CN101287750A - Albumin fusion proteins - Google Patents

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

Albumin fusion proteins

The introduction of the sequence table in the CD

The application relates to " sequence table " hereinafter listed, and it provides on three the identical CDs (CD-R) that indicate " copy 1 ", " copy 2 " and " copy 3 " as electronic file.Each all contains file " PF617PCT Sequence Listing.txt " (1,192,036 byte is built on July 28th, 2006) these CDs, with its complete being incorporated herein by reference.

Background technology

The present invention relates generally to the therapeutic protein (including but not limited at least a polypeptide, antibody, peptide or its fragment and variant) with white protein or albuminised fragment or variant fusion.Polynucleotide, therapeutic albumin fusion proteins, composition, pharmaceutical composition, preparation and the test kit of coding therapeutic albumin fusion proteins contained in the present invention.The polynucleotide transformed host cells through coding therapeutic albumin fusion proteins is also contained in the present invention, and uses these polynucleotide and/or host cell to prepare the method for albumin fusion proteins of the present invention.

Human serum albumin (HSA or HA), its mature form has 585 amino acid whose a kind of protein (shown in table 1 (SEQ ID NO:1)), and the serum osmotic pressure of significant proportion is made of it, but also works as the carrier of endogenous and exogenous ligand.At present, the HA of clinical use extracts from human blood and produces.It is open in EP 330 451 and EP 361991 to produce reorganization HA (rHA) in microorganism.

The therapeutic protein that native state or reorganization generate such as Interferon, rabbit and tethelin, normally presents the unstable molecule of of short duration preservation period, when particularly preparing in the aqueous solution.When preparation was used to use, the unstable of these molecules pointed out a lot of molecules must freeze-drying and refrigerate when storage always, thereby makes these molecules be difficult to transportation and/or storage.Must preserve beyond hospital environment and the branch timing when pharmaceutical formulations, it is sharp-pointed especially that storage problem just seems.

The rare proposition of practical solution for the storage problem of labile protein matter molecule.Therefore, need protein therapeutic molecular preparation stable, persistent, easy distribution, preferably need the simple formulations of inferior limit storage back operation.

After using in the body, the therapeutic protein that native state or reorganization generate such as Interferon, rabbit and tethelin, thereby presents of short duration plasma stability owing to removing rapidly from blood flow.Therefore, the result of treatment that provides of these protein also is a short-term.Therefore, in order to keep their expectation cylinder therapeutic effect, these protein are removed prompting rapidly from blood must be with higher frequency or high dosage administering therapeutic molecule more.Yet, increase reaction, side effect and toxicity increase that the proteinic dosage regimen of administering therapeutic usually causes patient's injection site.Similarly, also usually cause patient's toxicity and side effect to increase with high dosage administering therapeutic protein more.

The practical solution of the minority of the plasma stability of the raising therapeutic molecules of having proposed comprises chemically conjugatedly, has brought limited benefited for the patient.Usually, in most cases, the therapeutic molecules of these chemically modifieds is still used with frequent dosage regimen, still has serious injection site reaction, side effect and toxicity in the patient.Therefore, need the therapeutic molecules of stable form, it can keep also can using by lower frequency than the higher plasma stability of therapeutic protein of independent native state or reorganization generation in vivo, thereby reduces the potential side effect to the patient.

Summary of the invention

The albumin fusion proteins that comprises the therapeutic protein (as polypeptide, antibody or peptide or its fragment or variant) that merges with white protein or albuminised fragment (part) or variant is contained in the present invention.The polynucleotide of the nucleic acid molecule that comprises the therapeutic protein (as polypeptide, antibody or peptide or its fragment or variant) that coding and white protein or albuminised fragment (part) or variant merge or the polynucleotide of being made up of the coding and the nucleic acid molecule of the therapeutic protein (as polypeptide, antibody or peptide or its fragment or variant) of white protein or albuminised fragment (part) or variant fusion are also contained in the present invention.The polynucleotide that the present invention is also contained the polynucleotide that comprise the following proteinic nucleic acid molecule of encoding or is made up of the following proteinic nucleic acid molecule of coding: this protein comprises therapeutic protein with white protein or albuminised fragment (part) or variant fusion (as polypeptide, antibody, or peptide or its fragment or variant), this white protein or albuminised fragment (part) or variant are enough to the proteinic preservation period of extended treatment, with its not the fusion state compare the plasma stability that improves therapeutic protein, and/or in external and/or therapeutic protein and/or its activity of (or in pharmaceutical composition) in the stabilizing solution in vivo.The albumin fusion proteins by polynucleotide encoding of the present invention is also contained in the present invention, and through polynucleotide transformed host cells of the present invention, and use these polynucleotide of the present invention and/or host cell to prepare the method for albumin fusion proteins of the present invention.

Of the present invention one preferred aspect, albumin fusion proteins include but not limited to described in the table 2 those protein and the coding these type of proteinic polynucleotide.

The present invention is also contained and is comprised the pharmaceutical formulations that albumin fusion proteins of the present invention and pharmacopedics can be accepted diluent or carrier.These preparations can be contained in test kit or the container.This test kit or container can be packed with the specification sheets about the prolongation preservation period of this therapeutic protein.These preparations are used among the patient and treat, prevent, improve or diagnose the illness or the method for disease symptoms, comprise the step of the patient being used pharmaceutical formulations, and wherein patient's preferred mammal is most preferably human.

In other embodiments, disease or disorderly method are contained prevention, treat or improve in the present invention.In preferred embodiments, listed disease or disorderly method in treatment table 1 " preferred indication: the Y " row contained in the present invention, comprise with effective treatment, prevention or improve disease or disorderly amount to this treatment of needs, prevention or the patient who improves use albumin fusion proteins of the present invention, and this albumin fusion proteins comprises and table 1 " therapeutic protein: X " row (being arranged in same delegation with disease to be treated or disorder listed in table 1 " preferred indication: the Y " row) therapeutic protein or its part that disclosed therapeutic protein (or its fragment or variant) is corresponding.

In one embodiment, the albumin fusion proteins described in table 1 or the table 2 has increased shelf-life.

In another embodiment, not merge therapeutic molecules than corresponding described in the table 1 more stable for the albumin fusion proteins described in table 1 or the table 2.

The present invention also comprises the transgenic organism that comprises nucleic acid molecule of the present invention (include but not limited to table 1 and table 2 described in polynucleotide) after the modification, preferably modifies the transgenic organism that albumin fusion proteins of the present invention is expressed in the back.

The accompanying drawing summary

Figure 1A-D has shown the aminoacid sequence (SEQ ID NO:1) and its polynucleotide (SEQ ID NO:2) of coding of the albuminised mature form of people.The Nucleotide 1-1755 coding albuminised mature form of people (SEQ ID NO:1) of SEQ ID NO:2.

Fig. 2 has shown the restriction map of pPPC0005 cloning vector ATCC preservation thing PTA-3278.

Fig. 3 has shown the restriction map (people such as Sleep, BioTechnology 8:42,1990) of pSAC35 yeast saccharomyces cerevisiae expression vector.

Fig. 4 has compared by the IFN albumin fusion proteins (CID 3165 protein) of CID 3165 codings and the antiproliferative activity of IFNa (rIFNa) to the Hs294T melanoma cells of recombinating.Cell is cultivated in the substratum that contains different concns CID 3165 protein or rIFNa, and after cultivating 3 days, mixed the propagation situation of measuring by BrdU.CID 3165 protein on cell proliferation when concentration is higher than 10ng/ml has caused measurable restraining effect, has reached 50% and suppress when about 200ng/ml.(■)=and CID 3165 protein, (◆)=rIFNa.

The various extent of dilution that Fig. 5 has shown the IFNa albumin fusion proteins in ISRE-SEAP/293F report cell to the active influence of SEAP.Tested in a kind of prepared product (◆) of albuminised upstream fusion IFNa and two kinds of different prepared products (●) and (◆) of merging IFNa in albuminised downstream.

Fig. 6 has shown by the influence (seeing embodiment 76) to OAS (p41) mRNA level in treated monkey of the time of the coded IFNa albumin fusion proteins of the DNA that comprises in the construction 2249 (CID 2249 protein) and dosage.Each time point: article one is the vehicle contrast, second is intravenous injection in first day 30 μ g/kg CID 2249 protein, article three, be subcutaneous injection in first day 30 μ g/kg CID 2249 protein, article four, be subcutaneous injection in first day 300 μ g/kg CID 2249 protein, the 5th is subcutaneous injection in first and third, five day 40 μ g/kg reorganization IFNa.

Fig. 7 has shown the dosage-response relation (seeing embodiment 78 and 79) that in NPR-A/293F report cell activation cGMP is formed by the coded BNP albumin fusion proteins of the DNA that comprises in construction CID 3691 and 3618 (CID 3691 and 3618 protein).Two kinds of different prepared products () and (●) having tested BNP peptide (■) and merged BNP in albuminised upstream.

Fig. 8 has shown the influence (seeing embodiment 78) to mean arterial pressure in spontaneous hypertensive rat of BNP albumin fusion proteins.Vehicle (), BNP peptide (●) or BNP albumin fusion proteins (zero) are delivered by the tail vein injection.Systolic pressure and diastolic pressure carry out record by cover tail method (cuff-tail).

Fig. 9 has shown the blood plasma cGMP level (see embodiment 78) of male C57/BL6 mouse behind intravenous injection reorganization BNP peptide (●) or BNP albumin fusion proteins (zero) in 11-12 age in week.Several time points collection afterbody blood after intravenous injection prepare blood plasma and measure the cGMP level.

Figure 10 has shown BNP peptide and the dosage-response relation (seeing embodiment 80) that in NPR-A/293F report cell activation cGMP is formed by the coded BNP albumin fusion proteins of the DNA that comprises in construction CID 3796 and 3959.Test BNP peptide (■) and merged two kinds of different prepared products () of BNP in albuminised downstream and (◇).

Figure 11 A has shown have or do not have neutral lyase and handle under 24 hours the condition, BNP and ANP peptide in NPR-A/293F report cell to activating dosage-response relation (seeing embodiment 81) that cGMP forms.

Figure 11 B has shown after handling 20 minutes, 1 hour or 24 hours with neutral lyase or contrast MES damping fluid, dosage-response relation (seeing embodiment 81) that the ANP peptide forms activation cGMP in NPR-A/293F report cell.

Figure 11 C shown after handling 20 minutes, 1 hour or 24 hours with neutral lyase or contrast MES damping fluid, by the DNA that comprises among the construction CID 3484 ANP albumin fusion proteins coded, merge ANP in albuminised upstream in NPR-A/293F report cell to activating dosage-response relation (seeing embodiment 81) that cGMP forms.

Figure 11 D has shown the per-cent of complete natriuretic peptide (natriureticpeptide) after handling the specified time with neutral lyase.Tested two kinds of peptides of ANP and BNP and merge BNP (CID 3809) in albuminised upstream and merge two kinds of albumin fusion proteins (seeing embodiment 81) of ANP (CID 3484) in albuminised upstream through three glycine.

Figure 12 shown infected chronic hepatitis C virogene type 1 and before do not responded at least a PEGization interferon alpha associating ribavirin (ribavirin) treatment plan (PEG-RBV) patient's (not answering the person) in reduction with HSA-IFN α 2b associating ribavirin therapy 0-24 HCV RNA titre after week, change (log by intermediate value HCV RNA ₁₀IU/ml) measure.

Figure 13 A and B have shown that using 5mg/kg IV in normal, healthy pig (n=4-6 head/group) injects the influence of (bolus) HSA-BNP (construction ID#3959) back to blood plasma and urine cGMP level.Asterisk indicates the cGMP level with respect to vectorial significant difference (p＜0.05).

Figure 14 A has shown and has used the influence that intravenous push 2mg/kg or 6mg/kg HSA-BNP (construction ID#3959) change diastasis diameter (end-diastolicdiameter) in the experimental model in heart failure of pig (n=10 head/group).Heart failure is brought out in pig by ventricular pacemaking (ventricular pacing).The diastasis diameter is measured by ultrasonic cardiography.Indicated noticeable change (p＜0.05) (Fen Biewei ﹠amp with respect to vehicle or baseline; And #).

Figure 14 B has shown and has used intravenous push 2mg/kg or 6mg/kg HSA-BNP (construction ID#3959) to shortening the influence of mark (fractionalshortening) in the experimental model in heart failure of pig (n=10 head/group).Heart failure is brought out in pig by ventricular pacemaking.Indicated noticeable change (p＜0.05) (Fen Biewei ﹠amp with respect to vehicle or baseline; And #).

Figure 15 A-H has shown the haemodynamic effect of using single intravenous push 0.5mg/kg or 5mg/kgHSA-BNP (construction ID#3959) in the normal dogs model.Cardiac output (CO), mean arterial pressure (MAP), pulmonary capillary wedge pressure (PCWP) and Ppa pulmonary artery pressure (PAP) in the normal hybrid dog (mongrel) (n=8 only/group) of anesthesia, behind preceding baseline of intravenous push 0.5mg/kg or 5mg/kg HSA-BNP (construction ID#3959) and infusion, were measured in 30,60,90,150,210 and 270 minutes.Asterisk has indicated the statistics noticeable change (p＜0.05) with respect to baseline.

Figure 16 A-H has shown the kidney effect of using single intravenous push 0.5mg/kg or 5mg/kgHSA-BNP (construction ID#3959) in the normal dogs model.Urine stream (speed/30 minute collecting amount), sodium excretion, kidney blood flow and glomerular filtration rate(GFR (GFR) in the normal hybrid dog (n=8 only/group) of anesthesia, behind preceding baseline of intravenous push 0.5mg/kg or 5mg/kg HSA-BNP (construction ID#3959) and infusion, were measured in 30,60,90,150,210 and 270 minutes.Asterisk has indicated the statistics noticeable change (p＜0.05) with respect to baseline.

Figure 17 A-F has shown the hormone effect of using single intravenous push 0.5mg/kg or 5mg/kgHSA-BNP (construction ID#3959) in the normal dogs model.Plasma aldosterone, feritin and Angiotensin II level in the normal hybrid dog (n=8 only/group) of anesthesia, behind preceding baseline of intravenous push 0.5mg/kg or 5mg/kg HSA-BNP (construction ID#3959) and infusion, were measured in 30,60,90,150,210 and 270 minutes.Asterisk has indicated the statistics noticeable change (p＜0.05) with respect to baseline.

Figure 18 A-C has shown that single intravenous push 5mg/kg HSA-BNP (construction ID#3959) is to the influence of systolic pressure and mean arterial pressure in normal, healthy, clear-headed Bi Erge sleuth (beagle), Data Sciences International radiotelemetry projector has been implanted in operation in the animal, and it has the ability of collecting SAP, heart rate and ECG data.Presented systolic pressure with respect to the difference (Figure 18 B) of the variation (Figure 18 A) of baseline, average systolic and mean arterial pressure with respect to the infusion of the variation (Figure 18 C) of baseline after 48 hours continuous data recordings.Asterisk has indicated the statistically-significant difference (p＜0.05) of 5mg/kgHSA-BNP (construction ID#3959) than vectorial baseline adjustment average (baseline-adjustedvalue).

Figure 19 A and B have shown that injecting 0.02mg/kg in normal, healthy, clear-headed Bi Erge sleuth medium sized vein does not merge BNP peptide and subcutaneous injection 10mg/kg HSA-BNP (construction ID#3959) comparison to the influence of systemic blood pressure, Data SciencesInternational radiotelemetry projector has been implanted in operation in the animal, and it has the ability of collecting SAP, heart rate and ECG data.Presented and used BNP (Figure 19 A) and HSA-BNP (construction ID#3959) post shrinkage and press 48 hours continuous data recordings with respect to the variation of baseline.Asterisk has indicated the statistically-significant difference (p＜0.05) of 5mg/kgHSA-BNP (construction ID#3959) than vectorial baseline adjustment average.

Detailed Description Of The Invention

Definition

It is to run through employed some term of this specification for the ease of understanding that following definition is provided.

When being used for this paper, " polynucleotides " refer to have the nucleic acid molecules of the nucleotide sequence of the following fusion of coding, this fusion comprise at least one albumin (or its fragment or variant) molecule of being connected with at least one therapeutic protein X (or its fragment or variant) with identical reading frame or consisting of; The nucleic acid molecules of nucleotide sequence with the following fusion of coding, this fusion comprise the amino acid sequence of SEQ ID NO:Y (as described in table 2 the 6th row) or its fragment or variant or consisting of; Have comprise sequence shown in the SEQ ID NO:X or consisting of the nucleic acid molecules of nucleotide sequence; The nucleic acid molecules of nucleotide sequence with the following fusion of coding, this fusion comprise SEQ ID NO:Z amino acid sequence or consisting of; The nucleic acid molecules of the nucleotide sequence of the albumin fusion proteins of the present invention that having encodes generates described in table 2 or embodiment; Nucleic acid molecules with nucleotide sequence of code book invention therapeutic albumin fusion proteins; The nucleic acid molecules that has in the table 2 the contained nucleotide sequence of the albumin fusion construct described; The nucleic acid molecules that perhaps has the contained nucleotide sequence of the albumin fusion construct (as described in Table 3) that is preserved in ATCC.

When being used for this paper, " albumin fusion construct " refer to comprise at least one molecule of coding that is connected with at least one section polynucleotides of the therapeutic protein (or its fragment or variant) of at least one molecule of encoding with identical reading frame albumin (or its fragment or variant) polynucleotides or consisting of nucleic acid molecules; Comprise at least one molecule of coding that is connected with at least one section polynucleotides of the therapeutic protein that described in table 2 or embodiment, produces (or its fragment or variant) of at least one molecule of coding with identical reading frame albumin (or its fragment or variant) polynucleotides or consisting of nucleic acid molecules; Perhaps comprise at least one molecule of coding that is connected with at least one section polynucleotides of therapeutic protein (or its fragment or variant) of at least one molecule of coding with identical reading frame albumin (or its fragment or variant) polynucleotides or consisting of nucleic acid molecules, it also comprises for example one or more following elements: (1) functional autonomously replicationg vector (including but not limited to shuttle vector, expression vector, integration vector and/or dubbing system), (2) transcription initiation region (promoter region for example, such as for example can adjusting or inducible promoter, constitutive promoter), (3) transcription termination region, (4) targeting sequencing, and (5) selected marker. The polynucleotides of coding therapeutic protein and albumin protein, in case become the part of albumin fusion construct, each part can be described as " partly (portion) ", " district (territory) (region) " or " module (moiety) " of this albumin fusion construct so.

The present invention relates generally to the polynucleotides of coding albumin fusion proteins; Albumin fusion proteins; And the method for the treatment of, prevent or improve disease or disorder with the polynucleotides of albumin fusion proteins or coding albumin fusion proteins. When being used for this paper, " albumin fusion proteins " refers to by at least one albumin (or its fragment or variant) molecule and at least one therapeutic protein (or its fragment or variant) molecule are merged the protein that forms. Albumin fusion proteins of the present invention comprises at least one fragment of therapeutic protein or variant and human serum albumin's at least one fragment or variant, they merge by gene/heredity and link to each other (be that albumin fusion proteins produces by translating following nucleic acid, the polynucleotides of wherein encode complete or part therapeutic protein with identical reading frame with encode complete or the albuminised polynucleotides of part are connected). Therapeutic protein and albumin protein, in case become the part of albumin fusion proteins, each can be described as " part ", " district (territory) " or " module " (for example " therapeutic protein part " or " albumin protein part ") of this albumin fusion proteins. In highly preferred embodiment, albumin fusion proteins of the present invention comprises at least one molecule of therapeutic protein X or its fragment or variant (including but not limited to the mature form of therapeutic protein X) and at least one molecule of albumin or its fragment or variant (including but not limited to albuminised mature form).

In another preferred embodiment, albumin fusion proteins of the present invention is by host cell processing and in the culture medium around being secreted into. The processing to newborn albumin fusion proteins that occurs at the host's who be used for to express secretory pathway can include but not limited to that signal peptide cutting, disulfide bond form, the glycosylation that is connected with O-such as for example N-is connected in correct interpolation folding, carbohydrate with processing), the specific proteins hydrolysis cuts and is assembled into polymer protein. Albumin fusion proteins of the present invention preferably passes through form processing. In the most preferred embodiment, " the process form processing of albumin fusion proteins " refers to the albumin fusion proteins product through the terminal signal peptide cutting of N-, and this paper also is called " ripe albumin fusion proteins ".

In several situations, the representative clone who contains albumin fusion construct of the present invention is preserved in American type culture collection (this paper is called " ATCC "). In addition, by known in the art and other local technology of describing of this paper, might again obtain from preserved material the albumin fusion construct of appointment. ATCC is positioned at No. 10801 20110-2209 of Manassas town, Virginia, The United States state University Road (10801 University Boulevard, Manassas, Virginia 20110-2209, USA). The ATCC preserved material is to generate about the internationally recognized microbial preservation clause that is used for proprietary program according to budapest treaty.

In one embodiment, the invention provides that coding comprises therapeutic protein and serum albumin protein or by the polynucleotide of its albumin fusion proteins of forming.In another embodiment, the invention provides and comprise therapeutic protein and serum albumin protein or by its albumin fusion proteins of forming.In a preferred embodiment, the invention provides by the polynucleotide of describing in the table 2 coded, comprise therapeutic protein and serum albumin protein or by its albumin fusion proteins of forming.In another preferred embodiment, the invention provides the polynucleotide that its sequence of coding is shown in the albumin fusion proteins of SEQ ID NO:Y in the table 2.In other embodiments, the invention provides the biologic activity that comprises therapeutic protein and/or therapeutic active fragments and serum albumin protein or by its albumin fusion proteins of forming.In other embodiments, the invention provides the biologic activity that comprises therapeutic protein and/or therapeutic active variant and serum albumin protein or by its albumin fusion proteins of forming.In preferred embodiments, the serum albumin protein component of albumin fusion proteins is the maturing part of serum albumin.The polynucleotide of these albumin fusion proteins of encoding are also contained in the present invention.

In other embodiments, the invention provides the biologic activity that comprises therapeutic protein and serum albumin and/or therapeutic active fragments or by its albumin fusion proteins of forming.In other embodiments, the invention provides the biologic activity that comprises therapeutic protein and serum albumin and/or therapeutic active variant or by its albumin fusion proteins of forming.In a preferred embodiment, the therapeutic protein of albumin fusion proteins partly is the maturing part of therapeutic protein.In another preferred embodiment, the therapeutic protein of albumin fusion proteins partly is the outer solubility structural domain of born of the same parents of therapeutic protein.In a candidate's embodiment, the therapeutic protein of albumin fusion proteins partly is the activity form of therapeutic protein.The polynucleotide of these albumin fusion proteins of encoding are also contained in the present invention.

In other embodiments, the invention provides the biologic activity of the biologic activity that comprises therapeutic protein and/or therapeutic active fragments or variant and serum albumin and/or therapeutic active fragments or variant or by its albumin fusion proteins of forming.In preferred embodiments, the invention provides the maturing part of the maturing part that comprises therapeutic protein and serum albumin or by its albumin fusion proteins of forming.The polynucleotide of these albumin fusion proteins of encoding are also contained in the present invention.

Therapeutic protein

As mentioned above, polynucleotide encoding of the present invention comprises at least one fragment of at least one fragment of therapeutic protein or variant and human serum albumin or variant or by its protein of forming, described fragment or variant are connected with each other, and preferably are connected with each other by gene fusion.

Another embodiment comprises that coding comprises at least one fragment of therapeutic protein or variant and human serum albumin's at least one fragment or variant or by its proteinic polynucleotide of forming, described fragment or variant are connected with each other by chemically conjugated.

When being used for this paper, " therapeutic protein " refers to have protein, polypeptide, antibody, peptide or its fragment or the variant of one or more therapeutic and/or biologic activity.The therapeutic protein that the present invention is contained includes but not limited to protein, polypeptide, peptide, antibody and biological products.(term peptide, protein and polypeptide are used interchangeably in this article.) clearly imagined term " therapeutic protein " and contain antibody and fragment and variant.Therefore, protein of the present invention can contain at least one fragment of therapeutic protein or at least one fragment or the variant of variant and/or antibody.In addition, term " therapeutic protein " can refer to the endogenous or the naturally occurring related thing of therapeutic protein.

The protein that presents the polypeptide of " therapeutic activity " or have " therapeutic activity " is meant that having one or more known organisms as described herein all with therapeutic protein or that known one or more therapeutic proteins of other approach of this area are relevant learns and/or the active polypeptide of therapeutic.As limiting examples, " therapeutic protein " refers to can be used for treating, preventing or improve the protein of disease, situation or disorder.As limiting examples, " therapeutic protein " can be with particular cell types (normal (as lymphocyte) or unusual (as cancer cells)) specific combination and therefore be can be used for protein with compound (medicine or cytotoxic agent) this cell type of special target.

For example, can include but not limited to IFN α by " therapeutic protein " incomplete tabulation partly that albumin fusion proteins of the present invention comprises, ANP, BNP, LANP, VDP, KUP, CNP, DNP, HCC-1, β defensin (defensin)-2, fractalkine (CXXXC chemokine), secrete sour regulin (oxyntomodulin), kill and wound toxin peptide (killer toxin peptide), TIMP-4, PYY, adrenomedullin (adrenomedullin), ghrelin (ghrelin), CGRP, IGF-1, neuraminidase, hemagglutinin, butyrylcholine esterase, endothelin (endothelin), and Mecano growth factor (mechanogrowth factor).

Interferon, rabbit hybrid (hybrid) also can be blended in albuminised amino or C-terminal to form Interferon, rabbit hybrid albumin fusion proteins.The interferon activity that Interferon, rabbit hybrid albumin fusion proteins can have being enhanced or suppress is such as the regulation and control of antiviral response, cell growth and the adjusting of immunne response (people such as Lebleu, PNAS USA 73:3107-3111,1976; People such as Gresser, Nature 251:543-545,1974; And Johnson, Texas Reports Biol Med 35:357-369,1977).Every kind of Interferon, rabbit hybrid albumin fusion proteins can be used for treatment, prevention or improves virus infection (as hepatitis (as HCV); Or the infection of HIV virus), multiple sclerosis or cancer.

In one embodiment, the Interferon, rabbit hybrid of Interferon, rabbit hybrid albumin fusion proteins partly comprises interferon alpha-interferon alpha hybrid (being referred to herein as α-α hybrid).For example, the α of Interferon, rabbit hybrid albumin fusion proteins-α hybrid partly comprises the interferon alpha A that merges with interferon alpha D or is made up of it.In another embodiment, the A/D hybrid merges at total BglII restriction site place, and wherein the amino acid/11-62 of the N-terminal portions of A/D hybrid and interferon alpha A is corresponding and the C-terminal portions is corresponding with the amino acid 64-166 of interferon alpha D.For example, this A/D hybrid will comprise aminoacid sequence:

CDLPQTHSLGSRRTLMLLAQMRX ₁ISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFTTKDS?SAAWDEDLLDKFCTELYQQLNDLEACVMQEERVGETPLMNX ₂DSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIMRSLSLSTNLQERLRRKE(SEQ?ID?NO：99)，

X wherein ₁Be R or K and X ₂Be A or V.In another embodiment, the A/D hybrid merges at total PvuIII restriction site place, and wherein the amino acid/11-91 of the N-terminal portions of A/D hybrid and interferon alpha A is corresponding and the C-terminal portions is corresponding with the amino acid 93-166 of interferon alpha D.For example, this A/D hybrid will comprise aminoacid sequence:

CDLPQTHSLGSRRTLMLLAQMRX ₁ISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVMQEERVGETPLMNX ₂DSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIMRSLSLSTNLQERLRRKE(SEQ?ID?NO：100)，

X wherein ₁Be R or K and X ₂Be A or V.These hybrids are at United States Patent (USP) 4,414, further description are arranged in 510, with its complete being incorporated herein by reference.

In another embodiment, the α of Interferon, rabbit hybrid albumin fusion proteins-α hybrid partly comprises the interferon alpha A that merges with interferon alpha F or is made up of it.In another embodiment, the A/F hybrid merges at total PvuIII restriction site place, and wherein the amino acid/11-91 of the N-terminal portions of A/F hybrid and interferon alpha A is corresponding and the C-terminal portions is corresponding with the amino acid 93-166 of interferon alpha F.For example, this A/F hybrid will comprise aminoacid sequence:

CDLPQTHSLGSRRTLMLLAQMRXISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDMEACVIQEVGVEETPLMNVDSILAVKKYFQRITLYLTEKKYSPCAWEVVRAEIMRSFSLSKIFQERLRRKE(SEQ?ID?NO：101)，

Wherein X is R or K.These hybrids are at United States Patent (USP) 4,414, further description are arranged in 510, with its complete being incorporated herein by reference.In another embodiment, the α of Interferon, rabbit hybrid albumin fusion proteins-α hybrid partly comprises the interferon alpha A that merges with interferon alpha B or is made up of it.In another embodiment, the A/B hybrid merges at total PvuIII restriction site place, and wherein the amino acid/11-91 of the N-terminal portions of A/B hybrid and interferon alpha A is corresponding and the C-terminal portions is corresponding with the amino acid 93-166 of interferon alpha B.For example, this A/B hybrid will comprise aminoacid sequence: CDLPQTHSLGSRRTLMLLAQMRX ₁ISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDET LLDKFYTELYQQLNDLEX ₂X ₃X ₄X ₅QEVGVIESPLMYEDSILAVRKYFQRITLYLTEKKYSSCAWEVVRAEIMRSFSLSIN LQKRLKSKE (SEQ ID NO:102),

X wherein ₁Be R or K and X ₂To X ₅Be SCVM or VLCD.These hybrids are at United States Patent (USP) 4,414, further description are arranged in 510, with its complete being incorporated herein by reference.

In another embodiment, the Interferon, rabbit hybrid of Interferon, rabbit hybrid albumin fusion proteins partly comprises interferon beta-interferon alpha hybrid (being referred to herein as β-α hybrid).For example, the β of Interferon, rabbit hybrid albumin fusion proteins-α hybrid partly comprises the interferon beta-1 that merges with interferon alpha D (being also referred to as Alfacon-1) or is made up of it.In another embodiment, β-1/ α D hybrid is to merge like this, and wherein the amino acid/11-73 of N-terminal portions and interferon beta-1 is corresponding and the C-terminal portions is corresponding with the amino acid 74-167 of interferon alpha D.For example, this β-1/ α D hybrid will comprise aminoacid sequence:

MSYNLLGFLQRS?SNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDS?SAAWDEDLLDKFCTELYQQLNDLEACVMQEERVGETPLMNXDSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIMRSLSLSTNLQERLRRKE(SEQ?ID?NO：103)，

Wherein X is A or V.These hybrids are at United States Patent (USP) 4,758, further description are arranged in 428, with its complete being incorporated herein by reference.

In another embodiment, the Interferon, rabbit hybrid of Interferon, rabbit hybrid albumin fusion proteins partly comprises interferon alpha-interferon beta hybrid (being referred to herein as the alpha-beta hybrid).For example, the alpha-beta hybrid of Interferon, rabbit hybrid albumin fusion proteins partly comprises the interferon alpha D (being also referred to as Alfacon-1) that merges with interferon beta-1 or is made up of it.In another embodiment, α D/ β-1 hybrid is to merge like this, and wherein the amino acid/11-73 of N-terminal portions and interferon alpha D is corresponding and the C-terminal portions is corresponding with the amino acid 74-166 of interferon beta-1.For example, this α D/ β-1 hybrid will comprise aminoacid sequence:

MCDLPETHSLDNRRTLMLLAQMSRISPSSCLMDRHDFGFPQEEFDGNQFQKAPAISVLHELIQQIFNLFTTKDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN(SEQ?ID?NO：104)。

These hybrids are at U.S. Patent number 4,758, further description are arranged in 428, with its complete being incorporated herein by reference.

In other embodiments, the Interferon, rabbit hybrid of Interferon, rabbit hybrid albumin fusion proteins part can comprise other combination of α-interferon-alpha hybrid, alpha-beta Interferon, rabbit hybrid and β-interferon-alpha hybrid.In other embodiments, the Interferon, rabbit hybrid of Interferon, rabbit hybrid albumin fusion proteins part can modify with comprise sudden change, substitute Interferon, rabbit hybrid aminoacid sequence, deletion or add.These modifications to Interferon, rabbit hybrid albumin fusion proteins can be used for for example improving yield level, improve stability, improve or reduce active or give new biological characteristics.

Interferon, rabbit hybrid albumin fusion proteins mentioned above is contained in the present invention, and the host cell and the carrier that contain the polynucleotide of these polypeptide of encoding.In one embodiment, the Interferon, rabbit hybrid albumin fusion proteins by above-mentioned polynucleotide encoding has increased shelf-life.In another embodiment, by the Interferon, rabbit hybrid albumin fusion proteins of above-mentioned polynucleotide encoding with accordingly not fused interferon hybrid molecule compare external and/or in vivo in solution (or in pharmaceutical composition) have longer serum half-life and/or more stable activity.

In another limiting examples, " therapeutic protein " refers to have the protein that biologic activity particularly can be used for treating, preventing or improve the biologic activity of disease.The incomplete tabulation of the biologic activity that therapeutic protein can have comprises that the HIV-1 that suppresses cell infects, stimulate intestinal epithelial cells propagation, reduce the intestinal epithelial cells permeability, stimulate insulin secretion, induce bronchiectasis and vasorelaxation, suppress aldosterone and renin secretion, blood pressure regulation, promote neure growth, enhancing immunity is replied, strengthen inflammation, depress appetite, perhaps hereinafter described the and/or table 1 (secondary series) of " biologic activity " part is disclosed any or various biological is active to TA protein.

In one embodiment, IFN-α-HSA fusions is used for suppress ranging category-A-Filovirus (Filo) (Ebola (Ebola)), arenavirus (Arena) (Pichende), category-B-togavirus (Toga) (VEE) or the virokine of C class-bunyavirus (Bunya) (Pang Tatuolu (Punto toro)), flavivirus (Flavi) (yellow jack, Xi Niluo (West Nile)).For example, adopt CPE inhibition, toluylene red dyeing and virus yield assay method to assess the antiviral activity of the IFN-α (CID 3165 protein) that is blended in the HSA downstream.CID 3165 proteinic pharmacokinetics and pharmacodynamics are assessed in rhesus monkey and human experimenter.The result shows with favourable safety index and has realized antiviral activity at all RNA viruses of assessment.In the CPE assay method scope of IC50 value be＜0.1ng/ml (Pang Tatuolu A) is to 19ng/ml (VEE).In rhesus monkey, the 3165 proteinic transformation period of CID are 90 hours and still can detect in 14 days after reaching administration.In the human experimenter, CID 3165 protein be safe and tolerance good.C after the single injection administration _MaxProportional with dosage.Average C in 500 μ g group _MaxBe 22ng/ml, and average t _1/2It is 150 hours.Every 2-4 week or longer time are administered once and have obtained the support of pharmacokinetics.In single injection group (120-500 μ g), in most experimenters, observe antiviral response at hepatitis C virus.

In another embodiment, IFN-α-HSA fusions is used for the treatment of the patient of chronic hcv infection (HCV).Interferon alpha is also referred to as Interferon, rabbit alfa or LeIF, is the standard method that the patient of HCV has been infected in treatment.Term " interferon alpha " refers to have the family of the height homologous related polypeptide of antiviral activity.The interferon alpha of IFN-α-HSA fusions partly comprises any interferon alpha known in the art or its fragment or is made up of it.The limiting examples of the interferon alpha part in IFN-α of the present invention-HSA fusion rotein includes but not limited to disclosed interferon alpha protein in table 1 therapeutic protein one row.In specific embodiment, any commercialization form that interferon alpha partly comprises Intederon Alpha-2a, Interferon Alpha-2b, interferon c, total Interferon, rabbit, Interferon, rabbit alfacon-1, interferon alfa-n1, Alferon N, interferon alpha is such as for example

A (Schering Corp., Kenilworth, N.J.),

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, GreatBritian),

(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 purifying or its fragment or form by it.In further embodiment, the interferon alpha in IFN-α-HSA fusion rotein partly is included as release prolongation or controlled and forms through the interferon alpha of modification or preparation or by it.For example, interferon alpha partly comprises commercial prolongation release or controlled release interferon alpha or is made up of it, include but not limited to interferon alpha-XL (Flamel Technologies, France) and LOCTERON ^TM(BioLexTherapeutics/OctoPlus, Pittsboro, NC).In other embodiments, the interferon alpha of IFN-α-HSA fusion rotein part can be modified by adhering to chemical module.For example, the interferon alpha part can be modified by PEGization.Therefore, in other embodiments, the interferon alpha of IFN-α-HSA fusion rotein partly comprises the PEGization form of Intederon Alpha-2a, 2b or total Interferon, rabbit or is made up of it, includes but not limited to commercialization PEGization interferon alpha, such as for example PEG- (Schering Corp., Kenilworth, N.J.),

(Hoffman-L a Roche, Nutley, N.J.), PEG-OMNIFERON ^TM(Viragen, Inc., Plantation, FL) or its fragment.Yet when being used for this paper, " IFN-α-HSA " fusions refers to HSA or its fragment with any interferon alpha protein fusion known in the art.

Be used for the treatment of the Interferon, rabbit scheme that HCV infects according to before whether accepting, the patient who has infected HCV can be divided into two classes." patient who had not received treatment " refers to the patient of those never received Interferon, rabbit scheme treatments." patient who once received treatment " refers to that those once accepted or accepting the patient of Interferon, rabbit scheme treatment." do not answer the person " and refer to such patient who once received treatment, they had before accepted Interferon, rabbit scheme treatment but main purpose such as the early stage virus load that do not reach treatment reduces (early viralload reduction, EVR) or treat whole end reply ((end-of-treatment response, ETR)." recidivist " refers to such patient who once received treatment, and they had before accepted interferon therapy scheme treatment and had reached main purpose such as the EVR or the ETR of treatment, but becomes the positive to HCV at after a while time point subsequently.Yet, when being used for this paper, " HCV patient " though referred to infect the patient of HCV and he be do not received treatment or once received treatment.In addition, when being used for this paper, " the HCV patient " of " once receiving treatment " or do not answer the person or the recidivist.

In addition, hepatitis C virus can be divided into many genotype, and wherein four kinds of genotype are the most general, genotype 1,2,3 or 4.Usually, HCV infection patient's hepatitis C virus comprises the term single gene type.Yet hepatitis virus can comprise two or more genotypic combinations.In addition, the genotype of hepatitis C virus can also be the variant of one of known HCV genotype.In another embodiment, HCV patient's hepatitis C virus is genotype 1 or its variant.Yet when being used for this paper, " HCV " refers to any genotypic hepatitis C virus, or its combination or variant.

HCV patient's standard care scheme related to the agent of interferon alpha combination antiviral treat such as ribavirin.Usually, weekly twice or use interferon alpha once in a week and use ribavirin once a day once a day.Yet nearest research has also been used interferon alpha and has been united other antiviral agent of the HCV of being used for the treatment of known in the art.Therefore, in another embodiment, can with IFN-α-HSA fusions separately or the combination antiviral agent be applied to HCV patient such as for example ribavirin.In a preferred embodiment, IFN-α-HSA fusions can be united one or more antiviral agents such as for example ribavirin and other antiviral agent and be applied to HCV patient.

As mentioned above, CID 3165 proteinic pharmacokinetics are supported every 2-4 week or longer time dosage regimen once.Therefore, in another embodiment, antiviral agent once independent by every 2-4 week or the associating significant quantity is used IFN-α-HSA fusions and is treated HCV patient.In a preferred embodiment, use IFN-α-HSA fusions by one or more antiviral agents of once uniting significant quantity every 2-4 week and treat HCV patient.In another preferred embodiment, per 4 weeks are once used IFN-α-HSA fusions to HCV patient.In another preferred embodiment, per 4 weeks are above once HCV patient being used IFN-α-HSA fusions.In other embodiments, per 4 weeks or longer time are once used IFN-α-HSA fusions to HCV patient, and wherein treatment also comprises one or more antiviral agents of using significant quantity.

In another embodiment, IFN-α-HSA fusions monotherapy of can be used as low dosage is used for the supportive care of HCV.In another embodiment, IFN-α-HSA fusions can be united the treatment that ribavirin and one or more other antiviral agents are used for HCV.Perhaps, in another embodiment, IFN-α-HSA fusions can be united the treatment that one or more antiviral agents except that ribavirin are used for HCV.

In another embodiment, IFN-α-HSA fusions can be used for treating other virus infection.For example, in one embodiment, IFN-α-HSA fusions can be used for treating hepatitis B (HBV).In another embodiment, IFN-α-HSA fusions can be used for treating human papillomavirus (HPV).In another embodiment, IFN-α-HSA fusions 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 (chronic myelogenous leukemia), AIDS relevant Kaposi sarcoma (AIDS related Kaposi ' s Sarcoma), multiple myeloma or renal cell carcinoma.

In another embodiment, contain the HSA fusions of natriuretic peptide (natriuretic peptide), include but not limited to ANP-HSA fusions or BNP-HSA fusions, can be used for treating cardiovascular disorder.For example, in a preferred embodiment, contain the HSA fusions of natriuretic peptide, include but not limited to ANP-HSA fusions or BNP-HSA fusions, can be used for treating congestive heart failure.In another preferred embodiment, contain the HSA fusions of natriuretic peptide, include but not limited to ANP-HSA fusions or BNP-HSA fusions, can be used for the treatment (treatment ofpost-myocardial infarction) after the myocardial infarction.In other embodiments, the HSA fusions that contains natriuretic peptide, include but not limited to ANP-HSA fusions or BNP-HSA fusions, can be used for other cardiovascular disorder, include but not limited to hypertension, salt-sensitive hypertension, stenocardia, peripheral arterial disease, ypotension, Cardiac volume excess load (cardiac volume overload), cardiac decompensation, heart failure, left ventricular dysfunction, expiratory dyspnea, reperfusion injury of cardiac muscle or Left Ventricular Remodeling (left ventricularremodeling).In another embodiment, the HSA fusions that contains natriuretic peptide, include but not limited to ANP-HSA fusions or BNP-HSA fusions, can be used for treating the aldosterone level of rising, the aldosterone level of rising can cause the cardiac output and/or the hypertension of vasoconstriction, weakening.In other embodiments, the HSA fusions that contains natriuretic peptide, include but not limited to ANP-HSA fusions or BNP-HSA fusions, can be used for treating ephrosis, include but not limited to diabetic nephropathy, renal glomerulus hypertrophy, glomerular injury, renal glomerular disease, acute and/or chronic renal failure.In another embodiment, contain the HSA fusions of natriuretic peptide, include but not limited to ANP-HSA fusions or BNP-HSA fusions, it is excessive to can be used for treating apoplexy (stroke) or tissue juice.

In another embodiment, HSA and natriuretic peptide variant can be merged, include but not limited to the BNP-HSA fusions, wherein the BNP composition of fusion rotein is BNP amino-acid residue 1-29.In one embodiment, the BNP composition of HSA fusion rotein is made up of two placed in-line BNP variants (for example BNP amino-acid residue 1-29).In another embodiment, the BNP composition of HSA fusion rotein is made up of three, four, five or more a plurality of placed in-line BNP variant (for example BNP amino-acid residue 1-29).In a preferred embodiment, the HSA fusions that contains BNP variant (for example BNP amino-acid residue 1-29) can be used for treating congestive heart failure.In another preferred embodiment, the HSA fusions that contains BNP variant (for example BNP amino-acid residue 1-29) can be used for the treatment after the myocardial infarction.In another preferred embodiment, the HSA fusions that contains BNP variant (for example BNP amino-acid residue 1-29) can be used for treating other cardiovascular disorder, includes but not limited to hypertension, salt-sensitive hypertension, stenocardia, peripheral arterial disease, ypotension, Cardiac volume excess load, cardiac decompensation, heart failure, non-Hemodynamics CHF, left ventricular dysfunction, expiratory dyspnea, reperfusion injury of cardiac muscle or Left Ventricular Remodeling.In another preferred embodiment, the HSA fusions that contains BNP variant (for example BNP amino-acid residue 1-29) can be used for treating the aldosterone level of rising, and the aldosterone level of rising can cause the cardiac output and/or the hypertension of vasoconstriction, weakening.In a preferred embodiment, the HSA fusions that contains BNP variant (for example BNP amino-acid residue 1-29) can be used for treating the disorder or the disease of kidney, includes but not limited to diabetic nephropathy, renal glomerulus hypertrophy, glomerular injury, renal glomerular disease, acute and/or chronic renal failure.In another embodiment, the HSA fusions that contains BNP variant (for example BNP amino-acid residue 1-29) can be used for treating apoplexy or tissue juice is excessive.

In relevant but different embodiment, the present invention is devoted to not the natriuretic peptide variant that merges with HSA, includes but not limited to BNP amino-acid residue 1-29.In one embodiment, BNP variant of the present invention has the sequence of two placed in-line BNP variants (for example BNP amino-acid residue 1-29).In another embodiment, BNP variant of the present invention has the sequence of three, four, five or more a plurality of placed in-line BNP variant (for example BNP amino-acid residue 1-29).In a preferred embodiment, BNP variant of the present invention (for example BNP amino-acid residue 1-29) can be used for treating congestive heart failure.In another preferred embodiment, BNP variant of the present invention (for example BNP amino-acid residue 1-29) can be used for the treatment after the myocardial infarction.In another embodiment, BNP variant of the present invention (for example BNP amino-acid residue 1-29) can be used for treating other cardiovascular disorder, includes but not limited to hypertension, salt-sensitive hypertension, stenocardia, peripheral arterial disease, ypotension, Cardiac volume excess load, cardiac decompensation, heart failure, non-Hemodynamics CHF, left ventricular dysfunction, expiratory dyspnea, reperfusion injury of cardiac muscle or Left Ventricular Remodeling.In another embodiment, BNP variant of the present invention (for example BNP amino-acid residue 1-29) can be used for treating the aldosterone level of rising, and the aldosterone level of rising can cause the cardiac output and/or the hypertension of vasoconstriction, weakening.In another preferred embodiment, BNP variant of the present invention (for example BNP amino-acid residue 1-29) can be used for treating the disorder or the disease of kidney, includes but not limited to diabetic nephropathy, renal glomerulus hypertrophy, glomerular injury, renal glomerular disease, acute and/or chronic renal failure.In another embodiment, BNP variant of the present invention (for example BNP amino-acid residue 1-29) can be used for treating apoplexy or tissue juice is excessive.

In another embodiment, the present invention is devoted to include but not limited to BNP variant (for example BNP amino-acid residue 1-29) through modifying with prolong half-life, improving biologic activity and/or be convenient to the natriuretic peptide variant of purifying.According to this embodiment, natriuretic peptide variant (for example BNP amino-acid residue 1-29) can use technology known in the art to carry out PEGization, methylate or other chemically modified or coupling.Perhaps, can use methods known in the art that natriuretic peptide variant of the present invention and prolong half-life known in the art, other peptide sequences reorganization of improving biologic activity and/or being convenient to purifying are merged.For example, can be with natriuretic peptide variant of the present invention and antibody Fc district or its meromixis or coupling.The antibody moiety that merges with natriuretic peptide variant of the present invention (for example BNP amino-acid residue 1-29) can comprise any combination of constant region, hinge area, CH1 territory, CH2 territory and CH3 territory or entire structure territory or its part.Natriuretic peptide and above-mentioned antibody moiety can also be merged or coupling to form polymer.For example, and the Fc part that merges of polypeptide of the present invention (for example BNP amino-acid residue 1-29) can form dimer via the disulfide bonding between the Fc part.Can prepare more high-grade polymer form by meromixis with variant and IgA and IgM.Variant of the present invention and antibody moiety are merged or the link coupled method is known in the art.Referring to for example United States Patent (USP) 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCT publication WO96/04388; WO 91/06570; Ashkenazi et al., Proc.Natl.Acad.Sci.USA88:10535-10539 (1991); Zheng et al., J.Immunol.154:5590-5600 (1995); And Vilet al., Proc.Natl.Acad.Sci.USA 89:11337-11341 (1992) (being collected herein by reference described reference is complete).In another embodiment, the present invention has the sequence of two placed in-line BNP variants (for example BNP amino-acid residue 1-29) through the BNP variant of modifying.In another embodiment, the present invention has the sequence of three, four, five or more a plurality of placed in-line BNP variant (for example BNP amino-acid residue 1-29) through the BNP variant of modifying.In a preferred embodiment, the present invention can be used for treating congestive heart failure through BNP variant (for example BNP amino-acid residue 1-29) variant of modifying.In a preferred embodiment, the present invention can be used for the treatment after the myocardial infarction through BNP variant (for example BNP amino-acid residue 1-29) variant of modifying.In another embodiment, the present invention can be used for treating other cardiovascular disorder through BNP variant (for example BNP amino-acid residue 1-29) variant of modifying, and includes but not limited to hypertension, salt-sensitive hypertension, stenocardia, peripheral arterial disease, ypotension, Cardiac volume excess load, cardiac decompensation, heart failure, non-Hemodynamics CHF, left ventricular dysfunction, expiratory dyspnea, reperfusion injury of cardiac muscle or Left Ventricular Remodeling.In another embodiment, the present invention can be used for treating the aldosterone level of rising through BNP variant (for example BNP amino-acid residue 1-29) variant of modifying, and the aldosterone level of rising can cause the cardiac output and/or the hypertension of vasoconstriction, weakening.In a preferred embodiment, the present invention can be used for treating the illness or the disease of kidney through BNP variant (for example BNP amino-acid residue 1-29) variant of modifying, and includes but not limited to diabetic nephropathy, renal glomerulus hypertrophy, glomerular injury, renal glomerular disease, acute and/or chronic renal failure.In another embodiment, variant can be used for treating apoplexy or tissue juice is excessive for the BNP variant (for example BNP amino-acid residue 1-29) of the present invention's process modification.

In another embodiment, the CNP-HSA fusions can be used for regulating enchondral ossification.For example, in a preferred embodiment, the CNP-HSA fusions can be used for treating osteodysplasty, includes but not limited to fetal rickets (anchondroplasia), dyschondroplasia (hypochondroplasia) and thanatophoric dysplasia (thanatophoric dysplasia).

When being used for this paper, " therapeutic activity " or " activity " can refer to that its effect in human body meets the activity of expectation treatment achievement, perhaps refers to the desired effects in non-human mammal or other species or organism.Therapeutic activity can be in vivo or in in-vitro measurements.For example, can in cell culture, measure desired effects.These external or cell culture assays methods are that the public is obtainable for the described many therapeutic proteins in this area.The example of assay method includes but not limited to described in this paper embodiment part or table 1 " exemplary activation measurement " row (the 3rd row).

With the corresponding therapeutic protein of therapeutic protein part of albumin fusion proteins of the present invention,, modify by adhering to one or more oligosaccharides groups usually such as cell surface and secreted protein.This modification is called glycosylation, but the proteinic physical property of remarkably influenced, and may be important for proteinic stability, secretion and location.Glycosylation betides along the specific position of polypeptide main chain.Two kinds of main type of glycosylation are arranged usually: the glycosylation that O-connects oligosaccharides that is characterized as that is attached to Serine or threonine residues; Be attached to the glycosylation that is characterized as N-connection oligosaccharides of the asparagine residue in Asn-X-Ser or the Asn-X-Thr sequence, wherein X can be any amino acid except that proline(Pro).N-n acetylneuraminic acid n (being also referred to as sialic acid) normally N-connects the terminal residue that is connected oligosaccharides with O-.Influence the number and the essence of carbohydrate unit in the different glycosylation site chain such as parameters such as protein structure and cell types.The glycosylation isomer also usually is present in the same loci place in the designated cell type.

Therapeutic protein corresponding with the therapeutic protein part of albumin fusion proteins of the present invention and analogue thereof and variant can be modified, therefore owing to operation to its nucleotide sequence, change the glycosylation of one or more site by the host cell of expressing them, perhaps because their other expression condition.For example, can produce the glycosylation isomer by eliminating or introduce glycosylation site, for example substituting or deleting by amino-acid residue, such as substituting l-asparagine with glutamine, perhaps can by can not be with it in glycosylated host cell marking protein produce not glycosylated recombinant protein, for example in the yeast of intestinal bacteria or glycosylation defect.These methods have more detailed description hereinafter and are known in this area.

Therapeutic protein, particularly in the table 1 disclosed those, be well known in the art with their nucleic acid and aminoacid sequence, and can and provide database such as the GenSeq (as Derwent) of subscription to obtain from public database such as Chemical Abstracts Service database (Chemical Abstracts Services Databases) (as the CAS number of registration), GenBank.The exemplary therapeutic protein nucleotide sequence of polynucleotide of the present invention of can be used for deriving is shown in the 7th row " SEQ ID NO:X " of table 2.Sequence shown in the SEQ ID NO:X can be the wild-type polynucleotide sequence of coding TA protein (as total length or sophisticated), or this sequence can be the variant (polynucleotide of encoding wild type therapeutic protein for example of described wild-type polynucleotide sequence in some cases, the dna sequence dna of wherein said polynucleotide is optimized, and for example is used for expressing at specific species; Or the polynucleotide of the variant of encoding wild type therapeutic protein (are directed mutants; Allele variant)).Utilize sequence shown in the SEQID NO:X to derive with the construction described in the delegation fully within those of skill in the art's ability.For example, if SEQ ID NO:X corresponding to full length protein, but only some is used to generate specific CID to this protein, so according to increase specific fragment and it is cloned in the suitable carrier within art technology of Protocols in Molecular Biology such as PCR.

Other therapeutic protein corresponding with the therapeutic protein part of albumin fusion proteins of the present invention includes but not limited to disclosed one or more therapeutic proteins or peptide or its fragment or variant in table 1 " therapeutic protein X " row (the 1st row).

The tabulation that table 1 provides the therapeutic protein corresponding with the therapeutic protein of albumin fusion proteins of the present invention part or given an example by the non-limit of the albumin fusion proteins of polynucleotide encoding of the present invention.First row " therapeutic protein X " disclose proteinaceous therapeutic molecule, comprise in the possible subsequently parenthesis comprising this proteinaceous therapeutic molecule or its fragment or variant or by its proteinic formal name used at school and trade(brand)name of forming.When being used for this paper, " therapeutic protein X " can refer to the individualized treatment protein molecule, or whole group of relevant therapeutic protein of disclosed TA protein molecule in referring to be listed as therewith." biologic activity " row (the 2nd row) have been described the biologic activity relevant with proteinaceous therapeutic molecule.The 3rd row " exemplary activation measurement " provide to describe and can be used for testing therapeutic protein X or comprise the therapeutic of albumin fusion proteins of therapeutic protein X (or its fragment) part and/or the reference of the assay method of biologic activity.With complete being incorporated herein by reference of every piece of reference of being quoted in " exemplary activation measurement " row, particularly aspect the description that is used for the activation measurement separately of corresponding biologic activity shown in mensuration table 1 " biologic activity " row described in reference (for example seeing method part wherein).The 4th row " preferred indication: Y " have been described and can or have been comprised disease, disorder and/or the situation that therapeutic protein X (or its fragment) albumin fusion proteins is partly treated, prevents, diagnosed and/or improves by therapeutic protein X." construction ID " row (the 5th row) provide with table 2 in disclosed coding comprise therapeutic protein X (or its fragment) part mentioned or linking by the exemplary white protein fusion construct of its albumin fusion proteins of forming.

Table 2 provides the nucleic acid molecule that comprises the albumin fusion proteins of encoding among the present invention or by the non-exhaustive inventory of its polynucleotide of forming.The 1st row " fusions No. " have provided the fusions numbering of every kind of polynucleotide.The 2nd row " construction ID " provide unique numeric identifier for every kind of polynucleotide of the present invention.Construction ID can be used for the polynucleotide of identifier number albumin fusion proteins, wherein albumin fusion proteins comprises the therapeutic protein part corresponding with given therapeutic protein X or is made up of it, given therapeutic protein X is listed in the respective column of table 1, and wherein construction ID is listed in the 5th row.The 3rd row " construction title " provide title for given white protein fusion construct or polynucleotide.

The 4th row " descriptions " of table 2 provide general description for given white protein fusion construct, and the 5th row " expression vector " have been listed the nucleic acid molecule that comprises the given albumin fusion proteins of encoding or clone wherein carrier by its polynucleotide of forming.Carrier is known in the art, and can obtain by commercial sources or other local described approach.For example, as described in embodiment, can assemble the polynucleotide that comprise the given albumin fusion proteins of (1) coding in the cloning vector easily, (2) leader sequence, (3) promoter region, (4) wherein or multinomial or by its " expression cassette " of forming in translation termination transfers to it in other carrier then, such as the expression vector that for example comprises Yeast expression carrier for example or mammalian expression vector.In one embodiment, in order in yeast saccharomyces cerevisiae, to express, will to comprise the nucleic acid molecule of the albumin fusion proteins of encoding or be cloned among the pSAC35 by the expression cassette that it is formed.In another embodiment, in order in Chinese hamster ovary celI, to express, will to comprise the nucleic acid molecule of the albumin fusion proteins of encoding or be cloned among the pC4 by the expression cassette that it is formed.In another embodiment, will comprise the albumin fusion proteins of encoding therapeutic protein part nucleic acid molecule or be cloned among the pC4:HSA by the polynucleotide that it is formed.In another embodiment,, will comprise the nucleic acid molecule of the albumin fusion proteins of encoding or be cloned among the pEE12 by the expression cassette that it is formed in order in the NS0 cell, to express.Those of skill in the art also know clone and/or the expression vector that other is useful, and they also within the scope of the invention.

The 6th row " SEQ ID NO:Y " provide the full length amino acid sequence of albumin fusion proteins of the present invention.In most cases, SEQ ID NO:Y shows the undressed form of coded albumin fusion proteins, and in other words, SEQ ID NO:Y shows all by particular build thing encoded signals sequence, HSA part and therapeutic protein part.All polynucleotide of coding SEQ ID NO:Y are clearly contained in the present invention.When utilizing these polynucleotide by the coded protein of cell expressing, the natural secretion of cell and procedure of processing produce the protein of the signal sequence of listing in the 4th row that lack table 2 and/or the 11st row.The concrete aminoacid sequence of listed signal sequence is presented in the specification sheets of back, perhaps has been known in the art.Therefore, most preferred embodiment of the present invention comprises the albumin fusion proteins that produced by cell (it will lack shown leader sequence in the 4th row of table 2 and/or the 11st row).Same most preferred comprise SEQ ID NO:Y in addition but do not have the 4th row of table 2 and/or the 11st row in the polypeptide of the concrete leader sequence listed.Comprising the composition of these two preferred embodiments, comprise pharmaceutical composition, also is preferred.And, within the also complete limit of power of listing in the 4th row of describing in the specification sheets with different signal sequences such as back that help albumin fusion proteins excretory signal sequence substitution table 2 and/or the 11st row of signal sequence those of skill in the art through processing.

The 7th row " SEQ ID NO:X " provide the parental nucleic acid sequence of the therapeutic protein polynucleotide partly of the given albumin fusion proteins of coding of can deriving.In one embodiment, can the derive parental nucleic acid sequence of polynucleotide of therapeutic protein part of coding albumin fusion proteins comprises the wild type gene sequence of the therapeutic protein that shows in the coding schedule 1.In candidate's embodiment, the parental nucleic acid sequence of polynucleotide of therapeutic protein part of coding albumin fusion proteins of can deriving comprises the variant or the derivative of the wild type gene sequence of the therapeutic protein that shows in the coding schedule 1, such as the synthetic codon optimized variant of the wild type gene sequence of the therapeutic protein of for example encoding.

The 8th row " SEQ ID NO:Z " provide the prediction translation result of parental nucleic acid sequence (SEQ ID NO:X).This parental array can be used for deriving the particular build thing the proteinic maturing part of total length parent's protein, parent, wild-type protein variant or fragment or can be used for producing the artificial sequence of described construction.Those skilled in the art can utilize this aminoacid sequence that shows among the SEQ ID NO:Z to determine which amino-acid residue of the albumin fusion proteins of given construction coding is provided by therapeutic protein.And the sequence of utilizing SEQ ID NO:Z to show is derived with the construction of describing in the delegation also fully within those of skill in the art's limit of power.For example, if SEQ ID NO:Z is corresponding to full length protein, and only utilize this proteinic part to produce specific CID, and relying on increase specific fragment and it is cloned in the appropriate carriers of Protocols in Molecular Biology such as PCR so, this is within the technical scope of this area.

The 9th row and the 10th amplimer " SEQ ID NO:A " that provides respectively of row and " SEQ ID NO:B " be used for producing comprise the given albumin fusion proteins of encoding therapeutic protein partly nucleic acid molecule or by the demonstration primer of its polynucleotide of forming.In one embodiment of the invention, have the Oligonucleolide primers of sequence (SEQ ID NO:A and/or B) shown in the 9th row and/or the 10th row and be used for the polynucleotide of the therapeutic protein part of pcr amplification coding albumin fusion proteins, wherein utilize nucleotide sequence (SEQ ID NO:X) that the 7th row that comprise corresponding row provide or by its nucleic acid molecule of forming as template DNA.This area has been set up PCR method well.Those of ordinary skills can be easy to imagine and use other useful primer sequence.

In candidate's embodiment, Oligonucleolide primers can be used for producing sudden change in the template DNA sequence in overlapping PCR reaction.PCR method is known in the art.

As shown in table 3, disclosed some white protein fusion construct has been preserved in ATCC among the application.

Table 3

Construction ID	The construction title	ATCC preserving number/date
			2249	PSAC35:IFNa2-HSA is also referred to as pSAC23:IFN α 2-HSA	PTA-3763 2001-10-4
2343	pSAC35.INV-IFNA2.HSA	PTA-3940 2001-12-19
			2381	pC4:HSA-IFNa2(C17-E181)	PTA-3942 2001-12-19
2382	pC4:IFNa2-HSA	PTA-3939 2001-12-19
			3165	PSAC35:HSA.IFNa is also referred to as CID 3165, pSAC35:HSA.INF α	PTA-4670 2002-9-16

Again it is possible obtaining given white protein fusion construct by technology known in the art from the preservation thing, also is described (referring to embodiment 10) in other place of the application.ATCC be positioned at No. 10801 20110-2209 of the big ways for education in Virginia, USA Manassas town (10801 UniversityBoulevard, Manassas, Virginia 20110-2209, USA).Carried out the ATCC preservation according to budapest treaty about the microbial preservation clause of the international endorsement that is used for patented procedure.

In another embodiment of the present invention, the polynucleotide that comprise the given albumin fusion proteins of (1) coding, (2) leader sequence, (3) in promoter region and (4) translation termination wherein one or multinomial or by its " expression cassette " of forming can from a kind of carrier move or " subclone " to another kind of carrier.By method well-known in the art,, can produce the fragment that is used for subclone such as for example pcr amplification (for example utilizing Oligonucleolide primers) and/or restriction enzyme digestion with sequence shown in SEQ ID NO:A or the B.

In preferred embodiments, albumin fusion proteins of the present invention has therapeutic activity and/or corresponding therapeutic activity and/or the biologic activity of biologic activity with therapeutic protein, and wherein therapeutic protein is corresponding to the therapeutic protein part of the listed albumin fusion proteins of the corresponding row of table 1.In other preferred embodiment, the therapeutic activity protein portion of albumin fusion proteins of the present invention is proteinic fragment or the variant by the sequence encoding of the SEQ ID NO:X row demonstration of table 2, and has the therapeutic activity and/or the biologic activity of corresponding therapeutic protein.

Polypeptide and polynucleotide passage and variant

Fragment

The invention still further relates to fragment, albumin protein and/or the albumin fusion proteins of the present invention of the therapeutic protein of describing in the table 1.

The invention still further relates to the polynucleotide of fragment, albumin protein and/or the albumin fusion proteins of the present invention of the therapeutic protein of describing in the coding schedule 1.

Even can cause the modification or the forfeiture of one or more biological functions of therapeutic protein, albumin protein and/or albumin fusion proteins of the present invention from the one or more amino acid of the terminal deletion of proteinic N-, but still can keep other therapeutic activity and/or functionally active (as the ability of biologic activity, multimerization, the ability of binding partner).For example, when the N-end is removed the most residue that is less than complete polypeptide, can keep the ability of the antibody of the complete or mature form that polypeptid induction with the terminal deletion of N-and/or combination discern this peptide species usually.The ordinary method described by the application and other approach of this area is known can be easy to determine whether the specific polypeptide that lacks complete polypeptide N-terminal residue has kept this immunologic competence.The-terminal amino acid residue suffers that the mutain of a large amount of deletions keeps some biology or immunologic competence is not impossible.In fact, often can cause immunne response by few peptide that constitutes to six amino-acid residues.

Therefore, the fragment of the therapeutic protein corresponding with the therapeutic protein part of albumin fusion proteins of the present invention comprises full length protein, and deleted the polypeptide of one or more residues from the N-terminal of the aminoacid sequence of reference polypeptide (i.e. the therapeutic protein of mentioning the table 1 is perhaps by the therapeutic protein part of the albumin fusion proteins of polynucleotide of describing in the table 2 or white protein fusion construct coding).Particularly, the terminal deletion of N-can be described with the general formula of m to q, wherein q represents the reference polypeptide (therapeutic protein of mentioning in the table 1 for example, the perhaps therapeutic protein part of albumin fusion proteins of the present invention, perhaps by the therapeutic protein part of the albumin fusion proteins of polynucleotide of describing in the table 2 or white protein fusion construct coding) in the complete integer of amino-acid residue sum, and m to be defined as scope be 2 to q to subtract any integer of 6.The polynucleotide of these polypeptide of encoding are also contained in the present invention.

In addition, the fragment of the serum albumin polypeptide corresponding with the albumin protein part of albumin fusion proteins of the present invention comprises full length protein, and the polypeptide of having deleted one or more residues from the N-terminal of the aminoacid sequence of reference polypeptide (being serum albumin) perhaps by the serum albumin part of the albumin fusion proteins of polynucleotide of describing in the table 2 or white protein fusion construct coding.In preferred embodiments, the terminal deletion of N-can be described with the general formula of m to 585, and wherein 585 is complete integers of the ripe human serum albumin's of representative (SEQ ID NO:1) amino-acid residue sum, and m to be defined as scope be any integer of 2 to 579.The polynucleotide of these polypeptide of encoding are also contained in the present invention.In other embodiments, the terminal deletion of N-can be described with the general formula of m to 609, and wherein 609 is complete integers of representing total length human serum albumin's (SEQ ID NO:3) amino-acid residue sum, and m to be defined as scope be any integer of 2 to 603.The polynucleotide of these polypeptide of encoding are also contained in the present invention.

And, the fragment of albumin fusion proteins of the present invention comprises the total length albumin fusion proteins, and deleted the polypeptide of one or more residues from the N-terminal of albumin fusion proteins (, perhaps having the albumin fusion proteins of the disclosed aminoacid sequence of the 6th row of table 2) promptly by the albumin fusion proteins of polynucleotide of describing the table 2 or white protein fusion construct coding.Particularly, the terminal deletion of N-can be described with the general formula of m to q, and wherein q is a complete integer of representing the amino-acid residue sum of albumin fusion proteins, and m to be defined as scope be 2 to q to subtract any integer of 6.The polynucleotide of these polypeptide of encoding are also contained in the present invention.

Similarly, as described above, even can cause the modification or the forfeiture of proteinic one or more biological functions from the N-end or the one or more amino acid of the terminal deletion of C-of reference polypeptide (for example therapeutic protein, serum albumin protein or albumin fusion proteins of the present invention), but still can keep other functionally active (biological example is learned activity, the ability of multimerization, the ability of binding partner) and/or therapeutic activity.For example, when the C-end is removed the most residue that is less than complete or mature polypeptide, can keep the ability of the antibody of the complete or mature form that polypeptid induction with the terminal deletion of C-and/or combination discern this peptide species usually.The ordinary method described by the application and/or other approach of this area is known can be easy to determine whether the specific polypeptide that lacks reference polypeptide N-end and/or C-terminal residue has kept therapeutic activity.

The present invention also provides the polypeptide of having deleted one or more residues from the C-terminal of the aminoacid sequence of the therapeutic protein corresponding with the therapeutic protein of albumin fusion proteins of the present invention part (therapeutic protein of mentioning the table 1 for example, perhaps the therapeutic protein part of the albumin fusion proteins of being encoded by polynucleotide of describing in the table 2 or white protein fusion construct).Particularly, the terminal deletion of C-can be described with 1 to n general formula, wherein n is that scope is 6 to q to subtract 1 any complete integer, and q is a complete integer of representing amino-acid residue sum in the reference polypeptide (therapeutic protein of mentioning in the table 1 for example is perhaps by the therapeutic protein part of the albumin fusion proteins of polynucleotide of describing in the table 2 or white protein fusion construct coding).The polynucleotide of these polypeptide of encoding are also contained in the present invention.

In addition, the invention provides the polypeptide of having deleted one or more residues from the C-terminal of the aminoacid sequence of the albumin protein corresponding (serum albumin for example, perhaps the albumin protein part of the albumin fusion proteins of encoding by polynucleotide of describing in the table 2 or white protein fusion construct) with the albumin protein of albumin fusion proteins of the present invention part.Particularly, the terminal deletion of C-can be described with 1 to n general formula, and wherein n is that scope is 6 to 584 any complete integer, and 584 be that the ripe human serum albumin's of representative (SEQ ID NO:1) amino-acid residue sum subtracts 1 complete integer.The polynucleotide of these polypeptide of encoding are also contained in the present invention.Particularly, the terminal deletion of C-can be described with 1 to n general formula, and wherein n is that scope is 6 to 608 any complete integer, and 608 be to represent the amino-acid residue sum of serum albumin (SEQ ID NO:3) to subtract 1 complete integer.The polynucleotide of these polypeptide of encoding are also contained in the present invention.

And, the invention provides the polypeptide of having deleted one or more residues from the C-terminal of albumin fusion proteins of the present invention.Particularly, the terminal deletion of C-can be described with 1 to n general formula, and wherein n is that scope is 6 to q to subtract 1 any complete integer, and q is a complete integer of representing the amino-acid residue sum of albumin fusion proteins of the present invention.The polynucleotide of these polypeptide of encoding are also contained in the present invention.

In addition, can make up the terminal deletion of above-mentioned any N-or C-to produce the reference polypeptide of the terminal deletion of N-and C-.The present invention also provides from amino and two ends of carboxyl and has deleted one or more amino acid whose polypeptide, this can be described as usually and the have reference polypeptide (therapeutic protein of mentioning in the table 1 for example, the perhaps therapeutic protein part of albumin fusion proteins of the present invention, perhaps by the therapeutic protein part of polynucleotide of describing in the table 2 or white protein fusion construct coding, perhaps serum albumin (for example SEQ ID NO:1), the perhaps albumin protein part of albumin fusion proteins of the present invention, perhaps by the albumin protein part of polynucleotide of describing in the table 2 or white protein fusion construct coding, perhaps albumin fusion proteins, perhaps by the albumin fusion proteins of polynucleotide of the present invention or white protein fusion construct coding) residue m to n, wherein n and m are integers mentioned above.The polynucleotide of these polypeptide of encoding are also contained in the present invention.

The application also relates to and comprising and the listed reference polypeptide sequence of this paper (therapeutic protein of mentioning in the table 1 for example, the perhaps therapeutic protein part of albumin fusion proteins of the present invention, perhaps by the therapeutic protein part of polynucleotide of describing in the table 2 or white protein fusion construct coding, perhaps serum albumin (for example SEQ ID NO:1), the perhaps albumin protein part of albumin fusion proteins of the present invention, perhaps by the albumin protein part of polynucleotide of describing in the table 2 or white protein fusion construct coding, perhaps albumin fusion proteins, perhaps by the albumin fusion proteins of polynucleotide of the present invention or white protein fusion construct coding) or its fragment have at least 80%, 85%, 90%, 95%, 96%, 97%, the protein of the polypeptide of 98% or 99% identity.In preferred embodiments, the application relates to and comprises the protein that has the polypeptide of at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the reference polypeptide of the aminoacid sequence with the terminal deletion of N-mentioned above and C-.The polynucleotide of these polypeptide of encoding are also contained in the present invention.

Preferred polypeptide fragment of the present invention is to comprise the aminoacid sequence of the therapeutic activity that shows therapeutic protein or serum albumin protein and peptide sequence and/or functionally active (biological example is learned active) or by its fragment of forming, wherein said aminoacid sequence is the fragment of described therapeutic protein or the proteinic peptide sequence of serum albumin.

Other preferred polypeptide fragment has biological active fragment.Biological active fragment is that those demonstrate with the active similar of polypeptide of the present invention but active fragment that needn't be identical.Segmental biologic activity can comprise that improved expectation is active, the bad activity that perhaps weakens.

Variant

" variant " refers to different with reference nucleic acid or polypeptide but keeps the nucleic acid or the polypeptide of its intrinsic propesties.Usually, variant is closely similar and identical in many zones on the whole with reference nucleic acid or polypeptide.

When being used for this paper, " variant " refer to that sequence is different with therapeutic protein (for example referring to " therapeutic protein " of table 1 row), albumin protein and/or albumin fusion proteins respectively but keep its herein therapeutic protein part, the albumin fusion proteins of the present invention of the albumin fusion proteins of the present invention of other local at least a function that describe or that other approach of this area is known and/or treatment characteristic albumin protein partly or albumin fusion proteins of the present invention.Usually, variant with corresponding to the therapeutic protein of the therapeutic protein of albumin fusion proteins part, closely similar on the whole and identical with it in many zones corresponding to the aminoacid sequence of the albumin protein of the albumin protein part of albumin fusion proteins and/or albumin fusion proteins.The nucleic acid of these variants of encoding is also contained in the present invention.

The invention still further relates to comprise with for example corresponding to the therapeutic protein of the therapeutic protein of the albumin fusion proteins of the present invention part (aminoacid sequence of disclosed therapeutic protein X in the table 1 for example; Perhaps by the aminoacid sequence of the therapeutic protein part of the albumin fusion proteins of polynucleotide of describing in table 1 and the table 2 or white protein fusion construct coding; Or the aminoacid sequence of its fragment or variant), corresponding to the albumin protein of the albumin protein of albumin fusion proteins of the present invention part (for example by the aminoacid sequence of the albumin protein part of the albumin fusion proteins of polynucleotide of describing in table 1 and the table 2 or white protein fusion construct coding; The aminoacid sequence that shows among the SEQ ID NO:1; Or the aminoacid sequence of its fragment or variant) and/or the aminoacid sequence of albumin fusion proteins has the aminoacid sequence of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity or by its protein of forming.The fragment (fragment for example described herein) of these polypeptide also is provided.The present invention also comprises the polypeptide by such polynucleotide encoding, described polynucleotide under rigorous hybridization conditions (for example at about 45 degrees centigrade, in 6X sodium chloride/sodium citrate (SSC), hybridize with filter membrane bonded DNA, then at about 50-65 degree centigrade, at 0.2X SSC, wash one or many among the 0.1%SDS), under highly rigorous condition (for example at about 45 degrees centigrade, in 6X sodium chloride/sodium citrate (SSC), hybridize with filter membrane bonded DNA, then at about 68 degrees centigrade, at 0.1XSSC, wash one or many among the 0.2%SDS), or under other the rigorous hybridization conditions that those skilled in the art will know that (for example referring to Ausubel, F.M. wait the people, compile, 1989, " Current protocol inMolecular Biology ", Green publishing associates, Inc. and John Wiley ﹠amp; SonsInc., New York, 6.3.1-6.3.6 and 2.10.3) hybridize down with the complementary strand of the nucleic acid molecule of the albumin fusion proteins of the present invention of encoding.The polynucleotide of these polypeptide of encoding are also contained in the present invention.

Have that to have amino acid sequence of polypeptide and the search sequence of at least for example polypeptide feeling the pulse with the finger-tip of the aminoacid sequence of 95% " identity " identical with the inquiry aminoacid sequence, just the desired polypeptides sequence can comprise nearly five amino acid change in per 100 amino acid of inquiry aminoacid sequence.In other words, obtain to have the polypeptide that has the aminoacid sequence of at least 95% identity with the inquiry aminoacid sequence, can insert, delete or with the amino-acid residue that reaches 5% in the another kind of amino acid replacement aim sequence.These changes of reference sequences can occur in the amino or the C-terminal position of reference amino acid sequence, or between those terminal positions Anywhere, can individually be dispersed between the residue of reference sequences or as with reference to one or more continuous group in the sequence.

In fact, can utilize known computer program determine as usual any specific polypeptide and albumin fusion proteins of the present invention or its fragment (such as the therapeutic protein of albumin fusion proteins partly or the albumin part of albumin fusion proteins) aminoacid sequence whether have for example at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.Utilization is with people such as Brutlag (Comp.App.Biosci.6:237-245,1990) algorithm is also referred to as global sequence's comparison for the FASTDB computer program on basis can be identified for measuring the preferred method that the best overall between search sequence (sequence of the present invention) and the aim sequence is mated.In sequence alignment, inquiry and aim sequence can all be nucleotide sequences or all be aminoacid sequence.The result of described global sequence comparison represents with identity per-cent.The preferred parameter that is used for the comparison of FASTDB amino acid is: matrix=PAM 0, k-tuple=2, mispairing point penalty=1, connect point penalty=20, randomization group leader=0 is by score=1, window size=sequence length, breach point penalty=5, breach size point penalty=0.05, the shorter one in the length of window size=500 or purpose aminoacid sequence.

If aim sequence because the terminal deletion of N-or C-but not inner deletion is shorter than search sequence must manually revise the result so.This is because the FASTDB program not will consider the N-and the terminal brachymemma of C-of aim sequence when calculating overall identity per-cent.For with respect to the aim sequence of search sequence in the terminal brachymemma of N-and C-, following correction identity per-cent, calculate the N-that is positioned at aim sequence in the search sequence and C-end, not with the residue number of corresponding purpose residue coupling/comparison, as the per-cent of search sequence base sum.Whether residue mates/compare is that result by the FASTDB sequence alignment determines.Deduct this per-cent from the identity per-cent that utilizes special parameter to calculate by above-mentioned FASTDB program then, draw final identity per-cent score.This final identity per-cent score is used for score of the present invention exactly.Artificial N-that only considers to be positioned at aim sequence when adjusting identity per-cent score and C-end, the residue that does not match/compare with search sequence.That is to say, only consider to be positioned at aim sequence N-and C-terminal residue inquiry residue residue in addition farthest.

For example, the aim sequence of 90 amino-acid residues and the search sequence of 100 residues are compared to measure identity per-cent.Deletion occurs in the N-end of aim sequence, so the FASTDB comparison does not show the coupling/comparison result of terminal initial 10 residues of N-.10 unpaired residues account for 10% (the residue sum of the terminal unmatched residue number/search sequence of N-and C-) of sequence, therefore deduct 10% from the identity per-cent score that calculates by the FASTDB program.If remaining 90 residues mate fully, then final identity per-cent will be 90%.In another example, the aim sequence of 90 residues and the search sequence of 100 residues are compared.Current deletion is inner deletion, so the terminal residue that does not have not with search sequence coupling/comparison of the N-of aim sequence or C-.In this case, the identity per-cent that calculates by FASTDB need not manually to revise.Statement once more has only demonstration according to FASTDB comparison to be positioned at beyond the N-and C-end of aim sequence, manually not revise with the residue position needs of search sequence coupling/comparison.Do not carry out the artificial correction of other form in the present invention.

Variant normal HA or the therapeutic protein identical with it with length usually has the sequence identity of at least 75% (preferably at least about 80%, 90%, 95% or 99%).Utilization is for sequence similarity search and program blastp, the blastn, blastx, tblastn and the tblastx that revise (people such as Karlin, Proc.Natl.Acad.Sci.USA 87:2264-2268,1990 and Altschul, J.Mol.Evol.36:290-300,1993, complete being incorporated herein by reference) algorithm that is adopted is analyzed homology or the identity of measuring Nucleotide or amino acid sequence level by BLAST (the promptly basic local comparison research tool of Basic LocalAlignment Search Tool).

The method that blast program utilized is at first considered the similar section between search sequence and the database sequence, and all coupling assessment significance,statisticals that evaluation is drawn are only summed up the coupling that those satisfy predetermined significance threshold values at last then.About the discussion of the basic problem in the similarity searching of sequence library, referring to people such as Altschul, Nature Genetics 6:119-129,1994, with its complete being incorporated herein by reference.The search parameter of histogram, description, comparison, expected value (promptly reporting the significance,statistical threshold value at the coupling of database sequence), cutoff, matrix and strainer all adopts default setting.The acquiescence rating matrix that blastp, blastx, tblastn and tblastx adopted is BLOSUM62 matrix (people such as Henikoff, Proc.Natl.Acad.Sci.USA 89:10915-10919,1992, complete being incorporated herein by reference).For blastn, by the ratio of M (the prize branch that promptly mates residue) and N (point penalty of the residue that promptly do not match) rating matrix is set, wherein the default value of M and N is respectively 5 and-4.Can four blastn parameter: Q=10 of following adjustment (breach generation point penalty); R=10 (breach extension point penalty); Wink=1 (producing the speech hits in each winkth position) along search sequence; And gapw=16 (set window width, wherein generation contains the breach comparison).Equal blastp parameter is set to Q=9; R=2; Wink=1 and gapw=32.The DNA parameter that comparison program Bestfit uses between the sequence that can obtain from GCG software package version 10.0 is GAP=50 (breach generation point penalty) and LEN=3 (breach extension point penalty), and the protein comparison be set to GAP=8 and LEN=2 on an equal basis.

Polynucleotide variant of the present invention can contain in coding region, non-coding region or the two and changes.Especially preferably contain to produce and reticently substitute, add or deletion but do not change the characteristic of coded polypeptide or the polynucleotide variant of active change.Preferably the silence that is caused by the genetic code degeneracy substitutes the nucleotide variants that produces.And, preferred arbitrary combination also less than 50, less than 40, less than 30, less than 20, less than 10 or 5-50,5-25,5-10,1-5 or the 1-2 amino acid polypeptide variants that substituted, delete or add.Can be for multiple reason produces the polynucleotide variant, for example in order to express (codon of the codon among the people mRNA being changed into host bacterium such as yeast or intestinal bacteria preference) at the specific host optimizing codon.

In a preferred embodiment, in order in yeast or mammalian cell, to express, the polynucleotide of the present invention of the albumin part of coding albumin fusion proteins are optimized.In a further preferred embodiment, in order in yeast or mammalian cell, to express, the therapeutic protein polynucleotide of the present invention partly of coding albumin fusion proteins are optimized.In another preferred embodiment,, the polynucleotide of the albumin fusion proteins of the present invention of encoding are optimized in order in yeast or mammalian cell, to express.

In candidate's embodiment, the polynucleotide of the therapeutic protein of the coding albumin fusion proteins of codon optimization part are not hybridized with the wild-type polynucleotide of coding therapeutic protein under rigorous hybridization conditions described herein.In another embodiment, the polynucleotide of the albumin part of the coding albumin fusion proteins of codon optimization are not hybridized with the wild-type polynucleotide of coding albumin protein under rigorous hybridization conditions described herein.In another embodiment, the polynucleotide of the coding albumin fusion proteins of codon optimization are not hybridized with the wild-type polynucleotide of coding therapeutic protein part or albumin protein part under rigorous hybridization conditions described herein.

In another embodiment, the polynucleotide of the therapeutic protein part of coding albumin fusion proteins do not comprise the natural of therapeutic protein and have sequence or can't help its composition.In another embodiment, the polynucleotide of the albumin protein part of coding albumin fusion proteins do not comprise the natural of albumin protein and have sequence or can't help its composition.In candidate's embodiment, the polynucleotide of coding albumin fusion proteins do not comprise the therapeutic protein part or there is sequence in the natural of albumin protein part or can't help its composition.

The natural variant that exists is called " allele variant ", refers to that (B. compiles one of several replaceable forms that occupy the gene of given locus on the organism karyomit(e), John Wiley ﹠amp for Genes II, Lewin; Sons, New York, 1985).These allele variants can be different at polynucleotide and/or polypeptide level, and comprise in the present invention.Perhaps, can prepare the variant that non-natural exists by induced-mutation technique or direct synthetic technology.

Utilize the method for known protein matter engineering and recombinant DNA technology, can produce variant to improve or to change the feature of polypeptide of the present invention.For example, can delete one or more amino acid and not cause the substance of biological function to be lost from the N-end or the C-end of polypeptide of the present invention.For example, people such as Ron (J.Biol.Chem.268:2984-2988,1993) have reported even have still had the variant KGF protein of heparin binding activity behind 3,8 or 27 amino terminal amino acid residues of deletion.Similarly, IFN-shows higher activity people such as (, J.Biotechnology 7:199-216,1988) Dobeli that reaches ten times behind 8-10 amino-acid residue of proteinic C-terminal deletion from then on.

