Leu?Ser?Glu?Lys?Glu
530 535 540
Arg?Gln?Ile?Lys?Lys?Gln?Thr?Ala?Leu?Val?Glu?Leu?Val?Lys?His?Lys
545 550 555 560
Pro?Lys?Ala?Thr?Lys?Glu?Gln?Leu?Lys?Ala?Val?Met?Asp?Asp?Phe?Ala
565 570 575
Ala?Phe?Val?Glu?Lys?Cys?Cys?Lys?Ala?Asp?Asp?Lys?Glu?Thr?Cys?Phe
580 585 590
Ala?Glu?Glu?Gly?Lys?Lys?Leu?Val?Ala?Ala?Ser?Gln?Ala?Ala?Leu?Gly
595 600 605
Leu
<210>4
<211>15
<212>PRT
＜213〉artificial sequence
<220>
＜221〉corner
＜223〉be used in the joint peptide that connects VH and VL domain among the scFv
<400>4
Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser
1 5 10 15
<210>5
<211>394
<212>DNA
＜213〉human (Homo sapiens)
<400>5
gcggccgccg?gatgcaaggg?ttcgaatccc?ttagctctca?ttattttttg?ctttttctct?60
tgaggtcaca?tgatcgcaaa?atggcaaatg?gcacgtgaag?ctgtcgatat?tggggaactg?120
tggtggttgg?caaatgacta?attaagttag?tcaaggcgcc?atcctcatga?aaactgtgta?180
acataataac?cgaagtgtcg?aaaaggtggc?accttgtcca?attgaacacg?ctcgatgaaa?240
aaaataagat?atatataagg?ttaagtaaag?cgtctgttag?aaaggaagtt?tttccttttt?300
cttgctctct?tgtcttttca?tctactattt?ccttcgtgta?atacagggtc?gtcagataca?360
tagatacaat?tctattaccc?ccatccatac?aatg 394
<210>6
<211>21
<212>PRT
＜213〉human (Homo sapiens)
<400>6
Met?Lys?Val?Ser?Val?Ala?Ala?Leu?Ser?Cys?Leu?Met?Leu?Val?Thr?Ala
1 5 10 15
Leu?Gly?Ser?Gln?Ala
20
<210>7
<211>17
<212>PRT
＜213〉artificial sequence
<220>
＜223〉department's gland calcium protein (Stanniocalcin) signal peptide
<400>7
Met?Leu?Gln?Asn?Ser?Ala?Val?Leu?Leu?Leu?Leu?Val?Ile?Ser?Ala?Ser
1 5 10 15
Al?a
<210>8
<211>24
<212>PRT
＜213〉human (Homo sapiens)
<400>8
Met?Lys?Trp?Val?Thr?Phe?Ile?Ser?Leu?Leu?Phe?Leu?Phe?Ser?Ser?Ala
1 5 10 15
Tyr?Ser?Arg?Gly?Val?Phe?Arg?Arg
20
<210>9
<211>18
<212>PRT
＜213〉human (Homo sapiens)
<400>9
Met?Lys?Trp?Val?Thr?Phe?Ile?Ser?Leu?Leu?Phe?Leu?Phe?Ser?Ser?Ala
1 5 10 15
Tyr?Ser
<210>10
<211>18
<212>PRT
＜213〉human (Homo sapiens)
<400>10
Met?Lys?Trp?Val?Ser?Phe?Ile?Ser?Leu?Leu?Phe?Leu?Phe?Ser?Ser?Ala
1 5 10 15
Tyr?Ser
<210>11
<211>19
<212>PRT
＜213〉human (Homo sapiens)
<400>11
Met?Leu?Leu?Gln?Ala?Phe?Leu?Phe?Leu?Leu?Ala?Gly?Phe?Ala?Ala?Lys
1 5 10 15
Ile?Ser?Ala
<210>12
<211>86
<212>PRT
＜213〉human (Homo sapiens)
<220>
<221>MISC_FEATURE
<222>(84)
＜223〉Xaa equals Glu or Asp is wherein arbitrary
<400>12
Met?Arg?Phe?Pro?Ser?Ile?Phe?Thr?Ala?Val?Leu?Ala?Phe?Ala?Ala?Ser
1 5 10 15
Ser?Ala?Leu?Ala?Ala?Pro?Val?Asn?Thr?Thr?Thr?Glu?Asp?Glu?Thr?Ala
20 25 30
Gln?Ile?Pro?Ala?Glu?Ala?Val?Ile?Gly?Tyr?Ser?Asp?Leu?Glu?Gly?Asp
35 40 45
Phe?Asp?Val?Ala?Val?Leu?Pro?Phe?Ser?Asn?Ser?Thr?Asn?Asn?Gly?Leu
50 55 60
Leu?Phe?Ile?Asn?Thr?Thr?Ile?Ala?Ser?Ile?Ala?Ala?Lys?Glu?Glu?Gly
65 70 75 80
Val?Ser?Leu?Xaa?Lys?Arg
85
<210>13
<211>24
<212>PRT
＜213〉human (Homo sapiens)
<400>13
Met?Lys?Trp?Val?Ser?Phe?Ile?Ser?Leu?Leu?Phe?Leu?Phe?Ser?Ser?Ala
1 5 10 15
Tyr?Ser?Arg?Ser?Leu?Glu?Lys?Arg
20
<210>14
<211>24
<212>PRT
＜213〉human (Homo sapiens)
<400>14
Met?Lys?Trp?Val?Ser?Phe?Ile?Ser?Leu?Leu?Phe?Leu?Phe?Ser?Ser?Ala
1 5 10 15
Tyr?Ser?Arg?Ser?Leu?Asp?Lys?Arg
20
<210>15
<211>21
<212>PRT
＜213〉human (Homo sapiens)
<400>15
Met?Asn?Ile?Phe?Tyr?Ile?Phe?Leu?Phe?Leu?Leu?Ser?Phe?Val?Gln?Gly
1 5 10 15
Ser?Leu?Asp?Lys?Arg
20
<210>16
<211>19
<212>PRT
＜213〉human (Homo sapiens)
<400>16
Met?Gly?Trp?Ser?Cys?Ile?Ile?Leu?Phe?Leu?Val?Ala?Thr?Ala?Thr?Gly
1 5 10 15
Val?His?Ser
<210>17
<211>29
<212>PRT
＜213〉human (Homo sapiens)
<400>17
Met?Glu?Arg?Ala?Ala?Pro?Ser?Arg?Arg?Val?Pro?Leu?Pro?Leu?Leu?Leu
1 5 10 15
Leu?Gly?Gly?Leu?Ala?Leu?Leu?Ala?Ala?Gly?Val?Asp?Ala
20 25
<210>18
<211>22
<212>PRT
＜213〉human (Homo sapiens)
<400>18
Met?Met?Lys?Thr?Leu?Leu?Leu?Phe?Val?Gly?Leu?Leu?Leu?Thr?Trp?Glu
1 5 10 15
Ser?Gly?Gln?Val?Leu?Gly
20
<210>19
<211>21
<212>PRT
＜213〉human (Homo sapiens)
<400>19
Met?Leu?Pro?Leu?Cys?Leu?Val?Ala?Ala?Leu?Leu?Leu?Ala?Ala?Gly?Pro
1 5 10 15
Gly?Pro?Ser?Leu?Gly
20
<210>20
<211>24
<212>PRT
＜213〉human (Homo sapiens)
<400>20
Met?Lys?Trp?Val?Ser?Phe?Ile?Ser?Leu?Leu?Phe?Leu?Phe?Ser?Ser?Ala
1 5 10 15
Tyr?Ser?Arg?Gly?Val?Phe?Arg?Arg
20
<210>21
<211>18
<212>PRT
＜213〉artificial sequence
<220>
＜221〉mutagenic agent
<222>(14)to(18)
＜223〉variant of the natural targeting sequencing of HSA
<400>21
Met?Lys?Trp?Val?Thr?Phe?Ile?Ser?Leu?Leu?Phe?Leu?Phe?Ala?Gly?Val
1 5 10 15
Leu?Gly
<210>22
<211>18
<212>PRT
＜213〉artificial sequence
<220>
＜221〉mutagenic agent
<222>(14)to(18)
＜223〉variant of the natural targeting sequencing of HSA
<400>22
Met?Lys?Trp?Val?Thr?Phe?Ile?Ser?Leu?Leu?Phe?Leu?Phe?Ser?Gly?Val
1 5 10 15
Leu?Gly
<210>23
<211>18
<212>PRT
＜213〉artificial sequence
<220>
＜221〉mutagenic agent
<222>(14)to(18)
＜223〉variant of the natural targeting sequencing of HSA
<400>23
Met?Lys?Trp?Val?Thr?Phe?Ile?Ser?Leu?Leu?Phe?Leu?Phe?Gly?Gly?Val
1 5 10 15
Leu?Gly
<210>24
<211>18
<212>PRT
＜213〉human (Homo sapiens)
<400>24
Met?Lys?Trp?Val?Thr?Phe?Ile?Ser?Leu?Leu?Phe?Leu?Phe?Ala?Gly?Val
1 5 10 15
Ser?Gly
<210>25
<211>18
<212>PRT
＜213〉human (Homo sapiens)
<400>25
Met?Lys?Trp?Val?Thr?Phe?Ile?Ser?Leu?Leu?Phe?Leu?Phe?Gly?Gly?Val
1 5 10 15
Ser?Gly
<210>26
<211>18
<212>PRT
＜213〉artificial sequence
<220>
＜221〉mutagenic agent
<222>(14)to(18)
＜223〉variant of the natural targeting sequencing of HSA
<400>26
Met?Lys?Trp?Val?Thr?Phe?Ile?Ser?Leu?Leu?Phe?Leu?Phe?Ala?Gly?Val
1 5 10 15
Ser?Gly
<210>27
<211>18
<212>PRT
＜213〉artificial sequence
<220>
＜221〉mutagenic agent
<222>(14)to(18)
＜223〉variant of the natural targeting sequencing of HSA
<400>27
Met?Lys?Trp?Val?Thr?Phe?Ile?Ser?Leu?Leu?Phe?Leu?Phe?Ser?Gly?Val
1 5 10 15
Ser?Gly
<210>28
<211>18
<212>PRT
＜213〉artificial sequence
<220>
＜221〉mutagenic agent
<222>(14)to(18)
＜223〉variant of the natural targeting sequencing of HSA
<400>28
Met?Lys?Trp?Val?Thr?Phe?Ile?Ser?Leu?Leu?Phe?Leu?Phe?Gly?Gly?Val
1 5 10 15
Ser?Gly
<210>29
<211>23
<212>PRT
＜213〉artificial sequence
<220>
＜221〉mutagenic agent
<222>(14)to(23)
＜223〉variant of the natural targeting sequencing of HSA
<400>29
Met?Lys?Trp?Val?Thr?Phe?Ile?Ser?Leu?Leu?Phe?Leu?Phe?Gly?Gly?Val
1 5 10 15
Leu?Gly?Asp?Leu?His?Lys?Ser
20
<210>30
<211>22
<212>PRT
＜213〉artificial sequence
<220>
＜223〉composite signal peptide
<400>30
Met?Pro?Thr?Trp?Ala?Trp?Trp?Leu?Phe?Leu?Val?Leu?Leu?Leu?Ala?Leu
1 5 10 15
Tr?p?Ala?Pro?Ala?Arg?Gly
20
<210>31
<211>17
<212>PRT
＜213〉human (Homo sapiens)
<400>31
Met?Phe?Lys?Ser?Val?Val?Tyr?Ser?Ile?Leu?Ala?Ala?Ser?Leu?Ala?Asn
1 5 10 15
Ala
<210>32
<211>29
<212>PRT
＜213〉human (Homo sapiens)
<400>32
Met?Asn?Ile?Phe?Tyr?Ile?Phe?Leu?Phe?Leu?Leu?Ser?Phe?Val?Gln?Gly
1 5 10 15
Leu?Glu?His?Thr?His?Arg?Arg?Gly?Ser?Leu?Asp?Lys?Arg
20 25
<210>33
<211>23
<212>PRT
＜213〉human (Homo sapiens)
<400>33
Met?Lys?Leu?Ala?Tyr?Ser?Leu?Leu?Leu?Pro?Leu?Ala?Gly?Val?Ser?Ala
1 5 10 15
Ser?Val?Ile?Asn?Tyr?Lys?Arg
20
<210>34
<211>65
<212>PRT
＜213〉human (Homo sapiens)
<400>34
Met?Lys?Leu?Lys?Thr?Val?Arg?Ser?Ala?Val?Leu?Ser?Ser?Leu?Phe?Ala
1 5 10 15
Ser?Gln?Val?Leu?Gly?Gln?Pro?Ile?Asp?Asp?Thr?Glu?Ser?Gln?Thr?Thr
20 25 30
Ser?Val?Asn?Leu?Met?Ala?Asp?Asp?Thr?Glu?Ser?Ala?Phe?Ala?Thr?Gln
35 40 45
Thr?Asn?Ser?Gly?Gly?Leu?Asp?Val?Val?Gly?Leu?Ile?Ser?Met?Ala?Lys
50 55 60
Arg
65
<210>35
<211>70
<212>PRT
＜213〉human (Homo sapiens)
<400>35
Met?Lys?Leu?Lys?Thr?Val?Arg?Ser?Ala?Val?Leu?Ser?Ser?Leu?Phe?AIa
1 5 10 15
Ser?Gln?Val?Leu?Gly?Gln?Pro?Ile?Asp?Asp?Thr?Glu?Ser?Gln?Thr?Thr
20 25 30
Ser?Val?Asn?Leu?Met?Ala?Asp?Asp?Thr?Glu?Ser?Ala?Phe?Ala?Thr?Gln
35 40 45
Thr?Asn?Ser?Gly?Gly?Leu?Asp?Val?Val?Gly?Leu?Ile?Ser?Met?Ala?Glu
50 55 60
Glu?Gly?Glu?Pro?Lys?Arg
65 70
<210>36
<211>58
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used for generating the primer in XhoI and ClaI site at pPPC0006
<400>36
gcctcgagaa?aagagatgca?cacaagagtg?aggttgctca?tcgatttaaa?gatttggg 58
<210>37
<211>59
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used for generating the primer in XhoI and ClaI site at pPPC0006
<400>37
aatcgatgag?caacctcact?cttgtgtgca?tctcttttct?cgaggctcct?ggaataagc 59
<210>38
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used for generating the primer in XhoI and ClaI site at pPPC0006
<400>38
tacaaactta?agagtccaat?tagc 24
<210>39
<211>29
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used for generating the primer in XhoI and ClaI site at pPPC0006
<400>39
cacttctcta?gagtggtttc?atatgtctt 29
<210>40
<211>60
<212>DNA
＜213〉artificial sequence
<220>
<221>Misc_Structure
＜223〉be used for changing the synthetic oligonucleotide of restriction site at pPPC0007
<400>40
aagctgcctt?aggcttataa?taaggcgcgc?cggccggccg?tttaaactaa?gcttaattct?60
<210>41
<211>60
<212>DNA
＜213〉artificial sequence
<220>
<221>Misc_Structure
＜223〉be used for changing the synthetic oligonucleotide of restriction site at pPPC0007
<400>41
agaattaagc?ttagtttaaa?cggccggccg?gcgcgcctta?ttataagcct?aaggcagctt?60
<210>42
<211>32
<212>DNA
＜213〉artificial sequence
<220>
＜223〉can be used for generating the forward primer of albumin fusion proteins, wherein albumin module (moiety) is positioned at the N end of therapeutic protein
<220>
<221>misc_feature
<222>(18)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(19)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(20)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(21)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(22)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(23)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(24)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(25)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(26)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(27)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(28)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(29)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(30)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(31)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(32)
＜223〉n equals a, t, g or c
<400>42
aagctgcctt?aggcttannn?nnnnnnnnnn?nn 32
<210>43
<211>51
<212>DNA
＜213〉artificial sequence
<220>
＜223〉can be used for generating the reverse primer of albumin fusion proteins, wherein the albumin module is positioned at the N end of therapeutic protein
<220>
<221>misc_feature
<222>(37)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(38)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(39)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(40)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(41)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(42)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(43)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(44)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(45)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(46)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(47)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(48)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(49)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(50)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(51)
＜223〉n equals a, t, g or c
<400>43
gcgcgcgttt?aaacggccgg?ccggcgcgcc?ttattannnn?nnnnnnnnnn?n 51
<210>44
<211>4
<212>PRT
＜213〉human (Homo sapiens)
<400>44
Leu?Asp?Lys?Arg
1
<210>45
<211>4
<212>PRT
＜213〉human (Homo sapiens)
<400>45
Leu?Glu?Lys?Arg
1
<210>46
<211>33
<212>DNA
＜213〉artificial sequence
<220>
＜223〉can be used for generating the forward primer of albumin fusion proteins, wherein the albumin module is positioned at the C end of therapeutic protein
<220>
<221>misc_feature
<222>(19)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(20)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(21)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(22)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(23)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(24)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(25)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(26)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(27)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(28)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(29)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(30)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(31)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(32)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(33)
＜223〉n equals a, t, g or c
<400>46
aggagcgtcg?acaaaagann?nnnnnnnnnn?nnn 33
<210>47
<211>52
<212>DNA
＜213〉artificial sequence
<220>
＜223〉can be used for generating the reverse primer of albumin fusion proteins, wherein the albumin module is positioned at the C end of therapeutic protein
<220>
<221>misc_feature
<222>(38)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(39)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(40)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(41)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(42)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(43)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(44)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(45)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(46)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(47)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(48)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(49)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(50)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(51)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(52)
＜223〉n equals a, t, g or c
<400>47
ctttaaatcg?atgagcaacc?tcactctt?gtgtgcatcnnn?nnnnnnnnnn?nn 52
<210>48
<211>9
<212>PRT
＜213〉human (Homo sapiens)
<400>48
Asp?Ala?His?Lys?Ser?Glu?Val?Ala?His
1 5
<210>49
<211>11
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)to(11)
＜223〉Kozak sequence
<400>49
ccgccaccat?g 11
<210>50
<211>46
<212>DNA
＜213〉artificial sequence
<220>
＜223〉can be used for therapeutic protein is inserted the forward primer of pC4:HSA carrier
<220>
<221>misc_feature
<222>(29)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(30)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(31)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(32)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(33)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(34)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(35)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(36)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(37)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(38)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(39)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(40)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(41)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(42)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(43)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(44)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(45)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(46)
＜223〉n equals a, t, g or c
<400>50
ccgccgctcg?aggggtgtgt?ttcgtcgann?nnnnnnnnnn?nnnnnn 46
<210>51
<211>55
<212>DNA
＜213〉artificial sequence
<220>
＜223〉can be used for therapeutic protein is inserted the reverse primer of pC4:HSA carrier
<220>
<221>misc_feature
<222>(38)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(39)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(40)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(41)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(42)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(43)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(44)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(45)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(46)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(47)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(48)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(49)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(50)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(51)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(52)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(53)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(54)
＜223〉n equals a, t, g or c
<220>
<221>misc_feature
<222>(55)
＜223〉n equals a, t, g or c
<400>51
agtcccatcg?atgagcaacc?tcactcttgt?gtgcatcnnn?nnnnnnnnnn?nnnnn 55
<210>52
<211>733
<212>DNA
＜213〉human (Homo sapiens)
<400>52
gggatccgga?gcccaaatct?tctgacaaaa?ctcacacatg?cccaccgtgc?ccagcacctg 60
aattcgaggg?tgcaccgtca?gtcttcctct?tccccccaaa?acccaaggac?accctcatga 120
tctcccggac?tcctgaggtc?acatgcgtgg?tggtggacgt?aagccacgaa?gaccctgagg 180
tcaagttcaa?ctggtacgtg?gacggcgtgg?aggtgcataa?tgccaagaca?aagccgcggg 240
aggagcagta?caacagcacg?taccgtgtgg?tcagcgtcct?caccgtcctg?caccaggact 300
ggctgaatgg?caaggagtac?aagtgcaagg?tctccaacaa?agccctccca?acccccatcg 360
agaaaaccat?ctccaaagcc?aaagggcagc?cccgagaacc?acaggtgtac?accctgcccc 420
catcccggga?tgagctgacc?aagaaccagg?tcagcctgac?ctgcctggtc?aaaggcttct 480
atccaagcga?catcgccgtg?gagtgggaga?gcaatgggca?gccggagaac?aactacaaga 540
ccacgcctcc?cgtgctggac?tccgacggct?ccttcttcct?ctacagcaag?ctcaccgtgg 600
acaagagcag?gtggcagcag?gggaacgtct?tctcatgctc?cgtgatgcat?gaggctctgc 660
acaaccacta?cacgcagaag?agcctctccc?tgtctccggg?taaatgagtg?cgacggccgc 720
gact?ctagag?gat 733
<210>53
<211>5
<212>PRT
＜213〉human (Homo sapiens)
<220>
<221>MISC_FEATURE
<222>(3)
＜223〉Xaa equals any 20 kinds of natural L-of existence aminoacid
<400>53
Trp?Ser?Xaa?Trp?Ser
1 5
<210>54
<211>86
<212>DNA
＜213〉artificial sequence
<220>
＜223〉have the GAS binding site of in 1RF1 promoter people such as (, Immunity 1:457-468,1994) Rothman, finding 4 tandem copies, with the composition sequence of complementary 18 nucleotide of SV40 early promoter and Xho I restriction site.
<400>54
gcgcctcgag?atttccccga?aatctagatt?tccccgaaat?gatttccccg?aaatgatttc 60
cccgaaatat?ctgccatctc?aattag 86
<210>55
<211>27
<212>DNA
＜213〉artificial sequence
<220>
＜223〉with the complementary composition sequence of SV40 promoter; Comprise Hind III restriction site.
<400>55
gcggcaagct?ttttgcaaag?cctaggc 27
<210>56
<211>271
<212>DNA
＜213〉artificial sequence
<220>
<221>Protein_Bind
＜223〉be used for the synthetic promoter of biological assay; Be included in the GAS binding site of finding in the IRF1 promoter.
<400>56
ctcgagattt?ccccgaaatc?tagatttccc?cgaaatgatt?tccccgaaat?gatttccccg 60
aaatatctgc?catctcaatt?agtcagcaac?catagtcccg?cccctaactc?cgcccatccc 120
gcccctaact?ccgcccagtt?ccgcccattc?tccgccccat?ggctgactaa?ttttttttat 180
ttatgcagag?gccgaggccg?cctcggcctc?tgagctattc?cagaagtagt?gaggaggctt 240
ttttggaggc?ctaggctttt?gcaaaaagct?t 271
<210>57
<211>32
<212>DNA
＜213〉artificial sequence
<220>
＜223〉with the complementary synthetic primer of people's gene group EGR-1 promoter sequence; Comprise Xho I restriction site.
<400>57
gcgctcgagg?gat?gacagcg?atagaacccc?gg 32
<210>58
<211>31
<212>DNA
＜213〉artificial sequence
<220>
＜223〉with the complementary synthetic primer of people's gene group EGR-1 promoter sequence; Comprise Hind III restriction site.
<400>58
gcgaagcttc?gcgactcccc?ggatccgcct?c 31
<210>59
<211>12
<212>DNA
＜213〉human (Homo sapiens)
<400>59
ggggactttc?cc 12
<210>60
<211>73
<212>DNA
＜213〉artificial sequence
<220>
＜223〉have NF-KB binding site (GGGGACTTTCCC) 4 tandem copies, with SV40 early promoter sequence 5 ' end complementary 18 nucleotide,
Synthetic primer with Xho 1 restriction site.
<400>60
gcggcctcga?ggggactttc?ccggggactt?tccggggact?ttccgggact?ttccatcctg 60
ccatctcaat?tag 73
<210>61
<211>256
<212>DNA
＜213〉artificial sequence
<220>
<221>Protein_Bind
＜223〉be used for the synthetic promoter of biological assay; Be included in the NF-KB binding site
<400>61
ctcgagggga?ctttcccggg?gactttccgg?ggactttccg?ggactttcca?tctgccatct 60
caattagtca?gcaaccatag?tcccgcccct?aactccgccc?atcccgcccc?taactccgcc 120
cagttccgcc?cattctccgc?cccatggctg?actaattttt?tttatttatg?cagaggccga 180
ggccgcctcg?gcctctgagc?tattccagaa?gtagtgagga?ggcttttttg?gaggcctagg 240
cttttgcaaa?aagctt 256
<210>62
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase degeneracy VH forward primer of people VH domain
<400>62
caggtgcagc?tggtgcagtc?tgg 23
<210>63
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase degeneracy VH forward primer of people VH domain
<400>63
caggtcaact?taagggagtc?tgg 23
<210>64
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase degeneracy VH forward primer of people VH domain
<400>64
gaggtgcagc?tggtggagtc?tgg 23
<210>65
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase degeneracy VH forward primer of people VH domain
<400>65
caggtgcagc?tgcaggagtc?ggg 23
<210>66
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase degeneracy VH forward primer of people VH domain
<400>66
gaggtgcagc?tgttgcagtc?tgc 23
<210>67
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase degeneracy VH forward primer of people VH domain
<400>67
caggtacagc?tgcagcagtc?agg 23
<210>68
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase degeneracy JH reverse primer of people VH domain
<400>68
tgaggagacg?gtgaccaggg?tgcc 24
<210>69
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase degeneracy JH reverse primer of people VH domain
<400>69
tgaagagacg?gtgaccattg?tccc 24
<210>70
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase degeneracy JH reverse primer of people VH domain
<400>70
tgaggagacg?gtgaccaggg?ttcc 24
<210>71
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase degeneracy JH reverse primer of people VH domain
<400>71
tgaggagacg?gtgaccgtgg?tccc 24
<210>72
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase degeneracy V κ forward primer of people VL domain
<400>72
gacatccaga?tgacccagtc?tcc 23
<210>73
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase degeneracy V κ forward primer of people VL domain
<400>73
gatgttgtga?tgactcagtc?tcc 23
<210>74
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase degeneracy V κ forward primer of people VL domain
<400>74
gatattgtga?tgactcagtc?tcc 23
<210>75
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase degeneracy V κ forward primer of people VL domain
<400>75
gaaattgtgt?tgacgcagtc?tcc 23
<210>76
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase degeneracy V κ forward primer of people VL domain
<400>76
gacatcgtga?tgacccagtc?tcc 23
<210>77
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase degeneracy V κ forward primer of people VL domain
<400>77
gaaacgacac?tcacgcagtc?tcc 23
<210>78
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase degeneracy V κ forward primer of people VL domain
<400>78
gaaattgtgc?tgactcagtc?tcc 23
<210>79
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase degeneracy V of people VL domain
λForward primer
<400>79
cagtctgtgt?tgacgcagcc?gcc 23
<210>80
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase degeneracy V of people VL domain
λForward primer
<400>80
cagtctgccc?tgactcagcc?tgc 23
<210>81
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase degeneracy V of people VL domain
λForward primer
<400>81
tcctatgtgc?tgactcagcc?acc 23
<210>82
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase degeneracy V of people VL domain