And, there is competent evidence to prove, variant can keep and the natural similar biologic activity of protein that exists usually.For example, Gayle and colleague thereof (J.Biol.Chem.268:22105-22111,1993) have carried out mutation analysis widely to human cell factor IL-1a.They utilize random mutagenesis to produce above 3,500 kinds of unique IL-1a mutant, and promptly every kind of variant on average has 2.5 amino acid changes on the total length of molecule.At each possible amino acid position various mutations is checked.The researchist finds " most molecules can be very little to [combination or biologic activity] influence when being changed ".In fact, in surpassing in 3,500 kinds of nucleotide sequences of checking, have only 23 kinds of unique amino acid sequences to produce activity and the remarkable different protein of wild-type.

And, even can cause the modification or the forfeiture of one or more biological functions, but still can keep other biologic activity from the N-end or the one or more amino acid of the terminal deletion of C-of polypeptide.For example, when N-end or C-end are removed the most residue that is less than secreted form, might keep that the deletion variant is induced and/or in conjunction with the ability of the antibody of identification secreted form.The ordinary method of knowing by described herein and other approach of this area can be easy to determine whether the specific polypeptide that lacks proteinic N-end or C-terminal residue has kept this immunogenicity activity.

Therefore, the present invention also comprises and has functionally active the polypeptide variants of (biological example is learned activity and/or therapeutic activity).In one embodiment, the invention provides the albumin fusion proteins variant with the functionally active corresponding with one or more biology of therapeutic protein and/or therapeutic activity (biological example is learned activity and/or therapeutic activity), wherein therapeutic protein is corresponding to the therapeutic protein part of albumin fusion proteins.In another embodiment, the invention provides and have the functionally active corresponding with one or more biology of therapeutic protein and/or therapeutic activity the albumin fusion proteins variant of (biological example is learned active/or therapeutic activity), wherein therapeutic protein is corresponding to the therapeutic protein part of albumin fusion proteins.This variant comprise select according to general rule known in the art, to the very little deletion of activity influence, insertion, inversion, repetition with substitute.The polynucleotide of this variant of encoding are also contained in the present invention.

In preferred embodiments, variant of the present invention has conservative substituting." conservative substituting " refers to exchange in group, such as the replacement of aliphatics or hydrophobic amino acid Ala, Val, Leu and Ile; The replacement of hydroxyl residue Ser and Thr; The replacement of acidic residues Asp and Glu; The replacement of amide residues Asn and Gln; The replacement of alkaline residue Lys, Arg and His; The replacement of aromatic residue Phe, Tyr and Trp; And the replacement of small-sized amino acid Ala, Ser, Thr, Met and Gly.

For example, people such as Bowie, " Deciphering the Message in Protein Sequences:Tolerance to Amino Acid Substitutions ", Science 247:1306-1310, provide in 1990 about how carrying out phenotype silent amino acid alternate and instructed, wherein the author points out to study aminoacid sequence has two kinds of main strategies to the tolerance that changes.

First kind of strategy utilized the tolerance of the amino acid replacement of natural selection in the evolutionary process.Can identify conserved amino acid by the aminoacid sequence that compares different plant species.These conserved amino acids may be important to proteinic function.On the contrary, the amino acid position of the replacement that tolerated of natural selection shows that these positions are not vital to protein function.Therefore, can modify the position of tolerance amino acid replacement, still keep this proteinic biologic activity simultaneously.

Second kind of strategy utilizes genetic engineering to introduce the amino acid variation at the specific position of clone gene, to identify the vital zone of protein function.For example, can use site-directed mutagenesis or alanine scanning mutagenesis (each the residue place in molecule introduces single alanine mutation).Referring to Cunningham and Wells, Science 244:1081-1085,1989.Can test the biologic activity of the mutating molecule of generation like this then.

As the author said, these two kinds of strategies have disclosed the surprising tolerance amino acid replacement of albumen mass-energy.The author has pointed out that also it is that some amino acid position might be allowed in the protein which amino acid changes.For example, the amino-acid residue that great majority bury (in proteinic tertiary structure) needs non-polar sidechain, and the seldom characteristic of surface side chains is normally guarded.And the conserved amino acid of tolerance substitutes the replacement that relates to aliphatics or hydrophobic amino acid Ala, Val, Leu and Ile; The replacement of hydroxyl residue Ser and Thr; The replacement of acidic residues Asp and Glu; The replacement of amide residues Asn and Gln; The replacement of alkaline residue Lys, Arg and His; The replacement of aromatic residue Phe, Tyr and Trp; And the replacement of small-sized amino acid Ala, Ser, Thr, Met and Gly.Except that conserved amino acid substitutes, variant of the present invention comprises that (i) contains the alternate polypeptide of one or more non-conservative amino acid residues, wherein the alternate amino-acid residue can yes or no by the genetic code coding, or (ii) contain one or more alternate polypeptide with substituent amino-acid residue, or (iii) merge or chemically conjugated polypeptide such as the compound (for example polyoxyethylene glycol) that improves polypeptide stability and/or solubleness, or (iv) contain other amino acid such as for example polypeptide of IgG Fc district fusogenic peptide with another compound.According to the instruction of this paper, think that this variant polypeptide is within those skilled in the art's scope.

For example, contain the polypeptide variants that useful other electrically charged or neutral amino acids substitutes the amino acid replacement of charge residue and can produce protein, such as assembling still less with improved characteristics.The gathering of pharmaceutical formulations not only reduces activity, but also improves the removing that the immunogenicity because of aggregation causes.Referring to people such as Pinckard, Clin.Exp.Immunol.2:331-340,1967; People such as Robbins, Diabetes 36:838-845,1987; People such as Cleland, Crit.Rev.Therapeutic Drug Carrier Systems 10:307-377,1993.

In specific embodiment, polypeptide of the present invention comprises aminoacid sequence, therapeutic protein and/or human serum albumin's the fragment of aminoacid sequence of albumin fusion proteins or variant or is made up of it, wherein 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, substitute and/or deletion.In preferred embodiments, amino acid replacement is guarded.The nucleic acid of these polypeptide of encoding is also contained in the present invention.

Polypeptide of the present invention can be by each other by peptide bond or to modify peptide bond be that the amino acid that peptide isostere (isostere) links together constitutes, and can comprise the amino acid beyond the amino acid of 20 kinds of genes encodings.Can pass through natural process,, perhaps come modified polypeptide by chemical modification technology well-known in the art such as the translation post-treatment.Basic reader and more detailed monograph, and in long research document good description has been carried out in this modification.Modification can occur in the polypeptide Anywhere, comprises peptide backbone, amino acid side chain, and amino or C-terminal.Should be understood that the modification of same type can identical or different degree be present in several site of given polypeptide.And given polypeptide can contain polytype modification.Polypeptide can be a ramose, and for example because ubiquitin turns usefulness into, and they also can be to have or do not have the ramose ring-type.Can produce cyclic, ramose and branch's annular polypeptide by translation back natural process, perhaps can generate by synthetic method.Modification comprises acetylize; acidylate; the ADP-ribosylation; amidation; the covalent attachment of flavine; the covalent attachment of heme moiety; the covalent attachment of Nucleotide or nucleotide derivative; the covalent attachment of lipid or lipid derivate; the covalent attachment of phosphatidylinositols; crosslinked; cyclisation; disulfide linkage forms; demethylation; the formation of covalent cross-linking; the formation of halfcystine; the formation of Pyrrolidonecarboxylic acid; formylation; the γ carboxylation; glycosylation; the GPI anchor forms; hydroxylation; iodate; methylate; myristylization; oxidation; PEGization; proteolysis processing; phosphorylation; prenylization; racemization; selenizing (selenoylation); sulfation; interpolation amino acid such as the arginylization (arginylation) on protein of transfer RNA mediation; with ubiquitinization.(referring to for example " PROTEINS-STRUCTURE AND MOLECULAR PROPERTIES ", the 2nd edition, T.E.Creighton, W.H.Freeman and Company, New York, 1993; " POST-TRANSLATIONAL COVALENT MODIFICATION OF PROTEINS ", B.C.Johnson compiles, Academic Press, New York, 1-12 page or leaf, 1983; People such as Seifter, Meth.Enzymol.182:626-646,1990; People such as Rattan, Ann.N.Y.Acad.Sci.663:48-62,1992).

Functionally active

" polypeptide with functionally active " refers to show one or more known functions active polypeptide relevant with the total length of therapeutic protein, preceding albumen and/or mature form.This functionally active includes but not limited to biologic activity, antigenicity [in conjunction with the ability of (or combine with the polypeptide competition) anti-peptide antibody], immunogenicity (producing the ability with specific polypeptide bonded antibody of the present invention), form polymeric ability with polypeptide of the present invention, reach and the acceptor or the part bonded ability of polypeptide.

" polypeptide with biologic activity " refer to according to having or do not have the measurement of the particular biological assay method of dose-dependently, demonstrates the active polypeptide that comprises the active similar of mature form with therapeutic protein of the present invention but needn't be identical.In the situation that has dose-dependently really, compare with polypeptide of the present invention, given active dose-dependently needn't be identical with polypeptide, only need similar substantially (promptly with respect to polypeptide of the present invention, the candidate polypeptide will demonstrate bigger activity or be no more than about 25 times littler activity, preferably be no more than about 10 times littler activity, be most preferably not exceeding about 3 times littler activity).

Relevant biology and/or therapeutic activity when in preferred embodiments, albumin fusion proteins of the present invention has at least a and therapeutic protein part (or its fragment or variant) and do not merge with white protein.

In other preferred embodiment, albumin fusion proteins of the present invention has the plasma stability of comparing rising with the therapeutic protein part (or its fragment or variant) that does not merge state.Can utilize or conventional revise that assay method known in the art is measured albumin fusion proteins of the present invention or the plasma stability of the therapeutic protein part (or its fragment or variant) that do not merge.

Can utilize or conventional revise the functionally active (biological example is learned active) that assay method known in the art and assay method described herein are measured albumin fusion proteins of the present invention.In addition, those skilled in the art utilize its corresponding row (for example the 3rd of table 1 the row) at table 1 but in the therapeutic protein segmental activity of corresponding therapeutic protein partly of the assay method conventional determining mentioned and albumin fusion proteins.In addition, but those skilled in the art utilize the segmental activity of albumin protein known in the art and/or that hereinafter the assay method conventional determining partly described of embodiment is corresponding with the albumin protein part of albumin fusion proteins.

For example, measuring the albumin fusion proteins combination or competing in the embodiment of the ability that combines treatment-resistant polypeptide antibody and/or anti-white protein antibody with therapeutic protein, can use various immunoassay known in the art, include but not limited to utilize such as radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), " sandwich " immunoassay, immune radiating degree assay method, gel diffusion precipitation reaction, the immunodiffusion(ID) assay method, the original position immunoassay (is for example utilized Radioactive colloidal gold, enzyme or radioisotopic tracer), western blotting, precipitin reaction, agglutination assay (example gel agglutination assay, the haemagglutination assay method), complement fixation assay, immunofluorescence assay, the competitiveness of technology such as albumin A assay method and immunoelectrophoresis assay method and noncompetitive are measured system.In one embodiment, detect antibodies by detecting a marker that resists.In another embodiment, detect one with anti-combining and resist by detecting two anti-or reagent.In another embodiment, two is anti-through mark.This area is known and is used for detecting the many methods of bonded in immunoassay, and they within the scope of the present invention.

In a preferred embodiment of the binding partners (for example acceptor or part) of identifying therapeutic protein, for example can measure and comprise of the albumin fusion proteins of therapeutic protein part and the combining of this binding partners of this therapeutic protein as fusions by method well-known in the art such as for example reductibility and irreducibility gel chromatography, protein affinity chromatography and affine trace.Usually referring to people such as Phizicky, Microbiol.Rev.59:94-123,1995.In another embodiment, utilize technology known in the art can the conventional determining albumin fusion proteins in conjunction with the related ability of physiology of the substrate of the corresponding therapeutical peptide of the therapeutic protein part of fusions.

In candidate's embodiment of multimerization ability of assessment albumin fusion proteins, for example can measure association with other component of polymer by method well-known in the art such as for example reductibility and irreducibility gel chromatography, protein affinity chromatography and affine trace.Usually referring to people such as Phizicky, see above.

In preferred embodiments, the albumin fusion proteins that comprises the whole of the proteinic antibody of combined treatment or part has at least a biology and/or therapeutic activity (for example specific combination polypeptide or epi-position) relevant when not merging with white protein with the proteinic antibody of combined treatment (or its fragment or variant).In other preferred embodiment, comprise the biologic activity of albumin fusion proteins of the whole of the proteinic antibody of combined treatment or part and/or therapeutic activity and be the inhibition (being antagonistic action) of one or more biologic activity relevant and/or therapeutic activity or activate (being agonism) with the polypeptide that is subjected to the proteinic antibody specific combination of combined treatment.

Can characterize at least one fragment that comprises the proteinic antibody of combined treatment or the albumin fusion proteins of variant in many ways.Particularly, utilize technology described herein or conventional modification technology known in the art, can measure the albumin fusion proteins specificity in conjunction with the ability that is subjected to the same antigen of the proteinic antibody specific combination of combined treatment to the albumin fusion proteins of at least one fragment that comprises the proteinic antibody of combined treatment or variant, wherein said therapeutic protein is corresponding to the therapeutic protein part of albumin fusion proteins.

At albumin fusion proteins (at least one fragment or the variant that for example comprise the proteinic antibody of combined treatment) (special) in conjunction with the assay method of the ability of specified protein or epi-position can be in solution (Houghten for example, Bio/Techniques 13:412-421,1992), (Lam for example on pearl, Nature 354:82-84,1991), (Fodor for example on chip, Nature 364:555-556,1993), (for example U.S. Patent number 5 on bacterium, 223,409), (for example the patent No. 5 on spore, 571,698; 5,403,484; With 5,223,409), on plasmid (for example people such as Cull, Proc.Natl.Acad.Sci.USA 89:1865-1869,1992) or on phage (for example Scott and Smith, Science 249:386-390,1990; Devlin, Science 249:404-406,1990; People such as Cwirla, Proc.Natl.Acad.Sci.USA 87:6378-6382,1990; And Felici, J.Mol.Biol.222:301-310,1991) carry out (with whole complete being incorporated herein by reference of these reference).Utilize or conventionally revise technology described herein or that other approach of this area is known, also can measure its specificity and avidity at least one fragment that comprises therapeutic antibodies or the albumin fusion proteins of variant to specified protein or epi-position.

Utilize any method known in the art, can be at least one fragment or the albumin fusion proteins mensuration of variant and the cross reactivity of other antigen (molecule that for example has sequence/structure conservative property, wherein the antibodies therapeutic protein (or its fragment or variant) partly corresponding) that comprises the proteinic antibody of combined treatment with the therapeutic protein of albumin fusion proteins of the present invention with the molecule that is subjected to the antibody specific combination.

The immunoassay that can be used for analysis (immunologic opsonin) combination and cross reactivity includes but not limited to utilize such as western blotting, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), " sandwich " immunoassay, exempts to survey the competitiveness and the noncompetitive mensuration system of technology such as precipitation assay method, precipitin reaction, GDP reaction, immunodiffusion(ID) assay method, agglutination assay, complement fixation assay, immune radiating degree assay method, fluorescence immunoassay and albumin A immunoassay, has only mentioned some here.This assay method is conventional, and be well-known in the art (for example referring to people such as Ausubel, compile, 1994, " Current Protocols in Molecular Biology ", the 1st volume, John Wiley ﹠amp; Sons, Inc., NewYork is with its complete being incorporated herein by reference).Hereinafter sketched exemplary immunoassay (but being not intended to restriction).

The immunoprecipitation scheme is usually included in the dissolving damping fluid such as being supplemented with protein phosphatase and/or proteinase inhibitor (EDTA for example, PMSF, press down the enzyme peptide, vanadic acid sodium) RIPA damping fluid (1%NP-40 or Triton X-100,1% Sodium desoxycholate, 0.1%SDS, 0.15M NaCl, the 0.01M sodium phosphate of pH7.2, dissolved cell group 1%Trasylol), in the cytolysis thing, add albumin fusion proteins of the present invention (at least one fragment or the variant that for example comprise the proteinic antibody of combined treatment), in 40 degrees centigrade of incubation for some time (for example 1 to 4 hour), for example in the cytolysis thing, add sepharose pearl with anti-white protein antibody coupling, in about one hour of 40 degrees centigrade of incubations or longer time, in the dissolving damping fluid, wash pearl, and pearl is resuspended in the SDS/ sample buffer.Utilize western blot analysis for example can assess the ability of albumin fusion proteins immunoprecipitation specific antigen.Those skilled in the art understand can be increased albumin fusion proteins and combine and reduce background (for example clarifying the cytolysis thing in advance with the sepharose pearl) with antigenic by revising parameter., compile referring to people such as for example Ausubel about more discussion of immunoprecipitation scheme, 1994, " Current Protocols in Molecular Biology ", the 1st volume, John Wiley ﹠amp; Sons, Inc., New York, page number 10.16.1.

Western blot analysis generally includes the preparation protein example, electrophoresis protein example in polyacrylamide gel (for example selecting the SDS-PAGE of 8%-20%) according to antigen molecular, protein example is transferred to film such as nitrocellulose from polyacrylamide gel, PVDF or nylon, closed film in confining liquid (PBS that for example contains 3%BSA or skimmed milk), washing film in lavation buffer solution (for example PBS-Tween 20), albumin fusion proteins of the present invention (diluting in the sealing damping fluid) is applied on the film, in lavation buffer solution, wash film, be applied to that sealing dilutes in the damping fluid, with enzyme material (for example horseradish peroxidase or alkaline phosphatase) or Geigers (for example 32P or 125I) put together two anti-(it discerns albumin fusion proteins, AHS's white protein antibody for example), in lavation buffer solution, wash film, and detect antigenic existence.Those skilled in the art understand can be increased the signal of detection and reduce ground unrest by revising parameter., compile referring to people such as for example Ausubel about more discussion of western blotting scheme, 1994, " Current Protocols in MolecularBiology ", the 1st volume, John Wiley ﹠amp; Sons, Inc., New York, page number 10.8.1.

ELISA comprises preparation antigen, hole with antigen coated 96 hole microtiter plates, flush away not with hole bonded antigen, but Xiang Kongzhong adds the albumin fusion proteins of the present invention (at least one fragment or the variant that for example comprise the proteinic antibody of combined treatment) and the incubation for some time of puting together with detection compound such as enzyme material (for example horseradish peroxidase or alkaline phosphatase), the albumin fusion proteins of the unconjugated or non-specific combination of flush away, and detect and wrap by the existence of the antigen-specific bonded albumin fusion proteins in hole.In ELISA, but albumin fusion proteins needn't be puted together with detection compound; But but two anti-(it discerns albumin fusion proteins) of puting together with detection compound can be added in the hole.In addition, available albumin fusion proteins bag is replaced using antigen coated hole by the hole.In this case, but but detection molecules can be the antigen of puting together with detection compound such as enzyme material (for example horseradish peroxidase or alkaline phosphatase).Those skilled in the art understand can increase the signal of detection by revising parameter, and other ELISA that this area is known changes., compile referring to people such as Ausubel about more discussion of ELISA, 1994, " Current Protocols in Molecular Biology ", the 1st volume, John Wiley﹠amp; Sons, Inc., New York, page number 11.2.1.

Can measure the binding affinity and the interactional dissociation rate of albumin fusion proteins-protein/antigen/epi-position (off-rate) of albumin fusion proteins and protein, antigen or epi-position by the competitive binding assay method.An example of competitive binding assay method is a radioimmunoassay, it is included under the condition that has the cumulative unlabelled antigen of quantity, will be through labelled antigen (for example 3H or 125I) with albumin fusion proteins incubation of the present invention, and detect and through labelled antigen bonded antibody.The data determination albumin fusion proteins that can draw be analyzed by Scatchard is to the avidity of specified protein, antigen or epi-position with in conjunction with dissociation rate.Utilize radioimmunoassay can also measure and the second proteinic competition, described second protein and albumin fusion proteins are in conjunction with identical protein, antigen or epi-position.In this case, there are incubation under the unmarked second cumulative proteinic condition of quantity in protein, antigen or epi-position with the albumin fusion proteins of having puted together tagged compound (for example 3H or 125I), and described second protein and albumin fusion proteins of the present invention are in conjunction with identical protein, antigen or epi-position.

In a preferred embodiment, utilize the BIAcore dynamic analysis to measure combining and the speed of dissociating (binding on and offrate) of albumin fusion proteins of the present invention and protein, antigen or epi-position.The BIAcore dynamic analysis comprises analyzes combining and dissociating of the chip of having fixed specific polypeptide, antigen or epi-position or albumin fusion proteins on albumin fusion proteins or specific polypeptide, antigen or epi-position and its surface respectively.

Antibody in conjunction with the therapeutic protein corresponding with the therapeutic protein part of albumin fusion proteins also can be described or illustrate aspect the antigenic binding affinity of given protein or preferred their specific combination of antigen at it.Preferred binding affinity comprises that dissociation constant or Kd are less than 5x10 ^-2M, 10 ^-2M, 5x10 ^-3M, 10 ^-3M, 5x10 ^-4M, 10 ^-4Binding affinity.Preferred binding affinity comprises that dissociation constant or Kd are less than 5x10 ^-5M, 10 ^-5M, 5x10 ^-6M, 10 ^-6M, 5x10 ^-7M, 10 ^-7M, 5x10 ^-8M or 10 ^-8The binding affinity of M.Even preferred binding affinity comprises that dissociation constant or Kd are less than 5x10 ^-9M, 10 ^-9M, 5x10 ^-10M, 10 ^-10M, 5x10 ^-11M, 10 ^-11M, 5x10 ^-12M, 10 ^-12M, 5x10 ^-13M, 10 ^-13M, 5x10 ^-14M, 10 ^-14M, 5x10 ^-15M or 10 ^-15The binding affinity of M.In preferred embodiments, consider tiring of albumin fusion proteins (at least one fragment or the variant that comprise the proteinic antibody of combined treatment) and tiring of corresponding antibody, the albumin fusion proteins that comprises at least one fragment of the proteinic antibody of combined treatment or variant is similar with the avidity of the proteinic antibody of corresponding combined treatment (to the white protein fusion) to the avidity of given protein or epi-position.In addition, the assay method of can routine utilizing (referring to embodiment and table 1) described herein and other approach of this area to know measure albumin fusion proteins and fragment, variant and derivative cause with the therapeutic protein of albumin fusion proteins partly and/or the ability of the relevant biologic activity of albumin part and/or therapeutic activity (or external or in vivo).Those of skill in the art also know other method, and they within the scope of the present invention.

White protein

As mentioned above, albumin fusion proteins of the present invention comprises at least one fragment of therapeutic protein or variant and human serum albumin's at least one fragment or variant, and both are connected with each other, and preferably pass through gene fusion.

Another embodiment comprises at least one fragment of therapeutic protein or variant and human serum albumin's at least one fragment or variant, and both are connected with each other by chemically conjugated.

Term human serum albumin (HSA) and people's white protein (HA) are used interchangeably in this article.Term " white protein " and " serum albumin " scope are wideer, contain human serum albumin's (and fragment and variant) and from the white protein (and fragment and variant) of other species.

When being used for this paper, " white protein " refers to albumin protein or aminoacid sequence, perhaps has the overall of the white protein fragment of albuminised one or more functionally activies (biological example learn active) or variant.Particularly, " white protein " refers to people's white protein or its fragment (for example referring to EP 201 239, EP 322 094, WO 97/24445, WO 95/23857), especially the albuminised mature form of people shown in Fig. 1 and SEQ ID NO:1, perhaps from other vertebrate white protein or its fragment, perhaps these molecules or its segmental analogue or variant.

In preferred embodiments, the human serum albumin's protein that is used for albumin fusion proteins of the present invention contains a group or two groups of following two groups of point mutation combination: numbering sports Ala, Leu-408 with reference to SEQ ID NO:1:Leu-407 and sports that Val, Val-409 sport Ala and Arg-410 sports Ala; Perhaps Arg-410 sports that A, Lys-413 sport Gln and Lys-414 sports Gln (for example referring to international publication number WO 95/23857, complete being incorporated herein by reference).In addition preferred embodiment in, the albumin fusion proteins of the present invention that contains in above-mentioned two groups of point mutation one group or two groups has improved stability/to the resistivity of yeast Yap3p proteolysis cutting, allows the output of the reorganization albumin fusion proteins of expressing in yeast host cell to raise.

When being used for this paper, the part white protein that is enough to the proteinic therapeutic activity of extended treatment, plasma stability or preservation period refers to be enough on length or structure stable or prolongs proteinic therapeutic activity or plasma stability, so the therapeutic protein of albumin fusion proteins preservation period or plasma stability partly compared the part white protein that has obtained prolongation or extended with the preservation period or the plasma stability of non-fusion state.The albumin part of albumin fusion proteins can comprise aforesaid total length HA sequence, can comprise that perhaps it can stablize or the active one or more fragments of extended treatment.This segmental length can be 10 or amino acids more, perhaps can comprise about 15,20,25,30,50 or more from the continuous amino acid of HA sequence, perhaps can comprise the part in ad hoc structure territory of HA or whole.For example, can utilize one or more HA fragments of crossing over initial two immunoglobulin like domain.In a preferred embodiment, the HA fragment is the HA of mature form.

The albumin part of albumin fusion proteins of the present invention can be the variant of normal HA.The therapeutic protein part of albumin fusion proteins of the present invention also can be the variant of therapeutic protein described herein.Term " variant " comprises conservative or nonconservative insertion, deletion and substitute, wherein this variation does not change albuminised osmotic pressure (oncotic), useful part combination and in the non-immunogenic characteristic one or more basically, perhaps gives avtive spot or the active structure domain of therapeutic protein with therapeutic activity.

Particularly, albumin fusion proteins of the present invention can comprise albuminised natural polymorphie variant and the albuminised fragment of people of existing of people, for example disclosed fragment (be HA (Pn), wherein n is 369 to 419) among the EP 322 094.White protein can be derived from any vertebrates, especially any Mammals, for example people, ox, sheep or pig.The nonmammalian white protein includes but not limited to chicken and salmon.The albumin part of albumin fusion proteins can be from the animal different with the therapeutic protein part.

Generally speaking, HA fragment or variant are to 100 amino acid of the youthful and the elderly, preferably to 150 amino acid of the youthful and the elderly.The HA variant can comprise at least one complete structure territory of HA or be made up of it, for example structural domain 1 (amino acid/11-194 of SEQ ID NO:1), structural domain 2 (the amino acid/11 95-387 of SEQ ID NO:1), structural domain 3 (the amino acid 388-585 of SEQ ID NO:1), structural domain 1 and 2 (1-387 of SEQ ID NO:1), structural domain 2 and 3 (195-585 of SEQ ID NO:1) or structural domain 1 and 3 (the amino acid 388-585 of the amino acid/11-194 of SEQ ID NO:1 and SEQ ID NO:1).Each structural domain self is made up of two homology minor structure territories, be 1-105,120-194,195-291,316-387,388-491 and 512-585, and between the minor structure territory flexible joint district comprise residue Lys106 to Glu119, Glu292 to Va1315 and Glu492 to Ala511.

Preferably, the albumin part of albumin fusion proteins of the present invention comprises minor structure territory or structural domain or its conservative modification of at least one HA.If fusions based on the minor structure territory, preferably connects the therapeutic protein part with some or all of adjacent joints.

The antibody of specific combination therapeutic protein also is therapeutic protein

At least one fragment of the antibody that comprises disclosed therapeutic protein in the specific combination table 1 or the albumin fusion proteins of variant are also contained in the present invention.Take explicitly into account term " therapeutic protein " and contained the antibody of combined treatment protein (for example described in the 1st of table 1 the row) and fragment and variant.Therefore, albumin fusion proteins of the present invention can contain at least one fragment of therapeutic protein or at least one fragment or the variant of variant and/or the proteinic antibody of combined treatment.

Antibody structure and background

Known basic antibody structure unit comprises the tetramer.Each tetramer is made of two pairs of identical polypeptide chains, and each is to having one " light chain " (approximately 25kDa) and one " heavy chain " (approximately 50-70kDa).The N-terminal of every chain partly comprises about 100 to 110 or more a plurality of amino acid whose variable region, and it mainly is responsible for antigen recognition.The C-terminal of every chain partly is defined as constant region, and it mainly is responsible for effector function.People's light chain is divided into κ and lambda light chain.Heavy chain is divided into μ, δ, γ, α or ε, and antibody isotype is defined as IgM, IgD, IgG, IgA and IgE respectively.Generally referring to " FundamentalImmunology ", the 3-5 chapter, Paul, W. compiles, and the 4th edition, Raven Press, N.Y., 1998 (its complete being incorporated herein is used for all purposes).The variable region of every pair of light chain/heavy chain forms antibody combining site.

Therefore, complete IgG antibody has two binding sites.Except that difunctional or bi-specific antibody, these two binding sites are identical.

All chains all demonstrate identical one-piece construction, promptly by three hypervariable regions, are also referred to as complementary determining region or CDR, the conservative relatively framework region (FR) that couples together.The CDR district normally contacts and determines its specific part in the antibody with antigen.CDR from each right heavy chain and light chain aligns by framework region, thereby can combine with defined epitope.To the C-end, light chain and variable region of heavy chain be IncFlds FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 all from the N-end.The variable region links to each other with heavy chain or constant region of light chain.The amino acid of each structural domain distributes and meets Kabat, " Sequences ofProteins of Immunological Interest ", National Institutes of Health, Bethesda, Md., 1987 and 1991; Chothia and Lesk, J.Mol.Biol.196:901-917,1987; People such as Chothia, Nature 342:878-883,1989 definition.

When being used for this paper, " antibody " refers to the immunocompetence part of immunoglobulin molecules and immunoglobulin molecules, promptly contains the molecule (molecule that for example contains one or more CDR district of antibody) of the antigenic antigen binding site of specific combination.Can include but not limited to monoclonal corresponding to the therapeutic protein antibody partly of albumin fusion proteins, polyspecific, the people's, humanized or chimeric antibody, single-chain antibody (for example strand Fv), the Fab fragment, F (ab ') fragment, fragment by the generation of Fab expression library, antiidiotype (anti-Id) antibody (comprising for example special anti-Id antibody) to antibody of the present invention, with any above-mentioned epi-position binding fragment (VH structural domain for example, the VL structural domain, perhaps one or more CDR district).

The proteinic antibody of combined treatment

At least one fragment of the antibody that comprises combined treatment protein (for example disclosed in the table 1) or its fragment or variant or the albumin fusion proteins of variant are contained in the present invention.

The antibody of combined treatment protein (or its fragment or variant) can comprise birds and mammals from any animal origin.Preferably, antibody is the antibody of people, mouse (for example mouse and rat), donkey, sheep, rabbit, goat, cavy, camel, horse or chicken.Most preferably, antibody is people's antibody.When being used for this paper, " people's " antibody comprises the antibody of the aminoacid sequence with human normal immunoglobulin, and comprises that transgenic mice (xenomice) or other organism generation people antibody separate the antibody that obtains with passing through genetically engineered from the human normal immunoglobulin storehouse.

Combined treatment protein and can be any type (for example IgG, IgE, IgM, IgD, IgA and IgY), class (for example IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or the subclass of immunoglobulin molecules corresponding to the antibody molecule of the therapeutic protein of albumin fusion proteins of the present invention part.In preferred embodiments, combined treatment protein and can be IgG1 corresponding to the antibody molecule of the therapeutic protein of albumin fusion proteins part.In other embodiment preferred, combined treatment protein and can be IgG2 corresponding to the immunoglobulin molecules of the therapeutic protein of albumin fusion proteins part.In other embodiment preferred, combined treatment protein and can be IgG4 corresponding to the immunoglobulin molecules of the therapeutic protein of albumin fusion proteins part.

Most preferably, combined treatment protein and can be human antigen's binding antibody fragment of the present invention corresponding to the antibody of the therapeutic protein of albumin fusion proteins part includes but not limited to the Fv (sdFv) that Fab, Fab ' are connected with F (ab ') 2, Fd, strand Fv (scFv), single-chain antibody, disulfide linkage and comprises VL or the fragment of VH structural domain.Antigen binding antibody fragment comprises single-chain antibody, can only comprise the variable region, perhaps together with following whole or part: hinge region, CH1, CH2 and CH3 structural domain.

Combined treatment protein and can be antibody monospecific, dual specific, tri-specific or higher polyspecific corresponding to the antibody of the therapeutic protein of albumin fusion proteins part.Multi-specificity antibody can be that the different epi-positions to therapeutic protein have specificity, perhaps can be that the two all has specificity to therapeutic protein and allos epi-position such as heterologous polypeptide or solid support.Referring to for example PCT publication WO 93/17715; WO 92/08802; WO 91/00360; WO92/05793; People such as Tutt, J.Immunol.147:60-69,1991; U.S. Patent number 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; People such as Kostelny, J.Immunol.148:1547-1553,1992.

The antibody of combined treatment protein (or its fragment or variant) can be dual specific or bi-functional, this means that antibody is the artificial hybrid antibody with two pairs of different heavy chain/light chains and two different binding sites.Can utilize several different methods, comprise that hybridoma merges or the segmental connection of Fab ' generates bi-specific antibody.Referring to for example Songsivilai and Lachmann, Clin.Exp.Immunol.79:315-321,1990; People such as Kostelny, J.Immunol.148:1547-1553,1992.In addition, bi-specific antibody can be with " double antibody " (people such as Holliger, " ' Diabodies ': small bivalent andbispecific antibody fragments ", PNAS USA 90:6444-6448,1993) or " Janusins " (people such as Traunecker, " Bispecific single chain molecules (Janusins) targetcytotoxic lymphocytes on HIV infected cells ", EMBO J 10:3655-3659,1991 and people such as Traunecker, " Janusin:new molecular design for bispecific reagents ", Int.J.Cancer supplementary issue 7:51-52,1992) form forms.

The present invention also provides the fragment that comprises described herein or the antibody that other approach of this area is known or the albumin fusion proteins of variant (comprising derivative).Can utilize the standard technique that those skilled in the art will know that, comprise the site-directed mutagenesis and the PCR mediated mutagenesis that for example cause amino acid replacement, sudden change be introduced in the nucleotide sequence of code book invention molecule.Preferably, variant (comprising derivative) is less than 50 amino acid replacements, is less than 40 amino acid replacements, is less than 30 amino acid replacements, is less than 25 amino acid replacements, is less than 20 amino acid replacements, is less than 15 amino acid replacements, is less than 10 amino acid replacements, is less than 5 amino acid replacements, is less than 4 amino acid replacements, is less than 3 amino acid replacements or is less than 2 amino acid replacements with respect to reference VH structural domain, VHCDR1, VHCDR2, VHCDR3, VL structural domain, VLCDR1, VLCDR2 or VLCDR3 coding.In specific embodiment, variant coding VHCDR3 substitutes.In a preferred embodiment, it is alternative that variant has conserved amino acid at the non-key amino-acid residue place of one or more predictions.

Combined treatment protein and can be described or illustrate aspect the epi-position of the therapeutic protein of its identification or specific combination or the part corresponding to the antibody of the therapeutic protein of albumin fusion proteins part.The antibody that can also get rid of the defined epitope of specific combination therapeutic protein or therapeutic protein.Therefore, the antibody of specific combination therapeutic protein is contained in the present invention, and allows and get rid of described antibody.In preferred embodiments, comprise at least one fragment of the proteinic antibody of combined treatment or variant albumin fusion proteins and this antibody self do not merge fragment or variant in conjunction with identical epi-position.

Combined treatment protein and can also can being described or illustrating aspect its cross reactivity corresponding to the antibody of the therapeutic protein of albumin fusion proteins part.Any other analogue of debond therapeutic protein, directly in the antibody of homologue (ortholog) or homologue is also included within.Be also included among the present invention in conjunction with the antibody that has a polypeptide of at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55% and at least 50% sequence identity (utilize known in the art and described herein method calculate) with therapeutic protein.In specific embodiment, combined treatment protein and can be corresponding to the mouse of the antibody of the therapeutic protein of albumin fusion proteins part and human protein, rat/or rabbit homologue and corresponding epi-position generation cross reaction thereof.Debond and therapeutic protein have less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55% be also included among the present invention less than the antibody of the polypeptide of 50% sequence identity (utilizing method known in the art and described herein to calculate).In a specific embodiment, cross reactivity mentioned above relates to any single specific antigen disclosed herein or immunogenic polypeptide, and perhaps 2,3,4,5 or the combination of more kinds of specific antigen and/or immunogenic polypeptide.In preferred embodiments, the albumin fusion proteins that comprises at least one fragment of the proteinic antibody of combined treatment or variant has fragment or the similar or essentially identical cross reactivity feature of variant to this specific antibodies self.

The present invention also comprises the antibody in conjunction with the polypeptide of the polynucleotide encoding of being hybridized by the polynucleotide generation of (as described herein) and coding therapeutic protein under rigorous hybridization conditions.Combined treatment protein and can also can aspect the binding affinity of polypeptide of the present invention, being described or illustrating at it corresponding to the antibody of the therapeutic protein of albumin fusion proteins of the present invention part.Preferred binding affinity comprises that dissociation constant or Kd are less than 5x10 ^-2M, 10 ^-2M, 5x10 ^-3M, 10 ^-3M, 5x10 ^-4M, 10 ^-4Binding affinity.Preferred binding affinity comprises that dissociation constant or Kd are less than 5x10 ^-5M, 10 ^-5M, 5x10 ^-6M, 10 ^-6M, 5x10 ^-7M, 10 ^-7M, 5x10 ^-8M or 10 ^-8The binding affinity of M.Even preferred binding affinity comprises that dissociation constant or Kd are less than 5x10 ^-9M, 10 ^-9M, 5x10 ^-10M, 10 ^-10M, 5x10 ^-11M, 10 ^-11M, 5x10 ^-12M, 10 ^-12M, 5x10 ^-13M, 10 ^-13M, 5x10 ^-14M, 10 ^-14M, 5x10 ^-15M or 10 ^-15The binding affinity of M.In preferred embodiments, consider tiring of albumin fusion proteins (at least one fragment or the variant that comprise the proteinic antibody of combined treatment) and tiring of corresponding antibody, the albumin fusion proteins that comprises at least one fragment of the proteinic antibody of combined treatment or variant is similar with the avidity of the proteinic antibody of corresponding combined treatment (to the white protein fusion) to the avidity of given protein or epi-position.

The present invention also provides according to known in the art and has been used to measure for example mensuration of immunoassay described herein of any method of competitive bonded, and competitive inhibition antibody is to the bonded antibody of the epi-position of therapeutic protein.In preferred embodiments, antibody competition has suppressed the combination of 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% pair of epi-position.In preferred embodiments, comprise of the combination of the albumin fusion proteins competitive inhibition second antibody of at least one fragment of the proteinic antibody of combined treatment or variant to the epi-position of therapeutic protein.In other embodiment preferred, comprise the combination of the albumin fusion proteins competitive inhibition at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60% of at least one fragment of the proteinic antibody of combined treatment or variant or at least 50% second antibody to the epi-position of therapeutic protein.

Combined treatment protein and the agonist or the antagonist that can be used as therapeutic protein corresponding to the antibody of the therapeutic protein of albumin fusion proteins of the present invention part work.For example, the present invention includes and partially or completely interrupt receptor/ligand and the interactional antibody of polypeptide of the present invention.Characteristic of the present invention not only has receptor specific antibody, and ligand specificity's antibody is arranged.Characteristic of the present invention does not stop the part combination but the receptor specific antibody of prevention receptor activation in addition.Can utilize technology described herein or that other approach of this area is known to determine receptor activation (being the signal conduction).For example, can determine receptor activation by the phosphorylation (for example tyrosine or serine/threonine) of immunoprecipitation and western blot analysis subsequently (for example mentioned above) detection acceptor or its substrate.In specific embodiment, provide ligand activity or the receptor active activity when not having antibody has been suppressed 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.In preferred embodiments, the albumin fusion proteins that comprises at least one fragment of the proteinic antibody of combined treatment or variant stop have aspect part combination and the/receptor activation to the proteinic antibody of combined treatment do not merge fragment or variant is similar or similar substantially feature.

Characteristic of the present invention stop in addition part in conjunction with and the two receptor specific antibody of receptor activation, and the antibody of identification receptor-ligand complex, and preferably not unconjugated acceptor of specific recognition or unconjugated part.Similarly, the present invention includes binding partner and stop part and the neutrality antibody of receptors bind, and binding partner stops receptor activation thus but do not stop the antibody of part and receptors bind.The present invention also comprises the antibody of activated receptor.These antibody can be used as receptor stimulant, promptly strengthen or activate the whole or subclass of the biologic activity of ligand-mediated receptor activation, for example by inducing the dimerization of acceptor.Can specify antibody is the active agonist of particular biological, antagonist or the inverse agonist that biologic activity comprises therapeutic protein (for example disclosed in the table 1).Utilize methods known in the art can generate above-mentioned antibody agonist.For example referring to PCT publication WO 96/40281; U.S. Patent number 5,811,097; People such as Deng, Blood 92 (6): 1981-1988,1988; People such as Chen, Cancer Res.58 (16): 3668-3678,1998; People such as Harrop, J.Immunol.161 (4): 1786-1794,1998; People such as Zhu, Cancer Res.58 (15): 3209-3214,1998; People such as Yoon, J.Immunol.160 (7): 3170-3179,1998; People such as Prat, J.Cell.Sci.111 (Pt2): 237-147,1998; People such as Pitard, J.Immunol.Methods 205 (2): 177-190,1997; People such as Liautard, Cytokine9 (4): 233-241,1997; People such as Carlson, J.Biol.Chem.271 (17): 11295-11301,1997; People such as Taryman, Neuron 14 (4): 755-762,1995; People such as Muller, Structure6 (9): 1153-1167,1998; People such as Bartunek, Cytokine 8 (1): 14-20,1996 (with they all complete being incorporated herein by reference).In preferred embodiments, the albumin fusion proteins that comprises at least one fragment of the proteinic antibody of combined treatment or variant has not fusion fragment or similar or essentially identical agonist of variant or the antagonist properties to the proteinic antibody of combined treatment.

For example, can utilize combined treatment protein and can come purifying, detection and targeted therapeutic protein, comprise external and intravital diagnosis and methods of treatment corresponding to the therapeutic protein antibody partly of albumin fusion proteins of the present invention.For example, antibody can be used for the immunoassay of the level of therapeutic protein in qualitative and the quantitative measurment biological sample.For example referring to people such as Harlow, " Antibodies:ALaboratory Manual ", Cold Spring Harbor Laboratory Press, the 2nd edition, 1988, with its complete being incorporated herein by reference.Similarly, for example, can utilize at least one fragment that comprises the proteinic antibody of combined treatment or the albumin fusion proteins of variant to come purifying, detection and targeted therapeutic protein, comprise external and intravital diagnosis and methods of treatment.

Combined treatment protein and can comprise derivative through modifying corresponding to the antibody of the therapeutic protein of albumin fusion proteins part promptly is covalently attached to antibody by the molecule with any kind.For example and unrestricted, antibody derivatives for example comprise derivatize by glycosylation, acetylize, PEGization, phosphorylation, amidation, known protection/blocking groups, proteolysis cutting, with the proteinic antibody that is connected etc. through modification of cell ligand or other.Can utilize known technology to carry out number of chemical any in modifying, include but not limited to that the metabolism of specificity chemical chop, acetylize, formylation, tunicamycin is synthetic etc.In addition, derivative can contain one or more nonclassical amino acids.Also can modify albumin fusion proteins of the present invention as mentioned above.

Generate the method for the proteinic antibody of combined treatment

Can generate combined treatment protein and can be by any appropriate means known in the art corresponding to the antibody of the therapeutic protein of albumin fusion proteins of the present invention part.Can generate at the antigenic polyclonal antibody of purpose by various programs well-known in the art.For example, can give various host animals, include but not limited to rabbit, mouse, rat etc., administering therapeutic protein produces the serum that contains the polyclonal antibody of antigen-specific to induce.According to host species, can utilize various adjuvants to improve immunne response, include but not limited to Fu Shi (fully with incomplete) adjuvant, mineral coagulant agent such as aluminium hydroxide, surfactant such as lysolecithin, Pluronic polyvalent alcohol, polyanion, peptide, oil emulsion, key hole

People's adjuvant such as the BCG (bacille Calmette-Guerin vaccine) and the spillikin bacillus of hemocyanin, dinitrophenol and potentially useful.This adjuvant also is well-known in the art.

Can utilize multiple technologies known in the art, comprise and utilize hybridoma, recombinant chou and display technique of bacteriophage or its combination, prepare monoclonal antibody.For example, can utilize hybridoma technology, comprise known in the art and people such as for example Harlow, " Antibodies:A Laboratory Manual ", ColdSpring Harbor Laboratory Press, the 2nd edition, 1988; People such as Hammering, in " MonoclonalAntibodies and T-Cell Hybridomas ", page number 563-681, Elsevier, N.Y., the hybridoma technology of instruction generates monoclonal antibody in 1981 (being incorporated herein by reference described reference is complete).Term " monoclonal antibody " is not limited to the antibody by the hybridoma technology generation when being used for this paper.Term " monoclonal antibody " refers to the antibody derived from single clone, comprises any eucaryon, protokaryon or phage clone, rather than refers to generate its method.

Utilizing the method for hybridoma technology generation and screening specific antibody is routine and well-known in this area.In a limiting examples, can be with the cellular immunization mouse of therapeutic protein or its fragment or variant, albumin fusion proteins, this therapeutic protein of expression or its fragment or variant or albumin fusion proteins.In case detect immunne response, for example in mice serum, detect antibody to antigen-specific, just gather in the crops spleen and the separating Morr. cell of mouse.Then by well-known technology with splenocyte and any suitable myeloma cell, for example, merge from the cell of the clone SP20 that can obtain from ATCC.Select and the clone hybridization knurl by limiting dilution.Measure the hybridoma clone by methods known in the art then, can be to select secretion in conjunction with the cell of the antibody of polypeptide of the present invention.Can produce the ascites that contains high-level antibody usually with positive hybridoma clone immune mouse.

Therefore, the invention provides the generation monoclonal antibody method, and the antibody that generates by the method that comprises the following steps, promptly cultivate the hybridoma of secretory antibody, wherein preferably, hybridoma is by will generating by merging through the isolating splenocyte of the mouse of antigen immune of the present invention and myeloma cell, can clone in conjunction with the hybridoma of the antibody of polypeptide of the present invention merging the hybridoma screening secretion that produces then.

The another kind of well-known method that is used to generate two kinds of human B cells systems of polyclone and mono-clonal is the conversion that utilizes dust crust Er Shi virus (EBV) to carry out.Being used to generate the scheme that EBV transforms B clone is that this area is generally known, such as people such as for example Coligan, compiles " Current Protocols inImmunology ", 1994, John Wiley ﹠amp; Sons, NY, the scheme of 7.22 chapters general introduction is with its complete being incorporated herein by reference.The source that transforms with the B cell generally is a human peripheral, also can originate derived from other with the B cell but transform, and includes but not limited to lymphoglandula, tonsilla, spleen, tumor tissues and infected tissue.Before EBV transforms, earlier tissue is made single-cell suspension liquid usually.In addition, in the sample that contains the B cell, can take some step physical removals or deactivation T cell (for example handling), because the T cell of the individuality that is positive from the anti-EBV antibody of serum can suppress the B cell immortalityization that EBV causes with cyclosporin A.

Usually, contain the sample of human B cell with the EBV inoculation, and cultivate 3-4 week.The typical case source of EBV is the culture supernatants of B95-8 clone (ATCC numbers VR-1492).During approaching end of 3-4 week incubation period, can see the physical indication that EBV transforms usually.Show the emblem sem observation by differing, that transformant can show as is big, clear, hair shape and tend to closely assembling in the cell mass.At first, EBV system is normally polyclonal.Yet, prolonging cell cultures after the time, EBV system can become mono-clonal or polyclonal owing to the selective growth of particular B cell clone.Perhaps, can transform system to polyclonal EBV and carry out subclone (for example cultivating), perhaps be coated with to obtain monoclonal B clone with suitable fusion partner fusion and with limiting dilution by limiting dilution.The suitable fusion partner of EBV transformation cell lines comprises mouse myeloma cell line (for example SP2/0, X63-Ag8.653), assorted myelomatosis (heteromyeloma) clone (people x mouse; For example SPAM-8, SBC-H20 and CB-F7) and human cell line (for example GM1500, SKO-007, RPMI 8226 and KR4).Therefore, the present invention also provides the method that generates at polypeptide of the present invention or its segmental polyclone or monoclonal human antibody, comprises that the EBV of human B cell transforms.

Can generate the antibody fragment of identification defined epitope by known technology.For example, enzyme be can utilize,, Fab of the present invention and F (ab ') 2 fragments generated by the proteolysis cutting of immunoglobulin molecules such as papoid (producing the Fab fragment) or stomach en-(generation F (ab ') 2 fragments).F (ab ') 2 fragments contain the CH1 structural domain of variable region, constant region of light chain and heavy chain.

For example, can also utilize various phage display method known in the art to generate the proteinic antibody of combined treatment.In the phage display method, the functional antibodies structural domain is illustrated on the surface of the phage particle that carries the polynucleotides encoding them sequence.In a specific embodiment, this phage can be used for showing the antigen binding domains by complete or collected works or combinatorial antibody storehouse (for example people or mouse) expression.Can use antigen, for example labelled antigen or in conjunction with or be captured in antigen on solid surface or the pearl and select or identify the phage of expressing the antigenic antigen binding domains of binding purposes.The phage of using in these methods is typically the filobactivirus that comprises fd and M13, wherein merges by having the reorganization of the stable Fv antibody structure of Fab, Fv or the disulfide linkage binding domains territory, phage expression and phage gene III or gene VIII protein.The example that can be used for generating the phage display method of the proteinic antibody of combined treatment comprises people such as Brinkman, J.Immunol.Methods 182:41-50,1995; People such as Ames, J.Immunol.Methods 184:177-186,1995; People such as Kettleborough, Eur.J.Immunol.24:952-958,1994; People such as Persic, Gene187:9-18,1997; People such as Burton, Advances in Immunology 57:191-280,1994; PCT application number PCT/GB91/01134; PCT publication WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; With U.S. Patent number 5,698,426; 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; Disclosed method in 5,733,743 and 5,969,108 is incorporated herein by reference each piece is complete.

Described in above-mentioned reference, after phage is selected, can be from phage separation antibody coding region, and with generating complete antibody, comprise people's antibody or any Fab that other is wanted, and can in any desired host, express, comprise mammalian cell, insect cell, vegetable cell, yeast and bacterium, for example hereinafter described in detail.For example, utilize methods known in the art, such as PCT publication WO 92/22324; People such as Mullinax, BioTechniques 12 (6): 864-869,1992; People such as Sawai, AJRI 34:26-34,1995; And people such as Better, Science240:1041-1043, disclosed in 1988, also can adopt reorganization to generate Fab, Fab ' and F (ab ') 2 segmental technology (being incorporated herein by reference described reference is complete).

The example that can be used for generating the technology of strand Fv and antibody comprises United States Patent (USP) 4,946,778 and 5,258,498; People such as Huston, Methods in Enzymology 203:46-88,1991; People such as Shu, PNAS 90:7995-7999,1993; Reach people such as Skerra, Science 240:1038-1040, described in 1988.For some purposes, comprise purposes and vitro detection assay method in the body of antibody in human body, may preferably use chimeric, humanized or people's antibody.Chimeric antibody is the molecule of the different piece of antibody derived from the different animals species, such as having derived from the variable region of mouse monoclonal antibody and the antibody of human normal immunoglobulin constant region.The method that is used to generate chimeric antibody is known in the art.For example referring to Morrison, Science 229:1202,1985; People such as Oi, BioTechniques 4:214,1986; People such as Gillies, J.Immunol.Methods 125:191-202,1989; U.S. Patent number 5,807,715; 4,816,567 and 4,816397, with they complete being incorporated herein by reference.Humanized antibody be from conjunction with the antigenic inhuman species antibody of expectation, have one or more complementary determining regions (CDR) and from the antibody molecule of the framework region of human normal immunoglobulin molecule from inhuman species.Through being commonly used to the framework residue in the alternative people's framework region of the corresponding residue of CDR donor antibody, to change, the preferred improvement combining with antigenic.Utilize method well-known in the art can identify that these frameworks substitute, for example set up CDR and the interactional model of framework residue identifying antigen, and comparative sequences is to identify the uncommon framework residue of specific location in conjunction with important framework residue.(for example referring to people such as Queen, U.S. Patent number 5,585,089; People such as Riechmann, Nature 332:323,1988, with they complete being incorporated herein by reference.) can utilize several different methods known in the art with the antibody humanization, (EP 239,400 to comprise for example CDR grafting; PCT publication WO 91/09967; U.S. Patent number 5,225,539; 5,530,101 and 5,585,089), (EP 592,106 for facing (veneering) or resurfacing (resurfacing); EP 519,596; Padlan, Molecular Immunology 28 (4/5): 489-498,1991; People such as Studnicka, Protein Engineering 7 (6): 805-814,1994; People such as Roguska, PNAS 91:969-973,1994) and chain reorganization (U.S. Patent number 5,565,332).

People's antibody treatment human patients is wanted especially fully.Can be by several different methods known in the art, the phage display method that comprises the aforesaid antibody library that utilizes the derived from human immunoglobulin sequences antibody of being grown up next life.Also can be referring to U.S. Patent number 4,444,887 and 4,716,111; PCT publication WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741 are incorporated herein by reference each piece is complete.

Also can utilize the transgenic mice of can not the endogenous immunoglobulin (Ig) of expressive function but can the expressing human immunoglobulin gene antibody of being grown up next life.For example, can be at random or by homologous recombination people's heavy chain and light chain immunoglobulin gene mixture are imported mouse embryo stem cell.Perhaps, outside people's heavy chain and light chain gene, also people variable region, constant region and diversity zone can be imported in the mouse embryo stem cell.Import human immunoglobulin gene's seat by homologous recombination, can be respectively or make murine heavy chain and light chain immunoglobulin (Ig) not have function simultaneously.Particularly, the homozygous deletion in JH district stops the endogenous antibody of generation.The embryonic stem cell of amplification through modifying, and microinjection in the blastocyst to produce gomphosis mouse.Breed gomphosis mouse then to produce the offspring of isozygotying of expressing human antibody.With normal way, with the antigen of selecting, for example whole or partial immunity transgenic mice of polypeptide of the present invention.Utilize conventional hybridoma technology to obtain at antigenic monoclonal antibody from transgenic mice through immunity.The human normal immunoglobulin transgenosis that transgenic mice comprises is reset in the B cell differentiation procedure, experiences classification conversion and somatic mutation subsequently.Therefore, utilize this technology, might be created on treatment and go up useful IgG, IgA, IgM and IgE antibody.The general introduction that is used to generate this technology of people's antibody can be referring to Lonberg and Huszar, Int.Rev.Immunol.13:65-93,1995.Be used to generate the going through of this technology of people's antibody and human monoclonal antibodies, and be used to generate the scheme of this antibody can be referring to for example PCT publication WO 98/24893; WO92/01047; WO 96/34096; WO 96/33735; European patent number 0598877; U.S. Patent number 5,413,923; 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 and 6,114,598, with they complete being incorporated herein by reference.In addition, can employ such as Abgenix (Freemont, CA) and Genpharm (San Jose, CA) etc. company utilizes provides at selected antigenic people's antibody to similar technology mentioned above.

The technology that utilization is called " guiding is selected " can generate the antibody that the complete people of epi-position is selected in identification.In the method, utilize selected non-human monoclonal antibodies, mouse antibodies for example guides the complete people's who selects the identical epi-position of identification antibody people such as (, Bio/technology 12:899-903,1988) Jespers.

The polynucleotide of encoding antibody

The present invention also provides the polynucleotide that comprise encoding antibody and segmental nucleotide sequence thereof.The present invention also is encompassed in the polynucleotide that hybridization takes place with the polynucleotide of encoding antibody under the rigorous or low rigorous hybridization conditions for example defined above, the wherein preferred proteinic antibody of specificity combined treatment, the antibody of the polypeptide of the aminoacid sequence of disclosed " therapeutic protein X " in more preferably being listed as in conjunction with " SEQ ID NO:Z " with table 2.

Can obtain the nucleotide sequence of polynucleotide and mensuration polynucleotide by any method known in the art.For example, if the nucleotide sequence of antibody is known, can assemble the polynucleotide of this antibody of coding so from the oligonucleotide of chemosynthesis (for example as people such as Kutmeier, BioTechniques 17:242, described in 1994), in brief, it relates to the synthetic overlapping oligonucleotide that contains the partial sequence of encoding antibody, annealing also connects these oligonucleotide, the oligonucleotide after connecting by pcr amplification then.

Perhaps, can be from generate the polynucleotide of encoding antibody from the nucleic acid of appropriate sources.If can not obtain containing the clone of nucleic acid of specific antibodies of encoding, but the sequence of this antibody molecule is known, nucleic acid that so can the chemical synthesis coding immunoglobulin (Ig), perhaps utilize with 3 of sequence ' and hybridization takes place 5 ' end synthetic primer by pcr amplification and from appropriate sources (antibody cDNA storehouse for example, perhaps from expressing any tissue or the cell of this antibody, such as the cDNA storehouse of the hybridoma generation of selecting expressing antibodies or by its isolating nucleic acid, preferred polyA+RNA) obtains or utilize the clone that the special oligonucleotide probe of specific gene sequence is carried out to identify, for example from the cDNA clone in the cDNA storehouse of encoding antibody.Can utilize any method well-known in the art to be cloned into (referring to embodiment 65) in the reproducible cloning vector then by the amplification of nucleic acid that PCR generates.

In case measured the nucleotide sequence and the corresponding amino acid sequence of antibody, can utilize the method that is used to operate nucleotide sequence well-known in the art to operate the nucleotide sequence of antibody, recombinant DNA technology for example, site-directed mutagenesis, PCR or the like is (for example referring to people such as Sambrook, 1990, " MolecularCloning, A Laboratory Manual ", the 2nd edition, Cold Spring Harbor Laboratory, ColdSpring Harbor, people such as NY and Ausubel, compile, 1998, " Current Protocols in MolecularBiology ", John Wiley ﹠amp; Sons, the technology of describing among the NY is with they complete being incorporated herein by reference), with the antibody that generation has the different aminoacids sequence, for example produce amino acid replacement, deletion and/or insertion.

In a specific embodiment, by method well-known in the art, for example compare to determine the sequence hypervariable region by the known amino acid sequence with other heavy chain and variable region of light chain, the aminoacid sequence that can check heavy chain and/or light chain variable territory is to identify the sequence of complementary determining region (CDR).Utilize conventional recombinant DNA technology, one or more CDR can be inserted in the framework region, for example people's framework region makes non-human antibody's humanization, as mentioned above.Framework region can be naturally occurring or total framework region, preferred people's framework region (for example referring to people such as Chothia, J.Mol.Biol.278:457-479,1998 people's framework regions of listing).Preferably, the antibody of the polynucleotide encoding specific combination polypeptide of the present invention that produces by group frame district and CDR.Preferably, as mentioned above, can produce one or more amino acid replacements in framework region, preferably, amino acid replacement has improved antibody to its antigenic combination.In addition, can carry out amino acid replacement or delete the antibody molecule that lacks one or more intrachain disulfide bonds with generation the one or more variable region cysteine residues that participate in intrachain disulfide bond in this way.Other change to polynucleotide is contained in the present invention, and they are also in the technical scope of this area.

In addition, can utilize in order to generate " chimeric antibody " and technology (people such as Morrison, Proc.Natl.Acad.Sci.81:851-855,1984 of exploitation; People such as Neuberger, Nature 312:604-608,1984; People such as Takeda, Nature 314:452-454,1985), be about to from the gene with the specific mouse antibodies molecule of suitable antigen with from the gene splicing of human antibody molecules together with suitable biologic activity.As mentioned above, chimeric antibody is the molecule of different piece derived from the different animals species, has derived from the variable region of mouse mAb and the antibody of human normal immunoglobulin constant region, for example humanized antibody such as those.

Perhaps, technology (U.S. Patent number 4,946,778 that can make description be used to prepare single-chain antibody; Bird, Science 242:423-42,1988; People such as Huston, Proc.Natl.Acad.Sci.USA 85:5879-5883,1988; And people such as Ward, Nature 334:544-54,1989) be suitable for generating single-chain antibody.Couple together by the heavy chain and the light chain segments of amino acid bridge, produce single chain polypeptide, form single-chain antibody thus the Fv district.Also can use the segmental technology of assembling function Fv in intestinal bacteria (people such as Skerra, Science 242:1038-1041,1988).

The recombinant chou of antibody is expressed

The recombinant expressed expression vector that needs to make up the polynucleotide that contain encoding antibody of antibody or its fragment, derivative or analogue (for example heavy chain of antibody or light chain or single-chain antibody).The polynucleotide of antibody molecule of the present invention or heavy chain of antibody or light chain or its part (preferably containing heavy chain or light chain variable territory) in case obtain encoding just can utilize technology well-known in the art to generate the carrier that is used to produce antibody molecule by recombinant DNA technology.Therefore, this paper has described the polynucleotide that contain the nucleotide sequence of encoding antibody by expression and has prepared method of protein.Can utilize the well-known method of those skilled in the art to make up and contain antibody coding sequence and the suitable expression vector of transcribing and translate control signal.These methods comprise for example interior genetic recombination of extracorporeal recombinant DNA technology, synthetic technology and body.Therefore, the invention provides the replicable vector of the nucleotide sequence that comprises the coding that can be operatively connected antibody molecule of the present invention or its heavy chain or light chain or heavy chain or light chain variable territory with promotor.This carrier can comprise that the nucleotide sequence of encoding antibody molecule constant region is (for example referring to PCT publication WO 86/05807; PCT publication WO 89/01036; With U.S. Patent number 5,122,464), and in order to express whole heavy chain or light chain, the variable domain of antibody can be cloned in this carrier.

By routine techniques expression vector is transferred in the host cell, cultivated transfectional cell to produce antibody by ordinary method then.Therefore, the present invention includes the host cell of the polynucleotide that contain the coding that can be operatively connected antibody molecule of the present invention or its heavy chain or light chain or single-chain antibody with allogeneic promoter.In expressing the preferred embodiment of double-stranded antibody, can be in host cell the carrier of coexpression encoding heavy chain and light chain to express whole immunoglobulin molecules, as what hereinafter described in detail.

Can utilize multiple host-expression vector system to express antibody molecule of the present invention.This host-expression system has been represented and can have been generated and the media of subsequent purificn purpose encoding sequence, but has also represented the cell of expressed in situ antibody molecule of the present invention after with suitable nucleotide coding sequence conversion or transfection.They include but not limited to recombinant phage dna, plasmid DNA or cosmid DNA expression vector microorganism transformed such as bacterium (for example intestinal bacteria, subtilis) through containing antibody coding sequence; The yeast (for example saccharomyces, Pichia) that recombinant yeast expression vector through containing antibody coding sequence transforms; The insect cell system that recombinant virus expression vector through containing antibody coding sequence (for example baculovirus) infects; Recombinant virus expression vector through containing antibody coding sequence (cauliflower mosaic virus for example, CaMV; Tobacco mosaic virus (TMV), TMV) infection or the recombinant plasmid expression vector through containing antibody coding sequence (for example Ti-plasmids) plant transformed cell system; Or comprise the mammal cell line system (for example COS, CHO, BHK, 293,3T3 cell) that contains derived from the recombinant expression construct thing of the promotor of mammalian cell genome (for example metallothionein promoter) or mammalian virus (for example gland virus stage starting, vaccinia virus 7.5K promotor).Preferably, use bacterial cell, such as intestinal bacteria, more preferably eukaryotic cell especially when expressing whole recombinant antibody molecule, comes the expressing recombinant antibody molecule.For example, such as Chinese hamster ovary cell mammalian cells such as (CHO) together with such as carriers such as main immediate early gene promoter element being effective antibody expression system (people such as Foecking, Gene 45:101,1986 from the people cytomegalovirus; People such as Cockett, Bio/Technology 8:2,1990).

In bacterial system, according to the desired use of expressed antibody molecule, the multiple expression vector of selection that can be favourable.For example, when generating the pharmaceutical composition of antibody molecule, may want to instruct the carrier of the fusion protein product high level expression of easy purifying at a large amount of this protein of preparation.This carrier includes but not limited to coli expression carrier pUR278 (people such as Ruther, EMBO J.2:1791,1983), thereby wherein can be connected to antibody coding sequence in the carrier individually and the identical generation with the lacZ coding region of reading frame fusion rotein; PIN carrier (Inouye and Inouye, Nucleic Acids Res.13:3101-3109,1985; Van Heeke and Schuster, J.Biol.Chem.24:5503-5509,1989) or the like.Also can express the allogenic polypeptide that forms fusion rotein with glutathione S-transferase (GST) with the pGEX carrier.In general, this fusion rotein is soluble, and by with the absorption of matrix gsh-agarose pearl and combine and subsequently the wash-out when having free glutathione can be easy to that purifying comes out from dissolved cell.The pGEX carrier design is become to comprise zymoplasm or factor Xa proteolytic enzyme cutting site, the target gene product of DCRP can partly be discharged from GST.

In the insect system, utilize autographa california nuclear polyhedrosis virus (Autographacalifomica nuclear polyhedrosis virus) (AcNPV) to come expression alien gene as carrier.Virus is grown in fall army worm (Spondoptera frugiperda) cell.Antibody coding sequence can be cloned into individually in the nonessential region (for example polyhedron gene) of virus, and place under the control of AcNPV promotor (for example polyhedrin promotor).

In mammalian host cell, can use many expression systems based on virus.If use adenovirus as expression vector, the purpose antibody coding sequence can be transcribed with adenovirus/is translated control mixture for example late promoter be connected with tripartite leader[.This mosaic gene can be inserted in the adenoviral gene group by reorganization in external or the body then.Insertion in the viral genome nonessential region (for example E1 or E3 district) will produce can survive and can be in infected host the recombinant virus of expressed antibody molecule (for example referring to Logan and Shenk, Proc.Natl.Acad.Sci.USA 81:355-359,1984).Also may need specific start signal in order effectively to translate the antibody coding sequence that inserts.These signals comprise the sequence of ATG initiator codon and adjacency.And, initiator codon must with the expectation encoding sequence the reading frame homophase to guarantee the translation of whole inset.These external sources translation control signals and initiator codon can have multiple source, and natural the two all can with synthetic.Can strengthen expression efficiency (referring to people such as Bittner, Methods in Enzymol.153:51-544,1987) by comprising suitable transcriptional enhancer element, transcription terminator etc.

In addition, can select expression, perhaps the host cell strain of modification and processed gene product with the ad hoc fashion regulation and control insertion sequence of wanting.This modification of protein (for example glycosylation) and processing (for example cutting) may be important for proteinic function.Different host cells has distinctive and special protein and gene product translation post-treatment and modified mechanism.Can select suitable clone or host system to guarantee the correct modification and the processing of expressed foreign protein.For this reason, can use the eukaryotic host cell of cell mechanism with the initial transcript of correct processing, glycosylation and phosphorylation gene product.This mammalian host cell includes but not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293,3T3, W138, breast cancer cell line particularly, such as for example BT483, Hs578T, HTB2, BT20 and T47D, and MCF-10, such as for example CRL7030 and Hs578Bst.

For production recombinant protein long-term, high yield, preferred stable expression.For example, can transform the clone of stably express antibody molecule.Contain the expression vector of virus replication starting point with its utilization, not as good as with the DNA and the selective marker transformed host cell that are subjected to suitable expression controlling elements (for example promotor, enhanser, sequence, transcription terminator, polyadenylation site or the like) control.After importing foreign DNA, can allow in enriched medium, to grow 1-2 days through the cell of transforming, change the selection substratum then into.Selective marker in the recombinant plasmid is given at the resistance of selecting, and allows in the cell karyomit(e) that is incorporated into them that plasmid is stable and growth forms focus (foci), can clone and increase then to be clone.This method of utilizing that can be favourable is transformed the clone of expressed antibody molecule.This through the clone transformed directly or indirectly and particularly useful aspect the interactional compound of antibody molecule in screening and assessment.

Can utilize many selective systems, include but not limited to the herpes simplex virus thymidine kinase (people such as Wigler that can in tk-, hgprt-or aprt-cell, use respectively, Cell 11:223,1977), xanthoglobulin-guanine phosphoribosyltransferase (Szybalska and Szybalski, Proc.Nat1.Acad.Sci.USA 48:202,1992) and adenine phosphoribosyl transferase (people such as Lowy, Cell 22:817,1980) gene.And the metabolic antagonist resistance also can be used as the basis of selecting following gene: dhfr (people such as Wigler, Natl.Acad.Sci.USA 77:357,1980 of giving the methotrexate resistance; People such as O ' Hare, Proc.Natl.Acad.Sci.USA 78:1527,1981); Give the gpt (Mulligan and Berg, Proc.Natl.Acad.Sci.USA 78:2072,1981) of mycophenolic acid resistance; Give neo (the Clinical Pharmacy 12:488-505 of aminoglycoside G-418 resistance; Wu and Wu, Biotherapy 3:87-95,1991; Tolstoshev, Ann.Rev.Pharmacol.Toxicol.32:573-596,1993; Mulligan, Science 260:926-932,1993; And Morgan and Anderson, Ann.Rev.Biochem.62:191-217,1993; May, TIB TECH 11 (5): 155-215,1993); And give the hygro (people such as Santerre, Gene 30:147,1984) of hygromycin resistance.The method of the recombinant DNA technology that this area is generally known can routine be used to select conceivable recombinant clone, and these methods for example are described in people such as Ausubel, compiles " Current Protocols in MolecularBiology ", John Wiley ﹠amp; Sons, NY, 1993; Kriegler, " Gene Transfer andExpression, A Laboratory Manual ", Stockton Press, NY, 1990; Reach people such as Dracopoli, compile " Current Protocols in Human Genetics ", the 12nd and 13 chapters, John Wiley﹠amp; Sons, NY, 1994; People such as Colberre-Garapin, J.Mol.Biol.150:1,1981, with they complete being incorporated herein by reference.

Can increasing by carrier, (summary is referring to Bebbington and Hentschel for the expression level that improves antibody molecule, The use of vectors based on gene amplification for the expression ofcloned genes in mammalian cells in DNA cloning, the 3rd volume, Academic Press, New York, 1987).When the mark in the carrier system of expressing antibodies be can increase the time, the level that improves the inhibitor that exists in the host cell substratum will increase the copy number of marker gene.Because the zone of amplification is relevant with antibody gene, so the output of antibody also will improve people such as (, Mol.Cell.Biol.3:257,1983) Crouse.

Can when having medicine methionine sulphoximine (methionine sulphoximine) or methotrexate, increase respectively and utilize glutamine synthase (GS) or DHFR carrier as selective marker.Based on the advantage of the carrier of glutamine synthase be can utilize glutamine synthase negative cells system (rat bone marrow tumour cell system for example, NS0).The glutamine synthase expression system can also work in glutamine synthase express cell (for example Chinese hamster ovary (CHO) cell) by the extra inhibitor that prevents native gene performance function is provided.At PCT publication WO 87/04462; WO 86/05807; WO 89/01036; Among WO 89/10404 and the WO 91/06657 in detail glutamine synthase expression system and composition thereof have been described in detail, with they complete being incorporated herein by reference.In addition, the glutamine synthase expression vector that can use according to the present invention can be from for example comprising that (Portsmouth NH) buys in interior suppliers in Lonza Biologics company.People such as Bebbington, Bio/technology 10:169,1992 and Biblia and Robinson, Biolechnol.Prog.11:1, described in 1995 and utilized the GS expression system in rat bone marrow tumour cell, to express and the manufacture order clonal antibody, at this with they complete being incorporated herein by reference.

Can be with two kinds of expression vector cotransfection host cells of the present invention, first kind of vector encoded heavy chain polypeptides derived wherein, and the polypeptide of second kind of vector encoded derived light chain.These two kinds of carriers can contain the identical selective marker that makes that heavy chain and light chain polypeptide equivalent are expressed.Perhaps, also can use coding and can express the two single carrier of heavy chain and light chain polypeptide.In these cases, light chain should place before the heavy chain, to avoid poisonous free heavy chain excessive (Proudfoot, Nature 322:52,1986; Kohler, Proc.Natl.Acad.Sci.USA 77:2197,1980).The encoding sequence of heavy chain and light chain can comprise cDNA or genomic dna.

In case by animal generation, chemosynthesis or recombinant expressed antibody molecule of the present invention, just can utilize any method that is used for the purifying immunoglobulin molecules known in the art to carry out purifying, for example by chromatography (for example ion exchange chromatography, affinity chromatography particularly after the albumin A chromatography to the affinity chromatography and the size exclusion column chromatography of specific antigen), centrifugal, difference solubility or be used for any other standard technique of protein purification.In addition, can and can merge with the promotion purifying with allogeneic polypeptide sequence described herein or that other approach of this area is known combined treatment protein corresponding to antibody or its fragment of the therapeutic protein of albumin fusion proteins of the present invention part.

The modification of antibody

The antibody and flag sequence such as the peptide of combined treatment protein or its fragment or variant can be merged to promote purifying.In preferred embodiments, marker amino acid sequence is six polyhistidyl peptides, such as the label that provides in pQE carrier (9259Eton Avenue, Chatsworth, CA 91311 for QIAGEN, Inc.) etc., wherein many can the acquisition by commercial sources.As people such as for example Gentz, Proc.Natl.Acad.Sci.USA 86:821-824, described in 1989, six polyhistidyls provide purifying easily for fusion rotein.Other includes but not limited to corresponding to hemagglutinin label (being also referred to as " HA label ") (people such as Wilson, Cell37:767,1984) and " flag " label derived from the epi-position of influenza hemagglutinin protein matter the useful peptide tag of purifying.

Antibody or its fragment of puting together with diagnostic reagent or therapeutical agent also contained in the present invention.Antibody can be used for for example monitoring as the part of Clinical Laboratory program the development or the progress of tumour in diagnosis, thereby for example measures the effectiveness of given treatment plan.By antibody and detectable substance coupling can be promoted to detect.The example of detectable substance comprises positron emitting metal and the inactive paramagnetic metal ion that uses in various enzymes, prothetic group, fluorescent material, luminescent material, bioluminescent material, radioactive substance, the various positron emission tomography art.Utilize technology known in the art, can with detectable substance directly or by intermediate (such as for example joint known in the art) indirect with antibody (or its fragment) coupling or put together.Can be as the metal ion of diagnostic reagent thereby can put together referring to for example U.S. Patent number 4,741,900 according to the present invention with antibody.The example of suitable enzyme comprises horseradish peroxidase, alkaline phosphatase, beta-galactosidase enzymes or acetylcholinesterase; The example of suitable prothetic group mixture comprises strepto-affinity element/vitamin H and affinity element/vitamin H; The example of suitable fluorescent material comprises Umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazine amine fluorescein (dichlorotriazinylamine fluorescein), dansyl chloride or phycoerythrin; The example of luminescent material comprises luminol,3-aminophthalic acid cyclic hydrazide; The example of bioluminescent material comprises luciferase, luciferin and aequorin; And the example of suitable radioactive substance comprises ¹²⁵I, ¹³¹I, ¹¹¹I or ⁹⁹Tc.This paper has also described in other place other example of detectable substance.

In addition, antibody of the present invention and therapeutic module can be puted together,, for example suppress cell agent or cytocide such as cytotoxin, therapeutical agent or radioactive metal ion, alpha emitter for example is such as for example ²¹³Bi.Cytotoxin or cytotoxic agent comprise the deleterious any reagent of pair cell.Example comprises Pa Litasai (paclitaxol), Cytochalasin B, Gramicidin D, ethidium bromide, Hemometine, mitomycin, Etoposide, teniposide, vincristine(VCR), vincaleucoblastine, colchicine, Dx, daunorubicin, dihydroxyl anthracin diketone, mitoxantrone, mithramycin, dactinomycin, 1-boldenone, glucocorticosteroid, PROCAINE HCL, PHARMA GRADE, tetracaine, lignocaine, Proprasylyte and tetracycline and analogue or homologue.Therapeutical agent includes but not limited to metabolic antagonist (methotrexate for example, Ismipur, 6-thioguanine, cytosine arabinoside, 5 FU 5 fluorouracil, decarbazine (decarbazine)), alkylating agent (chlormethine (mustargen) for example, plug is for group, Chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), endoxan, busulfan, mitobronitol, streptozotocin, ametycin and suitable dichloro diamino platinum (II) be cis-platinum (DDP)), anthracycline antibiotics (for example daunorubicin (being called daunomycin in the past) and Dx), microbiotic (gengshengmeisu (being called actinomycin in the past) for example, bleomycin, mithramycin and anthramycin (AMC)) and antimitotic agent (for example vincristine(VCR) and vincaleucoblastine).

Conjugate of the present invention can be used for modifying given biological answer-reply, does not think that therapeutical agent or medicine module are limited to classical chemotherapeutic.For example, the medicine module can be protein or the polypeptide with expectation biologic activity.This protein can comprise for example toxin, such as abrin, ricin A, Pseudomonas exotoxin or diphtheria toxin; Protein, such as tumour necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, Thr6 PDGF BB, tissue plasminogen activator, apoptosis agent, for example TNF-α, TNF-β, AIM I (referring to international publication number WO 97/33899), AIM II (referring to international publication number WO 97/34911), Fas part (people such as Takahashi, Int.Immunol.6:1567-1574,1994), VEGI (referring to international publication number WO 99/23105), thrombosis agent or antiangiogenic agent, for example angiostatin or endostatin; Or the biological answer-reply modifier, such as for example lymphokine, il-1 (" IL-1 "), interleukin-2 (" IL-2 "), interleukin-6 (" IL-6 "), rHuGM-CSF (" GM-CSF "), granulocyte colony-stimulating factor (" G-CSF ") or other somatomedin.

Antibody can also be attached to solid support, this immunoassay or purifying to target antigen is useful especially.This solid support includes but not limited to glass, Mierocrystalline cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

The technology that is used for this therapeutic module and antibody are puted together is well-known.For example referring to people such as Amon, " Monoclonal Antibodies For Immunotargeting Of Drugs In CancerTherapy ", in " Monoclonal Antibodies And Cancer Therapy ", people such as Reisfeld, compile page number 243-56, Alan R.Liss, Inc., 1985; People such as Hellstrom, " AntibodiesFor Drug Delivery ", in " Controlled Drug Delivery ", the 2nd edition, people such as Robinson compile page number 623-53, Marcel Dekker, Inc., 1987; Thorpe, " Antibody CarriersOf Cytotoxic Agents In Cancer Therapy:A Review ", in " Monoclonal Antibodies ' 84:Biological And Clinical Applications ", people such as Pinchera, compile, page number 475-506,1985; " Analysis; Results; And Future Prospective Of The Therapeutic Use OfRadiolabeled Antibody In Cancer Therapy ", at " Monoclonal Antibodies ForCancer Detection And Therapy ", people such as Baldwin compile page number 303-16, AcademicPress, 1985; Reach people such as Thorpe, " The Preparation And Cytotoxic Properties OfAntibody-Toxin Conjugates ", Immunol.Rev.62:119-58,1982.

Perhaps,,, antibody and second antibody can be puted together to form antibody allos conjugate, with its complete being incorporated herein by reference described in 980 at U.S. Patent number 4,676 as Segal.

Separately or together with cytotoxic factor and/or cytokine antibody useful as therapeutics that use, that put together or do not put together the therapeutic module.

Antibody-white protein fusions

Combined treatment protein also can include but not limited to the antibody of disclosed therapeutic protein in associative list 1 " therapeutic protein X " row corresponding to the therapeutic protein antibody partly of albumin fusion proteins of the present invention, or its fragment or variant.

In specific embodiment, described immunologic opsonin combined treatment protein also comprises the VH structural domain or is made up of it corresponding to the fragment of the antibody of the therapeutic protein of albumin fusion proteins part or variant.In other embodiments, described immunologic opsonin combined treatment protein and comprise 1,2 or 3 VHCDR or form by it corresponding to the fragment of the antibody of the therapeutic protein part of albumin fusion proteins or variant.In other embodiments, described immunologic opsonin combined treatment protein and comprise VHCDR1 or form by it corresponding to the fragment of the antibody of the therapeutic protein part of albumin fusion proteins or variant.In other embodiments, described immunologic opsonin combined treatment protein and comprise VHCDR2 or form by it corresponding to the fragment of the antibody of the therapeutic protein part of albumin fusion proteins or variant.In other embodiments, described immunologic opsonin combined treatment protein and comprise VHCDR3 or form by it corresponding to the fragment of the antibody of the therapeutic protein part of albumin fusion proteins or variant.

In specific embodiment, described immunologic opsonin combined treatment protein also comprises the VL structural domain or is made up of it corresponding to the fragment of the antibody of the therapeutic protein of albumin fusion proteins part or variant.In other embodiments, described immunologic opsonin combined treatment protein and comprise 1,2 or 3 VLCDR or form by it corresponding to the fragment of the antibody of the therapeutic protein part of albumin fusion proteins or variant.In other embodiments, described immunologic opsonin combined treatment protein and comprise VLCDR1 or form by it corresponding to the fragment of the antibody of the therapeutic protein part of albumin fusion proteins or variant.In other embodiments, described immunologic opsonin combined treatment protein and comprise VLCDR2 or form by it corresponding to the fragment of the antibody of the therapeutic protein part of albumin fusion proteins or variant.In other embodiments, described immunologic opsonin combined treatment protein and comprise VLCDR3 or form by it corresponding to the fragment of the antibody of the therapeutic protein part of albumin fusion proteins or variant.

In other embodiments, described immunologic opsonin combined treatment protein and comprise 1,2,3,4,5 or 6 VH and/or VL CDR or form by it corresponding to the fragment of the antibody of the therapeutic protein part of albumin fusion proteins or variant.

In preferred embodiments, described immunologic opsonin combined treatment protein also comprises scFv or is made up of it corresponding to the fragment of the antibody of the therapeutic protein of albumin fusion proteins part or variant, and described scFv comprises by such as (Gly4Ser) 3 (the SEQ ID NO:4) peptide linker of etc.ing continuous therapeutic antibodies VL structural domain and therapeutic antibodies VH structural domain.

The immunophenotype somatotype

At least one fragment or the antibody of the present invention of variant or the immunophenotype somatotype that albumin fusion proteins of the present invention can be used for clone and biological sample that comprise the antibody of combined treatment protein (or its fragment or variant).Therapeutic protein of the present invention can be used as the cell-specific mark, or is exactly at the difference differentiation of particular cell types and/or the cell sign thing of stage of maturity differential expression in particular.To allow that at the monoclonal antibody of defined epitope or epi-position combination (or comprise at least one fragment of the proteinic antibody of combined treatment or the albumin fusion proteins of variant) screening expresses the cell mass of mark.Can utilize multiple technologies to use monoclonal antibody (or comprise at least one fragment of the proteinic antibody of combined treatment or the albumin fusion proteins of variant) to screen the cell mass of expressing mark, the magnetic that comprises the magnetic bead that uses antibody sandwich separates, with " elutriation " that be attached to the antibody on the solid substrate (being plate), and flow cytometry (referring to for example United States Patent (USP) 5,985,660 and people such as Morrison, Cell 96:737-49,1999).

These technology are allowed screening specific cells group, such as " non-self " cell in can finding haematological malignancies (being the minimal residual disease (MRD) in the acute leukemic patient) and transplanting to prevent graft versus host disease (GVH disease) (GVHD).Perhaps, these technology are allowed hemopoietic stem cell and the progenitor cell that screening can breed and/or break up, as finding in human cord blood.

Identify proteinic antibody of combined treatment and the fragment that comprises the proteinic antibody of combined treatment Or the albumin fusion proteins of variant

Antibody of the present invention or comprise at least one fragment of antibody of combined treatment protein (or its fragment or variant) or the albumin fusion proteins of the present invention of variant can be identified in many ways.Particularly, can use technology described herein or conventional modification technology known in the art, the albumin fusion proteins of the present invention of at least one fragment that comprises the proteinic antibody of combined treatment or variant is measured specificity in conjunction with the ability by the same antigen of following antibody specific combination, described antibodies and the therapeutic protein corresponding therapeutic protein of antibody partly in conjunction with albumin fusion proteins.

Be used for antibody of the present invention or comprise at least one fragment of antibody of combined treatment protein (or its fragment or variant) or the albumin fusion proteins of the present invention (specificity) of variant can be at solution (as Houghten in conjunction with the mensuration of the ability of specified protein or epi-position, Bio/Techniques 13:412-421,1992), on pearl (as Lam, Nature 354:82-84,1991), on chip (as Fodor, Nature 364:555-556,1993), on bacterium (as U.S. Patent number 5,223,409), on spore (as the patent No. 5,571,698; 5,403,484; With 5,223,409), on plasmid (as people such as Cull, Proc.Natl.Acad.Sci.USA 89:1865-1869,1992) or on phage (as Scott and Smith, Science 249:386-390,1990; Devlin, Science 249:404-406,1990; People such as Cwirla, Proc.Natl.Acad.Sci.USA 87:6378-6382,1990; And Felici, J.Mol.Biol.222:301-310,1991) carry out (with complete being incorporated herein by reference of each piece of these reference).Use or conventionally revise technology described herein or that other approach of this area is known, also can or comprise at least one fragment of antibody of combined treatment protein (or its fragment or variant) or the albumin fusion proteins of the present invention of variant is measured their specificity and avidity to specified protein or epi-position antibody of the present invention.