λForward primer
<400>82
tcttctgagc?tgactcagga?ccc 23
<210>83
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase degeneracy V of people VL domain
λForward primer
<400>83
cacgttatac?tgactcaacc?gcc 23
<210>84
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase degeneracy V of people VL domain
λForward primer
<400>84
caggctgtgc?tcactcagcc?gtc 23
<210>85
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase degeneracy V of people VL domain
λForward primer
<400>85
aattttatgc?tgactcagcc?cca 23
<210>86
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase degeneracy J κ reverse primer of people VL domain
<400>86
acgtttgatt?tccaccttgg?tccc 24
<210>87
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase degeneracy J κ reverse primer of people VL domain
<400>87
acgtttgatc?tccagcttgg?tccc 24
<210>88
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase degeneracy J κ reverse primer of people VL domain
<400>88
acgtttgata?tccactttgg?tccc 24
<210>89
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase degeneracy J κ reverse primer of people VL domain
<400>89
acgtttgatc?tccaccttgg?tccc 24
<210>90
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase degeneracy J κ reverse primer of people VL domain
<400>90
acgtttaatc?tccagtcgtg?tccc 24
<210>91
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase degeneracy J of people VL domain
λReverse primer
<400>91
cagtctgtgt?tgacgcagcc?gcc 23
<210>92
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase degeneracy J of people VL domain
λReverse primer
<400>92
cagtctgccc?tgactcagcc?tgc 23
<210>93
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase degeneracy J of people VL domain
λReverse primer
<400>93
tcctatgtgc?tgactcagcc?acc 23
<210>94
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase degeneracy J of people VL domain
λReverse primer
<400>94
tcttctgagc?tgactcagga?ccc 23
<210>95
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase degeneracy J of people VL domain
λReverse primer
<400>95
cacgttatac?tgactcaacc?gcc 23
<210>96
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase degeneracy J of people VL domain
λReverse primer
<400>96
caggctgtgc?tcactcagcc?gtc 23
<210>97
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used to the to increase degeneracy J of people VL domain
λReverse primer
<400>97
aattttatgc?tgactcagcc?cca 23
<210>98
<211>5
<212>PRT
＜213〉human (Homo sapiens)
<400>98
Cys?Asp?Leu?Pro?Gln
1 5
<210>99
<211>165
<212>PRT
＜213〉human (Homo sapiens)
<220>
<221>MISC_FEATURE
<222>(23)
＜223〉Xaa equals Arg or Lys
<220>
<221>MISC_FEATURE
<222>(113)
＜223〉Xaa equals Ala or Val
<400>99
Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?Arg?Thr?Leu?Met
1 5 10 15
Leu?Leu?Ala?Gln?Met?Arg?Xaa?Ile?Ser?Leu?Phe?Ser?Cys?Leu?Lys?Asp
20 25 30
Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe?Gln
35 40 45
Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Ile?Phe
50 55 60
Asn?Leu?Phe?Thr?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Asp?Leu
65 70 75 80
Leu?Asp?Lys?Phe?Cys?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu?Glu
85 90 95
Ala?Cys?Val?Met?Gln?Glu?Glu?Arg?Val?Gly?Glu?Thr?Pro?Leu?Met?Asn
100 105 110
Xaa?Asp?Ser?Ile?Leu?Ala?Val?Lys?Lys?Tyr?Phe?Arg?Arg?Ile?Thr?Leu
115 120 125
Tyr?Leu?Thr?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val?Arg
130 135 140
Ala?Glu?Ile?Met?Arg?Ser?Leu?Ser?Leu?Ser?Thr?Asn?Leu?Gln?Glu?Arg
145 150 155 160
Leu?Arg?Arg?Lys?Glu
165
<210>100
<211>165
<212>PRT
＜213〉human (Homo sapiens)
<220>
<221>MISC_FEATURE
<222>(23)
＜223〉Xaa equals Arg or Lys
<220>
<221>MISC_FEATURE
<222>(113)
＜223〉Xaa equals Ala or Val
<400>100
Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?Arg?Thr?Leu?Met
1 5 10 15
Leu?Leu?Ala?Gln?Met?Arg?Xaa?Ile?Ser?Leu?Phe?Ser?Cys?Leu?Lys?Asp
20 25 30
Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe?Gln
35 40 45
Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Ile?Phe
50 55 60
Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr?Leu
65 70 75 80
Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu?Glu
85 90 95
Ala?Cys?Val?Met?Gln?Glu?Glu?Arg?Val?Gly?Glu?Thr?Pro?Leu?Met?Asn
100 105 110
Xaa?Asp?Ser?Ile?Leu?Ala?Val?Lys?Lys?Tyr?Phe?Arg?Arg?Ile?Thr?Leu
115 120 125
Tyr?Leu?Thr?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val?Arg
130 135 140
Ala?Glu?Ile?Met?Arg?Ser?Leu?Ser?Leu?Ser?Thr?Asn?Leu?Gln?Glu?Arg
145 150 155 160
Leu?Arg?Arg?Lys?Glu
165
<210>101
<211>165
<212>PRT
＜213〉human (Homo sapiens)
<220>
<221>MISC_FEATURE
<222>(23)
＜223〉Xaa equals Arg or Lys
<400>101
Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?Arg?Thr?Leu?Met
1 5 10 15
Leu?Leu?Ala?Gln?Met?Arg?Xaa?Ile?Ser?Leu?Phe?Ser?Cys?Leu?Lys?Asp
20 25 30
Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe?Gln
35 40 45
Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Ile?Phe
50 55 60
Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr?Leu
65 70 75 80
Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Met?Glu
85 90 95
Ala?Cys?Val?Ile?Gln?Glu?Val?Gly?Val?Glu?Glu?Thr?Pro?Leu?Met?Asn
100 105 110
Val?Asp?Ser?Ile?Leu?Ala?Val?Lys?Lys?Tyr?Phe?Gln?Arg?Ile?Thr?Leu
115 120 125
Tyr?Leu?Thr?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val?Arg
130 135 140
Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Leu?Ser?Lys?Ile?Phe?Gln?Glu?Arg
145 150 155 160
Leu?Arg?Arg?Lys?Glu
165
<210>102
<211>165
<212>PRT
＜213〉human (Homo sapiens)
<220>
<221>MISC_FEATURE
<222>(23)
＜223〉Xaa equals Arg or Lys
<220>
<221>MISC_FEATURE
<222>(97)
＜223〉Xaa equals Ser or Val
<220>
<221>MISC_FEATURE
<222>(98)
＜223〉Xaa equals Cys or Leu
<220>
<221>MISC_FEATURE
<222>(99)
＜223〉Xaa equals Val or Cys
<220>
<221>MISC_FEATURE
<222>(100)
＜223〉Xaa equals Met or Asp
<400>102
Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?Arg?Thr?Leu?Met
1 5 10 15
Leu?Leu?Ala?Gln?Met?Arg?Xaa?Ile?Ser?Leu?Phe?Ser?Cys?Leu?Lys?Asp
20 25 30
Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe?Gln
35 40 45
Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Ile?Phe
50 55 60
Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr?Leu
65 70 75 80
Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu?Glu
85 90 95
Xaa?Xaa?Xaa?Xaa?Gln?Glu?Val?Gly?Val?Ile?Glu?Ser?Pro?Leu?Met?Tyr
100 105 110
Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr?Leu
115 120 125
Tyr?Leu?Thr?Glu?Lys?Lys?Tyr?Ser?Ser?Cys?Ala?Trp?Glu?Val?Val?Arg
130 135 140
Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Leu?Ser?Ile?Asn?Leu?Gln?Lys?Arg
145 150 155 160
Leu?Lys?Ser?Lys?Glu
165
<210>103
<211>167
<212>PRT
＜213〉human (Homo sapiens)
<220>
<221>MISC_FEATURE
<222>(115)
＜223〉Xaa equals Ala or Val
<400>103
Met?Ser?Tyr?Asn?Leu?Leu?Gly?Phe?Leu?Gln?Arg?Ser?Ser?Asn?Phe?Gln
1 5 10 15
Cys?Gln?Lys?Leu?Leu?Trp?Gln?Leu?Asn?Gly?Arg?Leu?Glu?Tyr?Cys?Leu
20 25 30
Lys?Asp?Arg?Met?Asn?Phe?Asp?Ile?Pro?Glu?Glu?Ile?Lys?Gln?Leu?Gln
35 40 45
Gln?Phe?Gln?Lys?Glu?Asp?Ala?Ala?Leu?Thr?Ile?Tyr?Glu?Met?Leu?Gln
50 55 60
Asn?Ile?Phe?Ala?Ile?Phe?Arg?Gln?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu
65 70 75 80
Asp?Leu?Leu?Asp?Lys?Phe?Cys?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp
85 90 95
Leu?Glu?Ala?Cys?Val?Met?Gln?Glu?Glu?Arg?Val?Gly?Glu?Thr?Pro?Leu
100 105 110
Met?Asn?Xaa?Asp?Ser?Ile?Leu?Ala?Val?Lys?Lys?Tyr?Phe?Arg?Arg?Ile
115 120 125
Thr?Leu?Tyr?Leu?Thr?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val
130 135 140
Val?Arg?Ala?Glu?Ile?Met?Arg?Ser?Leu?Ser?Leu?Ser?Thr?Asn?Leu?Gln
145 150 155 160
Glu?Arg?Leu?Arg?Arg?Lys?Glu
165
<210>104
<211>166
<212>PRT
＜213〉human (Homo sapiens)
<400>104
Met?Cys?Asp?Leu?Pro?Glu?Thr?His?Ser?Leu?Asp?Asn?Arg?Arg?Thr?Leu
1 5 10 15
Met?Leu?Leu?Ala?Gln?Met?Ser?Arg?Ile?Ser?Pro?Ser?Ser?Cys?Leu?Met
20 25 30
Asp?Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Asp?Gly?Asn?Gln
35 40 45
Phe?Gln?Lys?Ala?Pro?Ala?Ile?Ser?Val?Leu?His?Glu?Leu?Ile?Gln?Gln
50 55 60
Ile?Phe?Asn?Leu?Phe?Thr?Thr?Lys?Asp?Ser?Ser?Ser?Thr?Gly?Trp?Asn
65 70 75 80
Glu?Thr?Ile?Val?Glu?Asn?Leu?Leu?Ala?Asn?Val?Tyr?His?Gln?Ile?Asn
85 90 95
His?Leu?Lys?Thr?Val?Leu?Glu?Glu?Lys?Leu?Glu?Lys?Glu?Asp?Phe?Thr
100 105 110
Arg?Gly?Lys?Leu?Met?Ser?Ser?Leu?His?Leu?Lys?Arg?Tyr?Tyr?Gly?Arg
115 120 125
Ile?Leu?His?Tyr?Leu?Lys?Ala?Lys?Glu?Tyr?Ser?His?Cys?Ala?Trp?Thr
130 135 140
Ile?Val?Arg?Val?Glu?Ile?Leu?Arg?Asn?Phe?Tyr?Phe?Ile?Asn?Arg?Leu
145 150 155 160
Thr?Gly?Tyr?Leu?Arg?Asn
165
<210>105
<211>23
<212>PRT
＜213〉artificial sequence
<220>
＜223〉composite signal peptide
<400>105
Met?Arg?Pro?Thr?Trp?Ala?Trp?Trp?Leu?Phe?Leu?Val?Leu?Leu?Leu?Ala?Leu
1 5 10 15
Trp?Ala?Pro?Ala?Arg?Gly
20
<210>106
<211>0
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer (deletion)
<400>106
000
<210>107
<211>106
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used for the primer that total length IFNb increases, have BamH I cloning site
<400>107
gcgcggatcc?gaattccgcc?gccatgacca?acaagtgtct?cctccaaatt?gctctcctgt?60
tgtgcttctc?cactacagct?ctttccatga?gctacaactt?gcttgg 106
<210>108
<211>55
<212>DNA
＜213〉artificial sequence
<220>
＜223〉be used for the primer that total length IFNb increases, have Cla I cloning site
<400>108
gcgcgcatcg?atgagcaacc?teactcttgtgt?gcatcgttt?cggaggtaa?cctgt 55
<210>109
<211>41
<212>DNA
＜213〉human (Homo sapiens)
<400>109
cgcgcgcgtc?gacaaaagat?gtgatctgcc?tcaaacccac?a 41
<210>110
<211>59
<212>DNA
＜213〉human (Homo sapiens)
<400>110
gcgcgcatcg?atgagcaacc?tcactcttgt?gtgcatcttc?cttacttctt?aaactttct 59
<210>111
<211>21
<212>PRT
＜213〉human (Homo sapiens)
<400>111
Met?Trp?Trp?Arg?Leu?Trp?Trp?Leu?Leu?Leu?Leu?Leu?Leu?Leu?Leu?Trp
1 5 10 15
Pro?Met?Val?Trp?Ala
20
<210>112
<211>24
<212>PRT
＜213〉human (Homo sapiens)
<400>112
Met?Lys?Trp?Val?Ser?Phe?Ile?Ser?Leu?Leu?Phe?Leu?Phe?Ser?Ser?Ala
1 5 10 15
Tyr?Ser?Gly?Ser?Leu?Asp?Lys?Arg
20
<210>113
<211>1497
<212>DNA
＜213〉Hypocrea jecorina (Hypocrea jecorina)
<400>113
tctagagttg?tgaagtcggt?aatcccgctg?tatagtaata?cgagtcgcat?ctaaatactc?60
cgaagctgct?gcgaacccgg?agaatcgaga?tgtgctggaa?agcttctagc?gagcggctaa?120
attagcatga?aaggctatga?gaaattctgg?agacggcttg?ttgaatcatg?gcgttccatt?180
cttcgacaag?caaagcgttc?cgtcgcagta?gcaggcactc?attcccgaaa?aaactcggag?240
attcctaagt?agcgatggaa?ccggaataat?ataataggca?atacattgag?ttgcctcgac?300
ggttgcaatg?caggggtact?gagcttggac?ataactgttc?cgtaccccac?ctcttctcaa?360
cctttggcgt?ttccctgatt?cagcgtaccc?gtacaagtcg?taatcactat?taacccagac?420
tgaccggacg?tgttttgccc?ttcatttgga?gaaataatgt?cattgcgatg?tgtaatttgc?480
ctgcttgacc?gactggggct?gttcgaagcc?cgaatgtagg?attgttatcc?gaactctgct?540
cgtagaggca?tgttgtgaat?ctgtgtcggg?caggacacgc?ctcgaaggtt?cacggcaagg?600
gaaaccaccg?atagcagtgt?ctagtagcaa?cctgtaaagc?cgcaatgcag?catcactgga?660
aaatacaaac?caatggctaa?aagtacataa?gttaatgcct?aaagaagtca?tataccagcg?720
gctaataatt?gtacaatcaa?gtggctaaac?gtaccgtaat?ttgccaacgg?cttgtggggt?780
tgcagaagca?acggcaaagc?cccacttccc?cacgtttgtt?tcttcactca?gtccaatctc?840
agctggtgat?cccccaattg?ggtcgcttgt?ttgttccggt?gaagtgaaag?aagacagagg?900
taagaatgtc?tgactcggag?cgttttgcat?acaaccaagg?gcagtgatgg?aagacagtga?960
aatgttgaca?ttcaaggagt?atttagccag?ggatgcttga?gtgtatcgtg?taaggaggtt?1020
tgtctgccga?tacgacgaat?actgtatagt?cacttctggt?gaagtggtcc?atattgaaat?1080
gtaagtcggc?actgaacagg?caaaagattg?agttgaaact?gcctaagatc?tcgggccctc?1140
gggccttcgg?cctttgggtg?tacatgtttg?tgctccgggc?aaatgcaaag?tgtggtagga?1200
tcgaacacac?tgctgccttt?accaagcagc?tgagggtatg?tgataggcaa?atgttcaggg?1260
gccactgcat?ggtttcgaat?agaaagagaa?gcttagccaa?gaacaatagc?cgataaagat?1320
agcctcatta?aacggaatga?gctagtaggc?aaagtcagcg?aatgtgtata?tataaaggtt?1380
cgaggtccgt?gcctccctca?tgctctcccc?atctactcat?caactcagat?cctccaggag?1440
acttgtacac?catcttttga?ggcacagaaa?cccaatagtc?aaccgcggac?tggcatc 1497
<210>114
<211>366
<212>DNA
＜213〉aspergillus nidulans (Aspergillus nidulans)
<400>114
agatctggtt?cctgagtaca?tctaccgatg?cgcctcgatc?cccctcttag?ccgcatgaga?60
ttcctaccat?ttatgtccta?tcgttcaggg?tcctatttgg?accgctagaa?atagactctg?120
ctcgatttgt?ttccattatt?cacgcaatta?cgatagtatt?tggctctttt?cgtttggccc?180
aggtcaattc?gggtaagacg?cgatcacgcc?attgtggccg?ccggcgttgt?gctgctgcta?240
ttccccgcat?ataaacaacc?cctccaccag?ttcgttgggc?tttgcgaatg?ctgtactcta?300
tttcaagttg?tcaaaagaga?ggattcaaaa?aattataccc?cagatatcaa?agatatcaaa?360
gccatc 366
<210>115
<211>1646
<212>DNA
＜213〉Hypocrea jecorina (Hypocrea jecorina)
<400>115
tctagagttg?tgaagtcggt?aatcccgctg?tatagtaata?cgagtcgcat?ctaaatactc?60
cgaagctgct?gcgaacccgg?agaatcgaga?tgtgctggaa?agcttctagc?gagcggctaa?120
attagcatga?aaggctatga?gaaattctgg?agacggcttg?ttgaatcatg?gcgttccatt?180
cttcgacaag?caaagcgttc?cgtcgcagta?gcaggcactc?attcccgaaa?aaactcggag?240
attcctaagt?agcgatggaa?ccggaataat?ataataggca?atacattgag?ttgcctcgac?300
ggttgcaatg?caggggtact?gagcttggac?ataactgttc?cgtaccccac?ctcttctcaa?360
cctttggcgt?ttccctgatt?cagcgtaccc?gtacaagtcg?taatcactat?taacccagac?420
tgaccggacg?tgttttgccc?ttcatttgga?gaaataatgt?cattgcgatg?tgtaatttgc?480
ctgcttgacc?gactggggct?gttcgaagcc?cgaatgtagg?attgttatcc?gaactctgct?540
cgtagaggca?tgttgtgaat?ctgtgtcggg?caggacacgc?ctcgaaggtt?cacggcaagg?600
gaaaccaccg?atagcagtgt?ctagtagcaa?cctgtaaagc?cgcaatgcag?catcactgga?660
aaatacaaac?caatggctaa?aagtacataa?gttaatgcct?aaagaagtca?tataccagcg?720
gctaataatt?gtacaatcaa?gtggctaaac?gtaccgtaat?ttgccaacgg?cttgtggggt?780
tgcagaagca?acggcaaagc?cccacttccc?cacgtttgtt?tcttcactca?gtccaatctc?840
agctggtgat?cccccaattg?ggtcgcttgt?ttgttccggt?gaagtgaaag?aagacagagg?900
taagaatgtc?tgactcggag?cgttttgcat?acaaccaagg?gcagtgatgg?aagacagtga?960
aatgttgaca?ttcaaggagt?atttagccag?ggatgcttga?gtgtatcgtg?taaggaggtt?1020
tgtctgccga?tacgacgaat?actgtatagt?cacttctggt?gaagtggtcc?atattgaaat?1080
gtaagtcggc?actgaacagg?caaaagattg?agttgaaact?gcctaagatc?tcgggccctc?1140
gggccttcgg?cctttgggtg?tacatgtttg?tgctccgggc?aaatgcaaag?tgtggtagga?1200
tcgaacacac?tgctgccttt?accaagcagc?tgagggtatg?tgataggcaa?atgttcaggg?1260
gccactgcat?ggtttcgaat?agaaagagaa?gcttagcctg?cagcctctta?tcgagaaaga?1320
aattaccgtc?gctcgtgatt?tgtttgcaaa?aagaacaaaa?ctgaaaaaac?ccagacacgc?1380
tcgacttcct?gtcttcctat?tgattgcagc?ttccaatttc?gtcacacaac?aaggtcctag?1440
cttagccaag?aacaatagcc?gataaagata?gcctcattaa?acggaatgag?ctagtaggca?1500
aagtcagcga?atgtgtatat?ataaaggttc?gaggtccgtg?cctccctcat?gctctcccca?1560
tctactcatc?aactcagatc?ctccaggaga?cttgtacacc?atcttttgag?gcacagaaac?1620
ccaatagtca?accgcggact?ggcatc 1646
<210>116
<211>516
<212>DNA
＜213〉aspergillus nidulans (Aspergillus nidulans)
<400>116
agatctggtt?cctgagtaca?tctaccgatg?cgcctcgatc?cccctcttag?ccgcatgaga?60
ttcctaccat?ttatgtccta?tcgttcaggg?tcctatttgg?accgctagaa?atagactctg?120
ctcgatttgt?ttccattatt?cacgcaatta?cgatagtatt?tggctctttt?cgtttggccc?180
aggtcaattc?gggtaagacg?cgatcacgcc?attgtggccg?ccggcgctgc?agcctcttat?240
cgagaaagaa?attaccgtcg?ctcgtgattt?gtttgcaaaa?agaacaaaac?tgaaaaaacc?300
cagacacgct?cgacttcctg?tcttcctatt?gattgcagct?tccaatttcg?tcacacaaca?360
aggtcctacg?ccggcgttgt?gctgctgcta?ttccccgcat?ataaacaacc?cctccaccag?420
ttcgttgggc?tttgcgaatg?ctgtactcta?tttcaagttg?tcaaaagaga?ggattcaaaa?480
aattataccc?cagatatcaa?agatatcaaa?gccatc 516
<210>117
<211>495
<212>DNA
＜213〉human (Homo sapiens)
<400>117
tgtgatctgc?ctcaaaccca?cagcctgggt?tctagaagga?ccttgatgct?cctggcacag?60
atgaggagaa?tctctctttt?ctcctgcttg?aaggacagac?atgactttgg?atttccccag?120
gaggagtttg?gcaaccagtt?ccaaaaggct?gaaaccatcc?ctgtcctcca?tgagatgatc?180
cagcagatct?tcaatctctt?cagcacaaag?gactcatctg?ctgcttggga?tgagaccctc?240
ctagacaaat?tctacactga?actctaccag?cagctgaatg?acctggaagc?ctgtgtgata?300
cagggggtgg?gggtgacaga?gactcccctg?atgaaggagg?actccattct?ggctgtgagg?360
aaatacttcc?aaagaatcac?tctctatctg?aaagagaaga?aatacagccc?ttgtgcctgg?420
gaggttgtca?gagcagaaat?catgagatct?ttttctttgt?caacaaactt?gcaagaaagt?480
ttaagaagta?aggaa 495
<210>118
<211>495
<212>DNA
＜213〉human (Homo sapiens)
<400>118
tgtgatctgc?ctcaaaccca?cagcctgggt?tctagaagga?ccttgatgct?cctggcacag?60
atgaggagaa?tctctctttt?ctcctgcttg?aaggacagac?atgactttgg?atttccccag?120
gaggagtttg?gcaaccagtt?ccaaaaggct?gaaaccatcc?ctgtcctcca?tgagatgatc?180
cagcagatct?tcaatctctt?cagcacaaag?gactcatctg?ctgcttggga?tgagaccctc?240
ctagacaaat?tctacactga?actctaccag?cagctgaatg?acctggaagc?ctgtgtgata?300
cagggggtgg?gggtgacaga?gactcccctg?atgaaggagg?actccattct?ggctgtgagg?360
aaatacttcc?aaagaatcac?tctctatctg?aaagagaaga?aatacagccc?ttgtgcctgg?420
gaggttgtca?gagcagaaat?catgagatct?ttttctttgt?caacaaactt?gcaagaaagt?480
ttaagaagta?aggaa 495
<210>119
<211>495
<212>DNA
＜213〉human (Homo sapiens)
<400>119
tgtgatctgc?ctcaaaccca?cagcctgggt?tctagaagga?ccttgatgct?cctggcacag?60
atgaggagaa?tctctctttt?ctcctgcttg?aaggacagac?atgactttgg?atttccccag?120
gaggagtttg?gcaaccagtt?ccaaaaggct?gaaaccatcc?ctgtcctcca?tgagatgatc?180
cagcagatct?tcaatctctt?cagcacaaag?gactcatctg?ctgcttggga?tgagaccctc?240
ctagacaaat?tctacactga?actctaccag?cagctgaatg?acctggaagc?ctgtgtgata?300
cagggggtgg?gggtgacaga?gactcccctg?atgaaggagg?actccattct?ggctgtgagg?360
aaatacttcc?aaagaatcac?tctctatctg?aaagagaaga?aatacagccc?ttgtgcctgg?420
gaggttgtca?gagcagaaat?catgagatct?ttttctttgt?caacaaactt?gcaagaaagt?480
ttaagaagta?aggaa 495
<210>120
<211>498
<212>DNA
＜213〉human (Homo sapiens)
<400>120
tgtgatctgc?ctcaaaccca?cagcctgggt?tctagaagga?ccttgatgct?cctggcacag 60
atgaggagaa?tctctctttt?ctcctgcttg?aaggacagac?atgactttgg?atttccccag 120
gaggagtttg?gcaaccagtt?ccaaaaggct?gaaaccatcc?ctgtcctcca?tgagatgatc 180
cagcagatct?tcaatctctt?cagcacaaag?gactcatctg?ctgcttggga?tgagaccctc 240
ctagacaaat?tctacactga?actctaccag?cagctgaatg?acctggaagc?ctgtgtgata 300
cagggggtgg?gggtgacaga?gactcccctg?atgaaggagg?actccattct?ggctgtgagg 360
aaatacttcc?aaagaatcac?tctctatctg?aaagagaaga?aatacagccc?ttgtgcctgg 420
gaggttgtca?gagcagaaat?catgagatct?ttttctttgt?caacaaactt?gcaagaaagt 480
ttaagaagta?aggaataa 498
<210>121
<211>495
<212>DNA
＜213〉human (Homo sapiens)
<400>121
tgtgatctgc?ctcaaaccca?cagcctgggt?tctagaagga?ccttgatgct?cctggcacag 60
atgaggagaa?tctctctttt?ctcctgcttg?aaggacagac?atgactttgg?atttccccag 120
gaggagtttg?gcaaccagtt?ccaaaaggct?gaaaccatcc?ctgtcctcca?tgagatgatc 180
cagcagatct?tcaatctctt?cagcacaaag?gactcatctg?ctgcttggga?tgagaccctc 240
ctagacaaat?tctacactga?actctaccag?cagctgaatg?acctggaagc?ctgtgtgata 300
cagggggtgg?gggtgacaga?gactcccctg?atgaaggagg?actccattct?ggctgtgagg 360
aaatacttcc?aaagaatcac?tctctatctg?aaagagaaga?aatacagccc?ttgtgcctgg 420
gaggttgtca?gagcagaaat?catgagatct?ttttctttgt?caacaaactt?gcaagaaagt 480
ttaagaagta?aggaa 495
<210>122
<211>495
<212>DNA
＜213〉human (Homo sapiens)
<400>122
tgtgatctgc?ctcaaaccca?cagcctgggt?tctagaagga?ccttgatgct?cctggcacag 60
atgaggagaa?tctctctttt?ctcctgcttg?aaggacagac?atgactttgg?atttccccag 120
gaggagtttg?gcaaccagtt?ccaaaaggct?gaaaccatcc?ctgtcctcca?tgagatgatc 180
cagcagatct?tcaatctctt?cagcacaaag?gactcatctg?ctgcttggga?tgagaccctc 240
ctagacaaat?tctacactga?actctaccag?cagctgaatg?acctggaagc?ctgtgtgata 300
cagggggtgg?gggtgacaga?gactcccctg?atgaaggagg?actccattct?ggctgtgagg 360
aaatacttcc?aaagaatcac?tctctatctg?aaagagaaga?aatacagccc?ttgtgcctgg 420
gaggttgtca?gagcagaaat?catgagatct?ttttctttgt?caacaaactt?gcaagaaagt 480
ttaagaagta?aggaa 495
<210>123
<211>495
<212>DNA
＜213〉human (Homo sapiens)
<400>123
tgtgatctgc?ctcaaaccca?cagcctgggt?tctagaagga?ccttgatgct?cctggcacag 60
atgaggagaa?tctctctttt?ctcctgcttg?aaggacagac?atgactttgg?atttccccag 120
gaggagtttg?gcaaccagtt?ccaaaaggct?gaaaccatcc?ctgtcctcca?tgagatgatc 180
cagcagatct?tcaatctctt?cagcacaaag?gactcatctg?ctgcttggga?tgagaccctc 240
ctagacaaat?tctacactga?actctaccag?cagctgaatg?acctggaagc?ctgtgtgata 300
cagggggtgg?gggtgacaga?gactcccctg?atgaaggagg?actccattct?ggctgtgagg 360
aaatacttcc?aaagaatcac?tctctatctg?aaagagaaga?aatacagccc?ttgtgcctgg 420
gaggttgtca?gagcagaaat?catgagatct?ttttctttgt?caacaaactt?gcaagaaagt 480
ttaagaagta?aggaa 495
<210>124
<211>495
<212>DNA
＜213〉human (Homo sapiens)
<400>124
tgtgatctgc?ctcaaaccca?cagcctgggt?tctagaagga?ccttgatgct?cctggcacag 60
atgaggagaa?tctctctttt?ctcctgcttg?aaggacagac?atgactttgg?atttccccag 120
gaggagtttg?gcaaccagtt?ccaaaaggct?gaaaccatcc?ctgtcctcca?tgagatgatc 180
cagcagatct?tcaatctctt?cagcacaaag?gactcatctg?ctgcttggga?tgagaccctc 240
ctagacaaat?tctacactga?actctaccag?cagctgaatg?acctggaagc?ctgtgtgata 300
cagggggtgg?gggtgacaga?gactcccctg?atgaaggagg?actccattct?ggctgtgagg 360
aaatacttcc?aaagaatcac?tctctatctg?aaagagaaga?aatacagccc?ttgtgcctgg 420
gaggttgtca?gagcagaaat?catgagatct?ttttctttgt?caacaaactt?gcaagaaagt 480
ttaagaagta?aggaa 495
<210>125
<211>495
<212>DNA
＜213〉human (Homo sapiens)
<400>125
tgtgatctgc?ctcaaaccca?cagcctgggt?tctagaagga?ccttgatgct?cctggcacag 60
atgaggagaa?tctctctttt?ctcctgcttg?aaggacagac?atgactttgg?atttccccag 120
gaggagtttg?gcaaccagtt?ccaaaaggct?gaaaccatcc?ctgtcctcca?tgagatgatc 180
cagcagatct?tcaatctctt?cagcacaaag?gactcatctg?ctgcttggga?tgagaccctc 240
ctagacaaat?tctacactga?actctaccag?cagctgaatg?acctggaagc?ctgtgtgata 300
cagggggtgg?gggtgacaga?gactcccctg?atgaaggagg?actccattct?ggctgtgagg 360
aaatacttcc?aaagaatcac?tctctatctg?aaagagaaga?aatacagccc?ttgtgcctgg 420
gaggttgtca?gagcagaaat?catgagatct?ttttctttgt?caacaaactt?gcaagaaagt 480
ttaagaagta?aggaa 495
<210>126
<211>495
<212>DNA
＜213〉human (Homo sapiens)
<400>126
tgtgatctgc?ctcaaaccca?cagcctgggt?tctagaagga?ccttgatgct?cctggcacag?60
atgaggagaa?tctctctttt?ctcctgcttg?aaggacagac?atgactttgg?atttccccag?120
gaggagtttg?gcaaccagtt?ccaaaaggct?gaaaccatcc?ctgtcctcca?tgagatgatc?180
cagcagatct?tcaatctctt?cagcacaaag?gactcatctg?ctgcttggga?tgagaccctc?240
ctagacaaat?tctacactga?actctaccag?cagctgaatg?acctggaagc?ctgtgtgata?300
cagggggtgg?gggtgacaga?gactcccctg?atgaaggagg?actccattct?ggctgtgagg?360
aaatacttcc?aaagaatcac?tctctatctg?aaagagaaga?aatacagccc?ttgtgcctgg?420
gaggttgtca?gagcagaaat?catgagatct?ttttctttgt?caacaaactt?gcaagaaagt?480
ttaagaagta?aggaa 495
<210>127
<211>495
<212>DNA
＜213〉human (Homo sapiens)
<400>127
tgtgatctgc?ctcaaaccca?cagcctgggt?tctagaagga?ccttgatgct?cctggcacag?60
atgaggagaa?tctctctttt?ctcctgcttg?aaggacagac?atgactttgg?atttccccag?120
gaggagtttg?gcaaccagtt?ccaaaaggct?gaaaccatcc?ctgtcctcca?tgagatgatc?180
cagcagatct?tcaatctctt?cagcacaaag?gactcatctg?ctgcttggga?tgagaccctc?240
ctagacaaat?tctacactga?actctaccag?cagctgaatg?acctggaagc?ctgtgtgata?300
cagggggtgg?gggtgacaga?gactcccctg?atgaaggagg?actccattct?ggctgtgagg?360
aaatacttcc?aaagaatcac?tctctatctg?aaagagaaga?aatacagccc?ttgtgcctgg?420
gaggttgtca?gagcagaaat?catgagatct?ttttctttgt?caacaaactt?gcaagaaagt?480
ttaagaagta?aggaa 495
<210>128
<211>767
<212>PRT
＜213〉human (Homo sapiens)
<400>128
Met?Phe?Lys?Ser?Val?Val?Tyr?Ser?Ile?Leu?Ala?Ala?Ser?Leu?Ala?Asn
1 5 10 15
Ala?Asp?Ala?His?Lys?Ser?Glu?Val?Ala?His?Arg?Phe?Lys?Asp?Leu?Gly
20 25 30
Glu?Glu?Asn?Phe?Lys?Ala?Leu?Val?Leu?Ile?Ala?Phe?Ala?Gln?Tyr?Leu
35 40 45
Gln?Gln?Cys?Pro?Phe?Glu?Asp?His?Val?Lys?Leu?Val?Asn?Glu?Val?Thr
50 55 60
Glu?Phe?Ala?Lys?Thr?Cys?Val?Ala?Asp?Glu?Ser?Ala?Glu?Asn?Cys?Asp
65 70 75 80
Lys?Ser?Leu?His?Thr?Leu?Phe?Gly?Asp?Lys?Leu?Cys?Thr?Val?Ala?Thr
85 90 95
Leu?Arg?Glu?Thr?Tyr?Gly?Glu?Met?Ala?Asp?Cys?Cys?Ala?Lys?Gln?Glu
100 105 110
Pro?Glu?Arg?Asn?Glu?Cys?Phe?Leu?Gln?His?Lys?Asp?Asp?Asn?Pro?Asn
115 120 125
Leu?Pro?Arg?Leu?Val?Arg?Pro?Glu?Val?Asp?Val?Met?Cys?Thr?Ala?Phe
130 135 140
His?Asp?Asn?Glu?Glu?Thr?Phe?Leu?Lys?Lys?Tyr?Leu?Tyr?Glu?Ile?Ala
145 150 155 160
Arg?Arg?His?Pro?Tyr?Phe?Tyr?Ala?Pro?Glu?Leu?Leu?Phe?Phe?Ala?Lys
165 170 175
Arg?Tyr?Lys?Ala?Ala?Phe?Thr?Glu?Cys?Cys?Gln?Ala?Ala?Asp?Lys?Ala
180 185 190
Ala?Cys?Leu?Leu?Pro?Lys?Leu?Asp?Glu?Leu?Arg?Asp?Glu?Gly?Lys?Ala
195 200 205
Ser?Ser?Ala?Lys?Gln?Arg?Leu?Lys?Cys?Ala?Ser?Leu?Gln?Lys?Phe?Gly
210 215 220
Glu?Arg?Ala?Phe?Lys?Ala?Trp?Ala?Val?Ala?Arg?Leu?Ser?Gln?Arg?Phe
225 230 235 240
Pro?Lys?Ala?Glu?Phe?Ala?Glu?Val?Ser?Lys?Leu?Val?Thr?Asp?Leu?Thr
245 250 255
Lys?Val?His?Thr?Glu?Cys?Cys?His?Gly?Asp?Leu?Leu?Glu?Cys?Ala?Asp
260 265 270
Asp?Arg?Ala?Asp?Leu?Ala?Lys?Tyr?Ile?Cys?Glu?Asn?Gln?Asp?Ser?Ile
275 280 285
Ser?Ser?Lys?Leu?Lys?Glu?Cys?Cys?Glu?Lys?Pro?Leu?Leu?Glu?Lys?Ser
290 295 300
His?Cys?Ile?Ala?Glu?Val?Glu?Asn?Asp?Glu?Met?Pro?Ala?Asp?Leu?Pro
305 310 315 320
Ser?Leu?Ala?Ala?Asp?Phe?Val?Glu?Ser?Lys?Asp?Val?Cys?Lys?Asn?Tyr
325 330 335
Ala?Glu?Ala?Lys?Asp?Val?Phe?Leu?Gly?Met?Phe?Leu?Tyr?Glu?Tyr?Ala
340 345 350
Arg?Arg?His?Pro?Asp?Tyr?Ser?Val?Val?Leu?Leu?Leu?Arg?Leu?Ala?Lys
355 360 365
Thr?Tyr?Glu?Thr?Thr?Leu?Glu?Lys?Cys?Cys?Ala?Ala?Ala?Asp?Pro?His
370 375 380
Glu?Cys?Tyr?Ala?Lys?Val?Phe?Asp?Glu?Phe?Lys?Pro?Leu?Val?Glu?Glu
385 390 395 400
Pro?Gln?Asn?Leu?Ile?Lys?Gln?Asn?Cys?Glu?Leu?Phe?Glu?Gln?Leu?Gly
405 410 415
Glu?Tyr?Lys?Phe?Gln?Asn?Ala?Leu?Leu?Val?Arg?Tyr?Thr?Lys?Lys?Val
420 425 430
Pro?Gln?Val?Ser?Thr?Pro?Thr?Leu?Val?Glu?Val?Ser?Arg?Asn?Leu?Gly
435 440 445
Lys?Val?Gly?Ser?Lys?Cys?Cys?Lys?His?Pro?Glu?Ala?Lys?Arg?Met?Pro
450 455 460
Cys?Ala?Glu?Asp?Tyr?Leu?Ser?Val?Val?Leu?Asn?Gln?Leu?Cys?Val?Leu
465 470 475 480
His?Glu?Lys?Thr?Pro?Val?Ser?Asp?Arg?Val?Thr?Lys?Cys?Cys?Thr?Glu
485 490 495
Ser?Leu?Val?Asn?Arg?Arg?Pro?Cys?Phe?Ser?Ala?Leu?Glu?Val?Asp?Glu
500 505 510
Thr?Tyr?Val?Pro?Lys?Glu?Phe?Asn?Ala?Glu?Thr?Phe?Thr?Phe?His?Ala
515 520 525
Asp?Ile?Cys?Thr?Leu?Ser?Glu?Lys?Glu?Arg?Gln?Ile?Lys?Lys?Gln?Thr
530 535 540
Ala?Leu?Val?Glu?Leu?Val?Lys?His?Lys?Pro?Lys?Ala?Thr?Lys?Glu?Gln
545 550 555 560
Leu?Lys?Ala?Val?Met?Asp?Asp?Phe?Ala?Ala?Phe?Val?Glu?Lys?Cys?Cys
565 570 575
Lys?Ala?Asp?Asp?Lys?Glu?Thr?Cys?Phe?Ala?Glu?Glu?Gly?Lys?Lys?Leu
580 585 590
Val?Ala?Ala?Ser?Gln?Ala?Ala?Leu?Gly?Leu?Cys?Asp?Leu?Pro?Gln?Thr
595 600 605
His?Ser?Leu?Gly?Ser?Arg?Arg?Thr?Leu?Met?Leu?Leu?Ala?Gln?Met?Arg
610 615 620
Arg?Ile?Ser?Leu?Phe?Ser?Cys?Leu?Lys?Asp?Arg?His?Asp?Phe?Gly?Phe
625 630 635 640
Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe?Gln?Lys?Ala?Glu?Thr?Ile?Pro
645 650 655
Val?Leu?His?Glu?Met?lle?Gln?Gln?Ile?Phe?Asn?Leu?Phe?Ser?Thr?Lys
660 665 670
Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr?Leu?Leu?Asp?Lys?Phe?Tyr?Thr
675 680 685
Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu?Glu?Ala?Cys?Val?Ile?Gln?Gly
690 695 700
Val?Gly?Val?Thr?Glu?Thr?Pro?Leu?Met?Lys?Glu?Asp?Ser?Ile?Leu?Ala
705 710 715 720
Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr?Leu?Tyr?Leu?Lys?Glu?Lys?Lys
725 730 735
Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val?Arg?Ala?Glu?Ile?Met?Arg?Ser
740 745 750
Phe?Ser?Leu?Ser?Thr?Asn?Leu?Gln?Glu?Ser?Leu?Arg?Ser?Lys?Glu
755 760 765
<210>129
<211>769
<212>PRT
＜213〉human (Homo sapiens)
<400>129
Met?Leu?Leu?Gln?Ala?Phe?Leu?Phe?Leu?Leu?Ala?Cly?Phe?Ala?Ala?Lys
1 5 10 15
Ile?Ser?Ala?Asp?Ala?His?Lys?Ser?Glu?Val?Ala?His?Arg?Phe?Lys?Asp
20 25 30
Leu?Gly?Glu?Glu?Asn?Phe?Lys?Ala?Leu?Val?Leu?Ile?Ala?Phe?Ala?Gln
35 40 45
Tyr?Leu?Gln?Gln?Cys?Pro?Phe?Glu?Asp?His?Val?Lys?Leu?Val?Asn?Glu
50 55 60
Val?Thr?Glu?Phe?Ala?Lys?Thr?Cys?Val?Ala?Asp?Glu?Ser?Ala?Glu?Asn
65 70 75 80
Cys?Asp?Lys?Ser?Leu?His?Thr?Leu?Phe?Gly?Asp?Lys?Leu?Cys?Thr?Val
85 90 95
Ala?Thr?Leu?Arg?Glu?Thr?Tyr?Gly?Glu?Met?Ala?Asp?Cys?Cys?Ala?Lys
100 105 110
Gln?Glu?Pro?Glu?Arg?Asn?Glu?Cys?Phe?Leu?Gln?His?Lys?Asp?Asp?Asn
115 120 125
Pro?Asn?Leu?Pro?Arg?Leu?Val?Arg?Pro?Glu?Val?Asp?Val?Met?Cys?Thr
130 135 140
Ala?Phe?His?Asp?Asn?Glu?Glu?Thr?Phe?Leu?Lys?Lys?Tyr?Leu?Tyr?Glu
145 150 155 160
Ile?Ala?Arg?Arg?His?Pro?Tyr?Phe?Tyr?Ala?Pro?Glu?Leu?Leu?Phe?Phe
165 170 175
Ala?Lys?Arg?Tyr?Lys?Ala?Ala?Phe?Thr?Glu?Cys?Cys?Gln?Ala?Ala?Asp
180 185 190
Lys?Ala?Ala?Cys?Leu?Leu?Pro?Lys?Leu?Asp?Glu?Leu?Arg?Asp?Glu?Gly
195 200 205
Lys?Ala?Ser?Ser?Ala?Lys?Gln?Arg?Leu?Lys?Cys?Ala?Ser?Leu?Gln?Lys
210 215 220
Phe?Gly?Glu?Arg?Ala?Phe?Lys?Ala?Trp?Ala?Val?Ala?Arg?Leu?Ser?Gln
225 230 235 240
Arg?Phe?Pro?Lys?Ala?Glu?Phe?Ala?Glu?Val?Ser?Lys?Leu?Val?Thr?Asp
245 250 255
Leu?Thr?Lys?Val?His?Thr?Glu?Cys?Cys?His?Gly?Asp?Leu?Leu?Glu?Cys
260 265 270
Ala?Asp?Asp?Arg?Ala?Asp?Leu?Ala?Lys?Tyr?Ile?Cys?Glu?Asn?Gln?Asp
275 280 285
Ser?Ile?Ser?Ser?Lys?Leu?Lys?Glu?Cys?Cys?Glu?Lys?Pro?Leu?Leu?Glu
290 295 300
Lys?Ser?His?Cys?Ile?Ala?Glu?Val?Glu?Asn?Asp?Glu?Met?Pro?Ala?Asp
305 310 315 320
Leu?Pro?Ser?Leu?Ala?Ala?Asp?Phe?Val?Glu?Ser?Lys?Asp?Val?Cys?Lys
325 330 335
Asn?Tyr?Ala?Glu?Ala?Lys?Asp?Val?Phe?Leu?Gly?Met?Phe?Leu?Tyr?Glu
340 345 350
Tyr?Ala?Arg?Arg?His?Pro?Asp?Tyr?Ser?Val?Val?Leu?Leu?Leu?Arg?Leu
355 360 365
Ala?Lys?Thr?Tyr?Glu?Thr?Thr?Leu?Glu?Lys?Cys?Cys?Ala?Ala?Ala?Asp
370 375 380
Pro?His?Glu?Cys?Tyr?Ala?Lys?Val?Phe?Asp?Glu?Phe?Lys?Pro?Leu?Val
385 390 395 400
Glu?Glu?Pro?Gln?Asn?Leu?Ile?Lys?Gln?Asn?Cys?Glu?Leu?Phe?Glu?Gln
405 410 415
Leu?Gly?Glu?Tyr?Lys?Phe?Gln?Asn?Ala?Leu?Leu?Val?Arg?Tyr?Thr?Lys
420 425 430
Lys?Val?Pro?Gln?Val?Ser?Thr?Pro?Thr?Leu?Val?Glu?Val?Ser?Arg?Asn
435 440 445
Leu?Gly?Lys?Val?Gly?Ser?Lys?Cys?Cys?Lys?His?Pro?Glu?Ala?Lys?Arg
450 455 460
Met?Pro?Cys?Ala?Glu?Asp?Tyr?Leu?Ser?Val?Val?Leu?Asn?Gln?Leu?Cys
465 470 475 480
Val?Leu?His?Glu?Lys?Thr?Pro?Val?Ser?Asp?Arg?Val?Thr?Lys?Cys?Cys
485 490 495
Thr?Glu?Ser?Leu?Val?Asn?Arg?Arg?Pro?Cys?Phe?Ser?Ala?Leu?Glu?Val
500 505 510
Asp?Glu?Thr?Tyr?Val?Pro?Lys?Glu?Phe?Asn?Ala?Glu?Thr?Phe?Thr?Phe
515 520 525
His?Ala?Asp?Ile?Cys?Thr?Leu?Ser?Glu?Lys?Glu?Arg?Gln?Ile?Lys?Lys
530 535 540
Gln?Thr?Ala?Leu?Val?Glu?Leu?Val?Lys?His?Lys?Pro?Lys?Ala?Thr?Lys
545 550 555 560
Glu?Gln?Leu?Lys?Ala?Val?Met?Asp?Asp?Phe?Ala?Ala?Phe?Val?Glu?Lys
565 570 575
Cys?Cys?Lys?Ala?Asp?Asp?Lys?Glu?Thr?Cys?Phe?Ala?Glu?Glu?Gly?Lys
580 585 590
Lys?Leu?Val?Ala?Ala?Ser?Gln?Ala?Ala?Leu?Gly?Leu?Cys?Asp?Leu?Pro
595 600 605
Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?Arg?Thr?Leu?Met?Leu?Leu?Ala?Gln
610 615 620
Met?Arg?Arg?Ile?Ser?Leu?Phe?Ser?Cys?Leu?Lys?Asp?Arg?His?Asp?Phe
625 630 635 640
Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe?Gln?Lys?Ala?Glu?Thr
645 650 655
Ile?Pro?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Ile?Phe?Asn?Leu?Phe?Ser
660 665 670
Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr?Leu?Leu?Asp?Lys?Phe
675 680 685
Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu?Glu?Ala?Cys?Val?Ile
690 695 700
Gln?Gly?Val?Gly?Val?Thr?Glu?Thr?Pro?Leu?Met?Lys?Glu?Asp?Ser?Ile
705 710 715 720
Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr?Leu?Tyr?Leu?Lys?Glu
725 730 735
Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val?Arg?Ala?Glu?Ile?Met
740 745 750
Arg?Ser?Phe?Ser?Leu?Ser?Thr?Asn?Leu?Gln?Glu?Ser?Leu?Arg?Ser?Lys
755 760 765
Glu
<210>130
<211>779
<212>PRT
＜213〉human (Homo sapiens)
<400>130
Met?Asn?Ile?Phe?Tyr?Ile?Phe?Leu?Phe?Leu?Leu?Ser?Phe?Val?Gln?Gly
1 5 10 15
Leu?Glu?His?Thr?His?Arg?Arg?Gly?Ser?Leu?Asp?Lys?Arg?Asp?Ala?His
20 25 30
Lys?Ser?Glu?Val?Ala?His?Arg?Phe?Lys?Asp?Leu?Gly?Glu?Glu?Asn?Phe
35 40 45
Lys?Ala?Leu?Val?Leu?Ile?Ala?Phe?Ala?Gln?Tyr?Leu?Gln?Gln?Cys?Pro
50 55 60
Phe?Glu?Asp?His?Val?Lys?Leu?Val?Asn?Glu?Val?Thr?Glu?Phe?Ala?Lys
65 70 75 80
Thr?Cys?Val?Ala?Asp?Glu?Ser?Ala?Glu?Asn?Cys?Asp?Lys?Ser?Leu?His
85 90 95
Thr?Leu?Phe?Gly?Asp?Lys?Leu?Cys?Thr?Val?Ala?Thr?Leu?Arg?Glu?Thr
100 105 110
Tyr?Gly?Glu?Met?Ala?Asp?Cys?Cys?Ala?Lys?Gln?Glu?Pro?Glu?Arg?Asn
115 120 125
Glu?Cys?Phe?Leu?Gln?His?Lys?Asp?Asp?Asn?Pro?Asn?Leu?Pro?Arg?Leu
130 135 140
Val?Arg?Pro?Glu?Val?Asp?Val?Met?Cys?Thr?Ala?Phe?His?Asp?Asn?Glu
145 150 155 160
Glu?Thr?Phe?Leu?Lys?Lys?Tyr?Leu?Tyr?Glu?Ile?Ala?Arg?Arg?His?Pro
165 170 175
Tyr?Phe?Tyr?Ala?Pro?Glu?Leu?Leu?Phe?Phe?Ala?Lys?Arg?Tyr?Lys?Ala
180 185 190
Ala?Phe?Thr?Glu?Cys?Cys?Gln?Ala?Ala?Asp?Lys?Ala?Ala?Cys?Leu?Leu
195 200 205
Pro?Lys?Leu?Asp?Glu?Leu?Arg?Asp?Glu?Gly?Lys?Ala?Ser?Ser?Ala?Lys
210 215 220
Gln?Arg?Leu?Lys?Cys?Ala?Ser?Leu?Gln?Lys?Phe?Gly?Glu?Arg?Ala?Phe
225 230 235 240
Lys?Ala?Trp?Ala?Val?Ala?Arg?Leu?Ser?Gln?Arg?Phe?Pro?Lys?Ala?Glu
245 250 255
Phe?Ala?Glu?Val?Ser?Lys?Leu?Val?Thr?Asp?Leu?Thr?Lys?Val?His?Thr
260 265 270
Glu?Cys?Cys?His?Gly?Asp?Leu?Leu?Glu?Cys?Ala?Asp?Asp?Arg?Ala?Asp
275 280 285
Leu?Ala?Lys?Tyr?Ile?Cys?Glu?Asn?Gln?Asp?Ser?Ile?Ser?Ser?Lys?Leu
290 295 300
Lys?Glu?Cys?Cys?Glu?Lys?Pro?Leu?Leu?Glu?Lys?Ser?His?Cys?Ile?Ala
305 310 315 320
Glu?Val?Glu?Asn?Asp?Glu?Met?Pro?Ala?Asp?Leu?Pro?Ser?Leu?Ala?Ala
325 330 335
Asp?Phe?Val?Glu?Ser?Lys?Asp?Val?Cys?Lys?Asn?Tyr?Ala?Glu?Ala?Lys
340 345 350
Asp?Val?Phe?Leu?Gly?Met?Phe?Leu?Tyr?Glu?Tyr?Ala?Arg?Arg?His?Pro
355 360 365
Asp?Tyr?Ser?Val?Val?Leu?Leu?Leu?Arg?Leu?Ala?Lys?Thr?Tyr?Glu?Thr
370 375 380
Thr?Leu?Glu?Lys?Cys?Cys?Ala?Ala?Ala?Asp?Pro?His?Glu?Cys?Tyr?Ala
385 390 395 400
Lys?Val?Phe?Asp?Glu?Phe?Lys?Pro?Leu?Val?Glu?Glu?Pro?Gln?Asn?Leu
405 410 415
Ile?Lys?Gln?Asn?Cys?Glu?Leu?Phe?Glu?Gln?Leu?Gly?Glu?Tyr?Lys?Phe
420 425 430
Gln?Asn?Ala?Leu?Leu?Val?Arg?Tyr?Thr?Lys?Lys?Val?Pro?Gln?Val?Ser
435 440 445
Thr?Pro?Thr?Leu?Val?Glu?Val?Ser?Arg?Asn?Leu?Gly?Lys?Val?Gly?Ser
450 455 460
Lys?Cys?Cys?Lys?His?Pro?Glu?Ala?Lys?Arg?Met?Pro?Cys?Ala?Glu?Asp
465 470 475 480
Tyr?Leu?Ser?Val?Val?Leu?Asn?Gln?Leu?Cys?Val?Leu?His?Glu?Lys?Thr
485 490 495
Pro?Val?Ser?Asp?Arg?Val?Thr?Lys?Cys?Cys?Thr?Glu?Ser?Leu?Val?Asn
500 505 510
Arg?Arg?Pro?Cys?Phe?Ser?Ala?Leu?Glu?Val?Asp?Glu?Thr?Tyr?Val?Pro
515 520 525
Lys?Glu?Phe?Asn?Ala?Glu?Thr?Phe?Thr?Phe?His?Ala?Asp?Ile?Cys?Thr
530 535 540
Leu?Ser?Glu?Lys?Glu?Arg?Gln?Ile?Lys?Lys?Gln?Thr?Ala?Leu?Val?Glu
545 550 555 560
Leu?Val?Lys?His?Lys?Pro?Lys?Ala?Thr?Lys?Glu?Gln?Leu?Lys?Ala?Val
565 570 575
Met?Asp?Asp?Phe?Ala?Ala?Phe?Val?Glu?Lys?Cys?Cys?Lys?Ala?Asp?Asp
580 585 590
Lys?Glu?Thr?Cys?Phe?Ala?Glu?Glu?Gly?Lys?Lys?Leu?Val?Ala?Ala?Ser
595 600 605
Gln?Ala?Ala?Leu?Gly?Leu?Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly
610 615 620
Ser?Arg?Arg?Thr?Leu?Met?Leu?Leu?Ala?Gln?Met?Arg?Arg?Ile?Ser?Leu
625 630 635 640
Phe?Ser?Cys?Leu?Lys?Asp?Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu
645 650 655
Phe?Gly?Asn?Gln?Phe?Gln?Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu
660 665 670
Met?Ile?Gln?Gln?Ile?Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala
675 680 685
Ala?Trp?Asp?Glu?Thr?Leu?Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln
690 695 700
Gln?Leu?Asn?Asp?Leu?Glu?Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Thr
70 5710 715 720
Glu?Thr?Pro?Leu?Met?Lys?Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr
725 730 735
Phe?Gln?Arg?Ile?Thr?Leu?Tyr?Leu?Lys?Glu?Lys?Lys?Tyr?Ser?Pro?Cys
740 745 750
Ala?Trp?Glu?Val?Val?Arg?Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Leu?Ser
755 760 765
Thr?Asn?Leu?Gln?Glu?Ser?Leu?Arg?Ser?Lys?Glu
770 775
<210>131
<211>774
<212>PRT
＜213〉human (Homo sapiens)
<400>131
Met?Lys?Trp?Val?Ser?Phe?Ile?Ser?Leu?Leu?Phe?Leu?Phe?Ser?Ser?Ala
1 5 10 15
Tyr?Ser?Arg?Ser?Leu?Asp?Lys?Arg?Cys?Asp?Leu?Pro?Gln?Thr?His?Ser
20 25 30
Leu?Gly?Ser?Arg?Arg?Thr?Leu?Met?Leu?Leu?Ala?Gln?Met?Arg?Arg?Ile
35 40 45
Ser?Leu?Phe?Ser?Cys?Leu?Lys?Asp?Arg?His?Asp?Phe?Gly?Phe?Pro?Gln
50 55 60
Glu?Glu?Phe?Gly?Asn?Gln?Phe?Gln?Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu
65 70 75 80
His?Glu?Met?Ile?Gln?Gln?Ile?Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser
85 90 95
Ser?Ala?Ala?Trp?Asp?Glu?Thr?Leu?Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu
100 105 110
Tyr?Gln?Gln?Leu?Asn?Asp?Leu?Glu?Ala?Cys?Val?Ile?Gln?Gly?Val?Gly
115 120 125
Val?Thr?Glu?Thr?Pro?Leu?Met?Lys?Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg
130 135 140
Lys?Tyr?Phe?Gln?Arg?Ile?Thr?Leu?Tyr?Leu?Lys?Glu?Lys?Lys?Tyr?Ser
145 150 155 160
Pro?Cys?Ala?Trp?Glu?Val?Val?Arg?Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser
165 170 175
Leu?Ser?Thr?Asn?Leu?Gln?Glu?Ser?Leu?Arg?Ser?Lys?Glu?Asp?Ala?His
180 185 190
Lys?Ser?Glu?Val?Ala?His?Arg?Phe?Lys?Asp?Leu?Gly?Glu?Glu?Asn?Phe
195 200 205
Lys?Ala?Leu?Val?Leu?Ile?Ala?Phe?Ala?Gln?Tyr?Leu?Gln?Gln?Cys?Pro
210 215 220
Phe?Glu?Asp?His?Val?Lys?Leu?Val?Asn?Glu?Val?Thr?Glu?Phe?Ala?Lys
225 230 235 240
Thr?Cys?Val?Ala?Asp?Glu?Ser?Ala?Glu?Asn?Cys?Asp?Lys?Ser?Leu?His
245 250 255
Thr?Leu?Phe?Gly?Asp?Lys?Leu?Cys?Thr?Val?Ala?Thr?Leu?Arg?Glu?Thr
260 265 270
Tyr?Gly?Glu?Met?Ala?Asp?Cys?Cys?Ala?Lys?Gln?Glu?Pro?Glu?Arg?Asn
275 280 285
Glu?Cys?Phe?Leu?Gln?His?Lys?Asp?Asp?Asn?Pro?Asn?Leu?Pro?Arg?Leu
290 295 300
Val?Arg?Pro?Glu?Val?Asp?Val?Met?Cys?Thr?Ala?Phe?His?Asp?Asn?Glu
305 310 315 320
Glu?Thr?Phe?Leu?Lys?Lys?Tyr?Leu?Tyr?Glu?Ile?Ala?Arg?Arg?His?Pro
325 330 335
Tyr?Phe?Tyr?Ala?Pro?Glu?Leu?Leu?Phe?Phe?Ala?Lys?Arg?Tyr?Lys?Ala
340 345 350
Ala?Phe?Thr?Glu?Cys?Cys?Gln?Ala?Ala?Asp?Lys?Ala?Ala?Cys?Leu?Leu
355 360 365
Pro?Lys?Leu?Asp?Glu?Leu?Arg?Asp?Glu?Gly?Lys?Ala?Ser?Ser?Ala?Lys
370 375 380
Gln?Arg?Leu?Lys?Cys?Ala?Ser?Leu?Gln?Lys?Phe?Gly?Glu?Arg?Ala?Phe
385 390 395 400
Lys?Ala?Trp?Ala?Val?Ala?Arg?Leu?Ser?Gln?Arg?Phe?Pro?Lys?Ala?Glu
405 410 415
Phe?Ala?Glu?Val?Ser?Lys?Leu?Val?Thr?Asp?Leu?Thr?Lys?Val?His?Thr
420 425 430
Glu?Cys?Cys?His?Gly?Asp?Leu?Leu?Glu?Cys?Ala?Asp?Asp?Arg?Ala?Asp
435 440 445
Leu?Ala?Lys?Tyr?Ile?Cys?Glu?Asn?Gln?Asp?Ser?Ile?Ser?Ser?Lys?Leu
450 455 460
Lys?Glu?Cys?Cys?Glu?Lys?Pro?Leu?Leu?Glu?Lys?Ser?His?Cys?Ile?Ala
465 470 475 480
Glu?Val?Glu?Asn?Asp?Glu?Met?Pro?Ala?Asp?Leu?Pro?Ser?Leu?Ala?Ala
485 490 495
Asp?Phe?Val?Glu?Ser?Lys?Asp?Val?Cys?Lys?Asn?Tyr?Ala?Glu?Ala?Lys
500 505 510
Asp?Val?Phe?Leu?Gly?Met?Phe?Leu?Tyr?Glu?Tyr?Ala?Arg?Arg?His?Pro
515 520 525
Asp?Tyr?Ser?Val?Val?Leu?Leu?Leu?Arg?Leu?Ala?Lys?Thr?Tyr?Glu?Thr
530 535 540
Thr?Leu?Glu?Lys?Cys?Cys?Ala?Ala?Ala?Asp?Pro?His?Glu?Cys?Tyr?Ala
545 550 555 560
Lys?Val?Phe?Asp?Glu?Phe?Lys?Pro?Leu?Val?Glu?Glu?Pro?Gln?Asn?Leu
565 570 575
Ile?Lys?Gln?Asn?Cys?Glu?Leu?Phe?Glu?Gln?Leu?Gly?Glu?Tyr?Lys?Phe
580 585 590
Gln?Asn?Ala?Leu?Leu?Val?Arg?Tyr?Thr?Lys?Lys?Val?Pro?Gln?Val?Ser
595 600 605
Thr?Pro?Thr?Leu?Val?Glu?Val?Ser?Arg?Asn?Leu?Gly?Lys?Val?Gly?Ser
610 615 620
Lys?Cys?Cys?Lys?His?Pro?Glu?Ala?Lys?Arg?Met?Pro?Cys?Ala?Glu?Asp
625 630 635 640
Tyr?Leu?Ser?Val?Val?Leu?Asn?Gln?Leu?Cys?Val?Leu?His?Glu?Lys?Thr
645 650 655
Pro?Val?Ser?Asp?Arg?Val?Thr?Lys?Cys?Cys?Thr?Glu?Ser?Leu?Val?Asn
660 665 670
Arg?Arg?Pro?Cys?Phe?Ser?Ala?Leu?Glu?Val?Asp?Glu?Thr?Tyr?Val?Pro
675 680 685
Lys?Glu?Phe?Asn?Ala?Glu?Thr?Phe?Thr?Phe?His?Ala?Asp?Ile?Cys?Thr
690 695 700
Leu?Ser?Glu?Lys?Glu?Arg?Gln?lle?Lys?Lys?Gln?Thr?Ala?Leu?Val?Glu
705 710 715 720
Leu?Val?Lys?His?Lys?Pro?Lys?Ala?Thr?Lys?Glu?Gln?Leu?Lys?Ala?Val
725 730 735
Met?Asp?Asp?Phe?Ala?Ala?Phe?Val?Glu?Lys?Cys?Cys?Lys?Ala?Asp?Asp
740 745 750
Lys?Glu?Thr?Cys?Phe?Ala?Glu?Glu?Gly?Lys?Lys?Leu?Val?Ala?Ala?Ser
755 760 765
Gln?Ala?Ala?Leu?Gly?Leu
770
<210>132
<211>769
<212>PRT
＜213〉human (Homo sapiens)
<400>132
Met?Leu?Leu?Gln?Ala?Phe?Leu?Phe?Leu?Leu?Ala?Gly?Phe?Ala?Ala?Lys
1 5 10 15
Ile?Ser?Ala?Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?Arg
20 25 30
Thr?Leu?Met?Leu?Leu?Ala?Gln?Met?Arg?Arg?Ile?Ser?Leu?Phe?Ser?Cys
35 40 45
Leu?Lys?Asp?Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn
50 55 60
Gln?Phe?Gln?Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu?Met?Ile?Gln
65 70 75 80
Gln?Ile?Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp
85 90 95
Glu?Thr?Leu?Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn
100 105 110
Asp?Leu?Glu?Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Thr?Glu?Thr?Pro
115 120 125
Leu?Met?Lys?Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg
130 135 140
Ile?Thr?Leu?Tyr?Leu?Lys?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu
145 150 155 160
Val?Val?Arg?Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Leu?Ser?Thr?Asn?Leu
165 170 175
Gln?Glu?Ser?Leu?Arg?Ser?Lys?Glu?Asp?Ala?His?Lys?Ser?Glu?Val?Ala
180 185 190
His?Arg?Phe?Lys?Asp?Leu?Gly?Glu?Glu?Asn?Phe?Lys?Ala?Leu?Val?Leu
195 200 205
Ile?Ala?Phe?Ala?Gln?Tyr?Leu?Gln?Gln?Cys?Pro?Phe?Glu?Asp?His?Val
210 215 220
Lys?Leu?Val?Asn?Glu?Val?Thr?Glu?Phe?Ala?Lys?Thr?Cys?Val?Ala?Asp
225 230 235 240
Glu?Ser?Ala?Glu?Asn?Cys?Asp?Lys?Ser?Leu?His?Thr?Leu?Phe?Gly?Asp
245 250 255
Lys?Leu?Cys?Thr?Val?Ala?Thr?Leu?Arg?Glu?Thr?Tyr?Gly?Glu?Met?Ala
260 265 270
Asp?Cys?Cys?Ala?Lys?Gln?Glu?Pro?Glu?Arg?Asn?Glu?Cys?Phe?Leu?Gln
275 280 285
His?Lys?Asp?Asp?Asn?Pro?Asn?Leu?Pro?Arg?Leu?Val?Arg?Pro?Glu?Val
290 295 300
Asp?Val?Met?Cys?Thr?Ala?Phe?His?Asp?Asn?Glu?Glu?Thr?Phe?Leu?Lys
305 310 315 320
Lys?Tyr?Leu?Tyr?Glu?Ile?Ala?Arg?Arg?His?Pro?Tyr?Phe?Tyr?Ala?Pro
325 330 335
Glu?Leu?Leu?Phe?Phe?Ala?Lys?Arg?Tyr?Lys?Ala?Ala?Phe?Thr?Glu?Cys
340 345 350
Cys?Gln?Ala?Ala?Asp?Lys?Ala?Ala?Cys?Leu?Leu?Pro?Lys?Leu?Asp?Glu
355 360 365
Leu?Arg?Asp?Glu?Gly?Lys?Ala?Ser?Ser?Ala?Lys?Gln?Arg?Leu?Lys?Cys
370 375 380
Ala?Ser?Leu?Gln?Lys?Phe?Gly?Glu?Arg?Ala?Phe?Lys?Ala?Trp?Ala?Val
385 390 395 400
Ala?Arg?Leu?Ser?Gln?Arg?Phe?Pro?Lys?Ala?Glu?Phe?Ala?Glu?Val?Ser
405 410 415
Lys?Leu?Val?Thr?Asp?Leu?Thr?Lys?Val?His?Thr?Glu?Cys?Cys?His?Gly
420 425 430
Asp?Leu?Leu?Glu?Cys?Ala?Asp?Asp?Arg?Ala?Asp?Leu?Ala?Lys?Tyr?Ile
435 440 445
Cys?Glu?Asn?Gln?Asp?Ser?Ile?Ser?Ser?Lys?Leu?Lys?Glu?Cys?Cys?Glu
450 455 460
Lys?Pro?Leu?Leu?Glu?Lys?Ser?His?Cys?Ile?Ala?Glu?Val?Glu?Asn?Asp
465 470 475 480
Glu?Met?Pro?Ala?Asp?Leu?Pro?Ser?Leu?Ala?Ala?Asp?Phe?Val?Glu?Ser
485 490 495
Lys?Asp?Val?Cys?Lys?Asn?Tyr?Ala?Glu?Ala?Lys?Asp?Val?Phe?Leu?Gly
500 505 510
Met?Phe?Leu?Tyr?Glu?Tyr?Ala?Arg?Arg?His?Pro?Asp?Tyr?Ser?Val?Val
515 520 525
Leu?Leu?Leu?Arg?Leu?Ala?Lys?Thr?Tyr?Glu?Thr?Thr?Leu?Glu?Lys?Cys
530 535 540
Cys?Ala?Ala?Ala?Asp?Pro?His?Glu?Cys?Tyr?Ala?Lys?Val?Phe?Asp?Glu
545 550 555 560
Phe?Lys?Pro?Leu?Val?Glu?Glu?Pro?Gln?Asn?Leu?Ile?Lys?Gln?Asn?Cys
565 570 575
Glu?Leu?Phe?Glu?Gln?Leu?Gly?Glu?Tyr?Lys?Phe?Gln?Asn?Ala?Leu?Leu
580 585 590
Val?Arg?Tyr?Thr?Lys?Lys?Val?Pro?Gln?Val?Ser?Thr?Pro?Thr?Leu?Val
595 600 605
Glu?Val?Ser?Arg?Asn?Leu?Gly?Lys?Val?Gly?Ser?Lys?Cys?Cys?Lys?His
610 615 620
Pro?Glu?Ala?Lys?Arg?Met?Pro?Cys?Ala?Glu?Asp?Tyr?Leu?Ser?Val?Val
625 630 635 640
Leu?Asn?Gln?Leu?Cys?Val?Leu?His?Glu?Lys?Thr?Pro?Val?Ser?Asp?Arg
645 650 655
Val?Thr?Lys?Cys?Cys?Thr?Glu?Ser?Leu?Val?Asn?Arg?Arg?Pro?Cys?Phe
660 665 670
Ser?Ala?Leu?Glu?Val?Asp?Glu?Thr?Tyr?Val?Pro?Lys?Glu?Phe?Asn?Ala
675 680 685
Glu?Thr?Phe?Thr?Phe?His?Ala?Asp?Ile?Cys?Thr?Leu?Ser?Glu?Lys?Glu
690 695 700
Arg?Gln?Ile?Lys?Lys?Gln?Thr?Ala?Leu?Val?Glu?Leu?Val?Lys?His?Lys
705 710 715 720
Pro?Lys?Ala?Thr?Lys?Glu?Gln?Leu?Lys?Ala?Val?Met?Asp?Asp?Phe?Ala
725 730 735
Ala?Phe?Val?Glu?Lys?Cys?Cys?Lys?Ala?Asp?Asp?Lys?Glu?Thr?Cys?Phe
740 745 750
Ala?Glu?Glu?Gly?Lys?Lys?Leu?Val?Ala?Ala?Ser?Gln?Ala?Ala?Leu?Gly
755 760 765
Leu
<210>133
<211>835
<212>PRT
＜213〉human (Homo sapiens)
<400>133
Met?Arg?Phe?Pro?Ser?Ile?Phe?Thr?Ala?Val?Leu?Phe?Ala?Ala?Ser?Ser
1 5 10 15
Ala?Leu?Ala?Ala?Pro?Val?Asn?Thr?Thr?Thr?Glu?Asp?Glu?Thr?Ala?Gln
20 25 30
Ile?Pro?Ala?Glu?Ala?Val?Ile?Gly?Tyr?Ser?Asp?Leu?Glu?Gly?Asp?Phe
35 40 45
Asp?Val?Ala?Val?Leu?Pro?Phe?Ser?Asn?Ser?Thr?Asn?Asn?Gly?Leu?Leu
50 55 60
Phe?Ile?Asn?Thr?Thr?Ile?Ala?Ser?Ile?Ala?Ala?Lys?Glu?Glu?Gly?Val
65 70 75 80
Ser?Leu?Asp?Lys?Arg?Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser
85 90 95
Arg?Arg?Thr?Leu?Met?Leu?Leu?Ala?Gln?Met?Arg?Arg?Ile?Ser?Leu?Phe
100 105 110
Ser?Cys?Leu?Lys?Asp?Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe
115 120 125
Gly?Asn?Gln?Phe?Gln?Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu?Met
130 135 140
Ile?Gln?Gln?Ile?Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala
145 150 155 160
Trp?Asp?Glu?Thr?Leu?Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln
165 170 175
Leu?Asn?Asp?Leu?Glu?Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Thr?Glu
180 185 190
Thr?Pro?Leu?Met?Lys?Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe
195 200 205
Gln?Arg?Ile?Thr?Leu?Tyr?Leu?Lys?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala
210 215 220
Trp?Glu?Val?Val?Arg?Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Leu?Ser?Thr
225 230 235 240
Asn?Leu?Gln?Glu?Ser?Leu?Arg?Ser?Lys?Glu?Asp?Ala?His?Lys?Ser?Glu
245 250 255
Val?Ala?His?Arg?Phe?Lys?Asp?Leu?Gly?Glu?Glu?Asn?Phe?Lys?Ala?Leu
260 265 270
Val?Leu?Ile?Ala?Phe?Ala?Gln?Tyr?Leu?Gln?Gln?Cys?Pro?Phe?Glu?Asp
275 280 285
His?Val?Lys?Leu?Val?Asn?Glu?Val?Thr?Glu?Phe?Ala?Lys?Thr?Cys?Val
290 295 300
Ala?Asp?Glu?Ser?Ala?Glu?Asn?Cys?Asp?Lys?Ser?Leu?His?Thr?Leu?Phe
305 310 315 320
Gly?Asp?Lys?Leu?Cys?Thr?Val?Ala?Thr?Leu?Arg?Glu?Thr?Tyr?Gly?Glu
325 330 335
Met?Ala?Asp?Cys?Cys?Ala?Lys?Gln?Glu?Pro?Glu?Arg?Asn?Glu?Cys?Phe
340 345 350
Leu?Gln?His?Lys?Asp?Asp?Asn?Pro?Asn?Leu?Pro?Arg?Leu?Val?Arg?Pro
355 360 365
Glu?Val?Asp?Val?Met?Cys?Thr?Ala?Phe?His?Asp?Asn?Glu?Glu?Thr?Phe
370 375 380
Leu?Lys?Lys?Tyr?Leu?Tyr?Glu?Ile?Ala?Arg?Arg?His?Pro?Tyr?Phe?Tyr
385 390 395 400
Ala?Pro?Glu?Leu?Leu?Phe?Phe?Ala?Lys?Arg?Tyr?Lys?Ala?Ala?Phe?Thr
405 410 415
Glu?Cys?Cys?Gln?Ala?Ala?Asp?Lys?Ala?Ala?Cys?Leu?Leu?Pro?Lys?Leu
420 425 430
Asp?Glu?Leu?Arg?Asp?Glu?Gly?Lys?Ala?Ser?Ser?Ala?Lys?Gln?Arg?Leu
435 440 445
Lys?Cys?Ala?Ser?Leu?Gln?Lys?Phe?Gly?Glu?Arg?Ala?Phe?Lys?Ala?Trp
450 455 460
Ala?Val?Ala?Arg?Leu?Ser?Gln?Arg?Phe?Pro?Lys?Ala?Glu?Phe?Ala?Glu
465 470 475 480
Val?Ser?Lys?Leu?Val?Thr?Asp?Leu?Thr?Lys?Val?His?Thr?Glu?Cys?Cys
485 490 495
His?Gly?Asp?Leu?Leu?Glu?Cys?Ala?Asp?Asp?Arg?Ala?Asp?Leu?Ala?Lys
500 505 510
Tyr?Ile?Cys?Glu?Asn?Gln?Asp?Ser?Ile?Ser?Ser?Lys?Leu?Lys?Glu?Cys
515 520 525
Cys?Glu?Lys?Pro?Leu?Leu?Glu?Lys?Ser?His?Cys?Ile?Ala?Glu?Val?Glu
530 535 540
Asn?Asp?Glu?Met?Pro?Ala?Asp?Leu?Pro?Ser?Leu?Ala?Ala?Asp?Phe?Val
545 550 555 560
Glu?Ser?Lys?Asp?Val?Cys?Lys?Asn?Tyr?Ala?Glu?Ala?Lys?Asp?Val?Phe
565 570 575
Leu?Gly?Met?Phe?Leu?Tyr?Glu?Tyr?Ala?Arg?Arg?His?Pro?Asp?Tyr?Ser
580 585 590
Val?Val?Leu?Leu?Leu?Arg?Leu?Ala?Lys?Thr?Tyr?Glu?Thr?Thr?Leu?Glu
595 600 605
Lys?Cys?Cys?Ala?Ala?Ala?Asp?Pro?His?Glu?Cys?Tyr?Ala?Lys?Val?Phe
610 615 620
Asp?Glu?Phe?Lys?Pro?Leu?Val?Glu?Glu?Pro?Gln?Asn?Leu?Ile?Lys?Gln
625 630 635 640
Asn?Cys?Glu?Leu?Phe?Glu?Gln?Leu?Gly?Glu?Tyr?Lys?Phe?Gln?Asn?Ala
645 650 655
Leu?Leu?Val?Arg?Tyr?Thr?Lys?Lys?Val?Pro?Gln?Val?Ser?Thr?Pro?Thr
660 665 670
Leu?Val?Glu?Val?Ser?Arg?Asn?Leu?Gly?Lys?Val?Gly?Ser?Lys?Cys?Cys
675 680 685
Lys?His?Pro?Glu?Ala?Lys?Arg?Met?Pro?Cys?Ala?Glu?Asp?Tyr?Leu?Ser
690 695 700
Val?Val?Leu?Asn?Gln?Leu?Cys?Val?Leu?His?Glu?Lys?Thr?Pro?Val?Ser
705 710 715 720
Asp?Arg?Val?Thr?Lys?Cys?Cys?Thr?Glu?Ser?Leu?Val?Asn?Arg?Arg?Pro
725 730 735
Cys?Phe?Ser?Ala?Leu?Glu?Val?Asp?Glu?Thr?Tyr?Val?Pro?Lys?Glu?Phe
740 745 750
Asn?Ala?Glu?Thr?Phe?Thr?Phe?His?Ala?Asp?Ile?Cys?Thr?Leu?Ser?Glu
755 760 765
Lys?Glu?Arg?Gln?Ile?Lys?Lys?Gln?Thr?Ala?Leu?Val?Glu?Leu?Val?Lys
770 775 780
His?Lys?Pro?Lys?Ala?Thr?Lys?Glu?Gln?Leu?Lys?Ala?Val?Met?Asp?Asp
785 790 795 800
Phe?Ala?Ala?Phe?Val?Glu?Lys?Cys?Cys?Lys?Ala?Asp?Asp?Lys?Glu?Thr
805 810 815
Cys?Phe?Ala?Glu?Glu?Gly?Lys?Lys?Leu?Val?Ala?Ala?Ser?Gln?Ala?Ala
820 825 830
Leu?Gly?Leu
835
<210>134
<211>774
<212>PRT
＜213〉human (Homo sapiens)
<400>134