Any method that can know by this area, to at least one fragment or the albumin fusion proteins mensuration of the present invention of variant and the cross reactivity of other antigen (for example with the molecule that has sequence/structure conservative property by following antibodies specific bonded molecule, the therapeutic protein (or its fragment or variant) that described antibodies is partly corresponding with the therapeutic protein of albumin fusion proteins of the present invention) that comprises the proteinic antibody of combined treatment.

The immunoassay that can be used for analysis (immunologic opsonin) combination and cross reactivity includes but not limited to, the competitiveness of technology such as use such as western blotting, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), " sandwich " immunoassay, immunoprecipitation assay, precipitin reaction, GDP reaction, immunodiffusion(ID) assay method, agglutination assay, complement fixation assay, immune radiating degree assay method, fluorescence immunoassay and albumin A immunoassay and noncompetitive are measured system, only for several examples.Such assay method be conventional and in this area be well-known (compile referring to people such as for example Ausubel, 1994, " Current Protocols in Molecular Biology ", the 1st volume, John Wiley ﹠amp; Sons, Inc., New York is with its complete being incorporated herein by reference).Exemplary immunoassay is sketched in hereinafter (not limiting but be not intended to conduct).

The immunoprecipitation scheme is usually included in such as being supplemented with protein phosphatase and/or proteinase inhibitor (as EDTA, PMSF, press down the enzyme peptide, vanadic acid sodium) RIPA damping fluid (1%NP-40 or TritonX-100,1% sodium deoxycholate, 0.1%SDS, 0.15M NaCl, 0.01M sodium phosphate pH7.2,1%Trasylol) wait lysing cell group in the lysis buffer, in cell pyrolysis liquid, add antibody of the present invention or comprise at least one fragment of antibody of combined treatment protein (or its fragment or variant) or the albumin fusion proteins of the present invention of variant, in 40 ℃ of insulation for some time (for example 1-4 hour), in cell pyrolysis liquid, add albumin A and/or Protein G sepharose pearl (or in the situation of the albumin fusion proteins of at least one fragment that comprises the proteinic antibody of combined treatment or variant, pearl through suitable antiidiotypic antibody or anti-white protein antibody sandwich), in 40 ℃ of insulations about 1 hour or more of a specified duration, in lysis buffer, clean pearl and pearl is resuspended in the SDS/ sample buffer.Can assess the ability of antibody of the present invention or albumin fusion proteins immunoprecipitation specific antigen by for example western blot analysis.Those skilled in the art should know and can make amendment to increase antibody or albumin fusion proteins combined and reduced background with antigen parameter (as usefulness sepharose pearl preclearing cell pyrolysis liquid).Further discussion about the immunoprecipitation scheme is compiled referring to people such as for example Ausubel, and 1994, " Current Protocols inMolecular Biology ", the 1st volume, John Wiley ﹠amp; Sons, Inc., New York, 10.16.1.

Western blot analysis generally includes the preparation protein example, at polyacrylamide gel (8%-20%SDS-PAGE for example, depend on antigenic molecular weight) in protein example is carried out electrophoresis, protein example is transferred to such as soluble cotton from polyacrylamide gel, on the film such as PVDF or nylon, closing membrane in confining liquid (PBS that for example contains 3%BSA or skimmed milk), in cleaning buffer solution (for example PBS-Tween 20), clean film, antibody of the present invention or albumin fusion proteins (diluting in the sealing damping fluid) are applied on the film, in cleaning buffer solution, clean film, be applied to that sealing dilutes in the damping fluid be conjugated with enzyme substrates (for example horseradish peroxidase or alkaline phosphatase) or Geigers (for example ³²P or ¹²⁵I) two anti-(it discerns albumin fusion proteins, for example AHS's white protein antibody) are cleaned film, and are detected antigenic existence in cleaning buffer solution.Those skilled in the art should know and can make amendment with signal that improve to detect and the parameter that reduces ground unrest.Further discussion about the western blotting scheme is compiled referring to people such as for example Ausubel, and 1994, " Current Protocols in MolecularBiology ", the 1st volume, John Wiley ﹠amp; Sons, Inc., NewYork, 10.8.1.

ELISA comprises preparation antigen, hole with antigen coated 96 hole microtiter plates, flush away is not attached to the antigen on the hole, Xiang Kongzhong add be conjugated with such as enzyme substrates (for example horseradish peroxidase or alkaline phosphatase) but etc. detection compound antibody of the present invention or albumin fusion proteins (at least one fragment or the variant that comprise the proteinic antibody of combined treatment) and be incubated for some time, flush away not in conjunction with or the albumin fusion proteins of non-specific binding, and detection specificity in conjunction with bag by the antigenic antibody in hole or the existence of albumin fusion proteins.In ELISA, but antibody or albumin fusion proteins are not to put together detection compound; On the contrary, but but Xiang Kongzhong adds two anti-(it discerns antibody or albumin fusion proteins respectively) be conjugated with detection compound.In addition, available antibodies or albumin fusion proteins bag are used antigen coated hole with replacement to the hole.In this case, but detection molecules can be conjugated with such as enzyme substrates (for example horseradish peroxidase or alkaline phosphatase) but etc. the antigen of detection compound.Those skilled in the art should know the parameter that can make amendment with the signal that improve to detect, and other modification of the ELISA that knows of this area.Further discussion about ELISA is compiled referring to people such as for example Ausubel, and 1994, " Current Protocols in Molecular Biology ", the 1st volume, John Wiley ﹠amp; Sons, Inc., New York, 11.2.1.

Can measure binding affinity and the antibody or the interactional dissociation rate of albumin fusion proteins-protein/antigen/epi-position (off-rate) of albumin fusion proteins and protein, antigen or epi-position by the competitive binding assay method.An example of competitive binding assay method is a radioimmunoassay, when being included in the unlabelled antigen that has cumulative amount will through labelled antigen (as ³H or ¹²⁵I) be incubated with antibody of the present invention or albumin fusion proteins, and detect and through labelled antigen bonded antibody.Can measure the avidity of antibody of the present invention or albumin fusion proteins and specified protein, antigen or epi-position and combine dissociation rate by the data of Scatchard mapping analysis.Also can use radioimmunoassay to measure the second proteinic competition that combines same protein, antigen or epi-position with described antibody or albumin fusion proteins.In this case, exist cumulative amount combine unmarked second protein of same protein, antigen or epi-position with albumin fusion proteins of the present invention the time, with protein, antigen or epi-position and be conjugated with tagged compound (as ³H or ¹²⁵I) antibody of the present invention or albumin fusion proteins are incubated together.

In a preferred embodiment, use the BIAcore dynamic analysis to measure antibody of the present invention or albumin fusion proteins and protein, antigen or the epi-position bonded speed that combines and dissociate.The BIAcore dynamic analysis comprises analyzes combining and dissociating of antibody, albumin fusion proteins or specific polypeptide, antigen or epi-position and chip, is fixed with specific polypeptide, antigen or epi-position, antibody or albumin fusion proteins on the surface of its chips respectively.

Therapeutic use

The present invention also is devoted to the therapy based on antibody, it comprises antibody of the present invention or comprises at least one fragment of the proteinic antibody of combined treatment or the albumin fusion proteins of the present invention of variant is applied to animal, preferred mammal, the optimum patient that chooses is used for the treatment of one or more disclosed diseases, disorder or situation.Therapeutic compound of the present invention includes but not limited to the albumin fusion proteins of the present invention of nucleic acid, at least one fragment that comprises the proteinic antibody of combined treatment or variant of antibody of the present invention (comprising its fragment described herein, analogue and derivative), code book invention antibody (comprising its fragment described herein, analogue and derivative and antiidiotypic antibody) and the nucleic acid of such albumin fusion proteins of encoding.Antibody of the present invention or comprise at least one fragment of the proteinic antibody of combined treatment or the albumin fusion proteins of the present invention of variant can be used for treating, suppress or unconventionality expression and/or active diseases associated, disorder or the situation of prevention and therapeutic protein includes but not limited to any or multiple disease described herein, disorder or situation.Include but not limited to alleviate the symptom relevant with the unconventionality expression and/or the treating and/or preventing of active diseases associated, disorder or situation of therapeutic protein with those diseases, disorder or situation.Antibody of the present invention or comprise at least one fragment of the proteinic antibody of combined treatment or the albumin fusion proteins of the present invention of variant can be known in this area or pharmacopedics described herein can be accepted to provide in the composition.

In a concrete and embodiment preferred, the present invention is devoted to the therapy based on antibody, it comprises antibody of the present invention or comprises at least one fragment of the proteinic antibody of combined treatment or the albumin fusion proteins of the present invention of variant is applied to animal, preferred mammal, the optimum patient that chooses, be used for the treatment of one or more diseases, disorder or situation, include but not limited to: nervous disorders, immune system disorder, muscle disorder, the reproduction disorder, gastrointestinal dysfunction, the lung disorder, cardiovascular disorder, the kidney disorder, proliferative disorders, and/or Cancerous disease and situation, and/or other place of this paper is described.Therapeutic compound of the present invention includes but not limited to that antibody of the present invention is (for example at the antibody of the full length protein of expressing on mammalian cell surface; Antibody at the therapeutic protein epi-position) and the nucleic acid of code book invention antibody (comprise its fragment described herein, analogue and derivative and anti-unique antibody).Antibody of the present invention can be used for treating, suppress or unconventionality expression and/or active diseases associated, disorder or the situation of prevention and therapeutic protein, includes but not limited to any or multiple disease described herein, disorder or situation.Describedly include but not limited to alleviate the symptom relevant with those diseases, disorder or situation with the unconventionality expression and/or the treating and/or preventing of active diseases associated, disorder or situation of therapeutic protein.Antibody of the present invention or comprise at least one fragment of the proteinic antibody of combined treatment or the albumin fusion proteins of the present invention of variant can be known in this area or pharmacopedics described herein can be accepted to provide in the composition.

Antibody wherein of the present invention or comprise at least one fragment of the proteinic antibody of combined treatment or the summary of the mode that the albumin fusion proteins of the present invention of variant can use comprise local in vivo or systemic combined treatment protein in treatment, or the direct cytotoxicity by antibody, as by complement (CDC) or effector cell (ADCC) mediation.In these methods some is specified in hereinafter.Utilize instruction provided herein, how those of ordinary skills should know antibody of the present invention or comprise at least one fragment of the proteinic antibody of combined treatment or the albumin fusion proteins of the present invention of variant is used for diagnosis, monitoring or therapeutic purpose, and need not undo experimentation.

Antibody of the present invention or comprise at least one fragment of the proteinic antibody of combined treatment or the albumin fusion proteins of the present invention of variant can be favourable unites use with other mono-clonal or chimeric antibody or with lymphokine or hemopoieticgrowth factor (such as IL-2, IL-3 and IL-7) for example helps to increase number or activity with the interactional effector cell of antibody.

Antibody of the present invention or comprise at least one fragment of the proteinic antibody of combined treatment or the albumin fusion proteins of the present invention of variant can be used separately or co-administered with the treatment (for example radiotherapy, chemotherapy, hormonotherapy, immunotherapy and antineoplastic agent) of other type.Generally speaking, preferably use the product that belongs to the origin of species or the species reactivity (with regard to antibody) of same species with the patient.Therefore, in a preferred embodiment, people's antibody, fragment, derivative, analogue or nucleic acid are applied to people patient are used for the treatment of or prevent.

Preferably with at the high-affinity of therapeutic protein and/or effectively suppress in vivo and/or neutral antibody, its fragment or zone (or albumin fusion proteins relevant with such antibody) is used for comprising at polynucleotide of the present invention or polypeptide the treatment of its segmental immunoassay and relevant disorder with it.Such antibody, fragment or zone preferably comprise that to polynucleotide of the present invention or polypeptide its fragment has avidity.Preferred binding affinity comprises that dissociation constant or Kd are less than 5x10 ^-2M, 10 ^-2M, 5x10 ^-3M, 10 ^-3M, 5x10 ^-4M, 10 ^-4Those of M.Preferred binding affinity comprises that dissociation constant or Kd are less than 5x10 ^-5M, 10 ^-5M, 5x10 ^-6M, 10 ^-6M, 5x10 ^-7M, 10 ^-7M, 5x10 ^-8M or 10 ^-8Those of M.Even preferred binding affinity comprises that dissociation constant or Kd are less than 5x10 ^-9M, 10 ^-9M, 5x10 ^-10M, 10 ^-10M, 5x10 ^-11M, 10 ^-11M, 5x10 ^-12M, 10 ^-12M, 5x10 ^-13M, 10 ^-13M, 5x10 ^-14M, 10 ^-14M, 5x10 ^-15M or 10 ^-15Those of M.

Gene therapy

In a specific embodiment, by the mode of gene therapy, use and comprise the coding proteinic antibody of combined treatment or comprise at least one fragment of the proteinic antibody of combined treatment or the nucleic acid of the sequence of the albumin fusion proteins of variant is treated, suppressed or unconventionality expression and/or the active diseases associated or the disorder of prevention and therapeutic protein.Gene therapy refers to by the experimenter being used the treatment that nucleic acid expression or effable carries out.In this embodiment of the present invention, described nucleic acid produces its encoded protein matter of mediation result of treatment.

Any method of the available gene therapy in this area all can be used according to the invention.Other place that is described in the application that exemplary method is more detailed.

The real example of treatment or prophylactic activity

Before being used for the people, compound of the present invention or pharmaceutical composition are preferred earlier at vitro test, test required treatment or prophylactic activity then in vivo.For example, the external test method that is used to confirm the treatment of compound or pharmaceutical composition or prevention effectiveness comprises the effect of compound pair cell system or patient tissue samples.Can utilize the technology that those skilled in the art will know that to measure the effect of compound or composition pair cell system and/or tissue sample, include but not limited to that rosette forms assay method and cytolysis assay method.According to the present invention, sketched the external test method that can be used for determining whether to use specific compound, comprise that cell in vitro cultivates assay method, wherein cultivate patient's tissue sample and be exposed to or administered compound otherwise, and observe the effect of described compound tissue sample.

Therapeutic/preventative using and composition

The invention provides the The compounds of this invention by the experimenter being used significant quantity or treatment, inhibition and the prevention method of pharmaceutical composition.In a preferred embodiment, compound is (for example being substantially free of the material that limits its effect or produce undesired side effect) of purifying basically.The preferred animal of experimenter includes but not limited to such as animals such as ox, pig, horse, chicken, cat, dogs, and preferred mammal, and optimum is chosen.

When compound comprised nucleic acid or immunoglobulin (Ig), spendable preparation and application process were as mentioned above; Other appropriate formulation and route of administration can be selected from hereinafter described those.

Known multiple delivery system and can be used for using compound of the present invention, for example be encapsulated in liposome, particulate, the microcapsule, can express the reconstitution cell of this compound, receptor-mediated endocytosis (referring to for example Wu and Wu, J.Biol.Chem.262:4429-4432,1987), as structure of the nucleic acid of retrovirus or other carrier part etc.The method that imports includes but not limited in intracutaneous, intramuscular, intraperitoneal, intravenously, subcutaneous, the nose, epidural and oral route.Compound or composition can be used by any approach easily, for example by infusion or inject, by through the absorption of epithelium or mucocutaneous liner (as oral mucosa, rectum and intestinal mucosa etc.), and can be co-administered with other biologic activity reagent.But system or topical application.In addition, may wish medical compounds of the present invention or composition to be imported central nervous system, comprise Intraventricular and intrathecal injection by any suitable way; Can be convenient to intracerebral ventricle injection by intraventricular catheter, for example connect storage, such as the Ommaya storage.Also can adopt lung's dispenser, for example by use sucker or atomizer, and the formulation that contains propellant.

In a specific embodiment, may wish medical compounds of the present invention or composition topical application to the zone that needs treatment; This can by such as but not limited to perioperative local infusion, surface applications as in conjunction with postoperative wound dressings, by injection, via conduit, realize by suppository or by implant, described implant is porous, non-porous or spawn, comprise film, such as sialastic film or fiber.Preferably, when using protein of the present invention, comprise antibody, must be noted that employed material should be do not absorb proteinic.

In another embodiment, compound or composition can be delivered in vesica, particularly in liposome (referring to Langer, Science 249:1527-1533,1990; People such as Treat, in " Liposomesin the Therapy of Infectious Disease and Cancer ", Lopez-Berestein and Fidler compile, Liss, New York, pp.353-365,1989; Lopez-Berestein, the same, pp.317-327; Mainly referring to the same).

In another embodiment, compound or composition can be delivered in controlled release system.In one embodiment, can use pump (, to see before referring to Langer; Seflon, CRC Crit.Ref.Biomed.Eng.14:201,1987; People such as Buchwald, Surgery 88:507,1980; People such as Saudek, N.Engl.J.Med.321:574,1989).In another embodiment, (referring to " Medical Applications of Controlled Release ", Langer and Wise compile, CRCPres., Boca Raton, Florida, 1974 can to use polymeric material; " Controlled Drug Bioavailability, Drug ProductDesign and Performance ", Smolen and Ball compile, Wiley, New York, 1984; Ranger and Peppas, J.Macromol.Sci.Rev.Macromol.Chem.23:61,1983; Also referring to people such as Levy, Science 228:190,1985; People such as During, Ann.Neurol.25:351,1989; People such as Howard, J.Neurosurg.71:105,1989).In another embodiment, controlled release system can place treatment target such as brain near, therefore only need the part of system's dosage (referring to for example Goodson, in " Medical Applications of Controlled Release ", the same, the 2nd volume, pp.115-138,1984).

The summary (Science 249:1527-1533,1990) of Langer is seen in the discussion of other controlled release system.

At compound of the present invention is in the specific embodiments of nucleic acid of coded protein, the protein expression of administration of nucleic acid to promote that it is coded in vivo, can it be entered in the cell by it being made up as the part of suitable nucleic acid expression vector and using it, for example by utilizing retroviral vector (referring to U.S. Patent number 4,980,286), or by direct injection, or by utilizing microparticle bombardment (particle gun for example; Biolistic, Dupont), or with lipid or cell surface receptor or transfection agents bag quilt, or by connecting knownly enter nuclear homology frame sample peptide and use it (referring to people such as for example Joliot, Proc.Natl.Acad.Sci.USA 88:1864-1868,1991) etc.Perhaps, can will mix host cell DNA to express in the nucleic acid transfered cell and by homologous recombination.

The present invention also provides pharmaceutical composition.Such composition comprises the compound for the treatment of significant quantity, and pharmacopedics can be accepted carrier.In a specific embodiment, term " pharmacopedics is acceptable " refer to obtain the approval of federation or administration of state government list in American Pharmacopeia or pharmacopeia that other is generally acknowledged in for the animal use, people more specifically says so.Term " carrier " refers to thinner, adjuvant, vehicle or the media used with therapeutical agent.Such pharmacopedics carrier can be a sterile liquid, such as water and oil, comprises those of oil, animal, plant or synthetic source, such as peanut oil, soybean oil, mineral oil, sesame oil or the like.When intravenously drug administration composition, water is preferred carrier.Salt brine solution and moisture dextrose and glycerine solution also can be used as liquid vehicle, especially for injection liquid.Suitable pharmacopedics vehicle comprises starch, glucose, lactose, sucrose, gelatin, Fructus Hordei Germinatus, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talcum, sodium-chlor, skim-milk, glycerine, propylene, ethylene glycol, water, ethanol or the like.If desired, composition also can contain a spot of wetting agent or emulsifying agent or pH buffer reagent.These compositions can be taked forms such as solution, suspension, milk sap, tablet, pill, capsule, pulvis, sustained release forms.Composition can be mixed with suppository, contains traditional tackiness agent and such as carriers such as triglyceride level.Oral dosage form can comprise standard vector, such as pharmaceutical grade N.F,USP MANNITOL, lactose, starch, Magnesium Stearate, soluble saccharin, Mierocrystalline cellulose, magnesiumcarbonate etc.The example of suitable pharmacopedics carrier be described in E.W.Martin's " Remington ' s Pharmaceutical Sciences ".Such composition will contain the compound for the treatment of significant quantity, the form of preferred purifying, and the carrier of appropriate amount is to provide the form that the patient is used of being suitable for.Formulation should be fit to method of application.

In a preferred embodiment, according to old process composition is mixed with and is suitable for the pharmaceutical composition that people's intravenously is used.Usually, being used for composition that intravenously uses is solution at sterile isotonic water-based damping fluid.When needing, composition also can comprise solubilizing agent and such as local anesthetics such as lignocaines to ease the pain in the injection site.Generally speaking, composition be separately provide or with the unit dosage form blended, for example as the lyophilized powder in the hermetically sealed container or there is not aqueous concentrate, the ampoule or the pouch (sachette) of promoting agent quantity arranged such as sign.When composition will be used by infusion, available sterile pharmaceutical grade water or the brinish infusion bottle of being equipped with distributed.When composition is used by injection, can provide ampoule Injectable sterile water or salt solution so that composition can used preceding mixing.

Compound of the present invention can be mixed with neutrality or salt form.The acceptable salt of pharmacopedics comprises those that form with negatively charged ion, such as derived from those of hydrochloric acid, phosphoric acid, acetate, oxalic acid, tartrate etc., and form with positively charged ion those, such as derived from those of sodium hydroxide, potassium, ammonium, calcium, iron, Isopropylamine, triethylamine, 2-ethylamino-ethanol, Histidine, PROCAINE HCL, PHARMA GRADE etc.

Can by standard clinical techniques be determined at the unconventionality expression of therapeutic protein and/or active diseases associated or disorderly treatment, inhibition and prevention in the effective amount of The compounds of this invention.In addition, can choose employing external test method wantonly and help determine the optimal dose scope.The exact dosage desired that will adopt in the preparation also depends on route of administration and disease or disorderly severity, and should decide according to practitioner's judgement and every patient's situation.According to the dosage-response curve of external or the animal model test macro effective dose of can extrapolating.

For antibody, the dosage that the patient is used is the 0.1mg/kg-100mg/kg weight in patients normally.Preferably, the dosage that the patient is used is the 0.1mg/kg-20mg/kg weight in patients, more preferably the 1mg/kg-10mg/kg weight in patients.Generally speaking, owing to the immune response to external polypeptide, people's antibody has the longer transformation period than the antibody from other species in human body.Therefore, usually be possible than the people's antibody of low dosage and the dispenser of lower frequency.In addition, can be by strengthening the picked-up of antibody and application dosage and the frequency that tissue penetration (as entering brain) reduces antibody of the present invention such as modifications such as for example fatization.

Diagnosis and imaging

Can be used for unconventionality expression and/or active diseases associated, disorder and/or the situation of diagnostic purpose through the antibody of the combined treatment protein (or its fragment or variant) of mark and derivative thereof and analogue (comprising at least one fragment that comprises the proteinic antibody of combined treatment or the albumin fusion proteins of variant) with detection, diagnosis or monitoring and therapeutic protein.The invention provides the detection method of the unconventionality expression of therapeutic protein, comprise that (a) uses one or more expression to therapeutic protein in special TPPA individual cells of desired polypeptides or the body fluid, and (b) gene expression dose and standard gene expression level are compared, the therapeutic protein expression level that records thus is than the rising of standard expression level or reduce the indication unconventionality expression.

The invention provides and be used to diagnose disorderly diagnostic assay method, comprise that (a) uses one or more to the special antibody of therapeutic protein or comprise the expression of the albumin fusion proteins of at least one fragment of the special antibody of therapeutic protein or variant being measured therapeutic protein in individual cells or the body fluid, and (b) gene expression dose and standard gene expression level are compared, the therapeutic protein expression level that records thus is than the rising of standard expression level or reduce the indication particular disorder.With regard to cancer, in individual biopsy, exist the transcript of relative comparatively high amts can indicate easy the to be ill physique that forms disease, perhaps can be provided for before actual clinical symptom occurs, detecting the method for disease.Such more definite diagnosis tolerable medical worker more early takes preventive measures or attacks treatment, stops the development or the further progress of cancer thus.

Antibody of the present invention or comprise albumin fusion proteins at least one fragment of the special antibody of therapeutic protein or variant and can be used for measuring protein level in the biological sample, used the classical immunohistology method that those skilled in the art will know that (for example referring to people such as Jalkanen, J.Cell.Biol.101:976-985,1985; People such as Jalkanen, J.Cell.Biol.105:3087-3096,1987).Can be used for detecting other method that protein gene expresses and comprise immunoassay, such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) based on antibody.Suitable TPPA marker is that this area is known, comprises the enzyme labelling thing, such as glucose oxidase; Radio isotope, such as iodine ( ¹²⁵I, ¹²¹I), carbon ( ¹⁴C), sulphur ( ³⁵S), tritium ( ³H), indium ( ¹²¹In) and technetium ( ⁹⁹Tc); Luminous marker is such as luminol,3-aminophthalic acid cyclic hydrazide; Fluorescent marker is such as fluorescein and rhodamine; And vitamin H.

One aspect of the present invention is an animal, preferred mammal, most preferably the unconventionality expression diseases associated of philtrum and therapeutic protein or disorderly detection and diagnosis.In one embodiment, diagnosis comprises: the molecule of a) experimenter being used the specificity binding purposes polypeptide that passes through mark of (for example parenteral, subcutaneous or intraperitoneal) significant quantity; B) waiting for a period of time after the dispenser to allow the proteinic position of tagged molecule express therapeutic in the experimenter preferentially concentrated (and unconjugated tagged molecule is removed to background level); C) measure background level; And d) the detected result indication experimenter that the tagged molecule among the detection experimenter, tagged molecule surpass background level suffers from specified disease or the disorder relevant with the unconventionality expression of therapeutic protein.Can measure background level by several different methods, comprise that the amount of the tagged molecule that detection is obtained and the standard value of before particular system being measured compare.

This area will be understood, the amount that experimenter's build size and used imaging system will determine to produce the required image-forming module of diagnosis imaging.In the example of radio isotope module,, inject radioactive amount usually in about 5 to 20 millicuries for the human experimenter ^99mIn the scope of Tc.Then through antibody, the antibody fragment of mark or comprise at least one fragment of the proteinic antibody of combined treatment or the albumin fusion proteins of variant will preferentially contain the accumulation of the proteinic cell of particular treatment position.The description of in-vivo tumour imaging can be referring to people such as S.W.Burchiel, " Immunopharmacokinetics of RadiolabeledAntibodies and Their Fragments ", the 13rd chapter, " Tumor Imaging:The RadiochemicalDetection of Cancer ", S.W.Burchiel and B.A.Rhodes compile, Masson Publishing Inc., 1982.

According to some parameters, comprise the marker type and the dispenser pattern of use, allow after the dispenser that the position of tagged molecule in the experimenter preferentially concentrated and not the bonding mark molecule remove to the timed interval of background level be 6-48 hour or 6-24 hour or 6-12 hour.In another embodiment, the timed interval after the dispenser is 5-20 days or 5-10 days.

In one embodiment, diagnose the illness or disorderly method is carried out disease or disorderly monitoring by being recycled and reused for, for example after the initial diagnosis 1 month, at after the initial diagnosis 6 months, after initial diagnosis 1 year etc.

The method that is used for the body interscan that can use this area to know detects the keep the score existence of son of patient acceptance of the bid.These methods depend on employed marker type.The technician can be identified for detecting the appropriate method of particular marker.The method and apparatus that can be used for diagnostic method of the present invention includes but not limited to computerized tomography (CT), body scan such as positron emission tomography (PET), nuclear magnetic resonance (MRI) and ultrasonic examination.

In a specific embodiment, molecule uses the surgical instruments of radiation responsiveness to detect people such as (, U.S. Patent number 5,441,050) Thurston with labelled with radioisotope and in the patient.In another embodiment, molecule uses the scanning apparatus of fluorescence response to detect with the fluorescent chemicals mark and in the patient.In another embodiment, molecule uses positron emission tomography to detect with the positron emitting metal mark and in the patient.In another embodiment, molecule is with the spin labeling substance markers and use nuclear magnetic resonance (MRI) to detect in the patient.Specific detection albumin fusion proteins but not the independent white protein or the antibody of therapeutic protein are embodiment preferred.These can be used for detecting the albumin fusion proteins of describing in the specification sheets full text.

Test kit

The invention provides the test kit that can be used for aforesaid method.In one embodiment, test kit comprises antibody, and preferred antibody purified is contained in one or more containers.In a specific embodiment, test kit of the present invention contains isolated polypeptide basically, it comprise with test kit in included antibody play the epi-position of specific immune response.Preferably, test kit of the present invention also comprises control antibodies, and it does not react with desired polypeptides.In another specific embodiment, test kit of the present invention contains to be useful on and detects antibody and desired polypeptides bonded means (for example antibody can be conjugated with and can detect substrate, such as fluorescent chemicals, enzyme substrates, radioactive compound or luminophor, or discern one anti-two and anti-ly can be conjugated to and to detect on the substrate).

In another specific embodiment of the present invention, 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.Such test kit can comprise not the control antibodies that reacts with desired polypeptides.Such test kit can comprise isolated polypeptide antigen basically, and it comprises the epi-position that plays specific immune response with at least a anti-polypeptide antigen antibody.In addition, such test kit comprises and is used to detect described antibody and antigen bonded means (for example antibody can be conjugated with such as fluorescent chemicalses such as fluorescein or rhodamines, can pass through Flow cytometry).In specific embodiment, test kit can comprise polypeptide antigen recombinant production or chemosynthesis.The polypeptide antigen of test kit also can be attached on the solid support.

In a more particular embodiment, the detection means of mentioned reagent box comprises the solid support that is attached with described polypeptide antigen.Such test kit also can comprise the non-anti-people's antibody that adheres to the reporter molecule mark.In this embodiment, can detect combining of antibody and polypeptide antigen by the antibody of described reporter molecule mark.

In another embodiment, the present invention includes the diagnostic kit that is used to screen the antigenic serum that contains polypeptide of the present invention.Diagnostic kit comprises the isolated antibody basically that plays specific immune response with polypeptide or polynucleotide antigen, and the means that are used to detect polynucleotide or polypeptide antigen and antibodies.In one embodiment, antibody is attached on the solid support.In a specific embodiment, antibody can be monoclonal antibody.The detection means of test kit can comprise second kind of monoclonal antibody through mark.Perhaps/in addition, detection means can comprise the competitive antigen through mark.

In a kind of diagnostic form, test sera and the antigenic solid-phase reagent of surface bonding that has that obtains by the inventive method are reacted.After specific antigen-antibody combines with reagent and removes unconjugated serum composition by cleaning, the anti-people's antibody that makes reagent and reporter molecule mark react so that reporter molecule with solid support on the certain proportion binding reagents of the anti-antigen-antibody amount of bonded.Cleaning reagent to be removing unconjugated traget antibody once more, and the amount of mensuration and the associating reporter molecule of reagent.Usually, reporter molecule is an enzyme, and it is by (Sigma, St.Louis in the time of MO) are incubated solid phase and detect having suitable fluorescence, luminous or colorimetric substrates.

By being used for the known technology that proteinaceous substances is attached to solid support material such as polymeric beads, dipstick, 96 orifice plates or filter material is prepared the solid surface reagent of said determination method.These attachment meanss generally include the non-specific adsorption of protein and upholder, or protein is covalently attached to chemically reactive group on the solid support by free amino usually, such as activatory carboxyl, hydroxyl or aldehyde radical.Perhaps, can use the plate of the plain bag of strepto-affinity quilt together with biotinylated antigen.

So, the invention provides mensuration system or the test kit that is used to carry out this diagnostic method.Test kit generally includes the upholder of the recombinant antigen with surface bonding and is used to detect anti-people's antibody of reporter molecule mark of the anti-antigen-antibody of surface bonding.

Albumin fusion proteins

The present invention relates generally to albumin fusion proteins and treatment, prevention or improves disease or disorderly method.When being used for this paper, " albumin fusion proteins " refers to that the therapeutic protein (or its fragment or variant) of white protein (or its fragment or variant) and at least one molecule by at least one molecule merges the protein that forms.Albumin fusion proteins of the present invention comprises the fragment of therapeutic protein at least or variant and human serum albumin's fragment or variant at least, and they preferably are interconnected with one another by gene fusion (being that albumin fusion proteins is with identical reading frame is complete with coding or the albuminised polynucleotide of part are connected translated nucleic acid generation by the polynucleotide of wherein encode complete or part therapeutic protein).Therapeutic protein and white protein in case become the part of albumin fusion proteins, can be described as " part ", " zone " or " module " of albumin fusion proteins.

In a preferred embodiment, the invention provides the albumin fusion proteins of encoding by polynucleotide of describing in table 1 or the table 2 or white protein fusion constructs.The polynucleotide of these albumin fusion proteins of encoding are also contained in the present invention.

The preferred albumin fusion proteins of the present invention includes but not limited to the albumin fusion proteins by following nucleic acid molecule encoding, described nucleic acid molecule comprise at least one molecule of coding that is connected with at least one section polynucleotide of therapeutic protein (or its fragment or variant) of at least one molecule of coding with identical reading frame white protein (or its fragment or variant) polynucleotide or form by it; Described nucleic molecule comprise at least one molecule of coding that is connected with at least one section polynucleotide of the therapeutic protein that described in table 1, table 2 or embodiment, produces (or its fragment or variant) of at least one molecule of coding with identical reading frame white protein (or its fragment or variant) polynucleotide or form by it; Perhaps described nucleic acid molecule comprise at least one molecule of coding that is connected with at least one section polynucleotide of therapeutic protein (or its fragment or variant) of at least one molecule of coding with identical reading frame white protein (or its fragment or variant) polynucleotide or form by it, also comprise for example one or more following elements: (1) functional autonomously replicationg vector (includes, but are not limited to shuttle vectors, expression vector, integrative vector, and/or dubbing system), (2) transcription initiation region (promoter region for example, such as for example regulating or inducible promoter, constitutive promoter), (3) transcription termination region, (4) leader sequence and (5) selective marker.

In one embodiment, the invention provides and comprise therapeutic protein (for example described in the table 1) and serum albumin protein or by its albumin fusion proteins of forming.In other embodiments, the invention provides the biologic activity that comprises therapeutic protein and/or therapeutic activity fragment and serum albumin protein or by its albumin fusion proteins of forming.In other embodiments, the invention provides the biologic activity that comprises therapeutic protein and/or therapeutic activity variant and serum albumin protein or by its albumin fusion proteins of forming.In preferred embodiments, the serum albumin protein component of albumin fusion proteins is the maturing part of serum albumin.

In other embodiments, the invention provides the biologic activity that comprises therapeutic protein and serum albumin and/or therapeutic activity fragment or by its albumin fusion proteins of forming.In other embodiments, the invention provides the biologic activity that comprises therapeutic protein and serum albumin and/or therapeutic activity variant or by its albumin fusion proteins of forming.In preferred embodiments, the therapeutic protein of albumin fusion proteins partly is the maturing part of therapeutic protein.

In other embodiments, the invention provides the biologic activity of the biologic activity that comprises therapeutic protein and/or therapeutic activity fragment or variant and serum albumin and/or therapeutic activity fragment or variant or by its albumin fusion proteins of forming.In preferred embodiments, the invention provides the maturing part of the maturing part that comprises therapeutic protein and serum albumin or by its albumin fusion proteins of forming.

Preferably, albumin fusion proteins comprises HA as the N end parts, and comprises therapeutic protein as the C end parts.Perhaps, also can use and comprise HA as the C end parts and comprise the albumin fusion proteins of therapeutic protein as the N end parts.

In other embodiments, albumin fusion proteins all merges at albuminised N-terminal and C-terminal therapeutic protein.In a preferred embodiment, be identical therapeutic protein at N-terminal with the therapeutic protein that C-terminal merges.In another preferred embodiment, be different therapeutic proteins at N-terminal with the therapeutic protein that C-terminal merges.In another preferred embodiment, be to can be used for treatment or prevent different therapeutic proteins identical or relative disease, disorder or situation (for example cited in table 1 " the preferred indication Y " row) at the therapeutic protein that N-terminal and C-terminal merge.In another preferred embodiment, the therapeutic protein that N-terminal and C-terminal merge be can be used for treating, improve or prevent that this area knows usually in the patient simultaneously, concurrent or the disease of continued presence or existence associated with each other in the patient usually or the different therapeutic proteins of disorder (for example table 1 " preferred indication Y " is cited in being listed as).

Albumin fusion proteins of the present invention is contained the TA protein X of 1,2,3,4 or the more a plurality of molecules that contain fusion and/or the N-that merge at white protein or its variant and/or C-end terminal at the N-of albumin fusion proteins of the present invention or C-or the protein of its variant.The molecule of TA protein X or its variant can be the orientation of any number, include but not limited to " head to head " orientation (for example the N-end of one of them therapeutic protein X molecule is fused to the N-end of another therapeutic protein X molecule), or " head is to tail " orientation (for example the C-end of one of them therapeutic protein X molecule is fused to the N-end of another therapeutic protein X molecule).

In one embodiment, 1,2,3 or the therapeutic protein X polypeptide (or its fragment or variant) of more a plurality of series connection orientation be fused to the N-or the C-end of albumin fusion proteins of the present invention, and/or be fused to the N-and/or the C-end of white protein or its variant.

Albumin fusion proteins of the present invention is also contained the TA protein X of 1,2,3,4 or the more a plurality of molecules that contain fusion and/or the N-that merge at white protein or its variant and/or C-end terminal at the N-of albumin fusion proteins of the present invention or C-or the protein of its variant, and wherein said molecule connects by peptide linker.Example comprises U.S. Patent number 5,073, those peptide linkers of describing in 627 (being incorporated herein by reference).Can use conventional recombinant DNA technology to generate to comprise the albumin fusion proteins of a plurality of therapeutic protein X polypeptide that separate by peptide linker.When little peptide being fused on the big HSA molecule, joint is a particularly important.Peptide self can be by tandem copy peptide fusion and as joint, perhaps can use other known joint.The construct that mixes joint is described in table 2 or is conspicuous when checking SEQ ID NO:Y.

In addition, albumin fusion proteins of the present invention also can be by a kind ofly allowing that the mode that forms intramolecularly and/or intermolecular polymer form is with therapeutic protein X or its variant is fused to the N-end of white protein or its variant and/or C-is terminal produces with such.In one embodiment of the invention, albumin fusion proteins can be monomer or polymeric form (being dimer, tripolymer, the tetramer and Geng Gao polymer).In another embodiment of the invention, the therapeutic protein of albumin fusion proteins part can be monomer or polymeric form (being dimer, tripolymer, the tetramer and Geng Gao polymer).In a specific embodiment, the therapeutic protein of albumin fusion proteins partly is polymeric form (being dimer, tripolymer, the tetramer and Geng Gao polymer), and albumin protein partly is monomeric form.

Except albumin part wherein merges the albumin fusion proteins of the N-end of therapeutic protein part and/or C-end, albumin fusion proteins of the present invention also can pass through the interior region of therapeutic protein or purpose peptide (for example disclosed therapeutic protein X in the table 1, or the antibody of combined treatment protein or its fragment or variant) insertion HA and produce.For example, in the protein sequence of HA molecule, between the terminal point of alpha-helix and starting point, exist by disulfide linkage stable many rings or corner.According to the crystalline structure (PDB indications 1AO6,1BJ5,1BKE, 1BM0,1E7E to 1E71 and 1UOR) of HA, these rings are most of away from the molecule main body.These rings can be used for the insertion or inner fusion of therapeutic activity peptide, particularly need secondary structure that those of function are just arranged, or therapeutic protein, have the active white protein molecule of particular biological to produce in essence.

Can insert peptide or polypeptide in people's white protein structure comprises with the ring 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 a more preferred embodiment, peptide or polypeptide insert into ring Val54-Asn61, Gln170-Ala176 and/or the Lys560-Thr566 of acquaintance's white protein (SEQ ID NO:1).

The peptide that is inserted into can be derived from the phage display that the particular biological activity is screened or synthetic peptide library or from the active part of the molecule with desired function.In addition, can in specific ring, produce random peptide library, or peptide inserts in the specific ring of HA molecule by inciting somebody to action at random, wherein represented all possible amino acid combination.

Such library can produce on HA or HA structural domain fragment by one of following method:

(a) amino acid in HA or the segmental one or more peptide rings of HA structural domain is carried out random mutation.One of intra-annular, a plurality of or all residues by this way can suddenly change;

(b) in one or more rings of HA or HA structural domain fragment (promptly inner the fusion), carry out displacement or the insertion that length is the peptide at random of Xn (wherein X is an amino acid, and n is the residue number);

(c) except that (a) and/or N-terminal, C-terminal or N (b) and C-terminal peptide/protein merge.

Also can make HA or HA structural domain fragment become multi-functional by being transplanted in identical HA or the HA structural domain fragment the difference screening deutero-peptide that different rings carries out at different targets.

In preferred embodiments, the peptide in insertion human serum albumin's the ring is the peptide fragment or the peptide variant of disclosed therapeutic protein in the table 1.In particular, the present invention is contained and has been inserted the albumin fusion proteins of length 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 peptide fragment or peptide variant in the ring that is included in the human serum albumin.The present invention is also contained the N-terminal that is included in the human serum albumin and has been merged the albumin fusion proteins of length 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 peptide fragment or peptide variant.The present invention is also contained the C-terminal that is included in the human serum albumin and has been merged the albumin fusion proteins of length 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 peptide fragment or peptide variant.For example, the small peptide of describing in the table 1 and 2 (for example therapeutical agent Y) can be inserted in the albuminised ring.

Generally speaking, albumin fusion proteins of the present invention can have a HA derive zone and therapeutic protein zone of deriving.Yet every kind of proteinic a plurality of zones can be used for making up albumin fusion proteins of the present invention.Similar, surpass a kind of therapeutic protein and can be used for making up albumin fusion proteins of the present invention.For example, therapeutic protein can be fused to N-and two ends of C-of HA.In a kind of like this structure, the therapeutic protein part can be identical or different proteinaceous therapeutic molecule.The structure of difunctional 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 fusions is to pass through anti-BLyS ^TMScFv regulates the immunne response to IFN α-2b.Perhaps, can prepare two (or even many) functional agents of HA-fusions, HA-IFN α-2b fusions for example, and mix the anti-BLyS of HA-in varing proportions according to function, transformation period etc. ^TMScFv fusions or other HA-fusions.

Also can prepare the therapeutic protein part target target organ of fusions or the two or multi-functional albumin fusion proteins of cell type by protein or the peptide that is positioned at the HA the other end.

Alternative method as the fusions of known treatment molecule, can obtain peptide as the library that HA or HA structural domain fragment N-terminal, C-terminal or N and C-terminal fusions make up by screening, (wherein X is amino acid (aa) for their normally 6,8,12,20 or 25 or Xn, n is the residue number) random amino acid, wherein represented all possible amino acid combination.The special benefits of this method be can be on the HA molecule original position select peptide, so the characteristic of peptide is just as selecting, and can as situation about being attached to then by any other method derived peptide on the HA potential change not take place.

In addition, albumin fusion proteins of the present invention can comprise the joint peptide between merging partly, thereby bigger physical separation is provided between module, and makes the accessibility maximization of therapeutic protein part thus, for example combines with its associated receptor.The joint peptide can be made up of amino acid, makes its tool flexibility or has more rigidity.

Joint sequence can be by proteolytic enzyme or chemical ablation to produce the tethelin relevant portion.Preferably, proteolytic enzyme is the natural generation of host, for example the proteolytic enzyme of yeast saccharomyces cerevisiae proteolytic enzyme kex2 or equivalence.

Therefore, as mentioned above, albumin fusion proteins of the present invention can have following general formula: R1-L-R2; R2-L-R1 or R1-L-R2-L-R1, wherein R1 is at least a therapeutic protein, peptide or peptide sequence, and needs not to be identical therapeutic protein, L is a joint, and R2 is the serum albumin sequence.

In preferred embodiments, the albumin fusion proteins of the present invention that comprises therapeutic protein has and does not compare higher plasma stability with the proteinic plasma stability of identical treatment that white protein merges.When plasma stability is often referred in vivo administering therapeutic protein and enters blood flow with in the therapeutic protein degraded and from blood flow, removes and enter such as organs such as kidney or livers, final in body the timed interval between during removing.Transformation period according to therapeutic protein in the blood flow is calculated plasma stability.The transformation period of therapeutic protein can be easy to by the assay method commonly used that this area is known measure in the blood flow.

In preferred embodiments, the albumin fusion proteins of the present invention that comprises therapeutic protein has and does not compare with the proteinic quality guaranteed period of identical treatment that white protein merges the quality guaranteed period of prolongation.Quality guaranteed period be often referred in the solution or some other depot formulation in the therapeutic activity of therapeutic protein keep stable and do not have the excessively time bar of forfeiture of therapeutic activity.Many therapeutic proteins are highly not variable under the fusion state at it.As described below, these therapeutic proteins are common significant prolongation of its quality guaranteed period after introducing albumin fusion proteins of the present invention.

Quality guaranteed period " prolongation " albumin fusion proteins of the present invention shows bigger therapeutic activity with respect to the standard substance of accepting identical storage and treatment condition.Standard substance can be the total length therapeutic proteins that does not merge.When the therapeutic protein of albumin fusion proteins partly is this proteinic analogue, variant or when other change being arranged or not comprising complete sequence, the prolongation of therapeutic activity or can compare with the peptide of this analogue, variant, change or the not fusion equivalent of imperfect sequence.As an example, when preset time point compares, albumin fusion proteins of the present invention can keep the standard substance of accepting identical storage and treatment condition therapeutic activity greater than about 100% or greater than about 105%, 110%, 120%, 130%, 150% or 200% of therapeutic activity.

Quality guaranteed period also can be assessed according to storing the remaining therapeutic activity in back, and the therapeutic activity during at the storage beginning is carried out stdn.The albumin fusion proteins of the present invention that quality guaranteed period prolongs shows therapeutic activity and prolongs, can keep the equivalence of accepting the same terms do not merge therapeutic protein therapeutic activity greater than about 50%, about 60%, 70%, 80% or 90% or more.

Expression of Fusion Protein

Albumin fusion proteins of the present invention can be used as recombinant molecule and is produced by yeast, microorganism such as bacterium or the secretion of human or animal's clone.Optional is that polypeptide is by secretory host cell.

A specific embodiments of the present invention comprises that coding is for the DNA construct that instructs the effective signal sequence of secretion in yeast, yeast deutero-signal sequence (especially with the yeast host homologous) particularly, with the fusion molecule of first aspect present invention, between signal and mature polypeptide, there is not yeast deutero-former (pro) sequence.

Yeast saccharomyces cerevisiae saccharase signal is the preferred example of yeast deutero-signal sequence.

Do not imagine that class conjugate of people (FEBS Lett.239:18,1988) such as Poznansky preparation, wherein separately the polypeptide of preparation is connected by chemically crosslinked.

The present invention also comprises the cell of expressing albumin fusion proteins of the present invention through transforming, preferred yeast cell.Except through transformed host cell itself, the culture of those cells in nutritional medium also contained in the present invention, preferred mono-clonal (homogeneity on the clone) culture or by mono-clonal culture deutero-culture.If polypeptide is an excretory, then substratum will contain polypeptide, have cell, if or cell has been filtered or centrifugal removal then do not have cell.Many expression systems are known and spendable, comprise bacterium (for example colon bacillus (E.coli) and subtilis (Bacillus subtilis)), yeast (for example yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), Kluyveromyces lactis (Kluyveromyces lactis) and Pichia pastoris (Pichia pastoris)), filamentous fungus (for example Aspergillus), vegetable cell, zooblast and insect cell.

The yeast strain that is preferred for producing albumin fusion proteins is D88, DXY1 and BXP10.D88[leu2-3, leu2-122, can1, pra1, ubc4] be that parent strain AH22his+ (is also referred to as DB1; Referring to people such as for example Sleep, Biotechnology 8:42-46,1990) derivative.This bacterial strain contains the leu2 sudden change, and it allows that the plasmid based on 2 μ m that contains the LEU2 gene is carried out auxotroph to be selected.D88 also shows derepressing of PRB1 when glucose is excessive.The PRB1 promotor is subjected to the control of two kinds of check points of monitoring glucose level and growth phase usually.In wild-type yeast, this promotor exhausts to go forward side by side at glucose and activates into static after date.Bacterial strain D88 shows and is subjected to checking of glucose, but keeps inducing after entering stationary phase.PRA1 genes encoding yeast vacuole proteolytic enzyme, YscA endo-protease A, it is positioned ER.The UBC4 gene is in the ubiquitin approach and participates in making short-lived and unusual protein target ubiquitin dependency degraded.The separation of finding this ubc4 sudden change increases the copy number of expression plasmid in the cell and causes expectation protein expression level by plasmid expression raise (referring to for example international issue WO 99/00504, with its complete being incorporated herein by reference).

DXY1, a kind of derivative of D88 has following genotype: [leu2-3, leu2-122, can1, pra1, ubc4, ura3::yap3].The isolating sudden change, this bacterial strain also has knocking out of YAP3 proteolytic enzyme in D88.This proteolytic enzyme mainly causes the cutting of two alkaline residues (RR, RK, KR, KK) in the protein, but also can promote the cutting at single alkaline residue place.The separation of this yap3 sudden change causes high-caliber total length HSA product (referring to for example people such as U.S. Patent number 5,965,386 and Kerry-Williams, Yeast 14:161-169 is 1998, with its complete being incorporated herein by reference).

BXP10 has following genotype: leu2-3, leu2-122, can1, pra1, ubc4, ura3, yap3::URA3, lys2, hsp150::LYS2, pmt1::URA3.The isolating sudden change, this bacterial strain also has knocking out of PMT1 gene and HSP150 gene in DXY1.The PMT1 gene is the member of dolichol-phosphoric acid-D-Mannoproteins matter O-mannose transferase (Pmt) evolution conservative family.The Transmembrane Topology of Pmt1p shows that it is the integral protein of endoplasmic reticulum, connects in the glycosylation at O-and works.This sudden change helps the O-that reduces/eliminate the HSA fusions to connect glycosylation (referring to for example international issue WO 00/44772, with its complete being incorporated herein by reference).Research has disclosed by ion exchange chromatography and can not effectively separate Hsp150 protein in rHA.Sudden change in the HSP150 gene has been eliminated and has been confirmed to be difficult to the potential pollutant removed by the standard purification technology.Referring to for example U.S. Patent number 5,783,423, with its complete being incorporated herein by reference.

Produce desired protein with ordinary method, for example by inserting in the host chromosome or the generation of the encoding sequence on the free plasmid.With any common method for example electroporation with the encoding sequence transformed yeast of desired protein.Method by the electroporation transformed yeast is disclosed in Becker and Guarente, Methods Enzymol.194:182,1990.

The success cell transformed, the cell that promptly contains DNA construct of the present invention can be identified by well-known technology.For example, can cultivate by the cell that imports the expression construct generation to produce required polypeptide.But harvested cell, cracking, and using such as Southern, J.Mol.Biol.98:503,1975 or people such as Berent, Biotech.3:208,1985 methods of describing are to the existence of DNA content check DNA.Perhaps, can use antibody to detect proteinic existence in the supernatant liquor.

Useful yeast plasmid carrier comprises pRS403-406 and pRS413-416, and 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 have mixed yeast selective marker HIS3,7RP1, LEU2 and URA3.Plasmid pRS413-416 is yeast centromeric plasmid (YCp).

The carrier that is preferred for expressing in yeast with the preparation albumin fusion proteins comprises pPPC0005, pScCHSA, pScNHSA and pC4:HSA, and they are specified among the embodiment 1.Fig. 2 has shown the pPPC0005 plasmid map that can be used as underlying carrier, can be to the polynucleotide of clones coding therapeutic protein wherein to form the HA-fusions.It contains DNA (rHA) and the ADH1 yeast saccharomyces cerevisiae terminator sequence of PRB1 yeast saccharomyces cerevisiae promotor (PRB1p), fusion leader sequence (FL), coding HA.The sequence that merges leader sequence is made up of preceding 19 amino acid of human serum albumin (SEQ ID NO:3) signal peptide and last 5 amino acid of mating factor α 1 promotor (SLDKR is referring to EP-A-387319, with its complete being incorporated herein by reference).

Plasmid pPPC0005, pScCHSA, pScNHSA and pC4:HSA are preserved in U.S. typical case's culture collecting center April 11 calendar year 2001,10801 University Boulevard, Manassas, Virginia 20110-2209, and given son respectively preserving number ATCC PTA-3278, PTA-3276, PTA-3279 and PTA-3277.Be used in the another kind of carrier pSAC35 carrier of expressing albumin fusion proteins in the yeast and be described in people such as Sleep, BioTechnology 8:42,1990, with its complete being incorporated herein by reference.

The Yeast promoter that can be used for expressing albumin fusion proteins has the MET25 promotor.Referring to for example Dominik Mumburg, Rolf Muller and Martin Funk, Nucleic Acids Research 22 (25): 5767-5768,1994.The length of Met25 promotor is 383 bases (base-382 is to-1), and is also referred to as Met15, Met17 and YLR303W by the gene of this promoter expression.An embodiment preferred is used following sequence, is positioned at 5 ' the terminal Not I site that is used to clone in the wherein following sequence and indicates underscore, and be positioned at 3 ' terminal ATG initiator codon and indicate underscore:

GCGGCCGCCGGATGCAAGGGTTCGAATCCCTTAGCTCTCATTATTTTTTGCTTTTTCTCTTGAGGTCACATGATCGCAAAATGGCAAATGGCACGTGAAGCTGTCGATATTGGGGAACTGTGGTGGTTGGCAAATGACTAATTAAGTTAGTCAAGGCGCCATCCTCATGAAAACTGTGTAACATAATAACCGAAGTGTCGAAAAGGTGGCACCTTGTCCAATTGAACACGCTCGATGAAAAAAATAAGATATATATAAGGTTAAGTAAAGCGTCTGTTAGAAAGGAAGTTTTTCCTTTTTCTTGCTCTCTTGTCTTTTCATCTACTATTTCCTTCGTGTAATACAGGGTCGTCAGATACATAGATACAATTCTATTACCCCCATCCATACA A TG(SEQ?ID?NO：5)

Being used in other promotor of expressing albumin fusion proteins in the yeast comprises as follows:

A) cbh 1 promotor:

TCTAGAGTTGTGAAGTCGGTAATCCCGCTGTATAGTAATACGAGTCGCATCTAAATACTCCGAAGCTGCTGCGAACCCGGAGAATCGAGATGTGCTGGAAAGCTTCTAGCGAGCGGCTAAATTAGCATGAAAGGCTATGAGAAATTCTGGAGACGGCTTGTTGAATCATGGCGTTCCATTCTTCGACAAGCAAAGCGTTCCGTCGCAGTAGCAGGCACTCATTCCCGAAAAAACTCGGAGATTCCTAAGTAGCGATGGAACCGGAATAATATAATAGGCAATACATTGAGTTGCCTCGACGGTTGCAATGCAGGGGTACTGAGCTTGGACATAACTGTTCCGTACCCCACCTCTTCTCAACCTTTGGCGTTTCCCTGATTCAGCGTACCCGTACAAGTCGTAATCACTATTAACCCAGACTGACCGGACGTGTTTTGCCCTTCATTTGGAGAAATAATGTCATTGCGATGTGTAATTTGCCTGCTTGACCGACTGGGGCTGTTCGAAGCCCGAATGTAGGATTGTTATCCGAACTCTGCTCGTAGAGGCATGTTGTGAATCTGTGTCGGGCAGGACACGCCTCGAAGGTTCACGGCAAGGGAAACCACCGATAGCAGTGTCTAGTAGCAACCTGTAAAGCCGCAATGCAGCATCACTGGAAAATACAAACCAATGGCTAAAAGTACATAAGTTAATGCCTAAAGAAGTCATATACCAGCGGCTAATAATTGTACAATCAAGTGGCTAAACGTACCGTAATTTGCCAACGGCTTGTGGGGTTGCAGAAGCAACGGCAAAGCCCCACTTCCCCACGTTTGTTTCTTCACTCAGTCCAATCTCAGCTGGTGATCCCCCAATTGGGTCGCTTGTTTGTTCCGGTGAAGTGAAAGAAGACAGAGGTAAGAATGTCTGACTCGGAGCGTTTTGCATACAACCAAGGGCAGTGATGGAAGACAGTGAAATGTTGACATTCAAGGAGTATTTAGCCAGGGATGCTTGAGTGTATCGTGTAAGGAGGTTTGTCTGCCGATACGACGAATACTGTATAGTCACTTCTGGTGAAGTGGTCCATATTGAAATGTAAGTCGGCACTGAACAGGCAAAAGATTGAGTTGAAACTGCCTAAGATCTCGGGCCCTCGGGCCTTCGGCCTTTGGGTGTACATGTTTGTGCTCCGGGCAAATGCAAAGTGTGGTAGGATCGAACACACTGCTGCCTTTACCAAGCAGCTGAGGGTATGTGATAGGCAAATGTTCAGGGGCCACTGCATGGTTTCGAATAGAAAGAGAAGCTTAGCCAAGAACAATAGCCGATAAAGATAGCCTCATTAAACGGAATGAGCTAGTAGGCAAAGTCAGCGAATGTGTATATATAAAGGTTCGAGGTCCGTGCCTCCCTCATGCTCTCCCCATCTACTCATCAACTCAGATCCTCCAGGAGACTTGTACACCATCTTTTGAGGCACAGAAACCCAATAGTCAACCGCGGACTGGCATC(SEQ?ID?NO：113)

B) the cysD promotor of Aspergillus nidulans (Aspergillus nidulans):

AGATCTGGTTCCTGAGTACATCTACCGATGCGCCTCGATCCCCCTCTTAGCCGCATGAGATTCCTACCATTTATGTCCTATCGTTCAGGGTCCTATTTGGACCGCTAGAAATAGACTCTGCTCGATTTGTTTCCATTATTCACGCAATTACGATAGTATTTGGCTCTTTTCGTTTGGCCCAGGTCAATTCGGGTAAGACGCGATCACGCCATTGTGGCCGCCGGCGTTGTGCTGCTGCTATTCCCCGCATATAAACAACCCCTCCACCAGTTCGTTGGGCTTTGCGAATGCTGTACTCTATTTCAAGTTGTCAAAAGAGAGGATTCAAAAAATTATACCCCAGATATCAAAGATATCAAAGCCATC(SEQ?ID?NO：114)

C) modified cbh1 promotor has sequence:

TCTAGAGTTGTGAAGTCGGTAATCCCGCTGTATAGTAATACGAGTCGCATCTAAATACTCCGAAGCTGCTGCGAACCCGGAGAATCGAGATGTGCTGGAAAGCTTCTAGCGAGCGGCTAAATTAGCATGAAAGGCTATGAGAAATTCTGGAGACGGCTTGTTGAATCATGGCGTTCCATTCTTCGACAAGCAAAGCGTTCCGTCGCAGTAGCAGGCACTCATTCCCGAAAAAACTCGGAGATTCCTAAGTAGCGATGGAACCGGAATAATATAATAGGCAATACATTGAGTTGCCTCGACGGTTGCAATGCAGGGGTACTGAGCTTGGACATAACTGTTCCGTACCCCACCTCTTCTCAACCTTTGGCGTTTCCCTGATTCAGCGTACCCGTACAAGTCGTAATCACTATTAACCCAGACTGACCGGACGTGTTTTGCCCTTCATTTGGAGAAATAATGTCATTGCGATGTGTAATTTGCCTGCTTGACCGACTGGGGCTGTTCGAAGCCCGAATGTAGGATTGTTATCCGAACTCTGCTCGTAGAGGCATGTTGTGAATCTGTGTCGGGCAGGACACGCCTCGAAGGTTCACGGCAAGGGAAACCACCGATAGCAGTGTCTAGTAGCAACCTGTAAAGCCGCAATGCAGCATCACTGGAAAATACAAACCAATGGCTAAAAGTACATAAGTTAATGCCTAAAGAAGTCATATACCAGCGGCTAATAATTGTACAATCAAGTGGCTAAACGTACCGTAATTTGCCAACGGCTTGTGGGGTTGCAGAAGCAACGGCAAAGCCCCACTTCCCCACGTTTGTTTCTTCACTCAGTCCAATCTCAGCTGGTGATCCCCCAATTGGGTCGCTTGTTTGTTCCGGTGAAGTGAAAGAAGACAGAGGTAAGAATGTCTGACTCGGAGCGTTTTGCATACAACCAAGGGCAGTGATGGAAGACAGTGAAATGTTGACATTCAAGGAGTATTTAGCCAGGGATGCTTGAGTGTATCGTGTAAGGAGGTTTGTCTGCCGATACGACGAATACTGTATAGTCACTTCTGGTGAAGTGGTCCATATTGAAATGTAAGTCGGCACTGAACAGGCAAAAGATTGAGTTGAAACTGCCTAAGATCTCGGGCCCTCGGGCCTTCGGCCTTTGGGTGTACATGTTTGTGCTCCGGGCAAATGCAAAGTGTGGTAGGATCGAACACACTGCTGCCTTTACCAAGCAGCTGAGGGTATGTGATAGGCAAATGTTCAGGGGCCACTGCATGGTTTCGAATAGAAAGAGAAGCTTAGCCTGCAGCCTCTTATCGAGAAAGAAATTACCGTCGCTCGTGATTTGTTTGCAAAAAGAACAAAACTGAAAAAACCCAGACACGCTCGACTTCCTGTCTTCCTATTGATTGCAGCTTCCAATTTCGTCACACAACAAGGTCCTAGCTTAGCCAAGAACAATAGCCGATAAAGATAGCCTCATTAAACGGAATGAGCTAGTAGGCAAAGTCAGCGAATGTGTATATATAAAGGTTCGAGGTCCGTGCCTCCCTCATGCTCTCCCCATCTACTCATCAACTCAGATCCTCCAGGAGACTTGTACACCATCTTTTGAGGCACAGAAACCCAATAGTCAACCGCGGACTGGCATC(SEQ?ID?NO：115)

D) the cysD promotor of Aspergillus nidulans has sequence:

AGATCTGGTTCCTGAGTACATCTACCGATGCGCCTCGATCCCCCTCTTAGCCGCATGAGATTCCTACCATTTATGTCCTATCGTTCAGGGTCCTATTTGGACCGCTAGAAATAGACTCTGCTCGATTTGTTTCCATTATTCACGCAATTACGATAGTATTTGGCTCTTTTCGTTTGGCCCAGGTCAATTCGGGTAAGACGCGATCACGCCATTGTGGCCGCCGGCGCTGCAGCCTCTTATCGAGAAAGAAATTACCGTCGCTCGTGATTTGTTTGCAAAAAGAACAAAACTGAAAAAACCCAGACACGCTCGACTTCCTGTCTTCCTATTGATTGCAGCTTCCAATTTCGTCACACAACAAGGTCCTACGCCGGCGTTGTGCTGCTGCTATTCCCCGCATATAAACAACCCCTCCACCAGTTCGTTGGGCTTTGCGAATGCTGTACTCTATTTCAAGTTGTCAAAAGAGAGGATTCAAAAAATTATACCCCAGATATCAAAGATATCAAAGCCATC (SEQ?ID?NO：116)

Having developed several different methods is used for through complementary cohesive end DNA and carrier can being operatively connected.For example, complementary homopolymer bundle can be added into the DNA section that is inserted in the carrier DNA.Then, carrier and DNA section are connected to form recombinant DNA molecules by hydrogen bond between the complementary homopolymer tail.

The synthetic linker that contains one or more restriction sites provides the another kind of method that connects DNA section and carrier.To handle with phage T4DNA polysaccharase or e. coli dna polymerase I by the DNA section that the endonuclease restrictive diges-tion produces, these enzymes are eliminated the γ strand end that protrudes with its 3 '-5 ' exonuclease hydrolytic activity, and fill and lead up 3 recessed ' end with its polymerization activity.

Therefore, these are active in conjunction with the DNA section that produces end-filling.Then, when having enzyme such as the phage T4DNA ligase enzyme that can catalysis flush end dna molecular connects, the section of end-filling is incubated with the excessive greatly linkers of mole number.Thus, reaction product is to carry the DNA section of poly joint sequence at its end.Then, cut these DNA sections, and be connected with the expression vector that cut with the enzyme that produces the end compatible with the DNA segment ends with suitable restriction enzyme.

The synthetic linker that contains multiple restriction endonuclease site can be buied from many sources, comprises International Biotechnologies Inc, New Haven, CT, USA.

If for example will prepare the HA variant, a kind of Perfected process of modifying DNA is to use people such as Saiki according to the present invention, Science 239:487-491,1988 disclosed polymerase chain reactions.In this method, the flank of DNA that will enzymatic amplification is two kinds of special Oligonucleolide primers, and they itself mix among the DNA of amplification.Special primer can contain the restriction endonuclease recognition site, can be used for being cloned in the expression vector by the method that this area is known.

Imagination can be used as zymic illustration that the host who expresses albumin fusion proteins is used for the present invention's practice and belongs to Pichia (Pichia) (Hansenula Hansenula) is arranged, saccharomyces (Saccharomyces), genus kluyveromyces (Kluyveromyces), mycocandida (Candida), torulopsis (Torulopsis), spore torulopsis (Torulaspora) is arranged, fragmentation saccharomyces (Schizosaccharomyces), Citeromycesbaodingensis belongs to (Citeromyces), pipe capsule yeast belong (Pachysolen), Debaryomyces (Debaryomyces), the strange yeast belong of plum (Metschunikowia), red teliosporeae (Rhodosporidium), white winter spore yeast (Leucosporidium), Portugal's shape arthroderma (Botryoascus), lock is thrown yeast belong (Sporidiobolus), Endomycopsis (Endomycopsis) or the like.Is to be selected from down the genus of organizing preferred the genus: saccharomyces, fragmentation saccharomyces, genus kluyveromyces, Pichia and the spore torulopsis is arranged.The example of saccharomyces has Saccharomyces cerevisiae (S.cerevisiae), Italian sugar yeast (S.italicus) and Shandong formula sugar yeast (S.rouxii).

The example of genus kluyveromyces has the crisp kluyveromyces of wall (K.fragilis), Kluyveromyces lactis (K.lactis) and kluyveromyces marxianus (K.marxianus).The suitable spore torula species that have have Dell that spore torula (T.delbrueckii) is arranged.The example of pichia spp (debaryomyces hansenii) has P.angusta (being multiple-shaped nuohan inferior yeast H.polymorpha in the past), unusual pichia (P.anomala) (being unusual Hansenula anomala H.anomala in the past) and Pichia pastoris (P.pastoris).Be used for common instruction of method that yeast saccharomyces cerevisiae transforms, all be incorporated herein by reference from EP 251744, EP 258067 and WO 90/01063.

Preferred yeast saccharomyces cerevisiae example kind comprises that Saccharomyces cerevisiae (S.cerevisiae), Italian sugar yeast (S.italicus), saccharification sugar yeast (S.diastaticus) and Lu Shi engage sugar yeast (Zygosaccharomycesrouxii).Preferred kluyveromyces example kind comprises crisp kluyveromyces of wall (K.fragilis) and Kluyveromyces lactis (K.lactis).Preferred debaryomyces hansenii example kind comprises multiple-shaped nuohan inferior yeast (H.polymorpha) (being P.angusta now), unusual debaryomyces hansenii (H.anomala) (being unusual pichia spp P.anomala now) and broken capsule pichia spp (Pichia capsulata).Preferred in addition pichia spp example kind comprises Pichia pastoris (P.pastoris).Preferred aspergillus example kind comprises aspergillus niger (A.niger) and Aspergillus nidulans (A.nidulans).Preferred inferior sieve yeast (Yarrowia) example kind comprises separates the inferior sieve yeast (Y.lipolytica) of fat.Many preferred yeast species can obtain from ATCC.For example, following preferred yeast species can and can be used for expressing albumin fusion proteins from the ATCC acquisition: Saccharomyces cerevisiae Hansen, teleomorph strain BY4743 yap3 mutant (ATCC accession number 4022731); Saccharomyces cerevisiae Hansen, teleomorph strain BY4743hsp 150 mutant (ATCC accession number 4021266); Saccharomyces cerevisiae Hansen, teleomorph strain BY4743 pmt1 mutant (ATCC accession number 4023792); Saccharomycescerevisiae Hansen, teleomorph (ATCC accession number 20626; 44773; 44774 and 62995); Saccharomyces diastaticus Andrews et Gilliland ex van der Walt, teleomorph (ATCC accession number 62987); Kluyveromyces lactis (Dombrowski) van der Walt, teleomorph (ATCC accession number 76492); Pichia angusta (Teunisson et al.) Kurtzman, teleomorph is as Hansenula polymorpha de Morais et Maia, teleomorph preservation (ATCC accession number 26012); Aspergillus niger van Tieghem, anamorph (ATCC accession number 9029); Aspergillus niger van Tieghem, anamorph (ATCC accession number 16404); Aspergillus nidulans (Eidam) Winter, anamorph (ATCC accession number 48756); With Yarrowia lipolytica (Wickerham et al.) van der Walt et von Arx, teleomorph (ATCC accession number 201847).

The promotor that is applicable to yeast saccharomyces cerevisiae comprises the gene with PGK1, GAL1 or GAL10 gene, CYC1, PHO5, TRP1, ADH1, ADH2, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvic carboxylase, phosphofructokinase, triosephosphate isomerase, phosphoglucose isomerase, the gene of glucokinase, α-mating factor pheromone, (a mating factor pheromone) relevant promotor, the PRB1 promotor, the GUT2 promotor, the GPD1 promotor, and relate to part 5 ' control region and other promotor part 5 ' control region or with the hybrid promoter (for example promotor of EP-A-258 067) of the heterozygote of UAS.

The promotor of being convenient to regulate that is used for grain wine fragmentation sugar yeast (Schizosaccharomyces pombe) is as Maundrell, J.Biol.Chem.265:10857-10864,1990 VitB1s from the nmt gene of describing can be suppressed promotor, and as Hoffman and Winston, Genetics 124:807-816,1990 glucose of describing can suppress the jbp1 gene promoter.

Transform pichia spp and for example see people such as Cregg with the instruction of the method for expression alien gene, 1993 and multinomial Philips patent (for example US 4 857 467, be incorporated herein by reference), and the Pichia anomala expression test kit can be available from Invitrogen BV, Leek, Netherlands and Invitrogen Corp., SanDiego, California.Suitable promotor comprises AOX1 and AOX2.People such as Gleeson, J.Gen.Microbiol.132:3459-3465,1986 comprise the information about debaryomyces hansenii carrier and conversion, suitable promotor is MOX1 and FMD1; And EP 361 991, people such as Fleer, 1991 reach from other publication of Rhone-Poulenc Rorer to have instructed how to express foreign protein in kluyveromyces (Kluyveromycesspp.), and suitable promotor is PGK1.

3 ' flanking sequence of the preferred eukaryotic gene of transcription termination signal, it comprises the correct signal that is used for Transcription Termination and polyadenylation.3 ' suitable flanking sequence can be for example in the gene with natural those that are connected of used expression regulation sequence, can be corresponding to promotor.Perhaps, they can be different, in this case the termination signal of preferably saccharomyces cerevisiae ADH1 gene.

Required albumin fusion proteins can be expressed with the secretion leader sequence at first, and it can be effective any leader sequence in selected yeast.Useful leader sequence comprises any as follows in yeast:

A) MPIF-1 signal sequence (for example amino acid/11 of GenBank registration number AAB51134-21), MKVSVAALSCLMLVTALGSQA (SEQ ID NO:6)

B) department's gland calsequestrin signal sequence (MLQNSAVLLLLVISASA, SEQ ID NO:7)

C) the pre-pro district of HSA signal sequence (for example MKWVTFISLLFLFSSAYSRGVFRR, SEQ ID NO:8)

D) the pre district of HSA signal sequence (for example MKWVTFISLLFLFSSAYS, SEQ IDNO:9) or its variant are such as for example MKWVSFISLLFLFSSAYS (SEQ ID NO:10)

E) saccharase signal sequence (for example MLLQAFLFLLAGFAAKISA, SEQ ID NO:11)

F) yeast mating factor signal sequence (MRFPSIFTAVLAFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPF SNSTNNGLLFINTTIASIAAKEEGVSLEKR for example, SEQ ID NO:12 or MRFPSIFTAVLAFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPF SNSTNNGLLFINTTIASIAAKEEGVSLDKR, SEQ ID NO:12)

G) Kluyveromyces lactis (K.Lactis) kills and wounds the toxin leader sequence

H) heterozygosis signal sequence (for example MKWVSFISLLFLFSSAYSRSLEKR, SEQ IDNO:13)

I) HSA/MFa-1 heterozygosis signal sequence (being also referred to as HSA/kex2) (for example MKWVSFISLLFLFSSAYSRSLDKR, SEQ ID NO:14)

J) Kluyveromyces lactis kill and wound/MF α-1 merges leader sequence (for example MNIFYIFLFLLSFVQGSLDKR, SEQ ID NO:15)

K) immunoglobulin (Ig) Ig signal sequence (for example MGWSCIILFLVATATGVHS, SEQ IDNO:16)

L) fine protein B precursor signal sequence (for example MERAAPSRRVPLPLLLLGGLALLAAGVDA, SEQ ID NO:17)

M) bunch amyloid protein precursor signal sequence (for example MMKTLLLFVGLLLTWESGQVLG, SEQ ID NO:18)

N) IGFBP4 signal sequence (for example MLPLCLVAALLLAAGPGPSLG, SEQ ID NO:19)

O) variant in HSA signal sequence pre-pro district, for example

MKWVSFISLLFLFSSAYSRGVFRR(SEQ?ID?NO：20)，

MKWVTFISLLFLFAGVLG(SEQ?ID?NO：21)，

MKWVTFISLLFLFSGVLG(SEQ?ID?NO：22)，

MKWVTFISLLFLFGGVLG(SEQ?ID?NO：23)，

The leading HSA#64-MKWVTFISLLFLFAGVSG of modified HSA (SEQ IDNO:24);

The leading HSA#66-MKWVTFISLLFLFGGVSG of modified HSA (SEQ IDNO:25);

Modified HSA (A14) is leading-MKWVTFISLLFLFAGVSG (SEQ ID NO:26);

Leading (being also referred to as modified the HSA#65)-MKWVTFISLLFLFSGVSG of modified HSA (S14) (SEQ ID NO:27),

Modified HSA (G14) is leading-MKWVTFISLLFLFGGVSG (SEQ ID NO:28), or MKWVTFISLLFLFGGVLGDLHKS (SEQ ID NO:29)

P) shared signal sequence (MPTWAWWLFLVLLLALWAPARG, SEQ ID NO:30)

Q) acid phosphatase (PH05) leading (for example MFKSVVYSILAASLANA, SEQ IDNO:31)

R) the pre sequence of MFoz-1

S) the pre sequence of 0 dextranase (BGL2)

T) it is leading to kill and wound toxin

U) kill and wound the pre sequence of toxin

V) Kluyveromyces lactis kills and wounds toxin prepro (29 amino acid; 16 pre amino acid and 13 pro amino acid) MNIFYIFLFLLSFVQGLEHTHRRGSLDKR (SEQ ID NO:32)

W) saccharomyces diastaticus (S.diastaticus) grape saccharogenic amylase II secretion leader sequence

X) saccharomyces carlsbergensis (S.carlsbergensis) alpha-galactosidase (MEL1) secretion leader sequence

Y) candiyeast glucoamylase leader sequence

Z) disclosed heterozygosis leading (being incorporated herein by reference) among the EP-A-387319

Aa) gp67 signal sequence (with baculovirus expression system associating) (for example amino acid/11 of GenBank registration number AAA72759-19) or

Bb) therapeutic protein X's is natural leading;

Cc) yeast saccharomyces cerevisiae saccharase (SUC2) is leading, is disclosed in JP 62-096086 (grant number 911036516 is incorporated herein by reference); Or

Dd) multiple japanese bearbind powder enzyme (inulinase)-MKLAYSLLLPLAGVSASVINYKR (SEQ IDNO:33)

Ee) the leading variant #1-of the former peptide of modified TA57

MKLKTVRSAVLSSLFASQVLGQPIDDTESQTTSVNLMADDTESAFATQTNSGGLDVVGLISMAKR(SEQ?ID?NO：34)

Ff) the leading variant #2-MKLKTVRSAVLSSLFASQVLGQPIDDTESQTTSVNLMADDTESAFATQTNSGG of the former peptide of modified TA57 (propeptide) LDVVGLISMAEEGEPKR (SEQ ID NO:35)

Gg) shared signal peptide-MWWRLWWLLLLLLLLWPMVWA (SEQ ID NO:111)

Jj) modified HSA/kex2 signal sequence-MKWVSFISLLFLFSSAYSGSLDKR (SEQ ID NO:112)

Kk) shared signal peptide #2-MRPTWAWWLFLVLLLALWAPARG (SEQ ID NO:105)

Reorganization and synthetic other method of producing 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 technology.Carrier can be for example phage, plasmid, virus or retroviral vector.Retroviral vector can be to duplicate competent or replication defective.In the later case, Bing Du breeding can only take place in the complementary host cell usually.

The polynucleotide of code book invention albumin fusion proteins can with contain the carrier that is useful on the selective marker of in the host, duplicating and be connected.Usually, plasmid vector is introduced throw out, such as the calcium phosphate precipitation thing, or with the mixture of electrically charged lipid.If carrier is a virus, its available suitable package cell line carries out external packing so, in the host cell of transduceing then.

The polynucleotide inset should can be operatively connected with suitable promotor, early stage and late promoter and retrovirus LTR etc. such as lambda particles phage PL promotor, intestinal bacteria lac, trp, phoA and tac promotor, SV40.Other suitable promotor is known to those skilled in the art.Also will comprise in the expression construct and be used for transcription initiation and terminated site, and be used to the ribosome bind site translated in the transcriptional domain.The coding region of the transcript of being expressed by construct preferably comprises translation initiation codon in the starting point of polypeptide to be translated, comprises terminator codon (UAA, UGA or UAG) in the appropriate location of terminal point.

As mentioned above, expression vector preferably comprises at least a selective marker.Tsiklomitsin, kantlex or amicillin resistance that such mark comprises Tetrahydrofolate dehydrogenase, G418, glutamine synthase or is used for the neomycin resistance that eukaryotic cell cultivates and is used for cultivating on intestinal bacteria or other bacterium.Suitably host's representative example includes but not limited to: bacterial cell belongs to and Salmonella typhimurium (Salmonella typhimurium) cell such as intestinal bacteria, strepto-; The fungal cell is such as yeast cell (for example yeast saccharomyces cerevisiae or pichia pastoris phaff (Pichia pastoris, ATCC registration number 201178)); Insect cell is such as fruit bat S2 cell and noctuid Sf9 cell; Zooblast is such as CHO, COS, NSO, 293 and the Bao Si melanoma cells; And vegetable cell.The suitable culture medium and the culture condition of above-mentioned host cell are known in this area.

The preferred vector that is used for bacterium comprises pQE70, pQE60 and pQE-9, can buy from QIAGEN company; PBluescript carrier, Phagescript carrier, pNH8A, pNH16a, pNH18A, pNH46 can buy from Stratagene Cloning Systems company; Reach ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5, can buy from Pharmacia Biotech company.Preferred eukaryotic vector has pWLNEO, pSV2CAT, pOG44, pXT1 and pSG, can buy from Stratagene company; And pSVK3, pBPV, pMSG and pSVL, can buy from Pharmacia company.The preferred 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 (all can be from Invitrogen, Carlbad, CA buys).Other suitable carrier is conspicuous for those skilled in the art.

In one embodiment, the polynucleotide of code book invention albumin fusion proteins 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 the secretion of protokaryon or eukaryotic cell.For example, in intestinal bacteria, may wish protein expression is directed to periplasmic space.In order to guide the signal sequence that expression of polypeptides merges to the periplasmic space of bacterium and with albumin fusion proteins of the present invention or the example of protein (or its fragment) 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 heat-labile enterotoxin of E, coli B subunit and the signal sequence of alkaline phosphatase.Some carriers that are used to make up the localized fusion rotein of pilot protein matter can obtain by commercial sources, can buy from New England Biolabs company such as pMAL serial carrier (particularly pMAL-p series).In a specific embodiment, the polynucleotide of code book invention albumin fusion proteins and pelB pectin lyase signal sequence can be merged that this polypeptide is expressed and the efficient of purifying to improve in gram negative bacterium.Referring to U.S. Patent number 5,576,195 and 5,846,818, its contents intact is incorporated herein by reference.

In order to guide albumin fusion proteins of the present invention in mammalian cell, to secrete, can include but not limited to the example of the signal peptide of its fusion:

A) MPIF-1 signal sequence (for example amino acid/11 of GenBank registration number AAB51134-21), MKVSVAALSCLMLVTALGSQA (SEQ ID NO:6)

B) department's gland calsequestrin signal sequence (MLQNSAVLLLLVISASA, SEQ ID NO:7)

C) the pre-pro district of HSA signal sequence (for example MKWVTFISLLFLFSSAYSRGVFRR, SEQ ID NO:8)

D) the pre district of HSA signal sequence (for example MKWVTFISLLFLFSSAYS, SEQ IDNO:9) or its variant are such as for example MKWVSFISLLFLFSSAYS (SEQ ID NO:10)

E) saccharase signal sequence (for example MLLQAFLFLLAGFAAKISA, SEQ ID NO:11)

F) yeast mating factor signal sequence (MRFPSIFTAVLAFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPF SNSTNNGLLFINTTIASIAAKEEGVSLEKR for example, SEQ ID NO:12 or MRFPSIFTAVLAFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPF SNSTNNGLLFINTTIASIAAKEEGVSLDKR, SEQ ID NO:12)

G) Kluyveromyces lactis (K.Lactis) kills and wounds the toxin leader sequence

H) heterozygosis signal sequence (for example MKWVSFISLLFLFSSAYSRSLEKR, SEQ IDNO:13)

I) HSA/MFa-1 heterozygosis signal sequence (being also referred to as HSA/kex2) (for example MKWVSFISLLFLFSSAYSRSLDKR, SEQ ID NO:14)

J) Kluyveromyces lactis kill and wound/MF α-1 merges leader sequence (for example MNIFYIFLFLLSFVQGSLDKR, SEQ ID NO:15)

K) immunoglobulin (Ig) Ig signal sequence (for example MGWSCIILFLVATATGVHS, SEQ IDNO:16)

L) fine protein B precursor signal sequence (for example MERAAPSRRVPLPLLLLGGLALLAAGVDA, SEQ ID NO:17)

M) bunch amyloid protein precursor signal sequence (for example MMKTLLLFVGLLLTWESGQVLG, SEQ ID NO:18)

N) IGFBP4 signal sequence (for example MLPLCLVAALLLAAGPGPSLG, SEQ ID NO:19)

O) variant in HSA signal sequence pre-pro district, for example

MKWVSFISLLFLFSSAYSRGVFRR(SEQ?ID?NO：20)，

MKWVTFISLLFLFAGVLG(SEQ?ID?NO：21)，

MKWVTFISLLFLFSGVLG(SEQ?ID?NO：22)，

MKWVTFISLLFLFGGVLG(SEQ?ID?NO：23)，

The leading HSA#64-MKWVTFISLLFLFAGVSG of modified HSA (SEQ IDNO:24);

The leading HSA#66-MKWVTFISLLFLFGGVSG of modified HSA (SEQ IDNO:25);

Modified HSA (A14) is leading-MKWVTFISLLFLFAGVSG (SEQ ID NO:26);

Leading (being also referred to as modified the HSA#65)-MKWVTFISLLFLFSGVSG of modified HSA (S14) (SEQ ID NO:27),

Modified HSA (G14) is leading-MKWVTFISLLFLFGGVSG (SEQ ID NO:28), or MKWVTFISLLFLFGGVLGDLHKS (SEQ ID NO:29)

P) shared signal sequence (MPTWAWWLFLVLLLALWAPARG, SEQ ID NO:30)

Q) acid phosphatase (PH05) leading (for example MFKSVVYSILAASLANA, SEQ IDNO:31)

R) the pre sequence of MFoz-1

S) the pre sequence of 0 dextranase (BGL2)

T) it is leading to kill and wound toxin

U) kill and wound the pre sequence of toxin

V) Kluyveromyces lactis kills and wounds toxin prepro (29 amino acid; 16 pre amino acid and 13 pro amino acid) MNIFYIFLFLLSFVQGLEHTHRRGSLDKR (SEQ ID NO:32)

W) saccharomyces diastaticus (S.diastaticus) grape saccharogenic amylase II secretion leader sequence

X) saccharomyces carlsbergensis (S.carlsbergensis) alpha-galactosidase (MEL1) secretion leader sequence

Y) candiyeast glucoamylase leader sequence

Z) disclosed heterozygosis leading (being incorporated herein by reference) among the EP-A-387319

Aa) gp67 signal sequence (with baculovirus expression system associating) (for example amino acid/11 of GenBank registration number AAA72759-19) or

Bb) therapeutic protein X's is natural leading;

Cc) yeast saccharomyces cerevisiae saccharase (SUC2) is leading, is disclosed in JP 62-096086 (grant number 911036516 is incorporated herein by reference); Or

Dd) multiple japanese bearbind powder enzyme-MKLAYSLLLPLAGVSASVINYKR (SEQ ID NO:33)

Ee) the leading variant #1-MKLKTVRSAVLSSLFASQVLGQPIDDTESQTTSVNLMADDTESAFATQTNSGG of the former peptide of modified TA57 LDVVGLISMAKR (SEQ ID NO:34)

Ff) the leading variant #2-MKLKTVRSAVLSSLFASQVLGQPIDDTESQTTSVNLMADDTESAFATQTNSGG of the former peptide of modified TA57 LDVVGLISMAEEGEPKR (SEQ ID NO:35)

Gg) shared signal peptide-MWWRLWWLLLLLLLLWPMVWA (SEQ ID NO:111)

Jj) modified HSA/kex2 signal sequence-MKWVSFISLLFLFSSAYSGSLDKR (SEQ ID NO:112)

Kk) shared signal peptide #2-MRPTWAWWLFLVLLLALWAPARG (SEQ ID NO:105)

In a preferred embodiment, modified HSA/kex2 signal sequence (SEQ ID NO:112) is fused to the N-terminal of albumin fusion proteins, comprise the fusion rotein that comprises white protein and therapeutic protein described herein, and disclosed albumin fusion proteins among WO 93/15199, WO 97/24445, WO 03/60071, WO 03/59934 and the PCT/US04/01369, be incorporated herein by reference each piece is complete.Modified HSA/kex2 signal sequence is based on HSA/kex2 signal sequence (SEQ ID NO:14), and it is disclosed in for example people such as Sleep, Biotechnology 8:42-46,1990; With United States Patent (USP) 5,302,697, with these two pieces of complete being incorporated herein by reference.Modified HSA/kex2 leader sequence disclosed herein contains non-conserved amino acid and substitutes (Arg becomes Gly) at the 19th residue place of parent's signal peptide.When discovery was expressed in yeast, modified HSA/kex2 signal sequence was compared with unmodified HSA/kex2 signal sequence, made albumin fusion proteins produce unforeseeable better expression productive rate and/or better cutting efficiency.The variant of modified HSA/kex2 signal peptide is also contained in the present invention.Particularly, the 19th of SEQ ID NO:112 the Gly residue can substitute with the Pro residue.Also imagined other conservative alternative variations of modified HSA/kex2 signal sequence.The nucleic acid and the conservative alternative variations thereof of the modified HSA/kex2 signal sequence of coding SEQ ID NO:112 also contained in the present invention.