Met?Lys?Trp?Val?Thr?Phe?Ile?Ser?Leu?Leu?Phe?Leu?Phe?Ser?Ser?Ala
1 5 10 15
Tyr?Ser?Arg?Gly?Val?Phe?Arg?Arg?Asp?Ala?His?Lys?Ser?Glu?Val?Ala
20 25 30
His?Arg?Phe?Lys?Asp?Leu?Gly?Glu?Glu?Asn?Phe?Lys?Ala?Leu?Val?Leu
35 40 45
Ile?Ala?Phe?Ala?Gln?Tyr?Leu?Gln?Gln?Cys?Pro?Phe?Glu?Asp?His?Val
50 55 60
Lys?Leu?Val?Asn?Glu?Val?Thr?Glu?Phe?Ala?Lys?Thr?Cys?Val?Ala?Asp
65 70 75 80
Glu?Ser?Ala?Glu?Asn?Cys?Asp?Lys?Ser?Leu?His?Thr?Leu?Phe?Gly?Asp
85 90 95
Lys?Leu?Cys?Thr?Val?Ala?Thr?Leu?Arg?Glu?Thr?Tyr?Gly?Glu?Met?Ala
100 105 110
Asp?Cys?Cys?Ala?Lys?Gln?Glu?Pro?Glu?Arg?Asn?Glu?Cys?Phe?Leu?Gln
115 120 125
His?Lys?Asp?Asp?Asn?Pro?Asn?Leu?Pro?Arg?Leu?Val?Arg?Pro?Glu?Val
130 135 140
Asp?Val?Met?Cys?Thr?Ala?Phe?His?Asp?Asn?Glu?Glu?Thr?Phe?Leu?Lys
145 150 155 160
Lys?Tyr?Leu?Tyr?Glu?Ile?Ala?Arg?Arg?His?Pro?Tyr?Phe?Tyr?Ala?Pro
165 170 175
Glu?Leu?Leu?Phe?Phe?Ala?Lys?Arg?Tyr?Lys?Ala?Ala?Phe?Thr?Glu?Cys
180 185 190
Cys?Gln?Ala?Ala?Asp?Lys?Ala?Ala?Cys?Leu?Leu?Pro?Lys?Leu?Asp?Glu
195 200 205
Leu?Arg?Asp?Glu?Gly?Lys?Ala?Ser?Ser?Ala?Lys?Gln?Arg?Leu?Lys?Cys
210 215 220
Ala?Ser?Leu?Gln?Lys?Phe?Gly?Glu?Arg?Ala?Phe?Lys?Ala?Trp?Ala?Val
225 230 235 240
Ala?Arg?Leu?Ser?Gln?Arg?Phe?Pro?Lys?Ala?Glu?Phe?Ala?Glu?Val?Ser
245 250 255
Lys?Leu?Val?Thr?Asp?Leu?Thr?Lys?Val?His?Thr?Glu?Cys?Cys?His?Gly
260 265 270
Asp?Leu?Leu?Glu?Cys?Ala?Asp?Asp?Arg?Ala?Asp?Leu?Ala?Lys?Tyr?Ile
275 280 285
Cys?Glu?Asn?Gln?Asp?Ser?Ile?Ser?Ser?Lys?Leu?Lys?Glu?Cys?Cys?Glu
290 295 300
Lys?Pro?Leu?Leu?Glu?Lys?Ser?His?Cys?Ile?Ala?Glu?Val?Glu?Asn?Asp
305 310 315 320
Glu?Met?Pro?Ala?Asp?Leu?Pro?Ser?Leu?Ala?Ala?Asp?Phe?Val?Glu?Ser
325 330 335
Lys?Asp?Val?Cys?Lys?Asn?Tyr?Ala?Glu?Ala?Lys?Asp?Val?Phe?Leu?Gly
340 345 350
Met?Phe?Leu?Tyr?Glu?Tyr?Ala?Arg?Arg?His?Pro?Asp?Tyr?Ser?Val?Val
355 360 365
Leu?Leu?Leu?Arg?Leu?Ala?Lys?Thr?Tyr?Glu?Thr?Thr?Leu?Glu?Lys?Cys
370 375 380
Cys?Ala?Ala?Ala?Asp?Pro?His?Glu?Cys?Tyr?Ala?Lys?Val?Phe?Asp?Glu
385 390 395 400
Phe?Lys?Pro?Leu?Val?Glu?Glu?Pro?Gln?Asn?Leu?Ile?Lys?Gln?Asn?Cys
405 410 415
Glu?Leu?Phe?Glu?Gln?Leu?Gly?Glu?Tyr?Lys?Phe?Gln?Asn?Ala?Leu?Leu
420 425 430
Val?Arg?Tyr?Thr?Lys?Lys?Val?Pro?Gln?Val?Ser?Thr?Pro?Thr?Leu?Val
435 440 445
Glu?Val?Ser?Arg?Asn?Leu?Gly?Lys?Val?Gly?Ser?Lys?Cys?Cys?Lys?His
450 455 460
Pro?Glu?Ala?Lys?Arg?Met?Pro?Cys?Ala?Glu?Asp?Tyr?Leu?Ser?Val?Val
465 470 475 480
Leu?Asn?Gln?Leu?Cys?Val?Leu?His?Glu?Lys?Thr?Pro?Val?Ser?Asp?Arg
485 490 495
Val?Thr?Lys?Cys?Cys?Thr?Glu?Ser?Leu?Val?Asn?Arg?Arg?Pro?Cys?Phe
500 505 510
Ser?Ala?Leu?Glu?Val?Asp?Glu?Thr?Tyr?Val?Pro?Lys?Glu?Phe?Asn?Ala
515 520 525
Glu?Thr?Phe?Thr?Phe?His?Ala?Asp?Ile?Cys?Thr?Leu?Ser?Glu?Lys?Glu
530 535 540
Arg?Gln?Ile?Lys?Lys?Gln?Thr?Ala?Leu?Val?Glu?Leu?Val?Lys?His?Lys
545 550 555 560
Pro?Lys?Ala?Thr?Lys?Glu?Gln?Leu?Lys?Ala?Val?Met?Asp?Asp?Phe?Ala
565 570 575
Ala?Phe?Val?Glu?Lys?Cys?Cys?Lys?Ala?Asp?Asp?Lys?Glu?Thr?Cys?Phe
580 585 590
Ala?Glu?Glu?Gly?Lys?Lys?Leu?Val?Ala?Ala?Ser?Gln?Ala?Ala?Leu?Gly
595 600 605
Leu?Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?Arg?Thr?Leu
610 615 620
Met?Leu?Leu?Ala?Gln?Met?Arg?Arg?Ile?Ser?Leu?Phe?Ser?Cys?Leu?Lys
625 630 635 640
Asp?Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe
645 650 655
Gln?Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Ile
660 665 670
Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr
675 680 685
Leu?Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu
690 695 700
Glu?Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Thr?Glu?Thr?Pro?Leu?Met
705 710 715 720
Lys?Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr
725 730 735
Leu?Tyr?Leu?Lys?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val
740 745 750
Arg?Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Leu?Ser?Thr?Asn?Leu?Gln?Glu
755 760 765
Ser?Leu?Arg?Ser?Lys?Glu
770
<210>135
<211>773
<212>PRT
＜213〉human (Homo sapiens)
<400>135
Met?Ala?Leu?Thr?Phe?Ala?Leu?Leu?Val?Ala?Leu?Leu?Val?Leu?Ser?Cys
1 5 10 15
Lys?Ser?Ser?Cys?Ser?Val?Gly?Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu
20 25 30
Gly?Ser?Arg?Arg?Thr?Leu?Met?Leu?Leu?Ala?Gln?Met?Arg?Arg?Ile?Ser
35 40 45
Leu?Phe?Ser?Cys?Leu?Lys?Asp?Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu
50 55 60
Glu?Phe?Gly?Asn?Gln?Phe?Gln?Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His
65 70 75 80
Glu?Met?Ile?Gln?Gln?Ile?Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser
85 90 95
Ala?Ala?Trp?Asp?Glu?Thr?Leu?Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr
100 105 110
Gln?Gln?Leu?Asn?Asp?Leu?Glu?Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val
115 120 125
Thr?Glu?Thr?Pro?Leu?Met?Lys?Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys
130 135 140
Tyr?Phe?Gln?Arg?Ile?Thr?Leu?Tyr?Leu?Lys?Glu?Lys?Lys?Tyr?Ser?Pro
145 150 155 160
Cys?Ala?Trp?Glu?Val?Val?Arg?Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Leu
165 170 175
Ser?Thr?Asn?Leu?Gln?Glu?Ser?Leu?Arg?Ser?Lys?Glu?Asp?Ala?His?Lys
180 185 190
Ser?Glu?Val?Ala?His?Arg?Phe?Lys?Asp?Leu?Gly?Glu?Glu?Asn?Phe?Lys
195 200 205
Ala?Leu?Val?Leu?Ile?Ala?Phe?Ala?Gln?Tyr?Leu?Gln?Gln?Cys?Pro?Phe
210 215 220
Glu?Asp?His?Val?Lys?Leu?Val?Asn?Glu?Val?Thr?Glu?Phe?Ala?Lys?Thr
225 230 235 240
Cys?Val?Ala?Asp?Glu?Ser?Ala?Glu?Asn?Cys?Asp?Lys?Ser?Leu?His?Thr
245 250 255
Leu?Phe?Gly?Asp?Lys?Leu?Cys?Thr?Val?Ala?Thr?Leu?Arg?Glu?Thr?Tyr
260 265 270
Gly?Glu?Met?Ala?Asp?Cys?Cys?Ala?Lys?Gln?Glu?Pro?Glu?Arg?Asn?Glu
275 280 285
Cys?Phe?Leu?Gln?His?Lys?Asp?Asp?Asn?Pro?Asn?Leu?Pro?Arg?Leu?Val
290 295 300
Arg?Pro?Glu?Val?Asp?Val?Met?Cys?Thr?Ala?Phe?His?Asp?Asn?Glu?Glu
305 310 315 320
Thr?Phe?Leu?Lys?Lys?Tyr?Leu?Tyr?Glu?Ile?Ala?Arg?Arg?His?Pro?Tyr
325 330 335
Phe?Tyr?Ala?Pro?Glu?Leu?Leu?Phe?Phe?Ala?Lys?Arg?Tyr?Lys?Ala?Ala
340 345 350
Phe?Thr?Glu?Cys?Cys?Gln?Ala?Ala?Asp?Lys?Ala?Ala?Cys?Leu?Leu?Pro
355 360 365
Lys?Leu?Asp?Glu?Leu?Arg?Asp?Glu?Gly?Lys?Ala?Ser?Ser?Ala?Lys?Gln
370 375 380
Arg?Leu?Lys?Cys?Ala?Ser?Leu?Gln?Lys?Phe?Gly?Glu?Arg?Ala?Phe?Lys
385 390 395 400
Ala?Trp?Ala?Val?Ala?Arg?Leu?Ser?Gln?Arg?Phe?Pro?Lys?Ala?Glu?Phe
405 410 415
Ala?Glu?Val?Ser?Lys?Leu?Val?Thr?Asp?Leu?Thr?Lys?Val?His?Thr?Glu
420 425 430
Cys?Cys?His?Gly?Asp?Leu?Leu?Glu?Cys?Ala?Asp?Asp?Arg?Ala?Asp?Leu
435 440 445
Ala?Lys?Tyr?Ile?Cys?Glu?Asn?Gln?Asp?Ser?Ile?Ser?Ser?Lys?Leu?Lys
450 455 460
Glu?Cys?Cys?Glu?Lys?Pro?Leu?Leu?Glu?Lys?Ser?His?Cys?Ile?Ala?Glu
465 470 475 480
Val?Glu?Asn?Asp?Glu?Met?Pro?Ala?Asp?Leu?Pro?Ser?Leu?Ala?Ala?Asp
485 490 495
Phe?Val?Glu?Ser?Lys?Asp?Val?Cys?Lys?Asn?Tyr?Ala?Glu?Ala?Lys?Asp
500 505 510
Val?Phe?Leu?Gly?Met?Phe?Leu?Tyr?Glu?Tyr?Ala?Arg?Arg?His?Pro?Asp
515 520 525
Tyr?Ser?Val?Val?Leu?Leu?Leu?Arg?Leu?Ala?Lys?Thr?Tyr?Glu?Thr?Thr
530 535 540
Leu?Glu?Lys?Cys?Cys?Ala?Ala?Ala?Asp?Pro?His?Glu?Cys?Tyr?Ala?Lys
545 550 555 560
Val?Phe?Asp?Glu?Phe?Lys?Pro?Leu?Val?Glu?Glu?Pro?Gln?Asn?Leu?Ile
565 570 575
Lys?Gln?Asn?Cys?Glu?Leu?Phe?Glu?Gln?Leu?Gly?Glu?Tyr?Lys?Phe?Gln
580 585 590
Asn?Ala?Leu?Leu?Val?Arg?Tyr?Thr?Lys?Lys?Val?Pro?Gln?Val?Ser?Thr
595 600 605
Pro?Thr?Leu?Val?Glu?Val?Ser?Arg?Asn?Leu?Gly?Lys?Val?Gly?Ser?Lys
610 615 620
Cys?Cys?Lys?His?Pro?Glu?Ala?Lys?Arg?Met?Pro?Cys?Ala?Glu?Asp?Tyr
625 630 635 640
Leu?Ser?Val?Val?Leu?Asn?Gln?Leu?Cys?Val?Leu?His?Glu?Lys?Thr?Pro
645 650 655
Val?Ser?Asp?Arg?Val?Thr?Lys?Cys?Cys?Thr?Glu?Ser?Leu?Val?Asn?Arg
660 665 670
Arg?Pro?Cys?Phe?Ser?Ala?Leu?Glu?Val?Asp?Glu?Thr?Tyr?Val?Pro?Lys
675 680 685
Glu?Phe?Asn?Ala?Glu?Thr?Phe?Thr?Phe?His?Ala?Asp?Ile?Cys?Thr?Leu
690 695 700
Ser?Glu?Lys?Glu?Arg?Gln?Ile?Lys?Lys?Gln?Thr?Ala?Leu?Val?Glu?Leu
705 710 715 720
Val?Lys?His?Lys?Pro?Lys?Ala?Thr?Lys?Glu?Gln?Leu?Lys?Ala?Val?Met
725 730 735
Asp?Asp?Phe?Ala?Ala?Phe?Val?Glu?Lys?Cys?Cys?Lys?Ala?Asp?Asp?Lys
740 745 750
Glu?Thr?Cys?Phe?Ala?Glu?Glu?Gly?Lys?Lys?Leu?Val?Ala?Ala?Ser?Gln
755 760 765
Ala?Ala?Leu?Gly?Leu
770
<210>136
<211>769
<212>PRT
＜213〉human (Homo sapiens)
<400>136
Met?Leu?Leu?Gln?Ala?Phe?Leu?Phe?Leu?Leu?Ala?Gly?Phe?Ala?Ala?Lys
1 5 10 15
Ile?Ser?Ala?Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?Arg
20 25 30
Thr?Leu?Met?Leu?Leu?Ala?Gln?Met?Arg?Arg?Ile?Ser?Leu?Phe?Ser?Cys
35 40 45
Leu?Lys?Asp?Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn
50 55 60
Gln?Phe?Gln?Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu?Met?Ile?Gln
65 70 75 80
Gln?Ile?Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp
85 90 95
Glu?Thr?Leu?Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn
100 105 110
Asp?Leu?Glu?Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Thr?Glu?Thr?Pro
115 120 125
Leu?Met?Lys?Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg
130 135 140
Ile?Thr?Leu?Tyr?Leu?Lys?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu
145 150 155 160
Val?Val?Arg?Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Leu?Ser?Thr?Asn?Leu
165 170 175
Gln?Glu?Ser?Leu?Arg?Ser?Lys?Glu?Asp?Ala?His?Lys?Ser?Glu?Val?Ala
180 185 190
His?Arg?Phe?Lys?Asp?Leu?Gly?Glu?Glu?Asn?Phe?Lys?Ala?Leu?Val?Leu
195 200 205
Ile?Ala?Phe?Ala?Gln?Tyr?Leu?Gln?Gln?Cys?Pro?Phe?Glu?Asp?His?Val
210 215 220
Lys?Leu?Val?Asn?Glu?Val?Thr?Glu?Phe?Ala?Lys?Thr?Cys?Val?Ala?Asp
225 230 235 240
Glu?Ser?Ala?Glu?Asn?Cys?Asp?Lys?Ser?Leu?His?Thr?Leu?Phe?Gly?Asp
245 250 255
Lys?Leu?Cys?Thr?Val?Ala?Thr?Leu?Arg?Glu?Thr?Tyr?Gly?Glu?Met?Ala
260 265 270
Asp?Cys?Cys?Ala?Lys?Gln?Glu?Pro?Glu?Arg?Asn?Glu?Cys?Phe?Leu?Gln
275 280 285
His?Lys?Asp?Asp?Asn?Pro?Asn?Leu?Pro?Arg?Leu?Val?Arg?Pro?Glu?Val
290 295 300
Asp?Val?Met?Cys?Thr?Ala?Phe?His?Asp?Asn?Glu?Glu?Thr?Phe?Leu?Lys
305 310 315 320
Lys?Tyr?Leu?Tyr?Glu?Ile?Ala?Arg?Arg?His?Pro?Tyr?Phe?Tyr?Ala?Pro
325 330 335
Glu?Leu?Leu?Phe?Phe?Ala?Lys?Arg?Tyr?Lys?Ala?Ala?Phe?Thr?Glu?Cys
340 345 350
Cys?Gln?Ala?Ala?Asp?Lys?Ala?Ala?Cys?Leu?Leu?Pro?Lys?Leu?Asp?Glu
355 360 365
Leu?Arg?Asp?Glu?Gly?Lys?Ala?Ser?Ser?Ala?Lys?Gln?Arg?Leu?Lys?Cys
370 375 380
Ala?Ser?Leu?Gln?Lys?Phe?Gly?Glu?Arg?Ala?Phe?Lys?Ala?Trp?Ala?Val
385 390 395 400
Ala?Arg?Leu?Ser?Gln?Arg?Phe?Pro?Lys?Ala?Glu?Phe?Ala?Glu?Val?Ser
405 410 415
Lys?Leu?Val?Thr?Asp?Leu?Thr?Lys?Val?His?Thr?Glu?Cys?Cys?His?Gly
420 425 430
Asp?Leu?Leu?Glu?Cys?Ala?Asp?Asp?Arg?Ala?Asp?Leu?Ala?Lys?Tyr?Ile
435 440 445
Cys?Glu?Asn?Gln?Asp?Ser?Ile?Ser?Ser?Lys?Leu?Lys?Glu?Cys?Cys?Glu
450 455 460
Lys?Pro?Leu?Leu?Glu?Lys?Ser?His?Cys?Ile?Ala?Glu?Val?Glu?Asn?Asp
465 470 475 480
Glu?Met?Pro?Ala?Asp?Leu?Pro?Ser?Leu?Ala?Ala?Asp?Phe?Val?Glu?Ser
485 490 495
Lys?Asp?Val?Cys?Lys?Asn?Tyr?Ala?Glu?Ala?Lys?Asp?Val?Phe?Leu?Gly
500 505 510
Met?Phe?Leu?Tyr?Glu?Tyr?Ala?Arg?Arg?His?Pro?Asp?Tyr?Ser?Val?Val
515 520 525
Leu?Leu?Leu?Arg?Leu?Ala?Lys?Thr?Tyr?Glu?Thr?Thr?Leu?Glu?Lys?Cys
530 535 540
Cys?Ala?Ala?Ala?Asp?Pro?His?Glu?Cys?Tyr?Ala?Lys?Val?Phe?Asp?Glu
545 550 555 560
Phe?Lys?Pro?Leu?Val?Glu?Glu?Pro?Gln?Asn?Leu?Ile?Lys?Gln?Asn?Cys
565 570 575
Glu?Leu?Phe?Glu?Gln?Leu?Gly?Glu?Tyr?Lys?Phe?Gln?Asn?Ala?Leu?Leu
580 585 590
Val?Arg?Tyr?Thr?Lys?Lys?Val?Pro?Gln?Val?Ser?Thr?Pro?Thr?Leu?Val
595 600 605
Glu?Val?Ser?Arg?Asn?Leu?Gly?Lys?Val?Gly?Ser?Lys?Cys?Cys?Lys?His
610 615 620
Pro?Glu?Ala?Lys?Arg?Met?Pro?Cys?Ala?Glu?Asp?Tyr?Leu?Ser?Val?Val
625 630 635 640
Leu?Asn?Gln?Leu?Cys?Val?Leu?His?Glu?Lys?Thr?Pro?Val?Ser?Asp?Arg
645 650 655
Val?Thr?Lys?Cys?Cys?Thr?Glu?Ser?Leu?Val?Asn?Arg?Arg?Pro?Cys?Phe
660 665 670
Ser?Ala?Leu?Glu?Val?Asp?Glu?Thr?Tyr?Val?Pro?Lys?Glu?Phe?Asn?Ala
675 680 685
Glu?Thr?Phe?Thr?Phe?His?Ala?Asp?Ile?Cys?Thr?Leu?Ser?Glu?Lys?Glu
690 695 700
Arg?Gln?Ile?Lys?Lys?Gln?Thr?Ala?Leu?Val?Glu?Leu?Val?Lys?His?Lys
705 710 715 720
Pro?Lys?Ala?Thr?Lys?Glu?Gln?Leu?Lys?Ala?Val?Met?Asp?Asp?Phe?Ala
725 730 735
Ala?Phe?Val?Glu?Lys?Cys?Cys?Lys?Ala?Asp?Asp?Lys?Glu?Thr?Cys?Phe
740 745 750
Ala?Glu?Glu?Gly?Lys?Lys?Leu?Val?Ala?Ala?Ser?Gln?Ala?Ala?Leu?Gly
755 760 765
Leu
<210>137
<211>774
<212>PRT
＜213〉human (Homo sapiens)
<400>137
Met?Lys?Trp?Val?Ser?Phe?Ile?Ser?Leu?Leu?Phe?Leu?Phe?Ser?Ser?Ala
1 5 10 15
Tyr?Ser?Arg?Ser?Leu?Asp?Lys?Arg?Asp?Ala?His?Lys?Ser?Glu?Val?Ala
20 25 30
His?Arg?Phe?Lys?Asp?Leu?Gly?Glu?Glu?Asn?Phe?Lys?Ala?Leu?Val?Leu
35 40 45
Ile?Ala?Phe?Ala?Gln?Tyr?Leu?Gln?Gln?Cys?Pro?Phe?Glu?Asp?His?Val
50 55 60
Lys?Leu?Val?Asn?Glu?Val?Thr?Glu?Phe?Ala?Lys?Thr?Cys?Val?Ala?Asp
65 70 75 80
Glu?Ser?Ala?Glu?Asn?Cys?Asp?Lys?Ser?Leu?His?Thr?Leu?Phe?Gly?Asp
85 90 95
Lys?Leu?Cys?Thr?Val?Ala?Thr?Leu?Arg?Glu?Thr?Tyr?Gly?Glu?Met?Ala
100 105 110
Asp?Cys?Cys?Ala?Lys?Gln?Glu?Pro?Glu?Arg?Asn?Glu?Cys?Phe?Leu?Gln
115 120 125
His?Lys?Asp?Asp?Asn?Pro?Asn?Leu?Pro?Arg?Leu?Val?Arg?Pro?Glu?Val
130 135 140
Asp?Val?Met?Cys?Thr?Ala?Phe?His?Asp?Asn?Glu?Glu?Thr?Phe?Leu?Lys
145 150 155 160
Lys?Tyr?Leu?Tyr?Glu?Ile?Ala?Arg?Arg?His?Pro?Tyr?Phe?Tyr?Ala?Pro
165 170 175
Glu?Leu?Leu?Phe?Phe?Ala?Lys?Arg?Tyr?Lys?Ala?Ala?Phe?Thr?Glu?Cys
180 185 190
Cys?Gln?Ala?Ala?Asp?Lys?Ala?Ala?Cys?Leu?Leu?Pro?Lys?Leu?Asp?Glu
195 200 205
Leu?Arg?Asp?Glu?Gly?Lys?Ala?Ser?Ser?Ala?Lys?Gln?Arg?Leu?Lys?Cys
210 215 220
Ala?Ser?Leu?Gln?Lys?Phe?Gly?Glu?Arg?Ala?Phe?Lys?Ala?Trp?Ala?Val
225 230 235 240
Ala?Arg?Leu?Ser?Gln?Arg?Phe?Pro?Lys?Ala?Glu?Phe?Ala?Glu?Val?Ser
245 250 255
Lvs?Leu?Val?Thr?Asp?Leu?Thr?Lys?Val?His?Thr?Glu?Cys?Cys?His?Gly
260 265 270
Asp?Leu?Leu?Glu?Cys?Ala?Asp?Asp?Arg?Ala?Asp?Leu?Ala?Lys?Tyr?Ile
275 280 285
Cys?Glu?Asn?Gln?Asp?Ser?Ile?Ser?Ser?Lys?Leu?Lys?Glu?Cys?Cys?Glu
290 295 300
Lys?Pro?Leu?Leu?Glu?Lys?Ser?His?Cys?Ile?Ala?Glu?Val?Glu?Asn?Asp
305 310 315 320
Glu?Met?Pro?Ala?Asp?Leu?Pro?Ser?Leu?Ala?Ala?Asp?Phe?Val?Glu?Ser
325 330 335
Lys?Asp?Val?Cys?Lys?Asn?Tyr?Ala?Glu?Ala?Lys?Asp?Val?Phe?Leu?Gly
340 345 350
Met?Phe?Leu?Tyr?Glu?Tyr?Ala?Arg?Arg?His?Pro?Asp?Tyr?Ser?Val?Val
355 360 365
Leu?Leu?Leu?Arg?Leu?Ala?Lys?Thr?Tyr?Glu?Thr?Thr?Leu?Glu?Lys?Cys
370 375 380
Cys?Ala?Ala?Ala?Asp?Pro?His?Glu?Cys?Tyr?Ala?Lys?Val?Phe?Asp?Glu
385 390 395 400
Phe?Lys?Pro?Leu?Val?Glu?Glu?Pro?Gln?Asn?Leu?Ile?Lys?Gln?Asn?Cys
405 410 415
Glu?Leu?Phe?Glu?Gln?Leu?Gly?Glu?Tyr?Lys?Phe?Gln?Asn?Ala?Leu?Leu
420 425 430
Val?Arg?Tyr?Thr?Lys?Lys?Val?Pro?Gln?Val?Ser?Thr?Pro?Thr?Leu?Val
435 440 445
Glu?Val?Ser?Arg?Asn?Leu?Gly?Lys?Val?Gly?Ser?Lys?Cys?Cys?Lys?His
450 455 460
Pro?Glu?Ala?Lys?Arg?Met?Pro?Cys?Ala?Glu?Asp?Tyr?Leu?Ser?Val?Val
465 470 475 480
Leu?Asn?Gln?Leu?Cys?Val?Leu?His?Glu?Lys?Thr?Pro?Val?Ser?Asp?Arg
485 490 495
Val?Thr?Lys?Cys?Cys?Thr?Glu?Ser?Leu?Val?Asn?Arg?Arg?Pro?Cys?Phe
500 505 510
Ser?Ala?Leu?Glu?Val?Asp?Glu?Thr?Tyr?Val?Pro?Lys?Glu?Phe?Asn?Ala
515 520 525
Glu?Thr?Phe?Thr?Phe?His?Ala?Asp?Ile?Cys?Thr?Leu?Ser?Glu?Lys?Glu
530 535 540
Arg?Gln?Ile?Lys?Lys?Gln?Thr?Ala?Leu?Val?Glu?Leu?Val?Lys?His?Lys
545 550 555 560
Pro?Lys?Ala?Thr?Lys?Glu?Gln?Leu?Lys?Ala?Val?Met?Asp?Asp?Phe?Ala
565 570 575
Ala?Phe?Val?Glu?Lys?Cys?Cys?Lys?Ala?Asp?Asp?Lys?Glu?Thr?Cys?Phe
580 585 590
Ala?Glu?Glu?Gly?Lys?Lys?Leu?Val?Ala?Ala?Ser?Gln?Ala?Ala?Leu?Gly
595 600 605
Leu?Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?Arg?Thr?Leu
610 615 620
Met?Leu?Leu?Ala?Gln?Met?Arg?Arg?Ile?Ser?Leu?Phe?Ser?Cys?Leu?Lys
625 630 635 640
Asp?Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe
645 650 655
Gln?Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Ile
660 665 670
Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr
675 680 685
Leu?Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu
690 695 700
Glu?Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Thr?Glu?Thr?Pro?Leu?Met
705 710 715 720
Lys?Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr
725 730 735
Leu?Tyr?Leu?Lys?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val
740 745 750
Arg?Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Leu?Ser?Thr?Asn?Leu?Gln?Glu
755 760 765
Ser?Leu?Arg?Ser?Lys?Glu
770
<210>138
<211>774
<212>PRT
＜213〉human (Homo sapiens)
<400>138
Met?Lys?Trp?Val?Ser?Phe?Ile?Ser?Leu?Leu?Phe?Leu?Phe?Ser?Ser?Ala
1 5 10 15
Tyr?Ser?Gly?Ser?Leu?Asp?Lys?Arg?Asp?Ala?His?Lys?Ser?Glu?Val?Ala
20 25 30
His?Arg?Phe?Lys?Asp?Leu?Gly?Glu?Glu?Asn?Phe?Lys?Ala?Leu?Val?Leu
35 40 45
Ile?Ala?Phe?Ala?Gln?Tyr?Leu?Gln?Gln?Cys?Pro?Phe?Glu?Asp?His?Val
50 55 60
Lys?Leu?Val?Asn?Glu?Val?Thr?Glu?Phe?Ala?Lys?Thr?Cys?Val?Ala?Asp
65 70 75 80
Glu?Ser?Ala?Glu?Asn?Cys?Asp?Lys?Ser?Leu?His?Thr?Leu?Phe?Gly?Asp
85 90 95
Lys?Leu?Cys?Thr?Val?Ala?Thr?Leu?Arg?Glu?Thr?Tyr?Gly?Glu?Met?Ala
100 105 110
Asp?Cys?Cys?Ala?Lys?Gln?Glu?Pro?Glu?Arg?Asn?Glu?Cys?Phe?Leu?Gln
115 120 125
His?Lys?Asp?Asp?Asn?Pro?Asn?Leu?Pro?Arg?Leu?Val?Arg?Pro?Glu?Val
130 135 140
Asp?Val?Met?Cys?Thr?Ala?Phe?His?Asp?Asn?Glu?Glu?Thr?Phe?Leu?Lys
145 150 155 160
Lys?Tyr?Leu?Tyr?Glu?Ile?Ala?Arg?Arg?His?Pro?Tyr?Phe?Tyr?Ala?Pro
165 170 175
Glu?Leu?Leu?Phe?Phe?Ala?Lys?Arg?Tyr?Lys?Ala?Ala?Phe?Thr?Glu?Cys
180 185 190
Cys?Gln?Ala?Ala?Asp?Lys?Ala?Ala?Cys?Leu?Leu?Pro?Lys?Leu?Asp?Glu
195 200 205
Leu?Arg?Asp?Glu?Gly?Lys?Ala?Ser?Ser?Ala?Lys?Gln?Arg?Leu?Lys?Cys
210 215 220
Ala?Ser?Leu?Gln?Lys?Phe?Gly?Glu?Arg?Ala?Phe?Lys?Ala?Trp?Ala?Val
225 230 235 240
Ala?Arg?Leu?Ser?Gln?Arg?Phe?Pro?Lys?Ala?Glu?Phe?Ala?Glu?Val?Ser
245 250 255
Lys?Leu?Val?Thr?Asp?Leu?Thr?Lys?Val?His?Thr?Glu?Cys?Cys?His?Gly
260 265 270
Asp?Leu?Leu?Glu?Cys?Ala?Asp?Asp?Arg?Ala?Asp?Leu?Ala?Lys?Tyr?Ile
275 280 285
Cys?Glu?Asn?Gln?Asp?Ser?Ile?Ser?Ser?Lys?Leu?Lys?Glu?Cys?Cys?Glu
290 295 300
Lys?Pro?Leu?Leu?Glu?Lys?Ser?His?Cys?Ile?Ala?Glu?Val?Glu?Asn?Asp
305 310 315 320
Glu?Met?Pro?Ala?Asp?Leu?Pro?Ser?Leu?Ala?Ala?Asp?Phe?Val?Glu?Ser
325 330 335
Lys?Asp?Val?Cys?Lys?Asn?Tyr?Ala?Glu?Ala?Lys?Asp?Val?Phe?Leu?Gly
340 345 350
Met?Phe?Leu?Tyr?Glu?Tyr?Ala?Arg?Arg?His?Pro?Asp?Tyr?Ser?Val?Val
355 360 365
Leu?Leu?Leu?Arg?Leu?Ala?Lys?Thr?Tyr?Glu?Thr?Thr?Leu?Glu?Lys?Cys
370 375 380
Cys?Ala?Ala?Ala?Asp?Pro?His?Glu?Cys?Tyr?Ala?Lys?Val?Phe?Asp?Glu
385 390 395 400
Phe?Lys?Pro?Leu?Val?Glu?Glu?Pro?Gln?Asn?Leu?Ile?Lys?Gln?Asn?Cys
405 410 415
Glu?Leu?Phe?Glu?Gln?Leu?Gly?Glu?Tyr?Lys?Phe?Gln?Asn?Ala?Leu?Leu
420 425 430
Val?Arg?Tyr?Thr?Lys?Lys?Val?Pro?Gln?Val?Ser?Thr?Pro?Thr?Leu?Val
435 440 445
Glu?Val?Ser?Arg?Asn?Leu?Gly?Lys?Val?Gly?Ser?Lys?Cys?Cys?Lys?His
450 455 460
Pro?Glu?Ala?Lys?Arg?Met?Pro?Cys?Ala?Glu?Asp?Tyr?Leu?Ser?Val?Val
465 470 475 480
Leu?Asn?Gln?Leu?Cys?Val?Leu?His?Glu?Lys?Thr?Pro?Val?Ser?Asp?Arg
485 490 495
Val?Thr?Lys?Cys?Cys?Thr?Glu?Ser?Leu?Val?Asn?Arg?Arg?Pro?Cys?Phe
500 505 510
Ser?Ala?Leu?Glu?Val?Asp?Glu?Thr?Tyr?Val?Pro?Lys?Glu?Phe?Asn?Ala
515 520 525
Glu?Thr?Phe?Thr?Phe?His?Ala?Asp?Ile?Cys?Thr?Leu?Ser?Glu?Lys?Glu
530 535 540
Arg?Gln?Ile?Lys?Lys?Gln?Thr?Ala?Leu?Val?Glu?Leu?Val?Lys?His?Lys
545 550 555 560
Pro?Lys?Ala?Thr?Lys?Glu?Gln?Leu?Lys?Ala?Val?Met?Asp?Asp?Phe?Ala
565 570 575
Ala?Phe?Val?Glu?Lys?Cys?Cys?Lys?Ala?Asp?Asp?Lys?Glu?Thr?Cys?Phe
580 585 590
Ala?Glu?Glu?Gly?Lys?Lys?Leu?Val?Ala?Ala?Ser?Gln?Ala?Ala?Leu?Gly
595 600 605
Leu?Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?Arg?Thr?Leu
610 615 620
Met?Leu?Leu?Ala?Gln?Met?Arg?Arg?Ile?Ser?Leu?Phe?Ser?Cys?Leu?Lys
625 630 635 640
Asp?Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe
645 650 655
Gln?Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Ile
660 665 670
Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr
675 680 685
Leu?Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu
690 695 700
Glu?Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Thr?Glu?Thr?Pro?Leu?Met
705 710 715 720
Lys?Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr
725 730 735
Leu?Tyr?Leu?Lys?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val
740 745 750
Arg?Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Leu?Ser?Thr?Asn?Leu?Gln?Glu
755 760 765
Ser?Leu?Arg?Ser?Lys?Glu
770
<210>139
<211>165
<212>PRT
＜213〉human (Homo sapiens)
<400>139
Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?Arg?Thr?Leu?Met
1 5 10 15
Leu?Leu?Ala?Gln?Met?Arg?Arg?Ile?Ser?Leu?Phe?Ser?Cys?Leu?Lys?Asp
20 25 30
Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe?Gln
35 40 45
Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Ile?Phe
50 55 60
Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr?Leu
65 70 75 80
Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu?Glu
85 90 95
Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Thr?Glu?Thr?Pro?Leu?Met?Lys
100 105 110
Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr?Leu
115 120 125
Tyr?Leu?Lys?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val?Arg
130 135 140
Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Leu?Ser?Thr?Asn?Leu?Gln?Glu?Ser
145 150 155 160
Leu?Arg?Ser?Lys?Glu
165
<210>140
<211>165
<212>PRT
＜213〉human (Homo sapiens)
<400>140
Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?Arg?Thr?Leu?Met
1 5 10 15
Leu?Leu?Ala?Gln?Met?Arg?Arg?Ile?Ser?Leu?Phe?Ser?Cys?Leu?Lys?Asp
20 25 30
Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe?Gln
35 40 45
Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Ile?Phe
50 55 60
Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr?Leu
65 70 75 80
Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu?Glu
85 90 95
Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Thr?Glu?Thr?Pro?Leu?Met?Lys
100 105 110
Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr?Leu
115 120 125
Tyr?Leu?Lys?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val?Arg
130 135 140
Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Leu?Ser?Thr?Asn?Leu?Gln?Glu?Ser
145 150 155 160
Leu?Arg?Ser?Lys?Glu
165
<210>141
<211>165
<212>PRT
＜213〉class (Homo sapiens)
<400>141
Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?Arg?Thr?Leu?Met
1 5 10 15
Leu?Leu?Ala?Gln?Met?Arg?Arg?Ile?Ser?Leu?Phe?Ser?Cys?Leu?Lys?Asp
20 25 30
Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe?Gln
35 40 45
Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Ile?Phe
50 55 60
Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr?Leu
65 70 75 80
Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu?Glu
85 90 95
Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Thr?Glu?Thr?Pro?Leu?Met?Lys
100 105 110
Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr?Leu
115 120 125
Tyr?Leu?Lys?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val?Arg
130 135 140
Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Leu?Ser?Thr?Asn?Leu?Gln?Glu?Ser
145 150 155 160
Leu?Arg?Ser?Lys?Glu
165
<210>142
<211>165
<212>PRT
＜213〉class (Homo sapiens)
<400>142
Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?Arg?Thr?Leu?Met
1 5 10 15
Leu?Leu?Ala?Gln?Met?Arg?Arg?Ile?Ser?Leu?Phe?Ser?Cys?Leu?Lys?Asp
20 25 30
Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe?Gln
35 40 45
Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Ile?Phe
50 55 60
Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr?Leu
65 70 75 80
Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu?Glu
85 90 95
Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Thr?Glu?Thr?Pro?Leu?Met?Lys
100 105 110
Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr?Leu
115 120 125
Tyr?Leu?Lys?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val?Arg
130 135 140
Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Leu?Ser?Thr?A?sn?Leu?Gln?Glu?Ser
145 150 155 160
Leu?Arg?Ser?Lys?Glu
165
<210>143
<211>165
<212>PRT
＜213〉human (Homo sapiens)
<400>143
Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?Arg?Thr?Leu?Met
1 5 10 15
Leu?Leu?Ala?Gln?Met?Arg?Arg?Ile?Ser?Leu?Phe?Ser?Cys?Leu?Lys?Asp
20 25 30
Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe?Gln
35 40 45
Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Ile?Phe
50 55 60
Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr?Leu
65 70 75 80
Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu?Glu
85 90 95
Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Thr?Glu?Thr?Pro?Leu?Met?Lys
100 105 110
Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr?Leu
115 120 125
Tyr?Leu?Lys?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val?Arg
130 135 140
Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Leu?Ser?Thr?Asn?Leu?Gln?Glu?Ser
115 150 155 160
Leu?Arg?Ser?Lys?Glu
165
<210>144
<211>165
<212>PRT
＜213〉human (Homo sapiens)
<400>144
Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?Arg?Thr?Leu?Met
1 5 10 15
Leu?Leu?Ala?Gln?Met?Arg?Arg?Ile?Ser?Leu?Phe?Ser?Cys?Leu?Lys?Asp
20 25 30
Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe?Gln
35 40 45
Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Ile?Phe
50 55 60
Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr?Leu
65 70 75 80
Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu?Glu
85 90 95
Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Thr?Glu?Thr?Pro?Leu?Met?Lys
100 105 110
Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr?Leu
115 120 125
Tyr?Leu?Lys?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val?Arg
130 135 140
Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Leu?Ser?Thr?Asn?Leu?Gln?Glu?Ser
145 150 155 160
Leu?Arg?Ser?Lys?Glu
165
<210>145
<211>165
<212>PRT
＜213〉human (Homo sapiens)
<400>145
Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?Arg?Thr?Leu?Met
1 5 10 15
Leu?Leu?Ala?Gln?Met?Arg?Arg?Ile?Ser?Leu?Phe?Ser?Cys?Leu?Lys?Asp
20 25 30
Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe?Gln
35 40 45
Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Ile?Phe
50 55 60
Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr?Leu
65 70 75 80
Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu?Glu
85 90 95
Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Thr?Glu?Thr?Pro?Leu?Met?Lys
100 105 110
Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr?Leu
115 120 125
Tyr?Leu?Lys?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val?Arg
130 135 140
Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Leu?Ser?Thr?Asn?Leu?Gln?Glu?Ser
145 150 155 160
Leu?Arg?Ser?Lys?Glu
165
<210>146
<211>165
<212>PRT
＜213〉human (Homo sapiens)
<400>146
Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?Arg?Thr?Leu?Met
1 5 10 15
Leu?Leu?Ala?Gln?Met?Arg?Arg?Ile?Ser?Leu?Phe?Ser?Cys?Leu?Lys?Asp
20 25 30
Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe?Gln
35 40 45
Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Ile?Phe
50 55 60
Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr?Leu
65 70 75 80
Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu?Glu
85 90 95
Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Thr?Glu?Thr?Pro?Leu?Met?Lys
100 105 110
Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr?Leu
115 120 125
Tyr?Leu?Lys?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val?Arg
130 135 140
Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Leu?Ser?Thr?Asn?Leu?Gln?Glu?Ser
145 150 155 160
Leu?Arg?Ser?Lys?Glu
165
<210>147
<211>165
<212>PRT
＜213〉human (Homo sapiens)
<400>147
Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?Arg?Thr?Leu?Met
1 5 10 15
Leu?Leu?Ala?Gln?Met?Arg?Arg?Ile?Ser?Leu?Phe?Ser?Cys?Leu?Lys?Asp
20 25 30
Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe?Gln
35 40 45
Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Ile?Phe
50 55 60
Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr?Leu
65 70 75 80
Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu?Glu
85 90 95
Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Thr?Glu?Thr?Pro?Leu?Met?Lys
100 105 110
Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr?Leu
115 120 125
Tyr?Leu?Lys?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val?Arg
130 135 140
Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Leu?Ser?Thr?Asn?Leu?Gln?Glu?Ser
145 150 155 160
Leu?Arg?Ser?Lys?Glu
165
<210>148
<211>165
<212>PRT
＜213〉human (Homo sapiens)
<400>148
Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?Arg?Thr?Leu?Met
1 5 10 15
Leu?Leu?Ala?Gln?Met?Arg?Arg?Ile?Ser?Leu?Phe?Ser?Cys?Leu?Lys?Asp
20 25 30
Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe?Gln
35 40 45
Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Ile?Phe
50 55 60
Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr?Leu
65 70 75 80
Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu?Glu
85 90 95
Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Thr?Glu?Thr?Pro?Leu?Met?Lys
100 105 110
Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr?Leu
115 120 125
Tyr?Leu?Lys?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val?Arg
130 135 140
Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Leu?Ser?Thr?Asn?Leu?Gln?Glu?Ser
145 150 155 160
Leu?Arg?Ser?Lys?Glu
165
<210>149
<211>165
<212>PRT
＜213〉human (Homo sapiens)
<400>149
Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?Arg?Thr?Leu?Met
1 5 10 15
Leu?Leu?Ala?Gln?Met?Arg?Arg?Ile?Ser?Leu?Phe?Ser?Cys?Leu?Lys?Asp
20 25 30
Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe?Gln
35 40 45
Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Ile?Phe
50 55 60
Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr?Leu
65 70 75 80
Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu?Glu
85 90 95
Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Thr?Glu?Thr?Pro?Leu?Met?Lys
100 105 110
Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr?Leu
115 120 125
Tyr?Leu?Lys?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val?Arg
130 135 140
Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Leu?Ser?Thr?Asn?Leu?Gln?Glu?Ser
145 150 155 160
Leu?Arg?Ser?Lys?Glu
165
<210>150
<211>20
<212>DNA
＜213〉human (Homo sapiens)
<400>150
catacaaact?taagagtcca 20
<210>151
<211>56
<212>DNA
＜213〉human (Homo sapiens)
<400>151
ctttaaatcg?atgagcaacc?tcactcttgt?gtgcatcagc?gttagccaaa?gaagca 56
<210>152
<211>20
<212>DNA
＜213〉human (Homo sapiens)
<400>152
<210>153
<211>56
<212>DNA
＜213〉human (Homo sapiens)
<400>153
ctttaaatcg?atgagcaacc?tcactcttgt?gtgcatctgc?cgatattttg?gctgca 56
<210>154
<211>20
<212>DNA
＜213〉human (Homo sapiens)
<400>154
catacaaact?taagagtcca 20
<210>155
<211>56
<212>DNA
＜213〉human (Homo sapiens)
<400>155
ctttaaatcg?atgagcaacc?tcactcttgt?gtgcatctct?cttatccaaa?gaacct 56
<210>156
<211>41
<212>DNA
＜213〉human (Homo sapiens)
<400>156
cgcgcgcgtc?gacaaaagat?gtgatctgcc?tcaaacccac?a 41
<210>157
<211>59
<212>DNA
＜213〉human (Homo sapiens)
<400>157
gcgcgcatcg?atgagcaacc?tcactcttgt?gtgcatcttc?cttacttctt?aaactttct 59
<210>158
<211>27
<212>DNA
＜213〉human (Homo sapiens)
<400>158
gttagcagag?tagcagggct?ttcggct 27
<210>159
<211>59
<212>DNA
＜213〉human (Homo sapiens)
<400>159
gcgcgcatcg?atgagcaacc?tcactcttgt?gtgcatcttc?cttacttctt?aaactttct 59
<210>160
<211>41
<212>DNA
＜213〉human (Homo sapiens)
<400>160
cgcgcgcgtc?gacaaaagat?gtgatctgcc?tcaaacccac?a 41
<210>161
<211>59
<212>DNA
＜213〉human (Homo sapiens)
<400>161
gcgcgcatcg?atgagcaacc?tcactcttgt?gtgcatcttc?cttacttctt?aaactttct 59
<210>162
<211>38
<212>DNA
＜213〉human (Homo sapiens)
<400>162
caagctgcct?taggcttatg?tgatctgcct?caaaccca 38
<210>163
<211>39
<212>DNA
＜213〉human (Homo sapiens)
<400>163
gaattcggcg?cgccttattc?cttacttctt?aaactttct 39
<210>164
<211>107
<212>DNA
＜213〉human (Homo sapiens)
<400>164
gcgcgcggat?ccgccaccat?ggccttgacc?tttgctttac?tggtggccct?cctggtgctc 60
agctgcaagt?caagctgctc?tgtgggctgt?gatctgcctc?aaaccca 107
<210>165
<211>59
<212>DNA
＜213〉human (Homo sapiens)
<400>165
gcgcgcatcg?atgagcaacc?tcactcttgt?gtgcatcttc?cttacttctt?aaactttct 59
<210>166
<211>25
<212>DNA
＜213〉human (Homo sapiens)
<400>166
gcatgtgatc?tgcctcaaac?ccaca 25
<210>167
<211>59
<212>DNA
＜213〉human (Homo sapiens)
<400>167
gcgcgcatcg?atgagcaacc?tcactcttgt?gtgcatcttc?cttacttctt?aaactttct 59
<210>168
<211>20
<212>DNA
＜213〉human (Homo sapiens)
<400>168
catacaaact?taagagtcca 20
<210>169
<211>67
<212>DNA
＜213〉human (Homo sapiens)
<400>169
ctttaaatcg?atgagcaacc?tcactcttgt?gtgcatctct?cttatccaaa?gaaccggaat 60
aagccga 67
<210>170
<211>495
<212>DNA
＜213〉human (Homo sapiens)
<400>170
tgtgatctgc?ctcaaaccca?cagcctgggt?tctagaagga?ccttgatgct?cctggcacag 60
atgaggagaa?tctctctttt?ctcctgcttg?aaggacagac?atgactttgg?atttccccag 120
gaggagtttg?gcaaccagtt?ccaaaaggct?gaaaccatcc?ctgtcctcca?tgagatgatc 180
cagcagatct?tcaatctctt?cagcacaaag?gactcatctg?ctgcttggga?tgagaccctc 240
ctagacaaat?tctacactga?actctaccag?cagctgaatg?acctggaagc?ctgtgtgata 300
cagggggtgg?gggtgacaga?gactcccctg?atgaaggagg?actccattct?ggctgtgagg 360
aaatacttcc?aaagaatcac?tctctatctg?aaagagaaga?aatacagccc?ttgtgcctgg 420
gaggttgtca?gagcagaaat?catgagatct?ttttctttgt?caacaaactt?gcaagaaagt 480
ttaagaagta?aggaa 495
<210>171
<211>774
<212>PRT
＜213〉human (Homo sapiens)
<400>171
Met?Lys?Trp?Val?Ser?Phe?Ile?Ser?Leu?Leu?Phe?Leu?Phe?Ser?Ser?Ala
1 5 10 15
Tyr?Ser?Arg?Ser?Leu?Asp?Lys?Arg?Asp?Ala?His?Lys?Ser?Glu?Val?Ala
20 25 30
His?Arg?Phe?Lys?Asp?Leu?Gly?Glu?Glu?Asn?Phe?Lys?Ala?Leu?Val?Leu
35 40 45
Ile?Ala?Phe?Ala?Gln?Tyr?Leu?Gln?Gln?Cys?Pro?Phe?Glu?Asp?His?Val
50 55 60
Lys?Leu?Val?Asn?Glu?Val?Thr?Glu?Phe?Ala?Lys?Thr?Cys?Val?Ala?Asp
65 70 75 80
Glu?Ser?Ala?Glu?Asn?Cys?Asp?Lys?Ser?Leu?His?Thr?Leu?Phe?Gly?Asp
85 90 95
Lys?Leu?Cys?Thr?Val?Ala?Thr?Leu?Arg?Glu?Thr?Tyr?Gly?Glu?Met?Ala
100 105 110
Asp?Cys?Cys?Ala?Lys?Gln?Glu?Pro?Glu?Arg?Asn?Glu?Cys?Phe?Leu?Gln
115 120 125
His?Lys?Asp?Asp?Asn?Pro?Asn?Leu?Pro?Arg?Leu?Val?Arg?Pro?Glu?Val
130 135 140
Asp?Val?Met?Cys?Thr?Ala?Phe?His?Asp?Asn?Glu?Glu?Thr?Phe?Leu?Lys
145 150 155 160
Lys?Tyr?Leu?Tyr?Glu?Ile?Ala?Arg?Arg?His?Pro?Tyr?Phe?Tyr?Ala?Pro
165 170 175
Glu?Leu?Leu?Phe?Phe?Ala?Lys?Arg?Tyr?Lys?Ala?Ala?Phe?Thr?Glu?Cys
180 185 190
Cys?Gln?Ala?Ala?Asp?Lys?Ala?Ala?Cys?Leu?Leu?Pro?Lys?Leu?Asp?Glu
195 200 205
Leu?Arg?Asp?Glu?Gly?Lys?Ala?Ser?Ser?Ala?Lys?Gln?Arg?Leu?Lys?Cys
210 215 220
Ala?Ser?Leu?Gln?Lys?Phe?Gly?Glu?Arg?Ala?Phe?Lys?Ala?Trp?Ala?Val
225 230 235 240
Ala?Arg?Leu?Ser?Gln?Arg?Phe?Pro?Lys?Ala?Glu?Phe?Ala?Glu?Val?Ser
245 250 255
Lys?Leu?Val?Thr?Asp?Leu?Thr?Lys?Val?His?Thr?Glu?Cys?Cys?His?Gly
260 265 270
Asp?Leu?Leu?Glu?Cys?Ala?Asp?Asp?Arg?Ala?Asp?Leu?Ala?Lys?Tyr?Ile
275 280 285
Cys?Glu?Asn?Gln?Asp?Ser?Ile?Ser?Ser?Lys?Leu?Lys?Glu?Cys?Cys?Glu
290 295 300
Lys?Pro?Leu?Leu?Glu?Lys?Ser?His?Cys?Ile?Ala?Glu?Val?Glu?Asn?Asp
305 310 315 320
Glu?Met?Pro?Ala?Asp?Leu?Pro?Ser?Leu?Ala?Ala?Asp?Phe?Val?Glu?Ser
325 330 335
Lys?Asp?Val?Cys?Lys?Asn?Tyr?Ala?Glu?Ala?Lys?Asp?Val?Phe?Leu?Gly
340 345 350
Met?Phe?Leu?Tyr?Glu?Tyr?Ala?Arg?Arg?His?Pro?Asp?Tyr?Ser?Val?Val
355 360 365
Leu?Leu?Leu?Arg?Leu?Ala?Lys?Thr?Tyr?Glu?Thr?Thr?Leu?Glu?Lys?Cys
370 375 380
Cys?Ala?Ala?Ala?Asp?Pro?His?Glu?Cys?Tyr?Ala?Lys?Val?Phe?Asp?Glu
385 390 395 400
Phe?Lys?Pro?Leu?Val?Glu?Glu?Pro?Gln?Asn?Leu?Ile?Lys?Gln?Asn?Cys
405 410 415
Glu?Leu?Phe?Glu?Gln?Leu?Gly?Glu?Tyr?Lys?Phe?Gln?Asn?Ala?Leu?Leu
420 425 430
Val?Arg?Tyr?Thr?Lys?Lys?Val?Pro?Gln?Val?Ser?Thr?Pro?Thr?Leu?Val
435 440 445
Glu?Val?Ser?Arg?Asn?Leu?Gly?Lys?Val?Gly?Ser?Lys?Cys?Cys?Lys?His
450 455 460
Pro?Glu?Ala?Lys?Arg?Met?Pro?Cys?Ala?Glu?Asp?Tyr?Leu?Ser?Val?Val
465 470 475 480
Leu?Asn?Gln?Leu?Cys?Val?Leu?His?Glu?Lys?Thr?Pro?Val?Ser?Asp?Arg
485 490 495
Val?Thr?Lys?Cys?Cys?Thr?Glu?Ser?Leu?Val?Asn?Arg?Arg?Pro?Cys?Phe
500 505 510
Ser?Ala?Leu?Glu?Val?Asp?Glu?Thr?Tyr?Val?Pro?Lys?Glu?Phe?Asn?Ala
515 520 525
Glu?Thr?Phe?Thr?Phe?His?Ala?Asp?Ile?Cys?Thr?Leu?Ser?Glu?Lys?Glu
530 535 540
Arg?Gln?Ile?Lys?Lys?Gln?Thr?Ala?Leu?Val?Glu?Leu?Val?Lys?His?Lys
545 550 555 560
Pro?Lys?Ala?Thr?Lys?Glu?Gln?Leu?Lys?Ala?Val?Met?Asp?Asp?Phe?Ala
565 570 575
Ala?Phe?Val?Glu?Lys?Cys?Cys?Lys?Ala?Asp?Asp?Lys?Glu?Thr?Cys?Phe
580 585 590
Ala?Glu?Glu?Gly?Lys?Lys?Leu?Val?Ala?Ala?Ser?Gln?Ala?Ala?Leu?Gly
595 600 605
Leu?Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?Arg?Thr?Leu
610 615 620
Met?Leu?Leu?Ala?Gln?Met?Arg?Lys?Ile?Ser?Leu?Phe?Ser?Cys?Leu?Lys
625 630 635 640
Asp?Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe
645 650 655
Gln?Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Ile
660 665 670
Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr
675 680 685
Leu?Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu
690 695 700
Glu?Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Thr?Glu?Thr?Pro?Leu?Met
705 710 715 720
Lys?Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr
725 730 735
Leu?Tyr?Leu?Lys?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val
740 745 750
Arg?Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Leu?Ser?Thr?Asn?Leu?Gln?Glu
755 760 765
Ser?Leu?Arg?Ser?Lys?Glu
770
<210>172
<211>165
<212>PRT
＜213〉human (Homo sapiens)
<400>172
Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?Arg?Thr?Leu?Met
1 5 10 15
Leu?Leu?Ala?Gln?Met?Arg?Arg?Ile?Ser?Leu?Phe?Ser?Cys?Leu?Lys?Asp
20 25 30
Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe?Gln
35 40 45
Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Ile?Phe
50 55 60
Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr?Leu
65 70 75 80
Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu?Glu
85 90 95
Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Thr?Glu?Thr?Pro?Leu?Met?Lys
100 105 110
Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr?Leu
115 120 125
Tyr?Leu?Lys?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val?Arg
130 135 140
Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Leu?Ser?Thr?Asn?Leu?Gln?Glu?Ser
145 150 155 160
Leu?Arg?Ser?Lys?Glu
165
<210>173
<211>21
<212>DNA
＜213〉human (Homo sapiens)
<400>173
agaagtgctg?caaggctgac?g 21
<210>174
<211>20
<212>DNA
＜213〉human (Homo sapiens)
<400>174
acctgaccta?caggaaagag 20
<210>175
<211>501
<212>DNA
＜213〉human (Homo sapiens)
<400>175
tgtgatctgc?ctcaaaccca?cagcctgggt?tctagaagga?ccttgatgct?cctggcacag?60
atgaggagaa?tctctctttt?ctcctgcttg?aaggacagac?atgactttgg?atttccccag?120
gaggagtttg?gcaaccagtt?ccaaaaggct?gaaaccatcc?ctgtcctcca?tgagatgatc?180
cagcagatct?tcaatctctt?cagcacaaag?gactcatctg?ctgcttggga?tgagaccctc?240
ctagacaaat?tctacactga?actctaccag?cagctgaatg?acctggaagc?ctgtgtgata?300
cagggggtgg?gggtgacaga?gactcccctg?atgaaggagg?actccattct?ggctgtgagg?360
aaatacttcc?aaagaatcac?tctctatctg?aaagagaaga?aatacagccc?ttgtgcctgg?420
gaggttgtca?gagcagaaat?catgagatct?ttttctttgt?caacaaactt?gcaagaaagt?480
ttaagaagta?aggaataata?a 501
<210>176
<211>501
<212>DNA
＜213〉human (Homo sapiens)
<400>176
tgtgatctgc?ctcaaaccca?cagcctgggt?tctagaagga?ccttgatgct?cctggcacag?60
atgaggagaa?tctctctttt?ctcctgcttg?aaggacagac?atgactttgg?atttccccag?120
gaggagtttg?gcaaccagtt?ccaaaaggct?gaaaccatcc?ctgtcctcca?tgagatgatc?180
cagcagatct?tcaatctctt?cagcacaaag?gactcatctg?ctgcttggga?tgagaccctc?240
ctagacaaat?tctacacrga?actctaccag?cagctgaatg?acctggaagc?ctgtgtgata?300
cagggggtgg?gggtgacaga?gactcccctg?atgaaggagg?actccattct?ggctgtgagg?360
aaatacttcc?aaagaatcac?tctctatctg?aaagagaaga?aatacagccc?ttgtgcctgg?420
gaggttgtca?gagcagaaat?catgagatct?ttttctttgt?caacaaactt?gcaagaaagt?480
ttaagaagta?aggaataata?a 501
<210>177
<211>501
<212>DNA
＜213〉human (Homo sapiens)
<400>177
tgtgatctgc?ctcaaaccca?cagcctgggt?tctagaagga?ccttgatgct?cctggcacag?60
atgaggagaa?tctctctttt?ctcctgcttg?aaggacagac?atgactttgg?atttccccag?120
gaggagtttg?gcaaccagtt?ccaaaaggct?gaaaccatcc?ctgtcctcca?tgagatgatc?180
cagcagatct?tcaatctctt?cagcacaaag?gactcatctg?ctgcttggga?tgagaccctc?240
ctagacaaat?tctacactga?actctaccag?cagctgaatg?acctggaagc?ctgtgtgata?300
cagggggtgg?gggtgacaga?gactcccctg?atgaaggagg?actccattct?ggctgtgagg?360
aaatacttcc?aaagaatcac?tctctatctg?aaagagaaga?aatacagccc?ttgtgcctgg?420
gaggttgtca?gagcagaaat?catgagatct?ttttctttgt?caacaaactt?gcaagaaagt?480
ttaagaagta?aggaataata?a 501
<210>178
<211>501
<212>DNA
＜213〉human (Homo sapiens)
<400>178
tgtgatctgc?ctcaaaccca?cagcctgggt?tctagaagga?ccttgatgct?cctggcacag?60
atgaggagaa?tctcrctttt?ctcctgcttg?aaggacagac?atgactttgg?atttccccag?120
gaggagtttg?gcaaccagtt?ccaaaaggct?gaaaccatcc?ctgtcctcca?tgagatgatc?180
cagcagatct?tcaatctctt?cagcacaaag?gactcatctg?ctgcttggga?tgagaccctc?240
ctagacaaat?tctacactga?actctaccag?cagctgaatg?acctggaagc?ctgtgtgata?300
cagggggtgg?gggtgacaga?gactcccctg?atgaaggagg?actccattct?ggctgtgagg?360
aaatacttcc?aaagaatcac?tctctatctg?aaagagaaga?aatacagccc?ttgtgcctgg?420
gaggttgtca?gagcagaaat?catgagatct?ttttctttgt?caacaaactt?gcaagaaagt?480
ttaagaagta?aggaataata?a 501
<210>179
<211>501
<212>DNA
＜213〉human (Homo sapiens)
<400>179
tgtgatctgc?ctcaaaccca?cagcctgggt?tctagaagga?ccttgatgct?cctggcacag?60
atgaggagaa?tctctctttt?ctcctgcttg?aaggacagac?atgactttgg?atttccccag?120
gaggagtttg?gcaaccagtt?ccaaaaggct?gaaaccatcc?ctgtcctcca?tgagatgatc?180
cagcagatct?tcaatctctt?cagcacaaag?gactcatctg?ctgcttggga?tgagaccctc?240
ctagacaaat?tctacactga?actctaccag?cagctgaatg?acctggaagc?ctgtgtgata?300
cagggggtgg?gggtgacaga?gactcccctg?atgaaggagg?actccattct?ggctgtgagg?360
aaatacttcc?aaagaatcac?tctctatctg?aaagagaaga?aatacagccc?ttgtgcctgg?420
gaggttgtca?gagcagaaat?catgagatct?ttttctttgt?caacaaactt?gcaagaaagt?480
ttaagaagta?aggaataata?a 501
<210>180
<211>774
<212>PRT
＜213〉human (Homo sapiens)
<400>180
Met?Lys?Trp?Val?Ser?Phe?Ile?Ser?Leu?Leu?Phe?Leu?Phe?Ser?Ser?Ala
1 5 10 15
Tyr?Ser?Arg?Ser?Leu?Asp?Lys?Arg?Asp?Ala?His?Lys?Ser?Glu?Val?Ala
20 25 30
His?Arg?Phe?Lys?Asp?Leu?Gly?Glu?Glu?Asn?Phe?Lys?Ala?Leu?Val?Leu
35 40 45
Ile?Ala?Phe?Ala?Gln?Tyr?Leu?Gln?Gln?Ser?Pro?Phe?Glu?Asp?His?Val
50 55 60
Lys?Leu?Val?Asn?Glu?Val?Thr?Glu?Phe?Ala?Lys?Thr?Cys?Val?Ala?Asp
65 70 75 80
Glu?Ser?Ala?Glu?Asn?Cys?Asp?Lys?Ser?Leu?His?Thr?Leu?Phe?Gly?Asp
85 90 95
Lys?Leu?Cys?Thr?Val?Ala?Thr?Leu?Arg?Glu?Thr?Tyr?Gly?Glu?Met?Ala
100 105 110
Asp?Cys?Cys?Ala?Lys?Gln?Glu?Pro?Glu?Arg?Asn?Glu?Cys?Phe?Leu?Gln
115 120 125
His?Lys?Asp?Asp?Asn?Pro?Asn?Leu?Pro?Arg?Leu?Val?Arg?Pro?Glu?Val
130 135 140
Asp?Val?Met?Cys?Thr?Ala?Phe?His?Asp?Asn?Glu?Glu?Thr?Phe?Leu?Lys
145 150 155 160
Lys?Tyr?Leu?Tyr?Glu?Ile?Ala?Arg?Arg?His?Pro?Tyr?Phe?Tyr?Ala?Pro
165 170 175
Glu?Leu?Leu?Phe?Phe?Ala?Lys?Arg?Tyr?Lys?Ala?Ala?Phe?Thr?Glu?Cys
180 185 190
Cys?Gln?Ala?Ala?Asp?Lys?Ala?Ala?Cys?Leu?Leu?Pro?Lys?Leu?Asp?Glu
195 200 205
Leu?Arg?Asp?Glu?Gly?Lys?Ala?Ser?Ser?Ala?Lys?Gln?Arg?Leu?Lys?Cys
210 215 220
Ala?Ser?Leu?Gln?Lys?Phe?Gly?Glu?Arg?Ala?Phe?Lys?Ala?Trp?Ala?Val
225 230 235 240
Ala?Arg?Leu?Ser?Gln?Arg?Phe?Pro?Lys?Ala?Glu?Phe?Ala?Glu?Val?Ser
245 250 255
Lys?Leu?Val?Thr?Asp?Leu?Thr?Lys?Val?His?Thr?Glu?Cys?Cys?His?Gly
260 265 270
Asp?Leu?Leu?Glu?Cys?Ala?Asp?Asp?Arg?Ala?Asp?Leu?Ala?Lys?Tyr?Ile
275 280 285
Cys?Glu?Asn?Gln?Asp?Ser?Ile?Ser?Ser?Lys?Leu?Lys?Glu?Cys?Cys?Glu
290 295 300
Lys?Pro?Leu?Leu?Glu?Lys?Ser?His?Cys?Ile?Ala?Glu?Val?Glu?Asn?Asp
305 310 315 320
Glu?Met?Pro?Ala?Asp?Leu?Pro?Ser?Leu?Ala?Ala?Asp?Phe?Val?Glu?Ser
325 330 335
Lys?Asp?Val?Cys?Lys?Asn?Tyr?Ala?Glu?Ala?Lys?Asp?Val?Phe?Leu?Gly
340 345 350
Met?Phe?Leu?Tyr?Glu?Tyr?Ala?Arg?Arg?His?Pro?Asp?Tyr?Ser?Val?Val
355 360 365
Leu?Leu?Leu?Arg?Leu?Ala?Lys?Thr?Tyr?Glu?Thr?Thr?Leu?Glu?Lys?Cys
370 375 380
Cys?Ala?Ala?Ala?Asp?Pro?His?Glu?Cys?Tyr?Ala?Lys?Val?Phe?Asp?Glu
385 390 395 400
Phe?Lys?Pro?Leu?Val?Glu?Glu?Pro?Gln?Asn?Leu?Ile?Lys?Gln?Asn?Cys
405 410 415
Glu?Leu?Phe?Glu?Gln?Leu?Gly?Glu?Tyr?Lys?Phe?Gln?Asn?Ala?Leu?Leu
420 425 430
Val?Arg?Tyr?Thr?Lys?Lys?Val?Pro?Gln?Val?Ser?Thr?Pro?Thr?Leu?Val
435 440 445
Glu?Val?Ser?Arg?Asn?Leu?Gly?Lys?Val?Gly?Ser?Lys?Cys?Cys?Lys?His
450 455 460
Pro?Glu?Ala?Lys?Arg?Met?Pro?Cys?Ala?Glu?Asp?Tyr?Leu?Ser?Val?Val
465 470 475 480
Leu?Asn?Gln?Leu?Cys?Val?Leu?His?Glu?Lys?Thr?Pro?Val?Ser?Asp?Arg
485 490 495
Val?Thr?Lys?Cys?Cys?Thr?Glu?Ser?Leu?Val?Asn?Arg?Arg?Pro?Cys?Phe
500 505 510
Ser?Ala?Leu?Glu?Val?Asp?Glu?Thr?Tyr?Val?Pro?Lys?Glu?Phe?Asn?Ala
515 520 525
Glu?Thr?Phe?Thr?Phe?His?Ala?Asp?Ile?Cys?Thr?Leu?Ser?Glu?Lys?Glu
530 535 540
Arg?Gln?Ile?Lys?Lys?Gln?Thr?Ala?Leu?Val?Glu?Leu?Val?Lys?His?Lys
545 550 555 560
Pro?Lys?Ala?Thr?Lys?Glu?Gln?Leu?Lys?Ala?Val?Met?Asp?Asp?Phe?Ala
565 570 575
Ala?Phe?Val?Glu?Lys?Cys?Cys?Lys?Ala?Asp?Asp?Lys?Glu?Thr?Cys?Phe
580 585 590
Ala?Glu?Glu?Gly?Lys?Lys?Leu?Val?Ala?Ala?Ser?Gln?Ala?Ala?Leu?Gly
595 600 605
Leu?Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?Arg?Thr?Leu
610 615 620
Met?Leu?Leu?Ala?Gln?Met?Arg?Arg?Ile?Ser?Leu?Phe?Ser?Cys?Leu?Lys
625 630 635 640
Asp?Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe
645 650 655
Gln?Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Ile
660 665 670
Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr
675 680 685
Leu?Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu
690 695 700
Glu?Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Thr?Glu?Thr?Pro?Leu?Met
705 710 715 720
Lys?Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr
725 730 735
Leu?Tyr?Leu?Lys?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val
740 745 750
Arg?Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Leu?Ser?Thr?Asn?Leu?Gln?Glu
755 760 765
Ser?Leu?Arg?Ser?Lys?Glu
770
<210>181
<211>774
<212>PRT
＜213〉human (Homo sapiens)
<400>181
Met?Lys?Trp?Val?Ser?Phe?Ile?Ser?Leu?Leu?Phe?Leu?Phe?Ser?Ser?Ala
1 5 10 15
Tyr?Ser?Arg?Ser?Leu?Asp?Lys?Arg?Asp?Ala?His?Lys?Ser?Glu?Val?Ala
20 25 30
His?Arg?Phe?Lys?Asp?Leu?Gly?Glu?Glu?Asn?Phe?Lys?Ala?Leu?Val?Leu
35 40 45
Ile?Ala?Phe?Ala?Gln?Tyr?Leu?Gln?Gln?Cys?Pro?Phe?Glu?Asp?His?Val
50 55 60
Lys?Leu?Val?Asn?Glu?Val?Thr?Glu?Phe?Ala?Lys?Thr?Cys?Val?Ala?Asp
65 70 75 80
Glu?Ser?Ala?Glu?Asn?Cys?Asp?Lys?Ser?Leu?His?Thr?Leu?Phe?Gly?Asp
85 90 95
Lys?Leu?Cys?Thr?Val?Ala?Thr?Leu?Arg?Glu?Thr?Tyr?Gly?Glu?Met?Ala
100 105 110
Asp?Cys?Cys?Ala?Lys?Gln?Glu?Pro?Glu?Arg?Asn?Glu?Cys?Phe?Leu?Gln
115 120 125
His?Lys?Asp?Asp?Asn?Pro?Asn?Leu?Pro?Arg?Leu?Val?Arg?Pro?Glu?Val
130 135 140
Asp?Val?Met?Cys?Thr?Ala?Phe?His?Asp?Asn?Glu?Glu?Thr?Phe?Leu?Lys
145 150 155 160
Lys?Tyr?Leu?Tyr?Glu?Ile?Ala?Arg?Arg?His?Pro?Tyr?Phe?Tyr?Ala?Pro
165 170 175
Glu?Leu?Leu?Phe?Phe?Ala?Lys?Arg?Tyr?Lys?Ala?Ala?Phe?Thr?Glu?Cys
180 185 190
Cys?Gln?Ala?Ala?Asp?Lys?Ala?Ala?Cys?Leu?Leu?Pro?Lys?Leu?Asp?Glu
195 200 205
Leu?Arg?Asp?Glu?Gly?Lys?Ala?Ser?Ser?Ala?Lys?Gln?Arg?Leu?Lys?Cys
210 215 220
Ala?Ser?Leu?Gln?Lys?Phe?Gly?Glu?Arg?Ala?Phe?Lys?Ala?Trp?Ala?Val
225 230 235 240
Ala?Arg?Leu?Ser?Gln?Arg?Phe?Pro?Lys?Ala?Glu?Phe?Ala?Glu?Val?Ser
245 250 255
Lys?Leu?Val?Thr?Asp?Leu?Thr?Lys?Val?His?Thr?Glu?Cys?Cys?His?Gly
260 265 270
Asp?Leu?Leu?Glu?Cys?Ala?Asp?Asp?Arg?Ala?Asp?Leu?Ala?Lys?Tyr?Ile
275 280 285
Cys?Glu?Asn?Gln?Asp?Ser?Ile?Ser?Ser?Lys?Leu?Lys?Glu?Cys?Cys?Glu
290 295 300
Lys?Pro?Leu?Leu?Glu?Lys?Ser?His?Cys?Ile?Ala?Glu?Val?Glu?Asn?Asp
305 310 315 320
Glu?Met?Pro?Ala?Asp?Leu?Pro?Ser?Leu?Ala?Ala?Asp?Phe?Val?Glu?Ser
325 330 335
Lys?Asp?Val?Cys?Lys?Asn?Tyr?Ala?Glu?Ala?Lys?Asp?Val?Phe?Leu?Gly
340 345 350
Met?Phe?Leu?Tyr?Glu?Tyr?Ala?Arg?Arg?His?Pro?Asp?Tyr?Ser?Val?Val
355 360 365
Leu?Leu?Leu?Arg?Leu?Ala?Lys?Thr?Tyr?Glu?Thr?Thr?Leu?Glu?Lys?Cys
370 375 380
Cys?Ala?Ala?Ala?Asp?Pro?His?Glu?Cys?Tyr?Ala?Lys?Val?Phe?Asp?Glu
385 390 395 400