Utilize glutamine synthase (GS) or DHFR can when having medicine sulfo-methionine(Met) (methionine sulphoximine) or ammonia first dish purine, increase respectively as the carrier of selective marker.Based on the advantage of the carrier of glutamine synthase is that (for example rat bone marrow tumour cell system NSO) is easy to obtain for the clone of glutamine synthase feminine gender.The glutamine synthase expression system also can be by the extra inhibitor that stops native gene performance function is provided in glutamine synthase express cell (for example Chinese hamster ovary (CHO) cell) the performance function.It is open that glutamine synthase expression system and component thereof see PCT for details: WO 87/04462; WO 86/05807; WO 89/01036; WO 89/10404; With WO 91/06657, with its complete being incorporated herein by reference.In addition, the glutamine synthase expression vector can (Portsmouth NH) buys from Lonza Biologics company.Utilize the GS expression system in rat bone marrow tumour cell, to express and generate monoclonal antibody and see people such as Bebbington, Bio/technology 10:169,1992; And Biblia and Robinson, Biotechnol.Prog.11:1,1995, be introduced into this paper as a reference.

The invention still further relates to the host cell that contains above-mentioned vector construction body described herein, also contain the host cell that contains the nucleotide sequence of the present invention that can be operatively connected in addition by technology known in the art and one or more allos control regions (for example promotor and/or enhanser).Host cell can be a higher eucaryotic cells, such as mammalian cell (for example people's derived cell), or eukaryotic cell such as low, such as yeast cell, perhaps host cell can be a prokaryotic cell prokaryocyte, such as bacterial cell.Can select to regulate and control the expression of the gene order of inserting, perhaps with the AD HOC modification of expectation and host's strain of processed gene product.The existence of some inductor can improve the expression from some promotor; So expression of may command gene engineering polypeptide.In addition, different host cells have characteristics and specific mechanism to proteinic translation, translation post-treatment and modification (for example phosphorylation, cutting).Select suitable clone can guarantee modification and the processing that expressed exogenous protein is expected.

Nucleic acid of the present invention and nucleic acid construct importing host cell can be realized by the transfection of calcium phosphate transfection, the mediation of DEAE-dextran, transfection, electroporation, transduction, transfection or other method of cation lipid mediation.These methods are described in many standard laboratory handbooks, such as people such as Davis, and " Basic Methods In Molecular Biology ", 1986.Specifically imagined in fact and can express polypeptide of the present invention by the host cell that lacks recombinant vectors.Except containing the host cell that contains vector construction body described herein, the present invention is also contained through transforming deletion or having replaced endogenous substance of heredity (for example available albumin fusion proteins corresponding with therapeutic protein replaced and therapeutic protein corresponding codes sequence) and/or introduced genetic material and (for example can introduce the heterologous polynucleotide sequence, such as for example corresponding albumin fusion proteins of the present invention with therapeutic protein) vertebrates origin, particularly primary cell, subculture cell and the immortalized cells of Mammals origin.Can operate the genetic material that the links to each other endogenous polynucleotide that to activate, change and/or increase with endogenous polynucleotide.

In addition, available technology known in the art makes heterologous polynucleotide (polynucleotide of for example encode albumin protein or its fragment or variant) and/or allos control region (for example promotor and/or enhanser) can operate with the endogenous polynucleotide of coding therapeutic protein by homologous recombination to link to each other (referring to for example U.S. Patent number 5 of issue on June 24th, 1997,641,670; International publication number WO 96/29411; International publication number WO 94/12650; People such as Koller, Proc.Natl.Acad.Sci USA 86:8932-8935,1989; Reach people such as Zijlstra, Nature, 342:435-438,1989, with complete being incorporated herein by reference of disclosure of each piece).

Albumin fusion proteins of the present invention can reclaim and purifying from the reconstitution cell culture by well-known method, comprises ammonium sulfate or ethanol sedimentation, acid extraction, negatively charged ion or cation-exchange chromatography, phosphorylated cotton chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite, dewatering electric charge interaction chromatography and lectin chromatography.Most preferably, adopt high performance liquid chromatography (" HPLC ") to carry out purifying.

In preferred embodiments, albumin fusion proteins of the present invention carries out purifying with anion-exchange chromatography, includes but not limited to carry out chromatography on Q-Sepharose, DEAE Sepharose, poros HQ, porosDEAE, Toyopearl Q, Toyopearl QAE, Toyopearl DEAE, Resource/Source Q and DEAE, Fractogel Q and DEAE post.

In specific embodiment, albumin fusion proteins of the present invention carries out purifying with cation-exchange chromatography, includes but not limited to SP-Sepharose, CM Sepharose, poros HS, poros CM, Toyopearl SP, Toyopearl CM, Resource/Source S and CM, Fractogel S and CM post and equivalent thereof and comparable thing.

In specific embodiment, albumin fusion proteins of the present invention carries out purifying with hydrophobic interaction chromatography, include but not limited to phenyl, butyl, methyl, octyl group, hexyl-Sepharose, poros 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 equivalent thereof and comparable thing.

In specific embodiment, albumin fusion proteins of the present invention carries out purifying with size exclusion chromatography, includes but not limited to Sepharose S100, S200, S300, superdex resin column and equivalent thereof and comparable thing.

In specific embodiment, albumin fusion proteins of the present invention carries out purifying with affinity chromatography, includes but not limited to HSA or " fusion target " molecule selectively class dyestuff affinity column, peptide affinity column and antibody affinity column.

In preferred embodiments, albumin fusion proteins of the present invention carries out purifying with above-mentioned one or more chromatography methods.In other embodiment preferred, albumin fusion proteins of the present invention carries out purifying with following one or more chromatography columns: Q Sepharose FF post, SP Sepharose FF post, QSepharose high performance column, Blue Sepharose FF post, Blue post, phenyl Sepharose FF post, DEAE Sepharose FF or methyl post.

In addition, albumin fusion proteins of the present invention can carry out purifying with the method for describing among the international open WO 00/44772 of PCT, with its complete being incorporated herein by reference.Those skilled in the art can be easy to revise the method for wherein description to be used for purifying albumin fusion proteins of the present invention.

Albumin fusion proteins of the present invention can reclaim from the product of chemosynthesis flow process with by the product of recombinant technology from protokaryon or eucaryon host generation, and described host comprises for example bacterium, yeast, higher plant, insect and mammalian cell.According to the host who is adopted in the recombinant production flow process, polypeptide of the present invention can be glycosylated or not glycosylated.In addition, the result of the processing that in some situation, mediates as the host, albumin fusion proteins of the present invention also can comprise the methionine residues of initial modification.Therefore, this area is well-known, is efficiently eliminated from any protein after translation in all eukaryotic cells usually by the terminal methionine(Met) of the N-of translation initiation codon coding.Though the terminal methionine(Met) of most proteinic N-is also effectively removed in most prokaryotic cell prokaryocytes, for some protein, it is invalid that this protokaryon is removed processing, and this depends on and the covalently bound amino acid whose character of the terminal methionine(Met) of N-.

In one embodiment, use pichia pastoris phaff in eukaryotic system, to express albumin fusion proteins of the present invention.Pichia pastoris phaff is methyl alcohol can be carried out metabolic methylotrophy yeast as sole carbon source.Key step in the methanol metabolic pathway is to utilize O ₂Methanol oxidation is become formaldehyde.This reaction is by the oxidation of ethanol enzyme catalysis.In order to carry out metabolism with methyl alcohol as sole carbon source, pichia pastoris phaff must generate high-caliber alcohol oxidase, and partly cause is that alcohol oxidase is to O ₂Avidity relatively low.So in relying on the growth medium of methyl alcohol as main carbon source, the promoter region height of one of two kinds of alcohol oxidase genes (AOX1) has activity.When having methyl alcohol, the alcohol oxidase that produces from the AOX1 gene accounts for about 30% of pichia pastoris phaff all soluble protein.Referring to Ellis, people such as S.B., Mol.Cell.Biol.5:1111-21,1985; Koutz, people such as P.J., Yeast 5:167-77,1989; Tschopp, people such as J.F., Nucl.Acids Res.15:3859-76,1987.So, allogeneic coding sequence, such as for example polynucleotide of the present invention, under the transcriptional regulatory of whole or part A OX1 regulating and controlling sequence, in the pichia spp of when having methyl alcohol, cultivating with extra high horizontal expression.

In one embodiment, in essence as " Pichia Protocols:Methods in MolecularBiology ", D.R.Higgins and J.Cregg compile, The Humana Press, Totowa, NJ, the method for describing in 1998 uses plasmid vector pPIC9K to express the DNA of the listed code book invention of this paper albumin fusion proteins in Bichi yeast system.By the strong AOX1 promotor that is connected with pichia pastoris phaff alkaline phosphatase (PHO) secreting signal peptide (promptly leading) that is positioned at the multiple clone site upstream, this expression plasmid is allowed expression and is secreted polypeptide of the present invention.

Those skilled in the art will be easy to understand, can use many other yeast vectors to replace pPIC9K, such as pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K and PAO815, as long as the expression construct of suggestion provides the suitably localized signals such as (if necessary) of transcribing, translate, secrete, comprise the required AUG in the identical reading frame.

In another embodiment, allogeneic coding sequence can be realized such as culturing yeast culture among for example pGAPZ or the pGAPZalpha and when not having methyl alcohol by heterologous polynucleotide of the present invention being cloned into expression vector such as the high level expression of for example polynucleotide of code book invention albumin fusion proteins.

In addition, albumin fusion proteins of the present invention can utilize technology chemosynthesis known in the art (for example referring to Creighton, 1983, Proteins:Structures and Molecular Principles, W.H.Freeman; Co., N.Y.; Reach people such as Hunkapiller, Nature, 310:105-111,1984).For example, the segmental polypeptide that is equivalent to polypeptide can use Peptide synthesizer to synthesize.In addition, if necessary, nonclassical amino acid or chemical amino acid analogue can be used as and substitute or add and be incorporated in the peptide sequence.Nonclassical amino acid includes but not limited to the D-isomer of common amino acid, 2,4-diamino-butanoic, α-An Jiyidingsuan, 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, oxyproline, sarkosine, citrulline, Homocitrulline, cysteic acid, tertiary butyl glycine, tertiary butyl L-Ala, phenylglycocoll, Cyclohexylalanine, the b-L-Ala, fluoroamino acid, tape label amino acid is such as the b-methylamino acid, the Ca-methylamino acid, the Na-methylamino acid, and general amino acid analogue.In addition, amino acid can be D type (dextral) or L type (left-handed).

The present invention be encompassed in the translation or translation after carry out the albumin fusion proteins of the present invention of different modifying, for example glycosylation, acetylize, phosphorylation, amidation, by known protection/blocking groups derive, proteolysis cuts, with being connected of antibody molecule or other cell ligand etc.Can carry out any numerous chemically modified by known technology, include but not limited to cyanogen bromide, trypsinase, Quimotrase, papoid, V8 proteolytic enzyme, NaBH ₄Special chemical chop; Acetylize, formylation, oxidation, reduction; Metabolism when having tunicamycin is synthetic; Or the like.

Other posttranslational modification that the present invention is contained comprises that N-for example connects or the processing of carbohydrate chain, N-end or C-end that O-connects, on amino acid backbone, adhere to chemical module, N-connects or the chemically modified of the carbohydrate chain that O-connects, and add or deletion because prokaryotic host cell is expressed the N-end methionine residues that produces.The also available detectable of albumin fusion proteins is modified, and such as enzyme, fluorescein, isotropic substance or affinity tag, thereby can detect and isolated protein.

The example of suitable enzyme comprises horseradish peroxidase, alkaline phosphatase, beta-galactosidase enzymes or acetylcholinesterase; The example of suitable prothetic group mixture comprises strepto-affinity element/vitamin H and affinity element/vitamin H; The example of suitable fluorescent substance comprises Umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazine amine fluorescein, dansyl chloride or phycoerythrin; The example of luminophore comprises luminol,3-aminophthalic acid cyclic hydrazide; The noclilucence examples of substances comprises luciferase, fluorescein and aequorin; And the example of suitable radioactive substance comprise iodine ( ¹²¹I, ¹²³I, ¹²⁵I, ¹³¹I), carbon ( ¹⁴C), sulphur ( ³⁵S), tritium ( ³H), indium ( ¹¹¹In, ¹¹²In, ¹¹³In, ^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 embodiment, albumin fusion proteins of the present invention or its fragment or its variant are attached to and the associating macrocyclic chelants of radiometal ion, described radiometal ion includes but not limited to ¹⁷⁷Lu, ⁹⁰Y, ¹⁶⁶Ho and ¹⁵³Sm.In a preferred embodiment, with the associating radiometal ion of macrocyclic chelants be ¹¹¹In.In another preferred embodiment, with the associating radiometal ion of macrocyclic chelants be ⁹⁰Y.In specific embodiment, macrocyclic chelants is 1,4,7,10-tetraazacyclododecanand-N, N ', N ", N " '-tetraacethyl (DOTA).In other specific embodiment, DOTA is attached to antibody of the present invention or its fragment by linkers.Can be used for the example of the linkers of DOTA and conjugation of polypeptides is generally known in this area, referring to people such as for example DeNardo, Clin.Cancer Res.4 (10): 2483-90,1998; People such as Peterson, Bioconjug.Chem.10 (4): 553-7,1999; Reach people such as Zimmerman, Nucl.Med.Biol.26 (8): 943-50,1999; With its complete being incorporated herein by reference.

As mentioned above, albumin fusion proteins of the present invention can be modified by natural process, such as the translation post-treatment, perhaps modifies by chemical modification technology well-known in the art.Should understand, in given polypeptide, the modification of same type can exist with identical or different degree in several site.Polypeptide of the present invention can be branched, and for example because ubiquitin turns into the branch that causes, and they can be cyclic also, are with or without branch.Cyclic, branched and branch annular polypeptide can be caused by translation back natural process, also can be caused by synthetic method.Modification comprises acetylize, acidylate, the ADP-ribosylation, amidation, the covalent attachment of flavine, the covalent attachment of protoheme module, the covalent attachment of Nucleotide or nucleotide derivative, the covalent attachment of lipid or lipid derivate, the covalent attachment of phosphatidylinositols, crosslinked, cyclisation, disulfide linkage forms, demethylation, the formation of covalent cross-linking, the formation of halfcystine, the formation of Pyrrolidonecarboxylic acid, formylation, gamma-carboxylation, glycosylation, the formation of GPI anchor, hydroxylation, iodate, methylate, myristylization, oxidation, PEGization, proteolysis processing, phosphorylation, isoprenylation, racemize, the selenium acidylate, sulfation, interpolation amino acid such as the arginylization on protein of transfer RNA mediation, with ubiquitinization (referring to for example PROTEINS-STRUCTURE AND MOLECULAR PROPERTIES, the 2nd edition, T.E.Creighton, W.H.Freeman and Company, New York, 1993; POST-TRANSLATIONALCOVALENT MODIFICATION OF PROTEINS, B.C.Johnson compiles, AcademicPress, New York, pg.1-12,1983; People such as Seifter, Meth.Enzymol.182:626-646,1990; People such as Rattan, Ann.N.Y Acad.Sci.663:48-62,1992)

Can be with albumin fusion proteins of the present invention and the antibody and the fusion of mark sequence of combined treatment protein or its fragment or variant, such as the peptide of being convenient to purifying.In preferred embodiments, the mark aminoacid sequence is six Histidine peptides, such as label that provides in the pQE carrier (QIAGEN company, 9259EtonAvenue, Chatsworth, CA, 91311) etc., many can the acquisition by commercial sources in them.For example, as people such as Gentz, Proc.Natl.Acad.Sci.USA 86:821-824, described in 1989, six Histidines are that the purifying of fusion rotein facilitates.Other peptide tag that can be used for purifying includes but not limited to " HA " label, and it also has " flag " label corresponding to derived from the epi-position of influenza hemagglutinin protein matter people such as (, Cell 37:767,1984) Wilson.

In addition, albumin fusion proteins of the present invention and therapeutic module can be puted together,, for example suppress cell agent or cytocide such as cytotoxin, therapeutical agent or radioactive metal ion, alpha emitter for example is such as for example ²¹³Bi.Cytotoxin or cytotoxic agents comprise the deleterious any reagent of pair cell.Example comprises taxol, Cytochalasin B, Gramicidin D, ethidium bromide, ipecamine, mitomycin, Etoposide, teniposide, vincristine(VCR), vincaleucoblastine, colchicine, Zorubicin, daunorubicin, dihydroxyl anthracin diketone, mitoxantrone, Plicamycin, dactinomycin, 1-boldenone, glucocorticosteroid, PROCAINE HCL, PHARMA GRADE, tetracaine, lignocaine, Proprasylyte and tetracycline and analogue or homologue.Therapeutical agent includes but not limited to metabolic antagonist (methotrexate for example, Ismipur, 6-thioguanine, cytosine arabinoside, the 5 FU 5 fluorouracil decarbazine), alkylating agent (dichloromethyldiethylamine (mustargen) for example, plug is for group, Chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), endoxan, busulfan, mitobronitol, streptozotocin, ametycin and suitable dichloro diamino platinum (II) be cis-platinum (DDP)), anthracene nucleus class (for example daunorubicin (being called daunomycin in the past) and Zorubicin), microbiotic (gengshengmeisu (being called actinomycin in the past) for example, bleomycin, Plicamycin and anthramycin (AMC)) and antimitotic agent (for example vincristine(VCR) and vincaleucoblastine).

Conjugate of the present invention can be used for modifying given biological answer-reply, does not think that therapeutical agent or medicine module are limited to classical chemotherapeutic.For example, the medicine module can be protein or the polypeptide with expectation biologic activity.This protein can comprise for example toxin, such as abrin, ricin A, Pseudomonas exotoxin or diphtheria toxin; Protein, such as tumour necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, Thr6 PDGF BB, tissue plasminogen activator, apoptosis agent for example TNF-α, TNF-β, AIM I (referring to international publication number WO 97/33899), AIM II (referring to international publication number WO 97/34911), Fas part (people such as TakaHSAhi, Int.Immunol.6:1567-1574,1994), VEGI (referring to international publication number WO 99/23105), thrombosis agent or antiangiogenic agent, for example angiostatin or endostatin; Or the biological answer-reply modifier, such as for example lymphokine, il-1 (" IL-1 "), interleukin-2 (" IL-2 "), interleukin-6 (" IL-6 "), rHuGM-CSF (" GM-CSF "), granulocyte colony-stimulating factor (" G-CSF ") or other somatomedin.The technology that is used for these therapeutic modules are conjugated on the protein (for example albumin fusion proteins) is well-known in the art.

Albumin fusion proteins can also be attached to solid support, this is to albumin fusion proteins of the present invention institute bonded, with its bonded or the immunoassay or the purifying of associating polypeptide are useful especially with it.This solid support includes but not limited to glass, Mierocrystalline cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

Albumin fusion proteins is with or without with the therapeutic module and puts together, and can use separately or co-administered with the cytotoxic factor and/or the cytokine of useful as therapeutics.

Only comprise in the embodiment of VH structural domain of the proteinic antibody of combined treatment at albumin fusion proteins of the present invention, may must and/or wish the fusion rotein of the VL structural domain of the proteinic same antibody of coexpression combined treatment, make (covalently or non-covalently) combination after translation of described VH-albumin fusion proteins and VL protein.

Only comprise in the embodiment of VL structural domain of the proteinic antibody of combined treatment at albumin fusion proteins of the present invention, may must and/or wish the fusion rotein of the VH structural domain of the proteinic same antibody of coexpression combined treatment, make (covalently or non-covalently) combination after translation of described VL-albumin fusion proteins and VH protein.

Some therapeutic antibodies is a bi-specific antibody, this means that the proteinic antibody of combined treatment is artificial hybrid antibody, and it has two pairs of different heavy chain/light chains and two different binding sites.In order to produce the albumin fusion proteins corresponding with this therapeutic protein, might create such albumin fusion proteins, promptly it all merges at the N-of albumin protein module and two ends of C-the scFv fragment.In particular, the scFv that is blended in the white protein N-terminal will be corresponding to a pair of heavy chain/light chain (VH/VL) of the proteinic original antibody of combined treatment, will be corresponding to another of the proteinic original antibody of combined treatment to heavy chain/light chain (VH/VL) and be blended in the scFv of white protein C-terminal.

The present invention also provides the chemically modified derivative of albumin fusion proteins of the present invention, and it can provide extra advantage, such as solubility, stability and increase cycling time or the immunogenicity reduction (referring to U.S. Patent number 4,179,337) of polypeptide.The chemical module that is used for derivative can be selected from water-soluble polymers, such as polyoxyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol etc.Albumin fusion proteins can be modified at intramolecular random site or in intramolecular predetermined position, and can comprise one, two, the three or more chemical module that adheres to.

Polymkeric substance can be any molecular weight, and can be branched or not branched.For polyoxyethylene glycol, for easy handling and manufacturing, preferred molecular weight is (term " about " refers in the polyoxyethylene glycol goods, and some molecules may be bigger or little than the molecular weight of regulation) between about 1kDa and about 100kDa.Also can adopt other molecular weight, this depend on the desired therapeutic characteristic (for example Qi Wang slow-release time, if any to the influence of biologic activity, the easy degree of operation, antigenic degree or shortage, and polyoxyethylene glycol to other known effect of therapeutic protein or analogue).For example, the molecular-weight average of polyoxyethylene 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, polyoxyethylene glycol can have apparatus derivatorius.Branched polyoxyethylene glycol is described in for example U.S. Patent number 5,643,575; People such as Morpurgo, Appl.Biochem.Biotechnol.56:59-72,1996; People such as Vorobjev, Nucleosides Nucleotides 18:2745-2750,1999; And people such as Caliceti, Bioconjug.Chem.10:638-646,1999, the disclosure of each piece is incorporated herein by reference.

In the influence that peg molecule (or other chemical module) should be considered when being attached to protein to protein function or antigenicity zone.Those skilled in the art can utilize multiple attachment means, such as disclosed method (PEG is coupled on the G-CSF) among the EP 0401384 for example, are incorporated herein by reference; Also can be referring to people such as Malik, Exp.Hematol.20:1028-1035,1992, wherein reported with Halothane sulfonic acid (tresyl) muriate GM-GSF carry out PEGization.For example, polyoxyethylene glycol can be via amino-acid residue by reactive group such as free amine group or carboxyl covalent attachment.Reactive group is can be in conjunction with the group of activated polyethylene glycol molecule.Amino-acid residue with free amine group can comprise lysine residue and N-terminal amino-acid residue; Amino acid with free carboxy can comprise asparagicacid residue, glutaminic acid residue and C-terminal amino-acid residue.Sulfydryl also can be used as the reactive group that is used to adhere to peg molecule.Preferably be attached to amino for therapeutic purpose, such as being attached on N-terminal or the Methionin group.

As mentioned above, polyoxyethylene glycol can by with being connected of any multiple amino acids residue attached on the protein.For example, polyoxyethylene glycol can be by being connected with protein with the covalent linkage of Methionin, Histidine, aspartic acid, L-glutamic acid or cysteine residues.Can adopt one or more reactive chemistries that polyoxyethylene glycol is attached to proteinic particular amino acid residue (for example Methionin, Histidine, aspartic acid, L-glutamic acid or halfcystine) or the proteinic amino-acid residue (for example Methionin, Histidine, aspartic acid, L-glutamic acid, halfcystine and combination thereof) that surpasses a type.

May wish especially in N-terminal chemically modified protein matter.With the example of polyoxyethylene glycol as composition, can multiple peg molecule (according to molecular weight, branch situation, etc.), the type of the ratio in the reaction mixture between peg molecule and protein (polypeptide) molecule, pending PEGization reaction, and obtain to select in the selected N-terminal PEGization method of protein.The method (promptly where necessary the single PEGization module of this module and other being separated) that obtains selected N-terminal PEGization goods can be by purifying N-terminal PEGization material from PEGization protein molecule colony.Can realize that at N-terminal selective chemical modification protein the reductibility alkanisation utilizes specified protein to can be used for the difference reaction of the dissimilar primary aminos of deutero-(Methionin is to N-terminal) by the reductibility alkanisation.Under the appropriate reaction condition, realize proteinic selective derivatization basically at the N end with carbonyl bearing polymer.

As mentioned above, the PEGization of albumin fusion proteins of the present invention can realize by several different methods.For example, polyoxyethylene glycol directly or by intermediate head can be attached to albumin fusion proteins.Be used for polyoxyethylene glycol being attached to proteinic non junction system description in people such as Delgado, Crit.Rev.Thera.Drug Carrier Sys.9:249-304,1992; People such as Francis, Intern.J.of Hematol.68:1-18,1998; U.S. Patent number 4,002,531; U.S. Patent number 5,349,052; WO 95/06058; And WO 98/32466, the disclosure of each piece is incorporated herein by reference.

Be used for polyoxyethylene glycol directly being attached to proteinic amino-acid residue and not having a kind of system of intermediate head to adopt the sulfonated MPEG of Halothane, it is the muriate (ClSO that uses Halothane sulfonic acid ₂CH ₂CF ₃) single methoxy polyoxyethylene glycol (MPEG) modified generate.After protein and the sulfonated MPEG reaction of Halothane, polyoxyethylene glycol directly is attached to proteinic amino.So, the present invention includes by making protein of the present invention and having 2,2, the sulfonic peg molecule of 2-Halothane reacts and protein-polyoxyethylene glycol conjugate of generating.

Also available multiple different intermediate heads are attached to protein with polyoxyethylene glycol.For example, U.S. Patent number 5,612,460 disclose and are used for polyoxyethylene glycol is connected to urethane joint on the protein, and its complete open book is incorporated herein by reference.Wherein polyoxyethylene glycol is attached to proteinic protein-polyoxyethylene glycol conjugate by joint and also can generates by protein and following compounds are reacted, such as MPEG-succinimido succinate/ester, with 1,1 '-carbonyl dimidazoles activatory MPEG, MPEG-2,4,5-trichlorine amyl group carbonate/ester, MPEG-p-NP carbonate/ester, and various MPEG-succinate/ester derivatives.Described among the international publication number WO 98/32466 and be used for polyoxyethylene glycol is attached to proteinic many other polyethyleneglycol derivatives and reactive chemistry, its complete open book has been incorporated herein by reference.The PEGization protein that utilizes reactive chemistry described herein to generate comprises within the scope of the present invention.

The number of the polyoxyethylene glycol module of adhering on each albumin fusion proteins of the present invention (promptly replacing degree) also can change to some extent.For example, PEGization protein of the present invention can be connected with average 1,2,3,4,5,6,7,8,9,10,12,15,17,20 or poly glycol molecule more.Similarly, the scope that on average replaces degree is that each protein molecule is connected with 1-3,2-4,3-5,4-6,5-7,6-8,7-9,8-10,9-11,10-12,11-13,12-14,13-15,14-16,15-17,16-18,17-19 or 18-20 polyoxyethylene glycol module.Being used for measuring the replacement degree methods has discussion in the literature, for example referring to people such as Delgado, and Crit.Rev.Thera.Drug Carrier Sys.9:249-304,1992.

Polypeptide of the present invention can reclaim and purifying from chemosynthesis and reconstitution cell culture by standard method, includes but not limited to ammonium sulfate or ethanol sedimentation, acid extraction, negatively charged ion or cation-exchange chromatography, phosphorylated cotton chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite and lectin chromatography.Most preferably, adopt high performance liquid chromatography (" HPLC ") to carry out purifying.When polypeptide separating and/or purge process in during sex change, can adopt the well-known technology of protein refolding that makes to recover activity conformation.

The existence of albumin fusion proteins of the present invention and quantity can be that ELISA measures with immunoassay well-known in the art.In can be used for a kind of ELISA scheme of detected/quantified albumin fusion proteins of the present invention, may further comprise the steps: with AHS's white protein antibody sandwich elisa plate, plate is sealed to stop non-specific binding, clean elisa plate, (with one or more different concns) adds the solution that contains albumin fusion proteins of the present invention, it is anti-to add the proteinic specificity of the treatment-resistant be conjugated with detectable (as described herein or other approach of this area is known) two, and detects two anti-existence.In the optional form of this scheme, elisa plate can with the proteinic specific antibody bag of treatment-resistant by and anti-through mark two can be anti-people's white protein specific antibody.

The purposes of polynucleotide

Each polynucleotide that the present invention identifies can multiple mode be used as reagent.Below explanation should think exemplary and utilized known technology.

Polynucleotide of the present invention can be used for generating albumin fusion proteins of the present invention.As more detailed description hereinafter, (the coding albumin fusion proteins) polynucleotide of the present invention can be used for producing cell, clone or the tissue of expressing by the coded albumin fusion proteins of the polynucleotide of code book invention albumin fusion proteins by genetically engineered in recombinant DNA method.

Polynucleotide of the present invention also can be used for gene therapy.A purpose of gene therapy is to insert normal gene in the organism with dcc gene, to proofread and correct hereditary defect.Polynucleotide disclosed by the invention provide the method with these hereditary defectes of height accurate way target.Another purpose is to insert non-existent new gene in the host genome, thereby makes host cell produce new feature.The description that other place of this paper is more detailed other non-limitative example of the gene therapy that contains of the present invention (be the part of " gene therapy ", and embodiment 61 and 62) referring to for example title.

The purposes of polypeptide

Each polypeptide that the present invention identifies can multiple mode be used.Below explanation should think exemplary and utilized known technology.

Albumin fusion proteins of the present invention can be used for providing the immunology probe, be used for differential and differentiate that tissue is (sharp as the immunohistochemistry assay method, such as for example ABC immunoperoxidase (people such as Hsu, J.Histochem.Cytochem.29:577-580,1981) or cell type (for example immunocytochemical determination method).

Albumin fusion proteins can be used for using the classical immunohistology method that those skilled in the art will know that measure polypeptide level in the biological sample (for example referring to people such as Jalkanen, J.Cell.Biol.101:976-985,1985; People such as Jalkanen, J.Cell.Biol.105:3087-3096,1987).Can be used for detecting other method that protein gene expresses and comprise immunoassay, such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA).Suitable mensuration marker is road known in the art, comprises the enzyme labelling thing, such as glucose oxidase; Radio isotope, such as iodine ( ¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), carbon ( ¹⁴C), sulphur ( ³⁵S), tritium ( ³H), indium ( ^115mIn, ¹¹³In, ¹¹²In, ¹¹¹In), 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; Luminous marker is such as luminol,3-aminophthalic acid cyclic hydrazide; Fluorescent marker is such as fluorescein and rhodamine; And vitamin H.

Albumin fusion proteins of the present invention also can detect in vivo by imaging.Being used for the marker of protein in-vivo imaging or mark comprises and can pass through X-roentgenogpaphy, nucleus magnetic resonance (NMR) or spectrum (electron spin relaxtion, ESR) material of Jian Ceing.For the X-roentgenogpaphy, suitable marker comprises radio isotope, and such as barium or caesium, they send detectable radiation but the experimenter is not significantly injured.The suitable landmarks thing of NMR and ESR comprises the material with detectable characteristic spin, and such as deuterium, it can mix albumin fusion proteins to the nutrition that offers the clone of expressing albumin fusion proteins of the present invention by mark.

Will be through suitably detecting image-forming module, (for example such as radio isotope ¹³¹I, ¹¹²In, ^99mTc, iodine ( ¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), carbon ( ¹⁴C), sulphur ( ³⁵S), tritium ( ³H), indium ( ^115mIn, ^113mIn, ¹¹²In, ¹¹¹In), 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), the not penetrating material of ray, maybe can be by magnetic resonance detection the albumin fusion proteins of material mark import the Mammals that (for example parenteral, subcutaneous or intraperitoneal) has the examine immune system disorder.This area will be understood, and experimenter's size will determine to produce the required amount that becomes the shadow module of diagnosis imaging with used imaging system.In the example of radio isotope module,, inject radioactive amount usually in about 5 to 20 millicuries for the human experimenter ^99mIn the scope of Tc.Then the mark albumin fusion proteins will be preferentially existed in vivo position (for example organ, cell, extracellular space or the matrix) accumulation of one or more reporter molecules, part or substrate (corresponding) with reporter molecule, part or the substrate of the therapeutic protein that is used to generate albumin fusion proteins of the present invention.Perhaps, at least comprise in the situation of the fragment of therapeutic antibodies or variant at albumin fusion proteins, will preferentially in health, exist the position (for example organ, cell, extracellular space or matrix) of the polypeptide/epi-position corresponding to accumulate with (be used to generate albumin fusion proteins of the present invention) therapeutic antibodies institute bonded polypeptide/epi-position through the mark albumin fusion proteins.The description of in-vivo tumour imaging can be referring to people such as S.W.Burchiel, " Immunopharmacokinetics of Radiolabeled Antibodies andTheir Fragments ", the 13rd chapter, " Tumor Imaging:The Radiochemical Detection ofCancer ", S.W.Burchiel and B.A.Thodes compile, Masson Publishing Inc., 1982.Those skilled in the art can be easy to revise the scheme of wherein description to be used for albumin fusion proteins of the present invention.

In one embodiment, the invention provides by using with heterologous polypeptide or the associating albumin fusion proteins of the present invention of nucleic acid molecule the polynucleotide encoded polypeptide of code book invention albumin fusion proteins and/or antibody (for example by) and with the special method that is delivered to cell of albumin fusion proteins of the present invention.In one embodiment, the invention provides therapeutic protein is delivered to method in the target cell.In another embodiment, the invention provides single-chain nucleic acid (for example antisense or ribozyme) or double-strandednucleic acid (DNA that for example can be incorporated in the cellular genome or duplicate and can transcribe as episome) are delivered to the method in the target cell.

In another embodiment, the invention provides by using and specificity is destroyed the method for cell (for example destroying tumour cell) with toxin or the associating albumin fusion proteins of the present invention of cytotoxic drug precursor.

" toxin " refer in conjunction with and the catalytic subunit of one or more compounds of activation of endogenous cellulotoxic effect system, radio isotope, holotoxin, improvement toxin, toxin or cell in or non-existent usually on the surface, cause any molecule or the enzyme of necrocytosis under given conditions.Can include but not limited to radio isotope known in the art according to the toxin that method of the present invention is used, compound is such as for example in conjunction with the antibody (or it contains the complement fixation(CF) part) of inherent or inductive endogenous cell toxic effect system, thymidine kinase, endonuclease, alpha-toxin, ricin, toxalbumin, ETA, diphtheria toxin, the Saponaria officinalis toxalbumin, the balsam pear toxalbumin, spend more white tree toxalbumin, Pokeweed antiviral protein, α-broom aspergillin and Toxins,exo-, cholera." toxin " also comprise and suppress cell agent or cytocide, therapeutical agent or radioactive metal ion, and alpha emitter for example is such as for example ²¹³Bi, or other radio isotope are 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; Luminous marker is such as luminol,3-aminophthalic acid cyclic hydrazide; Fluorescent marker is such as fluorescein and rhodamine; And vitamin H.In a specific embodiment, the invention provides by using and radio isotope ⁹⁰Associating polypeptide of the present invention of Y or antibody and specificity are destroyed the method for cell (for example destroying tumour cell).In another specific embodiment, the invention provides by using and radio isotope ¹¹¹Associating polypeptide of the present invention of In or antibody and specificity are destroyed the method for cell (for example destroying tumour cell).In another concrete embodiment, the invention provides by using and radio isotope ¹³¹Associating polypeptide of the present invention of I or antibody and specificity are destroyed the method for cell (for example destroying tumour cell).

Can use technology known in the art to come mark polypeptide of the present invention.These technology include but not limited to difunctionally put together the application of agent (referring to for example U.S. Patent number 5,756,065; 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 5,808,003; The contents intact of each piece is incorporated herein by reference).

Albumin fusion proteins of the present invention can be used for diagnosing, treat, preventing and/or predict the preferred people's of Mammals various disorders.Such disorder includes but not limited to that herein hereinafter title is that " biologic activity " part is put down in writing.

So, the invention provides the disorderly method of diagnosis, comprise that (a) measures certain polypeptide expression level in individual cell or the body fluid with albumin fusion proteins of the present invention; And (b) the expression of polypeptides level that records is compared with standard expression of polypeptides level, the expression of polypeptides level that records is with respect to the rising of standard expression level or reduce as disorderly index.For cancer, the tendency that a large amount of relatively transcripts may be indicated disease progression appears in the individual biopsy, perhaps can be provided at the method that detects disease before actual clinical symptom occurs.Such more definite diagnosis can make the medical worker more early adopt preventive measures or attack treatment with the development that stops cancer or further increase the weight of.

In addition, albumin fusion proteins of the present invention can be used for treatment or prevents following disease or situation, such as for example nervous disorders, immune system disorder, muscle disorder, reproduction disorder, gastrointestinal dysfunction, lung disorder, cardiovascular disorder, kidney disorder, proliferative disorders and/or Cancerous disease and situation.For example, can use polypeptide of the present invention to the patient, to substitute the polypeptide (for example Regular Insulin) that disappearance or level descend, (for example Hb-S is to replenish oxyphorase B to replenish the lower not homopolypeptide of disappearance or level, SOD, catalase, dna repair protein), the activity (for example oncogene or tumor suppressor gene) that suppresses polypeptide, the activity of activated polypeptides (for example being attached on the acceptor), by competing the activity (for example when reducing inflammation, using soluble TNF acceptor) that free ligand reduces membrane-bound receptor with membrane-bound receptor, or cause that expectation replys and (for example suppress angiogenic growth, enhancing is to the immunne response of proliferative cell or tissue).

Particularly, comprise the fragment of therapeutic antibodies or the albumin fusion proteins of variant at least and can be used for treating disease (as above described) with other place of this paper.For example, use the fragment that comprises therapeutic antibodies at least or variant albumin fusion proteins can in conjunction with and/or neutralization be used to generate the polypeptide of the therapeutic antibodies institute specific combination of albumin fusion proteins, and/or reduce the excessive generation of the polypeptide of the therapeutic antibodies institute specific combination that is used to generate albumin fusion proteins.Similarly, the albumin fusion proteins of using the fragment that comprises therapeutic antibodies at least or variant can activate the polypeptide of the therapeutic antibodies institute specific combination that is used to generate albumin fusion proteins, and it is gone up the bonded polypeptide by binding film (acceptor) and realizes.

At least, use method well known to those skilled in the art, albumin fusion proteins of the present invention can be used as molecular weight marker on the SDS-PAGE gel or in the molecular sieve gel filtration column.Albumin fusion proteins of the present invention also can be used for producing antibody, antibody can be used for measuring the protein expression of therapeutic protein, albumin protein and/or albumin fusion proteins of the present invention from reconstitution cell then, as the method for conversion of assessment host cell or biological sample.In addition, albumin fusion proteins of the present invention can be used for testing biologic activity described herein.

The diagnostic assay method

Compound of the present invention can be used for diagnosing, treat, preventing and/or predicts particularly people's various disorders of Mammals.Such disorder includes but not limited to reach in table 1 corresponding row herein, and title is " immunocompetence ", " blood associated disorders ", " hyper-proliferative sexual disorder ", " kidney disorder ", " cardiovascular disorder ", " respiratory disorder ", " angiogenesis inhibitor activity ", " cell levels disease ", " wound healing and epithelial cell proliferation ", " neural activity and neurological disorder ", " endocrine regulation ", " reproductive system disorder ", " transmissible disease ", " regeneration ", and/or the disorder of putting down in writing for every kind of therapeutic protein in the part of " gastrointestinal dysfunction ".

For many disorders, can in the tissue of taking from the individuality of suffering from a kind of like this disorder, cell or body fluid (for example serum, blood plasma, urine, seminal fluid, synovia or spinal fluid), detect gene expression dose and promptly take from not the tissue of individuality that should disorder or the material alterations (rise or descend) of the expression level in the body fluid with respect to " standard " gene expression dose.Therefore, the invention provides diagnostic method useful in the diagnostic procedure of disorder, comprise the expression of gene level of taking from coded polypeptide in individual tissue, cell or the body fluid of measuring, and with the gene expression dose that records and standard gene expression level relatively, gene expression dose is with respect to the rising of standard or be reduced to disorderly indication.These diagnostic assay methods can be carried out in vivo or external, such as for example carrying out in blood sample, biopsy or autopsy tissue.

The present invention also can be used as the prediction indication, demonstrates the clinical effectiveness that genetic expression strengthens or the patient of reduction will suffer bad luck thus.

Qualitative or the quantitative measurment of the expression of gene level of coded polypeptide " measure " intention or estimate the level of the specific polypeptide of the present invention in first biological sample (for example with table 1 in the corresponding polypeptide of disclosed therapeutic protein) or the level of the mRNA of coded polypeptide, or directly (for example by measuring or the abswolute level of estimation protein or mRNA) or relative (for example by comparing) with polypeptide or mRNA level in second biological sample.Preferably, measure or estimate expression of polypeptides level or mRNA level in first biological sample, and compare with the polypeptide level or the mRNA level of standard, standard is from second biological sample of individuality that should not disorder, perhaps by individual crowd's that should not disorder mean level (ML) decision.Those skilled in the art will understand, in case known standard polypeptide level or mRNA level, it can repeat as comparative standard.

" biological sample " means any biological sample of taking from individuality, clone, tissue culture or containing other source of polypeptide of the present invention (comprising its part) or mRNA.Just as noted above, biological sample comprise body fluid (such as serum, blood plasma, urine, seminal fluid, synovia and spinal fluid) and find express polypeptide or the total length of mRNA or its segmental tissue-derived.The method that obtains biopsy and body fluid from Mammals is well known in the art.When biological sample comprised mRNA, biopsy was preferred source.

Can separate total cell RNA from biological sample with any suitable technology, such as Chomczynski and Sacchi, Anal.Biochem.162:156-159, one of record step guanidine thiocyanate-benzene phenol-chloroform method in 1987.Can adopt any appropriate means to measure the level of the mRNA of code book invention polypeptide then.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 (LCR) (RT-LCR).

The invention still further relates to and be used for detection of biological imitate product (for example cell and tissue) and albumin fusion proteins bonded of the present invention or by the diagnostic methods method of the level of its bonded or polypeptide associating with it, such as quantitative and diagnostic assay method, comprise the normal and abnormal level of measuring polypeptide.So, for example, be used to detect with the albumin fusion proteins bonded or can be used for detecting the existence of tumour by its bonded or polypeptide associating with respect to the diagnostic assay method of the abnormal level of normal control tissue sample with it according to of the present invention.Can be used for measuring derived from albumin fusion proteins bonded of the present invention or by the determination techniques of the level of its bonded or polypeptide associating those skilled in the art being known in host's the sample with it.Such measuring method comprises radioimmunoassay, competitive binding assay method, Western engram analysis and ELISA assay method.Available any method known in the art is measured the polypeptide level in the biological sample.

Can measure polypeptide level in the biological sample with multiple technologies.For example, can be with classical immunohistology method (people such as Jalkanen, J.Cell.Biol.101:976-985,1985; Jalkanen, people such as M., J.Cell.Biol.105:3087-3096,1987) come the expression of polypeptides in the research organization.Can be used for detecting other method that polypeptide gene expresses and comprise immunoassay, such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA).Suitable TPPA marker is road known in the art, comprises the enzyme labelling thing, such as glucose oxidase, with radio isotope, such as iodine ( ¹²⁵I, ¹²¹I), carbon ( ¹⁴C), sulphur ( ³⁵S), tritium ( ³H), indium ( ¹¹²In) and technetium ( ^99mTc), and fluorescent marker, such as fluorescein and rhodamine, and vitamin H.

Tissue to be analyzed or cell type generally include those known or those (such as for example cancers) suspection expression goal gene.The method for protein isolation that adopts among the present invention can be for example such as Harlow and Lane (Harlow, E. and Lane, D., 1988, " Antibodies:A Laboratory Manual ", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) put down in writing in, its complete being incorporated herein by reference.Isolated cells can be derived from cell culture or from the patient.To the cell analysis from culture may be to can be used as based on the cell of the part of the gene therapy technology of cell assess or test compounds to the steps necessary of the influence of genetic expression.

For example, albumin fusion proteins can be used for quantitatively or qualitative detection and albumin fusion proteins bonded of the present invention or by the existence of its bonded or polypeptide associating with it.This can realize that it uses fluorescently-labeled albumin fusion proteins by for example immunofluorescence technique, and detects coupling mutually with light microscopy, flow cytometry or fluorometry.

In a preferred embodiment, comprising the fragment of the antibody of the disclosed or therapeutic protein (for example disclosed therapeutic protein in the table 1) that other approach of this area is known among at least a the present invention of specific combination or the albumin fusion proteins of variant at least can be used for quantitatively or the existence of qualitative detection gene product or its conservative variant or peptide fragment.This can realize that it adopts fluorescently-labeled antibody by for example immunofluorescence technique, and detects coupling mutually with light microscopy, flow cytometry or fluorometry.

Albumin fusion proteins of the present invention also can be used for picture and be used in situ detection and albumin fusion proteins bonded of the present invention or by its bonded or polypeptide associating with it in immunofluorescence, immunoelectron microscope or non-immunologic assay method on histology.In situ detection can be by taking out histological specimen from the patient, and it is used through antibody or polypeptide of the present invention of mark realize.Preferably use albumin fusion proteins on the biological sample by covering through the albumin fusion proteins of mark.By a kind of like this program, not only might determine also may determine its distribution in being examined tissue with the albumin fusion proteins bonded or by the existence of its bonded or polypeptide associating with it.Utilize the present invention, those skilled in the art will be easy to discover any (such as the dyeing procedure) that can revise in the extremely multiple Histological method and realize in situ detection.

Detect with the albumin fusion proteins bonded or by the immunoassay and the non-immunoassay of its bonded or polypeptide associating and generally include sample with it, lysate such as biological fluid, tissue extract, the new cell of collecting or the cell that in cell cultures, had been incubated, exist can in conjunction with gene product or its conservative variant or peptide fragment detect through traget antibody the time be incubated, and by any bonded antibody that detects in the multiple technologies well-known in the art.

Biological sample is contacted with solid support or carrier and be fixed in the above, such as nitrocellulose or can fixed cell, other solid support of cell granulations or soluble protein its.Can handle with detecting subsequently with suitable buffer solution for cleaning upholder then through the albumin fusion proteins of the present invention of mark.Available then damping fluid cleans solid support once more to remove unconjugated antibody or polypeptide.Optional traget antibody subsequently.Can detect the quantity of bonded marker on the solid support then by ordinary method.

" solid support or carrier " means can be in conjunction with any upholder of polypeptide (for example albumin fusion proteins, or with albumin fusion proteins bonded of the present invention or by its bonded or polypeptide associating with it).Well-known upholder or carrier comprise glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and Mierocrystalline cellulose, polyacrylamide, gabbro and magnetite improvement.For the purposes of the present invention, the character of carrier can be to a certain degree soluble or insoluble.In fact upholder can have any possible construction profile, as long as the link coupled molecule can be in conjunction with polypeptide.Therefore, the profile of upholder can be a spheric, as pearl, or columniform, as the inwall of test tube, or the outside surface of bar.Perhaps, the surface can be flat, such as thin slice, test strip etc.Preferred upholder comprises polystyrene bead.Those skilled in the art also know and are suitable for binding antibody or antigenic many other carriers, perhaps can determine suitable carriers by normal experiment.

Can measuring according to well-known method of given batch multiple albumin fusion proteins in conjunction with activity.Those skilled in the art can determine the operation and the optimum determining condition of every kind of mensuration by normal experiment.

Except mensuration is taken from polypeptide level in the individual biological sample, also can detect polypeptide in vivo by imaging.For example, in one embodiment of the invention, use albumin fusion proteins of the present invention to come to ill cell or vegetation cell imaging.

The marker or the mark that are used for albumin fusion proteins in-vivo imaging of the present invention comprise the material that available X-roentgenogpaphy, NMR, MRI, CAT-scanning or ESR detect.For the X-roentgenogpaphy, suitable marker comprises radio isotope, and such as barium or caesium, they send detectable radiation but the experimenter is not significantly injured.The mark that is applicable to NMR and ESR comprises the material with detectable characteristic spin, and such as deuterium, it can mix albumin fusion proteins through the nutrition of engineered cells system (perhaps bacterium or yeast strain) by mark.

In addition, can use it and have detectable albumin fusion proteins of the present invention.For example, can use albumin fusion proteins of the present invention and imaging in vivo, as what above discuss about traget antibody through radiopacity compound or other suitable compound mark.In addition, such polypeptide also can be used for the in-vitro diagnosis program.

Will be through suitably (for example detecting image-forming module such as radio isotope ¹³¹I, ¹¹²In, ^99mTc), radio-opaque substance, maybe can be by magnetic resonance detection the polypeptid specificity antibody or the antibody fragment of material mark import the Mammals that (for example parenteral, subcutaneous or intraperitoneal) has the examine disorder.This area will be understood, and experimenter's size and used imaging system will determine to produce the amount of the required image-forming module of diagnosis imaging.In the example of radio isotope module,, inject radioisotopic amount usually in about 5 to 20 millicuries for the human experimenter ^99mIn the scope of Tc.To preferentially contain in vivo with albumin fusion proteins bonded of the present invention or by the position accumulation of its bonded or polypeptide associating or other material through the mark albumin fusion proteins then with it.The description of in-vivo tumour imaging can be referring to people such as S.W.Burchiel, " Immunopharmacokinetics of Radiolabeled Antibodies andTheir Fragments ", the 13rd chapter, " Tumor Imaging:The Radiochemical Detection ofCancer ", S.W.Burchiel and B.A.Rhodes compile, Masson Publishing Inc., 1982.

A kind of method of albumin fusion proteins of the present invention being carried out detectable label is that it is connected with the report enzyme, and will connect product and be used for enzyme immunoassay (EIA) (Voller, A., " The enzymeLinked Immunosorbent Assay (ELISA) ", 1978, Diagnostic Horizons 2:1-7, Microbiological Associates Quarterly Publication, Walkersville, MD); People such as Voller, J.Clin.Pathol.31:507-520,1978; Butler, J.E., Meth.Enzymol.73:482-523,1981; Maggio, E. compiles, and 1980, Enzyme Immunoassay, CRC Press, BocaRaton, FL; Ishikawa, people such as E. compile, and 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo.To react in such a way with the preferred chromogenic substrate of suitable substrate with the report enzyme of antibodies, promptly produce detectable chemical module, for example by spectrophotometry, fluorometry or range estimation mode.The report enzyme that can be used for detectable label antibody includes but not limited to malate dehydrogenase (malic acid dehydrogenase), staphylococcal nuclease, δ-5-steroid isomerase, yeast alcohol dehydrogenase, α-Gan Youlinsuantuoqingmei, triosephosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase enzymes, rnase, urase, catalase, glucose-6-phosphate dehydrogenase (G6PD), glucoamylase and acetylcholinesterase.In addition, can detect by colorimetry, it adopts the chromogenic substrate of report enzyme.Also can detect by the standard substance of the enzymatic reaction degree of substrate and similar preparation are estimated comparison.

Albumin fusion proteins also can carry out radio-labeling and be used for multiple other immunoassay.For example, by the radio-labeling albumin fusion proteins, albumin fusion proteins might be used for radioimmunoassay (RIA) (referring to for example Weintraub, B., Principles of radioimmunoass ay, Seventh training Course on Radioligand Assay Techniques, The EndocrineSociety, in March, 1986, be incorporated herein by reference).Can come the detection of radioactive isotropic substance by following means, include but not limited to gamma counter, scintillometer or radioautograph.

In addition, this area knows that chelating molecule and they can be used for the mark albumin fusion proteins.Chelating molecule can be attached to albumin fusion proteins of the present invention so that with the described protein of metal ion mark, and metal ion comprises radionuclide or fluorescent marker.For example, referring to Subramanian, R. and Meares, C.F., " Bifunctional Chelating Agents for Radiometal-labeled monoclonalAntibodies ", in " Cancer Imaging with Radiolabeled Antibodies ", D.M.Goldenberg compiles, Kluwer Academic Publications, Boston; Saji, H., " Targeteddelivery of radiolabeled imaging and therapeutic agents:bifunctionalradiopharmaceuticals ", Crit.Rev.Ther.Drug Carrier Syst.16:209-244,1999; Srivastava, S.C. and Mease, R.C., " Progress in research on ligands; nuclides andtechniques for labeling monoclonal antibodies ", Int.J.Rad.Appl.Instrum.B 18:589-603,1991; And Liu.S. and Edwards, D.S., " Bifunctional chelators for therapeuticlanthanide radiopharmaceuticals ", Bioconjug.Chem.12:7-34,2001.Can merge covalently bound any sequestrant with described white protein and all can be used for the present invention.Sequestrant also can comprise the joint module that chelating module and albumin fusion proteins are coupled together.

In one embodiment, albumin fusion proteins of the present invention is attached to acyclic sequestrant, such as diethylenetriamine-N, N, N ', N ", N "-analogue of pentaacetic acid (DPTA), DPTA, and the derivative of DPTA.As nonrestrictive example, sequestrant can be 2-(right-the isothiocyanato benzyl)-6-methyl diethylene triaminepentaacetic acid(DTPA) (IB4M-DPTA, be also referred to as MX-DTPA), 2-methyl-6-(ρ-nitrobenzyl)-1,4,7-triazaheptane-N, N, N '; N ", N "-pentaacetic acid (nitro-IB4M-DTPA or nitro-MX-DTPA); 2-(right-the isothiocyanato benzyl)-cyclohexyl diethylene triaminepentaacetic acid(DTPA) (CHX-DTPA), or N-[2-amino-3-(ρ-nitrophenyl) propyl group]-trans-cyclohexane-1,2-diamines-N, N ', N "-pentaacetic acid (nitro-CHX-A-DTPA).

In another embodiment, albumin fusion proteins of the present invention is attached to acyclic terpyridyl sequestrant, such as 6,6 " two [[N, N, N ", N " four (carboxymethyl) amino] methyl]-4 '-(3-amino-4-methoxyphenyl)-2,2 ': 6 ', 2 " terpyridyls (TMT-amine).

In a specific embodiment, the macrocyclic chelants that is attached on the albumin fusion proteins of the present invention is 1,4,7,10-tetraazacyclododecanand-N, N ', N ", N ...-tetraacethyl (DOTA).In other specific embodiment, DOTA is attached to albumin fusion proteins of the present invention by linkers.The example that can be used for DOTA is conjugated to the linkers on the polypeptide is that this area is generally known, referring to people such as for example DeNardo, Clin.Cancer Res.4 (10): 2483-90,1998; People such as Peterson, Bioconjug.Chem.10 (4): 553-7,1999; Reach people such as Zimmerman, Nucl.Med.Biol.26 (8): 943-50,1999, with its complete being incorporated herein by reference.In addition, United States Patent (USP) 5,652,361 and 5,756,065, sequestrant and the production and the using method that can be conjugated on the antibody are wherein disclosed, with its complete being incorporated herein by reference.Although United States Patent (USP) 5,652,361 and 5,756,065 focuses on sequestrant is conjugated on the antibody, thereby those skilled in the art can be easy to disclosed method wherein made amendment sequestrant is conjugated on other polypeptide.

Can be according to people such as M.Moi, J.Amer.Chem.Soc.49:2639,1989 (2-is right-nitrobenzyl-1,4,7, and 10-tetraazacyclododecanand-N, N ', N ", N " '-tetraacethyl); People such as S.V.Deshpande, J.Nucl.Med.31:473,1990; People such as G.Ruser, Bioconj.Chem.1:345,1990; People such as C.J.Broan, J.C.S.Chem.Comm.23:1739,1990; Reach people such as C.J.Anderson, J.Nucl.Med.36:850,1995 described employings are based on the bifunctional chelating agent of macrocyclic ligand, and it is attached on the part carbon skeleton by activation arm or functional group and realizes puting together.

In one embodiment,, choose wantonly and contain one or more carboxyls, amino, hydroxamic acid, phosphonic acids or phosphate group, be attached to albumin fusion proteins of the present invention macrocyclic chelants such as many nitrogen heterocyclic rings sequestrant.In another embodiment, sequestrant is the sequestrant that is selected from DOTA, DOTA analogue and DOTA derivative.

In one embodiment, the suitable chelator molecule that can be attached to albumin fusion proteins of the present invention comprises DOXA (1-oxygen-4,7,10-triazododecane nitrilotriacetic), NOTA (1,4,7-7-triazacyclononane nitrilotriacetic), TETA (1,4,8,11-tetraazacyclododecane tetradecane tetraacethyl) and THT (4 '-(3-amino-4-methoxy-phenyl)-6,6 " two (N ', N '-two carboxymethyls-N-methylhydrazine)-2; 2 ': 6 ', 2 " terpyridyl) and analogue and derivative.Referring to people such as for example Ohmono, J.Med.Chem.35:157-162,1992; People such as Kung, J.Nucl.Med.25:326-332,1984; People such as Jurisson, Chem.Rev.93:1137-1156,1993; And U.S. Patent number 5,367,080.Other suitable sequestrant comprises U.S. Patent number 4,647,447; 4,687,659; 4,885,363; EP-A-71564; WO 89/00557; And disclosed sequestrant among the EP-A-232751.

In another embodiment, can be used for suitable big ring carboxylic acid sequestrant of the present invention and comprise 1,4,7,10-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 " nitrilotriacetic (9N3); 1,5,9-triazododecane-N, N ', N " nitrilotriacetic (12N3); And 6-acetobrom amino-benzyl-1,4,8,11-tetraazacyclododecane tetradecane-N, N ', N ", N " '-tetraacethyl (BAT).

The preferred sequestrant that can be attached to albumin fusion proteins of the present invention is α-(5-isothiocyanato-2-methoxyphenyl)-1,4,7,10-tetraazacyclododecanand-1,4,7, and the 10-tetraacethyl is also referred to as MeO-DOTA-NCS.Also can use α-(5-isothiocyanato-2-methoxyphenyl)-1,4,7,10-tetraazacyclododecanand-1,4,7, the salt of 10-tetraacethyl or ester.

Covalent attachment the albumin fusion proteins of the present invention of above-mentioned sequestrant can (by the coordination site of sequestrant) with the radioisotope labeling that is suitable for treating, diagnosing or treat double diagnostic purpose.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 have 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 has ¹⁶⁶Ho, ¹⁶⁵Dy, ⁹⁰Y, ^115mIn, ⁵²Fe or ⁷²Ga.In one embodiment, the radionuclide that is used for diagnostic purpose is ¹⁶⁶Ho or ⁹⁰Y.The example that is used for the treatment of the radionuclide of the diagnostic purpose of holding concurrently comprises ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁷⁵Yb or ⁴⁷Sc.In one embodiment, radionuclide is ¹⁵³Sm, ¹⁷⁷Lu, ¹⁷⁵Yb or ¹⁵⁹Gd.

Preferred 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 reaches ¹⁴¹Ce.

In a specific embodiment, covalent attachment the albumin fusion proteins of the present invention of sequestrant can be with being selected from ⁹⁰Y, ¹¹¹In, ¹⁷⁷Lu, ¹⁶⁶Ho, ²¹⁵Bi and ²²⁵The metal ion of Ac carries out mark.

In addition, the radionuclide of emission γ, such as ^99mTc, ¹¹¹In, ⁶⁷Ga and ¹⁶⁹Yb has ratified to be used for diagnosing image or has investigated, and beta emitter, such as ⁶⁷Cu, ¹¹¹Ag, ¹⁸⁶Re and ⁹⁰Y can be used for the application in the oncotherapy.Other useful radionuclide comprises gamma emitter, such as ^99mTc, ¹¹¹In, ⁶⁷Ga reaches ¹⁶⁹Yb, and beta emitter, such as ⁶⁷Cu, ¹¹¹Ag, ¹⁸⁶Re, ¹⁸⁸Re and ⁹⁰Y, and other interested radionuclide, such as ²¹¹At, ²¹²Bi, ¹⁷⁷Lu, ⁸⁶Rb, ¹⁰⁵Rh, ¹⁵³Sm, ¹⁹⁸Au, ¹⁴⁹Pm, ⁸⁵Sr, ¹⁴²Pr, ²¹⁴Pb, ¹⁰⁹Pd, ¹⁶⁶Ho, ²⁰³Tl and ⁴⁴Sc.Covalent attachment the albumin fusion proteins of the present invention of sequestrant can carry out mark with above-mentioned radionuclide.

In another embodiment, covalent attachment the albumin fusion proteins of the present invention of sequestrant can carry out mark with paramagnetic metal ion, the ion that comprises transition metal and lanthanide series metal, such as ordination number is 21-29,42,43,44 or the ion of metal, particularly 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 57-71.The paramagnetic metal that is used for the nuclear magnetic resonance composition comprises that the atom sequence number is 22 to 29,42,44 and the element of 58-70.

In another embodiment, covalent attachment the albumin fusion proteins of the present invention of sequestrant can carry out mark with the fluorescence metal ion, comprise lanthanide series metal, particularly La, Ce, Pr, Nd, Pm, Sm, Eu are (for example ¹⁵²Eu), Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu.

In another embodiment, covalent attachment the albumin fusion proteins of the present invention of sequestrant can carry out mark with the reporter that contains heavy metal, can comprise the atom of Mo, Bi, Si and W.

Might use fluorescent chemicals mark albumin fusion proteins.In the light time that fluorescently-labeled antibody is exposed to suitable wavelength, because fluorescence can detect its existence.The most frequently used fluorescent mark compound has fluorescein isothiocyanate, rhodamine, phycoerythrin, Phycocyanins, C-, allophycocyanin, phthalaldehyde (ophthaldehyde) and glimmering amine.

The albumin fusion proteins also metal of available transmission fluorescence carries out detectable label, such as ¹⁵²Eu or other lanthanide series metal.These metals can be used such as diethylene triaminepentaacetic acid(DTPA) (DTPA) or ethylenediamine tetraacetic acid (EDTA) metal-chelating groups such as (EDTA) and be attached on the antibody.

Albumin fusion proteins also can be by carrying out detectable label with the coupling of chemiluminescence compound.Can determine the existence of the albumin fusion proteins of chemiluminescent labeling by detecting the cold light that produces in the chemical reaction process then.The example of useful especially chemiluminescent labeling compound has luminol,3-aminophthalic acid cyclic hydrazide, different luminol,3-aminophthalic acid cyclic hydrazide, theromatic acridinium ester, imidazoles, acridinium salt and barkite.

Equally, bioluminescent compound also can be used for mark albumin fusion proteins of the present invention.Noclilucence is a class chemoluminescence of finding in the biology system, and catalytic protein improves the efficient of chemiluminescence reaction in this system.The existence of bioluminescent protein is determined by the existence that detects cold light.The important biomolecule luminophor that is used for the mark purpose has luciferin, luciferase and aequorin.

Transgenic organism

The present invention also comprises the transgenic organism of expressing albumin fusion proteins of the present invention.Transgenic organism refers to wherein to shift reorganization, external source or the genetically modified organism, GMO of the genetic material of being cloned.Such genetic material often is called transgenosis.Genetically modified nucleotide sequence can comprise one or more transcription regulating nucleotide sequences and other nucleotide sequence such as intron, and they may be that coded proteinic optimum expression and secretion are necessary.Transgenosis can be designed to instruct coded protein to express by this way, promptly helps reclaiming this protein by organism or by the product of organism generation such as milk, blood, urine, ovum, hair or the seed of organism.Transgenosis can be by forming from the genome deutero-nucleotide sequence of species identical with the target animals species or different species.Transgenosis can be integrated into the common in other cases undiscovered locus of this specific nucleic acid sequence or this genetically modified normal gene seat in the genome.

Term " germ line cell is a transgenic organism " refers to such transgenic organism, wherein hereditary change or genetic information is imported germ line cell, thereby gives the ability that transgenic organism passes to genetic information the offspring.If in fact such offspring has some or all of these changes or genetic information, they also are transgenic organisms so.This change or genetic information can be external sources for the living species under the recipient, and external source with regard to the particular individual recipient, perhaps can be the genetic information that the recipient has had only.In the later case, expression of gene change or that introduce can be different with natural gene.

Transgenic organism can be transgenic animal or transgenic plant.Transgenic animal can produce by multiple different methods, comprise that gene targeting in transfection, electroporation, microinjection, the embryonic stem cell and recombinant virus and retroviral infection are (referring to for example U.S. Patent number 4,736,866; U.S. Patent number 5,602,307; People such as Mullins, Hypertension 22 (4): 630-633,1993; People such as Brenin, Surg.Oncol.6 (2): 99-110,1997; Tuan compiles, Recombinant Gene ExpressionProtocols, " Methods in Molecular Biology ", No.62, Humana Press, 1997).The method that nucleic acid fragment is imported the competent mammalian cell of reorganization can be any method that helps multiple nucleic acid molecule cotransformation.Those skilled in the art can obtain to be used to produce the detailed process of transgenic animal easily, comprise U.S. Patent number 5,489,743 and U.S. Patent number 5,602,307 in disclosed method.

Produced many reorganization or transgenic mice, comprised (U.S. Patent number 4,736,866) of expressing activate oncogene; Express ape SV40 T-antigenic (U.S. Patent number 5,728,915); Do not express (U.S. Patent number 5,731,490) of Interferon, rabbit regulatory factor 1 (IRF-1); Show dopaminergic handicapped (U.S. Patent number 5,723,719); Express (U.S. Patent number 5,731,489) of the people's gene of at least a participation controlling of blood pressure; Show (U.S. Patent number 5,720,936) of the symptom high similarity of showing with natural generation Alzheimer; (U.S. Patent number 5,602,307) that the mediated cell adhesive capacity descends; (people such as Clutter, Genetics 143 (4): 1753-1760,1996) who has bovine growth hormone gene; Perhaps, can produce that fully human antibodies replys (McCarthy, The Lancet 349 (9049): 405,1997) mouse.

Though most of transgenic experiments are still selected mouse and rat, preferred or even necessary other animal species that uses in some situation.The transgenosis process has been successfully applied to multiple non-mouse animal, comprises that sheep, goat, pig, dog, cat, monkey, chimpanzee, hamster, rabbit, ox and cavy are (referring to people such as for example Kim, Mol.Reprod.Dev.46 (4): 515-526,1997; Houdebine, Reprod.Nutr.Dev.35 (6): 609-617,1995; Petters, Reprod.Fertil.Dev.6 (5): 643-645,1994; People such as Schnieke, Science 278 (5346): 2130-2133,1997; And Amoah, J.AnimalScience 75 (2): 578-585,1997).

For the protein secreting of the present invention that guides transgenes encoding enters the milk of transgenic animal, can make under its control that is in promotor, this promotor preferentially activates in mammary epithelial cell.The promotor of the gene of preferred control coding milk protein matter, for example the promotor of casein, beta-lactoglobulin, whey acid protein or whey-protein (referring to for example DiTullio, BioTechnology 10:74-77,1992; People such as Clark, BioTechnology 7:487-492,1989; People such as Gorton, BioTechnology5:1183-1187,1987; Reach people such as Soulier, FEBS Letts.297:13,1992).The transgene mammal of selecting will produce a large amount of milk and have very long lactation period, for example goat, ox, camel or sheep.

Albumin fusion proteins of the present invention can also be expressed in transgenic plant, for example the DNA transgenosis is inserted into the plant in nucleus or plastid (plastidic) genome.Be used for the Plant Transformation flow process of external nucleic acid importing vegetable cell or protoplastis is known in this area.Participate in for example Methods inEnzymology Vol.153, " Recombinant DNA Part D ", 1987, Wu and Grossman compile, Academic Press and European patent application EP 693554.U.S. Patent number 5,283,184, also described the method that is used to produce the genetic engineering plant in U.S. Patent number 5,482,852 and the European patent application EP 693 554, they all are incorporated herein by reference.

Medicine or therapeutic composition

Can use this albumin fusion proteins or its preparation by any ordinary method, comprise that parenteral (for example subcutaneous or intramuscular) is injected or intravenously is inculcated.Treatment can be made of the multi-agent administration in single agent administration or one period.

Although might use albumin fusion proteins of the present invention separately, preferably can accept carrier with the form of pharmaceutical formulations with one or more provides.With regard to compatible with albumin fusion proteins and to its recipient harmless with regard to, carrier must be " acceptable ".Usually, carrier will be aseptic and pyrogen-free water or salt solution.Albumin fusion proteins of the present invention is particularly suitable for preparing in such as aseptic pyrogen-free water, salt solution or other isotonic solution at aqueous carrier, because their quality guaranteed perioves in solution have prolonged.For example, pharmaceutical composition of the present invention can prepare in aqueous form in advance in several weeks or several months before the distribution for example or more over a long time.

For example, can prepare the preparation that contains albumin fusion proteins considering the quality guaranteed period of albumin fusion proteins in aqueous formulation to prolong.Just as discussed above, the many quality guaranteed perioves in these therapeutic proteins significantly increase or have prolonged after merging with HA.

In being suitable for situation about using, can use standard program that albumin fusion proteins of the present invention is mixed with aerosol with aerosol.Term " aerosol " comprises any gas matchmaker suspending phase of the albumin fusion proteins of the present invention that can be drawn in bronchiole or the nasal meatus.Particularly, aerosol comprises the gas matchmaker suspended substance of the droplet of albumin fusion proteins of the present invention, and it can generate in metered dose inhaler upon actuation or spraying gun or in atomizer.Aerosol comprises that also The compounds of this invention is suspended in the dry powder composite in air or other vector gas, and it can for example be delivered by being blown into suction apparatus.Referring to Ganderton and Jones, " Drug Delivery to the Respiratory Tract ", Ellis Horwood, 1987; Gonda, Critical Reviews in Therapeutic Drug Carrier Systems 6:273-313,1990; Reach people such as Raebum, Pharmacol.Toxicol.Methods 27:143-159,1992.

Part since the composition of employed albumin fusion proteins derived from suitable species, preparation of the present invention is still non-immunogenicity usually.For example, be used for man-hour, the two all is the people usually for the therapeutic protein of albumin fusion proteins and albumin part.Two kinds of compositions are arbitrary therein is not in the certain situation of derived from human, can to make defined epitope demonstrate for human immune system be the people's rather than external source in essence by substituting key amino acid, and thus with this composition humanization.

Preparation can present by unit dosage form easily, and can prepare by the well-known any method of pharmacy field.Such method comprises the step that albumin fusion proteins is combined with the carrier that constitutes one or more supplementary components.Usually,, make product shaping then as required, so prepare described preparation by with activeconstituents and liquid carrier or finely-divided solid carrier or both homogeneous and closely combine.

The preparation that is suitable for parenteral administration comprises water-based or non-aseptic parenteral solution under water, can contain antioxidant, buffer reagent, fungistat and make described preparation be suitable for the solute of intended recipinent; And water-based or non-aqueous sterile suspensions, can contain suspension agent and thickening material.Preparation can present in unitary dose or multi-dose container, for example Mi Feng ampoule, phial or syringe, and can be stored under freeze-dried (freeze-drying) condition, only need face add before using aseptic liquid carrier for example water promptly can be used for injecting.Now joining injection liquid and suspension can be prepared by sterilized powder.Because many albumin fusion proteins of the present invention demonstrate the serum half-life of prolongation, dosage particles can contain and proteinic the compare low volumetric molar concentration or than the therapeutic protein part of low dosage of standard preparation that do not merge of TA.

As an example, when albumin fusion proteins of the present invention comprises one of listed protein of table 1 " therapeutic protein: X " row as one or more therapeutic proteins district, can be the basic calculation dosage form with respect to the effectiveness of independent therapeutic protein with the effectiveness of albumin fusion proteins, consider that simultaneously albumin fusion proteins compares the serum half-life and the quality guaranteed period of prolongation with natural therapeutic protein.For example, if therapeutic protein usually with 0.3 to 30.0IU/kg/ week or used in 0.9 to 12.0IU/kg/ week, 1 year or longer time divide and take for three times or seven times so.In the albumin fusion proteins of forming being merged by total length HA and therapeutic protein, the dosage of equivalence will make preparation present bigger weight with regard to unit, but administration frequency for example can be reduced to twice weekly, once in a week or still less.

Preparation of the present invention or composition can be packed or be included in the test kit with the specification sheets or the package insert that prolong about the albumin fusion proteins composition quality guaranteed period.For example, such specification sheets or package insert can provide the quality guaranteed period of considering that albumin fusion proteins of the present invention prolongs or expansion and the storage requirement of recommending, such as time, temperature and illumination.Such specification sheets or package insert also can provide the concrete advantage of albumin fusion proteins of the present invention, the preparation that may need in the open air such as being easy to store, use beyond the controlled hospital, clinical or clinic condition.As mentioned above, preparation of the present invention can be an aqueous form, and can store under less-than-ideal environment and not obviously forfeiture of therapeutic activity.

Albumin fusion proteins of the present invention also can be included in the healthcare products (nutraceuticals).For example, some albumin fusion proteins of the present invention can be used in natural product, comprises the breast or the milk-product that are obtained by the transgene mammal of expressing albumin fusion proteins.Such composition can also comprise plant or the plant prod that is obtained by the transgenic plant of expressing albumin fusion proteins.Can also provide albumin fusion proteins with medicinal powder or the tablet form that contains or do not contain other additives known, carrier, weighting agent and thinner.Healthcare products are described in Scott Hegenhart, " Food Product Design ", in December, 1993.

The present invention also provides by accepting in pharmacopedics in the carrier that the experimenter is used the method that the albumin fusion proteins of the present invention of significant quantity or the polynucleotide of code book invention albumin fusion proteins (" white protein fusions polynucleotide ") treat and/or prevent disease or disorder (such as for example any or multiple disease disclosed herein or disorder).

Consider individual patient clinical condition (side effect of using albumin fusion proteins and/or polynucleotide to treat especially separately), deliver the other factors that position, application method, dispenser scheme and practitioner know, prepare and use albumin fusion proteins and/or polynucleotide according to the mode consistent with good medical practice.Therefore, being used for " significant quantity " of the present invention will be determined by this class Consideration.

As general recommendation, total the pharmacopedics significant quantity of parenteral administration albumin fusion proteins will be at about 1 μ g/kg/ days to the scope of 10mg/kg/ days weight in patients, although this will treat judgement as mentioned above during each administration.More preferably, this dosage is 0.01mg/kg/ days at least, and for the people most preferably hormone about 0.01 and 1mg/kg/ days between.If successive administration is used albumin fusion proteins with about 1 μ g/kg/ hour to about 50 μ g/kg/ hours dose rates usually, or by injecting 1-4 time every day or by for example using continuous subcutaneous the inculcating of micropump.Can also use the packed solution of intravenously.Observe to change needed treatment length and treat the interval of replying the back and change according to required effect.

As mentioned above, albumin fusion proteins of the present invention is compared with independent therapeutic protein part (or its fragment or variant) and is had higher plasma stability.When using the significant quantity of albumin fusion proteins and administration application program, the each administration of decision should consider the increase of this plasma stability.Particularly, higher plasma stability can allow to use albumin fusion proteins in identical frequency of administration with lower dosage, perhaps can allow to use albumin fusion proteins with less number of times.Preferably, advantages of higher stability allow with less number of times not too frequent use albumin fusion proteins of the present invention.More preferably, can per two weeks use albumin fusion proteins one time.Still more preferably, can per three, four, five or use albumin fusion proteins more weeks one time, this depends on the pharmacokinetics of albumin fusion proteins.For example, just as discussed above, the pharmacokinetics of IFN-α-HSA fusion rotein is supported every 2-4 week or longer time dosage regimen once, even with 4 weeks or the above interval administration of 4 weeks.

Every dose of significant quantity with the albumin fusion proteins used can also be expressed as every dose of total albumin fusion proteins concentration of being prepared that is given.In one embodiment, every dose of total albumin fusion proteins range of concentrations of being prepared that is applied to the patient is that about 10 μ g/ agent are to about 2000 μ g/ agent.More preferably, the scope of total concn is greater than extremely about 1000 μ g/ agent of 100 μ g/ agent, and perhaps about 1000 μ g/ agent are to about 1200 μ g agent or extremely about 1800 μ g/ agent of about 900 μ g/ agent.

In a specific embodiment, IFN-α of the present invention-HSA fusion rotein (for example being produced by CID2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) carries out dosed administration with about 90 μ g/ agent to total compound concentration of about 2000 μ g/ agent.In a more preferred embodiment, IFN-α of the present invention-HSA fusion rotein (for example being produced) by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296 with about 900 μ g/ agent to about 2000 μ g/ agent, about 900 μ g/ agent to about 1200 μ g/ agent, about 900 μ g/ agent are to total compound concentration of about 1800 μ g/ agent and most preferredly carry out dosed administration with about 1200 μ g/ agent to total compound concentration of about 1800 μ g/ agent.In other preferred embodiment, IFN-α of the present invention-HSA fusion rotein (for example being produced by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) carries out dosed administration with total compound concentration of 600 μ g/ agent, 720 μ g/ agent, 800 μ g/ agent, 900 μ g/ agent, 1000 μ g/ agent, 1200 μ g/ agent, 1500 μ g/ agent, 1800 μ g/ agent or 2000 μ g/ agent.In other embodiment, total preparation medicament of IFN-α of the present invention-HSA fusion rotein (for example being produced by CID2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) or use separately or the combination antiviral compound, (ribavirin) uses such as ribavirin.In other preferred embodiment, total preparation medicament of IFN-α of the present invention-HSA fusion rotein (for example being produced by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) is united one or more antiviral compounds and is used, and described antiviral compound includes but not limited to ribavirin.

In another embodiment, IFN-α of the present invention-HSA fusion rotein (for example being produced by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) of using total compound concentration is treated the patient who has infected HCV.In a specific embodiment, IFN-α of the present invention-HSA fusion rotein (for example being produced by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) is applied to the HCV patient who had not received treatment, or antiviral compound independent or the associating significant quantity, use such as ribavirin, total compound concentration is that about 90 μ g/ agent are to about 2000 μ g/ agent.In a preferred embodiment, with IFN-α of the present invention-HSA fusion rotein (for example by CID2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295, or 4296 produced) be applied to the HCV patient that do not received treatment, or antiviral compound independent or the associating significant quantity, use such as ribavirin, total compound concentration is that about 900 μ g/ agent are to about 2000 μ g/ agent, about 900 μ g/ agent are to about 1200 μ g/ agent, about 900 μ g/ agent to about 1800 μ g/ agent and most preferred total compound concentration are that about 1200 μ g/ agent are to about 1800 μ g/ agent.In other preferred embodiment, with IFN-α of the present invention-HSA fusion rotein (for example by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295, or 4296 produced) be applied to the HCV patient that do not received treatment, or antiviral compound independent or the associating significant quantity, use such as ribavirin, total compound concentration is 600 μ g/ agent, 720 μ g/ agent, 800 μ g/ agent, 900 μ g/ agent, 1000 μ g/ agent, 1200 μ g/ agent, 1500 μ g/ agent, 1800 μ g/ agent, or 2000 μ g/ agent.

In another embodiment, the IFN-α of the present invention-HSA fusion rotein of total compound concentration is applied to the HCV patient who had not received treatment, one or more antiviral compounds of associating significant quantity, for example ribavirin is used, and total compound concentration is that about 90 μ g/ agent are to about 2000 μ g/ agent.In other preferred embodiment, with IFN-α of the present invention-HSA fusion rotein (for example by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295, or 4296 produced) be applied to the HCV patient that do not received treatment, one or more antiviral compounds of associating significant quantity, comprise that for example ribavirin is used, total compound concentration is that about 900 μ g/ agent are to about 2000 μ g/ agent, about 900 μ g/ agent are to about 1200 μ g/ agent, about 900 μ g/ agent to about 1800 μ g/ agent and most preferred total compound concentration are that about 1200 μ g/ agent are to about 1800 μ g/ agent.In other preferred embodiment, with IFN-α of the present invention-HSA fusion rotein (for example by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295, or 4296 produced) be applied to the HCV patient that do not received treatment, one or more antiviral compounds of associating significant quantity, comprise that for example ribavirin is used, total compound concentration is 600 μ g/ agent, 720 μ g/ agent, 800 μ g/ agent, 900 μ g/ agent, 1000 μ g/ agent, 1200 μ g/ agent, 1500 μ g/ agent, 1800 μ g/ agent, or 2000 μ g/ agent.

In another embodiment, IFN-α of the present invention-HSA fusion rotein (for example being produced by CID2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) is applied to the HCV patient who once received treatment, or antiviral compound independent or the associating significant quantity, use such as ribavirin, total compound concentration is that about 90 μ g/ agent are to about 2000 μ g/ agent.In a more preferred embodiment, with IFN-α of the present invention-HSA fusion rotein (for example by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295, or 4296 produced) be applied to the HCV patient that once received treatment, or antiviral compound independent or the associating significant quantity, use such as ribavirin, total compound concentration is that about 900 μ g/ agent are to about 2000 μ g/ agent, about 900 μ g/ agent are to about 1200 μ g/ agent, about 900 μ g/ agent to about 1800 μ g/ agent and most preferred total compound concentration are that about 1200 μ g/ agent are to about 1800 μ g/ agent.In other preferred embodiment, with IFN-α of the present invention-HSA fusion rotein (for example by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295, or 4296 produced) be applied to the HCV patient that once received treatment, or antiviral compound independent or the associating significant quantity, use such as ribavirin, total compound concentration is 600 μ g/ agent, 720 μ g/ agent, 800 μ g/ agent, 900 μ g/ agent, 1000 μ g/ agent, 1200 μ g/ agent, 1500 μ g/ agent, 1800 μ g/ agent, or 2000 μ g/ agent.

In another embodiment, IFN-α of the present invention-HSA fusion rotein (for example being produced by CID2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) is applied to the HCV patient who once received treatment, one or more antiviral compounds of associating significant quantity, comprise that for example ribavirin is used, total compound concentration is that about 90 μ g/ agent are to about 2000 μ g/ agent.In a more preferred embodiment, with IFN-α of the present invention-HSA fusion rotein (for example by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295, or 4296 produced) be applied to the HCV patient that once received treatment, one or more antiviral compounds of associating significant quantity, comprise that for example ribavirin is used, total compound concentration is that about 900 μ g/ agent are to about 2000 μ g/ agent, about 900 μ g/ agent are to about 1200 μ g/ agent, about 900 μ g/ agent to about 1800 μ g/ agent and most preferred total compound concentration are that about 1200 μ g/ agent are to about 1800 μ g/ agent.In other preferred embodiment, with IFN-α of the present invention-HSA fusion rotein (for example by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295, or 4296 produced) be applied to the HCV patient that once received treatment, one or more antiviral compounds of associating significant quantity, comprise that for example ribavirin is used, total compound concentration is 600 μ g/ agent, 720 μ g/ agent, 800 μ g/ agent, 900 μ g/ agent, 1000 μ g/ agent, 1200 μ g/ agent, 1500 μ g/ agent, 1800 μ g/ agent, or 2000 μ g/ agent.