Phe?Lys?Pro?Leu?Val?Glu?Glu?Pro?Gln?Asn?Leu?Ile?Lys?Gln?Asn?Cys
405 410 415
Glu?Leu?Phe?Glu?Gln?Leu?Gly?Glu?Tyr?Lys?Phe?Gln?Asn?Ala?Leu?Leu
420 425 430
Val?Arg?Tyr?Thr?Lys?Lys?Val?Pro?Gln?Val?Ser?Thr?Pro?Thr?Leu?Val
435 440 445
Glu?Val?Ser?Arg?Asn?Leu?Gly?Lys?Val?Gly?Ser?Lys?Cys?Cys?Lys?His
450 455 460
Pro?Glu?Ala?Lys?Arg?Met?Pro?Cys?Ala?Glu?Asp?Tyr?Leu?Ser?Val?Val
465 470 475 480
Leu?Ash?Gln?Leu?Cys?Val?Leu?His?Glu?Lys?Thr?Pro?Val?Ser?Asp?Arg
485 490 495
Val?Thr?Lys?Cys?Cys?Thr?Glu?Ser?Leu?Val?Asn?Arg?Arg?Pro?Cys?Phe
500 505 510
Ser?Ala?Leu?Glu?Val?Asp?Glu?Thr?Tyr?Val?Pro?Lys?Glu?Phe?Asn?Ala
515 520 525
Glu?Thr?Phe?Thr?Phe?His?Ala?Asp?Ile?Cys?Thr?Leu?Ser?Glu?Lys?Glu
530 535 540
Arg?Gln?Ile?Lys?Lys?Gln?Thr?Ala?Leu?Val?Glu?Leu?Val?Lys?His?Lys
545 550 555 560
Pro?Lys?Ala?Thr?Lys?Glu?Gln?Leu?Lys?Ala?Val?Met?Asp?Asp?Phe?Ala
565 570 575
Ala?Phe?Val?Glu?Lys?Cys?Cys?Lys?Ala?Asp?Asp?Lys?Glu?Thr?Cys?Phe
580 585 590
Ala?Glu?Glu?Gly?Lys?Lys?Leu?Val?Ala?Ala?Ser?Gln?Ala?Ala?Leu?Gly
595 600 605
Leu?Ser?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?Arg?Thr?Leu
610 615 620
Met?Leu?Leu?Ala?Gln?Met?Arg?Arg?Ile?Ser?Leu?Phe?Ser?Cys?Leu?Lys
625 630 635 640
Asp?Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe
645 650 655
Gln?Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Ile
660 665 670
Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr
675 680 685
Leu?Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu
690 695 700
Glu?Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Thr?Glu?Thr?Pro?Leu?Met
705 710 715 720
Lys?Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr
725 730 735
Leu?Tyr?Leu?Lys?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val
740 745 750
Arg?Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Leu?Ser?Thr?Asn?Leu?Gln?Glu
755 760 765
Ser?Leu?Arg?Ser?Lys?Glu
770
<210>182
<211>774
<212>PRT
＜213〉human (Homo sapiens)
<400>182
Met?Lys?Trp?Val?Ser?Phe?Ile?Ser?Leu?Leu?Phe?Leu?Phe?Ser?Ser?Ala
1 5 10 15
Tyr?Ser?Arg?Ser?Leu?Asp?Lys?Arg?Asp?Ala?His?Lys?Ser?Glu?Val?Ala
20 25 30
His?Arg?Phe?Lys?Asp?Leu?Gly?Glu?Glu?Asn?Phe?Lys?Ala?Leu?Val?Leu
35 40 45
Ile?Ala?Phe?Ala?Gln?Tyr?Leu?Gln?Gln?Cys?Pro?Phe?Glu?Asp?His?Val
50 55 60
Lys?Leu?Val?Asn?Glu?Val?Thr?Glu?Phe?Ala?Lys?Thr?Cys?Val?Ala?Asp
65 70 75 80
Glu?Ser?Ala?Glu?Asn?Cys?Asp?Lys?Ser?Leu?His?Thr?Leu?Phe?Gly?Asp
85 90 95
Lys?Leu?Cys?Thr?Val?Ala?Thr?Leu?Arg?Glu?Thr?Tyr?Gly?Glu?Met?Ala
100 105 110
Asp?Cys?Cys?Ala?Lys?Gln?Glu?Pro?Glu?Arg?Asn?Glu?Cys?Phe?Leu?Gln
115 120 125
His?Lys?Asp?Asp?Asn?Pro?Asn?Leu?Pro?Arg?Leu?Val?Arg?Pro?Glu?Val
130 135 140
Asp?Val?Met?Cys?Thr?Ala?Phe?His?Asp?Asn?Glu?Glu?Thr?Phe?Leu?Lys
145 150 155 160
Lys?Tyr?Leu?Tyr?Glu?Ile?Ala?Arg?Arg?His?Pro?Tyr?Phe?Tyr?Ala?Pro
165 170 175
Glu?Leu?Leu?Phe?Phe?Ala?Lys?Arg?Tyr?Lys?Ala?Ala?Phe?Thr?Glu?Cys
180 185 190
Cys?Gln?Ala?Ala?Asp?Lys?Ala?Ala?Cys?Leu?Leu?Pro?Lys?Leu?Asp?Glu
195 200 205
Leu?Arg?Asp?Glu?Gly?Lys?Ala?Ser?Ser?Ala?Lys?Gln?Arg?Leu?Lys?Cys
210 215 220
Ala?Ser?Leu?Gln?Lys?Phe?Gly?Glu?Arg?Ala?Phe?Lys?Ala?Trp?Ala?Val
225 230 235 240
Ala?Arg?Leu?Ser?Gln?Arg?Phe?Pro?Lys?Ala?Glu?Phe?Ala?Glu?Val?Ser
245 250 255
Lys?Leu?Val?Thr?Asp?Leu?Thr?Lys?Val?His?Thr?Glu?Cys?Cys?His?Gly
260 265 270
Asp?Leu?Leu?Glu?Cys?Ala?Asp?Asp?Arg?Ala?Asp?Leu?Ala?Lys?Tyr?Ile
275 280 285
Cys?Glu?Asn?Gln?Asp?Ser?Ile?Ser?Ser?Lys?Leu?Lys?Glu?Cys?Cys?Glu
290 295 300
Lys?Pro?Leu?Leu?Glu?Lys?Ser?His?Cys?Ile?Ala?Glu?Val?Glu?Asn?Asp
305 310 315 320
Glu?Met?Pro?Ala?Asp?Leu?Pro?Ser?Leu?Ala?Ala?Asp?Phe?Val?Glu?Ser
325 330 335
Lys?Asp?Val?Cys?Lys?Asn?Tyr?Ala?Glu?Ala?Lys?Asp?Val?Phe?Leu?Gly
340 345 350
Met?Phe?Leu?Tyr?Glu?Tyr?Ala?Arg?Arg?His?Pro?Asp?Tyr?Ser?Val?Val
355 360 365
Leu?Leu?Leu?Arg?Leu?Ala?Lys?Thr?Tyr?Glu?Thr?Thr?Leu?Glu?Lys?Cys
370 375 380
Cys?Ala?Ala?Ala?Asp?Pro?His?Glu?Cys?Tyr?Ala?Lys?Val?Phe?Asp?Glu
385 390 395 400
Phe?Lys?Pro?Leu?Val?Glu?Glu?Pro?Gln?Asn?Leu?Ile?Lys?Gln?Asn?Cys
405 410 415
Glu?Leu?Phe?Glu?Gln?Leu?Gly?Glu?Tyr?Lys?Phe?Gln?Asn?Ala?Leu?Leu
420 425 430
Val?Arg?Tyr?Thr?Lys?Lys?Val?Pro?Gln?Val?Ser?Thr?Pro?Thr?Leu?Val
435 440 445
Glu?Val?Ser?Arg?Asn?Leu?Gly?Lys?Val?Gly?Ser?Lys?Cys?Cys?Lys?His
450 455 460
Pro?Glu?Ala?Lys?Arg?Met?Pro?Cys?Ala?Glu?Asp?Tyr?Leu?Ser?Val?Val
465 470 475 480
Leu?Asn?Gln?Leu?Cys?Val?Leu?His?Glu?Lys?Thr?Pro?Val?Ser?Asp?Arg
485 490 495
Val?Thr?Lys?Cys?Cys?Thr?Glu?Ser?Leu?Val?Asn?Arg?Arg?Pro?Cys?Phe
500 505 510
Ser?Ala?Leu?Glu?Val?Asp?Glu?Thr?Tyr?Val?Pro?Lys?Glu?Phe?Asn?Ala
515 520 525
Glu?Thr?Phe?Thr?Phe?His?Ala?Asp?Ile?Cys?Thr?Leu?Ser?Glu?Lys?Glu
530 535 540
Arg?Gln?Ile?Lys?Lys?Gln?Thr?Ala?Leu?Val?Glu?Leu?Val?Lys?His?Lys
545 550 555 560
Pro?Lys?Ala?Thr?Lys?Glu?Gln?Leu?Lys?Ala?Val?Met?Asp?Asp?Phe?Ala
565 570 575
Ala?Phe?Val?Glu?Lys?Cys?Cys?Lys?Ala?Asp?Asp?Lys?Glu?Thr?Cys?Phe
580 585 590
Ala?Glu?Glu?Gly?Lys?Lys?Leu?Val?Ala?Ala?Ser?Gln?Ala?Ala?Leu?Gly
595 600 605
Leu?Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?Arg?Thr?Leu
610 615 620
Met?Leu?Leu?Ala?Gln?Met?Arg?Arg?Ile?Ser?Leu?Phe?Ser?Ser?Leu?Lys
625 630 635 640
Asp?Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe
645 650 655
Gln?Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Ile
660 665 670
Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr
675 680 685
Leu?Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu
690 695 700
Glu?Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Thr?Glu?Thr?Pro?Leu?Met
705 710 715 720
Lys?Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr
725 730 735
Leu?Tyr?Leu?Lys?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val
740 745 750
Arg?Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Leu?Ser?Thr?Asn?Leu?Gln?Glu
755 760 765
Ser?Leu?Arg?Ser?Lys?Glu
770
<210>183
<211>774
<212>PRT
＜213〉human (Homo sapiens)
<400>183
Met?Lys?Trp?Val?Ser?Phe?Ile?Ser?Leu?Leu?Phe?Leu?Phe?Ser?Ser?Ala
1 5 10 15
Tyr?Ser?Arg?Ser?Leu?Asp?Lys?Arg?Asp?Ala?His?Lys?Ser?Glu?Val?Ala
20 25 30
His?Arg?Phe?Lys?Asp?Leu?Gly?Glu?Glu?Asn?Phe?Lys?Ala?Leu?Val?Leu
35 40 45
Ile?Ala?Phe?Ala?Gln?Tyr?Leu?Gln?Gln?Cys?Pro?Phe?Glu?Asp?His?Val
50 55 60
Lys?Leu?Val?Asn?Glu?Val?Thr?Glu?Phe?Ala?Lys?Thr?Cys?Val?Ala?Asp
65 70 75 80
Glu?Ser?Ala?Glu?Asn?Cys?Asp?Lys?Ser?Leu?His?Thr?Leu?Phe?Gly?Asp
85 90 95
Lys?Leu?Cys?Thr?Val?Ala?Thr?Leu?Arg?Glu?Thr?Tyr?Gly?Glu?Met?Ala
100 105 110
Asp?Cys?Cys?Ala?Lys?Gln?Glu?Pro?Glu?Arg?Asn?Glu?Cys?Phe?Leu?Gln
115 120 125
His?Lys?Asp?Asp?Asn?Pro?Asn?Leu?Pro?Arg?Leu?Val?Arg?Pro?Glu?Val
130 135 140
Asp?Val?Met?Cys?Thr?Ala?Phe?His?Asp?Asn?Glu?Glu?Thr?Phe?Leu?Lys
145 150 155 160
Lys?Tyr?Leu?Tyr?Glu?Ile?Ala?Arg?Arg?His?Pro?Tyr?Phe?Tyr?Ala?Pro
165 170 175
Glu?Leu?Leu?Phe?Phe?Ala?Lys?Arg?Tyr?Lys?Ala?Ala?Phe?Thr?Glu?Cys
180 185 190
Cys?Gln?Ala?Ala?Asp?Lys?Ala?Ala?Cys?Leu?Leu?Pro?Lys?Leu?Asp?Glu
195 200 205
Leu?Arg?Asp?Glu?Gly?Lys?Ala?Ser?Ser?Ala?Lys?Gln?Arg?Leu?Lys?Cys
210 215 220
Ala?Ser?Leu?Gln?Lys?Phe?Gly?Glu?Arg?Ala?Phe?Lys?Ala?Trp?Ala?Val
225 230 235 240
Ala?Arg?Leu?Ser?Gln?Arg?Phe?Pro?Lys?Ala?Glu?Phe?Ala?Glu?Val?Ser
245 250 255
Lys?Leu?Val?Thr?Asp?Leu?Thr?Lys?Val?His?Thr?Glu?Cys?Cys?His?Gly
260 265 270
Asp?Leu?Leu?Glu?Cys?Ala?Asp?Asp?Arg?Ala?Asp?Leu?Ala?Lys?Tyr?Ile
275 280 285
Cys?Glu?Asn?Gln?Asp?Ser?Ile?Ser?Ser?Lys?Leu?Lys?Glu?Cys?Cys?Glu
290 295 300
Lys?Pro?Leu?Leu?Glu?Lys?Ser?His?Cys?Ile?Ala?Glu?Val?Glu?Asn?Asp
305 310 315 320
Glu?Met?Pro?Ala?Asp?Leu?Pro?Ser?Leu?Ala?Ala?Asp?Phe?Val?Glu?Ser
325 330 335
Lys?Asp?Val?Cys?Lys?Asn?Tyr?Ala?Glu?Ala?Lys?Asp?Val?Phe?Leu?Gly
340 345 350
Met?Phe?Leu?Tyr?Glu?Tyr?Ala?Arg?Arg?His?Pro?Asp?Tyr?Ser?Val?Val
355 360 365
Leu?Leu?Leu?Arg?Leu?Ala?Lys?Thr?Tyr?Glu?Thr?Thr?Leu?Glu?Lys?Cys
370 375 380
Cys?Ala?Ala?Ala?Asp?Pro?His?Glu?Cys?Tyr?Ala?Lys?Val?Phe?Asp?Glu
385 390 395 400
Phe?Lys?Pro?Leu?Val?Glu?Glu?Pro?Gln?Asn?Leu?Ile?Lys?Gln?Asn?Cys
405 410 415
Glu?Leu?Phe?Glu?Gln?Leu?Gly?Glu?Tyr?Lys?Phe?Gln?Asn?Ala?Leu?Leu
420 425 430
Val?Arg?Tyr?Thr?Lys?Lys?Val?Pro?Gln?Val?Ser?Thr?Pro?Thr?Leu?Val
435 440 445
Glu?Val?Ser?Arg?Asn?Leu?Gly?Lys?Val?Gly?Ser?Lys?Cys?Cys?Lys?His
450 455 460
Pro?Glu?Ala?Lys?Arg?Met?Pro?Cys?Ala?Glu?Asp?Tyr?Leu?Ser?Val?Val
465 470 475 480
Leu?Asn?Gln?Leu?Cys?Val?Leu?His?Glu?Lys?Thr?Pro?Val?Ser?Asp?Arg
485 490 495
Val?Thr?Lys?Cys?Cys?Thr?Glu?Ser?Leu?Val?Asn?Arg?Arg?Pro?Cys?Phe
500 505 510
Ser?Ala?Leu?Glu?Val?Asp?Glu?Thr?Tyr?Val?Pro?Lys?Glu?Phe?Asn?Ala
515 520 525
Glu?Thr?Phe?Thr?Phe?His?Ala?Asp?Ile?Cys?Thr?Leu?Ser?Glu?Lys?Glu
530 535 540
Arg?Gln?Ile?Lys?Lys?Gln?Thr?Ala?Leu?Val?Glu?Leu?Val?Lys?His?Lys
545 550 555 560
Pro?Lys?Ala?Thr?Lys?Glu?Gln?Leu?Lys?Ala?Val?Met?Asp?Asp?Phe?Ala
565 570 575
Ala?Phe?Val?Glu?Lys?Cys?Cys?Lys?Ala?Asp?Asp?Lys?Glu?Thr?Cys?Phe
580 585 590
Ala?Glu?Glu?Gly?Lys?Lys?Leu?Val?Ala?Ala?Ser?Gln?Ala?Ala?Leu?Gly
595 600 605
Leu?Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?Arg?Thr?Leu
610 615 620
Met?Leu?Leu?Ala?Gln?Met?Arg?Arg?Ile?Ser?Leu?Phe?Ser?Cys?Leu?Lys
625 630 635 640
Asp?Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe
645 650 655
Gln?Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Ile
660 665 670
Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr
675 680 685
Leu?Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu
690 695 700
Glu?Ala?Ser?Val?Ile?Gln?Gly?Val?Gly?Val?Thr?Glu?Thr?Pro?Leu?Met
705 710 715 720
Lys?Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr
725 730 735
Leu?Tyr?Leu?Lys?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val
740 745 750
Arg?Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Leu?Ser?Thr?Asn?Leu?Gln?Glu
755 760 765
Ser?Leu?Arg?Ser?Lys?Glu
770
<210>184
<211>774
<212>PRT
＜213〉human (Homo sapiens)
<400>184
Met?Lys?Trp?Val?Ser?Phe?Ile?Ser?Leu?Leu?Phe?Leu?Phe?Ser?Ser?Ala
1 5 10 15
Tyr?Ser?Arg?Ser?Leu?Asp?Lys?Arg?Asp?Ala?His?Lys?Ser?Glu?Val?Ala
20 25 30
His?Arg?Phe?Lys?Asp?Leu?Gly?Glu?Glu?Asn?Phe?Lys?Ala?Leu?Val?Leu
35 40 45
Ile?Ala?Phe?Ala?Gln?Tyr?Leu?Gln?Gln?Cys?Pro?Phe?Glu?Asp?His?Val
50 55 60
Lys?Leu?Val?Asn?Glu?Val?Thr?Glu?Phe?Ala?Lys?Thr?Cys?Val?Ala?Asp
65 70 75 80
Glu?Ser?Ala?Glu?Asn?Cys?Asp?Lys?Ser?Leu?His?Thr?Leu?Phe?Gly?Asp
85 90 95
Lys?Leu?Cys?Thr?Val?Ala?Thr?Leu?Arg?Glu?Thr?Tyr?Gly?Glu?Met?Ala
100 105 110
Asp?Cys?Cys?Ala?Lys?Gln?Glu?Pro?Glu?Arg?Asn?Glu?Cys?Phe?Leu?Gln
115 120 125
His?Lys?Asp?Asp?Asn?Pro?Asn?Leu?Pro?Arg?Leu?Val?Arg?Pro?Glu?Val
130 135 140
Asp?Val?Met?Cys?Thr?Ala?Phe?His?Asp?Asn?Glu?Glu?Thr?Phe?Leu?Lys
145 150 155 160
Lys?Tyr?Leu?Tyr?Glu?Ile?Ala?Arg?Arg?His?Pro?Tyr?Phe?Tyr?Ala?Pro
165 170 175
Glu?Leu?Leu?Phe?Phe?Ala?Lys?Arg?Tyr?Lys?Ala?Ala?Phe?Thr?Glu?Cys
180 185 190
Cys?Gln?Ala?Ala?Asp?Lys?Ala?Ala?Cys?Leu?Leu?Pro?Lys?Leu?Asp?Glu
195 200 205
Leu?Arg?Asp?Glu?Gly?Lys?Ala?Ser?Ser?Ala?Lys?Gln?Arg?Leu?Lys?Cys
210 215 220
Ala?Ser?Leu?Gln?Lys?Phe?Gly?Glu?Arg?Ala?Phe?Lys?Ala?Trp?Ala?Val
225 230 235 240
Ala?Arg?Leu?Ser?Gln?Arg?Phe?Pro?Lys?Ala?Glu?Phe?Ala?Glu?Val?Ser
245 250 255
Lys?Leu?Val?Thr?Asp?Leu?Thr?Lys?Val?His?Thr?Glu?Cys?Cys?His?Gly
260 265 270
Asp?Leu?Leu?Glu?Cys?Ala?Asp?Asp?Arg?Ala?Asp?Leu?Ala?Lys?Tyr?Ile
275 280 285
Cys?Glu?Asn?Gln?Asp?Ser?Ile?Ser?Ser?Lys?Leu?Lys?Glu?Cys?Cys?Glu
290 295 300
Lys?Pro?Leu?Leu?Glu?Lys?Ser?His?Cys?Ile?Ala?Glu?Val?Glu?Asn?Asp
305 310 315 320
Glu?Met?Pro?Ala?Asp?Leu?Pro?Ser?Leu?Ala?Ala?Asp?Phe?Val?Glu?Ser
325 330 335
Lys?Asp?Val?Cys?Lys?Asn?Tyr?Ala?Glu?Ala?Lys?Asp?Val?Phe?Leu?Gly
340 345 350
Met?Phe?Leu?Tyr?Glu?Tyr?Ala?Arg?Arg?His?Pro?Asp?Tyr?Ser?Val?Val
355 360 365
Leu?Leu?Leu?Arg?Leu?Ala?Lys?Thr?Tyr?Glu?Thr?Thr?Leu?Glu?Lys?Cys
370 375 380
Cys?Ala?Ala?Ala?Asp?Pro?His?Glu?Cys?Tyr?Ala?Lys?Val?Phe?Asp?Glu
385 390 395 400
Phe?Lys?Pro?Leu?Val?Glu?Glu?Pro?Gln?Asn?Leu?Ile?Lys?Gln?Asn?Cys
405 410 415
Glu?Leu?Phe?Glu?Gln?Leu?Gly?Glu?Tyr?Lys?Phe?Gln?Asn?Ala?Leu?Leu
420 425 430
Val?Arg?Tyr?Thr?Lys?Lys?Val?Pro?Gln?Val?Ser?Thr?Pro?Thr?Leu?Val
435 440 445
Glu?Val?Ser?Arg?Asn?Leu?Gly?Lys?Val?Gly?Ser?Lys?Cys?Cys?Lys?His
450 455 460
Pro?Glu?Ala?Lys?Arg?Met?Pro?Cys?Ala?Glu?Asp?Tyr?Leu?Ser?Val?Val
465 470 475 480
Leu?Asn?Gln?Leu?Cys?Val?Leu?His?Glu?Lys?Thr?Pro?Val?Ser?Asp?Arg
485 490 495
Val?Thr?Lys?Cys?Cys?Thr?Glu?Ser?Leu?Val?Asn?Arg?Arg?Pro?Cys?Phe
500 505 510
Ser?Ala?Leu?Glu?Val?Asp?Glu?Thr?Tyr?Val?Pro?Lys?Glu?Phe?Asn?Ala
515 520 525
Glu?Thr?Phe?Thr?Phe?His?Ala?Asp?Ile?Cys?Thr?Leu?Ser?Glu?Lys?Glu
530 535 540
Arg?Gln?Ile?Lys?Lys?Gln?Thr?Ala?Leu?Val?Glu?Leu?Val?Lys?His?Lys
545 550 555 560
Pro?Lys?Ala?Thr?Lys?Glu?Gln?Leu?Lys?Ala?Val?Met?Asp?Asp?Phe?Ala
565 570 575
Ala?Phe?Val?Glu?Lys?Cys?Cys?Lys?Ala?Asp?Asp?Lys?Glu?Thr?Cys?Phe
580 585 590
Ala?Glu?Glu?Gly?Lys?Lys?Leu?Val?Ala?Ala?Ser?Gln?Ala?Ala?Leu?Gly
595 600 605
Leu?Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?Arg?Thr?Leu
610 615 620
Met?Leu?Leu?Ala?Gln?Met?Arg?Arg?Ile?Ser?Leu?Phe?Ser?Cys?Leu?Lys
625 630 635 640
Asp?Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe
645 650 655
Gln?Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Ile
660 665 670
Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr
675 680 685
Leu?Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu
690 695 700
Glu?Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Thr?Glu?Thr?Pro?Leu?Met
705 710 715 720
Lys?Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr
725 730 735
Leu?Tyr?Leu?Lys?Glu?Lys?Lys?Tyr?Ser?Pro?Ser?Ala?Trp?Glu?Val?Val
740 745 750
Arg?Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Leu?Ser?Thr?Asn?Leu?Gln?Glu
755 760 765
Ser?Leu?Arg?Ser?Lys?Glu
770
<210>185
<211>165
<212>PRT
＜213〉human (Homo sapiens)
<400>185
Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?Arg?Thr?Leu?Met
1 5 10 15
Leu?Leu?Ala?Gln?Met?Arg?Arg?Ile?Ser?Leu?Phe?Ser?Cys?Leu?Lys?Asp
20 25 30
Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe?Gln
35 40 45
Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Ile?Phe
50 55 60
Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr?Leu
65 70 75 80
Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu?Glu
85 90 95
Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Thr?Glu?Thr?Pro?Leu?Met?Lys
100 105 110
Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr?Leu
115 120 125
Tyr?Leu?Lys?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val?Arg
130 135 140
Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Leu?Ser?Thr?Asn?Leu?Gln?Glu?Ser
145 150 155 160
Leu?Arg?Ser?Lys?Glu
165
<210>186
<211>165
<212>PRT
＜213〉human (Homo sapiens)
<400>186
Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?Arg?Thr?Leu?Met
1 5 10 15
Leu?Leu?Ala?Gln?Met?Arg?Arg?Ile?Ser?Leu?Phe?Ser?Cys?Leu?Lys?Asp
20 25 30
Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe?Gln
35 40 45
Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Ile?Phe
50 55 60
Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr?Leu
65 70 75 80
Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu?Glu
85 90 95
Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Thr?Glu?Thr?Pro?Leu?Met?Lys
100 105 110
Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr?Leu
115 120 125
Tyr?Leu?Lys?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val?Arg
130 135 140
Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Leu?Ser?Thr?Asn?Leu?Gln?Gl?u?Ser
145 150 155 160
Leu?Arg?Ser?Lys?Glu
165
<210>187
<211>165
<212>PRT
＜213〉human (Homo sapiens)
<400>187
Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?Arg?Thr?Leu?Met
1 5 10 15
Leu?Leu?Ala?Gln?Met?Arg?Arg?Ile?Ser?Leu?Phe?Ser?Cys?Leu?Lys?Asp
20 25 30
Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe?Gln
35 40 45
Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Ile?Phe
50 55 60
Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr?Leu
65 70 75 80
Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu?Glu
85 90 95
Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Thr?Glu?Thr?Pro?Leu?Met?Lys
100 105 110
Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr?Leu
115 120 125
Tyr?Leu?Lys?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val?Arg
130 135 140
Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Leu?Ser?Thr?Asn?Leu?Gln?Glu?Ser
145 150 155 160
Leu?Arg?Ser?Lys?Glu
165
<210>188
<211>165
<212>PRT
＜213〉human (Homo sapiens)
<400>188
Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?Arg?Thr?Leu?Met
1 5 10 15
Leu?Leu?Ala?Gln?Met?Arg?Arg?Ile?Ser?Leu?Phe?Ser?Cys?Leu?Lys?Asp
20 25 30
Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe?Gln
35 40 45
Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Ile?Phe
50 55 60
Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr?Leu
65 70 75 80
Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu?Glu
85 90 95
Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Thr?Glu?Thr?Pro?Leu?Met?Lys
100 105 110
Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr?Leu
115 120 125
Tyr?Leu?Lys?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val?Arg
130 135 140
Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Leu?Ser?Thr?Asn?Leu?Gln?Glu?Ser
145 150 155 160
Leu?Arg?Ser?Lys?Glu
165
<210>189
<211>165
<212>PRT
＜213〉human (Homo sapiens)
<400>189
Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?Arg?Thr?Leu?Met
1 5 10 15
Leu?Leu?Ala?Gln?Met?Arg?Arg?Ile?Ser?Leu?Phe?Ser?Cys?Leu?Lys?Asp
20 25 30
Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe?Gln
35 40 45
Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu?Met?Ile?Gln?Gln?Ile?Phe
50 55 60
Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr?Leu
65 70 75 80
Leu?Asp?Lys?Phe?Tyr?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu?Glu
85 90 95
Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Thr?Glu?Thr?Pro?Leu?Met?Lys
100 105 110
Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr?Leu
115 120 125
Tyr?Leu?Lys?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val?Arg
130 135 140
Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Leu?Ser?Thr?Asn?Leu?Gln?Glu?Ser
145 150 155 160
Leu?Arg?Ser?Lys?Glu
165
Claims (222)
1. albumin fusion proteins, it comprises and is selected from following member:
(a) fragment of human cytokines X or therapeutic X or variant and albumin or its albumin fragment or variant;
(b) fragment of human cytokines X or therapeutic X or variant and albumin or its albumin fragment or variant, wherein said albumin or its albumin fragment or variant comprise the aminoacid sequence of SEQ ID NO:1;
(c) (a) or the fragment of human cytokines X (b) or therapeutic X or variant and albumin or its albumin fragment or variant, the fragment of wherein said therapeutic X or variant have the biologic activity of human cytokines X, and wherein said its albumin fragment or variant have the albumin activity;
(d) fragment of human cytokines X (c) or therapeutic X or variant and albumin or its albumin fragment or variant, wherein said albumin activity are the abilities than the shelf life of the shelf life extended treatment albumin X of the human cytokines X that does not merge state;
(e) fragment of human cytokines X (c) or therapeutic X or variant and albumin or its fragment or variant, wherein said albumin activity are the abilities than the serum half-life of the serum half-life extended treatment albumin X of the human cytokines X that does not merge state;
(f) (a) human cytokines X-(e) or the fragment of therapeutic X or variant and albumin or its albumin fragment or variant, wherein said albumin fragment or variant comprise the amino acid whose aminoacid sequence of 1-387 of SEQ ID NO:1;
(g) (a) to the human cytokines X of (f) or fragment or variant and albumin or its albumin fragment or the variant of therapeutic X, the fragment of wherein said human cytokines X or therapeutic X or variant are blended in albuminised N-end, or are blended in the N-end of described its albumin fragment or variant;
(h) (a) to the human cytokines X of (f) or fragment or variant and albumin or its albumin fragment or the variant of therapeutic X, the fragment of wherein said human cytokines X or therapeutic X or variant are blended in albuminised C-end, or are blended in the C-end of described its albumin fragment or variant;
(i) (a) to the human cytokines X of (f) or fragment or variant and albumin or its albumin fragment or the variant of therapeutic X, the fragment of wherein said human cytokines X or therapeutic X or variant are blended in the terminal and C-end of albuminised N-, or are blended in the terminal and C-end of N-of described its albumin fragment or variant;
(j) (a) to the human cytokines X of (f) or fragment or variant and albumin or its albumin fragment or the variant of therapeutic X, it comprises the first human cytokines X or its fragment or variant and the second human cytokines X or its fragment or variant, and the wherein said first human cytokines X or its fragment or variant are different from the described second human cytokines X or its fragment or variant;
(k) (a) to the human cytokines X of (j) or fragment or variant and albumin or its albumin fragment or the variant of therapeutic X, the fragment of wherein said human cytokines X or therapeutic X or variant separate by joint and described albumin or described its albumin fragment or variant;
(l) (a) to the human cytokines X of (k) or fragment or variant and albumin or its albumin fragment or the variant of therapeutic X, wherein said albumin fusion proteins has following formula:
R1-L-R2; R2-L-R1; Or R1-L-R2-L-R1,
And further, wherein R1 is a human cytokines: X or its fragment or variant, and L is a peptide linker, and R2 is albumin or albuminised fragment or the variant that comprises the aminoacid sequence of SEQ ID NO:1;
(m) (a) to the human cytokines X of (l) or fragment or variant and albumin or its albumin fragment or the variant of therapeutic X, the shelf life of wherein said albumin fusion proteins is greater than the fragment of human cytokines X that does not merge state or therapeutic X or the shelf life of variant;
(n) (a) to the human cytokines X of (l) or fragment or variant and albumin or its albumin fragment or the variant of therapeutic X, the serum half-life of wherein said albumin fusion proteins is greater than the fragment of human cytokines X that does not merge state or therapeutic X or the serum half-life of variant;
(o) (a) to the human cytokines X of (l) or fragment or variant and albumin or its albumin fragment or the variant of therapeutic X, the extracorporeal biology activity of the human cytokines X of wherein said albumin fusion proteins or the fragment of therapeutic X or variant is greater than the fragment of human cytokines X that does not merge state or therapeutic X or the extracorporeal biology activity of variant; With
(p) (a) to the human cytokines X of (l) or fragment or variant and albumin or its albumin fragment or the variant of therapeutic X, biologic activity is greater than biologic activity in the body of the fragment of human cytokines X that does not merge state or therapeutic X or variant in the body of the human cytokines X of wherein said albumin fusion proteins or the fragment of therapeutic X or variant.
2. albumin fusion proteins according to claim 1, it is expressed in host cell, and wherein said host cell is yeast cells, mammalian cell or bacterial cell.
3. albumin fusion proteins according to claim 1, wherein said albumin fusion proteins further comprises the secretion targeting sequencing.
4. compositions, it comprises described albumin fusion proteins of claim 1 and pharmaceutically acceptable carrier.
5. medicine box, it comprises the described compositions of claim 4.
6. method for the treatment of disease of patient, illness or infection, it comprises the step of the described albumin fusion proteins of claim 1 of administering therapeutic effective dose.
7. method according to claim 6, wherein said disease, illness or infection comprise indication Y.
8. method according to claim 7, wherein said disease, illness or infection are selected from chronic hepatitis and infect; Hepatitis B infection; Chronic hepatitis B infects; Hepatitis C infection; Chronic hepatitis C infection; Hepatitis D infects; Chronic hepatitis D infects; Human papillomavirus infects; Herpes simplex infections; Outside condyloma acuminatum; HIV infects; Tumor; Cancer; Solid tumor; Melanoma; Malignant melanoma; Renal carcinoma; Pulmonary carcinoma; Colon cancer; Breast carcinoma; Hepatocarcinoma; Carcinoma of prostate; Bladder cancer; Gastric cancer; Sarcoma; The Kaposi sarcoma that AIDS is relevant; Lymphoma; T cell lymphoma; Cutaneous T cell lymphoma; Non_hodgkin lymphoma; The brain cancer; Glioma; Glioblastoma multiforme; Cervical atypical hyperplasia; Leukemia; Preleukemia; Myelopathy; Osteopathia; Hairy cell; Chronic granulocytic leukemia; Hematologic malignancies; Hematopathy; Multiple myeloma; Bacterial infection; Chemoproection; Thrombocytopenia; Multiple sclerosis; Pulmonary fibrosis; The macula lutea degenerative change relevant with the age; Degeneration of macula; Crohn disease; Neurological disorder; Arthritis; Rheumatoid arthritis; Ulcerative colitis; Osteoporosis, osteopenia, osteoclast generate; Fibromyalgia; Siogren's syndrome; Chronic fatigue syndrome; Heating; Hemorrhagic fever; Viral hemorrhagic fever; Hyperglycemia; Diabetes; Diabetes insipidus; Diabetes; Type 1 diabetes; Type 2 diabetes mellitus; Insulin resistant; Insulin deficit; Hyperlipemia; Hyperketonemia; Noninsulindependent diabetes (NIDDM); Insulin-dependent diabetes (IDDM); The infection that A, the B that serious acute respiratory organ syndrome (SARS) or the Center for Disease Control are identified or the C class factor cause, or its any combination.
9. according to claim 7 or 8 described methods, wherein said disease or illness are hepatitis C or hepatitis B infection.
10. according to each described method of claim 7 to 9, wherein said disease or illness are that hepatitis C or HIV infect.
11. according to claim 9 or 10 described methods, wherein said albumin fusion proteins is by the host cell expression that comprises the albumin fusion constructs, described albumin fusion constructs is selected from:
(a) construct ID 2249;
(b) construct ID 2343;
(c) construct ID 2366;
(d) construct ID 2381;
(e) construct ID 2382;
(f) construct ID 2410;
(g) construct ID 3165;
(h) construct ID 3422;
(i) construct ID 3423;
(j) construct ID 3424;
(k) construct ID 3476;
(l) construct ID 3960;
(m) construct ID 4290;
(n) construct ID 4291;
(o) construct ID 4292;
(p) construct ID 4295; With
(q) construct ID 4296.
12. method according to claim 11, wherein said albumin fusion constructs is (d).
13. method according to claim 11, wherein said albumin fusion constructs is (g).
14. method according to claim 11, wherein said albumin fusion constructs is (l).
15. method according to claim 13, wherein said patient suffer from hepatitis C infection and described patient be first treatment or live through treatment.
16. method according to claim 15, wherein said patient be live through treatment and be the nonresponder.
17. method according to claim 16 fails at least the combined treatment that comprises pegylated interferon alfa and ribavirin before the wherein said nonresponder.
18. method according to claim 15, wherein said hepatitis C infection are genotype 1 or genotype 2/3.
19. method according to claim 18, the described treatment effective dose of wherein said albumin fusion proteins is selected from:
(a) about 600 μ g/ agent;
(b) about 900 μ g/ agent;
(c) about 1000 μ g/ agent;
(d) about 1200 μ g/ agent;
(e) about 1800 μ g/ agent;
(f) about 2000 μ g/ agent; With
(g) about 2400 μ g/ agent.
20. method according to claim 19, wherein according to being selected from the following described albumin fusion proteins of drug dosage schedule administration:
(a) weekly;
(b) whenever biweekly;
(b) per three weeks once;
(c) whenever all around once; With
(d) per five weeks once.
21. method according to claim 19 is wherein according to comprising every once described albumin fusion proteins of drug dosage schedule administration of combination biweekly and around every.
Around before 22. method according to claim 21, wherein said albumin fusion proteins drug dosage schedule comprise whenever biweekly, every subsequently around the described albumin fusion proteins of applied once.
23. according to each described method of claim 20 to 22, wherein said drug dosage schedule continued for 24 to 48 weeks.
24. according to each described method of claim 15 to 23, wherein said albumin fusion proteins and one or more antiviral agent are co-administered.
25. method according to claim 24, wherein said antiviral agent is selected from:
(a) antiviral enzyme inhibitor;
(b) nucleoside analog antiviral AG14361;
(c) non-nucleoside is like thing antiviral AG14361;
(d) antisense oligonucleotide inhibitor;
(e)thiazolide；
(f) antiviral antibody;
(g) novel immunomodulator;
(h) cyclophilin inhibitor;
(i) liver protectant; With
(j) interferon enhancer.
26. method according to claim 24, wherein said antiviral agent are ribavirin or ribavirin analog.
27. method according to claim 24, wherein said albumin fusion proteins and two kinds, three kinds or four kinds of antiviral agent are co-administered.
28. method according to claim 27, one of wherein said antiviral agent are ribavirin or ribavirin analog.
29. method of suffering from the patient of hepatitis C infection with the albumin fusion proteins treatment of treatment effective dose, described albumin fusion proteins comprises the ripe Interferon Alpha-2b that merges with ripe albumin, wherein said ripe Interferon Alpha-2b is blended in ripe albuminised C-end, and further wherein (1) described patient is first treatment, (2) described hepatitis C infection is a genotype 1, (3) described treatment effective dose is extremely about 1800 μ g/ agent of about 900 μ g/ agent, and (4) are whenever biweekly with the described patient of described albumin fusion proteins administration.
30. method according to claim 29, wherein said treatment effective dose is selected from:
(a) about 900 μ g/ agent;
(b) about 1200 μ g/ agent; With
(c) about 1800 μ g/ agent.
31. according to claim 29 or 30 described methods, wherein said albumin fusion proteins and one or more antiviral agent are co-administered.
32. method according to claim 31, wherein said antiviral agent is selected from:
(a) antiviral enzyme inhibitor;
(b) nucleoside analog antiviral AG14361;
(c) non-nucleoside is like thing antiviral AG14361;
(d) antisense oligonucleotide inhibitor;
(e)thiazolide；
(f) antiviral antibody;
(g) novel immunomodulator;
(h) cyclophilin inhibitor;
(i) liver protectant; With
(j) interferon enhancer.
33. method according to claim 31, wherein said antiviral agent are ribavirin or ribavirin analog.
34. method according to claim 31, wherein said albumin fusion proteins and two kinds, three kinds or four kinds of antiviral agent are co-administered.
35. method according to claim 34, one of wherein said antiviral agent are ribavirin or ribavirin analog.
36. method of suffering from the patient of hepatitis C infection with the albumin fusion proteins treatment of treatment effective dose, described albumin fusion proteins comprises the ripe Interferon Alpha-2b that merges with ripe albumin, wherein said ripe Interferon Alpha-2b is blended in ripe albuminised C-end, and further wherein (1) described patient is first treatment, (2) described hepatitis C infection is a genotype 1, (3) described treatment effective dose is extremely about 1800 μ g/ agent of about 900 μ g/ agent, and (4) are every all around once with the described patient of described albumin fusion proteins administration.
37. method according to claim 36, wherein said treatment effective dose is selected from:
(a) about 900 μ g/ agent;
(b) about 1200 μ g/ agent; With
(c) about 1800 μ g/ agent.
38. according to claim 36 or 37 described methods, wherein said albumin fusion proteins and one or more antiviral agent are co-administered.
39. according to the described method of claim 38, wherein said antiviral agent is selected from:
(a) antiviral enzyme inhibitor;
(b) nucleoside analog antiviral AG14361;
(c) non-nucleoside is like thing antiviral AG14361;
(d) antisense oligonucleotide inhibitor;
(e)thiazolide；
(f) antiviral antibody;
(g) novel immunomodulator;
(h) cyclophilin inhibitor;
(i) liver protectant; With
(j) interferon enhancer.
40. according to the described method of claim 38, wherein said antiviral agent is ribavirin or ribavirin analog.
41. according to the described method of claim 38, wherein said albumin fusion proteins and two kinds, three kinds or four kinds of antiviral agent are co-administered.
42. according to the described method of claim 38, one of wherein said antiviral agent is ribavirin or ribavirin analog.
43. method of suffering from the patient of hepatitis C infection with the albumin fusion proteins treatment of treatment effective dose, described albumin fusion proteins comprises the ripe Interferon Alpha-2b that merges with ripe albumin, wherein said ripe Interferon Alpha-2b is blended in ripe albuminised C-end, and further wherein (1) described patient is first treatment, (2) described hepatitis C infection is a genotype 1, (3) described treatment effective dose is about 900 μ g/ agent to about 1800 μ g/ agent, and (4) around treatment preceding whenever biweekly, every subsequently all around once with the described patient of described albumin fusion proteins administration.
44. according to the described method of claim 43, wherein said albumin fusion proteins is applied to described patient and continues totally 24 to 48 weeks.
45. according to claim 43 or 44 described methods, wherein said treatment effective dose is selected from:
(a) about 900 μ g/ agent;
(b) about 1200 μ g/ agent; With
(c) about 1800 μ g/ agent.
46. according to each described method of claim 43 to 45, wherein said albumin fusion proteins and one or more antiviral agent are co-administered.
47. according to the described method of claim 46, wherein said antiviral agent is selected from:
(a) antiviral enzyme inhibitor;
(b) nucleoside analog antiviral AG14361;
(c) non-nucleoside is like thing antiviral AG14361;
(d) antisense oligonucleotide inhibitor;
(e)thiazolide；
(f) antiviral antibody;
(g) novel immunomodulator;
(h) cyclophilin inhibitor;
(i) liver protectant; With
(j) interferon enhancer.
48. according to the described method of claim 46, wherein said antiviral agent is ribavirin or ribavirin analog.
49. according to the described method of claim 46, wherein said albumin fusion proteins and two kinds, three kinds or four kinds of antiviral agent are co-administered.
50. according to the described method of claim 49, one of wherein said antiviral agent is ribavirin or ribavirin analog.
51. method of suffering from the patient of hepatitis C infection with the albumin fusion proteins treatment of treatment effective dose, described albumin fusion proteins comprises the ripe Interferon Alpha-2b that merges with ripe albumin, wherein said ripe Interferon Alpha-2b is blended in ripe albuminised C-end, and further wherein (1) described patient lives through treatment, (2) described hepatitis C infection is a genotype 1, (3) described treatment effective dose is extremely about 1800 μ g/ agent of about 1200 μ g/ agent, and (4) are whenever biweekly with the described patient of described albumin fusion proteins administration.
52. according to the described method of claim 51, wherein said albumin fusion proteins and one or more antiviral agent are co-administered.
53. according to the described method of claim 52, wherein said antiviral agent is selected from:
(a) antiviral enzyme inhibitor;
(b) nucleoside analog antiviral AG14361;
(c) non-nucleoside is like thing antiviral AG14361;
(d) antisense oligonucleotide inhibitor;
(e)thiazolide；
(f) antiviral antibody;
(g) novel immunomodulator;
(h) cyclophilin inhibitor;
(i) liver protectant; With
(j) interferon enhancer.
54. according to the described method of claim 52, wherein said antiviral agent is ribavirin or ribavirin analog.
55. according to the described method of claim 52, wherein said albumin fusion proteins and two kinds, three kinds or four kinds of antiviral agent are co-administered.
56. according to the described method of claim 55, one of wherein said antiviral agent is ribavirin or ribavirin analog.
57. method of suffering from the patient of hepatitis C infection with the albumin fusion proteins treatment of treatment effective dose, described albumin fusion proteins comprises the ripe Interferon Alpha-2b that merges with ripe albumin, wherein said ripe Interferon Alpha-2b is blended in ripe albuminised C-end, and further wherein (1) described patient lives through treatment, (2) described hepatitis C infection is a genotype 1, (3) described treatment effective dose is extremely about 1800 μ g/ agent of about 1200 μ g/ agent, and (4) are every all around once with the described patient of described albumin fusion proteins administration.
58. according to the described method of claim 57, wherein said albumin fusion proteins and one or more antiviral agent are co-administered.
59. according to the described method of claim 58, wherein said antiviral agent is selected from:
(a) antiviral enzyme inhibitor;
(b) nucleoside analog antiviral AG14361;
(c) non-nucleoside is like thing antiviral AG14361;
(d) antisense oligonucleotide inhibitor;
(e)thiazolide；
(f) antiviral antibody;
(g) novel immunomodulator;
(h) cyclophilin inhibitor;
(i) liver protectant; With
(j) interferon enhancer.
60. according to the described method of claim 58, wherein said antiviral agent is ribavirin or ribavirin analog.
61. according to the described method of claim 58, wherein said albumin fusion proteins and two kinds, three kinds or four kinds of antiviral agent are co-administered.
62. according to the described method of claim 61, one of wherein said antiviral agent is ribavirin or ribavirin analog.
63. method of suffering from the patient of hepatitis C infection with the albumin fusion proteins treatment of treatment effective dose, described albumin fusion proteins comprises the ripe Interferon Alpha-2b that merges with ripe albumin, wherein said ripe Interferon Alpha-2b is blended in ripe albuminised C-end, and further wherein (1) described patient lives through treatment, (2) described hepatitis C infection is a genotype 1, (3) described treatment effective dose is that about 1200 μ g/ agent are to about 1800 μ g/ agent, and (4) the treatment preceding around whenever biweekly, once with the described patient of described albumin fusion proteins administration, continued for 24 to 48 weeks around every subsequently.
64. according to the described method of claim 63, wherein said albumin fusion proteins is applied to described patient and continues totally 24 to 48 weeks.
65. according to claim 63 or 64 described methods, wherein said albumin fusion proteins and one or more antiviral agent are co-administered.
66. according to the described method of claim 65, wherein said antiviral agent is selected from:
(a) antiviral enzyme inhibitor;
(b) nucleoside analog antiviral AG14361;
(c) non-nucleoside is like thing antiviral AG14361;
(d) antisense oligonucleotide inhibitor;
(e)thiazolide；
(f) antiviral antibody;
(g) novel immunomodulator;
(h) cyclophilin inhibitor;
(i) liver protectant; With
(j) interferon enhancer.
67. according to the described method of claim 65, wherein said antiviral agent is ribavirin or ribavirin analog.
68. according to the described method of claim 65, wherein said albumin fusion proteins and two kinds, three kinds or four kinds of antiviral agent are co-administered.
69. according to the described method of claim 69, one of wherein said antiviral agent is ribavirin or ribavirin analog.
70. method of suffering from the patient of hepatitis C infection with the albumin fusion proteins treatment of treatment effective dose, described albumin fusion proteins comprises the ripe Interferon Alpha-2b that merges with ripe albumin, wherein said ripe Interferon Alpha-2b is blended in ripe albuminised C-end, and further wherein (1) described patient is first treatment, (2) described hepatitis C infection is genotype 2 or 3, (3) described treatment effective dose is extremely about 1800 μ g/ agent of about 900 μ g/ agent, and (4) are whenever biweekly with the described patient of described albumin fusion proteins administration.
71. according to the described method of claim 70, wherein said treatment effective dose is selected from:
(a) about 900 μ g/ agent;
(b) about 1200 μ g/ agent; With
(c) about 1800 μ g/ agent.
72. according to claim 70 or 71 described methods, wherein said albumin fusion proteins and one or more antiviral agent are co-administered.
73. according to the described method of claim 72, wherein said antiviral agent is selected from:
(a) antiviral enzyme inhibitor;
(b) nucleoside analog antiviral AG14361;
(c) non-nucleoside is like thing antiviral AG14361;
(d) antisense oligonucleotide inhibitor;
(e)thiazolide；
(f) antiviral antibody;
(g) novel immunomodulator;
(h) cyclophilin inhibitor;
(i) liver protectant; With
(j) interferon enhancer.
74. according to the described method of claim 72, wherein said antiviral agent is ribavirin or ribavirin analog.
75. according to the described method of claim 72, wherein said albumin fusion proteins and two kinds, three kinds or four kinds of antiviral agent are co-administered.
76. according to the described method of claim 75, one of wherein said antiviral agent is ribavirin or ribavirin analog.
77. method of suffering from the patient of hepatitis C infection with the albumin fusion proteins treatment of treatment effective dose, described albumin fusion proteins comprises the ripe Interferon Alpha-2b that merges with ripe albumin, wherein said ripe Interferon Alpha-2b is blended in ripe albuminised C-end, and further wherein (1) described patient is first treatment, (2) described hepatitis C infection is genotype 2 or 3, (3) described treatment effective dose is extremely about 1800 μ g/ agent of about 900 μ g/ agent, and (4) are every all around once with the described patient of described albumin fusion proteins administration.
78. according to the described method of claim 77, wherein said treatment effective dose is selected from:
(a) about 900 μ g/ agent;
(b) about 1200 μ g/ agent; With
(c) about 1800 μ g/ agent.
79. according to claim 77 or 78 described methods, wherein said albumin fusion proteins and one or more antiviral agent are co-administered.
80. according to the described method of claim 79, wherein said antiviral agent is selected from:
(a) antiviral enzyme inhibitor;
(b) nucleoside analog antiviral AG14361;
(c) non-nucleoside is like thing antiviral AG14361;
(d) antisense oligonucleotide inhibitor;
(e)thiazolide；
(f) antiviral antibody;
(g) novel immunomodulator;
(h) cyclophilin inhibitor;
(i) liver protectant; With
(j) interferon enhancer.
81. according to the described method of claim 79, wherein said antiviral agent is ribavirin or ribavirin analog.
82. according to the described method of claim 79, wherein said albumin fusion proteins and two kinds, three kinds or four kinds of antiviral agent are co-administered.
83. 2 described methods according to Claim 8, one of wherein said antiviral agent is ribavirin or ribavirin analog.
84. method of suffering from the patient of hepatitis C infection with the albumin fusion proteins treatment of treatment effective dose, described albumin fusion proteins comprises the ripe Interferon Alpha-2b that merges with ripe albumin, wherein said ripe Interferon Alpha-2b is blended in ripe albuminised C-end, and further wherein (1) described patient is first treatment, (2) described hepatitis C infection is genotype 2 or 3, (3) described treatment effective dose is about 900 μ g/ agent to about 1800 μ g/ agent, and (4) around treatment preceding whenever biweekly, every subsequently all around once with the described patient of described albumin fusion proteins administration.
85. 4 described methods according to Claim 8, wherein said albumin fusion proteins is applied to described patient and continues totally 24 to 48 weeks.
86. 4 or 85 described methods according to Claim 8, wherein said treatment effective dose is selected from:
(a) about 900 μ g/ agent;
(b) about 1200 μ g/ agent; With
(c) about 1800 μ g/ agent.
87. 4 to 86 each described methods according to Claim 8, wherein said albumin fusion proteins and one or more antiviral agent are co-administered.
88. 7 described methods according to Claim 8, wherein said antiviral agent is selected from:
(a) antiviral enzyme inhibitor;
(b) nucleoside analog antiviral AG14361;
(c) non-nucleoside is like thing antiviral AG14361;
(d) antisense oligonucleotide inhibitor;
(e)thiazolide；
(f) antiviral antibody;
(g) novel immunomodulator;
(h) cyclophilin inhibitor;
(i) liver protectant; With
(j) interferon enhancer.
89. 7 described methods according to Claim 8, wherein said antiviral agent is ribavirin or ribavirin analog.
90. 7 described methods according to Claim 8, wherein said albumin fusion proteins and two kinds, three kinds or four kinds of antiviral agent are co-administered.
91. according to the described method of claim 90, one of wherein said antiviral agent is ribavirin or ribavirin analog.
92. a treatment suffers from the patient's of disease, illness or infection method, wherein said method comprises:
(a) short-term,, the interferon with acceptable safety of maximal dose was used on ground to described patient; And
(b) use the described interferon of maintenance dose more for prolonged period of time to described patient.
93. according to the described method of claim 92, wherein said maximal dose is selected from:
(a) dosage identical with the standard dose of described interferon;
(b) be at least the dosage of the standard dose of described interferon;
(c) dosage higher 1 times than the standard dose of described interferon;
(d) dosage higher 2 times than the standard dose of described interferon;
(e) dosage higher 3 times than the standard dose of described interferon;
(f) dosage higher 4 times than the standard dose of described interferon;
(g) dosage higher 5 times than the standard dose of described interferon;
(d) dosage higher 6 times than the standard dose of described interferon;
(e) dosage higher 7 times than the standard dose of described interferon;
(f) dosage higher 8 times than the standard dose of described interferon;
(g) dosage higher 9 times than the standard dose of described interferon; With
(h) dosage higher 10 times than the standard dose of described interferon.
94. according to claim 92 or 93 described methods, wherein said maintenance dose is selected from:
(a) dosage identical with the standard dose of described interferon;
(b) be at least the dosage of the standard dose of described interferon;
(c) be the dosage of maximal dose 1/4 dosage;
(d) be the dosage of maximal dose 1/2 dosage;
(e) be the dosage of maximal dose 1/3 dosage;
(f) be the dosage of maximal dose 1/8 dosage;
(g) than the dosage of low 1 times of maximal dose;
(h) than the dosage of low 2 times of maximal dose;
(i) than the dosage of low 3 times of maximal dose;
(j) than the dosage of low 4 times of maximal dose;
(k) than the dosage of low 5 times of maximal dose; With
(l) than the dosage of low 6 times of maximal dose.
95., wherein use described maximal dose and described maintenance dose according to being selected from following drug dosage schedule according to each described method of claim 92 to 94:
(a) once a day;
(b) twice of every day;
(c) weekly;
(d) weekly twice;
(e) inferior on every Wendesdays;
(f) weekly;
(g) per 2 weeks once;
(h) per 3 weeks once; With
(i) per 4 weeks once.
96. according to each described method of claim 92 to 95, wherein said short-term, be selected from:
(a) week;
(b) two weeks;
(c) three weeks;
(d) all around;
(e) five weeks;
(f) six weeks;
(g) seven weeks; With
(h) eight weeks.
97., wherein saidly be selected from more over a long time according to each described method of claim 92 to 96:
(a) at least 16 weeks;
(b) at least 20 weeks;
(c) at least 24 weeks;
(d) at least 30 weeks;
(e) at least 36 weeks;
(f) at least 40 weeks;
(g) at least 46 weeks;
(h) at least 48 weeks;
(i) at least 50 weeks; With
(j) at least 60 weeks.
98. a treatment suffers from the patient's of disease, illness or infection method, wherein said method comprises:
(a) short-term,, the interferon of effective dose was used on ground to described patient with more frequent drug dosage schedule; And
(b) use described interferon with more not frequent drug dosage schedule to described patient more for prolonged period of time.
99. according to the described method of claim 98, wherein said more frequent drug dosage schedule is selected from:
(a) once a day;
(b) twice of every day;
(c) weekly;
(d) weekly twice;
(e) inferior on every Wendesdays; With
(f) per 2 weeks once.
100. according to claim 98 or 99 described methods, wherein said more not frequent drug dosage schedule is less than described more frequent drug dosage schedule and is selected from:
(a) weekly;
(b) weekly twice;
(c) per 2 weeks once;
(d) per 3 weeks once; With
(e) per 4 weeks once.
101. according to each described method of claim 98 to 100, wherein said short-term, be selected from:
(a) week;
(b) two weeks;
(c) three weeks;
(d) all around;
(e) five weeks;
(f) six weeks;
(g) seven weeks; With
(h) eight weeks.
102., wherein saidly be selected from more over a long time according to each described method of claim 98 to 101:
(a) at least 16 weeks;
(b) at least 20 weeks;
(c) at least 24 weeks;
(d) at least 30 weeks;
(e) at least 36 weeks;
(f) at least 40 weeks;
(g) at least 46 weeks;
(h) at least 48 weeks;
(i) at least 50 weeks; With
(j) at least 60 weeks.
103. a treatment suffers from the patient's of disease, illness or infection method, wherein said method comprises:
(a) short-term,, the interferon with acceptable safety of maximal dose was used on ground to described patient with more frequent drug dosage schedule; And
(b) use the described interferon of maintenance dose more for prolonged period of time to described patient with more not frequent drug dosage schedule.
104. according to the described method of claim 103, wherein said maximal dose is selected from:
(a) dosage identical with the standard dose of described interferon;
(b) be at least the dosage of the standard dose of described interferon;
(c) dosage higher 1 times than the standard dose of described interferon;
(d) dosage higher 2 times than the standard dose of described interferon;
(e) dosage higher 3 times than the standard dose of described interferon;
(f) dosage higher 4 times than the standard dose of described interferon;
(g) dosage higher 5 times than the standard dose of described interferon;
(d) dosage higher 6 times than the standard dose of described interferon;
(e) dosage higher 7 times than the standard dose of described interferon;
(f) dosage higher 8 times than the standard dose of described interferon;
(g) dosage higher 9 times than the standard dose of described interferon; With
(h) dosage higher 10 times than the standard dose of described interferon.
105. according to claim 103 or 104 described methods, wherein said maintenance dose is selected from:
(a) dosage identical with the standard dose of described interferon;
(b) be at least the dosage of the standard dose of described interferon;
(c) be the dosage of maximal dose 1/4 dosage;
(d) be the dosage of maximal dose 1/2 dosage;
(e) be the dosage of maximal dose 1/3 dosage;
(f) be the dosage of maximal dose 1/8 dosage;
(g) than the dosage of low 1 times of maximal dose;
(h) than the dosage of low 2 times of maximal dose;
(i) than the dosage of low 3 times of maximal dose;
(j) than the dosage of low 4 times of maximal dose;
(k) than the dosage of low 5 times of maximal dose; With
(l) than the dosage of low 6 times of maximal dose.
106. according to each described method of claim 103 to 105, wherein said more frequent drug dosage schedule is selected from:
(a) once a day;
(b) twice of every day;
(c) weekly;
(d) weekly twice;
(e) inferior on every Wendesdays; With
(f) per 2 weeks once.