Total compound concentration of albumin fusion proteins and the dosed administration of using albumin fusion proteins will change with desired effects and the concrete therapeutic protein of being used at interval.In one embodiment, every dose of total preparation albumin fusion proteins range of concentrations that is applied to the patient be about 10 μ g/ agent to about 2000 μ g/ agent, weekly, whenever biweekly, per three weeks once, every around or more weeks once.More preferably, the scope of total concn is that about 100 μ g/ agent are to about 1000 μ g/ agent, weekly, whenever biweekly, per three weeks once, every around or more weeks once, perhaps, about 1000 μ g/ agent are to about 1200 μ g/ agent or about 900 μ g/ agent to about 1800 μ g/ agent, weekly, whenever biweekly, per three weeks once, every around or more weeks once.

In a specific embodiment, use IFN-α of the present invention-HSA fusion rotein (for example being produced) with following total compound concentration by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296, about 90 μ g/ agent are to about 2000 μ g/ agent, per two weeks, three weeks, all around or five weeks once.In a more preferred embodiment, with following total compound concentration dosed administration IFN-α of the present invention-HSA fusion rotein (for example being produced) by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296, about 900 μ g/ agent are to about 2000 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; About 900 μ g/ agent are to about 1200 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; About 900 μ g/ agent are to about 1800 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; And most preferred, about 1200 μ g/ agent are to about 1800 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once.In other embodiment, use IFN-α of the present invention-HSA fusion rotein (for example being produced) with following total compound concentration by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296, about 600 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; 800 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; 900 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; 1000 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; 1200 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; 1500 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; 1600 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; 1800 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; Or 2000 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once.In a preferred embodiment, use IFN-α of the present invention-HSA fusion rotein (for example being produced) with following total compound concentration by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296,900 μ g/ agent, whenever biweekly; More preferably 1200 μ g/ agent, whenever biweekly; 1200 μ g/ agent, whenever all around once; Or 1800 μ g/ agent, every around once.In other embodiment, use total preparaton of IFN-α of the present invention-HSA fusion rotein (for example being produced) by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296, or independent or combination antiviral compound, use such as ribavirin.In other preferred embodiment, use total preparaton of IFN-α of the present invention-HSA fusion rotein (for example being produced) by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296, unite one or more antiviral compounds, comprise that for example ribavirin is used.

In specific embodiment, IFN-α of the present invention-HSA fusion rotein (for example being produced by CID2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) is applied to the HCV patient who had not received treatment, total compound concentration is that about 90 μ g/ agent are to about 2000 μ g/ agent, per two weeks, three weeks, all around or five weeks once, or independent or combination antiviral compound, use such as ribavirin.In a more preferred embodiment, IFN-α of the present invention-HSA fusion rotein (for example being produced by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) is applied to the HCV patient who had not received treatment, total compound concentration be about 900 μ g/ agent to about 2000 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; About 900 μ g/ agent are to about 1200 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; About 900 μ g/ agent are to about 1800 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; And most preferred about 1200 μ g/ agent are to about 1800 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once, or independent or combination antiviral compound comprises such as ribavirin and using.In other embodiment, IFN-α of the present invention-HSA fusion rotein (for example being produced by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) is applied to the HCV patient who had not received treatment, total compound concentration is about 600 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; 800 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; 900 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; 1000 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; 1200 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; 1500 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; 1600 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; 1800 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; Or 2000 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once, or independent or combination antiviral compound is used such as ribavirin.

In preferred specific embodiments, IFN-α of the present invention-HSA fusion rotein (for example being produced by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) is applied to the HCV patient who had not received treatment, total compound concentration is that about 90 μ g/ agent are to about 2000 μ g/ agent, per two weeks, three weeks, all around or five weeks once, unite one or more antiviral compounds, comprise that for example ribavirin is used.In a more preferred embodiment, IFN-α of the present invention-HSA fusion rotein (for example being produced by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) is applied to the HCV patient who had not received treatment, total compound concentration be about 900 μ g/ agent to about 2000 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; About 900 μ g/ agent are to about 1200 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; About 900 μ g/ agent are to about 1800 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; And most preferred about 1200 μ g/ agent are to about 1800 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once, unite one or more antiviral compounds, comprise that for example ribavirin is used.In other embodiment, IFN-α of the present invention-HSA fusion rotein (for example being produced by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) is applied to the HCV patient who had not received treatment, total compound concentration is about 600 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; 800 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; 900 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; 1000 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; 1200 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; 1500 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; 1600 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; 1800 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; Or 2000 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once, unite one or more antiviral compounds, comprise that for example ribavirin is used.

In a more preferred embodiment, IFN-α of the present invention-HSA fusion rotein (for example being produced by CID2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) is applied to the HCV patient who had not received treatment, total compound concentration is 900 μ g/ agent, whenever biweekly; And preferred 1200 μ g/ agent, per two kinds once; 1200 μ g/ agent, whenever all around once; Or 1800 μ g/ agent, every around once, or independent or combination antiviral compound is used such as ribavirin.In the most preferred embodiment, IFN-α of the present invention-HSA fusion rotein (for example being produced by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) is applied to the HCV patient who had not received treatment, total compound concentration is 900 μ g/ agent, whenever biweekly; And preferred 1200 μ g/ agent, whenever biweekly; 1200 μ g/ agent, whenever all around once; Or 1800 μ g/ agent, every around once, unite one or more antiviral compounds, comprise that for example ribavirin is used.

In other specific embodiments, IFN-α of the present invention-HSA fusion rotein (for example being produced by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) is applied to the HCV patient who once received treatment, total compound concentration is that about 90 μ g/ agent are to about 2000 μ g/ agent, per two weeks, three weeks, all around or five weeks once, or independent or combination antiviral compound, use such as ribavirin.In a more preferred embodiment, IFN-α of the present invention-HSA fusion rotein (for example being produced by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) is applied to the HCV patient who once received treatment, total compound concentration be about 900 μ g/ agent to about 2000 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; About 900 μ g/ agent are to about 1200 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; About 900 μ g/ agent are to about 1800 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; And most preferred about 1200 μ g/ agent are to about 1800 μ g/ agent, each week, three weeks, all around or five weeks once; Or most preferred whenever biweekly, or independent or combination antiviral compound is used such as ribavirin.In other embodiment, IFN-α of the present invention-HSA fusion rotein (for example being produced by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) is applied to the HCV patient who once received treatment, total compound concentration is about 600 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; 800 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; 900 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; 1000 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; 1200 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; 1500 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; 1600 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; 1800 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; Or 2000 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once, or independent or combination antiviral compound is used such as ribavirin.

In a more particular embodiment, IFN-α of the present invention-HSA fusion rotein (for example being produced by CID2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) is applied to the HCV patient who once received treatment, total compound concentration is that about 90 μ g/ agent are to about 2000 μ g/ agent, per two weeks, three weeks, all around or five weeks once, unite one or more antiviral compounds, comprise that for example ribavirin is used.In a more preferred embodiment, IFN-α of the present invention-HSA fusion rotein (for example being produced by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) is applied to the HCV patient who once received treatment, total compound concentration be about 900 μ g/ agent to about 2000 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; About 900 μ g/ agent are to about 1200 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; About 900 μ g/ agent are to about 1800 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; And most preferred about 1200 μ g/ agent are to about 1800 μ g/ agent, each week, three weeks, all around or five weeks once; Or most preferredly whenever biweekly unite one or more antiviral compounds, comprise that for example ribavirin is used.In other embodiment, IFN-α of the present invention-HSA fusion rotein (for example being produced by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) is applied to the HCV patient who once received treatment, total compound concentration is about 600 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; 800 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; 900 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; 1000 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; 1200 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; 1500 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; 1600 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; 1800 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once; Or 2000 μ g/ agent, each week, two weeks, three weeks, all around or five weeks once, unite one or more antiviral compounds, comprise that for example ribavirin is used.

In a more preferred embodiment, IFN-α of the present invention-HSA fusion rotein (for example being produced by CID2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) is applied to the HCV patient who once received treatment, total compound concentration is 900 μ g/ agent, whenever biweekly; And preferred 1200 μ g/ agent, whenever biweekly; 1200 μ g/ agent, whenever all around once; Or 1800 μ g/ agent, every around once, or independent or combination antiviral compound is used such as ribavirin.In the most preferred embodiment, IFN-α of the present invention-HSA fusion rotein (for example being produced by CID 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476,3960,4290,4291,4292,4295 or 4296) is applied to the HCV patient who once received treatment, total compound concentration is 900 μ g/ agent, whenever biweekly; And preferred 1200 μ g/ agent, whenever biweekly; 1200 μ g/ agent, whenever all around once; Or 1800 μ g/ agent, every around once, unite one or more antiviral compounds, comprise that for example ribavirin is used.

Albumin fusion proteins and/or polynucleotide can by in oral, rectum, parenteral, the brain pond, intravaginal, intraperitoneal, part (by pulvis, ointment, gelifying agent, drops or transdermal patch), suck or mouthful or nose spray and use." pharmacopedics can be accepted carrier " refers to any non-toxic solid, semisolid or liquid filling agent, thinner, coating material or pharmaceutical adjunct.Term " parenteral " refers to comprise when being used for this paper in intravenously, intramuscular, intraperitoneal, the breastbone, subcutaneous and intra-articular injection and be poured in mode of administration.

Albumin fusion proteins of the present invention and/or polynucleotide also are suitable for using by slow-released system.That the example of slowly-releasing albumin fusion proteins and/or polynucleotide has is oral, in the rectum, parenteral, brain pond, intravaginal, intraperitoneal, part (by pulvis, ointment, gelifying agent, drops or transdermal patch), suck or mouthful or the nose spray application." pharmacopedics can be accepted carrier " refers to non-toxic solid, semisolid or liquid filling agent, thinner, coating material or the pharmaceutical adjunct of any kind.Term " parenteral " refers to comprise when being used for this paper in intravenously, intramuscular, intraperitoneal, the breastbone, subcutaneous and intra-articular injection and be poured in mode of administration.Other example of slowly-releasing albumin fusion proteins and/or polynucleotide comprises suitable polymeric material (such as the semipermeability polymeric matrices of for example shaping prod form, for example film or micro-capsule), suitable hydrophobic material (for example can accept the milk sap in the oil) or ion exchange resin and a small amount of soluble derivative (such as for example a small amount of soluble salt).

Sustained-release matrix comprises polylactide (U.S. Patent number 3,773,919, EP 58,481), the multipolymer of L-L-glutamic acid and L-L-glutamic acid-γ-ethyl ester (people such as Sidman, Biopolymers 22:547-556,1983), poly-(2-hydroxyethyl meth acrylate) (people such as Langer, J.Biomed.Mater.Res.15:167-277,1981; And Langer, Chem.Tech.12:98-105,1982), ethene vinyl acetate people such as (, the same) Langer or poly--D-(-)-3-hydroxybutyric acid (EP 133,988).

Slowly-releasing albumin fusion proteins and/or polynucleotide also comprise albumin fusion proteins of the present invention that liposome is caught and/or polynucleotide (generally referring to Langer, Science 249:1527-1533,1990; People such as Treat, in " Liposomes in the Therapy of Infectious Disease and Cancer ", Lopez-Berestein and Fidler compile, Liss, New York, pp.317-327 and 353-365,1989).The liposome that contains albumin fusion proteins and/or polynucleotide prepares by method known per se: DE 3,218, and 121; People such as Epstein, Proc.Natl.Acad.Sci. (USA) 82:3688-3692,1985; People such as Hwang, 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; U.S. Patent number 4,485,045 and 4,544,545; And EP 102,324).Usually, liposome is little (about 200-800 dust) single layer type, and wherein lipid composition is greater than about 30mol% cholesterol, and adjusts selected ratio for optimum curative effect.

In another embodiment, albumin fusion proteins of the present invention and/or polynucleotide are delivered (referring to Langer, the same by pump; Sefton, CRC Crit.Ref.Biomed.Eng.14:201,1987; People such as Buchwald, Surgery 88:507,1980; People such as Saudek, N.Engl.J.Med.321:574,1989).

Langer, Science 249:1527-1533 has discussed other controlled release system in 1990 the summary.

About parenteral administration, in one embodiment, generally promptly nontoxic and compatible with other composition of preparation carrier mixes and prepares to the recipient in the dosage that is adopted and concentration by accepting carrier with pharmacopedics with the purity of required degree in unitary dose injectable forms (solution, suspension or milk sap) for albumin fusion proteins and/or polynucleotide.For example, the preferred oxygen-free agent of preparation and known to deleterious other compound of curative effect.

Usually, by with albumin fusion proteins and/or polynucleotide and liquid carrier or segmentation solid-state carrier or both homogeneous and closely contact and prepare preparation.Then, as required, product is shaped as required formulation.Preferably, carrier is the parenteral carrier, more preferably with the isoosmotic solution of recipient's blood.The example of such carrier medium comprises water, salt solution, RingerShi liquid and dextrose solution.The present invention also can use non-aqueous media, such as fixed oil and ethyl oleate, and liposome.

Carrier suitably contains minor amounts of additives, such as the material that strengthens isotonicity and chemical stability.Such material is nontoxic to the recipient at dosage that is adopted and concentration, comprises buffer reagent such as phosphoric acid salt, Citrate trianion, succinate, acetate and other organic acid or its salt; Antioxidant is such as xitix; Lower molecular weight (being less than about 10 residues) polypeptide, for example pR60 or tripeptides; Protein is such as serum albumin, gelatin or immunoglobulin (Ig); Hydrophilic polymer is such as polyvinylpyrrolidone; Amino acid is such as glycine, L-glutamic acid, aspartic acid or arginine; Monose, disaccharides and other carbohydrate comprise Mierocrystalline cellulose or derivatives thereof, glucose, seminose or dextrin; Sequestrant is such as EDTA; Sugar alcohol is such as N.F,USP MANNITOL or sorbyl alcohol; Counter ion are such as sodium; And/or nonionogenic tenside, such as polysorbate (comprising for example Tween-20), poloxamer or PEG.

Albumin fusion proteins usually in such media with the concentration of about 0.1mg/ml to 100mg/ml, preferred 1-10mg/ml is with about 3 to 8 pH preparation.Be appreciated that the formation of using some aforementioned excipients, carrier or stablizer will cause polypeptide salt.

Any medicine that being used for the treatment of property is used can be aseptic.Aseptic being easy to realized by aseptic filter membrane (for example 0.2 micron membranes) filtration.Usually albumin fusion proteins and/or polynucleotide are placed the container with aseptic access port, for example have the intravenous solution bag or the phial of the stopper that hypodermic needle can pierce through.

Albumin fusion proteins and/or polynucleotide are stored in unitary dose or multi-dose container as the aqueous solution or the freeze-dried preparation that is used for rehydration usually, for example in Mi Feng ampoule or the phial.As the example of freeze-dried preparation, 5ml is packed in the phial through 1% (w/v) albumin fusion proteins of sterile filtration and/or the polynucleotide aqueous solution, and with the freeze-drying of gained mixture.Make freeze dried albumin fusion proteins and/or polynucleotide rehydration with the preparation perfusion liquid with the injection bacteriostatic water.

In a concrete and embodiment preferred, the albumin fusion proteins preparation comprises 0.01M sodium phosphate, 0.15mM sodium-chlor, 0.16 micromole Sodium octoate/milligram fusion rotein, 15 mcg/ml polysorbate80s, pH7.2.At another in the concrete and embodiment preferred, the albumin fusion proteins preparation is by 0.01M sodium phosphate, 0.15mM sodium-chlor, 0.16 micromole Sodium octoate/milligram fusion rotein, 15 mcg/ml polysorbate80s, the pH7.2 composition.The pH and the buffer reagent of selection and physiological conditions coupling, and add salt as permeation promoter (tonicifier).Select Sodium octoate to be because its that reported increases the ability of the thermostability of protein in solution.At last, add polysorbate as the general purpose table surface-active agent, it reduces the surface tension of solution and reduces the non-special absorption of albumin fusion proteins to container closure system.

The present invention also provides packaged pharmaceuticals goods or the test kit that comprises one or more containers, and the composition of one or more albumin fusion proteins of the present invention and/or polynucleotide is housed in the described container.Announcement by production, use or the sale of the standard medicine of administrative organization of government promulgation or biology product can be provided with such container, and this announcement has reflected the approval of administrative organization to production, use or the sale of human body dispenser.In addition, described albumin fusion proteins and/or polynucleotide can be united use with other therapeutic compound.

Albumin fusion proteins of the present invention and/or polynucleotide can be used separately or unite adjuvant and use.Can include but not limited to the adjuvant that albumin fusion proteins of the present invention and/or polynucleotide are used alum (alum), alum add deoxycholate salt (ImmunoAg), MTP-PE (Biocine Corp.), QS21 (Genentech, Inc.), BCG (for example ), the non-living body goods of MPL and spillikin bacillus (Corynebacterium parvum).In a specific embodiment, albumin fusion proteins of the present invention and/or polynucleotide and alum are co-administered.In another specific embodiment, albumin fusion proteins of the present invention and/or polynucleotide and QS-21 are co-administered.Can include but not limited to monophosphoryl lipid matter immunomodulator, Adju Vax 100a, QS-21, QS-18, CRL1005, aluminium salt, MF-59 and virosome (Virosomal) adjuvant technology with co-administered other adjuvant of albumin fusion proteins of the present invention and/or polynucleotide.Can include but not limited to be devoted to (measles with the co-administered vaccine of albumin fusion proteins of the present invention and/or polynucleotide at MMR; parotitis; rubella); poliomyelitis (polio); varicella; tetanus/diphtheria; hepatitis A; hepatitis B; hemophilus influenzae (Haemophilus influenzae) B; Whooping cough (whooping cough); pneumonia; influenza; the Lai Mushi disease; rotavirus; cholera; yellow jack; Japanese encephalitis; poliomyelitis (poliomyelitis); rabies; typhoid fever; and Whooping cough (pertussis) provides the vaccine of protection.Co-administered both can be concomitant administration, for example as mixture, the preparation while of separating or parallel using; Also can be that order is used.This comprises wherein unites the situation that medicament is used together as the therapeutic mixture, comprises that also wherein uniting medicament is the preparation separately but program used simultaneously for example enters same individuality via the intravenously circuit that separates." associating " used and also comprised such separate administration, promptly gives a kind of in compound or the medicament earlier, gives second kind then.

Albumin fusion proteins of the present invention and/or polynucleotide can be used or unite other therapeutical agent separately and be used.Can include but not limited to chemotherapeutics, microbiotic, steroid and nonsteroidal anti-inflammatory, routine immunization therapeutical agent and/or therapeutic treatment described below with co-administered albumin fusion proteins and/or the polynucleotide reagent of albumin fusion proteins of the present invention and/or polynucleotide.Co-administered both can be concomitant administration, for example as mixture, the preparation that separates but simultaneously or parallel using; Also can be that order is used.This comprises wherein unites medicament as treatment mixture situation about using together, also comprises wherein uniting medicament separately but the program of using simultaneously for example enters same individuality via the intravenously circuit that separates." associating " used and also comprised such separate administration, promptly gives a kind of in compound or the medicament earlier, gives second kind then.

In one embodiment, albumin fusion proteins of the present invention and/or polynucleotide and antithrombotics are co-administered.Can (for example include but not limited to heparin, low molecular weight heparin, warfarin (warfarin) sodium with the co-administered antithrombotics of composition of the present invention

), temparin, 4 hydroxy coumarin, anisindione (MIRADON for example ^TM), Acenocoumarol (acenocoumarol) (Acenocoumarol (nicoumalone) for example, SINTHROME ^TM), 1,3-indenes diketone, phenprocoumon (MARCUMAR for example ^TM), temparin ethyl acetate (TROMEXA for example ^TM), and Asprin.In a specific embodiment, composition of the present invention and heparin and/or warfarin are co-administered.In another specific embodiment, composition of the present invention and warfarin are co-administered.In another specific embodiment, composition of the present invention and warfarin and Asprin are co-administered.In another specific embodiment, composition of the present invention and combination with heparin are used.In another specific embodiment, composition of the present invention and heparin and Asprin are co-administered.

In another embodiment, albumin fusion proteins of the present invention and/or polynucleotide and thrombolysis medicine are co-administered.Can include but not limited to Profibrinolysin, lys-Profibrinolysin, α 2-antiplasmin, streptokinase (KABIKINASE for example with the thrombolysis medicine that composition of the present invention is used ^TM), antiresplace (EMINASE for example ^TM), tissue-type plasminogen activator (t-PA, altevase, ACTIVASE ^TM), urokinase (ABBOKINASE for example ^TM), sauruplase, (uPA, single chain urokinase type plasminogen activator), and hexosamine (AMICAR for example ^TM).In a specific embodiment, composition of the present invention and tissue-type plasminogen activator and Asprin are co-administered.

In another embodiment, albumin fusion proteins of the present invention and/or polynucleotide and antiplatelet drug are co-administered.Can include but not limited to Asprin, Dipyridamole (dipyridamole) (PERSANTINE for example with the antiplatelet drug that composition of the present invention is used ^TM) and ticlopidine (ticlopidine) (TICLID for example ^TM).

In a specific embodiment, imagination is used for prevention, diagnosis and/or treats thrombosis, artery thrombosis, venous thrombosis, thromboembolism, pulmonary embolisms, atherosclerosis, myocardial infarction, transient ischemic attack, unstable angina pectoris by making uniting of antithrombotics, thrombolysis medicine and/or antiplatelet drug and albumin fusion proteins of the present invention and/or polynucleotide.In a specific embodiment, imagination is used for preventing saphenous vein graft obturation (occulsion ofsaphenous grafts) by making anti-coagulant, thrombolysis medicine and/or antiplatelet drug and uniting of albumin fusion proteins of the present invention and/or polynucleotide, reduce peri-operation period thrombosis (periprocedural thrombosis) risk that may follow with angioplasty, stroke risk, reduction and heart valve prosthesis and/or the relevant embolism risk of mitral valve disease that the reduction atrial fibrillation comprises non-rheumatic atrial fibrillation patient.Therapeutical agent of the present invention includes but not limited to prevent health to install obstruction in (for example the vascular access part flow arrangement in blood vessel inner sleeve, the hemodialysis patients, haemodialysis equipment and cardiopulmonary bypass device) outward separately or with other purposes of antiplatelet, anti-freezing and/or thrombolytic drug associating.

In certain embodiments, albumin fusion proteins of the present invention and/or polynucleotide and anti-retroviral agents, nucleoside/nucleotide reverse transcriptase inhibitors (NRTI), non-nucleoside reverse transcriptase inhibitor (NNRTI) and/or proteinase inhibitor (PI) are co-administered.Can include but not limited to RETROVIR with the co-administered NRTI of albumin fusion proteins of the present invention and/or polynucleotide ^TM(zidovudine, zidovudine/AZT), VIDEX ^TM(Didanosine, didanosine/ddI), HIVID ^TM(zalcitabine, zalcitabine/ddC), ZERIT ^TM(stavudine, stavudine/d4T), EPIVIR ^TM(lamivudine, lamivudine/3TC), and COMBIVIR ^TM(zidovudine/lamivudine).Can include but not limited to VIRAMUNE with the co-administered NNRTI of albumin fusion proteins of the present invention and/or polynucleotide ^TM(nevirapine, nevirapine), RESCRIPTOR ^TM(Delavirdine, delavirdine) and SUSTIVA ^TM(Sustiva, efavirenz).Can include but not limited to CRIXIVAN with the co-administered proteinase inhibitor of albumin fusion proteins of the present invention and/or polynucleotide ^TM(Indinavir, indinavir), NORVIR ^TM(ritonavir, ritonavir), INVIRASE ^TM(Saquinavir, saquinavir) and VIRACEPT ^TM(viracept see nelfinaivr, nelfinavir).In a specific embodiment, the arbitrary combination of antiretroviral agent, nucleoside reverse transcriptase inhibitor, non-nucleoside reverse transcriptase inhibitor and/or proteinase inhibitor and albumin fusion proteins of the present invention and/or polynucleotide can be used for the treatment of AIDS and/or prevention or treatment HIV infection.

Other NRTI comprises LODENOSINE ^TM(F-ddA; A kind of acid acceptance adenosine NRTI; Triangle/Abbott); COVIRACIL ^TM(emtricitabine, emtricitabine/FTC; With relevant on lamivudine (3TC) structure but external activity is big 3 to 10 times; Triangle/Abbott); DOTC (BCH-10652, also with the lamivudine structure on relevant but kept activity at the lamivudine resistance isolate of essence ratio; Biochem Pharma); (approval of FDA refusal is used for anti-HIV treatment to Adefovir (Adefovir); Gilead Sciences); (adefovir dipivoxil, the active parent drug of Adefovir; Its activity form is PMEA-pp); TENOFOVIR ^TM(bis-POCPMPA, the former medicine of PMPA; Gilead); DAPD/DXG (the active metabolite of DAPD; Triangle/Abbott); D-D4FC (relevant with 3TC, as to have activity) at AZT/3TC resistance virus; GW420867X (Glaxo Wellcome); ZIAGEN ^TM(Abacavir, abacavir/159U89; Glaxo Wellcome Inc.); CS-87 (3 '-nitrine-2 ', 3 '-di-deoxyuridine; WO 99/66936); And the former medicine form (WO98/17281) of carrying S-acyl group-2-second thioesters (SATE) of β-L-FD4C and β-L-FddC.

Other NNRTI comprises COACTINON ^TM(emivirine, Emivirine/MKC-442, effective NNRTI of HEPT class; Triangle/Abbott); CAPRAVIRINE ^TM(AG-1549/S-1153 has the NNRTI of future generation at the virus that contains the K130N sudden change; Agouron); PNU-142721 (has than high 20 to 50 times active of its predecessor's Delavirdine and to the K130N mutant activity is arranged; Pharmacia ﹠amp; Upjohn); DPC-961 and DPC-963 (s-generation derivative of Yi Feiweilun, design has activity to the virus with K103N sudden change; DuPont); GW-420867X (has than high 25 times active of HBY097 and to the K103N mutant activity is arranged; Glaxo Wellcome); CALANOLIDEA is (from the natural medicine that exists of rubber tree; One or both the virus that contains in Y181C and the K103N sudden change there is activity); And Propolis (WO99/49830).

Other proteinase inhibitor comprises LOPINAVIR ^TM(ABT378/r; Abbott Laboratories); BMS-232632 (a kind of azepine peptide; Bristol-Myres Squibb); TIPRANAVIR ^TM(PNU-140690, a kind of non-peptide dihydroxy pyrone; Pharmacia ﹠amp; Upjohn); PD-178390 (a kind of non-peptide dihydroxy pyrone; Parke-Davis); BMS 232632 (a kind of azepine peptide; Bristol-MyresSquibb); L-756,423 (Indinavir analogues; Merck); DMP-450 (cyclisation urea compounds; Avid﹠amp; DuPont); AG-1776 is (at the external active peptide mimics that has at proteinase inhibitor resistance virus; Agouron); VX-175/GW-433908 (An Ruinawei, the former medicine of the phosphoric acid salt of amprenavir; Vertex ﹠amp; Glaxo Welcome); CGP61755 (Ciba); And AGENERASE ^TM(An Ruinawei; Glaxo Welcome Inc.).

Other antiretroviral agent comprises fusion inhibitor/gp41 wedding agent.Fusion inhibitor/gp41 wedding agent comprises that (from the peptide of HIV gp41 transmembrane protein extracellular domain residue 643-678, this extracellular domain transforms to the fusion state in conjunction with gp41 and prevention in dormant state T-20; Trimeris) and T-1249 (s-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 (bicyclam), SDF-1 and analogue thereof and ALX40-4C (cationic peptide), T22 (18 amino acid peptides; Trimeris) and T22 analogue T134 and T140; The CCR5 antagonist is such as RANTES (9-68), AOP-RANTES, NNY--RANTES and TAK-779; And the CCR5/CXCR4 antagonist, such as NSC 651016 (distamycin analogue).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 (two caffeoyl winestone (DFQA) acid); Skyrin (QLC) and relevant anthraquinone; ZINTEVIR ^TM(AR 177, may act on cell surface but not the oligonucleotide of real integrase inhibitor; Arondex); And naphthols, such as disclosed among the WO 98/50347.

Other antiretroviral agent comprises hydroxyl urea sample compound, such as BCX-34 (purine nucleoside phosphatase inhibitor; Biocryst); The ribonucleotide reductase inhibitor is such as DIDOX ^TM(Moleculesfor Health); Trophicardyl list phosphate dehydrogenase (IMPDH) inhibitor is such as VX-497 (Vertex); And mycophenolic acid (mycopholic acid), such as CellCept (mycophenolic acid ethyl ester; Roche).

Other antiretroviral agent comprises the inhibitor of viral integrase enzyme, inhibitor such as the arylene Ketene dimethyl of viral genome nuclear translocation (arylene bis (methylketone)) compound; The inhibitor that HIV invades is such as the soluble complex of AOP-RANTES, NNY-RANTES, RANTES-IgG fusion rotein, RANTES and glycosaminoglycan (GAG), and AMD-3100; Nucleocapsid zinc refers to inhibitor, such as the dithiane compound; The target of HIV Tat and Rev; And drug effect toughener (pharmacoenhancer), such as ABT-378.

Other antiretroviral therapy and adjuvant therapy comprise cytokine and lymphokine, such as MIP-1 α, MIP-1 β, SDF-1 α, IL-2, PROLEUKIN ^TM(rIL-2, aldesleukin/L2-7001; Chiron), IL-4, IL-10, IL-12 and IL-13; Interferon, rabbit is such as IFN-α 2a, IFN-α 2b or IFN-β; The antagonist of TNF, NF κ B, GM-CSF, M-CSF and IL-10; Regulate the medicament of immuno-stimulating, such as ciclosporin and prednisone; Vaccine is such as Remune ^TM(HIV Immunogen), APL 400-003 (Apollon), reorganization gp120 and fragment, divalence (B/E) recombinant envelope glycoprotein, rgp120CM235, MN rgp120, SF-2rgp120, gp120/ solubility CD4 mixture, Delta JR-FL albumen, derived from the synthetic peptide of the branch of discontinuous gp120C3/C4 structural domain, merge competent immunogen, and Gag, Pol, Nef and Tat vaccine; Based on the therapy of gene, such as gene inhibition construction element (GSE; WO 98/54366) and born of the same parents' intrinsic factor (intrakine) (through the CC chemokine target ER of genetic modification with the surface expression of blocking new synthetic CCR5 (people such as Yang, PNAS 94:11567-72,1997; People such as Chen, Nat.Med.3:1110-16,1997)); Antibody, such as anti-CXCR4 antibody 12G5, antibodies against CCR 5 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 stimulant and antagonist, such as TCDD, 3,3 ', 4,4 ', 5-pentachlorodiphenyl, 3,3 ', 4,4 '-tetrachloro biphenyl and α-naphthoflavene (WO98/30213); And antioxidant, such as gamma-L-glutamine-cysteine ethyl ester (γ-GCE; WO99/56764).

In another embodiment, albumin fusion proteins of the present invention and/or polynucleotide and one or more antiviral drugs are co-administered.Can include but not limited to acyclovir (acyclovir), ribavirin (ribavirin), ribavirin analogue, amantadine (amantadine), remantidine, maxamine or Thymosin-Alpha1 (thymalfasin) with the co-administered antiviral drug of albumin fusion proteins of the present invention and/or polynucleotide.Particularly, the Interferon, rabbit albumin fusion proteins can be co-administered with any of these medicament.In addition, the interferon alpha albumin fusion proteins also can be co-administered with any of these medicament, and preferably, Intederon Alpha-2a or 2b albumin fusion proteins can be co-administered with any of these medicament.In addition, the interferon beta albumin fusion proteins also can be co-administered with any of these medicament.In addition, any IFN hybrid albumin fusion proteins can be co-administered with any of these medicament.

In a most preferred embodiment, Interferon, rabbit albumin fusion proteins of the present invention and ribavirin or ribavirin analogue are co-administered.In a preferred embodiment, can include but not limited to co-administered ribavirin or the ribavirin analogue of Interferon, rabbit albumin fusion proteins

(Hoffman-La Roche, Nutley, N.J.),

(Schering Corp., Kenilworth, N.J.),

(Valeant, Costa Mesa, CA), RIBAVIN ^TM(Lupin, Baltimore, MD), RIBAZID ^TM(Epla, Kirachi, Pakistan), tribavirin, VIRAMIDINE ^TM(Valeant, Costa Mesa, CA), RIBASPHERE ^TM(Three RiversPharmaceuticals, Cranberry Township, PA) co-administered.In another preferred embodiment, interferon alpha albumin fusion proteins and ribavirin or ribavirin analogue are co-administered.In another preferred embodiment, interferon alpha 2a albumin fusion proteins and ribavirin or ribavirin analogue are co-administered.In another preferred embodiment, interferon alpha 2 b albumin fusion proteins and ribavirin or ribavirin analogue are co-administered.In another preferred embodiment, interferon beta albumin fusion proteins and ribavirin or ribavirin analogue are co-administered.In another preferred embodiment, hydridization Interferon, rabbit (hybrid interferon) albumin fusion proteins and ribavirin or ribavirin analogue are co-administered.

In another embodiment, albumin fusion proteins of the present invention and/or polynucleotide can be used separately, perhaps unite one or more antiviral agents, are used for the treatment of virus infection.In a preferred embodiment, Interferon, rabbit-albumin fusion proteins of the present invention can be united one or more antiviral agents and used.In another preferred embodiment, virus infection is derived from the infection of hepatitis virus.In a most preferred embodiment, hepatitis virus is hepatitis C virus (HCV).Can include but not limited to the micromolecular inhibitor of viral enzyme, micromolecular inhibitor, antiviral agent, antisense oligonucleotide inhibitor, thiazolides, novel immunoregulation agent and the interferon enhancer of RNA polymerase 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 VX-950 (proteinase inhibitor with the co-administered antiviral enzyme inhibitors of albumin fusion proteins of the present invention and/or polynucleotide, Vertex, Cambridge, MA), VX-497 (merimepodib, oral IMPDH inhibitor, Vertex, Cambridge, MA), BILB 1941 (proteinase inhibitor, Boehringer Ingelheim, Germany), SCH7 (proteinase inhibitor, Schering Corp., Kenilworth, N.J.), MX-3253 (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, proteinase inhibitor, InterMune, Brisbane, CA), Celgosivir (MBI-3253, alpha-glucosidase inhibitor, Migenix, Inc., Vancouver, B.C.), SCH503034 (proteinase inhibitor, Schering Corp., Kenilworth, N.J.), ACH 806 (GS9132, oral protein enzyme inhibitors, Achillion, New Haven, CT/Gilead Sciences, Foster City, CA).Can be nucleoside analog or non-nucleosidic inhibitors (NNI) with the co-administered antiviral AG14361 of albumin fusion proteins of the present invention and/or polynucleotide.In a preferred embodiment, antiviral protease inhibitors suppresses HCV RNA proteolytic enzyme.In one embodiment, antiviral protease inhibitors can be a nucleoside analog, and (Cambride is MA) with 2 '-C-methyl nucleoside for the oral prodrug of 23 '-C-methyl-cytidine, Idenix to include but not limited to NM283.In another embodiment, antiviral AG14361 can be a non-nucleosidic inhibitors, includes 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) and XTL-2125 (BC2125, XTLbio, NewYork, NY).Can include but not limited to antisense oligonucleotide, ribozyme and siRNA or short hairpin RNA (shRNA) with the co-administered antiviral medicament of albumin fusion proteins of the present invention and/or polynucleotide based on nucleic acid.Can comprise with the co-administered antiviral antisense oligonucleotide inhibitor of albumin fusion proteins of the present invention and/or polynucleotide but be not limited to

AVI-4065 (AVIBiopharma, Portland, OR).In another embodiment, thiazolide can be co-administered with albumin fusion proteins of the present invention and/or polynucleotide.In a preferred embodiment, can include but not limited to the co-administered thiazolides of albumin fusion proteins of the present invention and/or polynucleotide

(nitazoxanide, Romark Laboratories, L.C., Tampa, FL).Can include but not limited to the co-administered antiviral immunity adjusting control agent of albumin fusion proteins of the present invention and/or polynucleotide

(thymosin, thymalfasin, SciClone PharmaceuticalsInt ' l, Hong Kong) and toll sample acceptor (TLR) agonist, include but not limited to ANA245 (TLR-7 agonist, Anadys Pharmaceuticals, San Diego, CA), ANA975 (the oral prodrug of ANA245, Anadys Pharmaceuticals, San Diego, CA) and CPG-10101 (ACTILON ^TM, the TLR-9 agonist, Coley Pharmaceutical Group, Wellesley, MA).Can with the co-administered interferon enhancer of albumin fusion proteins of the present invention and/or polynucleotide include but not limited to EMZ702 (Transition Therapeutics, Toronto, Ontario).In addition, can include but not limited to the Tarvacin (Humanized monoclonal antibodies of phosphatidylserine on the target tumor endothelial cell surface with the co-administered antiviral antibody of albumin fusion proteins of the present invention and/or polynucleotide, Peregrine Pharmaceuticals, Inc., Tustin, CA).

In a preferred embodiment, the present invention contain, can be separately or to unite the albumin fusion proteins that one or more antiviral agents use be Interferon, rabbit-albumin fusion proteins.In another embodiment, the Interferon, rabbit of Interferon, rabbit-albumin fusion proteins partly is an interferon alpha.The non-limitative example of the interferon alpha that the present invention is contained includes but not limited to the interferon alpha albumen disclosed in table 1 therapeutic protein one hurdle.In specific embodiment, any commercial form that interferon alpha partly comprises Intederon Alpha-2a, Interferon Alpha-2b, interferon c, total Interferon, rabbit (consensus interferon), Interferon, rabbit alfacon-1, interferon alfa-n1, Alferon N, interferon alpha is such as for example

ALPHA (Schering Corp., Kenilworth, N.J.),

ALPHA (Hoffman-LaRoche, Nutley, N.J.), 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 purifying or its fragment, perhaps is made up of it.In further embodiment, the interferon alpha of IFN-α-HSA fusion rotein partly is included as release prolongation or controlled and the interferon alpha modifying or prepare, perhaps is made up of it.For example, interferon alpha partly comprises commercially available prolongation release or controlled release interferon alpha or is made up of it, include but not limited to interferon-' alpha '-XL (Flamel Technologies, France) and LOCTERON ^TM(BioLex Therapeutics/OctoPlus, Pittsboro, NC).In another embodiment, the interferon alpha of IFN-α-HSA fusion rotein part can be modified by adhering to chemical module.For example, the interferon alpha part can be modified by PEGization.Accordingly, in other embodiment, the IFN-α-interferon alpha of HSA fusion rotein partly comprises Intederon Alpha-2a, 2b or the total Interferon, rabbit of PEGization form or is made up of it, includes but not limited to commercially available PEGization interferon alpha, such as for example PEG-

(Schering Corp., Kenilworth, N.J.),

(Hoffman-La Roche, Nutley, N.J.), PEG-OMNIFERON ^TM(Viragen, Inc., Plantation, FL) or its fragment.In another preferred embodiment, the Interferon, rabbit of albumin fusion proteins partly is interferon alpha 2a or 2b Interferon, rabbit.The Interferon, rabbit albumin fusion proteins can be united any of these medicament and be used.In addition, in another embodiment, the Interferon, rabbit of Interferon, rabbit-albumin fusion proteins partly is interferon beta or Interferon, rabbit hybrid.In further embodiment, the antiviral agent that one or more the present invention are contained can be used or unite to the not fused interferon part of Interferon, rabbit-albumin fusion proteins separately.

In other embodiments, albumin fusion proteins of the present invention and/or polynucleotide can be co-administered with anti-opportunistic infection medicine.Can include but not limited to TRIMETHOPRIM-SULFAMETHOXAZOLE with the co-administered anti-chance medicine of albumin fusion proteins of the present invention and/or polynucleotide ^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, filgrastim/G-CSF) and LEUKINE ^TM(Sargramostim, sargramostim/GM-CSF).In a specific embodiment, albumin fusion proteins of the present invention and/or polynucleotide and TRIMETHOPRIM-SULFAMETHOXAZOLE ^TM, DAPSONE ^TM, PENTAMIDINE ^TM, and/or ATOVAQUONE ^TMBe used for the treatment of or prevent opportunistic Pneumocystis carinii (Pneumocystis carinii) pneumonia so that arbitrary combination is preventative.In another specific embodiment, albumin fusion proteins of the present invention and/or polynucleotide and ISONIAZID ^TM, RIFAMPIN ^TM, PYRAZINAMIDE ^TM, and/or ETHAMBUTOL ^TMBe used for the treatment of or prevent opportunistic mycobacterium avium (Mycobacterium avium) multiplicity of infection so that arbitrary combination is preventative.In another specific embodiment, albumin fusion proteins of the present invention and/or polynucleotide and RIFABUTIN ^TM, CLARITHROMYCIN ^TM, and/or AZITHROMYCIN ^TMBe used for the treatment of or prevent opportunistic mycobacterium tuberculosis (Mycobacteriumtuberculosis) to infect so that arbitrary combination is preventative.In another specific embodiment, albumin fusion proteins of the present invention and/or polynucleotide and GANCICLOVIR ^TM, FOSCARNET ^TM, and/or CIDOFOVIR ^TMBe used for the treatment of or prevent opportunistic cytomegalovirus (cytomegalovirus) to infect so that arbitrary combination is preventative.In another specific embodiment, albumin fusion proteins of the present invention and/or polynucleotide and FLUCONAZOLE ^TM, ITRACONAZOLE ^TM, and/or KETOCONAZOLE ^TMBe used for the treatment of or prevent opportunistic fungal infection so that arbitrary combination is preventative.In another specific embodiment, albumin fusion proteins of the present invention and/or polynucleotide and ACYCLOVIR ^TMAnd/or FAMCICOLVIR ^TMBe used for the treatment of or prevent opportunistic I type and/or II herpes simplex virus type to infect so that arbitrary combination is preventative.In another specific embodiment, albumin fusion proteins of the present invention and/or polynucleotide and PYRIMETHAMINE ^TMAnd/or LEUCOVORIN ^TMBe used for the treatment of or prevent opportunistic mouse toxoplasma gondii (Toxoplasma gondii) to infect so that arbitrary combination is preventative.In another specific embodiment, albumin fusion proteins of the present invention and/or polynucleotide and LEUCOVORIN ^TMAnd/or NEUPOGEN ^TMBe used for the treatment of or prevent the opportunistic infectation of bacteria so that arbitrary combination is preventative.

In another embodiment, albumin fusion proteins of the present invention and/or polynucleotide and antibiotics are co-administered.Can include but not limited to the amoxycilline Trihydrate bp with the co-administered antibiotics of albumin fusion proteins of the present invention and/or polynucleotide, β-Nei Xiananmei, aminoglycoside, beta-lactam (glycopeptide), β-Nei Xiananmei, clindamycin, paraxin, cynnematin, Ciprofloxacin, erythromycin, fluoroquinolone, macrolide, metronidazole, penicillin, quinolone, rapamycin, Rifampin, Streptomycin sulphate, sulfanilamide (SN), tsiklomitsin, trimethoprim, trimethoprim-sulfamethoxazole, and vancomycin.

In other embodiments, albumin fusion proteins of the present invention and/or polynucleotide and immunologic stimulant are co-administered.Can include but not limited to LEVAMISOLE HCL (ERGAMISOL for example with the co-administered immunologic stimulant of albumin fusion proteins of the present invention and/or polynucleotide ^TM), different third creatinine (INOSIPLEX for example ^TM), Interferon, rabbit (for example interferon alpha) and interleukin (for example IL-2).

In other embodiments, albumin fusion proteins of the present invention and/or polynucleotide and immunosuppressor are co-administered.Can include but not limited to steroid, S-Neoral, cyclosporin analog, endoxan methyl prednisone, prednisone, imuran, FK-506,15-Gusperimus and other immunosuppressor that works by the function that suppresses responsiveness T cell with the co-administered immunosuppressor of albumin fusion proteins of the present invention and/or polynucleotide.Can include but not limited to Ultracortene-H, methotrexate, thalidomide (thalidomide), Methoxsalen (methoxsalen), rapamycin, leflunomide (leflunomide), mizoribine (mizoribine) (BREDININ with co-administered other immunosuppressor of albumin fusion proteins of the present invention and/or polynucleotide ^TM), brequinar (brequinar), Gusperimus and aza spiro alkane (azaspirane) (SKF 105685), ORTHOCLONE

3 (muromonab-CD3), SANDIMMUNE ^TM, NEORAL ^TM, SANGDYA ^TM(S-Neoral),

(FK506, tacrolimus),

(mycophenolic acid ethyl ester, active metabolite wherein is a mycophenolic acid), IMURAN ^TM(imuran), glucocorticosteroid, adrenocortical steroid are such as DELTASONE ^TM(prednisone) and HYDELTRASOL ^TM(Ultracortene-H), FOLEX ^TMAnd MEXATE ^TM(methotrexate), OXSORALEN-ULTRA ^TM(Methoxsalen) and RAPAMUNE ^TM(sirolimus).In a specific embodiment, immunosuppressor can be used for preventing the transplant rejection of organ or marrow.

In another embodiment, albumin fusion proteins of the present invention and/or polynucleotide are used separately or are co-administered with one or more intravenously immunoglobulin products.Can include but not limited to GAMMAR with the co-administered intravenously immunoglobulin product of albumin fusion proteins of the present invention and/or polynucleotide ^TM, IVEEGAM ^TM, SANDOGLOBULIN ^TM, GAMMAGARD S/D ^TM, ATGAM ^TM(antithymocyte globulin) and GAMIMUNE ^TMIn a specific embodiment, albumin fusion proteins of the present invention and/or polynucleotide and intravenously immunoglobulin product are co-administered in transplantation therapy (for example bone marrow transplantation).

In another embodiment, albumin fusion proteins of the present invention and/or polynucleotide are used separately or are used as the part of combination treatment, or are applied to the patient in vivo or are applied to cell external, with the treatment cancer.In a specific embodiment, albumin fusion proteins, particularly the IL-2-albumin fusion proteins is used during the passive immunotherapy of cancer repeatedly, the sexual cell transfer therapy of adopting such as metastatic melanoma, as people such as Dudley (Science Express, on September 19th, 2002, www.scienceexpress.org, complete being incorporated herein by reference) described.

In certain embodiments, albumin fusion proteins of the present invention and/or polynucleotide are used separately or are co-administered with antiphlogiston.Can include but not limited to reflunomide (Betamethasone Valerate for example with the co-administered antiphlogiston of albumin fusion proteins of the present invention and/or polynucleotide, budesonide, cortisone, dexamethasone, hydrocortisone, methyl meticortelone, Ultracortene-H, prednisone, and triamcinolone), non-steroid antiphlogiston (diclofenac for example, diflunisal, R-ETODOLAC, fenoprofen, floctafenine, flurbiprofen, Ibuprofen BP/EP, indomethacin, Ketoprofen, the meclofenamic acid ester, mefenamic acid, meloxicam, nabumetone, Naproxen Base, Taisho), Phenylbutazone (BUTE), piroxicam, sulindac, tenoxicam, tiaprofenic acid, and antihistaminic and tolmetin),, the aminoaryl carboxylic acid derivative, the Arylacetic acids derivative, arylbutyric acid derivatives, aryl carboxylic acid, aryl propionic acid derivatives, pyrazoles, pyrazolone, salicyclic acid derivatives, thiazine methane amide (thiazinecarboxamide), e-kharophen caproic acid (acetamidocaproic acid), S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, Bendazac, benzydamine, bucolome, Z-876, ditazole, nron, Guaiazulene, nabumetone, nimesulide, Proteins, orgoteins, oxaceprol, Renytoline, perisoxal, pifoxime, general Lu Kuizong, proxazole, and tenidap.

In another embodiment, composition of the present invention is used separately or is co-administered with the angiogenesis inhibitor medicine.Can include but not limited to angiostatin (Entremed with the co-administered angiogenesis inhibitor medicine of composition of the present invention, Rockville, MD), troponin-1 (Boston Life Sciences, Boston, MA), the anti-invasion factor, vitamin A acid and derivative thereof, taxol (Taxol), Suramine, metalloprotease-1 organize inhibition, metalloprotease-2 to organize inhibition, VEGI, plasminogen activator inhibitor-1, plasminogen activator inhibitor-2 and various forms of light " d group " transition metal.

Light " d group " transition metal comprises for example vanadium, molybdenum, tungsten, titanium, niobium and tantalum class.These transition metal-types can form transition metal composite.The suitable complexes of above-mentioned transition metal-type comprises oxo transition metal composite.

The representative example of vanadium mixture comprises oxo vanadium mixture, such as vanadate and vanadyl mixture.Suitable vanadate mixture comprises metavanadate and ortho-vanadate mixture, such as for example ammonium meta-vanadate, sodium metavanadate and sodium orthovanadate.Suitable vanadyl mixture comprises for example methyl ethyl diketone vanadyl and vanadylic sulfate, comprises the vanadylic sulfate hydrate, as one and three hydration vanadylic sulfates.

The representative example of tungsten and molybdenum mixture also comprises oxo mixture.Suitable oxo tungsten mixture comprises tungstate and Tungsten oxide 99.999 mixture.Suitable tungstate mixture comprises ammonium tungstate, calcium wolframate, Disodium tungstate (Na2WO4) dihydrate and wolframic acid.Suitable Tungsten oxide 99.999 comprises Tungsten oxide 99.999 (IV) and Tungsten oxide 99.999 (VI).Suitable oxo molybdenum mixture comprises molybdate, molybdenum oxide and molybdenyl mixture.Suitable molybdate mixture comprises ammonium molybdate and hydrate, Sodium orthomolybdate and hydrate thereof and potassium molybdate and hydrate thereof.Suitable molybdenum oxide comprises molybdenum oxide (VI), molybdenum oxide (VI) and molybdic acid.Suitable molybdenyl mixture comprises for example methyl ethyl diketone oxygen molybdenum.Tungsten that other is suitable and molybdenum mixture comprise derived from for example hydroxyl derivative of glycerine, tartrate and sugar.

Extremely multiple other angiogenesis inhibitor factor also can be used for content of the present invention.Representative example includes but not limited to platelet factor 4; Protamine sulfate; Sulfation chitin derivative (making) (people such as Murata, Cancer Res.51:22-26,1991) by the queen crab shell; Sulfation glycan peptidoglycan mixture (SP-PG) (existence of steroid such as oestrogenic hormon and citric acid tamoxifen can strengthen this compound functions); Staurosporine; The matrix metabolism conditioning agent comprises for example proline analogs, cis oxyproline, d, L-3,4-dehydroproline, Thiaproline, α, α-two pyridine, fumaric acid aminopropionitrile; 4-propyl group-5-(4-pyridyl)-2 (3H)-azolactones; Methotrexate; Mitoxantrone; Heparin; Interferon, rabbit; 2 macroglobulin-serum; ChIMP-3 (people such as Pavloff, J.Bio.Chem.267:17321-17326,1992); Chymotrypsin inhibitor (people such as Tomkinson, Biochem J.286:475-480,1992); Tetradecyl sulfuric acid cyclodextrin; Eponemycin; Camptothecine; Fumidil (people such as Ingber, Nature 348:555-557,1990); Disodium aurothiomalate (" GST "; Matsubara and Ziff, J.Clin.Invest.79:1440-1446,1987); Anticollagenase-serum; α 2-antiplasmin (people such as Holmes, J.Biol.Chem.262 (4): 1659-1664,1987); Bisantrene (NationalCancer Institute); Lobenzarit Disodium (N-(2)-carboxyphenyl-4-chrloroanthracene fennel acid (anthronilic acid) disodium or " CCA "; People such as Takeuchi, Agents Actions 36:312-316,1992); And inhibitors of metalloproteinase, such as BB94.

Other angiogenesis inhibitor factor that can be used for content of the present invention comprise Thalidomide (Celgene, Warren, NJ); The steroid that suppresses blood vessel; AGM-1470 (H.Brem and J.Folkman, J.Pediatr.Surg.28:445-51,1993); Integrin alpha v beta 3 antagonist (people such as C.Storgard, J.Clin.Invest.103:47-54,1999); The carboxyamino imidazoles; NSC 609974 (CAI) (National CancerInstitute, B ethesda, MD); Conbretastatin A-4 (CA4P) (OXiGENE, B oston, MA); Squalamine (Magainin Pharmaceuticals, Plymouth Meeting, PA); TNP-470 (Tap Pharmaceuticals, Deerfield, IL); ZD-0101AstraZeneca (London, UK); APRA (CT2584); Benefin, Byrostatin-1 (SC339555); CGP-41251 (PKC 412); CM101; Dexrazoxane (ICRF187); DMXAA; Endostatin; Flavopridiol; Genestein; GTE; ImmTher; Iressa (ZD1839); Sostatin (Somat); Panretin; Penacillamine; Photopoint; PI-88; Prinomastat (AG-3340); Purlytin; Suradista (FCE26644); Tamoxifen (Nolvadex); Tazarotene; Tetrathiomolybdate; Xeloda (capecitabine); And 5 FU 5 fluorouracil.

Can work by number of mechanisms with the co-administered angiogenesis inhibitor medicine of composition of the present invention, include but not limited to suppress proteolysis, the blocking-up endotheliocyte-extracellular matrix adhesion molecule of extracellular matrix function, antagonizing vessel generation inductor such as somatomedin function and be suppressed at the integrin receptor of expressing on the propagation endotheliocyte.Disturb the extracellular matrix protein hydrolysis and can include but not limited to AG-3340 (Agouron with the example of the co-administered angiogenesis inhibitor inhibitor of composition of the present invention, La Jolla, CA), BAY-12-9566 (Bayer, West Haven, CT), BMS-275291 (Bristol MyersSquibb, Princeton, NJ), CGS-27032A (Novartis, East Hanover, NJ), Marimastat (British Biotech, Oxford, UK) and Mei Tasita (Metastat) (Aetema, St-Foy, Quebec).Function by blocking-up endotheliocyte-extracellular matrix adhesion molecule works and can include but not limited to EMD-121974 (Merck KcgaA Darmstadt with the example of the co-administered angiogenesis inhibitor inhibitor of composition of the present invention, Germany) and Vitaxin (Ixsys, La Jolla, CA/Medimmune, Gaithersburg, MD).By direct antagonism or suppress that blood vessel generation inductor works and can include but not limited to Angiozyme (Ribozyme with the example of the co-administered antiangiogenic agent of composition of the present invention, Boulder, CO), VEGF antibody (Genentech, S.San Francisco, CA), PTK-787/ZK-225846 (Novartis, Basel, Switzerland), SU-101 (Sugen, S.San Francisco, CA), SU-5416 (Sugen/Pharmacia Upjohn, Bridgewater, NJ) and SU-6668 (Sugen).Other antiangiogenic agent takes place to work by indirect inhibition blood vessel.Can include but not limited to IM-862 (Cytran with the example of the co-administered blood vessel generation indirect inhibitor of composition of the present invention, Kirkland, WA), interferon alpha, IL-12 (Roche, Nutley, NJ) and many sulfuric acid piperylene (Georgetown University, Washington, DC).

In specific embodiment, imagination is used for treating, prevents by making uniting of composition of the present invention and antiangiogenic agent and/or improves autoimmune disease, such as for example autoimmune disease described herein.

In specific embodiment, imagination is used for treating, prevent and/or improving sacroiliitis by making uniting of composition of the present invention and antiangiogenic agent.In a more particular embodiment, imagination is used for treating, prevent and/or improving rheumatoid arthritis by making uniting of composition of the present invention and antiangiogenic agent.

In another embodiment, the polynucleotide of code book invention polypeptide and blood vessel generation protein or the proteinic polynucleotide of coding blood vessel generation are co-administered.Can include but not limited to acidity and Prostatropin with the co-administered proteinic example of blood vessel generation of composition of the present invention, VEGF-1, VEGF-2, VEGF-3, Urogastron α and β, platelet-derived endothelial cell growth factor (ECGF), Thr6 PDGF BB, tumor necrosis factor alpha, pHGF, rhIGF-1, G CFS, macrophage colony stimulating factor, granulocyte/macrophage colony stimulating factor, and nitricoxide synthase.

In other embodiments, composition of the present invention and chemotherapeutics are co-administered.Can include but not limited to alkylating agent with the co-administered chemotherapeutics of albumin fusion proteins of the present invention and/or polynucleotide, such as mustargen (chlormethine for example, endoxan, the endoxan ifosfamide, melphalan (Phenylalanin-Lost), and Chlorambucil), ethyleneimine and methylmelamine (for example altretamine and thio-tepa), alkylsulfonate (for example busulfan), nitrosourea (carmustine (BCNU) for example, lomustine (CCNU), semustine (Semustine), and streptozocin (streptozotocin)), triazene (Dacarbazine (DTIC for example; Dacarbazine)) folacin (for example methotrexate (methotrexate)), pyrimidine analogue (Fluracil (5 FU 5 fluorouracil for example; 5-FU), floxuridine (fluorodeoxyuridine; FudR), and cytosine arabinoside (cytarabin)), purine analogue and relevant inhibitor (mercaptopurine (Ismipur for example; 6-MP), Tioguanine (6-Tioguanine; TG), and pentostatin (2 '-deoxycoformycin)), vinca alkaloids (for example vinealeucoblastine(VLB) (VLB, Vinblastine sulphate)) and vincristine(VCR) (vincristine sulphate)), epipodophyllotoxin (for example Etoposide and teniposide), microbiotic (for example gengshengmeisu (dactinomycin), daunoblastin (daunomycin; Daunorubicin), Zorubicin, bleomycin, Plicamycin (mithramycin), and mitomycin (ametycin), enzyme (for example altheine enzyme), biological response modifier (for example interferon-' alpha ' and interferon-' alpha '-2b), iridium-platinum complex (for example cis-platinum (cis-DDP) and carboplatin), amerantrone (anthracenedione) (mitoxantrone), replace urea (for example hydroxyl urea), methylhydrazine derivative (Procarbazine (N-methylhydrazine for example; MIH)), adrenocortical steroid (for example prednisone), progesterone (caproic acid hydroxyl progestogen for example, medroxyprogesterone, medroxyprogesterone acetate, and Magace), oestrogenic hormon (stilboestrol (DES) for example, stilphostrol, estradiol, and Ethinylestradiol), estrogen antagonist (for example tamoxifen), male sex hormone (testosterone propionate and Fluoxymesterone), androgen antagonist (for example flutamide), gonadotropin releasing hormone analogues (for example Leuprolide), other hormone and hormone analogs (Synrotabs for example, estramustine, estramustine phosphate sodium, Chlortrianisoestrol, and testolactone), and other medicament (Dacarbazine for example, L-glutamic acid, and mitotane).

In one embodiment, composition of the present invention and one or more following medication combined using: 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 Rousse1), Kineret ^TM(the IL-1 receptor antagonist is also referred to as Anakinra, from Amgen, Inc.).

In a specific embodiment, composition of the present invention and CHOP (endoxan, Zorubicin, vincristine(VCR) and prednisone) or use with the combinatorial association of one or more compositions of CHOP.In one embodiment, composition of the present invention and anti-CD20 antibodies, human monoclonal anti-CD20 antibodies are co-administered.In another embodiment, particularly the arbitrary combination of endoxan and/or prednisone is co-administered for one or more compositions of composition of the present invention and anti-CD20 antibodies and CHOP or anti-CD20 antibodies and CHOP.In a specific embodiment, composition of the present invention and Rituximab are co-administered.In another embodiment, composition of the present invention and Rituximab and CHOP, or with Rituximab and one or more compositions of CHOP particularly the arbitrary combination of endoxan and/or prednisone use.In another embodiment, composition of the present invention with tositumomab and CHOP or tositumomab and one or more compositions of CHOP particularly the arbitrary combination of endoxan and/or prednisone use.Anti-CD20 antibodies can be chosen wantonly with the former medicine of radio isotope, toxin or cellular toxicity and link to each other.

In another specific embodiment, composition of the present invention and Zevalin ^TMCo-administered.In another embodiment, composition of the present invention and Zevalin ^TMWith CHOP or Zevalin ^TMWith one or more compositions of CHOP particularly the arbitrary combination of endoxan and/or prednisone use together.Zevalin ^TMCan link to each other with one or more radio isotope.Particularly preferred isotropic substance is ⁹⁰Y and ¹¹¹In.

In another embodiment, albumin fusion proteins of the present invention and/or polynucleotide and cytokine are co-administered.Can include but not limited to IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, ILI5, anti-CD40, CD40L, IFN-γ and TNF-α with the co-administered cytokine of albumin fusion proteins of the present invention and/or polynucleotide.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, IL4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, L-17, IL-18, IL-19, IL-20 and IL-21.

In one embodiment, albumin fusion proteins of the present invention and/or polynucleotide and TNF family member are co-administered.Can with albumin fusion proteins of the present invention and/or the co-administered TNF of polynucleotide, TNF associated molecule or TNF sample molecule include but not limited to the TNF-α of soluble form, lymphotoxin-α (LT-α is also referred to as TNF-β), LT-β (being found in compound allos trimer LT-α 2-β), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-γ (international publication number WO 96/14328), AIM-I (international publication number WO 97/33899), endokine-α (international publication number WO 98/07880), OPG, with neutrokine-α (international publication number WO98/18921), OX40, and nerve growth factor (NGF), Fas with soluble form, CD30, CD27, CD40 and 4-IBB, TR2 (international publication number WO 96/34095), DR3 (international publication number WO 97/33904), DR4 (international publication number WO 98/32856), TR5 (international publication number WO98/30693), TRANK, TR9 (international publication number WO 98/56892), TR10 (international publication number WO 98/54202), 312C2 (international publication number WO 98/06842), and TR12, and the CD154 of soluble form, CD70, and CD153.

In another embodiment, albumin fusion proteins of the present invention and/or polynucleotide and blood vessel generation protein are co-administered.Can include but not limited to neuroglia derivative growth factor (GDGF) with the co-administered blood vessel generation protein of albumin fusion proteins of the present invention and/or polynucleotide, be disclosed in european patent number EP-399816; Thr6 PDGF BB (PDGF-A) is disclosed in european patent number EP-682110; Thr6 PDGF BB-B (PDGF-B) is disclosed in european patent number EP-282317; Placenta growth factor (PIGF) is disclosed in international publication number WO 92/06194; Placenta growth factor-2 (PIGF-2) is disclosed in people such as Hauser, Growth Factors 4:259-268,1993); Vascular endothelial growth factor (VEGF) is disclosed in international publication number WO 90/13649; Vascular endothelial growth factor-A (VEGF-A) is disclosed in european patent number EP-506477; Vascular endothelial growth factor-2 (VEGF-2) is disclosed in international publication number WO 96/39515; Vascular endothelial growth factor B (VEGF-3); Vascular endothelial growth factor B-186 (VEGF-B186) is disclosed in international publication number WO 96/26736; Vascular endothelial growth factor-D (VEGF-D) is disclosed in international publication number WO98/02543; Vascular endothelial growth factor-D (VEGF-D) is disclosed in international publication number WO98/07832; And vascular endothelial growth factor-E (VEGF-E), be disclosed in German patent DE19639601.Be incorporated herein by reference above-mentioned reference is complete.

In another embodiment, albumin fusion proteins of the present invention and/or polynucleotide and fibroblast growth factor are co-administered.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 co-administered fibroblast growth factor of albumin fusion proteins of the present invention and/or polynucleotide.

In another embodiment, albumin fusion proteins of the present invention and/or polynucleotide and hemopoieticgrowth factor are co-administered.Can include but not limited to rHuGM-CSF (GM-CSF) (Sargramostim, sargramostim, LEUKINE with the co-administered hemopoieticgrowth factor of albumin fusion proteins of the present invention and/or polynucleotide ^TM, PROKINE ^TM), granulocyte colony-stimulating factor (G-CSF) (filgrastim, filgrastim, NEUPOGEN ^TM), macrophage colony stimulating factor (M-CSF, CSF-1), erythropoietin (Epoetin Alfa, epoetin alfa, EPOGEN ^TM, PROSCRIT ^TM), STEM CELL FACTOR (SCF, c-kit part, steel factor (steel factor)), megakaryocyte colony stimulating factor, PIXY321 (GMCSF/IL-3 fusion rotein), interleukin any one or multiple, interferon-gamma or thrombopoietin of IL-1 to IL-12 especially.

In certain embodiments, albumin fusion proteins of the present invention and/or polynucleotide and adrenergic blocking drug are co-administered, such as for example acebutolol (acebutolol), atenolol USP 23 (atenolol), betaxolol (betaxolol), bisoprolol (bisoprolol), carteolol (carteolol), Trate (labetalol), metoprolol (metoprolol), nadolol (nadolol), oxprenolol (oxprenolol), penbutolol (penbutolol), pindolol (pindolol), Proprasylyte (propranolol), sotalol (sotalol) and timolol (timolol).

In another embodiment, the co-administered (adenosine for example of albumin fusion proteins of the present invention and/or polynucleotide and antiarrhythmics, amidoarone, bromine Bian ammonium (bretylium), purple foxglove (digitalis), digoxin (digoxin), digoxigenin (digitoxin), diliazem, norpace (disopyramide), esmolol (esmolol), flecainide (flecainide), lignocaine (lidocaine), mexiletine (mexiletine), Moracizine (moricizine), Phenytoin Sodium Salt (phenytoin), pronestyl (procainamide), N-acetyl pronestyl, Propafenone (propafenone), Proprasylyte, Quinidine (quinidine), sotalol, tocainide (tocainide), and verapamil (verapamil)).

In another embodiment, albumin fusion proteins of the present invention and/or polynucleotide and diuretic(s) are co-administered, such as carbonic anhydrase inhibitor (for example acetazolamide, daranide (dichlorphenamide) and methazolamide), osmotic diuretic (for example glycerine, Isosorbide, N.F,USP MANNITOL and urea), inhibition Na ⁺-K ⁺-2Cl ^-The diuretic(s) of symport (Furosemide for example, bumetanide, azosemide, piretanide, tripamide, Ethacrynic Acid, Muzolimine, and torsemide), thiazine and thiazine sample diuretic(s) (Hydrex for example, benzthiazide, chlorothiazide, hydrochlorothiazide, Hydroflumethiazide, Methyclothiazide, many thiazines, trichlormethiazide, chlorthalidone, indapamide, metolazone, and quinethazone), Potassium-sparing diuretic (for example guanamprazine and triamterene), with mineralocorticoid receptor antagonist (spironolactone for example, canrenone, and potassium canrenoate).

In one embodiment, the treatment of albumin fusion proteins of the present invention and/or polynucleotide and internal secretion and/or hormone imbalances disorder is co-administered.The treatment of internal secretion and/or hormone imbalances disorder includes but not limited to ¹²⁷The radio isotope of I, iodine such as ¹³¹I and ¹²³I; Recombinant human growth hormone is such as HUMATROPE ^TM(reorganization somatotropin); Growth hormone analogs is such as PROTROPIN ^TM(somatrem); Dopamine agonist is such as PARLODEL ^TM(bromocriptine); The Somat analogue is such as SANDOSTATIN ^TM(Sostatin); The gonad-stimulating hormone goods are such as PREGNYL ^TM, A.P.L. ^TMAnd PROFASI ^TM(chorionic-gonadotropin hormone (CG)), PERGONAL ^TM(Menotrophins) and METRODIN ^TM(Urofollitropin (uFSH)); Synthetic human gonadotropin's releasing hormone goods are such as FACTREL ^TMAnd LUTREPULSE ^TM(Factrel); Synthetic gonad-stimulating hormone agonist is such as LUPRON ^TM(leuprorelin acetate), SUPPRELIN ^TM(histrelin acetate), SYNAREL ^TM(Synarela) and ZOLADEX ^TM(goserelin acetate); The throtropin releasing hormone synthetic products is such as RELEFACT TRH ^TMAnd THYPINONE ^TM(Protirelin); Recombinant human TSH is such as THYROGEN ^TMTriiodothyronine natural isomer sodium salt synthetic products is such as L-T ₄ ^TM, SYNTHROID ^TMAnd LEVOTHROID ^TM(levothyroxine sodium, levothyroxine), L-T ₃ ^TM, CYTOMEL ^TMAnd TRIOSTAT ^TM(Cynomel, liothyroine sodium), and THYROLAR ^TM(liotrix, liotrix); The antithyroid compound is dredged basic imidazoles and TAPAZOLE such as 6-n-propylthiouracil (propylthiouracil), 1-methyl-2- ^TM(methimazole), NEO-MERCAZOLE ^TM(carbimazole); The B-adrenergic receptor antagonist is such as Proprasylyte (Propranolol) and esmolol (esmolol); Ca ²⁺Channel blocker; Dexamethasone and iodate radiology contrast medium are such as TELEPAQUE ^TM(iopanoic acid) and ORAGRAFIN ^TM(sodium lopodate).

Other treatment of internal secretion and/or hormone imbalances disorder includes but not limited to oestrogenic hormon or puts together oestrogenic hormon, such as ESTRACE ^TM(estradiol), ESTINYL ^TM(Ethinylestradiol), PREMARIN ^TM, ESTRATAB ^TM, ORTHO-ES ^TM, OGEN ^TMAnd estropipate (oestrone), ESTROVIS ^TM(quinestrol), ESTRADERM ^TM(estradiol), DELESTROGEN ^TMAnd VALERGEN ^TM(Estradiol Valerate), DEPO-ESTRADIOL CYPIONATE ^TMWith ESTROJECT LA ^TM(estradiol cypionate); Estrogen antagonist is such as NOLVADEX ^TM(tamoxifen), SEROPHENE ^TMAnd CLOMID ^TM(Chloramiphene); Progesterone is such as DURALUTIN ^TM(caproic acid hydroxyprogesterone), MPA ^TMAnd DEPO-PROVERA ^TM(depomedroxy progesterone acetate), PROVERA ^TMAnd CYCRIN ^TM(MPA), MEGACE ^TM(Magace), NORLUTIN ^TM(Norethisterone), and NORLUTATE ^TMAnd AYGESTIN ^TM(Norethisterone Acetate); The progesterone implant is such as NORPLANT SYSTEM ^TM(hypodermic implant of norgestrel); Mifepristone is such as RU 486 ^TM(mifepristone); Hormonal contraceptive is such as ENOVID ^TM(norethynodrel adds mestranol), PROGESTASERT ^TM(discharging the intra-uterine contraceptive device of progesterone), LOESTRIN ^TM, BREVICON ^TM, MODICON ^TM, GENORA ^TM, NELONA ^TM, NORINYL ^TM, OVACON-35 ^TMAnd OVACON-50 ^TM(Ethinylestradiol/Norethisterone), LEVLEN ^TM, NORDETTE ^TM, TRI-LEVLEN ^TMAnd TRIPHSAIL-21 ^TM(Ethinylestradiol/Levonorgestrel), LO/OVRAL ^TMAnd OVRAL ^TM(Ethinylestradiol/norgestrel), DEMULEN ^TM(Ethinylestradiol/ethynodiol diacetate), NORINYL ^TM, ORTHO-NOVUM ^TM, NORETHIN ^TM, GENORA ^TM, and NELOVA ^TM(Norethisterone/mestranol), DESOGEN ^TMAnd ORTHO-CEPT ^TM(Ethinylestradiol/desogestrel), ORTHO-CYCLEN ^TMAnd ORTHO-TRICYCLEN ^TM(Ethinylestradiol/norgestimate), MICRONOR ^TMAnd NOR-QD ^TM(Norethisterone) and OVRETTE ^TM(norgestrel).

Other treatment of internal secretion and/or hormone imbalances disorder includes but not limited to the testosterone ester, such as metenolone acetate and testosterone undecanoate; Parenteral and oral androgenic are such as TESTOJECT-50 ^TM(testosterone), TESTEX ^TM(testosterone propionate), DELATESTRYL ^TM(Testoviron-Depot), DEPO-TESTOSTERONE ^TM(depo-testosterone), DAQNOCRINE ^TM(danazol), HALOTETIN ^TM(Fluoxymesterone), ORETON METHYL ^TM, TESTRED ^TMAnd VIRILON ^TM(Synrotabs) and OXANDRIN ^TM(Oxrolone); The transdermal testosterone system is such as TESTODERM ^TMAndrogen receptor antagonists and 5-alpha-reductase inhibitors are such as ANDROCUR ^TM(cyproterone acetate), EULEXIN ^TM(flutamide) and PROSCAR ^TM(finasteride); The thyroliberin goods are such as CORTROSYN ^TM(cosyntropin); Adrenocortical steroid and synthetic analogues thereof are such as ACLOVATE ^TM(Sch-22219), CYCLOCORT ^TM(amcinonide), BECLOVENT ^TMAnd VANCERIL ^TM(Viarox), CELESTONE ^TM(Betamethasone Valerate), BENISONE ^TMAnd UTICORT ^TM(W-5975), DDIPROSONE ^TM(Sch-11460), CELESTONEPHOSPHATE ^TM(betamethasone sodium phosphate), CELESTONE SOLUSPAN ^TM(Betamethasone Valerate phosphoric acid and sodium-acetate), BETA-VAL ^TMAnd VALISONE ^TM(Valisone), TEMOVATE ^TM(clobetasol propionate), CLODERM ^TM(trimethylacetic acid clocortolone), CORTEF ^TMAnd HYDROCORTONE ^TM(hydrocortisone (hydrocortisone)), HYDROCORTONE ACETATE ^TM(acetic acid hydrocortisone (hydrocortisone)), LOCOID ^TM(butyric 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(cortisone acetate), DESOWEN ^TMAnd TRIDESILON ^TM(Hydroxyprednisolone Acetonide), TOPICORT ^TM(desoximetasone), DECADRON ^TM(dexamethasone), DECADRON LA ^TM(dexamethasone acetate), DECADRON PHOSPHATE ^TMWith HEXADROL PHOSPHATE ^TM(dexamethasone sodium phosphate), FLORONE ^TMAnd MAXIFLOR ^TM(oxalic acid diflorasone), FLORINEF ACETATE ^TM(fludrocortisone acetate), AEROBID ^TMAnd NASALIDE ^TM(flunisolide), FLUONID ^TMAnd SYNALAR ^TM(fluocinolone acetonide), LIDEX ^TM(fluocinonide), FLUOR-OP ^TMAnd FML ^TM(fluorometholone), CORDRAN ^TM(flurrenolone), HALOG ^TM(halcinonide), HMS LIZUIFILM ^TM(Zpoflogin), MEDROL ^TM(methylprednisolone), DEPO-MEDROL ^TMWith MEDROL ACETATE ^TM(methylprednisolone acetate), A-METHAPRED ^TMAnd SOLUMEDROL ^TM(Urbason Solubile), ELOCON ^TM(furoic acid momisone), HALDRONE ^TM(paramethasone acetate), DELTA-CORTEF ^TM(prednisolone), ECONOPRED ^TM(prednisolone acetate), HYDELTRASOL ^TM(prednisolone phosphate disodium), HYDELTRA-T.B.A ^TM(tertiary butyl prednisolone acetate), DELTASONE ^TM(prednisone), ARISTOCORT ^TMAnd KENACORT ^TM(triamcinolone), KENALOG ^TM(Triamcinolone Acetonide), ARISTOCORT ^TMAnd KENACORTDIACETATE ^TM(two Ledercort A) and ARISTOSPAN ^TM(triamcinolone hexacetonide); The inhibitor of adrenocortical steroid biosynthesizing and effect is such as CYTADREN ^TM(aminoglutethimide), NIZORAL ^TM(KETOKONAZOL), MODRASTANE ^TM(Win-24540) and METOPIRONE ^TM(metyrapone); Ox, pig or insulin human 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(P-607), TOLAMIDE ^TMAnd TOLINASE ^TM(tolazamide), DYMELOR ^TM(Acetohexamide), Glyburide, MICRONASE ^TM, DIBETA ^TMAnd GLYNASE ^TM(U26452), GLUCOTROL ^TM(Glipizide) and DIAMICRON ^TM(gliclazide), GLUCOPHAGE ^TM(metformin), ciglitazone, pioglitazone and alpha-glucosidase inhibitor; Ox or pig hyperglycemic-glycogenolytic factor; Somatostatin is such as SANDOSTATIN ^TM(Sostatin); And diazoxide, such as PROGLYCEM ^TM(diazoxide).

In one embodiment, the treatment of albumin fusion proteins of the present invention and/or polynucleotide and uterine activity sexual disorder is co-administered.The treatment of uterine activity sexual disorder includes but not limited to estrogenic, such as (for example puting together oestrogenic hormon

With

), estradiol (for example

With

), estropipate and Chlortrianisoestrol; The progesterone medicine (for example

(medroxyprogesterone),

(Norethisterone Acetate),

Progesterone and Magace); And oestrogenic hormon/progesterone conjoint therapy, such as for example puting together oestrogenic hormon/medroxyprogesterone (PREMPRO for example ^TMWith

) and Norethisterone Acetate/Ethinylestradiol (FEMHRT for example ^TM).

In another embodiment, albumin fusion proteins of the present invention and/or polynucleotide and treatment iron deficiency and anemia with low hemoglobin be medication combined using effectively, includes but not limited to ferrous sulfate (iron sulfate, FEOSOL ^TM), Ferrous Fumarate (FEOSTAT for example ^TM), Ferrous Gluconate (FERGON for example ^TM), polysaccharide-iron complex (NIFEREX for example ^TM), iron dextran injection liquid (INFED for example ^TM), blue vitriol, pyridoxol (pyroxidine), riboflavin, vitamins B ₁₂, Vitral injection liquid (REDISOL for example ^TM, RUBRAMIN PC ^TM), hydroxocobalamine, folic acid (FOLVITE for example ^TM), formyl tetrahydrofolic acid (folinic acid, 5-CHOH4PteGlu, Calciumlevofolinate) or WELLCOVORIN (calcium salt of formyl tetrahydrofolic acid), Transferrins,iron complexes or ferritin.

In certain embodiments, albumin fusion proteins of the present invention and/or polynucleotide and to be used for the treatment of the medicament of abalienation co-administered.Can include but not limited to tranquilizer (chlorpromazine for example with the co-administered psychosis medicine of albumin fusion proteins of the present invention and/or polynucleotide, chlorprothixene, leoponex, Fluphenazine, haloperidol, loxapine, mesoridazine, molindone, olanzapine, trilafon, pimozide, Kui sulphur is flat, risperidone, thioridazine, for thioxanthene difficult to understand, trifluoperazine, and Triflupromazine), anti-manic medicine (Carbamzepine for example, Sodium hydrogen divalproate, Quilonum Retard, and Lithium Citrate de), thymoleptic (amitriptyline for example, amoxapine, Wellbutrin, citalopram, clomipramine, Desipramine, doxepin, fluvoxamine, fluoxetine, imipramine, Isocarboxazid, maprotiline, mirtazapine, nefazodone, nortriptyline, paroxetine, Phenelzine, protriptyline, Sertraline, Tranylcypromine, trazodone, Trimipramine, and Venlafaxine), anxiolytic (alprazolam for example, buspirone, Chlordiazepoxidum, chlordiazepoxide, diazepam, halazepam, lorazepam, oxazepam, and prazepam), and stimulant (d-amphetamine for example, Methylphenidylacetate, and pemoline).

In other embodiments, albumin fusion proteins of the present invention and/or polynucleotide and to be used for the treatment of the medicament of neuroscience disorder co-administered.Can include but not limited to antiepileptic drug (Carbamzepine for example with the co-administered neuroscience medicament of albumin fusion proteins of the present invention and/or polynucleotide, clonazepam, ethosuximide, phenylethyl barbituric acid, Phenytoin Sodium Salt, primidone, valproic acid, Sodium hydrogen divalproate, felbamate, gabapentin, lamotrigine, Levetiracetam, oxcarbazepine, tiagabine, topiramate, zonisamide, diazepam, lorazepam, and clonazepam), antiparkinsonism drug (levodopa/carbidopa for example, selegiline, amantadine, bromocriptine, pergolide, Ropinirole, pramipexole, Benzatropine; Biperiden; The Ai Puba piperazine; Procyclidine; Trihexyphenidyl, tolcapone) and ALS therapeutical agent (for example Riluzole).

In another embodiment, albumin fusion proteins of the present invention and/or polynucleotide and vasodilator and/or calcium channel blocker are co-administered.Can include but not limited to Zinc metallopeptidase Zace1 (ACE) inhibitor (Papaverine for example with the co-administered vasodilator of albumin fusion proteins of the present invention and/or polynucleotide, isoxsuprine, benazepril, captopril, Yipingshu, enalapril, enalaprilat, fosinopril, lisinopril, moexipril, perindopril, quinapril, Ramipril, spirapril, Trolapril, and Nylidrine), and nitric ether (Dilatrate-SR for example, isosorbide mononitrate, and pannonit).Can include but not limited to the example of the co-administered calcium channel blocker of albumin fusion proteins of the present invention and/or polynucleotide amlodipine, Bepridil, Er Zhuoer, felodipine, flunarizine, Isrodipine, nicardipine, nifedipine, nimodipine and verapamil.

In certain embodiments, the therapeutical agent of albumin fusion proteins of the present invention and/or polynucleotide and gastrointestinal dysfunction is co-administered.Can include but not limited to H2 histamine receptor antagonists (TAGAMET for example with the therapeutical agent of the co-administered gastrointestinal dysfunction of albumin fusion proteins of the present invention and/or polynucleotide ^TM(cimitidine), ZANTAC ^TM(Ranitidine HCL), PEPCTD ^TM(famotidine) and AXID ^TM(nizatidine)); H ⁺, K ⁺Atpase inhibitor (PREVACID for example ^TM(lansoprazole) and PRILOSEC ^TM(omeprazole)); Bismuth compound (PEPTO-BISMOL for example ^TM(bismuth subsalicylate) and DE-NOL ^TM(alkali formula bismuth citrate)); Various antacid; Sucralfate; Prostaglandin analogue (for example, CYTOTEC ^TM(Misoprostol)); The muscarine cholinergic antagonist; Laxative (for example tensio-active agent laxative, pungency laxative, salt solution and perviousness laxative); Diarrhea (LOMOTIL for example ^TM(diphenoxylate), MOTOFEN ^TM(diphenoxin) and IMODIUM ^TM(loperamide hydrochloride)), the synthetic analogues of somatostatin, such as SANDOSTATIN ^TM(Sostatin), antiemetic (ZOFRAN for example ^TM(ondansetron), KYTRIL ^TM(Granisetron Hydrochloride), tropisetron, dolasetron, metoclopramide, chlorpromazine, trilafon, prochlorperazine, promethazine, Tietylperazine, Triflupromazine, domperidone, haloperidol, droperidol, trimethobenzamide, dexamethasone, methylprednisolone, dronabinol and hemp dragon); D2 antagonist (for example metoclopramide, trimethobenzamide and chlorpromazine); Cholate; Chenodiol; Ursodesoxycholic acid; With the pancreas enzyme preparation, such as pancreatin and steapsase.

In other embodiments, albumin fusion proteins of the present invention and/or polynucleotide and other therapeutic or preventative scheme are co-administered such as for example radiotherapy.

The present invention also provides packaged pharmaceuticals goods or the test kit that comprises one or more containers, and the composition that one or more comprise the pharmaceutical composition of albumin fusion proteins of the present invention is housed in the described container.Optional is, provides announcement by production, use or the sale of the standard medicine of administrative organization of government promulgation or biology product with such container, and this announcement has reflected the approval of administrative organization to production, use or the sale of human body dispenser.

Gene therapy

The part that the construct of code book invention albumin fusion proteins can be used as the gene therapy scheme is used to deliver the albumin fusion proteins of treatment effective dose.The preferred method that is used in vivo the nucleic acid transfered cell is the virus vector that comprises the nucleic acid of code book invention albumin fusion proteins by use.Advantage with the viral vector infection cell is that most of target cell can both obtain described nucleic acid.In addition, molecule effective expression in the cell of picked-up virus vector nucleic acid of for example encoding in the virus vector by the contained cDNA of virus vector.

Retrovirus and adeno-associated virus vector can be used as the exogenous nucleic acid molecule that the recombination delivery system is used for shifting in vivo the coding albumin fusion proteins.These carriers effectively are delivered to nucleic acid in the cell, and the transfection nucleic acid stability is incorporated in the host chromosome DNA.The exploitation that only produces the retroviral special-purpose clone of replication defect type (being called " packing cell ") has increased the effectiveness of retrovirus in gene therapy, and the defective type retrovirus of being identified be used for gene therapy purposes transgenosis (summary referring to Miller, A.D., Blood 76:271,1990).By standard technique, the replication defect type retrovirus can be packaged into virus particle, and it can be used for target cell infection by the use of helper virus.Be used to generate recombinant retrovirus and be used for that the scheme with this virus infected cell can be referring to " Current Protocols in Molecular Biology " in external or body, Ausubel, F.M. wait the people to compile, Greene Publishing Associates, 1989, Sections 9.10-9.14 and other standard test guide.

Can be used for another kind of virogene delivery system of the present invention and use adenovirus deutero-carrier.The genome that can operate adenovirus like this makes it coding and expresses the goal gene product, but its ability of in normal molten born of the same parents' venereal disease poison life cycle, duplicating of deactivation.Referring to people such as for example Berkner, BioTechniques 6:616,1988; People such as Rosenfeld, Science 252:431-434,1991; Reach people such as Rosenfeld, Cell 68:143-155,1992.Derived from the suitable adenovirus carrier of adenopathy strain Ad5 type d1324 or other adenopathy strain (for example Ad2, Ad3, Ad7 etc.) is known to those skilled in the art.Recombinant adenovirus has superiority in some cases, because they can not infect not somatoblast, and can be used for infecting diversified cell type, comprises epithelial cell (people such as Rosenfeld, 1992, quote the same).In addition, virion is relatively stable, and is easy to purifying and concentrated, and as mentioned above, can transform to influence its pattern of infection.In addition, the adenovirus DNA that imports (and wherein contained foreign DNA) unconformability is in the host cell gene group, still as episome, thereby avoided being incorporated in the situation of (for example retrovirus DNA) in the host genome because of inserting the potential problems that mutagenesis takes place at the DNA that imports.In addition, the ability that the adenoviral gene group is carried foreign DNA is that big (can reach 8 kilobase) (people such as Berkner quotes the same with respect to other gene delivery carrier; People such as Haj-Ahmand, J.Virol.57:267,1986).

In another embodiment, non-viral gene delivery system of the present invention depends on the cell endocytic approach of target cell picked-up theme nucleic acid molecule.Such exemplary genes delivery system comprises liposome flavor, many Methionin conjugate, reaches artificial viral envelope.In a representative embodiment, the nucleic acid molecule of code book invention albumin fusion proteins can be captured its surface positively charged and (choosing wantonly) use (people such as Mizuno at (for example lipofectin) in the tagged liposome of the antibody of target tissue cell-surface antigens, No Shinkei Geka 20:547-551,1992; PCT announces WO 91/06309; Japanese patent application 1047381; And European patent publication EP-A-43075).

Can the gene delivery system of the gene of code book invention albumin fusion proteins be imported to the patient by several different methods.For example, pharmaceutical preparation that can be by for example intravenous injection system quiding gene delivery system, and the transfection specificity that proteinic specialized transduction is mainly provided by the gene delivery media in the target cell, be attributable to the cell type of the transcription regulating nucleotide sequence that the control acceptor gene expresses or types of organization expresses or its combination and taking place.In other embodiments, the initial delivery of recombination is limited to quite partial importing animal.For example, can pass through conduit (referring to United States Patent (USP) 5,328,470) or come quiding gene to deliver media by directed injection (for example people such as Chen, PNAS 91:3054-3057,1994).The pharmaceutical preparation of gene therapy construct can be made up of the gene delivery system that can accept in the thinner in essence, perhaps can comprise the sustained-release matrix that wherein is embedded with the gene delivery media.In the time can generating complete albumin fusion proteins by reconstitution cell, retroviral vector for example, pharmaceutical preparation can comprise one or more cells that generate albumin fusion proteins.

Other gene therapy

The gene therapy that is used for the treatment of or prevents disorder, disease and situation is also contained in the present invention.Gene therapy relates to nucleic acid (DNA, RNA and antisense DNA or RNA) sequence importing animal to realize the expression of albumin fusion proteins of the present invention.The polynucleotide of this method requirement code book invention albumin fusion proteins and promotor and necessary any other gene element of target tissue expressed fusion protein can be operatively connected.Such gene therapy and delivery technology are known in the art, referring to for example WO90/11092, are introduced into this paper as a reference.

Therefore, for example, the polynucleotide (DNA or RNA) of the promotor that can be operatively connected with the polynucleotide that comprise with code book invention albumin fusion proteins exsomatize and transform cell from the patient, will offer the patient that will treat with albumin fusion proteins of the present invention then through the cell of transforming.Such method is well-known in the art.For example, referring to Belldegrun, people such as A., J.Natl.Cancer Inst.85:207-216,1993; Ferrantini, people such as M., Cancer Research 53:1107-1112,1993; Ferrantini, people such as M., J.Immunology 153:4604-4615,1994; Kaido, people such as T., Int.J.Cancer 60:221-229,1995; Ogura, people such as H., Cancer Research 50:5102-5106,1990; Santodonato, people such as L., Human Gene Therapy 7:1-10,1996; Santodonato, people such as L., Gene Therapy 4:1246-1255,1997; And Zhang, people such as J.-F., Cancer GeneTherapy 3:31-38,1996, be introduced into this paper as a reference.In one embodiment, the cell of transforming is an arterial cell.Can be by being injected directly in the artery, in the tissue around the artery or by tube injection arterial cell being imported the patient again.

As hereinafter discussing in more detail, can polynucleotide constructs be delivered to zooblast by any method of delivering injectable materials, such as being expelled to tissue (heart, muscle, skin, lung, liver or the like) intercellular space.Polynucleotide constructs can be delivered in pharmacopedics acceptable liquid or aqueous carrier.

In one embodiment, the polynucleotide of code book invention albumin fusion proteins are delivered as exposed polynucleotide.The polynucleotide that term " exposes ", DNA or RNA refer to that sequence is free on any delivery media auxiliary, that promote or quicken to enter cell, comprises virus sequence, virion, Liposomal formulation, fat transfection or precipitation reagent etc.Yet the polynucleotide of code book invention albumin fusion proteins also can be delivered in Liposomal formulation that can prepare by method well known to those skilled in the art and fat transfecting formulations etc.For example U.S. Patent number 5,593, described such method in 972,5,589,466 and 5,580,859, are introduced into this paper as a reference.

The polynucleotide carrier construction that is used for gene therapy preferably neither can be incorporated into host genome and also 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; And can be from pEF1/V5, pcDNA3.1 and the pRc/CMV2 of Invitrogen acquisition.Other suitable carriers is conspicuous for those of skill in the art.

Any strong promoter that those skilled in the art will know that all can be used for driving the expression of polynucleotide sequence.Suitable promotor comprises adenovirus promoter, such as adenovirus major late promoter; Or allogeneic promoter, such as cytomegalovirus (CMV) promotor; Respiratory syncytial virus (RSV) promotor; Inducible promoter is such as MMT promotor, metallothionein promoter; The heat-shocked promotor; The white protein promotor; The ApoAI promotor; The human immunoglobulin promotor; Viral thymidine kinase promoter is such as herpes simplex thymidine kinase promoter; Retrovirus LTR; The b-actin promoter; And human growth hormone promotor.Promotor also can be natural promotor for the gene corresponding with the therapeutic protein part of albumin fusion proteins of the present invention.

Different with other gene therapy technology, the major advantage that the naked nucleotide sequence that falls is imported target cell is the of short duration character of polynucleotide synthetic in the cell.Studies show that can be with non-repetition DNA sequence transfered cell, thus be 6 months during the generation of expectation polypeptide is provided.

The polynucleotide construction can be delivered to the intercellular space of organizing of animal, comprise muscle, skin, brain, lung, liver, spleen, marrow, thymus gland, the heart, lymph, blood, bone, cartilage, pancreas, kidney, gall-bladder, stomach, intestines, testis, ovary, uterus, rectum, neural system, eye, gland and reticular tissue.Organize the intercellular space to comprise the collegen filament of spandex fiber in mucopolysaccharide layer, tube wall or the locular wall between the reticulin fiber of iuntercellular, liquid, organ-tissue, fibrous tissue or include myocyte's reticular tissue or the same matrix in the bone crack in.Also similar by the space that the lymph liquid of circulating plasma and lymph pipeline occupies.Owing to the reason of discussing below preferably is delivered to the muscle tissue intercellular space.They can be delivered to the tissue that comprises these cells easily by injection.They preferably are delivered to persistent, nondividing noble cells and express therein, can be although deliver and express at not differentiation or the more incomplete cell of differentiation, and such as finishing in the stem cell of for example blood or the skin flbroblast.Muscle cell is effectively competent especially aspect the ability of their picked-ups and expression polynucleotide in vivo.

For exposed nucleotide sequence injection, the effective dose of DNA or RNA will be in about 0.05mg/kg body weight to the scope of about 50mg/kg body weight.Preferably, dosage will be about 0.005mg/kg to about 20mg/kg and 0.05mg/kg about 5mg/kg extremely more preferably from about.Certainly, those of ordinary skills will understand, and this dosage will change along with the tissue site of injection.Suitable and the effective dosage of nucleotide sequence can be easy to determine, and may depend on the situation and the drug delivery route of treatment by those of ordinary skills.

Preferred drug delivery route is to organizing in the intercellular space by the parenteral injection approach.Yet, also can use other parenteral route, suck such as aerosol, be specially adapted to be delivered to lung or bronchial tissue, larynx or nasal mucosa.In addition, the conduit that can be in the angioplasty process uses in by program of naked DNA construction is delivered to artery.

Deliver exposed polynucleotide by any method that this area is known, comprise that the direct pin injection, intravenous injection, topical application, the conduit that are not limited to deliver the position are inculcated, and so-called " particle gun ".These delivering methods are known in the art.

Also can deliver construct with delivering media such as virus sequence, virion, Liposomal formulation, fat transfection, precipitation reagent etc.These delivering methods are known in the art.

In certain embodiments, polynucleotide constructs is compound in the liposome preparation thing.Be used for liposome preparation thing of the present invention and comprise positively charged ion (positively charged), negatively charged ion (electronegative) and neutral prepared product.Yet special preferred cationic liposome is because can form charge recombination body closely between cationic-liposome and polyanionic nucleic acid.Shown that cationic-liposome is with functional form mediation plasmid DNA (people such as Feigner, Proc.Natl.Acad.Sci.USA 84:7413-7416 1987, are introduced into this paper as a reference); MRNA (people such as Malone, Proc.Natl.Acad.Sci.USA 86:6077-6081,1989, be introduced into this paper as a reference); With deliver in the cell of the transcription factor of purifying (people such as Debs, J.Biol.Chem.265:10189-10192 1990, are introduced into this paper as a reference).

Cationic-liposome is easy to obtain.For example, N[1-2,3-two oil base oxygen] propyl group]-N, N, N-triethyl ammonium (DOTMA) liposome is particularly useful, and can be from GIBCO BRL, Grand Island, N.Y. obtains, trade mark is that Lipofectin (also can be referring to people such as Felgner, Proc.Natl.Acad.Sci.USA84:7413-7416,1987, be introduced into this paper as a reference).Other commercialization liposome comprises transfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).

Other cationic-liposome can use technology well-known in the art by the material preparation that is easy to obtain.Referring to synthesizing of the described DOTAP of for example PCT publication No. WO 90/11092 (being introduced into this paper as a reference) (1,2-two (oleoyl oxygen)-3-(TMA (TriMethylAmine)) propane) liposome.Described the preparation of DOTMA liposome in the document, referring to people such as for example P.Felgner, Proc.Natl.Acad.Sci.USA84:7413-7417 is introduced into this paper as a reference.Similar approach can be used for by other cation lipid material preparation liposome.

Similarly, negatively charged ion and neutral fat plastid also are easy to obtain, such as (Birmingham Ala.) obtains, and perhaps can be easy to preparation with the material that is easy to obtain from Avanti Polar Lipids.Such material comprises phosphatidylcholine, cholesterol, phosphatidylethanolamine, dioleoyl phospholipid phatidylcholine (DOPC), DOPG (DOPG), DOPE (DOPE) etc.These materials also can mix with suitable proportion with DOTMA and DOTAP parent material.Method with these material preparation liposomes is well-known in the art.

For example, can be used to prepare conventional liposome by various combination, can add or not add cholesterol in commercial dioleoyl phospholipid phatidylcholine (DOPC), DOPG (DOPG) and DOPE (DOPE).Therefore, for example, can by with each 50mg DOPG and DOPC under the nitrogen gas stream in the supersound process bottle drying prepare the DOPG/DOPC liposome.Sample placed under the vacuum pump spend the night, used the deionized water hydration in second day.Then sample is used to be equipped with 350 type Heat Systems ultrasonic apparatus of inverted cup (water-bath type) probe supersound process to be set 2 hours with maximum in the bottle of lid lid, water-bath simultaneously is with 15 ℃ of circulations.Perhaps, can produce multilamellar vesicle or prepare electronegative vesica without supersound process by the unilamellar vesicle of extruding nucleopore membranes generation different sizes.Those skilled in the art also know and can utilize other method.

Liposome can comprise multilamellar vesicle (MLV), little unilamellar vesicle (SUV) or big unilamellar vesicle (LUV), preferred SUV.Use method well-known in the art to prepare various liposome-nucleic acid complexes.Referring to people such as for example Straubinger, Methods ofImmunology 101:512-527,1983, be introduced into this paper as a reference.For example, can be by phospholipid membrane being deposited on the glass test tube wall, the solution aquation with the material that will adorn capsule prepares the MLV that contains nucleic acid subsequently.SUV prepares by the unilamellar liposome group that prolongation supersound process MLV produces homogeneous.The material of desiring to catch is added in the treated MLV suspension supersound process then.When using the liposome of cation lipid, the exsiccant lipid film is resuspended in suitable solution such as sterilized water or waits damping fluid a such as 10mMTris/NaCl, supersound process is directly mixed treated liposome then with DNA.Because positively charged liposome combines with cation DNA, thereby liposome and DNA form highly stable mixture.SUV can be used for the nucleic acid small segment.Many method preparations that LUV knows by this area.Method commonly used comprises Ca ²⁺-EDTA chelating (people such as Papahadjopoulos, Biochim.Biophys.Acta 394:483,1975; People such as Wilson, Cell 17:77,1979); Ether injection (Deamer, D. and Bangham, A., Biochim.Biophys.Acta 443:629,1976; People such as Ostro, Biochem.Biophys.Res.Commun.76:836,1977; People such as Fraley, Proc.Natl.Acad.Sci.USA76:3348,1979); Detergent dialysis (Enoch, H. and Strittmatter, P., Proc.Natl.Acad.Sci.USA 76:145,1979); With anti-phase distillation (REV) (people such as Fraley, J.Biol.Chem.255:10431,1980; Szoka, F. and Papahadjopoulos, D., Proc.Natl.Acad.Sci.USA75:145,1978; People such as Schaefer-Ridder, Science 215:166,1982), be introduced into this paper as a reference.

Generally speaking, the ratio of DNA and liposome is about 10: 1 to about 1: 10.Preferably, ratio is about 5: 1 to about 1: 5.More preferably, ratio is about 3: 1 to about 1: 3.Still more preferably, ratio is about 1: 1.

U.S. Patent number 5,676,954 (being introduced into this paper as a reference) have been reported and have been injected in mice and cationic-liposome carrier compound genetic stocks.U.S. Patent number 4,897,355,4,946,787,5,049,386,5,459,127,5,589,466,5,693,622,5,580,859,5,703,055 and international publication number WO 94/9469 (being introduced into this paper as a reference) provide and be used for the cation lipid of DNA transfection to cell and Mammals.U.S. Patent number 5,589,466,5,693,622,5,580,859,5,703,055 and international publication number WO 94/9469 provide DNA-cation lipid mixture be delivered to mammiferous method.

In certain embodiments, exsomatize or in vivo with the retroviral particle engineered cells of the RNA that contains the sequence that comprises code book invention albumin fusion proteins.Can include but not limited to Moroni murine leukemia virus, spleen necrosis virus, Rous sarcoma virus, harvey sarcoma virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, bone marrow proliferative sarcoma virus and mammary tumor virus by the retrovirus of its retroviral plasmid vector of deriving.

Use retroviral plasmid vector transduction package cell line to form producer's clone.But the example of the packing cell of transfection includes but not limited to Miller, Human Gene Therapy 1:5-14, PE501, PA317, R-2, R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12 and the DAN clone described in 1990 are with its complete being incorporated herein by reference.Any method carrier transduction packing cell that can know by this area.Such method includes but not limited to electroporation, utilizes liposome and CaPO ₄Precipitation.In a possibility, retroviral plasmid vector can be wrapped in the liposome, perhaps, be applied to the host then with the lipid coupling.

Producer's clone produces the infectious retroviral vector particle of the polynucleotide that comprise code book invention albumin fusion proteins.Then can be with such retroviral vector particle at the external or eukaryotic cell of transduceing in vivo.Eukaryotic cell through transduction will be expressed fusion rotein of the present invention.

In some other embodiment, exsomatize or in vivo with the polynucleotide engineered cells that is included in the adenovirus carrier.Can operate adenovirus like this and make it coding and express fusion rotein of the present invention, its ability of in normal molten born of the same parents' venereal disease poison life cycle, duplicating of deactivation simultaneously.Do not need viral DNA is incorporated into and just realize gland virus expression in the host cell chromosome, alleviated thus inserting the worry of mutagenesis.In addition, adenovirus has fabulous security feature people such as (, Am.Rev.Respir.Dis.109:233-238,1974) Schwartz as live body enteron aisle vaccine for many years.At last, demonstrate many examples of adenovirus mediated transgenosis, comprised the lung (Rosenfeld, people such as M.A., Science 252:431-434,1991 that α-1-antitrypsin and CFTR are transferred to cotton mouse; People such as Rosenfeld, Cell68:143-155,1992).In addition, the broad research of attempting adenovirus is defined as the human cancer virulence factor all is (Green, people such as M., Proc.Natl.Acad.Sci.USA 76:6606,1979) of negating without exception.

Can be used for suitable adenovirus carrier of the present invention and be described in for example Kozarsky and Wilson, Curr.Opin.Genet.Devel.3:499-503,1993; People such as Rosenfeld, Cell 68:143-155,1992; People such as Engelhardt, Human Genet.Ther.4:759-769,1993; People such as Yang, Nature Genet.7:362-369,1994; People such as Wilson, Nature 365:691-692,1993; And U.S. Patent number 5,652,224, be introduced into this paper as a reference.For example, adenovirus carrier Ad2 is useful, and can cultivate in people's 293 cells.These cells contain the E1 district of adenovirus, and constructive expression E1a and E1b, and they are by the product that the gene of deleting in the carrier the is provided type adenovirus that supplies a gap.Except that Ad2, other multiple adenovirus (for example Ad3, Ad5 and Ad7) also can be used for the present invention.

Preferably, being used for adenovirus of the present invention is replication defect type.Replication-defective adenoviral needs the help of helper virus and/or package cell line to form infectious particles.The virus that so obtains can cells infected, and can express the polynucleotide of interest that can be operatively connected with promotor, but in most cells reproducible not.Replication-defective adenoviral can have been deleted the whole or part of one or more following genes: E1a, E1b, E3, E4, E2a or L1 to L5.

In some other embodiment, exsomatize or use adeno associated virus (AAV) engineered cells in vivo.AAV needs helper virus to produce the natural defective virus (Muzyczka, N., Curr.Topics in Microbiol.Immunol.158:97,1992) that exists of infectious particles.It still can be incorporated into its DNA one of minority virus in the Unseparated Cell.Contain the few of AAV and just can pack and integrate, still supply the space constraint of foreign DNA at about 4.5kb to the carrier of 300 base pairs.Being used to produce and use the method for such AAV is that this area is known.Referring to for example U.S. Patent number 5,139,941,5,173,414,5,354,678,5,436,146,5,474,935,5,478,745 and 5,589,377.

For example, be used for suitable AAV carrier of the present invention will comprise dna replication dna, encapsidate, and host cell integrate necessary all sequences.Use standard cloning process inserts the AAV carrier with polynucleotide constructs, such as people such as Sambrook, " Molecular Cloning:A Laboratory Manual ", Cold Spring Harbor Press is found in 1989.Use any standard technique then, comprise fat transfection, electroporation, calcium phosphate precipitation etc., will recombinate the transfection of AAV carrier in the package cell line that infects with helper virus.Suitable helper virus comprises adenovirus, cytomegalovirus, vaccinia virus or simplexvirus.In case packing cell is subjected to transfection or infection, they comprise generation the infectious AAV virion of polynucleotide constructs.Exsomatize or the eukaryotic cell of transduceing in vivo with these virions then.Cell through transduction will contain the polynucleotide constructs that is incorporated in its genome, and will express fusion rotein of the present invention.

Another kind of gene therapy method relates to be made the allos control region can operate with endogenous polynucleotide sequence (polypeptide of the present invention of for example encoding) by homologous recombination to link to each other (referring to the U.S. Patent number of for example delivering on June 24th, 1,997 5,641,670; The international publication number WO96/29411 that on September 26th, 1996 published; The international publication number WO 94/12650 that on August 4th, 1994 published; People such as Koller, Proc.Natl.Acad.Sci.USA 86:8932-8935,1989; Reach people such as Zijlstra, Nature 342:435-438,1989, be introduced into this paper as a reference).This method relate to exist in the target cell but in cell, do not express under the normal circumstances or to be lower than the activation of the gene that aspiration level expresses.

The standard technique of knowing with this area prepares polynucleotide constructs, and it comprises promotor and this promotor flank is the target sequence.This paper has described suitable promotor.Target sequence and endogenous sequence are fully complementary to allow the homologous recombination of promotor-target sequence and endogenous sequence.The target sequence will make promotor can be operatively connected by homologous recombination and endogenous sequence fully near 5 ' end of the endogenous polynucleotide sequence of expectation.

Can pcr amplification promotor and target sequence.Preferably, the promotor of amplification contains distinct restriction enzyme sites at 5 ' and 3 ' end.Preferably, 3 ' end of the first target sequence contains the identical restriction enzyme sites of 5 ' end with the amplification promotor, and 5 ' end of the second target sequence contains with 3 ' of the promotor that increases and holds identical restriction enzyme sites.The promotor of digest amplification is with the target sequence and connect together.

Promotor-target sequence construct body is delivered to cell, perhaps as exposed polynucleotide, perhaps with above associatings such as short in greater detail transfection agents such as liposome, virus sequence, virion, totivirus, fat transfection, precipitation agent.Can deliver P promotor-target sequence by any method, comprise that direct pin injection, intravenous injection, topical application, conduit are inculcated, particle accelerator etc.Hereinafter more detailed description these methods.

Promotor-target sequence construct body is by cellular uptake.Between construct and the endogenous sequence homologous recombination taking place, makes endogenous sequence place under the control of promotor.The expression of promoters driven endogenous sequence then.

The polynucleotide of code book invention albumin fusion proteins can contain the secretory signal sequence that promotes protein secreting.Typically, signal sequence wait to express in the polynucleotide encoding district towards or be positioned at 5 ' end of coding region.Signal sequence can be a homologous or allogenic for polynucleotide of interest, and for treating that transfectional cell can be a homologous or allogenic.In addition, signal sequence can use the method chemosynthesis that this area is known.

Can use any pattern to use any above-mentioned polynucleotide constructs, as long as this pattern causes one or more molecules to be enough to the providing quantity of result of treatment to express.This comprises that the injection of direct pin, system's injection, conduit are inculcated, the injection of biological projectile, particle accelerator (i.e. " particle gun "), gelfoam sponge bank (depot), other commercialization bank material, osmotic pump (for example Alza micropump), oral or suppository solid (tablet or pill) pharmaceutical formulations, reach intra-operative and pour into or topical application.For example, exposed calcium phosphate precipitation plasmid is injected directly in rats'liver and the rat spleen or the plasmid of protein bag quilt is injected directly into and cause the genetic expression of foreign gene in rats'liver (people such as Kaneda in the portal vein, Science 243:375,1989).

A kind of preferred method of topical application is to pass through direct injection.Preferably, will with deliver media compound albumin fusion proteins of the present invention and be administered in the artery by direct injection or locally apply in the arteriosomes.Composition locally applied in the arteriosomes refer to composition is expelled to several centimetres preferred several millimeters of intra-arterial.

The another kind of method of topical application is to place polynucleotide constructs of the present invention among the wound or on every side.For example, can perform a surgical operation, and polynucleotide constructs can be covered on the tissue surface in the wound, perhaps construct can be expelled in the tissue regions in the wound to the patient.

The therapeutic composition that can be used for systemic application comprises with target of the present invention delivers media compound fusion rotein of the present invention.The delivery media that is applicable to systemic application comprises the liposome that comprises the part of media target privileged site.In specific embodiment, be applicable to that the delivery media of systemic application comprises the liposome that comprises the albumin fusion proteins of the present invention of media target privileged site.

The preferred method of systemic application comprises intravenous injection, aerosol, oral and deliver through skin (part).Intravenous injection can be carried out with the standard method of this area.Aerosol is delivered the also standard method of available this area and is carried out (referring to people such as for example Stribling, Proc.Natl.Acad.Sci.USA 189:11277-11281 1992, is introduced into this paper as a reference).Oral delivery can be by with polynucleotide constructs of the present invention and compound the carrying out of carrier that can keep out the degraded of digestive ferment in the animal intestinal.The example of such carrier comprises plastic capsule or tablet, contracts such as this area and knows.Local delivery can be by mixing polynucleotide constructs of the present invention to carry out with the lipotropy medicament that can pass skin (for example DMSO).

The significant quantity that the decision material is delivered can be depending on multiple factor, comprise the age of the chemical structure of these materials for example and biologic activity, animal and body weight, needs treatment definite situation and severity thereof, reach drug delivery route.The frequency of treatment depends on multiple factor, the quantity of the polynucleotide construction of using such as each administration and experimenter's healthy state and medical history.Attending doctor or animal doctor will determine definite consumption, administration number of times and administration time.

Albumin fusion proteins of the present invention can be applied to any animal, preferred mammal and birds.Preferred Mammals comprises people, dog, cat, mouse, rat, rabbit, sheep, ox, horse and pig, and the people is particularly preferred.

Biologic activity

The polynucleotide of albumin fusion proteins of the present invention and/or coding albumin fusion proteins can be used for testing the assay method of one or more biologic activity.If albumin fusion proteins and/or polynucleotide demonstrate activity in the particular assay method, those therapeutic proteins corresponding with fusion rotein relate to and this biologic activity diseases associated probably.Therefore, described fusion rotein can be used for treating relative disease.

In preferred embodiments, listed disease or disorderly method in treatment table 1 " the preferred indication Y " row contained in the present invention, comprise with effective treatment, prevention or improve described disease or disorderly amount is used albumin fusion proteins of the present invention to the patient of this treatment of needs, prevention or improvement that this albumin fusion proteins comprises with table 1 " therapeutic protein X " and is listed as the corresponding therapeutic protein part of disclosed therapeutic protein (with table 1 " preferred indication Y " row listed disease or disorder are positioned at same delegation).

In another preferred embodiment, the present invention is contained treatment table 1 " preferred indication Y " row to particular treatment protein listed disease or disorderly method, comprise with effective treatment, prevention or improve described disease or disorderly amount is used albumin fusion proteins of the present invention to the patient of this treatment of needs, prevention or improvement that this albumin fusion proteins comprises the corresponding therapeutic protein part of the therapeutic protein relevant with the indication among the embodiment.

The present invention has taken explicitly into account by the polynucleotide encoding of coding SEQ ID NO:Y the time albumin fusion proteins by cell generated.When using these polynucleotide by the coded protein of cell expressing, the secretion naturally of cell and procedure of processing have generated and have lacked the protein that table 2 the 4th and/or 11 is listed the signal sequence of really listing.The concrete aminoacid sequence of listed signal sequence has demonstration or well-known in the art in specification sheets.So, the most preferred embodiment of the present invention comprises the albumin fusion proteins (it lacks table 2 the 4th and/or the listed leader sequence of 11 row) that is generated by cell.Most preferredly comprise SEQ ID NO:Y in addition but do not have table 2 the 4th and/or the polypeptide of the listed leader sequence of 11 row.Comprising the composition of these two preferred embodiments, comprise pharmaceutical composition, also is preferred.Taken explicitly into account and used these albumin fusion proteins to treat, prevent or improve table 1 " preferred indication: Y " and classify listed disease of particular treatment protein or disorder as.

In preferred embodiments, fusion rotein of the present invention can be used for diagnosis, prediction, prevent and/or treat and endocrine system (referring to " endocrine regulation " part for example hereinafter), neural system (referring to " neuroscience disorder " part for example hereinafter), immunity system (referring to " immunocompetence " part for example hereinafter), respiratory system (referring to " respiratory disorder " part for example hereinafter), cardiovascular systems (referring to " cardiovascular disorder " part for example hereinafter), reproductive system (referring to " reproductive system disorder " part for example hereinafter), disease and the disorderly diseases associated and/or the disorder of Digestive tract (referring to " gastrointestinal dysfunction " part for example hereinafter), with cell proliferation diseases associated and/or disorder (referring to " hyper-proliferative sexual disorder " part for example hereinafter), and/or with blood diseases associated or disorder (referring to " blood associated disorders " part for example hereinafter).

In certain embodiments, albumin fusion proteins of the present invention can be used for diagnosing and/or predicts and wherein organize diseases associated and/or disorder with the corresponding gene of the therapeutic protein part of fusion rotein of the present invention is expressed.

Therefore, the polynucleotide of fusion rotein of the present invention and code book invention albumin fusion proteins can be used for diagnosing, detecting and/or treat and include but not limited to the active diseases associated and/or the disorder of prohormone activation, neurotransmitter activity, cell signal, cell proliferation, cytodifferentiation and cell migration.

More general, the polynucleotide of fusion rotein of the present invention and code book invention albumin fusion proteins can be used for diagnosing, predict, prevent and/or treat and following system diseases associated and/or disorder.

Immunocompetence

The polynucleotide of albumin fusion proteins of the present invention and code book invention albumin fusion proteins can be used for treating, prevent, diagnose and/or predict immune disease, disorder and/or situation by propagation, differentiation or the reactivity (chemotaxis) of for example activation or inhibition immunocyte.Immunocyte is grown in being called the process of hemoposieis, produces medullary cell (thrombocyte, red corpuscle, neutrophilic granulocyte and scavenger cell) and lymphoidocyte (B and T lymphocyte) from multipotential stem cell.The cause of disease of these Immunological diseases, disorder and/or situation may be hereditary, somatic (such as cancer and some autoimmune diseases), acquired (for example being obtained by chemotherapy or toxin) or infective.In addition, the polynucleotide of fusion rotein of the present invention and code book invention albumin fusion proteins can be used as specific disease of immune system or disorderly mark or detection 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 with disorderly and/or suppress or strengthen the immunne response that is produced by the relevant cell of the tissue of expressing with polypeptide of the present invention wherein.

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for treatment, prevention, diagnosis and/or prediction immune deficiency, comprise two kinds of geneogenous and acquired immune deficiencies.Wherein the example of the B cellular immunity deficiency that reduces of immunoglobulin level B cell function and/or B cell number comprises: the chain agammaglobulinemia of X-(the Bu Ludunshi disease (Bruton ' sdisease); the infantile agammaglobulinemia that X-is chain; the high IgM immune deficiency that X-is chain; the high IgM immune deficiency that non-X-is chain; the lymphopoiesis syndromes (XLP) that X-is chain; agammaglobulinemia comprises congenital and acquired agammaglobulinemia disease; the agammaglobulinemia of adulthood outbreak; tardy property agammaglobulinemia; dysgammaglobulinemia; hypogammag lobulinemia; not qualitative hypogammag lobulinemia (unspecified hypogammaglobulinemia); recessive agammaglobulinemia (Swiss cut); selective IgM deficiency; selective IgA deficiency; selectivity IgG subclass defective; IgG subclass defective (having or do not have the IgA defective); Ig defective IgM simultaneously increases; IgG and IgA defective IgM simultaneously increase; normal or the rising of Ig simultaneously of antibody defective; the Ig heavy chain deletion; kappa chain deficiency; B cell lymphocyte propagation disorderly (BLPD); common variable immunodeficiency (CVID); common variable immunodeficiency (CVI) (acquired); and transient hypogammaglobulinemia of infancy.

In specific embodiment, use the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins to treat, prevent, diagnose and/or predict ataxia-telangiectasis or the situation relevant with ataxia-telangiectasis.

Wherein the example of the innate immunity defective of T cell and/or B cell function and/or number reduction includes but not limited to: enlightening George unusual (DiGeorge anomaly), Reconstruction in Sever Combined Immunodeciency (SCID) (includes but not limited to the SCID that X-is chain, autosomal recessive SCID, the adenosine deaminase defective, purine nucleoside phosphorylase (PNP) defective, II class MHC defective (bare lymphocyte syndrome), Wei-Ao two syndromes (Wiskott-Aldrich syndrome), ataxia-telangiectasia), thymic aplasia, third and fourth pharyngealpouch syndrome, 22q11.2 disappearance, the chronic mucocutaneous candidiasis, natural killer cell defective (NK), the special property sent out CD4+T-lymphopenia, the immune deficiency of dominance T cell defect (not qualitative), immune deficiency with not qualitative cell-mediated immunity.

In specific embodiment, use the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins treat, prevent, diagnose and/or predict the enlightening George unusual or with the unusual relevant situation of enlightening George.

The polynucleotide treatment of available fusion rotein of the present invention and/or code book invention albumin fusion proteins, prevention, other immune deficiency of diagnosis and/or prediction includes but not limited to chronic granulomatous disease, Qie-Xi two syndromes (Chediak-Higashi syndrome), myeloperoxidase deficiency, white corpuscle glucose-6-phosphate dehydrogenase (G6PD) defective, the lymphopoiesis syndromes (XLP) that X-is chain, 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, immune deficiency with thymoma, the severe congenital oligoleukocythemia, heteroplasia with immune deficiency, newborn infant's neutrophil reduces, micromelic dwarf disease, and Nei Ziluofu syndromes (Nezelof syndrome)-merging Ig immune deficiency.

In a preferred embodiment, with polynucleotide are treated, prevent, diagnosed and/or prediction is relevant with above-mentioned immune deficiency the immune deficiency and/or the situation of fusion rotein of the present invention and/or code book invention albumin fusion proteins.

In a preferred embodiment, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins can be used as the medicament of enhancing immunity responsiveness in the immune deficiency individuality.In specific embodiment, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins can be used as the medicament of enhancing immunity responsiveness in B cell and/or T cellular immunity deficiency individuality.

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for treatment, prevention, diagnosis and/or prediction autoimmune disorder.Many autoimmune disorders all be since the immunocyte mistake self is identified as foreign matter.This wrong identification causes and causes host tissue to suffer the destructive immunne response.Therefore, use can suppress immunne response particularly the polynucleotide of propagation, differentiation or the chemotactic fusion rotein of the present invention of T cell and/or code book invention albumin fusion proteins may be effective methods of treatment of prevention autoimmune disorder.

Can treat with the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins, prevention, the autoimmune disorder of diagnosis and/or prediction and disorderly include but not limited to following one or more: systemic lupus erythematous, rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis, autoimmune thyroiditis, struma lymphomatosa, autoimmune hemolytic anemia, hemolytic anemia, thrombopenia, autoimmune thrombocytopenic purpura, autoimmunity newborn infant thrombopenia, idiopathic thrombocytopenic purpura, purpura (for example prosperous-permitted Er Shi purpura (Henloch-Scoenlein purpura)), the autoimmunity hemocytopenia, the Goodpasture syndrome (Goodpasture ' s dyndrome), pemphigus vulgaris, myasthenia gravis, Graves' disease (Grave ' sdisease) (hyperthyroidism), with the insulin resistance diabetes.

The polynucleotide treatment that may have the albumin fusion proteins available of the present invention and/or the code book invention albumin fusion proteins of autoimmunization factor, other disorder of prevention and/or diagnosis includes but not limited to II Collagen Type VI inductive sacroiliitis, anti-phosphatide syndromes, dermatitis, allergic encephalitis, myocarditis, relapsing polychondritis, rheumatic heart disease, neuritis, the uveitis ophthalmia, many incretopathys, Lay Te Shi disease, stiff people's syndromes, the autoimmunity pneumonia, autism, Ge-Ba two syndromes, insulin-dependent diabetes, with the autoimmunity inflammatory eye disease.

The polynucleotide treatment that may have the albumin fusion proteins available of the present invention and/or the code book invention albumin fusion proteins of autoimmunization factor, prevention, other disorder of diagnosis and/or prediction includes but not limited to have the scleroderma (being characterized as for example kernel and other antinuclear antibodies usually) of anticol original antibody, mixed connective tissue disease's (being characterized as usually) for example at the antibody that can extract nuclear antigen (for example nucleoprotein), polymyositis (being characterized as for example nonhistones ANA usually), pernicious anemia (is characterized as for example anti-parietal cell usually, microsome and intrinsic factor antibody), the special property sent out Addison's disease (being characterized as for example body fluid and cell-mediated adrenal cells toxicity usually), infertility (being characterized as for example antisperm antibody usually), glomerulonephritis (being characterized as for example GBM Antibody or immunocomplex usually), bulla pemphigoid (being characterized as IgG and complement in the basilar membrane for example usually), the Si Yegelun syndrome (is characterized as for example antibody of organizing usually, and/or the nonhistones ANA of specificity (SS-B)), diabetes (be characterized as usually for example cell-mediated with body fluid ICA), with adrenergic resistance (the adrenergic resistance that comprises concomitant asthma or cystic fibrosis) (being characterized as for example B-adrenergic receptor antibody usually).

The polynucleotide treatment that may have the albumin fusion proteins available of the present invention and/or the code book invention albumin fusion proteins of autoimmunization factor, prevention, other disorder of diagnosis and/or prediction includes but not limited to chronic active hepatitis (being characterized as for example smooth muscle antibody (SMA) usually), primary gall-bladder sclerosis (being characterized as for example mitochondrial antibody usually), other endocrine gland failure (in some cases, being characterized as for example particular organization's antibody usually), vitiligo (being characterized as for example melanocyte antibody usually), vasculitis (being characterized as Ig and complement and/or low serum complement in the tube wall for example usually), back MI (being characterized as for example heart antibody usually), cardiotomy syndromes (being characterized as for example heart antibody usually), urticaria (for example being characterized as IgG and IgM antibody usually) at IgE, atopic dermatitis (for example being characterized as IgG and IgM antibody usually) at IgE, asthma (for example being characterized as IgG and IgM antibody usually) at IgE, with many other inflammatories, granulomatous, degenerative and atrophic disorder.

In a preferred embodiment, use the polynucleotide of fusion rotein for example of the present invention and/or code book invention albumin fusion proteins to treat, prevent, diagnose and/or prediction and above-mentioned disease and disorderly relevant autoimmune disorder and disorder and/or situation.In a concrete preferred embodiment, use the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins treat, prevent and/diagnostics classes rheumatic arthritis.

In another concrete preferred embodiment, use the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins treat, prevent and/the diagnositc system lupus erythematosus.In another concrete preferred embodiment, use the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins treat, prevent and/the diagnosis idiopathic thrombocytopenic purpura.

In another concrete preferred embodiment, use the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins treat, prevent and/the diagnosis of iga ephrosis.

In a preferred embodiment, use the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins to treat, prevent, diagnose and/or prediction and above-mentioned disease and disorderly relevant autoimmune disorder and disorder and/or situation.

In preferred embodiments, the polynucleotide with fusion rotein of the present invention and/or code book invention albumin fusion proteins are used as immunosuppressor.

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for treating, prevent, predicting and/or diagnose disease, disorder and/or the situation of hematopoietic cell.The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are used in treatment or prevention reduces relevant those diseases, disorder and/or situation with some (perhaps many) type hematopoietic cell, include but not limited in oligoleukocythemia, neutrophilic granulocyte minimizing, anaemia and the thrombocytopenic effort, strengthen differentiation and propagation that hematopoietic cell comprises multipotential stem cell.Perhaps, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins are used in treatment or prevention increases relevant those diseases, disorder and/or situation with some (perhaps many) type hematopoietic cell, include but not limited in the effort of histiocytosis, strengthen differentiation and propagation that hematopoietic cell comprises multipotential stem cell.

Transformation reactions and situation are treated, prevent, diagnose and/or predicted to the polynucleotide of also available fusion rotein of the present invention and/or code book invention albumin fusion proteins, such as asthma (particularly atopic asthma) or other respiratory problems.In addition, these molecules can be used for treating, prevent, predicting and/or diagnose supersensitivity, incompatible to the hypersensitivity or the blood group of antigenicity molecule.

In addition, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins can be used for treating, prevent, diagnosing and/or predict the transformation reactions of IgE mediation.Such transformation reactions includes but not limited to asthma, rhinitis and eczema.In specific embodiment, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins are used in external or regulate IgE concentration in vivo.

In addition, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins can be used for diagnosing, predict, prevent and/or treat the inflammatory situation.For example, because the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins can suppress to relate to activation, propagation and/or the differentiation of the cell of inflammatory response, thereby these molecules can be used for preventing and/or treating chronic and the acute inflammation situation.These inflammatory situations include but not limited to for example excessively generate with the hyperacute rejection, ephritis, cytokine or the chemokine inductive pulmonary lesion that infect relevant inflammation (for example septic shock, septicemia or systemic inflammatory response syndrome), ischemia-reperfusion injury, intracellular toxin lethality, complement-mediated, inflammatory bowel, Crohn disease, cytokine (for example TNF or IL-1), respiratory disorder (for example asthma and transformation reactions); Gastrointestinal dysfunction (for example inflammatory bowel); Cancer (for example stomach, ovary, lung, bladder, liver and breast); CNS disorder (multiple sclerosis for example; Ischemic brain injury and/or apoplexy, traumatic brain injury, nerve degenerative diseases (for example Parkinson's disease and Alzheimer); AIDS is relevant dull-witted; And prion disease); Cardiovascular disorder (for example atherosclerosis, myocarditis, cardiovascular diseases and cardiopulmonary bypass complication); And be many other diseases, situation and the disorder (for example hepatitis, rheumatoid arthritis, gout, wound, pancreatitis, sarcoidosis, dermatitis, renal ischaemia-reperfusion injury, Graves' disease, systemic lupus erythematous, diabetes and allograft rejection) of feature with the inflammation.

Because inflammation is basic defense mechanism, so the inflammatory disorder in fact can influence any tissue of body.Therefore, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins can be used for treated tissue specific inflammation sexual disorder, include but not limited to paranephritis, pulmonary alveolitis (alveolitis), angiocholecystitis, ecphyaditis, balanitis, blepharitis, bronchitis, bursitis, carditis, phlegmon, cervicitis, cholecystitis, chorditis, cochleitis, colitis, conjunctivitis, urocystitis, dermatitis, diverticulitis, encephalitis, endocarditis, esophagitis, syringitis, fibrositis, folliculitis, gastritis, gastro-enteritis, oulitis, glossitis, hepatosplenitis, keratitis, labyrinthitis, laryngitis, lymphangitis, mazoitis, otitis media, meningitis, metritis, mucositis, myocarditis, myositis, myringitis, ephritis, neuritis, testitis, osteochondritis, otitis, pericarditis, tenosynovitis, peritonitis, pharyngitis, phlebitis, poliomyelitis, prostatitis, pulpitis, the retinitis, rhinitis, salpingitis, scleritis, sclerochoroiditis, scrotitis, sinusitis, spondylitis, pimelitis, stomatitis, synovitis, syringitis, tendonitis, tonsillitis, urethritis, and vaginitis.

In specific embodiment, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins can be used for diagnosing, predict, prevent and/or treat organ-graft refection and graft versus host disease (GVH disease).The organ rejection destroys transplanted tissue by the host immune cell through immune response and takes place.Similarly, GVHD also relates to immune response, but in the case, external transplantation immunity cytoclasis host tissue.Suppress effective methods of treatment that immune response, the particularly activation of T cell, propagation, differentiation or chemotactic polypeptide of the present invention, antibody or polynucleotide and/or its agonist or antagonist may be prevention organ rejection or GVHD.In specific embodiment, the polynucleotide that suppress immune response, the particularly activation of T cell, propagation, differentiation or chemotactic fusion rotein of the present invention and/or code book invention albumin fusion proteins may be effective methods of treatment that prophylactic tria allergic and super acute heterograft repel.

In other embodiments, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins can be used for diagnosing, predicting, prevent and/or treat immune-complex disease (ICD), include but not limited to serum sickness, post-streptococcal glomerulonephritis, polyarteritis nodosa and immunocomplex inductive vasculitis.

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for treatment, detection and/or the preventing infection factor.For example,, particularly strengthen propagation, activation and/or the differentiation of B and/or T cell, can treat, detect and/or keep off infection by the enhancing immunity reaction.Immune response can be by strengthening existing immune response or strengthening by starting new immune response.Perhaps, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins also can directly suppress infectant (listing the part of infectant etc. in the application reference book), and need not to cause immune response.

In another embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are used for strengthening at antigenic immune responsiveness as vaccine adjuvant.In a specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are used to strengthen the tumour-specific immune response as adjuvant.

In another specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are used to strengthen the antiviral immunity reaction as adjuvant.Can use composition of the present invention as adjuvant and enhanced antiviral immunity reaction comprises virus and viral relative disease or symptom described herein or that other approach of this area is known.In specific embodiment, composition of the present invention is used to strengthen immune response at the virus, disease or the symptom that are selected from down group as adjuvant: AIDS, meningitis, singapore hemorrhagic fever, EBV and hepatitis (for example hepatitis B).In another specific embodiment, composition of the present invention is used to strengthen immune response at the virus, disease or the symptom that are selected from down group as adjuvant: HIV/AIDS, respiratory syncytial virus, dengue fever virus, rotavirus, Type B Japanese encephalitis, A and Type B influenza, parainfluenza, measles, cytomegalovirus, rabies, Junin virus (Junin), chikungunya disease (Chikungunya), Rift valley fever (Rift Valley Fever), herpes simplex and yellow jack.

In another specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are used to strengthen antibacterium or antifungal immunity reaction as adjuvant.Available composition of the present invention is as adjuvant and the reaction of enhanced antibacterium or antifungal immunity comprises bacterium or fungi or bacterium and fungi bacterium or fungi relative disease or symptom described herein or that other approach of this area is known.In specific embodiment, composition of the present invention is used to strengthen at the bacterium that is selected from down group or the immune response of fungi, disease or symptom as adjuvant: tetanus, diphtheria, sausage poisoning and Type B meningitis.

In another specific embodiment, composition of the present invention is used to strengthen bacterium or the fungi of organizing at being selected from down as adjuvant, the immune response of disease or symptom: vibrio cholerae (Vibrio cholerae), Mycobacterium leprae (Mycobacterium leprae), salmonella typhi (Salmonella typhi), salmonella paratyphi (Salmonella paratyphi), Neisseria meningitidis (Neisseriameningitidis), streptococcus pneumoniae (Streptococcus pneumoniae), the B group B streptococcus B, Shigellae (Shigella spp.), enterotoxigenic dust Xi Shi intestinal bacteria (Escherichia coli), enterohemorrhagic Escherichia coli, and B. burgdorferi (Borrelia burgdorferi).

In another specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are used to strengthen the antiparasitic immunity reaction as adjuvant.Available composition of the present invention is as adjuvant and enhanced antiparasitic immunity reaction comprises parasite and parasite relative disease or symptom described herein or that other approach of this area is known.In specific embodiment, composition of the present invention is used for strengthening at parasitic immune response as adjuvant.In another specific embodiment, composition of the present invention is used for the immune response of enhancing at plasmodium (malaria) or Leishmania as adjuvant.

In another specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins also can be used for treating transmissible disease, comprise silicosis, sarcoidosis and idiopathic pulmonary fibrosis, for example by stoping raising and activating of mononuclear phagocyte.

In another specific 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 to generate antibody as antigen, to suppress or to strengthen immune-mediated reaction at polypeptide of the present invention.

In one embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are applied to animal (mouse for example, rat, rabbit, hamster, cavy, pig, miniature pig, chicken, camel, goat, horse, ox, sheep, dog, cat, non-human primates, and people, optimum is chosen) produce one or more antibody (IgG for example of greater amt with the enhancing immunity system, IgA, IgM and IgE), induce the antibody that produces high-affinity more and immunoglobulin class conversion (IgG for example, IgA, IgM and IgE), and/or the enhancing immunity reaction.

In another specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are used as the stimulator at the B cellular response of pathogenic agent.

In another specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are as the activator of T cell.

In another specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are as the medicament that promotes its immune state before individuality is accepted immunosuppressant therapy.

In another specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are as inducing the more medicament of high-affinity antibody.

In another specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are as the medicament that improves serum immune globulin concentration.

In another specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are as accelerating the medicament that immunocompromised individuals is recovered.

In another specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are as the medicament that strengthens the reaction of old group and/or neonatal immunity.

In another specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins as bone marrow transplantation and/or other transplanting (for example allogeneic or xenotransplant) before, during or immune-system enhancers afterwards.For transplanting, composition of the present invention can be before transplanting, use simultaneously and/or afterwards.In a specific embodiment, composition of the present invention after transplanting, the T cell colony uses before beginning to recover.In another specific embodiment, composition of the present invention but is used before the B cell colony recovers fully at first after T cell colony after the transplanting begins to recover.

In another specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are used as the medicament of enhancing immunity responsiveness in the individuality with acquired B cell function forfeiture.The situation of the acquired B cell function forfeiture that causes improving by the polynucleotide of using albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins or to treat includes but not limited to HIV infection, AIDS, bone marrow transplantation and B cell lymphocytic leukemia (CLL).

In another specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are used as the medicament of enhancing immunity responsiveness in having the individuality of temporary immune deficiency.Cause the situation of the temporary immune deficiency that can improve by the polynucleotide of using albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins or treat include but not limited to from virus infection (for example influenza) recover, with malnutritive relevant situation, from infectious mononucleosis recoverys, pressure-dependent situation, from measles recover, from the blood transfusion recovery, reach from surgery recovery.

In another specific embodiment, the conditioning agent that the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are presented as monocyte, dendritic cell and/or B cell antigen.In one embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins external or in vivo enhancement antigen present or the antagonism antigen presentation.In addition, in related embodiment, the enhancing of this antigen presentation or antagonism can be used as antineoplaston or are used to regulate immunity system.

In another specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins as guiding individual immunity system to humoral response (being TH2) but not the medicament of TH1 cell response development.

In another specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are used as the means of induced tumor propagation, thereby make it more to be subject to the influence of anti-superfluous crude drug.For example, multiple myeloma is slow splitted disease, and therefore in fact all anti-superfluous treatment plans of giving birth to all are difficult to control.If force the faster propagation of these cells, their susceptible spectrum changes probably so.

In another specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are as the stimulant that generates such as B cell in the pathology such as AIDS, chronic lymphocytic disorder and/or common variable immunodeficiency.

In another specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins with operate, Lymphoid tissue produces and/or the regenerated methods of treatment after wound or the hereditary defect.In another specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are used to transplant preceding marrow The pretreatment.

In another specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are as the methods of treatment based on gene that causes such as the congenital heredity sexual disorder of viewed immune incapability/immune deficiency among the SCID patient.

In another specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are resisted the monocytic parasitosis of influence such as leishmanial means as activated mononuclear cell/scavenger cell.

In another specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are as the means of regulating the secretion sexual cell factor that is caused by polypeptide of the present invention.

In another embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are used for one or more application described herein, can be used for for animals as them.

In another specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are as the means of blocking-up at adventitious agents or the immune response different aspect of self.The example that may wish to block the disease of some aspect of immune response wherein or situation comprise autoimmune disorder such as lupus and sacroiliitis and to the immune responsiveness of skin allergic reaction, inflammation, enteropathy, damage and with pathogenic agent diseases associated/disorder.

In another specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are as stoping B cell proliferation and the Ig excretory methods of treatment relevant with autoimmune disease such as idiopathic thrombocytopenic purpura, systemic lupus erythematous and multiple sclerosis.

In another specific embodiment, polypeptide, antibody, polynucleotide and/or the agonist of the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins or antagonist are as the inhibitor of B in the endotheliocyte and/or T cell migration.This active disorganize framework or similar reaction, and can be used for for example destroying immune response and stop septicemia.

In another specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins as such as not qualitative MG ((monoclonal gammopathy of undetermined significance, MGUS), waldenstrom's disease (Waldenstrom ' s disease), the relevant special methods of treatment of sending out tangible chronic hypergammaglobulinemia in the diseases such as property MG and plasmoma.

In another specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for for example suppressing the activation of polypeptide chemotaxis and scavenger cell and precursor, neutrophilic granulocyte, basophilic granulocyte, bone-marrow-derived lymphocyte and some T cell subclass such as activatory and cd8 cell toxicity T cell and natural killer cell in some autoimmunity and chronic inflammatory and communicable disease.This paper has described the example of autoimmune disease, comprises multiple sclerosis and insulin-dependent diabetes.

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can also be treated the high eosinophile granulocyte syndromes of the special property sent out by the generation and the migration that for example stop eosinophile granulocyte.

In another specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are used to strengthen or suppress the cytolysis of complement-mediated.

In another specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are used for strengthening or suppressing antibody dependent cellular cytotoxicity.

In another specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins also can be used for treating atherosclerosis, for example by stoping the monocyte infiltration in the arterial wall.

In another specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for treating adult respiratory distress syndrome (ARDS).

In another specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for stimulating wound and tissue repair, stimulation blood vessel to take place and/or stimulate blood vessel or lymphatic vessel disease or disorderly reparation.In addition, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins also can be used for the regeneration on stimulating mucosal surface.

In a specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are used to diagnose, predict, treat and/or prevent the disorder that is characterized as primary or acquired immunodeficiency, serum immune globulin generation deficiency, recurrent infection and/or function of immune system obstacle.In addition, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins can be used for treatment or prevention joint, bone, skin, and/or the infection of the parotid gland, the blood matchmaker infects (septicemia for example, meningitis, septic arthritis, and/or osteomyelitis), autoimmune disease (for example disclosed herein), the inflammatory disorder, and malignant tumour, and/or with these infection, disease, any disease that disorder and/or malignant tumour are relevant or disorder or situation include but not limited to CVID, other primary immunodeficiency, the HIV disease, CLL, the recurrent bronchitis, sinusitis, otitis media, conjunctivitis, pneumonia, hepatitis, meningitis, zoster (for example serious zoster), and/or Pneumocystis carinii (Pneumocystiscarnii).Other disease and the disorder of polynucleotide prevention, diagnosis, prediction and/or the treatment of available fusion rotein of the present invention and/or code book invention albumin fusion proteins include but not limited to HIV infection, HTLV-BLV infection, lymphopenia, phagocyte bactericidal dysfunction anaemia, thrombopenia and hemoglobinuria.

In another embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are used for the treatment of and/or diagnose and have common variable immunodeficiency (" CVID "; Be also referred to as " acquired agammaglobulinemia disease " and " acquired hypogammaglobulinemia ") or the individuality of the subclass of this disease.

In a specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for diagnosing, predicting, prevent and/or treat cancer or vegetation, comprise immunocyte or immuning tissue's associated cancer or vegetation.The polynucleotide prevention of available fusion rotein of the present invention and/or code book invention albumin fusion proteins, the cancer or the excrescent example of diagnosis or treatment include but not limited to acute myeloid leukaemia, chronic myelogenous leukemia, lymphogranulomatosis, the non-Hodgkin lymphomas, acute lymphocytic anemia (ALL), lymphocytic leukemia, plasmoma, multiple myeloma, Bai Jiteshi (Burkitt ' s) lymphoma, the disease that EBV transforms, and/or other local title of this paper is disease and disorder that " hyper-proliferative sexual disorder " part is described.

In another specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are as the methods of treatment that reduces large-scale B cell lymphoma cell proliferation.

In another specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are as reducing B cell relevant with chronic myelogenous leukemia and the means that involve of Ig.

In specific embodiment, composition of the present invention as B cellular immunity deficiency individuality such as the individuality of for example having accepted part or all of splenectomy in the medicament of enhancing immunity responsiveness.

The blood associated disorders

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for regulating hemostasis (stopping hemorrhage) or thrombolysis (clot dissolution) activity.For example, by strengthening hemostasis or thrombolysis activity, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins can be used for treatment or preclude blood condense disease, disorder and/or situation (for example afibrinogenemia, the factor lack, hemophilia), blood platelet disorder, disorder and/or situation (for example thrombopenia) or come from the damage of wound, operation or other reason.Perhaps, can reduce the fusion rotein of the present invention of hemostasis or thrombolysis activity and/or the polynucleotide of code book invention albumin fusion proteins can be used for suppressing or the dissolving grumeleuse.These molecules can play an important role in heart attack (infarct), apoplexy or the treatment that scabs or prevention.

In specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for prevention, diagnosis, prediction and/or treatment thrombosis, artery thrombosis, venous thrombosis, thromboembolism, pulmonary embolisms, atherosclerosis, myocardial infarction, transient ischemic attack, unstable angina pectoris.In specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for preventing saphenous vein graft obturation (occulsion of saphenous grafts), reduce the peri-operation period thrombosis risk that may follow with angioplasty, reduce stroke risk, reduction and heart valve prosthesis and/or the relevant embolism risk of mitral valve disease that atrial fibrillation comprises non-rheumatic atrial fibrillation patient.Other purposes of the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins includes but not limited to prevent health to install obstruction in (for example the vascular access part flow arrangement in blood vessel inner sleeve, the hemodialysis patients, haemodialysis equipment and cardiopulmonary bypass device) outward.

In another embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for preventing, diagnose, predict and/or treat disease and the disorder that the relevant blood of the tissue of expressing with polypeptide of the present invention wherein and/or blood form organ.

The polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins can be used for regulating hematopoiesis activity (formation of hemocyte).For example, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for increasing all hemocytes or hemocyte subclass such as for example red corpuscle, lymphocyte (B or T cell), medullary cell (for example basophilic cell, eosinocyte, neutrophil(e) cell, mastocyte, scavenger cell) and hematoblastic quantity.The ability that reduces hemocyte or hemocyte subclass quantity can be used for preventing, detect, diagnose and/or treating anaemia described below and oligoleukocythemia.Perhaps, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for reducing all hemocytes or hemocyte subclass such as for example red corpuscle, lymphocyte (B or T cell), medullary cell (for example basophilic cell, eosinocyte, neutrophil(e) cell, mastocyte, scavenger cell) and hematoblastic quantity.The ability that reduces hemocyte or hemocyte subclass quantity can be used for prevention, detects, diagnoses and/or treats leukocytosis such as for example eosinophilia.

The polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins can be used for prevention, treatment or diagnosis blood cachexy.

Anaemia is that red corpuscle number or oxyphorase wherein (carrying the protein of oxygen) quantity are lower than the situation of normal level.Anaemia may be reduced or hematoclasis (haemolysis) increases and causes by excessively hemorrhage, erythropoiesis.The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for the treatment, the prevention and/diagnosis anaemia.The polynucleotide treatment of available albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins, the anaemia of prevention or diagnosis comprises hypoferric anemia, anemia with low hemoglobin, microcytic anemia, chlorosis (chlorosis), the heredity sideroblastic anemia, the acquired sideroblastic anemia of the special property sent out, red blood cell development is incomplete, megaloblastic anemia (pernicious anemia for example, (vitamin B12 deficiency) and folic acid deficiency anemia), aplastic anemia, hemolytic anemia (autoimmune hemolytic anemia for example, microangiopathic hemolytic anemia, and paroxysmal nocturnal hemoglobinuria).The anaemia that the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for treating, prevent and/or diagnosis is relevant with following disease, described disease includes but not limited to and systemic lupus erythematous, cancer, lymphoma, chronic nephropathy and splenauxe relevant anaemia.The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for treating, preventing and/or diagnose the anaemia that is caused by pharmacological agent, such as the anaemia relevant with methyldopa, dapsone and/or sulfa drug.In addition, the anaemia that the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins can be used for treating, prevent and/or diagnosis is relevant with the abnormal erythrocyte framework includes but not limited to that the heredity spherule cell increases, the heredity cameloid cell increases, glucose-6-phosphate dehydrogenase (G6PD) defective and sicklemia.

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for treatment, prevention and/or diagnosis haemoglobin anomaly (for example relevant with sicklemia, Hemoglobin C disease, Hemoglobin S-C disease and Hemoglobin E disease).In addition, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for diagnosing, predict, prevent and/or treat thalassemia (thalassemia), include but not limited to heavy and light-duty α-Di Zhonghaipinxue and β-thalassemia.

In another embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for diagnosis, prediction, prevent and/or treat the hemorrhagic disorder, include but not limited to thrombopenia (idiopathic thrombocytopenic purpura for example, with the thrombus essential thrombopenia), Feng's von Willebrand disease, (storage pool disease for example is such as Qie-Xi two syndromes and He-Pu two syndromes for the heredity thrombopathia, the thromboxane A2 dysfunction, Thrombasthenia, and Bai-Su two syndromes), the hemolytic uremic syndrome syndromes, hemophilia such as hemophilia A or factor VII deficiency and Ji Shi disease (hemophilia B) or factors IX lack, Osler Weber Rendu, be also referred to as Lang-Ao-Wei three syndromes (Rendu-Osler-Weber syndrome), condense in anaphylactoid purpura (prosperous-permitted Er Shi purpura (Henoch Schonlein purpura)) and the diffusive blood vessel.

Can use any blood coagulation method of testing known in the art to monitor of the influence of the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins, include but not limited to whole blood partial thromboplastin time (PTT), activated partial thromboplastin time (aPTT), activated clotting time (ACT), multiple calcification activated clotting time or Lee-White clotting time blood coagulating time.

Several diseases and multiple medicine can cause dysfunction of platelet.Therefore, in a specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for diagnosis, prediction, 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 of systemic lupus erythematous, comprise Asprin with high dosage with pharmacological agent, ticlopidine, non-steroid antiphlogiston (is used for sacroiliitis, pain, with sprain), the treatment of carrying out with penicillin.

In another embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for diagnosing, predict, prevent and/or treat and be characterized as that the white corpuscle number increases or reduce or diseases associated and disorder with it.Leukopenia takes place when being lower than normal level when the white corpuscle number is reduced to.Oligoleukocythemia includes but not limited to that neutrophil reduces and lymphopenia.The white corpuscle number is called leukocytosis with respect to the increase of normal level.Body produces the white corpuscle that number increases between period of infection.Therefore, leukocytosis may only be the normal physiological mathematic(al) parameter that reflection is infected.Perhaps, leukocytosis may be the index of damage or other diseases such as cancer.Leukocytosis includes but not limited to eosinophilia disease and scavenger cell accumulation.In specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for diagnosing, predict, prevent and/or treat oligoleukocythemia.In other specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for diagnosing, predict, prevent and/or treat leukocytosis.

Oligoleukocythemia can be the leukocytic generally minimizing of all types, perhaps can be the leukocytic special loss of particular type.Therefore, in specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for diagnosing, predict, prevent and/or treat the neutrophilic granulocyte reduced number that is called the neutrophil minimizing.The polynucleotide diagnosis of available albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins, prediction, the neutrophil minimizing that prevents and/or treats includes but not limited to infant's heredity agranulocytosis, the familial neutrophil reduces, periodically neutrophil reduces, come from diet and lack (for example vitamin B12 deficiency or folic acid deficiency) or relevant with it neutrophil minimizing, come from pharmacological agent (regimen of antibiotics for example, treat such as penicillin, the sulfonamide treatment, the anti-coagulant treatment, anticonvulsant drug, antithyroid drug and cancer chemotherapy) or relevant with it neutrophil minimizing, with come from the neutropenia that neutrophilic granulocyte destroy to increase, neutrophilic granulocyte destroy increase may with some bacteriums or virus infection, allergic disorder, autoimmune disease, splenomegaly (Fil base of a fruit syndromes (Felty syndrome) for example, malaria and sarcoidosis) individual situation, relevant with some therapeutic schemes.

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for diagnosis, prediction, prevent and/or treat lymphopenia (B and/or T lymphocyte number reduce), include but not limited to come from pressure, pharmacological agent (for example corticosteroid medication treatment, cancer chemotherapy, and/or radiotherapy), AIDS infection and/or other disease are such as for example cancer, rheumatoid arthritis, systemic lupus erythematous, chronic infection, some virus infectiones and/or heredity disorder (enlightening George syndromes for example, Wei-Ao two syndromes (Qiskott-Aldrich syndrome), serious compressibility immune deficiency, ataxia-telangiectasia) or relevant with it lymphopenia.

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for diagnosing, predict, prevent and/or treat and scavenger cell number and/or macrophage function diseases associated and disorder, include but not limited to familial splenic anemia (Gaucher ' s disease), niemann-Pick disease (Niemann-Pick disease), letterer-Siwe disease (Letterer-Siwe disease) and Hand-Schuiller-Christian Disease (Hand-Schuller-Christian disease).

In another embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for diagnosing, predict, prevent and/or treat with the eosinophile granulocyte number and/eosinophile granulocyte function diseases associated and disorder, include but not limited to that the special property eosinophile granulocyte of sending out increases syndromes (idiopathic hypereosinophilic syndrome), eosinophile granulocyte-myalgia syndromes and Hand-Schuiller-Christian Disease (Hand-Schuller-Christian disease).

In another embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for diagnosis, prediction, prevent and/or treat leukemia and lymphoma, include but not limited to acute lymphocytic (lymphocytoblast property) leukemia (ALL), acute myeloid sample (myeloid, marrow, myeloblast property, or bone marrow mononuclear cell) leukemia, lymphocytic leukemia (B cell leukemia for example, the T chronic myeloid leukemia, the Sai Zhali syndromes, and hairy cell), chronic myeloid (myelocyte sample, marrow, or granulocytic) leukemia, hodgkin's lymphomas, the non-Hodgkin lymphomas, burkitt's lymphoma, and cutaneous T cell lymphoma.

In other embodiments, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for diagnosing, predicting, prevent and/or treat plasmacytic disease and disorder, include but not limited to plasma cell dyscrasia, MG, not qualitative MG, multiple myeloma, macroglobulinemia, Walden Si Telunshi macroglobulinemia, cryoglobulinemia and Lei Nuoshi (Raynaud ' s) phenomenon.

In other embodiments, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for treatment, prevention and/or diagnosis bone marrow proliferation sexual disorder, include but not limited to polycythemia vera, relative polycythemia, secondary polycythemia, myelofibrosis, acute myelofibrosis, the life of agnogenic medullization, thrombocythemia (comprising primary and Secondary cases thrombocythemia) and chronic granulocytic leukemia.

In other embodiments, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for preoperative processing to increase hemopoietic.

In other embodiments, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used as that the migration, phagolysis, the super-oxide that strengthen neutrophilic granulocyte, eosinophile granulocyte and scavenger cell generate, the medicament of antibody dependent cellular cytotoxicity.

In other embodiments, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used as the medicament that increases stem cell number in the circulation before stem cell is extracted.In another specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used as the medicament that increases stem cell number in the circulation before thrombocyte extracts.

In other embodiments, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used as increases the medicament that cytokine generates.

In other embodiments, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for prevention, diagnosis and/or treatment primary hematopoiesis disorder.

The hyper-proliferative sexual disorder

In certain embodiments, the polynucleotide of fusion rotein of the present invention and/or albumin fusion proteins of the present invention can be used for treatment or detect the hyper-proliferative sexual disorder, comprise vegetation.The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can suppress disorderly diffusion (proliferation) by directly or indirectly interacting.Perhaps, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins can enable to suppress other cell proliferation of hyper-proliferative sexual disorder.

For example, can particularly improve the antigenic quality of hyper-proliferative sexual disorder, perhaps, treat the hyper-proliferative sexual disorder by making T cell proliferation, differentiation or mobilizing by improving immunne response.Or by the existing immunne response of enhancing, or, can improve this immunne response by starting new immunne response.Perhaps, reduce the method that immunne response is also treated the hyper-proliferative sexual disorder, such as chemotherapeutics.

The example of the polynucleotide treatment of available fusion rotein of the present invention and/or code book invention albumin fusion proteins or the hyper-proliferative sexual disorder that detects includes but not limited to be positioned at the vegetation at following position: colon, belly, bone, breast, Digestive tract, liver, pancreas, peritonaeum, incretory gland (suprarenal gland, Parathyroid, hypophysis, testis, ovary, thymus gland, Tiroidina), eye, head and neck, neural (maincenter and on every side), lymphsystem, pelvis, skin, soft tissue, spleen, the thoracic cavity, and urogenital tract.

Similarly, the polynucleotide of also available fusion rotein of the present invention of other hyper-proliferative sexual disorder and/or code book invention albumin fusion proteins are treated or are detected.The example of these hyper-proliferative sexual disorders includes but not limited to: the lymphoblastic leukemia Childhood of acute, acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, adult's (primary) hepatocellular carcinoma, adult's (primary) liver cancer, adult's acute lymphoblastic leukemia, adult's acute myeloid leukemia, adult's lymphogranulomatosis, adult's hodgkin's lymphomas, adult lymphoid cellularity leukemia, adult's non-Hodgkin lymphomas, become the human primary liver cancer, adult soft tissue sarcoma, the AIDS lymphoma of being correlated with, the AIDS associated malignancies, anus cancer, astrocytoma, cholangiocarcinoma, bladder cancer, osteocarcinoma, the brain stem neurospongioma, brain tumor, breast cancer, renal plevis and carcinoma of ureter, central nervous system (primary) lymphoma, central nervous system lymphoma, cerebellar astrocytoma, big cerebral astrocytoma, cervical cancer, the Childhood (primary) hepatocellular carcinoma, the Childhood (primary) liver cancer, the Childhood acute lymphoblastic leukemia, the Childhood acute myeloid leukemia, the Childhood brain stem neurospongioma, the Childhood cerebellar astrocytoma, the Childhood big cerebral astrocytoma, the Childhood extracranial germ cell knurl, the Childhood lymphogranulomatosis, the Childhood hodgkin's lymphomas, the Childhood hypothalamus and look the road neurospongioma, the Childhood lymphocytoblast leukemia, the Childhood medulloblastoma, the Childhood non-Hodgkin lymphomas, the Childhood pineal gland and curtain go up an original neuroectodermal tumor, the Childhood primary hepatocarcinoma, the Childhood rhabdosarcoma, the Childhood soft tissue sarcoma, the Childhood look road and hypothalamus neurospongioma, lymphocytic leukemia, chronic granulocytic leukemia, colorectal carcinoma, cutaneous T cell lymphoma, the internal secretion islet-cell carcinoma, carcinoma of endometrium, ependymoma, epithelial cancer, the esophageal carcinoma, ewing's sarcoma and related neoplasms, the exocrine pancreas cancer, the extracranial germ cell knurl, the outer sexual cell knurl of sexual gland, cholangiocarcinoma, cancer eye, women's breast cancer, familial splenic anemia, carcinoma of gallbladder, cancer of the stomach, the stomach and intestine carcinoid tumor, the stomach and intestine knurl, gonioma, gestational trophoblastic neoplasms, hairy cell, head and neck cancer, hepatocellular carcinoma, lymphogranulomatosis, hodgkin's lymphomas, hypergammaglobulinemia, hypopharyngeal cancer, intestinal cancer, intraocular melanoma, islet-cell carcinoma, the islet cells carcinoma of the pancreas, Kaposi sarcoma, kidney, laryngocarcinoma, lip and oral carcinoma, liver cancer, lung cancer, the lymphocytic hyperplasia sexual disorder, macroglobulinemia, women's breast cancer, malignant mesothe, malignant thymoma, medulloblastoma, melanoma, mesothelioma, the disguised primary squamous cell of transitivity neck cancer, transitivity primary squamous cell neck cancer, transitivity squamous cell neck cancer, multiple myeloma, multiple myeloma/plasmocyte vegetation, myelodysplastic syndrome, myelocytic leukemia, myelocyte sample leukemia, the bone marrow proliferation sexual disorder, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, the Gestation period non-Hodgkin lymphomas, non-melanoma skin cancer, nonsmall-cell lung cancer, disguised primary transitivity squamous cell neck cancer, the oropharynx cancer, bone-/the malignant fibrous sarcoma, osteosarcoma/malignant fibrous histiocytoma, the malignant fibrous histiocytoma of osteosarcoma/bone, epithelial ovarian cancer, the ovarian germ cell knurl, the rudimentary pernicious potential tumor of ovary, carcinoma of the pancreas, paraproteinemia, purpura, the Parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumor, plasmocyte vegetation/multiple myeloma, primary central nervous system lymphoma, primary hepatocarcinoma, prostate cancer, the rectum cancer, renal cell carcinoma, renal plevis and carcinoma of ureter, retinoblastoma, rhabdosarcoma, salivary-gland carcinoma, the sarcoidosis sarcoma, the Sai Zhali syndromes, skin carcinoma, small cell lung cancer, carcinoma of small intestine, soft tissue sarcoma, squamous cell neck cancer, cancer of the stomach, original neuroderm and pinealoma on the curtain, t cell lymphoma, carcinoma of testis, thymoma, thyroid carcinoma, renal plevis and ureteral transitional cell carcinoma, transition type renal plevis and carcinoma of ureter, trophoblastic tumor, ureter and renal plevis cell carcinoma, urethral carcinoma, uterus carcinoma, sarcoma of uterus, carcinoma of vagina, look road and hypothalamus neurospongioma, the vaginal orifice cancer, Walden Si Telunshi macroglobulinemia, the Wei Ermusishi tumour, and be arranged in above listed tract any other excess proliferative disease except that vegetation.

In another preferred embodiment, situation before the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are used for diagnosis, prediction, prevention and/or treat deterioration, and be used for preventing to develop into vegetation or malignant state, include but not limited to above-described those disorders.These purposes are known or aforementioned status of development to vegetation or cancer needed (indicated) has taken place suspection, particularly hyperplasia, the change that is made of non-neoplastic cell growth given birth to, or the most special other be that (summary about these misgrowth situations can be consulted Robbins and Angell to heteroplasia, 1976, " Basic Pathology ", the 2nd edition, W.B.Saunders company, Philadelphia, pp.68-79).

Hyperplasia is a kind of form of controlled cell propagation, relate to the increase of cell number in tissue or the organ, but structure or function does not have great change.The polynucleotide diagnosis of available fusion rotein of the present invention and/or code book invention albumin fusion proteins, prediction, prevention, and/or the hyperplasia sexual disorder of treatment includes but not limited to blood vessel folliculus mediastinal lymph nodes hyperplasia, blood vessel lymphocytic hyperplasia with eosinophilia, atypical melanocytic hyperplasia, basal cell hyperplasia, optimum giant lymph node hyperplasia, hypercementosis, adrenal,congenital hyperplasia, congenital hyperplasia of sebacous glands, cystic hyperplasia, the cystic hyperplasia of breast, denture hypertrophy, ductal hyperplasia, endometrial hyperplasia, fibromuscular hyperplasia, focal epithelial hyperplasia, gingival hyperplasia, fibrous inflammatory hyperplasia, the inflammatory papillary hyperplasia, intravascular papillary endothelial hyperplasia, prostatic joint knot property hyperplasia, joint knot property regenerative proliferation, pseudoepitheliomatous hyperplasia, senile sebaceous gland hyperplasia, and verrucous hyperplasia.

Changing life is a kind of form of controlled cell growth, wherein a class another kind of sophisticated cell of cell replacement sophisticated or that break up fully.The polynucleotide diagnosis of available fusion rotein of the present invention and/or code book invention albumin fusion proteins, prediction, prevention, and/or the change natural disposition disorder of treatment includes but not limited to the special property sent out myeloid metaplasia, Apocrine metaplasia, atypicalization given birth to, self realize and give birth to (autoparenchymatousmetaplasia), reticular tissueization is given birth to, epithelial metaplasia, intestinesization are given birth to, metaplastic anemia, metaplastic ossification, metaplastic polyp, myeloid metaplasia, the primary myeloid metaplasia, the Secondary cases myeloid metaplasia, squamous metaplasia, the squamous metaplasia of amnion, with Symptomatic myeloid metaplasia.

Heteroplasia usually is the omen of cancer, and mainly finds in epithelium; It is the most of disorderly form of non-neoplastic cell growth, relates to the forfeiture of cell individual consistence and cell structure orientation.The heteroplasia cell usually has unusual large-scale, dense painted nuclear, and shows pleomorphism.Heteroplasia is distinctive to be occurred in the situation that has chronic stimulation (irritation) or inflammation.The polynucleotide diagnosis of available fusion rotein of the present invention and/or code book invention albumin fusion proteins, prediction, prevention, and/or the heteroplasia sexual disorder of treatment includes but not limited to anhidrotic ectodermal dysplasia, anterofacialdysplasia, asphyxiating thoracic dysplasia, atrium finger heteroplasia, bronchopulmonary dysplasia, encephalodysplasia, dysplasia of cervix, chondroectodermal dysplasia, clavicle skull heteroplasia, congenital ectodermal dysplasia, skull is done heteroplasia, cranium wrist instep heteroplasia, cranium metaphysis heteroplasia, dentinal dysplasia, diaphyseal sclerosis, ectodermal dysplasia, enamel heteroplasia, the brain eyeball development is unusual, inclined to one side side seam epiphysis heteroplasia, dysplasia epiphysealis multiplex, stippled epiphyses heteroplasia, epithelial development is unusual, face refers to that (toe) genital development is unusual, the familial fiber heteroplasia of jaw (jaw), familial white fold heteroplasia, fiber flesh sexual abnormality, the fiber heteroplasia of bone, vigorous bone sexual abnormality, heredity kidney retinal development is unusual, the sweat ectodermal dysplasia is arranged, the hypohidrosis ectodermal dysplasia, the lymphopenia thymus development is unusual, mammogenesis is unusual, lower jaw face heteroplasia, metaphysis heteroplasia, Mondini heteroplasia, single bone fiber heteroplasia, mucous epithelium heteroplasia, dysplasia epiphysealis multiplex, oculoauricular dysplasia, eye vertebra heteroplasia, tooth source sexual abnormality, eye ramus of mandible heteroplasia, the all dentinal dysplasias of point, boniness fiber heteroplasia, false cartilage forms backbone epiphyseal dysplasia (pseudoachondroplasticspondyloepiphysial dysplasia), retinal development is unusual, every dysplasia of eye, the backbone epiphyseal dysplasia, with ventricles of the brain radius heteroplasia.

Other superfluous disorder before death of polynucleotide diagnosis, prediction, prevention and/or the treatment of available fusion rotein of the present invention and/or code book invention albumin fusion proteins includes but not limited to optimum abnormality proliferation sexual disorder (as innocent tumour, fibrous capsule proterties condition, tissue hypertrophy, polyp intestinal, polyp of colon and esophagus heteroplasia), leukoplasia, keratosis, bowen's disease, farmer's skin, solar cheilitis and solar keratosis.

In another embodiment, the relevant disorder of tissue that the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for diagnosing and/or prediction and polypeptide of the present invention are expressed therein.

In another embodiment, the albumin fusion proteins of the present invention that as described herein and toxin or radio isotope are puted together and/or the polynucleotide of code book invention albumin fusion proteins can be used for treating cancer and vegetation, include but not limited to described herein.In still another preferred embodiment, the albumin fusion proteins of the present invention puted together of as described herein and toxin or radio isotope and/or the polynucleotide of code book invention albumin fusion proteins can be used for treating acute myelogenous leukemia.

In addition, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins can influence apoptosis, therefore can be used for treating with cell survival enhancing or apoptosis suppressing relevant numerous disease.For example, available polynucleotide of the present invention, polypeptide, and/or agonist or antagonist diagnosis, prediction, prevention, and/or treatment strengthen with cell survival or apoptosis suppresses diseases associated and comprises that cancer is (such as follicular lymphoma, cancer with p53 sudden change, and hormone-dependent tumor, include but not limited to colorectal carcinoma, cardiac tumor, carcinoma of the pancreas, melanoma, retinoblastoma, Glioblastoma, lung cancer, intestinal cancer, carcinoma of testis, cancer of the stomach, neuroblastoma, myxoma, myomata, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi sarcoma, and ovarian cancer), autoimmune disorder is (such as multiple sclerosis, the Si Yegelun syndrome, struma lymphomatosa, cholehepatocirrhosis, Behcet, Crohn disease, polymyositis, systemic lupus erythematous and immunity relevant glomerulonephritis and rheumatoid arthritis) and virus infection (such as simplexvirus, poxvirus, and adenovirus), inflammation, graft versus host disease (GVH disease), acute grafing is got rid of, and chronic transplanting rejection.

In preferred embodiments, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins are used to suppress the growth, development of cancer and/or shift, particularly above cited.

The polynucleotide diagnosis of available fusion rotein of the present invention and/or code book invention albumin fusion proteins, prediction, prevention, and/or treatment strengthen development and/or the transfer that other relevant disease or situation include but not limited to malignant tumour and associated disorders with cell survival, (comprise that acute leukemia is (as acute lymphoblastic leukemia such as leukemia, acute myelocytic leukemia (comprises myeloblast, promyelocyte (promyelocyte), myelomonocyte, monocyte and erythroleukemia)) and chronic leukemia (as chronic myeloid (granulocytic) leukemia and lymphocytic leukemia)), polycythemia vera, lymphoma (as lymphogranulomatosis and Fei Huoqijinshi disease), multiple myeloma, Walden Si Telunshi macroglobulinemia, heavy chain disease, and solid tumor, include but not limited to sarcoma and cancer, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovioma, mesothelioma, the You Wenshi tumour, leiomyosarcoma, rhabdosarcoma, colorectal carcinoma, carcinoma of the pancreas, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, rodent cancer, gland cancer, syringocarcinoma, sebaceous carcinoma, papillary carcinoma, papillary carcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, spermocytoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, neurospongioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic tumor, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

What the polynucleotide of available fusion rotein of the present invention and/or code book invention albumin fusion proteins were diagnosed, predict, prevented and/or treated comprises AIDS with apoptosis enhancing diseases associated; Neurodegenerative disorders (such as Alzheimer, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration and brain tumor or aforementioned relative disease); Autoimmune disorder (such as multiple sclerosis, Si Yegelun syndrome, struma lymphomatosa, cholehepatocirrhosis, Behcet, Crohn disease, polymyositis, systemic lupus erythematous and relevant glomerulonephritis of immunity and rheumatoid arthritis); Development of bone marrow abnormal syndrome (such as aplastic anemia), graft versus host disease (GVH disease), ischemia injury (such as causing), liver injury (as the relevant liver injury of hepatitis, ischemia/reperfusion injury, cholestasis (bile duct injury) and liver cancer) by myocardial infarction, apoplexy and reperfusion injury; The hepatopathy of toxin-induced (such as what cause), septic shock, cachexia and anorexia by alcohol.

The polynucleotide diagnosis of available fusion rotein of the present invention and/or code book invention albumin fusion proteins, prediction, prevention, and/or the excess proliferative disease of treatment and/or the disorderly vegetation that includes but not limited to be positioned at following position: liver, belly, bone, breast, Digestive tract, pancreas, peritonaeum, incretory gland (suprarenal gland, Parathyroid, hypophysis, testis, ovary, thymus gland, Tiroidina), eye, head and neck, neural system (maincenter and on every side), lymphsystem, pelvis, skin, soft tissue, spleen, the thoracic cavity, and urogenital tract.

Similarly, the polynucleotide of also available fusion rotein of the present invention of other hyper-proliferative sexual disorder and/or code book invention albumin fusion proteins are diagnosed, predict, are prevented and/or treat.The example of these hyper-proliferative sexual disorders includes but not limited to: hypergammaglobulinemia, lymphocytic hyperplasia sexual disorder, paraproteinemia, purpura, sarcoidosis, plug are pricked Richter scale syndromes, Walden Si Telunshi macroglobulinemia, familial splenic anemia, histiocytosis and are arranged in above listed tract any other excess proliferative disease except that vegetation.

Another embodiment preferred utilizes the polynucleotide of code book invention albumin fusion proteins to suppress abnormal division, gene therapy of its application of the invention and/or protein blend compound or its fragment.

Thus, by the polynucleotide of code book invention albumin fusion proteins being inserted the cell of abnormality proliferation, wherein said polynucleotide are suppressed described expression, the invention provides the method that is used for the treatment of cell proliferative disorders.

Another embodiment of the invention provides the method that is used at individuality treatment cell proliferative disorders, comprises the cell of abnormality proliferation is used one or more active gene copies of the present invention.In a preferred embodiment, polynucleotide of the present invention are the DNA constructions that comprise recombinant expression vector, the encode dna sequence dna of described polynucleotide of this recombinant expression vector effective expression.In another preferred embodiment of the present invention, utilize retrovirus or preferred adenovirus carrier that the DNA construction insertion cell to be treated of code book invention fusion rotein (is consulted people such as G.J.Nabel, PNAS, 1999,96:324-326 is introduced into this paper as a reference).In a most preferred embodiment, virus vector is a defective type, and can not transform non-proliferative cell, only transforms proliferative cell.In addition, in a preferred embodiment, can regulate and control with the outside stimulus (being magnetic, specific small molecules, chemical preparations or medicament administration etc.) of inducing coded protein to express via the promotor that acts on described polynucleotide upstream then with polynucleotide of the present invention or separately or associating or merge other polynucleotide and insert proliferative cell.So, can clearly regulate and control useful result of treatment of the present invention (promptly improve, reduce or suppress expression of the present invention) according to described outside stimulus.