107. according to each described method of claim 103 to 106, wherein said more not frequent drug dosage schedule is less than described more frequent drug dosage schedule and is selected from:
(a) weekly;
(b) weekly twice;
(c) per 2 weeks once;
(d) per 3 weeks once; With
(e) per 4 weeks once.
108. according to each described method of claim 103 to 107, wherein said short-term, be selected from:
(a) week;
(b) two weeks;
(c) three weeks;
(d) all around;
(e) five weeks;
(f) six weeks;
(g) seven weeks; With
(h) eight weeks.
109., wherein saidly be selected from more over a long time according to each described method of claim 103 to 108:
(a) at least 16 weeks;
(b) at least 20 weeks;
(c) at least 24 weeks;
(d) at least 30 weeks;
(e) at least 36 weeks;
(f) at least 40 weeks;
(g) at least 46 weeks;
(h) at least 48 weeks;
(i) at least 50 weeks; With
(j) at least 60 weeks.
110. according to each described method of claim 92 to 109, wherein said disease, illness or infection are selected from chronic hepatitis and infect; Hepatitis B infection; Chronic hepatitis B infects; Hepatitis C infection; Chronic hepatitis C infection; Hepatitis D infects; Chronic hepatitis D infects; Human papillomavirus infects; Herpes simplex infections; Outside condyloma acuminatum; HIV; HIV infects; Tumor; Cancer; Solid tumor; Melanoma; Malignant melanoma; Renal carcinoma; Pulmonary carcinoma; Colon cancer; Breast carcinoma; Hepatocarcinoma; Carcinoma of prostate; Bladder cancer; Gastric cancer; Sarcoma; The Kaposi sarcoma that AIDS is relevant; Lymphoma; T cell lymphoma; Cutaneous T cell lymphoma; Non_hodgkin lymphoma; The brain cancer; Glioma; Glioblastoma multiforme; Cervical atypical hyperplasia; Leukemia; Preleukemia; Myelopathy; Osteopathia; Hairy cell; Chronic granulocytic leukemia; Hematologic malignancies; Hematopathy; Multiple myeloma; Bacterial infection; Chemoproection; Thrombocytopenia; Multiple sclerosis; Pulmonary fibrosis; The macula lutea degenerative change relevant with the age; Degeneration of macula; Crohn disease; Neurological disorder; Arthritis; Rheumatoid arthritis; Ulcerative colitis; Osteoporosis, osteopenia, osteoclast generate; Fibromyalgia; Siogren's syndrome; Chronic fatigue syndrome; Heating; Hemorrhagic fever; Viral hemorrhagic fever; Hyperglycemia; Diabetes; Diabetes insipidus; Diabetes; Type 1 diabetes; Type 2 diabetes mellitus; Insulin resistant; Insulin deficit; Hyperlipemia; Hyperketonemia; Noninsulindependent diabetes (NIDDM); Insulin-dependent diabetes (IDDM); The infection that A, the B that serious acute respiratory organ syndrome (SARS) and the Center for Disease Control are identified or the C class factor cause, or its any combination.
111. according to the described method of claim 110, wherein said disease, illness or infection are hepatitis C or hepatitis B infection.
112. according to the described method of claim 111, wherein said disease or illness are that hepatitis C or HIV infect.
113. according to claim 110 or 111 described methods, wherein said hepatitis C is to be selected from following genotype:
(a) genotype 1;
(b) genotype 2; With
(c) genotype 3.
114. according to each described method of claim 92 to 113, wherein said interferon is interferon-ALPHA, interferon beta, interferon gamma, interferon ω or its fragment.
115. according to the described method of claim 114, wherein said interferon-ALPHA is selected from:
(a) Intederon Alpha-2a;
(b) Interferon Alpha-2b;
(c) interferon c;
(d) total interferon;
(c) interferon alfacon-1;
(d) interferon alfa-n1;
(e) Alferon N; With
(f) any commercially available form that obtains of interferon-ALPHA.
116. according to the described method of claim 115, wherein said commercially available obtainable interferon-ALPHA is selected from:
(a)
A；
(b)
A；
(c) Berofor interferon-ALPHA;
(d)OMNIFERON
TM；
(e)MULTIFERON
TM；
(f)
(g)
(h)
With
(i)MAXY-ALPHA
TM。
117. according to the described method of claim 114, wherein said interferon is long lasting.
118. according to the described method of claim 117, wherein said long lasting interferon is selected from:
(a) comprise and albumin or its fragment or the interferon of variant fusion or the albumin fusion proteins of its fragment or variant;
(b)
(c) interferon-' alpha '-XL;
(d)LOCTERON
TM；
(e) pegylated interferon alfa-2a;
(f) pegylated interferon alfa-2b; With
(g) the total interferon of Pegylation.
119. according to the described method of claim 118, wherein said albumin fusion proteins is by the host cell expression that comprises the albumin fusion constructs, described albumin fusion constructs is selected from:
(a) construct ID 2249;
(b) construct ID 2343;
(c) construct ID 2366;
(d) construct ID 2381;
(e) construct ID 2382;
(f) construct ID 2410;
(g) construct ID 3165;
(h) construct ID 3422;
(i) construct ID 3423;
(j) construct ID 3424;
(k) construct ID 3476;
(l) construct ID 3960;
(m) construct ID 4290;
(n) construct ID 4291;
(o) construct ID 4292;
(p) construct ID 4295; With
(q) construct ID 4296.
120. according to each described method of claim 92 to 119, wherein said interferon and one or more antiviral agent are co-administered.
121. according to the described method of claim 120, wherein said antiviral agent is selected from:
(a) ribavirin or ribavirin analog;
(b) antiviral enzyme inhibitor;
(c) nucleoside analog antiviral AG14361;
(d) non-nucleoside is like thing antiviral AG14361;
(e) antisense oligonucleotide inhibitor;
(f)thiazolide；
(g) antiviral antibody;
(h) novel immunomodulator;
(i) cyclophilin inhibitor;
(j) liver protectant; With
(k) interferon enhancer.
122. according to claim 120 or 121 described methods, wherein said antiviral agent is applied to described patient every day.
123. fragment or the shelf life of variant or the method for serum half-life of extended treatment albumin X or therapeutic X, this method comprises the step that the fragment of described human cytokines X or therapeutic X or variant is blended in albumin or its albumin fragment or variant, and comparing described albumin or its albumin fragment or variant with shelf life of the fragment of described human cytokines X that does not merge state or therapeutic X or variant or serum half-life is enough to prolong the fragment of described human cytokines X or therapeutic X or the shelf life or the serum half-life of variant.
124. nucleic acid molecules that comprises the polynucleotide sequence of the described albumin fusion proteins of coding claim 123.
125. carrier that comprises the described nucleic acid molecules of claim 124.
126. host cell that comprises the described nucleic acid molecules of claim 124.
127. method of suffering from the patient of hepatitis C infection with the albumin fusion proteins treatment of treatment effective dose, described albumin fusion proteins comprises the ripe interferon-ALPHA that merges with ripe albumin, wherein said ripe interferon-ALPHA is blended in ripe albuminised C-end, and further wherein (1) described patient is first treatment, (2) described weight in patients is at least 75kg, (3) described hepatitis C infection is a genotype 1, described treatment effective dose is extremely about 2400 μ g/ agent of about 900 μ g/ agent, and (4) are whenever biweekly with the described patient of described albumin fusion proteins administration.
128. according to the described method of claim 127, wherein said treatment effective dose is selected from:
(a) about 900 μ g/ agent;
(b) about 1200 μ g/ agent;
(c) about 1800 μ g/ agent;
(d) about 2100 μ g/ agent; With
(e) about 2400 μ g/ agent.
129. according to claim 127 or 128 described methods, wherein said albumin fusion proteins and one or more antiviral agent are co-administered.
130. according to the described method of claim 129, wherein said antiviral agent is selected from:
(a) antiviral enzyme inhibitor;
(b) nucleoside analog antiviral AG14361;
(c) non-nucleoside is like thing antiviral AG14361;
(d) antisense oligonucleotide inhibitor;
(e)thiazolide；
(f) antiviral antibody;
(g) novel immunomodulator;
(h) cyclophilin inhibitor;
(i) liver protectant; With
(j) interferon enhancer.
131. according to the described method of claim 129, wherein said antiviral agent is ribavirin or ribavirin analog.
132. according to the described method of claim 129, wherein said albumin fusion proteins and two kinds, three kinds or four kinds of antiviral agent are co-administered.
133. according to the described method of claim 132, one of wherein said antiviral agent is ribavirin or ribavirin analog.
134. method of suffering from the patient of hepatitis C infection with the albumin fusion proteins treatment of treatment effective dose, described albumin fusion proteins comprises the ripe interferon-ALPHA that merges with ripe albumin, wherein said ripe interferon-ALPHA is blended in ripe albuminised C-end, and further wherein (1) described patient is first treatment, (2) described weight in patients is at least 75kg, (3) described hepatitis C infection is a genotype 1, described treatment effective dose is extremely about 2400 μ g/ agent of about 900 μ g/ agent, and (4) are every all around once with the described patient of described albumin fusion proteins administration.
135. according to the described method of claim 134, wherein said treatment effective dose is selected from:
(a) about 900 μ g/ agent;
(b) about 1200 μ g/ agent;
(c) about 1800 μ g/ agent;
(d) about 2100 μ g/ agent; With
(e) about 2400 μ g/ agent.
136. according to claim 134 or 135 described methods, wherein said albumin fusion proteins and one or more antiviral agent are co-administered.
137. according to the described method of claim 136, wherein said antiviral agent is selected from:
(a) antiviral enzyme inhibitor;
(b) nucleoside analog antiviral AG14361;
(c) non-nucleoside is like thing antiviral AG14361;
(d) antisense oligonucleotide inhibitor;
(e)thiazolide；
(f) antiviral antibody;
(g) novel immunomodulator;
(h) cyclophilin inhibitor;
(i) liver protectant; With
(j) interferon enhancer.
138. according to the described method of claim 136, wherein said antiviral agent is ribavirin or ribavirin analog.
139. according to the described method of claim 136, wherein said albumin fusion proteins and two kinds, three kinds or four kinds of antiviral agent are co-administered.
140. according to the described method of claim 139, one of wherein said antiviral agent is ribavirin or ribavirin analog.
141. method of suffering from the patient of hepatitis C infection with the albumin fusion proteins treatment of treatment effective dose, described albumin fusion proteins comprises the ripe interferon-ALPHA that merges with ripe albumin, wherein said ripe interferon-ALPHA is blended in ripe albuminised C-end, and further wherein (1) described patient is first treatment, (2) described weight in patients is at least 75kg, (3) described hepatitis C infection is a genotype 1, described treatment effective dose is about 900 μ g/ agent to about 2400 μ g/ agent, and (4) around treatment preceding whenever biweekly, every subsequently all around once with the described patient of described albumin fusion proteins administration.
142. according to the described method of claim 141, wherein said albumin fusion proteins is applied to described patient and continues totally 24 to 48 weeks.
143. according to claim 141 or 142 described methods, wherein said treatment effective dose is selected from:
(a) about 900 μ g/ agent;
(b) about 1200 μ g/ agent;
(c) about 1800 μ g/ agent;
(d) about 2100 μ g/ agent; With
(e) about 2400 μ g/ agent.
144. according to each described method of claim 141 to 143, wherein said albumin fusion proteins and one or more antiviral agent are co-administered.
145. according to the described method of claim 144, wherein said antiviral agent is selected from:
(a) antiviral enzyme inhibitor;
(b) nucleoside analog antiviral AG14361;
(c) non-nucleoside is like thing antiviral AG14361;
(d) antisense oligonucleotide inhibitor;
(e)thiazolide；
(f) antiviral antibody;
(g) novel immunomodulator;
(h) cyclophilin inhibitor;
(i) liver protectant; With
(j) interferon enhancer.
146. according to the described method of claim 144, wherein said antiviral agent is ribavirin or ribavirin analog.
147. according to the described method of claim 144, wherein said albumin fusion proteins and two kinds, three kinds or four kinds of antiviral agent are co-administered.
148. according to the described method of claim 147, one of wherein said antiviral agent is ribavirin or ribavirin analog.
149. method of suffering from the patient of hepatitis C infection with the albumin fusion proteins treatment of treatment effective dose, described albumin fusion proteins comprises the ripe interferon-ALPHA that merges with ripe albumin, wherein said ripe interferon-ALPHA is blended in ripe albuminised C-end, and further wherein (1) described patient lives through treatment, (2) described weight in patients is at least 75kg, (3) described hepatitis C infection is a genotype 1, described treatment effective dose is extremely about 2400 μ g/ agent of about 900 μ g/ agent, and (4) are whenever biweekly with the described patient of described albumin fusion proteins administration.
150. according to the described method of claim 149, wherein said treatment effective dose is selected from:
(a) about 900 μ g/ agent;
(b) about 1200 μ g/ agent;
(c) about 1800 μ g/ agent;
(d) about 2100 μ g/ agent; With
(e) about 2400 μ g/ agent.
151. according to claim 149 or 150 described methods, wherein said albumin fusion proteins and one or more antiviral agent are co-administered.
152. according to the described method of claim 151, wherein said antiviral agent is selected from:
(a) antiviral enzyme inhibitor;
(b) nucleoside analog antiviral AG14361;
(c) non-nucleoside is like thing antiviral AG14361;
(d) antisense oligonucleotide inhibitor;
(e)thiazolide；
(f) antiviral antibody;
(g) novel immunomodulator;
(h) cyclophilin inhibitor;
(i) liver protectant; With
(j) interferon enhancer.
153. according to the described method of claim 151, wherein said antiviral agent is ribavirin or ribavirin analog.
154. according to the described method of claim 151, wherein said albumin fusion proteins and two kinds, three kinds or four kinds of antiviral agent are co-administered.
155. according to the described method of claim 154, one of wherein said antiviral agent is ribavirin or ribavirin analog.
156. method of suffering from the patient of hepatitis C infection with the albumin fusion proteins treatment of treatment effective dose, described albumin fusion proteins comprises the ripe interferon-ALPHA that merges with ripe albumin, wherein said ripe interferon-ALPHA is blended in ripe albuminised C-end, and further wherein (1) described patient lives through treatment, (2) described weight in patients is at least 75kg, (3) described hepatitis C infection is a genotype 1, described treatment effective dose is extremely about 2400 μ g/ agent of about 900 μ g/ agent, and (4) are every all around once with the described patient of described albumin fusion proteins administration.
157. according to the described method of claim 156, wherein said treatment effective dose is selected from:
(a) about 900 μ g/ agent;
(b) about 1200 μ g/ agent;
(c) about 1800 μ g/ agent;
(d) about 2100 μ g/ agent; With
(e) about 2400 μ g/ agent.
158. according to claim 156 or 157 described methods, wherein said albumin fusion proteins and one or more antiviral agent are co-administered.
159. according to the described method of claim 158, wherein said antiviral agent is selected from:
(a) antiviral enzyme inhibitor;
(b) nucleoside analog antiviral AG14361;
(c) non-nucleoside is like thing antiviral AG14361;
(d) antisense oligonucleotide inhibitor;
(e)thiazolide；
(f) antiviral antibody;
(g) novel immunomodulator;
(h) cyclophilin inhibitor;
(i) liver protectant; With
(j) interferon enhancer.
160. according to the described method of claim 158, wherein said antiviral agent is ribavirin or ribavirin analog.
161. according to the described method of claim 158, wherein said albumin fusion proteins and two kinds, three kinds or four kinds of antiviral agent are co-administered.
162. according to the described method of claim 161, one of wherein said antiviral agent is ribavirin or ribavirin analog.
163. method of suffering from the patient of hepatitis C infection with the albumin fusion proteins treatment of treatment effective dose, described albumin fusion proteins comprises the ripe interferon-ALPHA that merges with ripe albumin, wherein said ripe interferon-ALPHA is blended in ripe albuminised C-end, and further wherein (1) described patient lives through treatment, (2) described weight in patients is at least 75kg, (3) described hepatitis C infection is a genotype 1, described treatment effective dose is about 900 μ g/ agent to about 2400 μ g/ agent, and (4) around treatment preceding whenever biweekly, every subsequently all around once with the described patient of described albumin fusion proteins administration.
164. according to the described method of claim 163, wherein said albumin fusion proteins is applied to described patient and continues totally 24 to 48 weeks.
165. according to claim 163 or 164 described methods, wherein said treatment effective dose is selected from:
(a) about 900 μ g/ agent;
(b) about 1200 μ g/ agent;
(c) about 1800 μ g/ agent;
(d) about 2100 μ g/ agent; With
(e) about 2400 μ g/ agent.
166. according to each described method of claim 163 to 165, wherein said albumin fusion proteins and one or more antiviral agent are co-administered.
167. according to the described method of claim 169, wherein said antiviral agent is selected from:
(a) antiviral enzyme inhibitor;
(b) nucleoside analog antiviral AG14361;
(c) non-nucleoside is like thing antiviral AG14361;
(d) antisense oligonucleotide inhibitor;
(e)thiazolide；
(f) antiviral antibody;
(g) novel immunomodulator;
(h) cyclophilin inhibitor;
(i) liver protectant; With
(j) interferon enhancer.
168. according to the described method of claim 166, wherein said antiviral agent is ribavirin or ribavirin analog.
169. according to the described method of claim 166, wherein said albumin fusion proteins and two kinds, three kinds or four kinds of antiviral agent are co-administered.
170. according to the described method of claim 169, one of wherein said antiviral agent is ribavirin or ribavirin analog.
171. method of suffering from the patient of hepatitis C infection with the albumin fusion proteins treatment of treatment effective dose, described albumin fusion proteins comprises the ripe interferon-ALPHA that merges with ripe albumin, wherein said ripe interferon-ALPHA is blended in ripe albuminised C-end, and further wherein (1) described patient is first treatment, (3) described weight in patients is at least 75kg, (4) described hepatitis C infection is genotype 2 or 3, (5) described treatment effective dose is extremely about 2400 μ g/ agent of about 900 μ g/ agent, and (4) are whenever biweekly with the described patient of described albumin fusion proteins administration.
172. according to the described method of claim 171, wherein said treatment effective dose is selected from:
(a) about 900 μ g/ agent;
(b) about 1200 μ g/ agent;
(c) about 1800 μ g/ agent;
(d) about 2100 μ g/ agent; With
(e) about 2400 μ g/ agent.
173. according to claim 171 or 172 described methods, wherein said albumin fusion proteins and one or more antiviral agent are co-administered.
174. according to the described method of claim 173, wherein said antiviral agent is selected from:
(a) antiviral enzyme inhibitor;
(b) nucleoside analog antiviral AG14361;
(c) non-nucleoside is like thing antiviral AG14361;
(d) antisense oligonucleotide inhibitor;
(e)thiazolide；
(f) antiviral antibody;
(g) novel immunomodulator;
(h) cyclophilin inhibitor;
(i) liver protectant; With
(j) interferon enhancer.
175. according to the described method of claim 173, wherein said antiviral agent is ribavirin or ribavirin analog.
176. according to the described method of claim 173, wherein said albumin fusion proteins and two kinds, three kinds or four kinds of antiviral agent are co-administered.
177. according to the described method of claim 176, one of wherein said antiviral agent is ribavirin or ribavirin analog.
178. method of suffering from the patient of hepatitis C infection with the albumin fusion proteins treatment of treatment effective dose, described albumin fusion proteins comprises the ripe interferon-ALPHA that merges with ripe albumin, wherein said ripe interferon-ALPHA is blended in ripe albuminised C-end, and further wherein (1) described patient is first treatment, (3) described weight in patients is at least 75kg, (4) described hepatitis C infection is genotype 2 or 3, (5) described treatment effective dose is extremely about 2400 μ g/ agent of about 900 μ g/ agent, and (6) are every all around once with the described patient of described albumin fusion proteins administration.
179. according to the described method of claim 178, wherein said treatment effective dose is selected from:
(a) about 900 μ g/ agent;
(b) about 1200 μ g/ agent;
(c) about 1800 μ g/ agent;
(d) about 2100 μ g/ agent; With
(e) about 2400 μ g/ agent.
180. according to claim 178 or 179 described methods, wherein said albumin fusion proteins and one or more antiviral agent are co-administered.
181. according to the described method of claim 180, wherein said antiviral agent is selected from:
(a) antiviral enzyme inhibitor;
(b) nucleoside analog antiviral AG14361;
(c) non-nucleoside is like thing antiviral AG14361;
(d) antisense oligonucleotide inhibitor;
(e)thiazolide；
(f) antiviral antibody;
(g) novel immunomodulator;
(h) cyclophilin inhibitor;
(i) liver protectant; With
(j) interferon enhancer.
182. according to the described method of claim 180, wherein said antiviral agent is ribavirin or ribavirin analog.
183. according to the described method of claim 180, wherein said albumin fusion proteins and two kinds, three kinds or four kinds of antiviral agent are co-administered.
184. according to the described method of claim 183, one of wherein said antiviral agent is ribavirin or ribavirin analog.
185. method of suffering from the patient of hepatitis C infection with the albumin fusion proteins treatment of treatment effective dose, described albumin fusion proteins comprises the ripe interferon-ALPHA that merges with ripe albumin, wherein said ripe interferon-ALPHA is blended in ripe albuminised C-end, and further wherein (1) described patient is first treatment, (3) described weight in patients is at least 75kg, (4) described hepatitis C infection is genotype 2 or 3, (5) described treatment effective dose is about 900 μ g/ agent to about 2400 μ g/ agent, and (6) around treatment preceding whenever biweekly, every subsequently all around once with the described patient of described albumin fusion proteins administration.
186. according to the described method of claim 185, wherein said albumin fusion proteins is applied to described patient and continues totally 24 to 48 weeks.
187. according to claim 185 or 186 described methods, wherein said treatment effective dose is selected from:
(a) about 900 μ g/ agent;
(b) about 1200 μ g/ agent;
(c) about 1800 μ g/ agent;
(d) about 2100 μ g/ agent; With
(e) about 2400 μ g/ agent.
188. according to each described method of claim 185 to 187, wherein said albumin fusion proteins and one or more antiviral agent are co-administered.
189. according to the described method of claim 188, wherein said antiviral agent is selected from:
(a) antiviral enzyme inhibitor;
(b) nucleoside analog antiviral AG14361;
(c) non-nucleoside is like thing antiviral AG14361;
(d) antisense oligonucleotide inhibitor;
(e)thiazolide；
(f) antiviral antibody;
(g) novel immunomodulator;
(h) cyclophilin inhibitor;
(i) liver protectant; With
(j) interferon enhancer.
190. according to the described method of claim 188, wherein said antiviral agent is ribavirin or ribavirin analog.
191. according to the described method of claim 188, wherein said albumin fusion proteins and two kinds, three kinds or four kinds of antiviral agent are co-administered.
192. according to the described method of claim 191, one of wherein said antiviral agent is ribavirin or ribavirin analog.
193. method of suffering from the patient of hepatitis C infection with the albumin fusion proteins treatment of treatment effective dose, described albumin fusion proteins comprises the ripe interferon-ALPHA that merges with ripe albumin, wherein said ripe interferon-ALPHA is blended in ripe albuminised C-end, and further wherein (1) described patient lives through treatment, (3) described weight in patients is at least 75kg, (4) described hepatitis C infection is genotype 2 or 3, (5) described treatment effective dose is extremely about 2400 μ g/ agent of about 900 μ g/ agent, and (6) are whenever biweekly with the described patient of described albumin fusion proteins administration.
194. according to the described method of claim 193, wherein said treatment effective dose is selected from:
(a) about 900 μ g/ agent;
(b) about 1200 μ g/ agent;
(c) about 1800 μ g/ agent;
(d) about 2100 μ g/ agent; With
(e) about 2400 μ g/ agent.
195. according to claim 193 or 194 described methods, wherein said albumin fusion proteins and one or more antiviral agent are co-administered.
196. according to the described method of claim 195, wherein said antiviral agent is selected from:
(a) antiviral enzyme inhibitor;
(b) nucleoside analog antiviral AG14361;
(c) non-nucleoside is like thing antiviral AG14361;
(d) antisense oligonucleotide inhibitor;
(e)thiazolide；
(f) antiviral antibody;
(g) novel immunomodulator;
(h) cyclophilin inhibitor;
(i) liver protectant; With
(j) interferon enhancer.
197. according to the described method of claim 195, wherein said antiviral agent is ribavirin or ribavirin analog.
198. according to the described method of claim 195, wherein said albumin fusion proteins and two kinds, three kinds or four kinds of antiviral agent are co-administered.
199. according to the described method of claim 198, one of wherein said antiviral agent is ribavirin or ribavirin analog.
200. method of suffering from the patient of hepatitis C infection with the albumin fusion proteins treatment of treatment effective dose, described albumin fusion proteins comprises the ripe interferon-ALPHA that merges with ripe albumin, wherein said ripe interferon-ALPHA is blended in ripe albuminised C-end, and further wherein (1) described patient lives through treatment, (3) described weight in patients is at least 75kg, (4) described hepatitis C infection is genotype 2 or 3, (5) described treatment effective dose is extremely about 2400 μ g/ agent of about 900 μ g/ agent, and (6) are every all around once with the described patient of described albumin fusion proteins administration.
201. according to the described method of claim 200, wherein said treatment effective dose is selected from:
(a) about 900 μ g/ agent;
(b) about 1200 μ g/ agent;
(c) about 1800 μ g/ agent;
(d) about 2100 μ g/ agent; With
(e) about 2400 μ g/ agent.
202. according to claim 200 or 201 described methods, wherein said albumin fusion proteins and one or more antiviral agent are co-administered.
203. according to the described method of claim 202, wherein said antiviral agent is selected from:
(a) antiviral enzyme inhibitor;
(b) nucleoside analog antiviral AG14361;
(c) non-nucleoside is like thing antiviral AG14361;
(d) antisense oligonucleotide inhibitor;
(e)thiazolide；
(f) antiviral antibody;
(g) novel immunomodulator;
(h) cyclophilin inhibitor;
(i) liver protectant; With
(j) interferon enhancer.
204. according to the described method of claim 202, wherein said antiviral agent is ribavirin or ribavirin analog.
205. according to the described method of claim 202, wherein said albumin fusion proteins and two kinds, three kinds or four kinds of antiviral agent are co-administered.
206. according to the described method of claim 205, one of wherein said antiviral agent is ribavirin or ribavirin analog.
207. method of suffering from the patient of hepatitis C infection with the albumin fusion proteins treatment of treatment effective dose, described albumin fusion proteins comprises the ripe interferon-ALPHA that merges with ripe albumin, wherein said ripe interferon-ALPHA is blended in ripe albuminised C-end, and further wherein (1) described patient lives through treatment, (3) described weight in patients is at least 75kg, (4) described hepatitis C infection is genotype 2 or 3, (5) described treatment effective dose is about 900 μ g/ agent to about 2400 μ g/ agent, and (6) around treatment preceding whenever biweekly, every subsequently all around once with the described patient of described albumin fusion proteins administration.
208. according to the described method of claim 207, wherein said albumin fusion proteins is applied to described patient and continues totally 24 to 48 weeks.
209. according to claim 207 or 208 described methods, wherein said treatment effective dose is selected from:
(a) about 900 μ g/ agent;
(b) about 1200 μ g/ agent;
(c) about 1800 μ g/ agent;
(d) about 2100 μ g/ agent; With
(e) about 2400 μ g/ agent.
210. according to each described method of claim 207 to 209, wherein said albumin fusion proteins and one or more antiviral agent are co-administered.
211. according to the described method of claim 210, wherein said antiviral agent is selected from:
(a) antiviral enzyme inhibitor;
(b) nucleoside analog antiviral AG14361;
(c) non-nucleoside is like thing antiviral AG14361;
(d) antisense oligonucleotide inhibitor;
(e)thiazolide；
(f) antiviral antibody;
(g) novel immunomodulator;
(h) cyclophilin inhibitor;
(i) liver protectant; With
(j) interferon enhancer.
212. according to the described method of claim 210, wherein said antiviral agent is ribavirin or ribavirin analog.
213. according to the described method of claim 210, wherein said albumin fusion proteins and two kinds, three kinds or four kinds of antiviral agent are co-administered.
214. according to the described method of claim 213, one of wherein said antiviral agent is ribavirin or ribavirin analog.
215. method of suffering from the patient of hepatitis C infection with the albumin fusion proteins treatment of treatment effective dose, described albumin fusion proteins comprises the ripe Interferon Alpha-2b that merges with ripe albumin, wherein said ripe Interferon Alpha-2b is blended in ripe albuminised C-end, and further wherein (1) described patient is first treatment, (2) described hepatitis C infection is a genotype 1, (3) described treatment effective dose is extremely about 1800 μ g/ agent of about 1200 μ g/ agent, and (4) are every all around once with the described patient of described albumin fusion proteins administration and at the described albumin fusion proteins of using back second all administration extra dose.
216. according to the described method of claim 215, wherein said albumin fusion proteins is applied to described patient and continues totally 24 to 48 weeks.
217. according to claim 215 or 216 described methods, wherein said albumin fusion proteins and one or more antiviral agent are co-administered.
218. according to the described method of claim 217, wherein said antiviral agent is selected from:
(a) antiviral enzyme inhibitor;
(b) nucleoside analog antiviral AG14361;
(c) non-nucleoside is like thing antiviral AG14361;
(d) antisense oligonucleotide inhibitor;
(e)thiazolide；
(f) antiviral antibody;
(g) novel immunomodulator;
(h) cyclophilin inhibitor;
(i) liver protectant; With
(j) interferon enhancer.
219. according to the described method of claim 217, wherein said antiviral agent is ribavirin or ribavirin analog.
220. according to the described method of claim 217, wherein said albumin fusion proteins and two kinds, three kinds or four kinds of antiviral agent are co-administered.
221. according to the described method of claim 220, one of wherein said antiviral agent is ribavirin or ribavirin analog.
222. according to each described method of claim 215 to 221, wherein said weight in patients is at least 75kg.
Applications Claiming Priority (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US84434906P | 2006-09-14 | 2006-09-14 | |
| US60/844,349 | 2006-09-14 | ||
| US60/858,410 | 2006-11-13 | ||
| US60/902,039 | 2007-02-20 | ||
| US60/942,647 | 2007-06-07 | ||
| US60/960,039 | 2007-09-12 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101557817A true CN101557817A (en) | 2009-10-14 |
Family
ID=41175575
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2007800423220A Pending CN101557817A (en) | 2006-09-14 | 2007-09-13 | Albumin fusion proteins |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101557817A (en) |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103376280A (en) * | 2012-04-20 | 2013-10-30 | 佳佑企业有限公司 | Method and apparatus for detecting glycated hemoglobin |
| CN110357968A (en) * | 2018-04-08 | 2019-10-22 | 颜浩为 | Antineoplastic amalgamation protein and its preparation method and application |
| CN111836646A (en) * | 2017-08-15 | 2020-10-27 | 儿童医学中心公司 | APOM-FC fusion protein and application thereof |
| CN112654635A (en) * | 2018-05-14 | 2021-04-13 | 狼人治疗公司 | Activatable cytokine polypeptides and methods of use thereof |
| CN113286817A (en) * | 2018-09-25 | 2021-08-20 | 哈普恩治疗公司 | DLL3 binding proteins and methods of use |
| CN113325179A (en) * | 2021-04-14 | 2021-08-31 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Immunochromatographic test strip based on Au @ Pt enzyme and preparation method thereof |
| CN113831414A (en) * | 2020-06-24 | 2021-12-24 | 上海中鹰科技实业有限公司 | Porcine circovirus type 2b Capsid-Fc fusion protein, and preparation method, gene, construction method and application thereof |
| CN114144194A (en) * | 2019-04-19 | 2022-03-04 | 斯纳凯恩制药私人有限责任公司 | Fusion protein containing IL13 |
| CN114149508A (en) * | 2021-11-25 | 2022-03-08 | 苏州普乐康医药科技有限公司 | Fusion protein combined with CD40L and application thereof |
| US12084518B2 (en) | 2015-05-21 | 2024-09-10 | Harpoon Therapeutics, Inc. | Trispecific binding proteins and methods of use |
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- 2007-09-13 CN CNA2007800423220A patent/CN101557817A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103376280A (en) * | 2012-04-20 | 2013-10-30 | 佳佑企业有限公司 | Method and apparatus for detecting glycated hemoglobin |
| US12084518B2 (en) | 2015-05-21 | 2024-09-10 | Harpoon Therapeutics, Inc. | Trispecific binding proteins and methods of use |
| CN111836646A (en) * | 2017-08-15 | 2020-10-27 | 儿童医学中心公司 | APOM-FC fusion protein and application thereof |
| CN110357968B (en) * | 2018-04-08 | 2023-08-25 | 吉林省汇融生物科技有限公司 | Anti-tumor fusion protein and preparation method and application thereof |
| CN110357968A (en) * | 2018-04-08 | 2019-10-22 | 颜浩为 | Antineoplastic amalgamation protein and its preparation method and application |
| CN112654635A (en) * | 2018-05-14 | 2021-04-13 | 狼人治疗公司 | Activatable cytokine polypeptides and methods of use thereof |
| CN113286817A (en) * | 2018-09-25 | 2021-08-20 | 哈普恩治疗公司 | DLL3 binding proteins and methods of use |
| CN114144194A (en) * | 2019-04-19 | 2022-03-04 | 斯纳凯恩制药私人有限责任公司 | Fusion protein containing IL13 |
| CN113831414A (en) * | 2020-06-24 | 2021-12-24 | 上海中鹰科技实业有限公司 | Porcine circovirus type 2b Capsid-Fc fusion protein, and preparation method, gene, construction method and application thereof |
| CN113831414B (en) * | 2020-06-24 | 2023-08-01 | 上海中鹰科技实业有限公司 | Porcine circovirus 2b type Capid-Fc fusion protein, preparation method, gene and construction method and application thereof |
| CN113325179B (en) * | 2021-04-14 | 2024-02-27 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Immunochromatography test strip based on Au@Pt enzyme and preparation method thereof |
| CN113325179A (en) * | 2021-04-14 | 2021-08-31 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Immunochromatographic test strip based on Au @ Pt enzyme and preparation method thereof |
| CN114149508A (en) * | 2021-11-25 | 2022-03-08 | 苏州普乐康医药科技有限公司 | Fusion protein combined with CD40L and application thereof |
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