Polynucleotide of the present invention can be used for suppressing oncogene or antigenic expression." expression of compacting oncogene " means containment genetic transcription, degrading genes transcript (premessenger RNA), suppresses montage, destroys messenger RNA(mRNA), stops the protein translation post-treatment, destroys protein or arrestin matter normal function.

For being locally applied to the abnormality proliferation cell, can use polynucleotide of the present invention by any method that those skilled in the art will know that, include but not limited to the transfection, electroporation, microinjection of cell or in such as media such as liposomes, fat transfection or any other method of describing in full as exposed polynucleotide or specification sheets.Can deliver polynucleotide of the present invention by known gene delivery system, such as, but not limited to retrovirus (Gilboa, J., Virology, 44:845,1982; Hocke, Nature, 320:275,1986; People such as Wilson, Proc.Natl.Acad.Sci.U.S.A., 85:3014), (people such as Chakrabarty of vaccinia virus system, Mol.Cell Biol., 5:3403,1985) or other efficient DNA delivery system (people such as Yates, the Nature that those skilled in the art will know that, 313:812,1985).These reference are exemplary, and are incorporated herein by reference.Avoid nondividing cell for the cell of special delivery or transfection abnormality proliferation, preferred retrovirus or adenovirus (described) delivery system that those skilled in the art will know that adopt as this area and this paper other places.Because the integration of retrovirus DNA needs host DNA to duplicate, and retrovirus for want of its life cycle needed reverse transcription virus gene and can not self-replacation, therefore adopt this retrovirus delivery system will make the cell of described gene and construction target abnormality proliferation for polynucleotide of the present invention, and avoid nondividing normal cell.

Be used to instruct entry needle to arrive the imaging device of disease location just by use, polynucleotide of the present invention directly can be delivered to the cell proliferative disorders/disease location in internal organ, body cavity or the like.Polynucleotide of the present invention also can be applied to disease location when surgical intervention.

" cell proliferation disorders " means invasion and attack any organ, chamber or body portion or its arbitrary combination, is characterized as anyone or Animal diseases or disorder of the single or multiple local abnormality proliferation (no matter being benign or virulent) of cell, cell mass or tissue.

Can use any amount of polynucleotide of the present invention, as long as it has biology inhibition effect to the propagation of treatment cell.In addition, might be applied to same area simultaneously with surpassing a kind of polynucleotide of the present invention." biology inhibition " mean part or growth-inhibiting completely, and the reduction of cell proliferation or growth velocity.Biology suppress dosage can by assessment polynucleotide of the present invention to target in the tissue culture effect of tumor growth and cell cultures in pernicious or abnormal cell growth, the animal, perhaps any other method of knowing of those of ordinary skills is determined.

In addition, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins can be used for suppressing the blood vessel generation of proliferative cell or tissue, or separately as the protein blend compound, or directly or indirectly unite other polypeptide, just as described elsewhere herein.In a most preferred embodiment, described angiogenesis inhibitor effect can be indirectly by for example suppress hematopoiesis, tomour specific cell such as tumor-associated macrophages realizes (consulting Joseph, I.B. wait the people, J.Natl.Cancer Inst., 90 (21): 1648-53,1998, be introduced into this paper as a reference).

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for by apoptosis-induced proliferative cell or the tissue of suppressing.These fusion roteins and/or polynucleotide can or direct or indirect acting on induce the apoptosis of proliferative cell and tissue, for example the activation at dead domain receptor exists, such as the protein (TRAMP) of tumour necrosis factor (TNF) acceptor-1, CD95 (Fas/APO-1), the mediation of TNF acceptor related apoptosis, and TNF apoptosis induction ligand related (TRAIL) acceptor-1 and-2 (consult Schulze-Osthoff, K. wait the people, Eur.J.Biochem., 254 (3): 439-59,1998, be introduced into this paper as a reference).In addition, in another preferred embodiment, these fusion roteins and/or polynucleotide can come apoptosis-induced by other mechanism, such as in other the proteinic activation that activates apoptosis, perhaps by stimulating these protein expressions, or independent or associating small-molecule drug or adjuvant, (consult for example Mutat.Res. such as apoptonin, gala lectin, Trx, anti-inflammatory albumen, 400 (1-2): 447-55,1998; Med.Hypotheses, 50 (5): 423-33,1998; Chem.Biol.Interact., April 24,111-112:23-34,1998; J.Mol.Med., 76 (6): 402-12,1998; Int.J.Tissue React., 20 (1): 3-15,1998, it all is incorporated herein by reference).

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins 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, perhaps indirect, such as activating the protein expression that known inhibition is shifted, for example alpha-4 integrin (is consulted for example Curr.Top.Microbiol.Immunol., 231:125-41,1998, be introduced into this paper as a reference).This result of treatment of the present invention can separately or associating small-molecule drug or adjuvant realize.

In another embodiment, the invention provides the method that the composition that will contain the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins is delivered to the target cell of expression albumin fusion proteins of the present invention institute bonded, its combination or associating polypeptide.Albumin fusion proteins of the present invention can associate via hydrophobicity, wetting ability, 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 the immunogenicity and/or the antigenicity of proliferative cell or tissue, or directly, if will take place when causing at proliferative antigen and immunogen such as " vaccination " of albumin fusion proteins of the present invention, or indirect, such as in activating the expression of known enhancing at the protein (as chemokine) of described antigen and immunogenic immunne response.

Kidney disease

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for treating, prevent, diagnosing and/or predict the disorder of kidney system.The kidney disorder of available composition diagnosis of the present invention, prediction, prevention and/or treatment includes but not limited to urinary disturbance, autoimmune disorder, sclerosis and necrosis, electrolyte imbalance and the kidney of the blood vessel disorder of renal failure, ephritis, kidney, metabolic and congenital kidney disorder, kidney.

Available composition diagnosis of the present invention, prediction, prevention, and/or the ephrosis of treatment includes but not limited to acute renal failure, chronic renal failure, congee sample embolic renal failure, end-stage renal disease, the inflammatory diseases of kidney is (as acute glomerulonephritis, infect the back glomerulonephritis, fast-developing glomerulonephritis, nephrotic syndrome, membranous glomerulonephritis, the familial nephrotic syndrome, I and II type membranoprolifer ative glomerulonephritis, the mesentery proliferative glomerulonephritis, chronic glomerulonephritis, the acute tubular interstitial nephritis, the chronic renal tubulointerstitial nephritis, acute poststreptococcal glomerulonephritis (PSGN), pyelonephritis, systemic lupus erythematosus, chronic nephritis, interstitial nephritis, and post-streptococcal glomerulonephritis), the blood vessel disorder of kidney is (as renal infarction, congee sample embolic ephrosis, cortical necrosis, malignant nephrosclerosis, renal venous thrombosis, the low perfusion of kidney, renal retinopathy, renal ischaemia-perfusion again, thrombosis of renal artery, and renal artery stenosis), with the kidney disorder that causes by urinary tract disorder (as pyelonephritis, nephrohydrosis, urolithiasis (urinary stone disease, nephrolithiasis), reflux nephropathy, urinary tract infection, urinary retention, with acute or chronic one-sided obstructive uropathy).

In addition, composition of the present invention can be used for diagnosis, prediction, prevention, and/or the treatment metabolism of kidney and congenital disorder are (as uremia, renal amyloidosis, renal osteodystrophy, renal tubular acidosis, renal glycosuria, diabetes insipidus,nephrogenic, cystinuria, Fanconi's syndrome, kidney fibrous capsule osteosis (renal rickets), Hart is received the Pu Shi disease, Bart's syndrome, the Li Deer syndrome, POLYCYSTIC KIDNEY DISEASE, MCD, medullary sponge kidney, the Ahlport syndrome, nail kneecap syndromes, congenital nephrotic syndrome, the extruding syndromes, horseshoe kidney, diabetic nephropathy, diabetes insipidus,nephrogenic, analgesic nephropathy, urinary stone disease, and membranous nephropathy), with the autoimmune disorder of kidney (as systemic lupus erythematous (SLE), the Goodpasture syndrome, IgA nephropathy, with IgM mesentery proliferative glomerulonephritis).

Composition of the present invention can be used for diagnosis, prediction, prevention, and/or the sclerosis of treatment kidney or downright bad disorderly (as glomerulosclerosis, diabetic nephropathy, FSG (FSGS), necrotizing glomerulonephritis, and necrosis of renal papillae), kidney is (as nephroncus, hypernephroma, the nephroblastoma, renal cell carcinoma, transitional cell carcinoma, renal adenocarcinoma, squamous cell carcinoma, and Wilms' tumor), and electrolyte imbalance is (as nephrocalcinosis, pyuria, oedema, nephrohydrosis, albuminuria, hyponatremia, hypernatremia, hypokalemia, hyperpotassemia, hypocalcemia, hypercalcemia, hypophosphatemia, and hyperphosphatemia).

Any method that composition of the present invention can use this area to know is used, include but not limited to that direct pin injection, intravenous injection, topical application, the conduit of delivering the position are inculcated, in the solid-state pharmaceutical formulations of biological projectile injector, particle accelerator, gelfoam stock, other commercialization storage material, osmotic pump, oral or suppository, surgical procedure topple over or topical application, aerosol are delivered.These methods are roads known in the art.Composition of the present invention can be used as the part of therapeutical agent and uses, and more detailed description is hereinafter arranged.The method of delivering polynucleotide of the present invention has more detailed description in this article.

Cardiovascular disorder

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for treatment, prevention, diagnosis and/or predicting cardiovascular disease, include but not limited to the peripheral arterial disease, such as limb ischemia.

Cardiovascular disorder includes but not limited to Cardiovascular abnormality, such as artery-fistula of artery, artery-vein fistula, cerebral arteries deformity, congenital heart defect, pulmonary atresia and tulwar syndromes.Congenital heart defect includes but not limited to aortic stenosis, cor triatriatum, coronary vasodilator is unusual, the cross heart, dextrocardia, patent ductus arteriosus, the Ai Busitanshi deformity, the compound disease of Ai Senmangeershi, the HLH syndromes, the sinistrocardia, method Rockwell tetra logy, transposition of great vessels, double outlet of right ventricle, tricuspid atresia, persistent truncus arteriosus, heart septum defect is such as aortopulmonary septal defect, endocardial cushion defect, Bach's syndromes is risen in the Shandong, method Rockwell triad, the ventricle heart septum defect.

Cardiovascular disorder also includes but not limited to heart trouble, such as irregular pulse, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocarditis (comprising bacteroidal), cardiac aneurysm, asystole, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, cardiac hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, cardiac rupture after the infarct, rupture of interventricular septum, valvular heart disease, cardiomyopathy, myocardial ischemia, pericardial effusion, pericarditis (comprising constriction and tuberculosis), pneumopericardium, pericardial incision postoperative syndromes, pulmonary heart disease, rheumatic heart disease, the ventricle dysfunction, congested, cardiovascular pregnancy complications, the tulwar syndromes, cardiovascular syphilis, with the cardiovascular tuberculosis disease.

Irregular pulse includes but not limited to sinus arrhythmia, atrial fibrillation, auricular flutter, bradyrhythmia, premature contraction, Adam Mu-Stokes syndromes, bundle branch block, sinoatrial block, long QT syndromes, parasystole, Lang-Gan-Lai three syndromes, Ma Haimu type pre-excitation syndrome, Wo-Pa-Huai syndromes, sick sinus syndrome, tachycardia and ventricular fibrillation.Tachycardia comprises paroxysmal tachycardia, supraventricular tachycardia, accelerated idioventricular rhythm, A-V nodal reentry tachycardia, atopy atrial tachycardia, atopy junctional tachycardia, sinus node reentry tachycardia, sinus tachycardia, torsade de pointes and ventricular tachycardia.

Valvular heart disease includes but not limited to aortic incompetence, aortic stenosis, heart murmur, 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 alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, subaortic stenosis, subvalvular pulmonary stenosis, restrictive cardiomyopathy, looks into Ge Sishi myocardosis, endocardial fibroelastosis, endomyocardial fibrosis, Ka Erensi syndrome, reperfusion injury of cardiac muscle and myocarditis.

Myocardial ischemia includes but not limited to coronary artery disease, such as stenocardia, coronary aneurysm, coronary sclerosis, Coronary thrombosis, coronary vasospasm, myocardial infarction and myocardial stunning.

Cardiovascular disorder also comprises vascular disease, such as aneurysma, angiodysplasia, vascular tumor, the bacilus vascular tumor, hippel-Lindau disease, Ke-Te-Wei three syndromes, Sturge-Weber syndrome, vasodilation, arotic disease, high iS-One arteritis, aortitis, Amur syndromes in reining in, arteriosclerosis obliterans, arteritis, enarteritis, polyarteritis nodosa, cerebrovascular disorder, diabetic angiopathy, diabetic retinopathy, embolism, thrombosis, erythromelalgia, hemorrhoid, hepatic veno-occulusive disease, hypertension, ypotension, local asphyxia, peripheral vascular disease, phlebitis, pulmonary veno-occlusive disease, Raynaud disease, the CREST syndromes, retinal vein occlusion, the tulwar syndromes, superior vena cava syndrome, telangiectasis, ataxia telangiectasis, hereditary hemorrhagic telangiectasia, varicocele, varix, varicose ulcer, vasculitis, and venous insufficiency.

Aneurysma includes but not limited to dissecting aneurysm, false aneurysm, infectious aneurysm, ruptured aneurysm, aortic aneurysm, cerebral aneurysm, coronary artery aneurysma, arterio-cardiac aneurysm and iliac artery knurl.

Arteriosclerosis obliterans includes but not limited to arteriosclerosis, intermittent claudication, carotid artery stenosis, fiber flesh sexual abnormality, mesenteric vascular occlusion, moyamoya, Renal artery obturation, retinal arterial obstruction and thromboangiitis obliterans.

Cerebrovascular disorder 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, the carotid artery thrombosis, thrombosis of venous sinus, Wahlen Burger syndrome, hematencephalon, epidural hematoma, subdural hematoma, subarachnoid hemorrhage, cerebral infarction, cerebral ischemia (comprising temporary), subclavian artery is stolen the blood syndromes, periventricular leukomalacia, vascular headache, cluster headache, migraine, and VBI.

Embolism includes but not limited to air embolism, amniotic fluid embolism, cholesterol embolism, blue toe syndrome, fat embolism, pulmonary infarction and thromboembolism.Thrombosis includes but not limited to Coronary thrombosis, hepatic vein thrombosis formation, retinal vein occlusion, carotid artery thrombosis, thrombosis of venous sinus, Wahlen Burger syndrome and thrombophlebitis.

The local asphyxia sexual disorder includes but not limited to cerebral ischemia, ischemic colitis, interval syndromes (compartment syndrome), tibialis anterior syndromes (anterior compartment syndrome), myocardial ischemia, reperfusion injury and limb ischemia (peripheral limb ischemia).Vasculitis includes but not limited to aortitis, arteritis, Bei Qiete syndrome, Qiu-Si two syndromes, mucocutaneous lymphoglandula syndromes, thromboangiitis obliterans, hypersensitive vasculitis, schonlein-Henoch purpurs, allergic cutaneous vasculitis and Wa Genashi granuloma.

Any method that the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can use this area to know is used, include but not limited to that direct pin injection, intravenous injection, topical application, the conduit of delivering the position are inculcated, in the solid-state pharmaceutical formulations of biological projectile injector, particle accelerator, gelfoam stock, other commercialization storage material, osmotic pump, oral or suppository, surgical procedure topple over or topical application, aerosol are delivered.These methods are roads known in the art.The method of delivering polynucleotide has more detailed description in this article.

Respiratory system disease

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for treating, prevent, diagnosing and/or predict the disease and/or the disorder of respiratory system.

The disease and the disorder of respiratory system include but not limited to nasal vestibulitis, the anallergic rhinitis is (as acute rhinitis, chronic rhinitis, atrophic rhinitis, vasomotor rhinitis), nasal polyp and sinusitis paranasal sinusitis, the juvenile form hemangiofibroma, rhinocarcinoma and juvenile form papilloma, polyp of vocal cord, brief summary (singer's brief summary), contact ulcer, paralysis vocal cord, the larynx bulging, pharyngitis (as viral and bacillary), tonsillitis, the tonsilla phlegmon, parapharyngeal abscess, laryngitis, the larynx bulging, and laryngocarcinoma is (as nasopharyngeal carcinoma, carcinoma of tonsil, laryngocarcinoma), lung cancer is (as squamous cell carcinoma, minicell (oat cells) cancer, large cell carcinoma, and gland cancer), allergic disorder (eosinocyte pneumonia, the hypersensitivity pneumonia is (as the extrinsic allergic alveolitis, allergic interstitial pneumonitis, the particulate organic matter pneumoconiosis, the allergy bronchopneumonic aspergillosis, asthma, Wa Genashi granuloma (granuloma vasculitis), the Goodpasture syndrome)), pneumonia is (as bacterial pneumonia (as streptococcus pneumoniae Streptococcus pneumoniae (pneumococcal pneumonia), streptococcus aureus Staphylococcus aureus (staphylococcal pneumonia), gram negative bacterium property pneumonia (causing) by for example Klebsiella Klebsiella and Rhodopseudomonas Pseudomonas, Mycoplasma pneumoniae Mycoplasma pneumoniae pneumonia, Haemophilus influenzae Hemophilus influenzae pneumonia, legionella Legionella pneumonia (legionnaires disease), with chlamydia psittaci Chlamydia psittaci (psittacosis)), and virus pneumonia is (as influenza, fowl pox (varicella)).

Other respiratory system disease and disorder include but not limited to bronchiolitis, poliomyelitis, croup, respiratory syncytial virus infection, parotitis, erythema infectiosum (erythema infectiosum), roseola infantum, progressive rubella panencephalitis, Rubella (rubella), and subacute sclerosing panencephalitis, fungal pneumonia is (as histoplasmosis, coccidioidomycosis, blastomycosis, immunity system is subjected to the fungi infestation of serious philtrum of containing (as the torulosis that is caused by novel Cryptococcus Cryptococcus neoformans; The aspergillosis that causes by Aspergillus Aspergillus; The candidiasis that causes by mycocandida Candida; And mucormycosis)), Pneumocystis carinii Pneumocystis carinii (pneumccystis pneumonia), severe acute respiratory syndrome (as mycoplasma and chlamydiaceae), the opportunistic infection pneumonia, nosocomial pneumonia, chemical pneumonitis, and aspiration pneumonitis, the pleura disorder is (as pleuritis, hydrothorax, and pneumothorax is (as simple spontaneous pneumothorax, complicated spontaneous pneumothorax, tension pneumothorax)), obstructive respiratory disease is (as asthma, chronic obstructive pulmonary disease (COPD), pulmonary emphysema, chronic or acute bronchitis), occupational tuberculosis is (as silicatosis, black lung (miner lung coniosis), asbestosis, berylliosis, occupational asthma, byssinosis, with optimum Pneumonoconiosis), wetting property tuberculosis is (as pulmonary fibrosis (as FA, common interstitial pneumonia), idiopathic pulmonary fibrosis, desquamative interstitial pneumonia, LIP, histiocytosis X is (as Letterer-Siwe disease, Hand-Sch, eosinophilic granuloma), idiopathic pulmonary hemosiderosis, sarcoidosis and pulmonary alveolar proteinosis), acute respiratory distress syndrome (also becoming for example adult respiratory distress syndrome), oedema, pulmonary infarction, bronchitis is (as viral, bacillary), bronchiectasis, pulmonary atelectasis, pulmonary abscess (by for example streptococcus aureus or invade lung legionella Legionella pneumophila cause), and cystic fibrosis.

The angiogenesis inhibitor activity

Natural between endogenous stimulator that blood vessel takes place and inhibition exists the influence that suppresses in the balance to preponderate.People such as Rastinejad, Cell, 56:345-355,1989.Take place under the normal physiologic situation in the rare situation of neovascularization, such as wound healing, neomorph, fetal development and female reproduction process, blood vessel is subjected to strict regulation and control and defines room and time.Under the situation that pathologic vessels takes place, such as showing the solid tumor growth, these regulation and control controls have been failed.Not modulated blood vessel becomes morbid state and supports many superfluous lifes and non-superfluous living advancing of disease.Many serious diseases are subjected to the domination of unusual neovascularization, comprise the ophthalmology disorder and the psoriatic of solid tumor growth and transfer, sacroiliitis, some type.Consult for example following summary: people such as Moses, Biotech., 9:630-634,1991; People such as Folkman, N.Engl.J.Med., 333:1757-1763,1995; People such as Auerbach, J.Microvasc.Res., 29:401-411,1985; Folkman, " Advances in CancerResearch ", Klein and Weinhouse compile, Academic Press, New York, pp.175-203,1985; Patz, Am.J.Opthalmol., 94:715-743,1982; Reach people such as Folkman, Science, 221:719-725,1983.In many pathologic situations, the blood vessel generating process helps morbid state.For example, accumulated a large amount of evidences and shown that the growth of solid tumor depends on blood vessel and takes place.Folkman and Klagsbrun, Science, 235:442-447,1987.

The invention provides polynucleotide pair and neovascularization diseases associated or disorderly treatment by using fusion rotein of the present invention and/or code book invention albumin fusion proteins.Pernicious and the transfer condition of available polynucleotide of the present invention and polypeptide or agonist or antagonist for treating include but not limited to malignant tumour, solid tumor, and described herein and cancer that other approach of this area is known (summary about these disorders is consulted people such as Fishman, " Medicine ", the 2nd edition, J.B.Lippincott Co., Philadelphia, 1985).Thus, the invention provides treatment blood vessel generation relative disease and/or disorderly method, comprise the albumin fusion proteins of the present invention that needed individual administering therapeutic significant quantity is arranged and/or the polynucleotide of code book invention albumin fusion proteins.For example, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins can be used for multiple other method, thus treatment cancer or treatment in treatment.The cancer of the polynucleotide treatment of available fusion rotein of the present invention and/or code book invention albumin fusion proteins includes but not limited to solid tumor, comprises prostate gland, lung, breast, ovary, stomach, pancreas, larynx, esophagus, testis, liver, the parotid gland, bile duct, colon, rectum, uterine neck, uterus, uterine endometrium, kidney, bladder, thyroid carcinoma; Primary tumor and transfer; Melanoma; Glioblastoma; Kaposi sarcoma; Leiomyosarcoma; The non-cancer of non-small cell; Colorectal carcinoma; Senior malignant tumour; With the blood propagation tumour, such as leukemia.For example, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins can locally be delivered with the treatment cancer, such as skin carcinoma, head and neck tumour, lacteal tumor and Kaposi sarcoma.

In others, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins can be used the bladder cancer that is used for the treatment of shallow sheet form by for example intravesical.The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can directly be delivered in the tumour or near the tumor locus by injection or conduit.Certainly, will understand as those of ordinary skill, suitable mode of administration will change according to cancer to be treated.This paper has also discussed other delivery pattern.

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for treating other disorder that blood vessel takes place that relates to except that cancer.These disorders include but not limited to: innocent tumour, for example vascular tumor, acoustic tumor, neurofibroma, trachoma and botryomycosis hominis; Atherosclerotic plaque; The blood vessel generation disease of eye, for example uveitis and pteryium (the abnormal vascular growth) of diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, Terry's sign, rubescent, retinoblastoma, eye; Rheumatoid arthritis; Psoriatic; Wound healing is slow; Endometriosis; Angiogenesis (vasculogenesis); Granulation forms; Hypertrophic cicatrix (keloid); Disunited fracture; Scleroderma; Trachoma; Blood vessel adheres to; Myocardial vascular takes place; CC; The brain side shoot; Arteriovenous malformotion; (ischemic limb angiogenesis) takes place in the ischemia limb vessel; Ao-Wei two syndromes; Plaque neovessels forms; Telangiectasis; Bleeder's joint; Hemangiofibroma; Fibrillar muscle heteroplasia; The wound granulation forms; Crohn disease; And atherosclerosis.

For example, in one aspect of the invention, the method that is used for the treatment of hypertrophic cicatrix and keloid is provided, has comprised the step of hypertrophic cicatrix or keloid being used the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins.

In one embodiment of the invention, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins are injected directly in hypertrophic cicatrix or the keloid to prevent the development of these damages.This therapy is valuable especially in the known prophylactic treatment that causes hypertrophic cicatrix and keloid (as burn) status of development, and (after the initial damage about 14 days) but startup before hypertrophic cicatrix or keloid development preferably in propagation phase free development back.As mentioned above, the present invention also provides the method for the neovascular disorders that is used for the treatment of eye, comprises for example cornea neovascularization, neovascular glaucoma, proliferating diabetic retinopathy, Terry's sign and macular degeneration.

In addition, the ophthalmology disorder relevant with neovascularization of the polynucleotide of available albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins treatments includes but not limited to: neovascular glaucoma, diabetic retinopathy, retinoblastoma, Terry's sign, uveitis, retinopathy of prematurity, macular degeneration, corneal transplantation neovascularization and with choroid or relevant ophthalmology inflammatory diseases, optic tubercle and the disease of iris neovascularization.Consult for example following summary: people such as Waltman, Am.J.Ophthal., 85:704-710,1978; Reach people such as Gartner, Surv.Ophthal., 22:291-312,1978.

Thus, in one aspect of the invention, the method of the neovascular disorders that is used for the treatment of eye is provided, such as cornea neovascularization (comprising the corneal transplantation neovascularization), comprise the step of compound (as the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins), make the formation of blood vessel be suppressed patients with corneal administering therapeutic significant quantity.In brief, cornea is the tissue that under normal circumstances lacks blood vessel.Yet in some pathological condition, kapillary can extend into cornea by the cornea surrounding blood vessel clump of cornea and sclera junction.After cornea became vascularization, it also just thickened, and caused patient's visual deterioration.If it is fully opaque that cornea becomes, then can completely lose eyesight.Extremely multiple disorder can cause the cornea neovascularization, comprises for example corneal infection (as trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis), immunology process (as transplant rejection and Si-Yue two syndromes), alkali burn, wound, inflammation (any reason), poisoning and nutritive deficiency state, and as the complication of wearing contact lens.

In particularly preferred embodiment of the present invention, preparation is used for topical application can (to unite any sanitas and the biocide that are usually used in the ophthalmology prepared product) in salt solution, and uses with the form of eye drop.Solution or suspension can the preparations of its pure form, and every day administered several times.Perhaps, the angiogenesis inhibitor composition of preparation also can directly be applied to cornea as mentioned above.In preferred embodiments, the angiogenesis inhibitor composition prepares with the mucoadhesive polymers in conjunction with cornea.In other embodiments, the angiogenesis inhibitor factor or angiogenesis inhibitor composition can be used as the adjuvant therapy of conventional Steroid treatment.Local treatment also can be used for the known corneal injury that induction of vascular is replied the high probability of (such as chemical burn) that has in prevention.In these situations, begin treatment might be united steroid immediately, to help the follow-up complication of prevention.

In other embodiments, compound mentioned above can be injected directly in the corneal stroma under microscope instructs by the eye doctor.Preferred injection site can change with the morphology of indivedual damages, but the target of using will be the forward position (promptly interspersing among between blood vessel and the normal cornea) that composition is placed blood vessel structure.In most applications, this cornea injection that will be referred to around cornea and the sclera junction avoids propulsive blood vessel with " protection " cornea.This method also can not only be used after corneal injury, thus the neovascularization of preventative prevention cornea.In this case, material can be expelled in the cornea on every side of cornea and sclera junction, intersperse among between corneal injury and undesired potential cornea and the blood supply of sclera junction.This method can also similar fashion be used to prevent the kapillary invasion of corneal transplant.In sustained release form, may only need 2-3 injection every year.Also can in injection liquid, add steroid to reduce the inflammation that causes by injection itself.

In another aspect of the present invention, the method that is used for the treatment of neovascular glaucoma is provided, comprise the step to the polynucleotide of the albumin fusion proteins of the present invention of eye administering therapeutic significant quantity and/or code book invention albumin fusion proteins, make the formation of blood vessel be suppressed the patient.In one embodiment, but the compound topical application to the eye with the treatment old model a neovascular glaucoma.In other embodiments, compound can beat in the anterior chamber angle zone by injection.In other embodiments, compound also can place any position, makes compound be discharged in the aqueous humor continuously.In another aspect of the present invention, the method that is used for the treatment of proliferating diabetic retinopathy is provided, comprise the step to the polynucleotide of the albumin fusion proteins of the present invention of eye administering therapeutic significant quantity and/or code book invention albumin fusion proteins, make the formation of blood vessel be suppressed the patient.

In particularly preferred embodiment of the present invention, can treat proliferating diabetic retinopathy to improve polynucleotide, polypeptide, antagonist and/or the partial concn of agonist in retina by being expelled in aqueous humor or the vitreum.Preferably, this treatment should start before acquisition needs the serious disease of photocoagulation.

In another aspect of the present invention, the method that is used for the treatment of Terry's sign is provided, comprise the step to the polynucleotide of the albumin fusion proteins of the present invention of eye administering therapeutic significant quantity and/or code book invention albumin fusion proteins, make the formation of blood vessel be suppressed the patient.Compound can be implanted topical application by intravitreal injection and/or intraocular.

In addition, the disorder of the polynucleotide of available fusion rotein of the present invention and/or code book invention albumin fusion proteins treatments includes but not limited to that vascular tumor, sacroiliitis, psoriatic, hemangiofibroma, atherosclerotic plaque, wound healing are slow, granulation formation, bleeder's joint, hypertrophic cicatrix, disunited fracture, Ao-Wei two syndromes, botryomycosis hominis, scleroderma, trachoma and blood vessel adhere to.

In addition, the polynucleotide of available albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins treatment, prevention, diagnosis, and/or the disorder and/or the state of prediction include but not limited to solid tumor, the blood propagation tumour is such as leukemia, metastases, Kaposi sarcoma, innocent tumour is vascular tumor for example, acoustic tumor, neurofibroma, trachoma, and botryomycosis hominis, rheumatoid arthritis, psoriatic, ophthalmology blood vessel generation disease is diabetic retinopathy for example, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, Terry's sign, rubescent, retinoblastoma, and uveitis, wound healing is slow, endometriosis, angiogenesis, granulation forms, hypertrophic cicatrix (keloid), disunited fracture, scleroderma, trachoma, blood vessel adheres to, myocardial vascular takes place, CC, the brain side shoot, arteriovenous malformotion, the ischemia limb vessel takes place, Ao-Wei two syndromes, plaque neovessels forms, telangiectasis, bleeder's joint, hemangiofibroma, fibrillar muscle heteroplasia, the wound granulation forms, Crohn disease, atherosclerosis, by preventing that the embryo from implanting the needed angiopoietic contraceptive bian of control menstruation, have disease such as the cat scratch disease (Rochele minalia quintosa) of blood vessel generation as pathological consequences, ulcer (helicobacter pylori Helicobacter pylori), bartonellosis, and BA.

Aspect of method of birth control, the amount of using before or after sexual intercourse and fertilization generation is enough to block the compound that the embryo implants, and effective method of birth control is provided thus, may be " being still drank after a night " method.The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins also can be used for controlling menstruation, perhaps in the film ectopic treatment in uterus or as peritoneal lavage fluid or being used for the abdominal cavity implants and use.

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can mix surgical sutures with prevention stitch granuloma.

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for extremely multiple surgical procedure.For example, in one aspect of the invention, composition (for example form of sprays or film) is used in before the tumor resection the smearing or spray of specific region, thereby normal surrounding tissue is separated with malignant tissue, and/or preventing disease diffuses to surrounding tissue.In others of the present invention, composition (for example form of sprays) can be delivered by endoscopic procedure, thus the blood vessel generation that covers tumour or transplant desired area.Aspect other, can be used for to utilize any program of surgery reticulated of the present invention through the surgery reticulated of angiogenesis inhibitor composition bag quilt of the present invention.For example, in one embodiment of the invention, the surgery reticulated that has loaded angiogenesis inhibitor composition of the present invention can be used for abdomen carcinectomy process (for example colectomy postoperative), thereby provides the support of structure and discharge a certain amount of angiogenesis inhibitor factor.

In others of the present invention, the method that is used for the treatment of the tumor resection position is provided, be included in the polynucleotide of the margins of excision of tumour being used after the surgical blanking albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins, make original place (local) recurrence of cancer and the formation of this position neovascularity be suppressed.In one embodiment of the invention, the angiogenesis inhibitor composition is applied directly to tumor resection position (for example using by the margins of excision of smearing, brushing with the angiogenesis inhibitor compound or alternate manner covers tumour).Perhaps, the angiogenesis inhibitor compound can mix known surgery patch before using.In particularly preferred embodiment of the present invention, the angiogenesis inhibitor compound is used after the hepatectomy of malignant tumour and after neurosurgery.

In one aspect of the invention, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins can be applied to the extremely margins of excision of kinds of tumors, comprise for example breast, colon, brain and liver tumor.For example, in one embodiment of the invention, the angiogenesis inhibitor compound can be applied to the neuroscience tumor locus after surgical blanking, makes the formation of this position neovascularity be suppressed.

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins also can be used with other angiogenesis inhibitor factor.The representative example of other angiogenesis inhibitor factor comprises: anti-invade the factor, vitamin A acid and derivative thereof, taxol (Paclitaxel), Suramine, metalloprotease-1 organize inhibition, metalloprotease-2 organize inhibition, Type 1 plasminogen activator inhibitor-1, Type 1 plasminogen activator inhibitor-2 and various forms of light " d group " transition metal.

Light " d group " transition metal comprises for example vanadium, molybdenum, tungsten, titanium, niobium and tantalum nucleic.These transition metal nucleic can form transition metal composite.The suitable complexes of above-mentioned transition metal nucleic comprises oxo transition metal composite.

The representative example of vanadium mixture comprises oxo vanadium mixture, such as vanadate and vanadylic mixture.Suitable vanadate mixture comprises metavanadate and positive vanadate mixture, such as for example ammonium meta-vanadate, sodium metavanadate and sodium vanadate.Suitable vanadylic mixture comprises that for example methyl ethyl diketone vanadyl and vanadylic sulfate comprise that the vanadylic sulfate hydrate is such as one and three hydration vanadylic sulfates.

The representative example of tungsten and molybdenum mixture also comprises oxo mixture.Suitable oxo tungsten mixture comprises tungstate and Tungsten oxide 99.999 mixture.Suitable tungstate mixture comprises ammonium tungstate, calcium wolframate, Disodium tungstate (Na2WO4) dihydrate and wolframic acid.Suitable Tungsten oxide 99.999 comprises Tungsten oxide 99.999 (IV) and Tungsten oxide 99.999 (VI).Suitable oxo molybdenum mixture comprises molybdate, molybdenum oxide and molybdyl mixture.Suitable molybdate mixture comprises ammonium molybdate and hydrate, Sodium orthomolybdate and hydrate thereof and potassium molybdate and hydrate thereof.Suitable molybdenum oxide comprises molybdenum oxide (VI), molybdenum oxide (VI) and molybdic acid.Suitable molybdyl mixture copies for example methyl ethyl diketone oxygen molybdenum.Tungsten that other is suitable and molybdenum mixture comprise by for example glycerine, tartrate and carbohydrate deutero-coordination hydroxyl derivative.

Extremely multiple other angiogenesis inhibitor factor also can be used for content of the present invention.Representational example comprises platelet factor 4; Protamine sulfate; Sulfuric acid chitin derivatives (by queen crab shell preparation) people such as (, Cancer Res., 51:22-26,1991) Murata; Sulfated polysaccharide peptidoglycan mixture (SP-PG) (function of this mixture can strengthen by the existence of steroid, such as oestrogenic hormon and citric acid tamoxifen); Staurosporine; The modulator of matrix metabolism comprises for example proline analogs, cis hydroxyl groups proline(Pro), d, L-3,4-dehydroproline, thioproline, α, α-bipyridyl aminopropionitrile fumaric acid esters, 4-propyl group-5-(4-pyridyl)-2 (3H)-azolactones; Methotrexate; Mitoxantrone; Heparin; Interferon, rabbit; 2 macroglobulin-white protein; ChIMP-3 (people such as Pavloff, J.Bio.Chem., 267:17321-17326,1992); Chymostatin (people such as Tomkinson, Biochem.J., 286:475-480,1992); Cyclodextrin 14 sulfuric esters; Eponemycin; Camptothecine; Fumidil (people such as Ingber, Nature, 348:555-557,1990); Gold sodium thiomalate (" GST "; Matsubara and Ziff, J.Clin.Invest., 79:1440-1446,1987); Anticollagenase serum; α 2-antiplasmin (people such as Holmes, J.Biol.Chem., 262 (4): 1659-1664,1987); Orang Crush (NationalCancer Institute); Lobenzarit Disodium (N-(2)-carboxyphenyl-4-chrloroanthracene fennel acid (anthronilic acid) disodium or " CCA "; People such as Takeuchi, Agents Actions, 36:312-316,1992); Thalidomide; Suppress the steroid that blood vessel takes place; AGM-1470; The carboxyamino imidazoles; With the metalloprotein enzyme inhibitor, such as BB94.

The disease of cell levels

The polynucleotide treatment of available fusion rotein of the present invention and/or code book invention albumin fusion proteins, prevention, diagnosis, and/or prediction strengthen with cell survival or apoptosis suppresses diseases associated and comprises that cancer is (such as follicular lymphoma, cancer with p53 sudden change, and hormone-dependent tumor, include but not limited to colorectal carcinoma, cardiac tumor, carcinoma of the pancreas, melanoma, retinoblastoma, Glioblastoma, lung cancer, intestinal cancer, carcinoma of testis, cancer of the stomach, neuroblastoma, myxoma, myomata, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi sarcoma, and ovarian cancer); Autoimmune disorder (such as multiple sclerosis, Si Yegelun syndrome, struma lymphomatosa, cholehepatocirrhosis, Behcet, Crohn disease, polymyositis, systemic lupus erythematous and relevant glomerulonephritis of immunity and rheumatoid arthritis) and virus infection (such as simplexvirus, poxvirus and adenovirus), inflammation, graft versus host disease (GVH disease), acute grafing are got rid of and chronic transplanting rejection.

In preferred embodiments, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins are used to suppress the growth, development of cancer and/or shift, particularly above cited.

Development and/or transfer that the polynucleotide treatment of available fusion rotein of the present invention and/or code book invention albumin fusion proteins or other disease relevant with the cell survival enhancing that detects or situation include but not limited to malignant tumour and associated disorders, (comprise that acute leukemia is (as acute lymphoblastic leukemia such as leukemia, acute myelocytic leukemia (comprises myeloblast, promyelocyte (promyelocyte), myelomonocyte, monocyte and erythroleukemia)) and chronic leukemia (as chronic myeloid (granulocytic) leukemia and lymphocytic leukemia)), polycythemia vera, lymphoma (as lymphogranulomatosis and Fei Huoqijinshi disease), multiple myeloma, Walden Si Telunshi macroglobulinemia, heavy chain disease, and solid tumor, include but not limited to sarcoma and cancer, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovioma, mesothelioma, the You Wenshi tumour, leiomyosarcoma, rhabdosarcoma, colorectal carcinoma, carcinoma of the pancreas, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, rodent cancer, gland cancer, syringocarcinoma, sebaceous carcinoma, papillary carcinoma, papillary carcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, spermocytoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, neurospongioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic tumor, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

What the polynucleotide of available fusion rotein of the present invention and/or code book invention albumin fusion proteins were treated, prevent, diagnosed and/or predicted includes but not limited to AIDS with apoptosis enhancing diseases associated; Neurodegenerative disorders (such as Alzheimer, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration and brain tumor or aforementioned relative disease); Autoimmune disorder (such as multiple sclerosis, Si Yegelun syndrome, struma lymphomatosa, cholehepatocirrhosis, Behcet, Crohn disease, polymyositis, systemic lupus erythematous and relevant glomerulonephritis of immunity and rheumatoid arthritis); Development of bone marrow abnormal syndrome (such as aplastic anemia), graft versus host disease (GVH disease), ischemia injury (such as causing), liver injury (as the relevant liver injury of hepatitis, ischemia/reperfusion injury, cholestasis (bile duct injury) and liver cancer) by myocardial infarction, apoplexy and reperfusion injury; Hepatopathy (such as what cause), septic shock, cachexia and the anorexia of the hepatopathy of toxin-induced (such as what cause), toxin-induced by alcohol by alcohol.

Wound healing and epithelial cell proliferation

According to another aspect of the present invention, the method that the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins is used for the treatment of purpose is provided, for example the purpose for wound healing is used to stimulate epithelial cell proliferation and substrate keratinocyte, and is used for hair follicle stimulating generation and dermal wounds healing.The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for stimulating wound healing clinically, comprise surgical wound, the surgical blanking wound, the gash that relates to corium and epidermis injury, ocular tissue's wound, the dental tissue wound, the oral cavity wound, diabetic ulcer, corium ulcer, elbow ulcer, ulcer of artery, venous stasis ulcer, by the burn that heat exposes or pharmaceutical chemicals causes, and other unusual wound healing situation, such as uremia, malnutritive, vitamin deficiency, and and steroid, radiotherapy, systemic treatment complications associated with arterial system with anti-superfluous crude drug and metabolic antagonist.The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for promoting the corium after the corium loss to rebuild.

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for improving skin graft to the adhesion of wound face be used to stimulate the reepithelialization of wound face.It is the adherent graft type of the polynucleotide raising of available fusion rotein of the present invention and/or code book invention albumin fusion proteins below: autograft (autograft) to wound face, artificial skin, allograft (allograft), the auto derma graft, from the body surface skin graft, avascular graft, blair-Brown graft, bone graft, brephoplastic graft, skin graft (cutis graft), delayed graft, dermis graft (dermic graft), epidermic graft, fascial graft, full thickness graft, heterograft (heterologous graft), heterograft (xenograft), allograft (homologous graft), activated graft, cornea thin layer graft, mesh graft, mucosal graft, ollier-Thiersch graft, omental grafts, patch graft, double-end graft, full thickness corneal graft, split-skin graft, thick-split graft.The outward appearance that the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for promoting skin intensity and improve old and feeble skin.

We think that the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins also will cause variation in the epithelial cell proliferation in hepatocyte growth and lung, breast, pancreas, stomach, small intestine and the large intestine.The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can promote epithelial cell proliferation, such as other epithelial cell and the ancestors thereof contained in sebaceous cell (sebocyte), hair follicle, liver cell, II type pneumonocyte, the goblet cell that generates mucoitin and skin, lung, liver and the gi tract.The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can promote the propagation of endotheliocyte, keratinocyte and substrate keratinocyte.

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins also can be used for reducing internal organ (gut) toxic side effects that is caused by radiotherapy, chemotherapy or virus infection.The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can have the cytoprotective effect to mucous membrane of small intestine.The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins also can stimulate the rehabilitation of the mucositis (stomatocace) that is caused by chemotherapy and virus infection.

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins also can be used for fully and the segment thickness skin injury comprises that the holomorphosis, other skin injury of burn (be hair follicle, sweat gland and sebiferous gland heavily in) are such as psoriatic treatment.The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for treating epidermolysis bullosa, be the be stained with defective of epidermis, cause frequent, open and bubble pain by the reepithelialization that quickens these damages to following corium.The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins also can be used for treating gastric duodenal ulcer, and help faster healing by the cicatrization of mucous membrane lining and the regeneration of gland mucous membrane and duodenal mucosa lining.Inflammatory bowel such as Crohn disease and ulcerative colitis, refers to cause respectively small intestine or large intestine mucomembranous surface destructive disease.Thus, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins can be used for promoting that the new surface of mucomembranous surface forms (resurfacing) to help the development of more piece healing and prophylaxis of inflammatory bowel disease.The treatment of carrying out with the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins is estimated whole GI mucus generated and is had unusual effect, and can be used for protecting intestinal mucosa avoid the objectionable impurities of absorbing or postoperative injury.The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for treatment and express not enough diseases associated.

In addition, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins can be used for preventing and treat the infringement of various pathological states to lung.The albumin fusion proteins of the present invention that can stimulate alveolar and bronchiolar epithelium propagation and differentiation and promote to repair and/or the polynucleotide of code book invention albumin fusion proteins can be used for prevention or treat acute or the chronic pulmonary damage.For example, cause the pulmonary emphysema of alveolar progressive loss and the inhalation injury that causes bronchiolar epithelium and alveolar necrosis (promptly causing) can use polynucleotide of the present invention or polypeptide, agonist or antagonist effectively to treat by suction cigarette and burn.Equally, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins can be used for stimulating the propagation and the differentiation of II type pneumonocyte, this helps treatment or prevention such as diseases such as hyaline membrane diseases, such as infant respiratory distress syndrome among the premature infant and bronchopulmonary dysplasia.

But the propagation and the differentiation of the polynucleotide cell cultured supernatant of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins, can be used for alleviating or treating hepatopathy and pathology thus, the liver injury that causes such as the explosive liver failure that causes by liver cirrhosis, by viral hepatitis and toxic substance (being acetaminophen, tetracol phenixin (carbon tetraholoride) and other hepatotoxin known in the art).

In addition, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins can be used for treating or the outbreak of prevent diabetes.Be diagnosed as I type and type ii diabetes recently, wherein keeping among the patient of some islet cell function, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins can be used for keeping islet function, thereby alleviate, postpone or prophylactic permanent performance.Equally, the polynucleotide of fusion rotein of the present invention and/or the code book invention albumin fusion proteins adjuvant therapy that can be used as islet cell transplantation is to improve or to promote islet cell function.

Neural activity and neurological disorder

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for the diagnosis and/or the treatment of brain and/or neural disease, disorder, damage or infringement.The neurological disorder of available composition of the present invention (as the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins) treatment includes but not limited to nervous system injury, and causes that aixs cylinder disconnects, neurone is subdued or sex change or demyelination or disorder.Can include but not limited to following maincenter (comprising spinal cord, brain) or peripheral nervous system lesions according to the nervous system injury that method of the present invention is treated in patient's (comprising people and non-human mammal patient): (1) ischemia injury, wherein partial nerve system anoxic causes neuronal damage or death, comprises cerebral infarction or ischemic or spinal infarction or ischemic; (2) traumatic injury comprises damage that caused by physical injury or relevant with operation, and for example neural damage of severed segment perhaps weighs wounded; (3) malignant lesion, wherein the partial nerve system suffers the destruction or the damage of malignant tissue, this malignant tissue or neural system associated malignancies or by non-neural system tissue deutero-malignant tumour; (4) infectious damage, wherein the partial nerve system is destroyed or is damaged because infect, for example by trachoma or with human immunodeficiency virus, zoster or herpes simplex infections or relevant with Lyme disease, tuberculosis or syphilis; (5) sex change damage, wherein the partial nerve system is destroyed or is damaged because of denaturation process, includes but not limited to and the relevant sex change of Parkinson's disease, Alzheimer, huntington's chorea or amyotrophic lateral sclerosis (ALS); (6) with nutritive disease or disorderly relevant damage, wherein the partial nerve system includes but not limited to vitamin B12 deficiency, folic acid deficiency, acute hemorrhagic polioencephalitis, tobacco and wine amblyopia, marchiafava-Bignami disease (primary degeneration of corpus callosum) and alcoholic cerebellar degeneration because of metabolic nutritive disease or disorderly destroyed or damage; (7) neurological damage relevant with systemic disease includes but not limited to diabetes (diabetic neuropathy, Bei Ershi facial paralysis), systemic lupus erythematous, cancer or sarcoidosis; (8) damage that is caused by toxic substance comprises alcohol, lead or specific neurotoxin; (9) demyelination damage, wherein the partial nerve system is destroyed or is damaged because of demyelination, includes but not limited to multiple sclerosis, human immunodeficiency virus relevant myelopathy, transverse myelopathy or various nosetiology, progressive multifocal leukoencephalopathy and central pontine myelinolysis.

In one embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are used for the detrimental effect that the neuroprotective cell avoids hypoxemia.In another preferred embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are used to comprise the detrimental effect that neurocyte avoids cerebral anoxia.In this embodiment, the neural cell injury that composition of the present invention is used for the treatment of or prevention is relevant with cerebral anoxia.Aspect a nonexcludability of this embodiment, the neural cell injury that polynucleotide are used for the treatment of or prevention is relevant with cerebral ischemia of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins.Aspect another nonexcludability of this embodiment, the neural cell injury that polynucleotide are used for the treatment of or prevention is relevant with cerebral infarction of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins.

In another preferred embodiment, the neural cell injury that polynucleotide are used for the treatment of or prevention is relevant with apoplexy of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins.In a specific embodiment, the damage of the cranial nerve cell that polynucleotide are used for the treatment of or prevention is relevant with apoplexy of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins.

In another preferred embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are used for the treatment of or prevent and the relevant neural cell injury of having a heart attack.In a specific embodiment, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins 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 present composition of neurological disorder to select by the biologic activity of test in promoting neuronal survival or differentiation.For example, but not the conduct restriction, the composition that causes any following effect can use according to the present invention: there be or lacking the increase survival time of neurone in cultivation in anoxic or the hypoxia condition in (1); (2) in cultivation or increase neuronic germination in vivo; (3) in cultivation or increase the generation of neurone associated molecule in vivo, as be cholinacetyltranslase or acetylcholinesterase with regard to motor neuron; Or (4) reduce neuron dysfunction in vivo.These effects can be measured by any method that this area is known.In preferred non-limiting embodiments, increasing neuronic survival can use method cited herein or that other approach of this area is known to come routine measurement, such as people such as for example Zhang, Proc.Natl.Acad.Sci.USA, 973637-42,2000 or people such as Arakawa, J.Neurosci., 10:3507-15,1990; Increasing neuronic germination can measure by the method that this area is known, such as people such as for example Pestronk, and Exp.Neurol., 70:65-82,1980 or people such as Brown, Ann.Rev.Neurosci., 4:1742, the method for enumerating in 1981; The generation of increase neurone associated molecule can be used that this area is known and depend on that the technology of molecule to be measured waits and measures by bioassay method, enzymatic assay method, antibodies, Northern trace assay method; And the motor neuron dysfunction can be measured by the physical manifestations (physicalmanifestation) of assessment motor neuron disorder, for example weakness, motor neuron conduction of velocity or functional disability.

In specific embodiment, can include but not limited to such as following disorder according to the motor neuron disorder of the present invention's treatment, infarction, infect, be exposed to toxin, wound, surgical injury, can influence the degenerative disease or the malignant tumour of motor neuron and other composition of neural system, and selectivity affect the nerves the unit disorder such as amyotrophic lateral sclerosis, include but not limited to progressive spinal muscular atrophy, PBP, primary lateral sclerosis, infantilism and juvenile muscular atrophy, the Childhood PBP (Fa Qiao-Longde two syndromes), syndromes after poliomyelitis and the poliomyelitis, and hereditary motor and sensory neuropathy (Xia Ke-Mali-Tu Si San Shi disease).

In addition, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins can play a role in neuronal survival, cynapse formation, conduction, Neural Differentiation etc.Thus, composition of the present invention (polynucleotide that comprise fusion rotein of the present invention and/or code book invention albumin fusion proteins) can be used for diagnosis and/or treatment or prevention and these effect diseases associated or disorder, includes but not limited to study and/or cognitive disorders.Composition of the present invention also can be used for treatment or prevention neurodegenerative disease state and/or conduct disorder.These neurodegenerative disease states and/or conduct disorder include but not limited to Alzheimer, Parkinson's disease, Huntington's disease, the many syndromess of tourette, schizophrenia, mania, dementia, paranoia, mandatory disorder, terrified sexual disorder, learning disability, ALS, psychosis, autism and behavior change, comprise feed, sleep pattern, balance and disturbance of perception.In addition, composition of the present invention also can play a role in treatment, prevention and/or the detection of growth disorder relevant with embryo in the growth or the chain disorder of sex.

In addition; the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins can be used for the neuroprotective cell and avoid and the cerebrovascular disorder diseases associated; damage; disorderly; or infringement, include but not limited to that carotid disease is (as the 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 the carotid artery thrombosis; thrombosis of venous sinus; or Wahlen Burger syndrome); hematencephalon is (as exterior dura or subdural hematoma; or subarachnoid hemorrhage); cerebral infarction; cerebral ischemia is (as transient cerebral ischemia; subclavian artery is stolen the blood syndromes; or VBI); vascular dementia (as multiple cerebral); periventricular leukomalacia; and vascular headache (as cluster headache or migraine).

According to another aspect of the present invention, the method for utilizing the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins for therapeutic purpose is provided, cell proliferation and/or differentiation for example are used to excite nerve.Therefore, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins can be used for treatment and/or detect sacred disease.In addition, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins can be used as specific nervous system disorders or disorderly mark or detection thing.

The example of the polynucleotide treatment of available fusion rotein of the present invention and/or code book invention albumin fusion proteins or the nervous disorders that detects comprises cerebral disorders, such as metabolic encephalopathy, comprise that phenylketonuria is such as the parent phenylketonuria, pyruvate carboxylase deficiency, pyruvate dehydrogenase complex deficiency, wernicke encephalopathy, cerebral edema, brain vegetation comprises vegetation in the curtain such as cerebellum vegetation, ventricles of the brain vegetation is such as choroid plexus vegetation, hypothalamus vegetation, vegetation on the curtain, canavan's disease, little disease of brain, such as cerebellar ataxia, comprise that spinocerebellar degeneration is such as ataxia telangiectasis, cerebellum dyssynergia, family ataxia, macado-Joseph disease, olivopontocerebellar atrophy, cerebellum vegetation is such as vegetation in the curtain, the all encephalitis of diffuse cerebrosclerosis such as axle, globoid cell leukodystrophy, metachromatic leukodystrophy, and subacute sclerosing panencephalitis.

The polynucleotide treatment of available fusion rotein of the present invention and/or code book invention albumin fusion proteins or other sacred disease that detects comprise that cerebrovascular disorder (such as carotid disease, comprises the carotid artery thrombosis, 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 the carotid artery thrombosis, thrombosis of venous sinus, with Wahlen Burger syndrome, hematencephalon is such as epidural hematoma, subdural hematoma, and subarachnoid hemorrhage, cerebral infarction, cerebral ischemia is such as transient cerebral ischemia, subclavian artery is stolen the blood syndromes, and VBI, vascular dementia is such as multi infarct dementia, periventricular leukomalacia, vascular headache such as cluster headache and migraine.

The polynucleotide treatment of available fusion rotein of the present invention and/or code book invention albumin fusion proteins or other sacred disease that detects comprise dull-witted such as the dull-witted compound disease of AIDS, presenile dementia such as Alzheimer and Ke-Ya two syndromes, senile dementia such as Alzheimer and stein-leventhal syndrome, vascular dementia is such as multi infarct dementia, encephalitis comprises all encephalitis of axle, 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 syndromes, Parkinson's disease after the encephalitis, and subacute sclerosing panencephalitis, encephalomalacia is such as periventricular leukomalacia, epilepsy comprises infantile spasm such as generalized epilepsy, inattentive epilepsy, myoclonic epilepsy comprises the MERRF syndromes, tetanic clonic epilepsy, the localized epilepsy of localized epilepsy such as complexity, frontal epilepsy and temporal-lobe epilepsy, post-traumatic epilepsy, epileptic state is such as EPC, and Ha-Si two syndromes.

The polynucleotide treatment of available fusion rotein of the present invention and/or code book invention albumin fusion proteins or other sacred disease that detects comprise hydrocephalus such as Dandy-Walker syndrome and normal pressure hydrocephalus, the hypothalamus disease is such as hypothalamus vegetation, cerebral malaria, narcolepsy comprises cataplexy, bulbar poliomyelitis, pseudotumor cerebri, auspicious special syndrome, the thunder syndrome, the ganglion cerebral disease, the cerebral toxoplasmosis disease, encephalic tuberculoma and Ze Weige syndromes, the dull-witted compound disease of central nervous system infection such as AIDS, cerebral abscess, subdural empyema, encephalomyelitis is such as equine encephalomyelitis, Venezuelan equine encephalomyelitis, necrotizing hemorrhagic encephalomyelitis, visna, and cerebral malaria.

The polynucleotide treatment of available fusion rotein of the present invention and/or code book invention albumin fusion proteins or other sacred disease that detects comprise that meningitis is such as arachnoiditis, aseptic meningitis comprises lymphocytic choriomeningitis such as viral meningitis, bacterial meningitis comprises Haemophilus meningitis, the listeria meningitis, epidemic cerebrospinal meningitis is such as Waterhouse-Friderichsen syndrome, pneumococcal meningitis and meningeal tuberculosis, fungal meningitis is such as crypotococcal, subdural effusion, meningoencephalitis is such as the uveomeningoencephalitis syndromes, myelitis is such as transverse myelitis, neurosyphilis is such as myelophthisis, poliomyelitis comprises syndromes after bulbar poliomyelitis and the poliomyelitis, prion disease is (such as Ke-Ya two syndromes, bovine spongiform encephalopathy, Ge-Si two syndromes, Kuru disease, itch), with the cerebral toxoplasmosis disease.

The polynucleotide treatment of available fusion rotein of the present invention and/or code book invention albumin fusion proteins or other sacred disease that detects comprise central nervous system vegetation, such as brain vegetation, comprise that cerebellum vegetation is such as vegetation in the curtain, ventricles of the brain vegetation is such as choroid plexus vegetation, hypothalamus vegetation and curtain are gone up vegetation, meninx vegetation, spinal cord vegetation comprises exterior dura vegetation, demyelination is such as canavan's disease, diffuse cerebrosclerosis comprises adrenoleukodystrophy, the all encephalitis of axle, globoid cell leukodystrophy, diffuse cerebrosclerosis is such as metachromatic leukodystrophy, allergic encephalitis, necrotizing hemorrhagic encephalomyelitis, progressive multifocal leukoencephalopathy, multiple sclerosis, central pontine myelinolysis, transverse myelitis, optic neuromyelitis, itch, hollow back, chronic fatigue syndrome, visna, High Pressure Nervous Syndrome, pseudomeningitis, myelopathy is such as congenital amyotonia, amyotrophic lateral sclerosis, Duchenne-Arandisease is such as Werdnig Hoffmann, compression of spinal cord, spinal cord vegetation is such as exterior dura vegetation, syringomyelia, myelophthisis, stiff body syndromes, mental retardation is such as angel's syndromes, cat's cry syndrome, the De Langre syndrome, Down's syndrome, gangliosidosis is such as gangliosidosis G (M1), sandhoff disease, Tay-Sach disease, how Hart pounces on disease, homocystinuria, Lao-Mu-ratio three syndromes, Lai-Ni two syndromes, maple syrup urine disease, Mucolipidosis is such as Fucosidosis, neuronal ceroid lipofuscinosis, eye brain renal syndrome, phenylketonuria is such as the parent phenylketonuria, pula moral-Willie syndromes, auspicious special syndrome, Rubinstein-Taybi syndrome, tuberous sclerosis, the WAGR syndromes, nervous system abnormality is such as holoprosencephaly, neural tube defect comprises hydranencephaly such as anencephalia, A-Cha Er Shi deformity, the brain bulging, the meninx bulging, the bulging of spinal cord spinal meninges, spinal dysraphism such as spina bifida cystica and hemirachischisis.

The polynucleotide treatment of available fusion rotein of the present invention and/or code book invention albumin fusion proteins or other sacred disease that detects comprise heredity motion and esthesioneurosis, comprise Xia Ke-Malian Er Shi disease, hereditary optic atrophy, Refsum's disease, hereditary spastic paraplegia, Werdnig Hoffmann, heredity sensation and autonomic neuropathy such as congenital absence of pain and dysautonomia, neural performance (such as agnosia, comprises the special Man syndromes of Gus, amnesia is such as retrograde amnesia, apraxia, neurogenic bladder, cataplexy, social disorder comprises deafness such as auditory disorder, the part hearing disability, loudness recruitment and tinnitus, the language disorder comprises disease of failing in writing such as aphasia, anomia, Broca's aphasia and Wernicke's aphasia, dislexia is such as the acquired character dislexia, the language development disorder, the language disorder comprises anomia such as aphasia, Broca's aphasia and Wernicke's aphasia, the structure language is disorderly, disorder comprises dysarthria such as language in social disorder, echophrasia, mutism and stutter, get muddled such as crying out and trachyphonia, decerebrate state, delirium, facsiculation, illusion, pseudomeningitis, dyskinesia is such as angel's syndromes, ataxia, mobile spasm, chorea, dystonia, hypokinesis, muscle tone is low excessively, myoclonus, twitch, torticollis and trembling, muscle tone is too high to ossify such as stiff body syndromes such as muscle, the muscle spasm state, paralysis comprises herpes auris such as facial paralysis, gastroparesis, hemiplegia, ophthalmoplegia is such as diplopia, the Du An syndrome, the Horner syndromes, chronic progressive external ophthalmoplegia is such as the Ji Ensi syndrome, bulbar paralysis, torrid zone Spastic Paraplegia, paraplegia such as Blang-fork clip that syndromes, tetraplegia, respiratory paralysis and paralysis vocal cord, paresis, phantom limb, sense of taste disorder such as ageusia and dysgeusia, the vision disorder is such as amblyopia, blind, color defect, diplopia, hemianopsia, blind spot and subnormal vision, somnopathy such as hypersomnia comprises the Kleine-Levin syndromes, insomnia and somnambulism, spasm is such as teeth clenched, unconscious of stupor, persistent vegetable state and faintness and dizzy, neuromuscular disease is such as congenital amyotonia, amyotrophic lateral sclerosis, Lambert-Eton myasthenic syndrome, motor neurone disease, myatrophy is such as Duchenne-Arandisease, Xia-Ma Er Shi disease and Werdnig Hoffmann, syndromes after the poliomyelitis, muscular dystrophy, myasthenia gravis, myotonia atrophica, congenital myotonia, nemaline myopathy, familial periodic paralysis, multiple (pair) myoclonus, torrid zone Spastic Paraplegia and stiff body syndromes, peripheral nervous disease is such as dactylalgia, the amyloid neuropathy, autonomic nervous system diseases is such as the Ai Di syndrome, Ba-Li two syndromes, the familial autonomic nerve is unusual, the Horner syndromes, reflex sympathetic dystrophy and Xia Yi-De Leige syndromes, cranial nerve diseases comprises neurofibromatosis 2 such as auditory nerve disease such as acoustic tumor, the facial nerve disease is such as face ache, Mel Ke Song-Rosenthal syndromes, moving eye disorderly (ocular motility disorder) comprises amblyopia, nystagmus, oculomotor paralysis, ophthalmoplegia is such as the Du An syndrome, the Horner syndromes, chronic progressive external ophthalmoplegia comprises the Ji Ensi syndrome, stravismus such as esotropia and external strabismus, oculomotor paralysis, optic nerve disease comprises hereditary optic atrophy such as optic atrophy, drusen of optic disc, the optic nerve eye is such as optic neuromyelitis, papilloedema, trigeminal neuralgia, paralysis vocal cord, demyelination such as optic neuromyelitis and hollow back, with diabetic neuropathy such as diabetic foot.

The polynucleotide treatment of available fusion rotein of the present invention and/or code book invention albumin fusion proteins or other sacred disease that detects comprise that the neurothlipsis syndromes is such as carpal tunnel syndrome, instep pipe syndromes, the thoracic outlet syndromes is such as scalenussyndrome, ulnar nerve compressing syndromes, neurodynia is such as causalgia, cervico-brachial neuralgia, face ache and trigeminal neuralgia, neuritis is such as the EAN, optic neuritis, polyneuritis, polyradiculoneuropathy and radiculitis are such as polyradiculitis, heredity motion and esthesioneurosis such as Xia Ke-Malian Er Shi disease, hereditary optic atrophy, Refsum's disease, hereditary spastic paraplegia and Werdnig Hoffmann, heredity sensation and autonomic neuropathy comprise congenital absence of pain and dysautonomia, the POEMS syndromes, sciatica, gustatory sweating and tetany.

Endocrine regulation

The disorder that the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for treating, prevent, diagnose and/or prediction is relevant with hormone imbalances and/or the disorder or the disease of disease and/or endocrine system.

Hormone by the glandular secretion of endocrine system is being controlled physical growth, sexual function, metabolism and other function.Disorder can two kinds of methods be classified: hormonogenic disturbance and reply the tissue incapability of hormone.(as by chemotherapy, damage or toxin) that the cause of disease of these hormone imbalances or endocrine system disease, disorder or situation can be hereditary, the human body (such as cancer and some autoimmune disease), obtain or communicable.In addition, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins can be used as specified disease relevant with endocrine system and/or hormone imbalances or disorderly mark or detection thing.

Endocrine system and/or hormone imbalances disorder and/or disease contain the disorder of uterus movement, include but not limited to: the complication of gestation and childbirth (as premature labor childbirth, postterm pregnancy, spontaneous abortion and childbirth slowly or stop); And disorder and/or the disease (as dysmenorrhoea and endometriosis) of menstrual cycle.

Endocrine system and/or hormone imbalances disorder and/or disease comprise the disorder and/or the disease of pancreas, such as for example diabetes, diabetes insipidus, congenital pancreas underdevelopment, pheochromocytoma-islet cell tumor syndromes; Adrenal disorder and/or disease are such as for example addison's disease, reflunomide deficiency disease, manlike disease, hirsutism, Cushing syndrome, high aldosterone disease, pheochromocytoma; The disorder of pituitary gland and/or disease are such as for example hyperpituitarism, hypopituitarism, growth hormone deficiency dwarfism, pituitary adenoma, panhypopituitarism, acromegaly, gigantosoma; Thyroid disorder and/or disease include but not limited to hyperthyroidism, thyroprivia, general Lu's Mo's disease, Graves' disease (graves disease), toxicity joint knot goiter, thyroiditis (struma lymphomatosa, subacute granulomatous thyroiditis, with the tranquillization lymphocytic thyroiditis), Pan De Randt syndrome, solid edema, cretinism, thyrotoxicosis, thyroid hormone coupling defect, thymic aplasia, thyroid H, the Tiroidina cancer, thyroid carcinoma, medullary thyroid carcinoma; Parathyroid disorder and/or disease are such as for example hyperparathyroidism, hypoparathyroidism; Hypothalamic disorder and/or disease.

In addition, endocrine system and/or hormone imbalances disorder and/or disease also can comprise the disorder and/or the disease of testis or ovary, comprise cancer.Other disorder of testis or ovary and/or disease comprise that also for example the vegetation of ovarian cancer, polycystic ovary syndrome, Klinefelter syndrome, testis ease mistake syndromes (bilateral anorchia), interstitial cells congenital absence, cryptorchidism, the capillary hemangioma (optimum) of exerting southern syndrome, myotonic dystrophy, testis, testis forms and new testis (neo-testis).

In addition, endocrine system and/or hormone imbalances disorder and/or disease can comprise that also disorder and/or disease form such as for example polyadenous deficiency symptoms, pheochromocytoma, neuroblastoma, multiple internal secretion vegetation, and the disorder and/or the cancer of endocrine tissue.

In another embodiment, relevant endocrinopathy and/or the disorder of tissue that the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for diagnosing, predict, prevent and/or treatment and the pairing therapeutic protein of therapeutic protein part of albumin fusion proteins of the present invention are expressed therein.

The reproductive system disorder

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for diagnosing, treating or prevent the disease and/or the disorder of reproductive system.The reproductive system disorder of available combination treatment of the present invention includes but not limited to reproductive system damage, infection, superfluous natural disposition disorder, birth defects and causes sterile disease or the complication and the difficulty in postpartum (postpartum difficulties) of disorder, gestation, childbirth or production.

Reproductive system disorder and/or disease comprise the disease and/or the disorder of testis, comprise testicular atrophy, testicular feminization, cryptorchidism (one-sided and bilateral), anorchia, ectopic testis, epididymitis and testitis (are caused by infection, usually such as for example gonorrhoea, parotitis, tuberculosis, and syphilis), testicular torsion, joint knot shape vasitis, germinoma is (as spermocytoma, the cells,primordial cancer, teratocarcinoma, choriocarcinoma, yolk sac tumor, and teratoma), stromal tumors (as the interstitial cells tumour), the hydrocele of tunica vaginalis, vaginal hematocele, varicocele, spermatocele, inguinal hernia, with the spermatogenesis disorder (as immotile cuia syndrome, azoospermia, azoospermia, azoospermia, oligospermia, and teratozoospermia).

The reproductive system disorder also comprises prostatic disorder, such as acute nonbacterial prostatitis, chronic nonbacterial prostatitis, acute bacterial prostatitis, chronic bacterial prostatitis, prostate gland dystonia, prostatosis, granulomatous prostatitis, malakoplakia, benign prostatauxe or regeneration, and the superfluous natural disposition disorder of prostate gland, comprise gland cancer, transitional cell carcinoma, duct carcinoma and squamous cell carcinoma.

In addition, composition of the present invention can be used for diagnosis, treatment and/or the prevention of penis and urethra disorder or disease, comprise the inflammatory disorder, such as balanoposthitis, balanitis xerotica obliterans, phimosis, paraphimosis, syphilis, hsv, gonorrhoea, non gonococcal urethritis, chlamydozoan, mycoplasmas, trichomonas, HIV, AIDS, Lai Teer syndrome, pointed condyloma, flat condyloma and pearly penile papules; Urethra is unusual, such as hypospadia, epispadia and phimosis; Damage before worsening comprises erythroplasia of Queyrat, bowen's disease, rich warm bowenoid papulosis, Bu-huge condyloma of Le Er Shi and verrucous carcinoma; Penile cancer comprises squamous cell carcinoma, carcinoma in situ, verrucous carcinoma cancer and dispersivity penile cancer; The urethra natural disposition disorder of going to live in the household of one's in-laws on getting married comprises penis urethral carcinoma, bulb urethrae film cancer (bulbomembranous urethralcarcinoma) and prostate-urethra cancer; And it is disorderly to erect, such as priapism, Perun Nie Shi disease, erective dysfunction and impotence.

In addition, deferential disease and/or disorder comprise vasculitis and CBAVD (vas deferens congenital bilateral lack as); In addition, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for seminal vesicle disease and/or disorderly diagnosis, treatment and/or prevention, comprise cysticercosis, congenital chloride diarrhea and POLYCYSTIC KIDNEY DISEASE.

Other disorder of male reproductive system and/or disease comprise for example Klinefelter syndrome, Young syndromes, premature ejaculation, diabetes, cystic fibrosis, Ka Tagena syndrome, high heat, multiple sclerosis and gynecomastia.

In addition, polynucleotide of the present invention, the polynucleotide of fusion rotein and/or code book invention albumin fusion proteins can be used for vagina and vaginal orifice disease and/or disorderly diagnosis, treatment, and/or prevention, comprise bacterial vaginosis, the candiyeast vaginitis, hsv, venereal ulcer, granuloma inguinale, lymphogranuloma venereum, scabies, human papillomavirus, the vagina wound, the vaginal orifice wound, adenopathy, the chlamydozoan vaginitis, gonorrhoea, the trichomonas vaginitis, pointed condyloma, syphilis, molluscum contagiosum, atrophic vaginitis, osteitis deformans, lichen sclerosus, lichen planus, vulvodynia, toxic shock syndrome, vulvismus, vulvovaginitis, vulvar vestibulitis, with superfluous natural disposition disorder, such as the squamous cell hyperplasia, clear cell carcinoma, rodent cancer, melanoma, Ba Tuolin gland cancer, form with intracutaneous vegetation on the vulva.

The disorder in uterus and/or disease comprise dysmenorrhoea, retroversion, endometriosis, fibroma, gland myopathy (adenomyosis), anovulatory bleeding, amenorrhoea, Cushing syndrome, hydatidiform mole (hydatidiform mole), Ah thank'sing Man syndromes, premature menopause, sexual prematurity, metropolypus, anovulatory dysfunctional uterine hemorrhage (as because unusual hormone signal) and superfluous natural disposition disorder, such as gland cancer, leiomyosarcoma and sarcoma.In addition, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used as the mark of congenital abnormal uterus or detect thing, and be used for diagnosis, treatment and/or prevention, such as bicornute uterus, uterus septus, simple uterus unicornis, uterus unicornis, uterus unicornis, uterus unicornis, uterus arcuatus, duplex uterus and T shape uterus with connected chamber angle with no connected chamber rudimentary horn with no chamber rudimentary horn.

The disease of ovary and/or disorder comprise does not ovulate, polycystic ovary syndrome (Si Tan-Leventhal two syndromes), ovarian cysts, hypovarionism, ovary is insensitive to gonad-stimulating hormone, ovary excessively generates male sex hormone, the right ovarian vein syndromes, amenorrhoea, hirutism, and ovarian cancer (includes but not limited to the carcinous growth of primary and Secondary cases, Sai Ertuoli-Lai Dixi Er Shi tumour, the ovary endometrioid carcinoma, the ovary papillary serous adenocarcinoma, the ovary mucinous adenocarcinoma, with the ovary krukenberg's tumor).

The disease of uterine neck and/or disorder comprise cervicitis, chronic cervicitis, sticking purulence cervicitis, dysplasia of cervix, cervical polyp, naboths cysts, cervical erosion, incompetence,cervical and uterine neck vegetation (comprising that for example cervical cancer, squamous metaplasia, squamous cell carcinoma, glandular scale shape cell vegetation form and mast cell's vegetation forms).

In addition, the disease of reproductive system and/or disorder comprise pregnant disorder and/or disease, comprise miscarriage and stillbirth, such as early abortion, late abortion, spontaneous abortion, artificial abortion, therapeutic(al) abortion, threatened abortion, missed abortion, incomplete abortion, artificial abortion, habitual abortion, missed abortion and abortive infection; Ectopic pregnancy, anemia, Rh uncompatibility, pregnancy duration vaginal hemorrhage, gestational diabetes, intrauterine growth retardation, polyhydramnios, HELLP syndromes, placental abruption, placenta previa, hypermesis, preeclampsia, eclampsia, herpes gestationis and pregnant urticaria.In addition, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for the diagnosis that gestation can concurrent disease, treatment, and/or prevention, comprise heart trouble, in heart failure, rheumatic heart disease, congenital heart disease, mitral valve prolapse, hypertension, anaemia, ephrosis, communicable disease is (as rubella, cytomegalovirus, toxoplasmosis, infectious hepatitis, chlamydozoan, HIV, AIDS, and genital herpes), diabetes, Graves' disease, thyroiditis, thyroprivia, struma lymphomatosa, chronic active hepatitis, liver cirrhosis, primary biliary cirrhosis, asthma, systemic lupus erythematous, rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, ecphyaditis, ovarian cysts, the gall-bladder disorder, and intestinal obstruction.

With childbirth and production complications associated with arterial system comprise premature rupture of amniotic membrane, premature labor childbirth, postterm pregnancy, postmaturity, progress of labor too slowly, fetal distress (as abnormal cardiac rate (fetus or mother), breathing problem and abnormal presentation), shoulder dystocia, prolapsus umbilical cord, amniotic fluid embolism and abnormal uterine bleeding.

In addition, divide the disease in period in puerperium and/or disorder to comprise endometritis, myometritis, other (tissue) inflammation in uterus, peritonitis, pelvis thrombophlebitis, pulmonary infarction, endotoxemia, pyelonephritis, thrombophlebitis of saphenous vein, mazoitis, urocystitis, postpartum hemorrhage and be inverted the uterus.

Other disorder and/or the disease of the female reproductive system of polynucleotide diagnosis, treatment and/or the prevention of available albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins comprise for example Turner's synodrome, pseudohermaphroditism, premenstrual syndrome, pelvic inflammatory disease, pelvic congestion (congestion of blood vessel), frigidity, ahedonia, dyspareunia, salpingorrhexis and middle pain.

Infectious diseases

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for treatment or detect Vector of infection.For example, can be by improving immunne response, transmissible disease is treated in the propagation and the differentiation that particularly improve B and/or T cell.Or by the existing immunne response of enhancing, or, can improve immunne response by starting new immunne response.Perhaps, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins also can directly suppress Vector of infection, need not to cause immunne response.

Virus is a kind of example that can cause the Vector of infection of the polynucleotide treatment of available albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins or disease that detects or symptom.The example of virus includes but not limited to following DNA and RNA viruses and Viraceae: arboviruses (Arbovirus), Adenoviridae (Adenoviridae), Arenaviridae (Arenaviridae), Arteriviridae (Arterivirus), birnavirus section (Birnaviridae), Bunyaviridae (Bunyaviridae), Caliciviridae (Caliciviridae), PCV-II section (Circoviridae), coronaviridae (Coronaviridae), dengue virus, EBV, HIV, flaviviridae (Flaviviridae), Hepadnaviridae (Hepadnaviridae) (hepatitis virus), herpetoviridae (Herpesviridae) is (such as cytomegalovirus, hsv, varicella zoster virus), Mononegavirus is (as Paramyxoviridae (Paramyxoviridae), Measles virus, Rhabdoviridae (Rhabdoviridae)), orthomyxoviridae family (Orthomyxoviridae) is (as influenza virus A, influenza virus B, and parainfluenza virus), papilloma virus, papovaviridae (Papovaviridae), Parvoviridae (Parvoviridae), Picornaviridae (Picornaviridae), Poxviridae (Poxviridae) (such as smallpox or cowpox), Reoviridae (Reoviridae) (as rotavirus), Retroviridae (Retroviridae) (HTLV-I, HTLV-II, slow virus), and Togaviridae (Togaviridae) (as rubella virus).The virus that belongs to these sections can cause multiple disease or symptom, includes but not limited to: sacroiliitis, bronchiolitis, respiratory syncytial virus, encephalitis, ocular infection is (as conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (first type, B-mode, third type, penta type, chronic active, the fourth type), Japan's Type B encephalitis, junin fever, cut elder brother Gong Yare, Rift Valley fever, yellow jack, meningitis, opportunistic infection (as AIDS), pneumonia, burkitt's lymphoma, fowl pox, hemorrhagic fever, measles, parotitis, parainfluenza, rabies, flu, poliomyelitis, leukemia, rubella, sexually transmitted disease (STD), tetter is (as Ka Boxishi, wart), and viremia.The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for treatment or detect any of these symptom or disease.In specific embodiment, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins are used for the treatment of: meningitis, singapore hemorrhagic fever, EBV and/or hepatitis (as hepatitis B).In another embodiment, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins are used for the treatment of the patient that one or more other commercialization hepatitis vaccines are not responded.In another specific embodiments, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins are used for the treatment of AIDS.

Similarly, can cause that the polynucleotide treatment of available albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins or the disease that detects or the bacterium and the fungi media of symptom include but not limited to following Gram-negative and gram positive bacterium, bacterium section, and fungi: actinomyces (Actinomyces) (as Nocardia (Nocardia)), acinetobacter (Acinetobacter), novel Cryptococcus (Cryptococcus neoformans), Aspergillus (Aspergillus), Bacillaceae (Bacillaceae) (as Bacillus anthracis (Bacillus anthracis)), Bacteroides (Bacteroides) (as bacteroides fragilis (Bacteroides fragilis)), Blastomyces (Blastomyces), bordetella belongs to (Bordetella), Borrelia (Borrelia) (as B. burgdorferi (Borrelia burgdorferi)), Brucella (Brucella), mycocandida (Candida), campylobacter (Campylobacter), chlamydiaceae (Chlamydia), Clostridium (Clostridium) is (as clostridium botulinum (Clostridium botulinum), difficulty is distinguished clostridium (Clostridium diffcile), bacillus aerogenes capsulatus (Clostridium perfringens), clostridium tetanus (Clostridiumtetani)), ball spore Pseudomonas (Coccidioides), corynebacterium (Corynebacterium) (as diphtheria corynebacterium (Corynebacterium diphtheriae)), Cryptococcus (Cryptococcus), tinea (Dermatocycoses), bacillus coli (E.coli) (as enterotoxigenic intestinal bacteria and enterohemorrhagic Escherichia coli), enterobacter (Enterobacter) (as enteroaerogen (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), hemophilus (Haemophilus) (as Type B hemophilus influenzae (Haemophilus influenzae)), Helicobacterium (Helicobacter), legionella (Legionella) (as legionella pneumophilia (Legionella pneumophila)), Spirochaetes (Leptospira), Listera belongs to (Listeria) (as Listeria monocytogenes (Listeria monocytogenes)), mycoplasma (Mycoplasma), Mycobacterium (Mycobacterium) (as Mycobacterium leprae (Mycobacteriumleprae) and mycobacterium tuberculosis (Mycobacterium tuberculosis)), Vibrio (Vibrio) (as cholera chytrid (Vibrio cholerae)), eisseriaceae (Neisseriaceae) is (as Diplococcus gonorrhoeae (Neisseria gonorrheae), Neisseria meningitidis (Neisseria meningilidis)), Pasteurellaceae (Pasteurellaceae), proteus (Proteus), Rhodopseudomonas (Pseudomonas) (as Pseudomonas aeruginosa (Pseudomonas aeruginosa)), Rickettsiaceae (Rickettsiaceae), spirochete (Spirochetes) is (as treponema (Treponema spp.), leptospira (Leptospiraspp.), Borrelia (Borrelia spp.)), Shigella (Shigella spp.), Staphylococcus (Staphylococcus) (as streptococcus aureus (Staphylococcus aureus)), meningococcus (Meningiococcus), streptococcus pneumoniae (Pneumococcus) and streptococcus (Streptococcus) are (as streptococcus pneumoniae (Streptococcus pneumoniae) and A, B, with the C group streptococcus), with urine mycoplasma (Ureaplasmas).These bacteriums, parasite, can cause disease or symptom with fungi section, include but not limited to: antibiotics resistance infects, microbemia, endocarditis, septicemia, ocular infection (as conjunctivitis), uveitis, tuberculosis, oulitis, bacterial diarrhea, opportunistic infection (as the AIDS infections relating), paronychia, the prosthese infections relating, dental caries, the Lai Teershi disease, respiratory tract infection such as Whooping cough or pyothorax, Sepsis, Lyme disease, cat scratch disease, dysentery, paratyphoid, food poisoning, legionnaires disease, chronic and acute inflammation, erythema, yeast infection, typhoid fever, pneumonia, lymph, meningitis (as A type and Type B meningitis), chlamydozoan, syphilis, diphtheria, leprosy, brucellosis, peptide ulceration, anthrax, spontaneous abortion, inborn defect, pneumonia, pulmonary infection, ear infection, deaf, blind, drowsiness, uncomfortable, vomiting, chronic diarrhoea, Crohn disease, colitis, vaginitis, sterile, pelvic inflammatory disease, candidiasis, paratuberculosis, tuberculosis, lupus, sausage poisoning, gangrene, tetanus, pustulosis, rheumatic fever, scarlet fever, sexually transmitted disease (STD), tetter is (as phlegmon, tinea), toxicaemia, urinary tract infection, wound infection, hospital infection.The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for treatment or detect any of these symptom or disease.In specific embodiment, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins are used for the treatment of: tetanus, diphtheria, sausage poisoning and/or Type B meningitis.

In addition, cause the polynucleotide treatment of available fusion rotein of the present invention and/or code book invention albumin fusion proteins, prevention, and/or the diagnosis disease or the parasite media of symptom include but not limited to following section or class: amoeba worm (disease), crust is this worm (disease) doubly, coccidia (disease), Cryptosporidium (disease), double-core amoeba worm (disease), covering diseases such as horse, epizoa (disease), giardia lamblia (disease), worm (disease), Leishmania (disease), schistosomicide (schistosomicide) disease, Taylor's that piroplasm (disease), toxoplasma gondii (disease), taper worm (disease), and trichomonad (disease) and sporozoite are (as Plasmodium vivax Plasmodium virax, plasmodium falciparum Plasmodium falciparium, malariae Plasmodium malariae and Plasmodium ovale Plasmodium ovale).These parasites can cause multiple disease or symptom, include but not limited to: scabies, chigger disease, ocular infection, enteropathy (as dysentery, giardiasis), hepatopathy, tuberculosis, opportunistic infection (relevant as AIDS), malaria, pregnancy complications and toxoplasmosis.The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for treatment, prevention and/or diagnosis any of these symptom or disease.In specific embodiment, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins are used for the treatment of, prevent and/or diagnose malaria.

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used like this, or the patient is used the albumin fusion proteins of the present invention of significant quantity, or gather cell by the patient, supply polynucleotide of the present invention to cell, and will return patient's (therapy exsomatizes) through the cell of transforming.In addition, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used as antigen in vaccine, be used to cause the immunne response at transmissible disease.

Regeneration

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for differentiation, the propagation of cell and attract, and cause tissue regeneration (consulting Science, 276:59-87,1997).Tissue regeneration can be used for repairing, replaces or is responsible to replace because of birth defects, wound (wound, burn, otch or ulcer), aging, disease (as osteoporosis, osteoarthritis, periodontopathy, liver failure), performing the operation comprises lift face Cosmetics Surgery, fibrosis, reperfusion injury or the impaired tissue of systemic cytokine damage.

Can use regenerated tissue of the present invention to comprise organ (as pancreas, liver, intestines, kidney, skin, endothelium), muscle (unstriated muscle, skeletal muscle or cardiac muscle), vascular system (comprising blood vessel and lymphatic vessel), nerve, hematopoiesis and bone (bone, cartilage, tendon and ligament) tissue.Preferably, outgrowth do not have scar or scar to reduce.Survive again and can comprise that blood vessel takes place.

In addition, the polynucleotide of fusion rotein of the present invention and/or the code book invention albumin fusion proteins regeneration that can improve the tissue that is difficult to cure.For example, the time of recovery after raising tendon/ligament regeneration will quickening damage.The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins also can be used to avoid the effort that damages in prevention.Medicable disease specific comprises that tendonitis, carpal tunnel syndrome and other tendon or ligament are damaged.Another example of the tissue regeneration of disunion wound comprise pressure ulcer, with complete relevant ulcer, operation and the trauma wounds of vascular function.

Similarly, neural and cerebral tissue also can use the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins to regenerate, and makes neurocyte proliferation and differentiation.Can use the disease of this method treatment to comprise maincenter and peripheral nervous disease, neuropathy or mechanicalness and traumatic disorder (as spinal cord disorder, injury of head, cerebrovascular disease and apoplexy).Particularly, all can use the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins to treat with peripheral nerve injury, peripheral neuropathy (as causing), localized neuropathy and central nervous system disease (as Alzheimer, Parkinson's disease, Huntington disease, amyotrophic lateral sclerosis and Xia Yi-De Leige syndromes) diseases associated by chemotherapy or other medical therapy.

Gastrointestinal dysfunction

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for treatment, prevention, diagnosis and/or prediction gastrointestinal dysfunction, comprise inflammatory diseases and/or situation, infection, cancer (as intestines vegetation (enteric carcinoid tumour, small intestine non-Hodgkin lymphomas, enteric lymphoma)) and ulcer, such as peptide ulceration.

Gastrointestinal dysfunction comprises dysphagia, odynophagia, the esophagus inflammation, peptic esophagitis, gastric reflux, submucosal fibrosis and narrow, Ma Luolai-Wei Si damage, leiomyoma, lipoma, epidermal carcinoma, gland cancer, the gastric retention disorder, gastro-enteritis, gastratrophy, cancer of the stomach, polyp of stomach, autoimmune disorder is such as pernicious anemia, pyloristenosis, gastritis is (bacillary, viral, eosinocyte, pressure inducement, chronic aggressiveness, atrophic, plasmocyte, with the Menetrey Erichsen), and disease of peritoneum is (as chyloperitoneum (chyloperioneum), hemoperitoneum, mesenteric cyst, mesenteric lymphadenitis, the mesentery angiemphraxis, pimelitis, vegetation, peritonitis, pneumoperitoneum, bubphrenic abscess).

Gastrointestinal dysfunction also comprises the disorder relevant with small intestine, such as malabsorption syndrome, expand, irritable bowel syndrome, sugar is not anti-, celiac disease, duodenal ulcer, duodenitis, ceylon sore mouth, whipple's disease, intestinal lymphangiectasia, Crohn disease, ecphyaditis, ileocleisis, Meckel's diverticulum, multiple diverticulum, the complete operational failure of small intestine and large intestine, lymphoma, with bacterium and parasitic disease (such as traveler's diarrhea, typhoid fever and paratyphoid, cholera, roundworm (ascariasis Ascariasislumbricoides), hookworm (Ancylostoma duodenale Ancylostoma duodenale), nematode (shape worm Enterobius vermicularis of wriggling) in the gutstring, tapeworm (taeniasis bovis Taenia saginata, Echinococcus granulosus Echinococcus granulosus, Bothriocephalus Diphyllobothrium spp., with taeniasis suis T.solium) infect.

Disease of liver and/or disorder comprise intrahepatic cholestasis (my gill syndrome, cholehepatocirrhosis), fatty liver (alcoholic fatty liver, the Lay syndrome), hepatic vein thrombosis forms, the sex change of liver beans shape grain, hepatomegaly, liver lung syndromes, hepatorenal syndrome, portal hypertension (esophagus and stomach varix), liver abscess (amebic liver abscess), liver cirrhosis (alcohol, bile, with experimental), alcoholic liver disease (fatty liver, hepatitis, sclerosis), parasite (hepatic echinococcosis, fascioliasis, amebic liver abscess), jaundice (hemolytic, liver cell property, with cholestasis), cholestasis, portal hypertension, liver enlarges, ascites, hepatitis (alcoholic hepatitis, animal hepatitis, chronic hepatitis (autoimmunization, hepatitis B, third liver, the fourth liver, drug-induced), toxic hepatitis, viral people's hepatitis (hepatitis A, hepatitis B, third liver, the fourth liver, viral hepatitis type E), Wilson's disease, granulomatous hepatitis, the Secondary cases cholehepatocirrhosis, hepatogenic encephalopathy, portal hypertension, varix, hepatogenic encephalopathy, primary biliary cirrhosis, primary sclerosing cholangitis, adenoma, vascular tumor, cholelith, liver failure (hepatogenic encephalopathy, acute hepatic failure), with liver vegetation (angiomyoliopma, calcified liver metastasis, the capsule metastatic liver cancer, epithelial tumor, fibre plate liver cancer, kitchen range joint knot property hyperplasia, liver gland cancer, hepatobiliary cystadenoma, hepatoblastoma, hepatocellular carcinoma, hepatoma, liver cancer, angioendothelioma of liver, mesenchymal hamartoma, the mesenchymal cell knurl of liver, joint knot regenerative proliferation, optimum liver tumor (hepatic cyst [simple tumour, polycystic liver disease, hepatobiliary cystadenoma, choledochal cyst], mesenchymal cell knurl [mesenchymal hamartoma, child's hemangioendothelioma, vascular tumor, peliosis hepatis, lipoma, inflammatory pseudotumor, mix type], epithelioma [epithelial duct (bile duct progonoma, cholangioadenoma), liver cell (adenoma, kitchen range joint knot property hyperplasia, joint knot regenerative proliferation)], malignancy hepatic tumor [liver cell, hepatoblastoma, hepatocellular carcinoma, bile duct cell, cholangiocarcinoma, cystadenocarcinoma, vascular tumor, angiosarcoma, Kaposi sarcoma, hemangioendothelioma, other tumour, embryonal sarcoma, fibrosarcoma, leiomyosarcoma, rhabdosarcoma, sarcocarcinoma, teratoma, carcinoid, squamous cell carcinoma, the primary lymphoma]), peliosis hepatis, the erythropoiesis hepatic porphyria, hepatic porphyria (acute intermittent porphyria, porphyria cutanea tarda), the Ze Weige syndromes).

The disease of pancreas and/or disorder comprise acute pancreatitis, chronic pancreatitis (acute necrotizing pancreatitis, alcoholic pancreatitis), vegetation (gland cancer of pancreas, cystadenocarcinoma, nesidioblastoma, gastrinoma and glucagonoma, capsule vegetation, islet cells tumour, pancreas blastoma) and other pancreatic disease (as cystic fibrosis, tumour (pancreatic pseudocyst, pancreas fistula, insufficiency)).

Gallbladder disease comprises cholelith (cholelithiasis and choledocholithiasis), postcholecystectomy syndromes, diverticulum of gallbladder disease, acute cholecystitis, chronic cholecystitis, tumor of bile duct and mucous cyst.

The disease of large intestine and/or disorder comprise antibiotics resistance colitis, diverticulitis, ulcerative colitis, acquired megacolon, abscess, fungi and infectation of bacteria, anal orifice and rectal intestine disorder (as anal fissure, hemorrhoid), colon diseases (colitis, colon vegetation [colorectal carcinoma, adenoma shape polyp of colon (as villous adenoma), colorectal carcinoma, colorectal carcinoma], diverticulitis of colon, diverticulosis of colon, megacolon [Hirschsprung disease, toxic megacolon]; Sigmoid diseases [rectocolitis, sigmoid colon vegetation]), constipation, Crohn disease, diarrhoea (infantile diarrhea, dysentery), dudenal disease (duodenum vegetation, duodenal obstruction, duodenal ulcer, duodenitis), enteritis (enterocolitis), the FIN enteropathy, ileum disease (ileum vegetation, ileitis), the immunoproliferation disease of intestine, inflammatory bowel (ulcerative colitis, Crohn disease), intestinal occlusion, parasitosis (anisakiasis, balantidiasis, yeast infection (blastocystis infection), cryptosporidiosis, double-core amoeba parasitosis, amebic dysentery, giardiasis), intestinal fistula (rectal fistula), intestines vegetation (caecum vegetation, colon vegetation, duodenum vegetation, ileum vegetation, polyp intestinal, jejunum vegetation rectum vegetation), intestinal obstruction (input loop syndromes, duodenal obstruction, ight soil blocks, the pseudoileus proctoptosis), peptide ulceration (duodenal ulcer, peptic esophagitis, hemorrhage, perforation, stomach ulcer, Zollinger Ellison syndrome), syndromes after the gastrectomy (dumping syndrome), stomach trouble is (as achlorhydria, duodenogastric reflux (bile regurgitation), the vasodilation of stomach hole, gastric fistula, gastric outlet obstruction, gastritis (atrophic or plumpness), gastroparesis, gastric dilatation, gastric diverticulum, stomach vegetation (cancer of the stomach, polyp of stomach, adenocarcinoma of stomach, the hyperplasia polyp of stomach), gastric rupture, stomach ulcer, gastric volvulus), tuberculosis, visceroptosis, vomiting is (as spitting blood, hyperemesis gravidarum, postoperative nausea and vomiting), and hemorrhagic colitis.

Other disease and/or the disorder of gastro-intestinal system comprise bile duct disease, split such as abdomen, fistula is (as leak, the esophagus fistula, gastric fistula, intestinal fistula, the pancreas fistula), vegetation is (as bile duct vegetation, esophagus vegetation is such as esophageal adenocarcinoma, the esophagus squamous cell carcinoma, stomach and intestine vegetation, the gland cancer of pancreas vegetation such as pancreas, the Saliva Orthana capsule vegetation of pancreas, pancreatic capsule vegetation, the pancreas blastoma, with peritonaeum vegetation), esophagus disease is (as big kitchen disease, candidiasis, glycogenesis property acanthosis, ulcer, Barrett esophagus varix, locking, tumour, diverticulum (as divicine), fistula (as tracheo esophageal fistula), the motility disorder is (as the CREST syndromes, dysphgia, achalasia, spasm, gastroesophageal reflux), vegetation, perforation (is breathed out not syndromes as boolean, Mallory-Weiss syndrome), narrow, esophagitis, diaphragmatocele (as hiatal hernia); Gastrointestinal illness is such as gastro-enteritis (infecting as eholera morbus, Norwalk virus), hemorrhage (as spitting blood, melaena, digestive ulcerative bleeding), stomach vegetation (cancer of the stomach, polyp of stomach, adenocarcinoma of stomach, cancer of the stomach)), hernia (as congenital diaphragmatic hernia, femoral hernia, inguinal hernia, oodeocele, umbilical hernia, laparacele) and intestinal disease (as caecum disease (ecphyaditis, caecum vegetation)).

Chemotaxis

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can have chemotactic activity.Chemotactic molecule attracts cell (as monocyte, inoblast, neutrophilic granulocyte, T cell, mastocyte, eosinocyte, epithelial cell and/or endotheliocyte) or mobilizes privileged site to the health, such as inflammation, infection or hyper-proliferative position.Particular trauma or unusual can be resisted and/or treat to the cell of Dong Yuaning then.

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can improve the chemotactic activity of specific cells.Thereby these chemotactic molecules can be used for treating inflammation, infection, hyper-proliferative sexual disorder or any immune system disorder by the cell number that improves privileged site in the target health.For example, chemotactic molecule can be used for treating wound or original to other of tissue by immunocyte being attracted to the injury.Chemotactic molecule of the present invention also can attract inoblast, and it can be used for treating wound.

The polynucleotide of also having imagined fusion rotein of the present invention and/or code book invention albumin fusion proteins can suppress chemotactic activity.It is disorderly that these molecules also can be used for treatment.Thus, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins can be used as chemotactic inhibitor.

In conjunction with active

Albumin fusion proteins of the present invention can be used for screening in conjunction with the molecule of the therapeutic protein part of fusion rotein or the therapeutic protein part bonded molecule of fusion rotein.Fusion rotein can stimulate (agonist) with combining of this molecule, improve, suppresses the activity of (antagonist) or reduction fusion rotein or institute's binding molecule.The example of this molecule comprises antibody, oligonucleotide, protein (as acceptor) or small molecules.

Preferably, the native ligand of the therapeutic protein part of this molecule and fusion rotein of the present invention is closely related, fragment as part, or natural substrate, part, structure or functional analogue thing (are consulted people such as Coligan, Current Protocols in Immunology, 1 (2): the 5th chapter, 1991).Similarly, this molecule can be closely related with the therapeutic protein part bonded natural receptor of albumin fusion proteins of the present invention, perhaps is can be by the therapeutic protein part bonded receptor fragments (as avtive spot) of albumin fusion proteins of the present invention at least.In either case, can use this molecule of known technology design and rational.

Preferably, the screening of these molecules relates to the suitable cell that generates expression albumin fusion proteins of the present invention.Preferred cell comprises from Mammals, yeast, fruit bat or colibacillary cell.

Assay method can only be tested combining of candidate compound and albumin fusion proteins of the present invention, wherein in conjunction with being to detect by marker or relating in the assay method of the competition of mark competition thing.In addition, assay method can be tested candidate compound by producing signal with combining of fusion rotein.

Perhaps, assay method can be used acellular prepared product, the fusion rotein/molecule, chemical libraries or the natural product mixture that are attached on the solid support carry out.Assay method also can only comprise the following steps: candidate compound and contain albumin fusion proteins solution to mix; Measurement fusion albumen/molecular activity or combination; And with standard substance fusion rotein/molecular activity or combine relatively.

Preferably, the ELISA assay method can be used fusion rotein level or the activity in mono-clonal or the polyclonal antibody measure sample (as biological sample).Or by combining with the direct or indirect of albumin fusion proteins, or by with albumin fusion proteins competition substrate, but antibody measurement fusion protein level or activity.

In addition, can identify the therapeutic protein part bonded acceptor of albumin fusion proteins of the present invention, for example part elutriation and FACS sorting (people such as Coligan by the many methods that those skilled in the art will know that, Current Protocols in Immun., 1 (2), the 5th chapter, 1991).For example, in the situation of therapeutic protein part corresponding to FGF of fusion rotein, can adopt cloning by expression, wherein by the cell preparation poly+RNA that responds albumin fusion proteins, for example known NIH3T3 cell and the SC-3 cell that contains the multiple acceptor of FGF family protein, and will be thus the cDNA library that makes up of RNA be divided into several set and be used for rotaring redyeing COS cell or do not respond other cell of albumin fusion proteins.That will cultivate on wave carrier piece is exposed to albumin fusion proteins of the present invention through transfectional cell, in advance they is carried out mark.Can come the mark albumin fusion proteins by several different methods, comprise the recognition site of iodate or introducing site-specific protein kinase.

Behind the fixing and incubation, wave carrier piece is carried out radioautographic analysis.Identify positive set, and use repeatedly the branch subclass and once more screening process prepare subclass and transfection once more, the encode monospecific polyclonal of putative receptor of final generation.

Alternative method as the acceptor evaluation, can will be connected by light is affine with the cytolemma or the extraction prepared product of the acceptor molecule of the therapeutic protein part of expressing albumin fusion proteins of the present invention through the mark albumin fusion proteins, the material of connection can be analyzed by PAGE and resolve and make the X-ray film exposure.Can downcut contain the fusion rotein acceptor through labeled complex, resolve to peptide fragment, and carry out the protein microsequencing.The aminoacid sequence that is obtained by microdetermination will be used to design one group of degenerate oligonucleotide probe, be used to screen the gene of cDNA library with the identification code putative receptor.

In addition, can adopt gene reorganization, motif reorganization, exon reorganization and/or codon reorganization (being referred to as " DNA reorganization ") technology to regulate and control the therapeutic protein part of fusion rotein and/or albumin fusion proteins of the present invention or the activity of white protein composition, efficiently generate the agonist and the antagonist of albumin fusion proteins of the present invention thus.Usually consult U.S. Patent number 5,605,793,5,811,238,5,830,721,5,834,252 and 5,837,458 and Patten, people such as P.A., Curr.Opinion Biotechnol., 8:724-33,1997; Harayama, S., Trends Biotechnol., 16 (2): 76-82,1998; Hansson, people such as L.O., J.Mol.Biol., 287:265-76,1999; And Lorenzo, M.M. and Blasco, R., Biotechniques, 24 (2): 308-13,1998; Each piece of writing of these patents and publication is incorporated herein by reference.In one embodiment, can reorganize the polynucleotide of realizing code book invention albumin fusion proteins and thereby by the change of the albumin fusion proteins of its coding by DNA.DNA reorganization relates to by homology or site-specific reorganization two or more DNA sections is assembled into desired molecule.In another embodiment, can by carry out random mutagenesis change code book invention albumin fusion proteins polynucleotide and thereby by the albumin fusion proteins of its coding, and random mutagenesis can by before reorganization, carry out erroneous tendancy PCR, random nucleotide inserts or other method realize.In another embodiment, can be with one or more compositions of albumin fusion proteins of the present invention, motif, parts, partly, one or more compositions of structural domain, fragment etc. and one or more heterologous molecule, motif, parts, partly, reorganization such as structural domain, fragment.In preferred embodiments, heterologous molecule is the family member.In embodiment preferred more, heterologous molecule is a somatomedin, such as for example Thr6 PDGF BB (PDGF), rhIGF-1 (IGF-1), transforming growth factor (TGF)-α, Urogastron (EGF), fibroblast growth factor (FGF), TGF-β, Delicious peptide (BMP)-2, BMP-4, BMP-5, BMP-6, BMP-7, activator A and B, decapentaplegic (dpp), 60A, OP-2, back of the body albumen, growth and differentiation factor (GDF), nodal, MIS, statin-α, TGF-β 1, TGF-β 2, TGF-β 3, TGF-β 5, with neurogliocyte derived neurotrophic factor (GDNF).

Other preferred fragment has the therapeutic protein part of albumin fusion proteins of the present invention and/or the biological active fragment of white protein composition.Biological active fragment refers to the active similar of the therapeutic protein part of the activity of showing and albumin fusion proteins of the present invention and/or white protein composition but needn't identical segments.Segmental biologic activity can comprise the undesired activity of the active or reduction of wanting of raising.

In addition, the invention provides the method for SCREENED COMPOUND with the compound of evaluation regulation and control albumin fusion proteins effect of the present invention.The example of this assay method comprise with Mammals inoblast, albumin fusion proteins of the present invention and compound to be screened and ³[H] thymidine is mixed under with normal proliferating cells culture condition inoblast.The blank determination method can be carried out under shortage is treated the condition of SCREENED COMPOUND, and by measuring in every kind of situation ³[H] thymidine picked-up quantity of fibroblast proliferation when having compound compares to determine whether this compound stimulates proliferation.The quantity of fibroblast proliferation is by the liquid scintillation laminar analysis measurement, and it is measured ³Mixing of [H] thymidine.Two kinds of compounds of agonist and antagonist all can be identified by this program.

In another approach, there is incubation under the condition of compound in the mammalian cell or the membrane prepare thing of acceptor of expressing the therapeutic protein composition of fusion rotein of the present invention with the fusion rotein of the present invention through mark.Can measure the compound enhancing then or block this interactional ability.Perhaps, measure replying of the known second messenger system treat behind SCREENED COMPOUND and the acceptor interaction, and measure the compound bind receptor and cause whether the ability that the second messenger replys is the potential fusion rotein with definite this compound.This second messenger system includes but not limited to cAMP guanylate cyclase, ionic channel or phosphoinositide hydrolysis.

All these said determination methods all can be used as diagnosis or prediction indication.By activation or inhibition fusion rotein/molecule, the molecule that uses these assay methods to find can be used for treating disease or produce particular result (as angiogenic growth) in the patient.In addition, these assay methods cell or tissue that can find to suppress or to strengthen proper operation generates the reagent of albumin fusion proteins of the present invention.

Therefore, the present invention includes the method for evaluation in conjunction with the compound of albumin fusion proteins of the present invention, comprise the following steps: (a) with candidate's binding compounds with albumin fusion proteins incubation of the present invention; And (b) measure whether combination has taken place.In addition, the present invention includes the method for identifying agonist/antagonist, comprise the following steps: (a) with candidate compound with albumin fusion proteins incubation of the present invention, (b) measure biologic activity, and whether the biologic activity of (c) measuring fusion rotein change has taken place.

Target is delivered

In another embodiment, the invention provides the method that composition is delivered to the target cell of the acceptor of expressing a kind of composition of albumin fusion proteins of the present invention.

As discussed in this article, fusion rotein of the present invention can associate via hydrophobicity, wetting ability, ion and/or covalent interaction and heterologous polypeptide, heterologous nucleic acids, toxin or prodrug.In one embodiment, the invention provides by using with heterologous polypeptide or the associating fusion rotein of the present invention of nucleic acid (comprising antibody) and with the special method that is delivered to cell of composition of the present invention.In one embodiment, the invention provides the method that is used for therapeutic protein is delivered to target cell.In another embodiment, the invention provides and be used for single-chain nucleic acid (as antisense or ribozyme) or double-strandednucleic acid (DNA that if can be incorporated into cellular genome or duplicate and can transcribe as episome) are delivered to the method in the target cell.

In another embodiment, the invention provides the method for coming special destruction cell (as the destruction tumour cell) with toxin or the associating albumin fusion proteins of the present invention of cytotoxic agents (as polypeptide of the present invention or antibody of the present invention) by using.

" toxin " mean in conjunction with and activate endogenous cell toxic effect system system compound, radio isotope, holotoxin, improvement toxin, toxin catalytic subunit or usually in cell or at any molecule or enzyme non-existent on the cell surface, that under qualifications, cause necrocytosis.Can include but not limited to radio isotope known in the art according to the toxin that the present invention uses, such as for example in conjunction with the antibody compounds such as (or it contain the complement fixation(CF) part) of intrinsic or inductive endogenous cell toxic effect system system, thymidine kinase, endonuclease, the RNA enzyme, alpha-toxin, ricin, toxalbumin, ETA, diphtheria toxin, the Saponaria officinalis toxalbumin, the balsam pear toxalbumin, spend more white tree toxalbumin, Pokeweed antiviral protein, α-broom aspergillin, and Toxins,exo-, cholera." cytotoxicity prodrug " means the non-toxic compound that is become cytotoxic compound in cell by the enzymic transformation that exists usually.Can include but not limited to that the phosphate derivative of glutamyl derivative, etoposide or the ametycin of phenylformic acid mustard seed alkylating agent, arabinose born of the same parents are sweet according to the cytotoxicity prodrug that method of the present invention is used, the phenoxy-acetamide derivative of daunoblastin and Zorubicin.

Drug screening

The polynucleotide of also having imagined albumin fusion proteins of the present invention or these fusion roteins of encoding are used for the purposes that the molecule of (modify) albumin fusion proteins of the present invention or the activity of proteins corresponding with the therapeutic protein part of albumin fusion proteins is modified in screening.This method will comprise makes the fusion rotein contact suspect the selected compound with antagonist or agonist activity, and measures the activity of fusion rotein after combination.

By use albumin fusion proteins of the present invention or its binding fragment in multiple drug screening technology, the present invention is particularly useful for screening therapeutic compound.The albumin fusion proteins that is adopted in this test can be attached to solid support, be expressed in cell surface, be free in the solution or be positioned in the cell.The eucaryon or the prokaryotic host cell of the recombinant nucleic acid stable conversion of a kind of drug screening method utilization through expressing albumin fusion proteins.In the competitive binding assay method, through cell transformed or by cultivating the supernatant liquor that this cell obtains medicine is screened at this.Can measure for example formation of mixture between the institute's test agent and albumin fusion proteins of the present invention.

Thus, the invention provides screening of medicaments or influence active any other compositions and methods by albumin fusion proteins mediation of the present invention.These methods comprise by method well-known in the art makes this reagent contact albumin fusion proteins of the present invention or its fragment, and measures the existence of mixture between reagent and albumin fusion proteins or its fragment.In this competitive binding assay method, reagent to be screened has carried out mark usually.Behind the incubation, free reagent is separated with the reagent that exists with combining form, and free or not the quantity of the marker of complexing be the tolerance of particular agent in conjunction with the ability of albumin fusion proteins of the present invention.

Another kind of drug screening technology provides the high flux screening that albumin fusion proteins of the present invention is had the compound of appropriate combination avidity, and in the european patent application of announcing on September 13rd, 1,984 84/03564, carried out very detailed description, be introduced into this paper as a reference.In brief, synthetic a large amount of different little peptide test compounds on solid phase substrate such as plastic pins or some other surfaces.Peptide test compounds and albumin fusion proteins of the present invention are reacted, and clean.Detect the bonded peptide by method well-known in the art then.Can with the albumin fusion proteins direct coated of purifying on flat board to be used for the said medicine triage techniques.In addition, nonneutralizing antibody can be used for catching peptide and is fixed on the solid support.

The present invention has also imagined the purposes of competitive drug screening assay assay method, wherein can be in conjunction with the neutralizing antibody and the special competition of test compounds and albumin fusion proteins or its segmental combination of albumin fusion proteins of the present invention.So, antibody is used to detect the existence of sharing any peptide of one or more antigenic epitopes with albumin fusion proteins of the present invention.

Binding peptide and other molecule

The present invention is also contained and is used to identify in conjunction with the polypeptide of albumin fusion proteins of the present invention and the screening method of non-polypeptide, and identifies the binding molecule that obtains thus.These binding molecules can be used as for example agonist and the antagonist of albumin fusion proteins of the present invention.The treatment embodiment that this agonist and antagonist can be used for hereinafter describing in detail according to the present invention.

This method comprises the following steps: to make albumin fusion proteins of the present invention to contact multiple molecule; And evaluation is in conjunction with the molecule of albumin fusion proteins.

The step that makes albumin fusion proteins of the present invention contact multiple molecule can be carried out in many ways.For example, can imagine albumin fusion proteins is fixed on the solid support, and make the solution of multiple molecule contact immobilized polypeptide.A kind of like this flow process will be similar to the affinity chromatography process, and wherein affinity matrix is made of immobilized albumin fusion proteins of the present invention.Can come purifying albumin fusion proteins to be had the molecule of selective affinity by affine selection then.The essence of solid support, the condition that is used for albumin fusion proteins is attached to process, solvent and the affine separation or the selection of solid support are very easily, and are well known to those of ordinary skill in the art.

Perhaps, also multiple polypeptides can be separated into the fraction of separating basically, give self-contained indivedual polypeptide or its subclass.For example, can being used for of knowing the similar approach that polypeptide separates be separated multiple polypeptides by gel electrophoresis, column chromatography or those of ordinary skills.Can also a kind of like this mode by generate indivedual polypeptide through transformed host cell, promptly be expressed on its outside surface or (as recombinant phage) on every side.Available then albumin fusion proteins of the present invention " is detected " the individual separation thing, chooses wantonly to exist under the condition of expressing needed inductor, to determine whether there is the affine interaction of any selectivity between albumin fusion proteins and the indivedual clone.Before making the albumin fusion proteins contact comprise each fraction of indivedual polypeptide, at first polypeptide can be transferred to solid support so that extra facility to be provided.This solid support can only be a slice filter membrane, such as what made by nitrocellulose or nylon.So, can identify positive colony by gathering through transformed host cell of expression library, it comprises coding has the polypeptide of selective affinity to albumin fusion proteins of the present invention DNA construction.In addition, can have the amino acid sequence of polypeptide of selective affinity to albumin fusion proteins of the present invention, perhaps usually can measure the encoding sequence of the DNA of coded polypeptide more easily by direct mensuration of ordinary method.Then can be by the corresponding DNA sequence primary sequence of deriving.If measure aminoacid sequence, then can adopt the microsequencing technology with polypeptide self.Sequencing technologies can comprise mass spectroscopy.

In some cases, may wish before attempting to measure or detect the affine interactional existence of selectivity by any unconjugated polypeptide of the mixture flush away of albumin fusion proteins of the present invention and multiple polypeptides.When albumin fusion proteins of the present invention or multiple polypeptides are incorporated into solid support, may need this cleaning step especially.

The multiple molecule that provides according to this method can provide by the mode of diverse libraries, such as the molecule that can be used for screening specific combination albumin fusion proteins of the present invention at random or combined peptide or non-peptide library.Many available library is known in this area, for example chemically synthesized library, reorganization storehouse (as the phage display storehouse) and based in vitro translated library.People such as Fodor, Science, 251:767-773,1991; People such as Houghten, Nature, 354:84-86,1991; People such as Lam, Nature, 354:82-84,1991; Medynski, Bio/Technology, 12:709-710,1994; People such as Gallop, J.MedicinalChemistry, 37 (9): 1233-1251,1994; People such as Ohlmeyer, Proc.Natl.Acad.Sci.USA, 90:10922-10926,1993; People such as Erb, Proc.Natl.Acad.Sci.USA, 91:11422-11426,1994; People such as Houghten, Biotechniques, 13:412,1992; People such as Jayawickreme, Proc.Natl.Acad.Sci.USA, 91:1614-1618,1994; People such as Salmon, Proc.Natl.Acad.Sci.USA, 90:11708-11712,1993; PCT publication No. WO 93/20242; And Brenner and Lemer, Proc.Natl.Acad.Sci.USA, 89:5381-5383 has described the example of chemically synthesized library in 1992.

People such as Scott, Science, 249:386-390,1990; People such as Devlin, Science, 249:404-406,1990; People such as Christian, J.Mol.Biol., 227:711-718,1992; Lenstra, J.Immunol.Meth., 152:149-157,1992; People such as Kay, Gene, 128:59-65,1993; And PCT publication No. WO has described the example in phage display storehouse in 18,94/18318,1994 on Augusts.

Include but not limited to PCT publication No. WO on April 18th, 91/05058,1991 based in vitro translated library; Reach people such as Mattheakis, Proc.Natl.Acad.Sci.USA, 91:9022-9026, described in 1994.

As the example of non-peptide library, benzodiazepines library (consulting for example people such as Bunin, Proc.Natl.Acad.Sci.USA, 91:4708-4712,1994) can adapt to this purposes.Also can use class peptide library (people such as Simon, Proc.Natl.Acad.Sci.USA, 89:9367-9371,1992).People such as Ostresh, Proc.Natl.Acad.Sci.USA, 91:11138-11142,1994 have described the another kind of example in available library, wherein the amide functionality of peptide permethylated to produce the combinatorial libraries of chemical conversion.

The kind that can be used for non-peptide library of the present invention is extremely many.For example; Ecker and Crooke; Bio/Technology; 13:351-360,1995 have listed benzodiazepines, glycolylurea, piperazinedione, biphenyl, sugar analogue, β-sulfydryl ketone, Arylacetic acids, acylpiperidine, chromene, cubane, xanthine, aminimide, with azolactone in the chemical species that constitutes basis, various library.

Non-peptide library can be rough be divided into two classes: decorate monomer and oligopolymer.Decorate the monomer storehouse and adopt simple relatively supporting structure, add multiple functional group thereon.Support usually is the molecule with known useful pharmacological activity.For example, support can be benzodiazepines (benzodiazepine) structure.

Non-peptide oligopolymer library utilizes a large amount of monomers and it is assembled into together, produces new shape according to monomeric order.Carbamate, pyrrolinone and morpholino are arranged in the monomer unit that has adopted.The class peptide, promptly wherein side chain is attached to alpha-amino group but not the peptide sample oligopolymer of alpha-carbon, has constituted the basis in the non-peptide oligopolymer library of another kind of form.Initial non-peptide oligopolymer library utilizes the monomer of single type, thereby contains the multiple main chain.Nearest library has utilized and has surpassed a kind of monomer, makes the handiness (flexibility) in library increase.

The screening library can be undertaken by the several different methods of generally knowing.Consult for example following reference, they disclose the screening in peptide storehouse: people such as Parsley, Adv.Exp.Med.Biol., 251:215-218,1989; People such as Scott, Science, 249:386-390,1990; People such as Fowlkes, BioTechniques, 13:422-427,1992; People such as Oldenburg, Proc.Natl.Acad.Sci.USA, 89:5393-5397,1992; People such as Yu, Cell, 76:933-945,1994; People such as Staudt, Science, 241:577-580,1988; People such as Bock, Nature, 355:564-566,1992; People such as Tuerk, Proc.Natl.Acad.Sci.USA, 89:6988-6992,1992; People such as Ellington, Nature, 355:850-852,1992; U.S. Patent number 5,096,815, U.S. Patent number 5,223,409 and U.S. Patent number 5,198,346, all authorized people such as Ladner; People such as Rebar, Science, 263:671-673,1993; And PCT publication No. WO 94/18318.

In a specific embodiments, can carry out with those libraries of albumin fusion proteins bonded member by albumin fusion proteins of the present invention and results that library member contact is fixed on the solid support in order to identify the screening of carrying out in conjunction with the molecule of albumin fusion proteins of the present invention.People such as Parmley for example, Gene, 73:305-318,1988; People such as Fowlkes, BioTechniques, 13:422-427,1992; PCT publication No. WO 94/18318; And the example of these screening methods that are called " elutriation " technology described in the reference of quoting

In another embodiment, be used for two-hybrid system (people such as Fields, Nature, 340:245-246,1989 at yeast screening interacting protein; People such as Chien, Proc.Natl.Acad.Sci.USA, 88:9578-9582,1991) can be used for identifying the molecule of specific combination polypeptide of the present invention.

When binding molecule is polypeptide, can select polypeptide easily by any peptide storehouse, comprise random peptide library, combined peptide storehouse or preference peptide storehouse.Term " preference " refers to be used to make up the library when being used for this paper method has applied restriction to one or multiple parameters when operation, and this parameter has determined the diversity of molecule (the being peptide in this case) set that obtains thus.

Thus, real random peptide library will produce such peptide set, promptly wherein find that at the specified location of peptide the probability of specific amino acids is identical for all 20 seed amino acids.Yet, be fixed as by the position 4,8 and 9 of specifying for example per 5 amino acid Methionin or decapeptide storehouse to occur and only comprise arginine, can in the library, introduce preference.Obviously, can imagine the preference of many types, and the invention is not restricted to any concrete preference.In addition, the present invention has imagined the peptide storehouse of particular type, such as phage display peptide library with adopt and to comprise the peptide storehouse that λShi Juntizaiti and DNA insert segmental DNA construction.

As mentioned above, be in the situation of polypeptide at binding molecule, polypeptide can have about 6 to being less than about 60 amino-acid residues, preferred about 6 to about 10 amino-acid residues, and most preferably from about 6 to about 22 amino acid.In another embodiment, has 15-100 amino acid or 20-50 amino acid in conjunction with polypeptide.

Selected can be by chemosynthesis or recombinant expressed the acquisition in conjunction with polypeptide.

Other activity

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are used in the treatment of the ischemic tissue's moderate stimulation neovascularization (re-vascularization) that causes because of multiple disease condition such as thrombosis, arteriosclerosis and other cardiovascular status.The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins also can be used for stimulating blood vessel to take place and limb regeneration, and are just as discussed above.

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins also can be used for treating the wound that evil, burn, postoperative tissue repair and ulcer due to wound cause, because they promote the mitotic division of the various kinds of cell of Different Origin, such as inoblast and Skeletal Muscle Cell, and promote the reparation or the displacement of impaired or illing tissue thus.

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins also can be used for stimulating neuronal growth, and the neuronal damage that is used for the treatment of and prevents in some neurone disorder or neurodegenerative conditions, to take place, such as Alzheimer, Parkinson's disease and AIDS AIDS-related complex AIDS.The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can have the ability that stimulates chondrocyte's growth, so they can be used for strengthening bone and periodontal regenerative, and offer help in tissue transplantation or bone are transplanted.

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins also can be used for the skin aging that causes because of sunburn by stimulating keratinocyte to grow to prevent.

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins also can be used for pre-losing fibre preventing (hair loss).Equally, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for the growth and the differentiation of hemopoietic cell and medullary cell when other cytokine of associating is used.

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins also are used in the cell culture of keeping organ before the transplanting or supporting prior structure.The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins also are used in induces the tissue of mesoderm origin to break up in the body early embryo.

Except that hematopoietic lineage discussed above, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins also can improve or reduce the differentiation or the propagation of embryonic stem cell.

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins also can be used for regulating and control mammiferous feature, such as ratio, pigmentation, build and the bodily form (as cosmetic surgery) of height, body weight, hair color, eye color, skin, fatty tissue.Similarly, the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for regulating and control mammiferous metabolism, influence processing, utilization and the storage of katabolism, anabolism, energy.

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins can be used for changing the mammiferous mental status or physical state by influencing biorhythm, the rhythm of the heart, depression (comprising dysthymia disorders), violent tenet, pain tolerance level, reproductive performance (preferably by activator or statin sample activity), hormone or internal secretion level, appetite, sexual desire, memory, pressure or other cognitive quality.

The polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins also can act on foodstuff additive or sanitas, such as being used for improving or reducing storage capacity, lipid content, lipid, protein, carbohydrate, microorganism, mineral substance, the spoke factor or other nutritive ingredient.

Above-mentioned application can be used for host extremely widely.These hosts include but not limited to people, mouse, rabbit, goat, cavy, camel, horse, mouse, rat, hamster, pig, miniature pig, chicken, goat, ox, sheep, dog, cat, non-human primates and people.In specific embodiment, the host is mouse, rabbit, goat, cavy, chicken, rat, hamster, pig, sheep, dog or cat.In preferred embodiments, the host is a Mammals.In the most preferred embodiment, the host is the people.

In general description behind the present invention, understand the present invention with reference to the following examples with easier.These embodiment provide as illustration, but not intention is as restriction.

We think do not having under the condition of more descriptions, rely on aforementioned description and following exemplary embodiments, and those of ordinary skills can generate and utilize the change of finding among the present invention, and put into practice the method for being advocated.Therefore, following work embodiment has specifically noted the preferred embodiments of the invention, but not is interpreted as the rest part of limit publicity book by any way.

Embodiment

The generation of embodiment 1:pScNHSA and pScCHSA

Carrier pScNHSA (ATCC preserving number PTA-3279) and pScCHSA (ATCC preserving number PTA-3276) are the derivatives of pPPC0005 (ATCC preserving number PTA-3278), and be used to insert the polynucleotide of coding therapeutic protein or its fragment or variant as cloning vector, with coding human serum albumin's " HSA " polynucleotide in abutting connection with and in same translation box.PScCHSA can be used for producing therapeutic protein-HSA fusions, and pScNHSA can be used for producing HSA-therapeutic protein fusions.

The generation of pScCHSA: albumin part is positioned at the white protein fusions of therapeutic portion C-end

Nucleotide sequence by changing the chimeric HSA signal peptide of coding among the pPPC0005 is convenient to the dna clone of the coding therapeutic protein carrier to the N-end of the DNA of encoding mature albumin protein to comprise that Xho I and Cla I restriction site prepare.

The first step by remove pPPC0005 inherent XhoI and Cla I site (being positioned at 3 ' end of ADH1 terminator sequence) with Xho I and ClaI digestion pPPC0005, is mended flat cohesive end with the T4 archaeal dna polymerase, and is reconnected flush end to produce pPPC0006.

In second step, use two-wheeled PCR that Xho I and Cla I restriction site are added in the nucleotide sequence (HSA leader sequence with from the kex2 site of mating factor α " MAF " mosaic) of coding HSA signal peptide among the pPPC006.In first round PCR, increase with primer shown in SEQ ID NO:36 and the SEQ ID NO:37.The primer of sequence shown in SEQ ID NO:36 comprise encoding part HSA signal peptide sequence, from the kex2 site of mating factor α leader sequence and the aminoterminal nucleotide sequence of section H SA mature form.Introduce four point mutation in the sequence, be created in the Xho I and the Cla I site of the junction discovery of chimeric signal peptide and HSA mature form.These four sudden changes indicate underscore in sequence as follows.In pPPC0005, the Nucleotide from 5 ' to 3 ' of these four positions is T, G, T and G.

5 '-GC CT CGA GAAAAGAGATGCACACAAGAGTGAGGTTGCTCATCG ATTTAAAGATTTGGG-3 ' (SEQ ID NO:36) and 5 '-AATCGATGAGCAACCTCACTCTTGTGTGCATCTCTTTTCTCGAGGCTCCTGGAATA AGC-3 ' (SEQ ID NO:37).Use upstream flank primer then, 5 '-TACAAACTTAAGAGTCCAATTAGC-3 ' (SEQ ID NO:38) and downstream flank primer 5 '-CACTTCTCTAGAGTGGTTTCATATGTCTT-3 ' (SEQ ID NO:39) carries out second and takes turns PCR.The PCR product that so produces of purifying then with Afl II and Xba I digestion, and is connected in the same loci of pPPC0006 to produce pScCHSA.So the plasmid that produces has Xho I and the Cla I site that is incorporated in the signal sequence.Having produced single amino acids in the end that exists in signal sequence in Xho I site changes promptly from LDKR to LEKR.After the nucleotide sequence that will have 5 ' Sal I site (it is compatible with the XhoI site) and 3 ' Cla I site, comprise the polynucleotide of coding albumin fusion proteins therapeutic part is connected in the Xho I of pScCHSA and the Cla I site, D will can not be present in the final albumin fusion proteins expression plasmid to the change of E.Sal I has recovered the original amino acid of signal peptide sequence to the connection of Xho I.The DNA of coding albumin fusion proteins therapeutic part can be inserted into after the Kex2 site before (Kex2 cuts after two basic aminoacids sequence KR of signal peptide end) and Cla I site.

The generation of pScNHSA: albumin part is positioned at the white protein fusions of therapeutic part N-end

Prepare and be convenient to by in pScCHSA, adding three eight base pair restriction sites the dna clone of coding therapeutic protein part carrier to the C-end of the DNA of encoding mature albumin protein.Asc I, Fse I and Pme I restriction site are added between the Bsu36 I and Hind III site of encoding mature HSA nucleic acid sequences to proteins end.This is to contain Asc I, Fse I that underscore indicates and two complementary synthetic primers (SEQ ID NO:40 and SEQ ID NO:41) of Pme I restriction site are realized by use.

5 '-AAGCTGCCTTAGGCTTATAATAA GGCGCGCCGGCCGGCCGTTTAAACTAAGCTTAATTCT-3 ' (SEQ ID NO:40) and 5 '-AGAATTAAGCTTA GTTTAAACGGCCGGCCGGCGCGCCTTATTATAAGCCTAAGGCAGCTT-3 ' (SEQ ID NO:41).With these primer annealings,, and be connected in the same loci of pScCHSA to produce pScNHSA with Bsu36I and HindIII digestion.

Embodiment 2: be used for the generation of the general construction of yeast conversion

Carrier pScNHSA and pScCHSA can be used as the polynucleotide that cloning vector is used to insert coding therapeutic protein or its fragment or variant, with encoding mature human serum albumin's " HSA " polynucleotide adjacency.PScCHSA is used to produce therapeutic protein-HSA fusions, and pScNHSA can be used to produce HSA-therapeutic protein fusions.

Comprise the generation of the white protein fusion construct of HSA-therapeutic protein fusion product

The DNA (shown in SEQ ID NO:X or sequence known in the art) of coding therapeutic protein can use the primer (for example by adding restriction site, the seamless fusions of encoding, coding joint sequence etc.) of being convenient to produce fusion construct to carry out pcr amplification.For example, those skilled in the art's polynucleotide that can design last four amino acid (with containing the Bsu36I site) of the HSA mature form of will encoding add the 5 ' primer of 5 ' end of the DNA of coding therapeutic protein to; And the 3 ' primer that terminator codon and suitable cloning site is added to 3 ' end of therapeutic protein encoding sequence.For example, the forward primer that is used for the DNA of amplification coding therapeutic protein can have sequence, 5 '-aagctG CCTTAGGCTTA (N) ₁₅-3 ' (SEQ ID NO:42), wherein the underscore sequence is the Bsu36I site, ripe proteinic last four amino acid of HSA (ALGL) of the nucleotide coding of capitalization, and (N) ₁₅Identical with proteinic 15 Nucleotide at first of coding therapeutic interest.Similarly, the reverse primer that is used for the DNA of amplification coding therapeutic protein can have sequence, 5 '-GCGCGCGTTTAAAC

GGCGCGCC

(N) ₁₅-3 ' (SEQ IDNO:43), wherein the italic sequence is Pme I site, and the double underline sequence is Fse I site, and single underscore sequence is Asc I site, and square frame Nucleotide is the reverse complementary sequence of two series connection terminator codons, and (N) ₁₅Identical with the reverse complementary sequence of proteinic last 15 Nucleotide of coding therapeutic interest.In case amplification obtains the PCR product, it can cut with one of Bsu36I and (Asc I, Fse I or Pme I), and is connected among the pScNHSA.

In the chimeric leader sequence of HSA Xho I site exist in chimeric signal sequence, promptly produced the single amino acids change in the end of HSA-kex2 signal sequence, from LDKR (SEQ ID NO:44) to LEKR (SEQ ID NO:45).

Comprise the generation of the white protein fusion construct of gene-HSA fusion product

Similar with method mentioned above, the DNA of coding therapeutic protein can use following primer to carry out pcr amplification: the 5 ' primer of 5 ' end that the polynucleotide that contain SalI site and coding HSA leader sequence last three amino acid DKR is added to the DNA of coding therapeutic protein; With add 3 ' primer of 3 ' end of the DNA of coding therapeutic protein to containing ClaI site, the amino acid whose polynucleotide of encoding mature HSA first few.For example, the forward primer that is used for the DNA of amplification coding therapeutic protein can have sequence, 5 '-aggagc GtcGACAAAAGA (N) ₁₅-3 ' (SEQ ID NO:46), wherein the underscore sequence is Sal I site, last three amino acid (DKR) of capitalization nucleotide coding HSA leader sequence, and (N) ₁₅Identical with proteinic 15 Nucleotide at first of coding therapeutic interest.Similarly, the reverse primer that is used for the DNA of amplification coding therapeutic protein can have sequence, 5 '-CTTTAAATCG ATGAGCAACCTCACTCTTGTGTGCATC(N) ₁₅-3 ' (SEQ IDNO:47), wherein the italic sequence is Cla I site, and underscore Nucleotide is the reverse complementary sequence of DNA of initial 9 amino acid (DAHKSEVAH, SEQ ID NO:48) of coding HSA mature form, and (N) ₁₅Identical with the reverse complementary sequence of proteinic last 15 Nucleotide of coding therapeutic interest.In case amplification obtains the PCR product, it can cut with Sal I and Cla I, and is connected in the pScCHSA of Xho I and Cla I digestion.May need different signals or leader sequence, for example can be by standard method known in the art with saccharase " INV " (Swiss-Prot AccessionP00724), mating factor α " MAF " (Genbank Accession AAA18405), MPIF (GeneseqAAF82936), fine protein B (Swiss-Prot Accession P23142), bunch albumen (Swiss-ProtAccession P10909), conversion (permutation) subclone of IGFBP4 (Swiss-Prot AccessionP22692) and HSA leader sequence is in suitable carrier.

The generation of the white protein fusion construct that is adapted at expressing in the yeast saccharomyces cerevisiae

Contain the Not I fragment that coding N holds or C holds the DNA of albumin fusion proteins from pScNHSA or pScCHSA generation, can be cloned into then in the Not I site of the pSAC35 with LEU2 selective marker.Then the carrier that will so produce is used for the conversion of yeast saccharomyces cerevisiae expression system.

Embodiment 3: the general expression in the yeast saccharomyces cerevisiae

The expression vector compatible with yeast expression can be by Lithium Acetate conversion, electroporation or known in the art and/or Sambrook, Fritsch and Maniatis, 1989, " Molecular Cloning:A LaboratoryManual ", the 2nd edition, people such as volume 1-3 and Ausubel, 2000, Massachusetts General Hospitaland Harvard Medical School, " Current Protocols in Molecular Biology ", other method of describing among the volume 1-4 is transformed in the yeast saccharomyces cerevisiae.Expression vector imports among Wine brewing yeast strain DXY1, D88 or the BXP10 by transforming, indivedual transformant can for example be cultivated 3 days among 10ml YEPD (1%w/v yeast extract, 2%w/v peptone, 2%w/v glucose) in 30 ℃, and cultivation after 60 hours in collecting cell stationary phase.By cell was collected supernatant liquor in centrifugal 10 minutes with 3000g.

Except that the LEU2 selective marker, pSAC35 (people such as Sleep, 1990, Biotechnology 8:42 and referring to Fig. 3) comprise whole yeast 2 μ m plasmids, PRB1 promotor and ADH1 termination signal that copy function is provided.

Embodiment 4: the general purifying of the albumin fusion proteins of being expressed by the white protein fusions in the yeast saccharomyces cerevisiae

In preferred embodiments, albumin fusion proteins of the present invention comprises mature form or the N-of its part (for example mature form of the therapeutic protein of listing in the table 1, or the mature form of the therapeutic protein that shows with SEQ ID NO:Z in the table 2) or the mature form of the HSA that C-holds that is blended in therapeutic protein.In one embodiment of the invention, albumin fusion proteins of the present invention also is included in the signal sequence of the host's who is used for expressing the nascent fusion polypeptide of Secretory Pathway guidance.In preferred embodiments, excised by the signal peptide of signal sequence encoding, and sophisticated albumin fusion proteins direct secretion is in substratum.Albumin fusion proteins of the present invention preferably comprises allos signal sequence (for example proteinic non-natural signal sequence of particular treatment), includes but not limited to MAF, INV, Ig, fine protein B, bunch albumen, IGFBP4, the variant HSA leader sequence that includes but not limited to chimeric HSA/MAF leader sequence or other allos signal sequence known in the art.Especially those signal sequences listed in the preferred table 2 and/or the signal sequence of above listing in specification sheets " Expression of Fusion Protein " and/or " reorganization and synthetic other method of producing albumin fusion proteins " part.In preferred embodiments, fusion rotein of the present invention also comprises N end methionine residues.The polynucleotide of these polypeptide of encoding are also contained in the present invention, comprise fragment and/or variant.

The albumin fusion proteins of expressing in yeast as mentioned above can followingly carry out small scale purification on Dyax peptide affinity column.From the zymic supernatant liquor of expressing albumin fusion proteins with 3mM phosphate buffered saline buffer pH 6.2,20mM NaCl and 0.01%Tween 20 diafiltrations to reduce volume and removing pigment.Making solution pass through 0.22 μ m device then filters.Filtrate is loaded on the Dyax peptide affinity column.Carry out wash-out with 100mM Tris/HCl pH 8.2 damping fluid coupled columns.Collection contains proteinic peak fraction, and analyzes on SDS-PAGE after concentrating 5 times.

For large scale purification, can use following method.The diafiltration and be concentrated into 500ml in 20mMTris/HCl pH8.0 of supernatant liquor that will be above 2 liters.Spissated protein soln is loaded into through on the 50ml DEAE-Sepharose Fast Flow post of pre-balance, washes post, and with among the 20mM Tris/HClpH8.0 from the linear gradient NaCl elute protein of 0 to 0.4M NaCl.Merge and contain proteinic fraction, with 0.5M sodium phosphate (NaH ₂PO ₄) transfer to pH6.8.(the NH that in protein soln, adds final concentration 0.9M ₄) ₂SO ₄, and whole solution is loaded into through on the 50ml Butyl650S post of pre-balance.Ammonium sulfate (0.9 to 0M NH with linear gradient ₄) ₂SO ₄) elute protein.Merge the fraction that contains the white protein fusions once more, use 10mM Na ₂HPO ₄/ citrate buffer solution pH5.75 carries out diafiltration, and is loaded into 50ml through on the SP-Sepharose Fast Flow post of pre-balance.NaCl linear gradient elution protein with 0 to 0.5M.Merge the fraction that contains target protein matter, change damping fluid into 10mM Na with the Amicon thickener ₂HPO ₄/ citric acid pH6.25, specific conductivity＜2.5mS/cm.This protein soln is loaded into 15ml through on the Q-Sepharose high performance column of pre-balance, washes post, and use NaCl linear gradient elution protein from 0 to 0.15M NaCl.Can be mixed with specific buffer composition by the protein that damping fluid is changed purifying then.

Embodiment 5: be used for the generation of the general construction of mammalian cell transfection

The generation of the white protein fusion construct that is adapted at expressing in the mammal cell line

Can produce the white protein fusion construct at the expression vector that is used for mammalian cell culture system.N end or the C end of dna clone that can be by standard method known in the art (for example pcr amplification, restrictive diges-tion and the be connected) therapeutic protein of will encoding HSA in the mammalian expression vector.In case made up expression vector, can carry out transfection to mammalian expression system.Suitable carriers is known in the art, for example includes but not limited to pC4 carrier and/or can be from Lonza Biologics, Inc. (Portsmouth, the carrier of NH) buying.

Coding human serum albumin's DNA has been cloned in the pC4 carrier that is suitable for the Mammals culture systems, produces plasmid pC4:HSA (ATCC preserving number PTA-3277).This carrier has Tetrahydrofolate dehydrogenase, and " DHFR " gene is allowed when having methotrexate and selected.

The pC4:HSA carrier is suitable for expressing albumin fusion proteins in Chinese hamster ovary celI.In order in other mammalian cell culture system, to express, may wish to comprise encode the DNA of albumin fusion proteins or by its fragment subclone of forming in candidate's expression vector.For example, can with comprise the DNA of encoding mature albumin fusion proteins or by its fragment subclone of forming in another kind of expression vector, include but not limited to any mammalian expression vector described herein.

In preferred embodiments, will encode the DNA subclone of white protein fusion construct to by Lonza Biologics by program known in the art, (Portsmouth in the carrier that NH) provides, is used for the expression of NSO cell to Inc..

Comprise the generation of the white protein fusion construct of HSA-therapeutic protein fusion product

Use pC4:HSA (ATCC preserving number PTA-3277), can produce the white protein fusion construct that therapeutic protein wherein partly is positioned at ripe white protein sequence C end.For example, can be with the dna clone of coding therapeutic protein or its fragment or variant between the Bsu 36I and Asc I restriction site of carrier.In the time of in being cloned into Bsu 36I and Asc I, can adopt in order to be cloned in the yeast vector system and the same primers as (SEQ ID NO:42 and 43) (seeing embodiment 2) of design.

Comprise the generation of the white protein fusion construct of gene-HSA fusion product

Use pC4:HSA (ATCC preserving number PTA-3277), can produce the white protein fusion construct that therapeutic protein wherein partly is cloned into ripe white protein sequence N end.For example, the dna clone of therapeutic protein of signal sequence that coding can be had oneself is between the Bam HI (or HindIII) and Cla I site of pC4:HSA.In the time of in being cloned into Bam HI or Hind III site, preferably before the translation initiation codon of DNA of coding therapeutic protein, comprise Kozak sequence (CCGCCACC ATG, SEQID NO:49).If therapeutic protein does not have signal sequence, so can with the coding this therapeutic protein dna clone between the Xho I and Cla I site of pC4:HSA.When using Xho I site, can use the exemplary PCR primer of following 5 ' (SEQ ID NO:50) and 3 ' (SEQID NO:51):

5′-CCGCCG CTCGAGGGGTGTGTTTCGTCGA(N) ₁₈-3′(SEQ?ID?NO：50)

5′-AGTCCC ATCGATGAGCAACCTCACTCTTGTGTGCATC(N) ₁₈-3′(SEQ?IDNO：51)

In 5 ' primer (SEQ ID NO:50), the underscore sequence is Xho I site; And the last seven amino acid of the dna encoding natural human serum albumin leader sequence behind Xho I site and the Xho I site.In SEQ ID NO:50, " (N) ₁₈" identical with proteinic 18 Nucleotide at first of coding therapeutic interest.In 3 ' primer (SEQ ID NO:51), the underscore sequence is Cla I site; And Cla I site and DNA thereafter are encoding mature HSA protein (the SEQ ID NO:1) reverse complementary sequences of 10 amino acid whose DNA at first.In SEQ ID NO:51, " (N) ₁₈" be the reverse complementary sequence of last 18 Nucleotide of coding therapeutic interest protein DNA.Use this two primers, but pcr amplification therapeutic interest protein, and purified pcr product digests it with Xho I and Cla I restriction enzyme, and it is cloned among the Xho I and Cla I site of pC4:HSA carrier.

Candidate's leader sequence can replace to chimeric white protein leader sequence with the native albumin leader sequence by standard method known in the art so if desired, i.e. HSA-kex2 signal peptide, or other optional leader sequence.(for example, those skilled in the art can be conventional pcr amplification replace with leader sequence and with PCR product subclone in the white protein fusion construct to replace the white protein leader sequence, keep reading frame simultaneously).

Embodiment 6: the general expression in the mammal cell line

Can pass through calcium phosphate precipitation, fat transfection amine reagent, electroporation, or known in the art and/or Sambrook, Fritsch and Maniatis, 1989, " Molecular Cloning:A LaboratoryManual ", the 2nd edition reaches people such as Ausubel, 2000, Massachusetts General Hospital andHarvard Medical School, " Current Protocols in Molecular Biology ", the white protein fusion construct transfection that other transfection method of describing among the volume 1-4 will produce in the expression vector that is suitable for expressing in mammal cell line is in suitable clone.Then by selecting transfectional cell by the existence of the selective agent of the selective marker in expression vector decision.

PC4 expression vector (ATCC Accession No.209646) is the derivative of plasmid pSV2-DHFR (ATCCAccession No.37146).PC4 contains strong promoter long terminal repeat " the LTR " (people such as Cullen of Rous sarcoma virus, March 1985, Molecular and Cellular Biology, 438-447) and the fragment of cytomegalovirus " CMV " enhanser (people such as Boshart, 1985, Cell41:521-530).This carrier also contains 3 ' intron, polyadenylic acidization and the termination signal of rat preproinsulin (preproinsulin) gene, and the mouse DHFR gene under the control of SV40 early promoter.Chinese hamster ovary " CHO " cell or other clone of lacking active DHFR gene are used for transfection.By methods known in the art the white protein fusion construct transfection among the pC4 will be allowed and in Chinese hamster ovary celI, to express albumin fusion proteins that excise leader sequence subsequently, justacrine is in supernatant liquor in the Chinese hamster ovary celI.Be further purified albumin fusion proteins by supernatant liquor then.

The pEE12.1 expression vector is by Lonza Biologics, and (Portsmouth NH) provides Inc., and it is the derivative of pEE6 (Stephens and Cockett, 1989, Nucl.Acids Res.17:7110).This carrier comprises promotor, enhanser and the complete 5 ' non-translational region (international publication number WO89/01036) of the main immediate early gene of people cytomegalovirus " hCMV-MIE ", the upstream of aim sequence, and glutamine synthetase gene (people such as Murphy, 1991, Biochem J.227:277-279; People such as Bebbington, 1992, Bio/Technology 10:169-175; U.S. Pat 5,122,464), its objective is in the substratum that optionally contains sulfo-methionine(Met) (methionine sulphoximine) and select transfectional cell.The white protein fusion construct transfection that will produce in pEE12.1 by methods known in the art is allowed in the NSO cell in the NSO cell (international publication number WO86/05807) and is expressed albumin fusion proteins, excise leader sequence subsequently, justacrine is in supernatant liquor.Can use technology described herein or that other approach of this area is known to be further purified albumin fusion proteins then by supernatant liquor.

The expression of albumin fusion proteins can be analyzed by for example SDS-PAGE and Western trace, reversed-phase HPLC analysis or other method known in the art.

Produce and select stable CHO and NSO clone through the transfection of white protein fusion construct, the cultivation when for example selecting to have Tetrahydrofolate dehydrogenase " DHFR " gene by methods known in the art (for example fat transfection amine reagent transfection) as the carrier of selective marker or by the shortage glutamine with the 100nM methotrexate.Expression level can be checked by for example immunoblotting, and is first-selected anti-as one with anti-HSA serum, and perhaps time choosing is anti-as one to contain at the serum of the antibody of the therapeutic protein part of given albumin fusion proteins.

With anti-HSA serum is one anti-, checks expression level by immunoblotting.Concrete productivity is measured by ELISA, wherein capture antibody can be the monoclonal antibody at the therapeutic protein part of white protein fusions, and detect antibody can be monoclonal anti HSA biotinylated antibody (or vice versa), subsequently according to the scheme of manufacturers in conjunction with horseradish peroxidase/Streptavidin and analyze.

Embodiment 7: the expression of albumin fusion proteins in mammalian cell

Can in mammalian cell, express albumin fusion proteins of the present invention.Typical mammalian expression vector contains promoter element, protein coding sequence and Transcription Termination and the needed signal of transcript polyadenylic acidization of mediation mRNA transcription initiation.Other element comprises that enhanser, Kozak sequence and flank are the intervening sequence of RNA donor splicing site and acceptor site.Use early stage and late promoter,, and realized efficiently transcribing from the early promoter of cytomegalovirus (CMV) from the long terminal repeat (LTR) of retrovirus such as RSV, HTLVI, HIVI from SV40.Yet, also can use cell element (for example human actin promotor).

Be applicable to that carrying out expression vector of the present invention comprises for example such as following carrier, pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC37146), pBC12MI (ATCC 67109), pCMVSport 2.0 and pCMVSport 3.0.Operable mammalian host cell includes but not limited to people Hela, 293, H9 and Jurkat cell, mouse NIH 3T3 and C127 cell, Cos 1, Cos 7 and CV1, quail QC1-3 cell, mouse Lcell and Chinese hamster ovary (CHO) cell.

Perhaps, can in the stable cell lines of the polynucleotide that contain the coding albumin fusion proteins that is incorporated in the karyomit(e), express albumin fusion proteins.Allow evaluation and separate transfectional cell with the cotransfection of selective marker such as DHFR, gpt, Xin Meisu or Totomycin.

Transfection polynucleotide that can also the amplification coding fusion rotein are to express a large amount of coded fusion roteins.DHFR (Tetrahydrofolate dehydrogenase) mark can be used for setting up carry hundreds of or even the clone of the goal gene of several thousand copies (referring to people such as for example Alt, J.Biol.Chem.253:1357-1370,1978; People such as Hamlin, Biochem.et Biophys.Acta 1097:107-143,1990; People such as Page, Biotechnology 9:64-68,1991).Another kind of useful selective marker is an enzyme glutamine synthetase (GS) (people such as Murphy, Biochem J.227:277-279,1991; People such as Bebbington, Bio/Technology 10:169-175,1992).Use these marks, in selective medium, cultivate mammalian cell and select to have the cell of high resistance.These clones contain the amplification gene that is incorporated in the karyomit(e).Chinese hamster ovary (CHO) and NSO cell are usually used in proteinic production.

The derivative of plasmid pSV2-dhfr (ATCC Accession No.37146), expression vector pC4 (ATCC Accession No.209646) and pC6 (ATCC Accession No.209647) contain strong promoter (the LTR) (people such as Cullen of Rous sarcoma virus, Molecular and CellularBiology, 438-447, March, 1985) add the fragment (people such as Boshart, Cell 41:521-530,1985) of cmv enhancer.Multiple clone site for example has restriction enzyme site BamHI, XbaI and Asp718, is convenient to the clone of goal gene.Carrier also contains 3 ' intron, polyadenylic acidization and the termination signal of rat proinsulin protogene, and the mouse DHFR gene under the control of SV40 early promoter.

Specifically, for example use suitable restriction enzyme digested plasmid pC6, use calf intestinal phosphatase enzyme dephosphorylation by program known in the art then.Then from 1% sepharose carrier of separating.

Use technology known in the art to produce the polynucleotide of code book invention albumin fusion proteins, and use round pcr known in the art this polynucleotide that increase.If use naturally occurring signal sequence to produce fusion rotein of the present invention, carrier does not need second kind of signal peptide so.Perhaps, if do not use naturally occurring signal sequence, carrier can be modified to comprise allos signal sequence (referring to for example international publication number WO96/34891) so.

(La Jolla Ca.) separates the amplified fragments of code book invention fusion rotein to the commodity in use test kit from 1% sepharose for " Geneclean ", BIO 101 Inc..Digest this fragment with suitable restriction enzyme then, and purifying on 1% sepharose once more.

Then with the amplified fragments of identical restriction enzyme digestion code book invention albumin fusion proteins, and on 1% sepharose purifying.Then connect isolating fragment and dephosphorylized carrier with the T4 dna ligase.Subsequent transformation intestinal bacteria HB101 or XL-1 Blue cell, and identify by for example restriction enzyme analysis and to contain the segmental bacterium that inserts among the plasmid pC6.

The Chinese hamster ovary cell that lacks active DHFR gene is used for transfection.Use lipofectin reagent people such as (, the same) Felgner with 5 μ g expression plasmid pC6 or pC4 and 0.5 μ g plasmid pSVneo cotransfection.Plasmid pSV2-neo contains dominant selectable marker, gives the neo gene at the enzyme of the one group of antibiotic resistance that comprises G418 from Tn5, coding.Seed cells among the α-MEM that replenishes 1mg/mlG418.Two days later, with cell with tryptic digestion and be inoculated into hybridoma clone plate (Greiner, replenish in Germany) 10,25 or the 50ng/ml methotrexate add among α-MEM of 1mg/ml G418.After about 10-14 days, single clone is used tryptic digestion, be inoculated into then in the 6 hole petri disses or 10ml flask that use different concns methotrexate (50nM, 100nM, 200nM, 400nM, 800nM).Subsequently the clone that will in the maximum concentration methotrexate, grow transfer to new, contain in addition the 6 hole plates of greater concn methotrexate (1 μ M, 2 μ M, 5 μ M, 10mM, 20mM) in.Repeat same program, up to the clone who obtains in 100-200 μ M concentration, to grow.By for example SDS-PAGE and Western trace or by the reversed-phase HPLC analysis expectation Expression of Fusion Protein is analyzed.

Embodiment 8: the albumin fusion proteins of expressing from the white protein fusion construct in the mammal cell line General purifying

In preferred embodiments, albumin fusion proteins of the present invention comprises mature form or the N-end of its part (for example mature form of the therapeutic protein of listing in the table 1, or the mature form of the therapeutic protein that shows with SEQ ID NO:Z in the table 2) or the mature form of the HSA that C-holds that is blended in therapeutic protein.In one embodiment of the invention, albumin fusion proteins of the present invention also is included in the signal sequence of the host's who is used for expressing the nascent fusion polypeptide of Secretory Pathway guidance.In preferred embodiments, excised by the signal peptide of signal sequence encoding, and sophisticated albumin fusion proteins direct secretion is in substratum.Albumin fusion proteins of the present invention preferably comprises allos signal sequence (for example proteinic non-natural signal sequence of particular treatment), includes but not limited to MAF, INV, Ig, fine protein B, bunch albumen, IGFBP4, the variant HSA leader sequence that includes but not limited to chimeric HSA/MAF leader sequence or other allos signal sequence known in the art.Especially those signal sequences listed in the preferred table 2 and/or the signal sequence of above listing in specification sheets " Expression of Fusion Protein " and/or " reorganization and synthetic other method of producing albumin fusion proteins " part.In preferred embodiments, fusion rotein of the present invention also comprises N end methionine residues.The polynucleotide of these polypeptide of encoding are also contained in the present invention, comprise fragment and/or variant.

Adopt different schemes by mammal cell line supernatant liquor purifying albumin fusion proteins according to employed expression system.

From CHO and 293T clone purifying

Can comprise by the 293T cell conditioned medium liquid purifying albumin fusion proteins of Chinese hamster ovary celI supernatant liquor or transient transfection and at first to use sodium phosphate buffer to catch with negatively charged ion HQ resin, and use the phosphoric acid salt gradient elution, on Blue Sepharose FF post, carry out affinity chromatography subsequently, use the salt gradient wash-out.BlueSepharose FF removes main BSA/ Pp63 glycophosphoproteins pollutent.Being further purified that employing phosphoric acid salt gradient is carried out on Poros PI50 resin can remove and reduce endotoxin contaminants and concentrate albumin fusion proteins.

From NSO clone purifying

Can comprise the Q-Sepharose anion-exchange chromatography by NSO cell conditioned medium liquid purifying albumin fusion proteins, follow SP-sepharose purifying by stepwise elution, be the Phenyl-650M purifying of stepwise elution and last diafiltration then.

Then can be by the protein of damping fluid conversion preparation purifying.

Embodiment 9: the bacterial expression of albumin fusion proteins

Use the PCR Oligonucleolide primers amplification corresponding to comprise the polynucleotide of bacterium signal sequence, code book invention albumin fusion proteins, insert fragment to synthesize with 5 ' and 3 ' end of dna sequence dna.For with extension amplification outcome in expression vector, be used for the primer that amplification coding inserts segmental polynucleotide and preferably contain restriction site, such as BamHI and XbaI at 5 ' end of primer.For example, BamHI and XbaI are corresponding to bacterial expression vector pQE-9 (Qiagen, Inc., Chatsworth, CA) restriction enzyme sites on.This plasmid vector coding antibiotics resistance (Amp ^r), bacterium replication orgin (ori), the adjustable promotor/operon of IPTG (P/O), ribosome bind site (RBS), hexahistidine tag (6-His) and restriction enzyme cloning site.

Digest the pQE-9 carrier with BamHI and XbaI, and amplified fragments is connected in the pQE-9 carrier, keep originating in the reading frame of bacterium RBS.To connect then mixed solution be used for transformed into escherichia coli bacterial strain M15/rep4 (Qiagen, Inc.), it contains the plasmid pREP4 of multiple copied, the latter expresses lacI and suppresses son and give kalamycin resistance (Kan ^r).Identify transformant by its ability of on the LB plate, growing, and select penbritin/kalamycin resistance bacterium colony.Isolated plasmid dna is also confirmed by restriction analysis.

To contain being cloned in the LB substratum that replenishes Amp (100 μ g/ml) and Kan (25 μ g/ml) of expectation construction with liquid culture mode overnight incubation (O/N).The O/N culture is used for inoculating big culture with 1: 100 to 1: 250 ratio.Cell cultures is arrived optical density(OD) 600 (O.D. ⁶⁰⁰) between 0.4 and 0.6.Add the final concentration of IPTG (sec.-propyl-B-D-thiogalactoside) subsequently to 1mM.IPTG suppresses the removing that son is induced P/O by deactivation lacI, causes genetic expression to raise.

Cell was cultivated 3-4 hour again.Then by centrifugal (6000Xg, 20 minutes) collecting cell.The cell precipitation thing was dissolved in 3-4 hour chaotropic agent 6M guanidine-HCl or preferred 8M urea and concentration above (referring to people such as for example Burton, Eur.J.Biochem.179:379-387,1989) in the 2 mercapto ethanol of 0.14M by stirring in 4 ℃.By the centrifugal cell debris of removing, and the supernatant liquor that will contain polypeptide is loaded on the affine resin column of nickel-nitrilotriacetic acid(NTA) (" Ni-NTA ") (can be from QIAGEN, Inc. obtains, and is the same).Protein with 6xHis label with high-affinity in conjunction with the Ni-NTA resin, and can be in a simple step program purifying (details are referring to The QIAexpressionist, 1995, QIAGEN, Inc., the same).

Briefly, supernatant liquor is loaded on the post among 6M guanidine-HCl pH8.Post at first cleans with the 6M guanidine-HCl pH8 of 10 times of volumes, and the 6M guanidine-HCl pH6 with 10 times of volumes cleans then, uses 6M guanidine-HCl pH5 wash-out polypeptide at last.

Dialyse and make the protein renaturation of purifying by it is added 200mM NaCl to phosphate buffered saline (PBS) (PBS) or 50mM sodium-acetate pH6 damping fluid subsequently.Perhaps, make the refolding of protein success in the time of can be on being fixed on the Ni-NTA post.Exemplary condition is as follows: renaturation is used the linear 6M-1M urea gradient among 500mM NaCl, 20% glycerine, the 20mM Tris/HCl pH7.4, wherein contains protease inhibitor.Renaturation should be carried out 1.5 hours or the longer time.After the renaturation, come elute protein by adding the 250mM imidazoles.Remove imidazoles by the dialysis step that at last PBS or 50mM sodium-acetate pH6 damping fluid is added 200mMNaCl.The protein of purifying is stored in 4 ℃ or frozen in-80 ℃.

Except that above-mentioned expression vector, the present invention also comprises expression vector (the ATCCAccession Number 209645 that is called pHE4a, in preservation on February 25 in 1998), it contains phage operator gene and the promoter element that the polynucleotide with code book invention albumin fusion proteins can be operatively connected, be called pHE4a (ATCC Accession Number 209645 was in preservation on February 25 in 1998).This carrier contains: 1) as the neomycin phosphotransferase gene of selective marker, 2) intestinal bacteria replication orgin, 3) T5 bacteriophage promoter sequences, 4) two lac operator gene sequences, 5) Shine-Delgarno sequence and 6) lactose operon repressor thing gene (lacIq).Replication orgin (oriC) derived from pUC19 (LTI, Gaithersburg, MD).Promotor and operon sequence are synthetic.

Can following DNA be inserted among the pHE4a, promptly use NdeI and XbaI, BamHI, XhoI or Asp718 restrictive diges-tion carrier,, and separate big fragment (stuffer should be about 310 base pairs) restriction enzyme digestion product electrophoresis on gel.Use the PCR primer of restriction site to produce the DNA inset according to PCR scheme described herein or that other approach of this area is known with NdeI (5 ' primer) and XbaI, BamHI, XhoI or Asp718 (3 ' primer).With PCR inset gel-purified, and carry out restrictive diges-tion with compatible enzyme.The secundum legem scheme connects inset and carrier.

Transformation carrier in the replaceable such scheme is with marking protein in bacterial system.

Embodiment 10: from the selected cDNA clone of the sample separation of preservation

As shown in table 3, many white protein fusion construct of the present invention are by the ATCC preservation.The white protein fusion construct can comprise any one following expression vector: yeast saccharomyces cerevisiae expression vector pSAC35, mammalian expression vector pC4 or mammalian expression vector pEE12.1.

PSAC35 (people such as Sleep, 1990, Biotechnology 8:42), pC4 (ATCC AccessionNo.209646; People such as Cullen, Molecular and Cellular Biology, 438-447,1985; People such as Boshart, Cell 41:521-530,1985) and pEE12.1 (Lonza Biologies, Inc.; Stephens and Cockett, Nucl.Acids Res.17:7110,1989; International publication number WO89/01036; People such as Murphy, Biochem J.227:277-279,1991; People such as Bebbington, Bio/Technology 10:169-175,1992; U.S. Pat 5,122,464; International publication number WO86/05807) carrier comprises ampicillin resistance gene to cultivate in bacterial cell.The technology that can use this area to describe, such as Hanahan, be coated on the Luria-Broth agar plate that contains 100 μ g/ml penbritins, and in 37 ℃ of overnight incubation, these carriers and/or the white protein fusion construct that comprises them are transformed in the coli strain, such as Stratagene XL-1 Blue (Stratagene Cloning Systems, Inc., 11011 N.Torrey Pines Road, La Jolla, CA, 92037).

Preserved material in the specified sample of the ATCC preserving number of quoting in the table 3 also can contain one or more extra white protein fusion construct, every kind of albumin fusion proteins that coding is different.Therefore, the preservation thing of shared same ATCC preserving number contains the white protein fusion construct of determining at least a table 3 corresponding line.

There are two kinds of methods to can be used for this white protein fusion construct of preservation sample separation of the plasmid DNA from table 3, quoted for specific white protein fusion construct.

Method 1: screening

At first, can directly separate the white protein fusion construct, promptly use methods known in the art to use the plasmid DNA sample of the polynucleotide probes screening preservation corresponding with the SEQ ID NO:X of indivedual construction ID numberings in the table 1.For example, can use Applied Biosystems dna synthesizer according to the synthetic special polynucleotide of the sequence of report with 30-40 Nucleotide.Oligonucleotide can for example use the T4 polynucleotide kinase to use according to ordinary method ³²P-γ-ATP carries out mark and carries out purifying (for example people such as Maniatis, " Molecular Cloning:A Laboratory Manual ", Cold Spring Harbor Press, Cold Spring, NY, 1982).Use technology well known by persons skilled in the art, such as by carrier supplying merchant or the technology that in relevant publication of above quoting or patent, provides, will from the white protein fusion construct of specifying ATCC preservation thing be transformed into suitable host mentioned above (such as XL-1 Blue, Stratagene) in.The density of transformant with about 150 transformant of each plate (bacterium colony) is applied on 1.5% agar plate (containing suitable selective agent, for example penbritin).According to the ordinary method of bacterial colony screening (people such as Sambrook for example, " Molecular Cloning:A LaboratoryManual ", the 2nd edition, 1989, Cold Spring Harbor Laboratory Press, pp 1.93-1.104) or other technology well known by persons skilled in the art use nylon membrane to screen these flat boards.

Method 2:PCR

Perhaps, coding specifies the DNA of albumin fusion proteins to increase from the sample of the white protein fusion construct of preservation with SEQ ID NO:X, for example uses in 5 of the white protein fusion construct of preservation ' and 3 ' specify the DNA of albumin fusion proteins that two kinds of primers of 17-20 the Nucleotide of hybridizing take place with coding.Carry out under normal condition the polymerase chain reaction, for example in containing 25 μ l reaction mixtures of the above-mentioned cDNA template of 0.5 μ g.Reaction mixture is 1.5-5mM MgCl easily ₂, 0.01% (w/v) gelatin, dATP, dCTP, every kind 20 μ M of dGTP, dTTP, every kind of 25pmol of primer, and the Taq polysaccharase of 0.25 unit.Carry out 35 PCR circulation (94 ℃ of sex change 1 minute with the automatic thermal cycler of Perkin-Elmer Cetus; Annealed 1 minute for 55 ℃; 72 ℃ prolong 1 minute).Amplified production is analyzed by agarose gel electrophoresis, downcuts DNA band and purifying with expection molecular weight.Confirm that by subclone and order-checking this PCR product is the sequence of selecting to the DNA product.

Have several method can be used for identifying among the preservation clone may non-existent gene 5 ' or 3 ' non-encoding part.These methods include but not limited to filter membrane detect (filter probing), use specific probe clone's enrichment and with known in the art 5 ' and 3 ' " RACE " schemes are similar or the scheme that is equal to.For example, there are a kind of and the similar method of 5 ' RACE to can be used for producing the disappearance of expectation total length transcript 5 ' terminal people such as (, Nucleic Acids Res.21 (7): 1683-1684,1993) Fromont-Racine.

Briefly, specific RNA oligonucleotide is connected to 5 ' end of the RNA colony that may contain full-length gene rna transcription thing.To contain the special primer of connection RNA oligonucleotide and the primer sets of the special primer of the known array of goal gene will be used for 5 ' part of pcr amplification expectation full-length gene.Can check order to this amplified production then, and be used to produce full-length gene.

This aforesaid method still also can use polyA+RNA from the isolating total RNA in expection source.If desired, can handle the RNA prepared product to remove 5 ' phosphate group on RNA degraded or impaired with Phosphoric acid esterase subsequently, they may influence the RNA ligase enzyme step of back.Then should the deactivation Phosphoric acid esterase, and handle RNA is positioned at messenger RNA(mRNA) 5 ' end with removal cap structure with the acid Pyrophosphate phosphohydrolase of tobacco.This 5 ' end that is reflected at the RNA of excision cap stays 5 ' phosphate group, and it can use the T4RNA ligase enzyme to be connected with the RNA oligonucleotide subsequently.

This modified RNA prepared product as template, is used the synthetic article one cDNA chain of oligonucleotide of gene specific.Article one chain synthesis reaction as template, is used special primer of connects RNA oligonucleotide and the 5 ' end that the special primer PCR of the known array of goal gene is increased and expects.Then to the order-checking of the product of generation like this with analyze to confirm that this 5 ' terminal sequence belongs to the expection gene.

Embodiment 11: (Multifusion) fusions that merge more

Albumin fusion proteins (for example containing the therapeutic protein (or its fragment or variant) that merges with white protein (or its fragment or variant)) can merge to produce " many fusion roteins " with other protein is extra.These many fusion roteins can be used for using widely.For example, the fusion of albumin fusion proteins of the present invention and His label, HA label, albumin A, IgG structural domain and maltose binding protein will help purifying (to see for example EP A 394,827; People such as Traunecker, Nature 331:84-86,1988).The nuclear localization signal that merges with polypeptide of the present invention can be with the specific Subcellular Localization of protein target, and covalency heterodimer or homodimer can improve or reduce the activity of albumin fusion proteins.In addition, the fusion of additional proteins sequence and albumin fusion proteins of the present invention can further improve the solvability and/or the stability of fusion rotein.Above-mentioned fusion rotein can use or the conventional following scheme of revising technology known in the art and/or merging by modification general introduction polypeptide and IgG molecule generates.

Briefly, can use the Fc part of crossing over sequence 5 ' hereinafter described and 3 ' terminal primer PCR amplification human IgG molecule.These primers also should have restriction enzyme sites easily, so that be cloned in the expression vector preferred mammal or Yeast expression carrier.

For example, if use pC4 (ATCC Accession No.209646), people Fc partly can be connected in the BamHI cloning site so.Note, should destroy 3 ' BamHI site.Then, with BamHI once more restrictive diges-tion contain the carrier of people Fc part, the polynucleotide of linearized vector and code book invention albumin fusion proteins (use technology known in the art to produce and separate) are connected in this BamHI site.Notice that the polynucleotide of code book invention fusion rotein are cloned under the situation of terminator codon not having, otherwise will can not produce the fusion rotein that contains Fc.

If use naturally occurring signal sequence to produce albumin fusion proteins of the present invention, pC4 does not need second kind of signal peptide so.Perhaps, if need not naturally occurring signal sequence, carrier can be modified to comprise allos signal sequence (referring to for example international publication number WO 96/34891) so.

Human IgG Fc district:

GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT(SEQ?ID?NO：52)

Embodiment 12: produce antibody by albumin fusion proteins

Hybridoma technology

Can be prepared by a number of procedures (referring to " Current Protocols ", the 2nd chapter) with part (as the therapeutic protein part or the albumin part of fusion rotein) the bonded antibody of albumin fusion proteins of the present invention and albumin fusion proteins of the present invention.An example as these methods has prepared the prepared product of the part of albumin fusion proteins of the present invention or albumin fusion proteins of the present invention, and has carried out purifying so that it is substantially free of natural pollutant.Then such prepared product is imported animal and have the more active polyclonal antiserum of high specific with generation.

Use hybridoma technology to prepare (people such as Kohler, Nature 256:495,1975 to the special monoclonal antibody of the part of albumin fusion proteins of the present invention or albumin fusion proteins of the present invention; People such as Kohler, Eur.J.Immunol.6:511,1976; People such as Kohler, Eur.J.Immunol.6:292,1976; People such as Hammering, in " Monoclonal Antibodies and T-Cell Hybridomas ", Elsevier, N.Y., pp.563-681,1981).Usually, with the partial immunity animal of albumin fusion proteins of the present invention or albumin fusion proteins of the present invention (preferred mouse).Extract the splenocyte of these mouse and merge with suitable myeloma cell line.Can adopt any suitable myeloma cell line according to the present invention; Yet preferred use can be from the parent myeloma cell lines (SP20) of ATCC acquisition.After the fusion, selectivity is kept the hybridoma of generation like this in the HAT substratum, and the limiting dilution of describing by people such as Wands (Gastroenterology 80:225-232,1981) is subsequently cloned.Measure the hybridoma that obtains by this selection subsequently to identify that secrete can be in conjunction with the clone of the antibody of the part of albumin fusion proteins of the present invention or albumin fusion proteins of the present invention.

Perhaps, can use the antiidiotypic antibody can be in conjunction with the additional antibody of the part of albumin fusion proteins of the present invention or albumin fusion proteins of the present invention with the programs preparation of two steps.Therefore it also is antigen that this method is utilized antibody itself, might obtain the fact with second kind of antibody of certain antibodies.According to this method, protein-specific antibody is used for immune animal, preferred mouse.Splenocyte with this animal is used to produce hybridoma then, and screen hybridoma to identify the clone who produces following antibody, the ability of this antibodies albumin fusion proteins of the present invention (or part of albumin fusion proteins of the present invention) specific antibody can be blocked by the part of fusion rotein of the present invention or albumin fusion proteins of the present invention.These antibody comprise the antiidiotypic antibody at fusion rotein of the present invention (or part of albumin fusion proteins of the present invention) specific antibody, and are used for immune animal to induce the formation of other fusion rotein of the present invention (or part of albumin fusion proteins of the present invention) specific antibody.

In order to use in the body of antibody in human body, with antibody " humanization ".Can use by the hybridoma deutero-gene constructs that produces monoclonal antibody and produce these antibody.It is known in the art being used to produce chimeric and method humanized antibody, and has argumentation (to look back referring to Morrison Science 229:1202,1985 in this article; People such as Oi, BioTechniques 4:214,1986; People such as Cabilly, U.S. Patent number 4,816,567; People such as Taniguchi, EP 171496; People such as Morrison, EP 173494; People such as Neuberger, WO 8601533; People such as Robinson, international publication number WO8702671; People such as Boulianne, Nature 312:643,1984; People such as Neuberger, Nature 314:268,1985).

From the antibody fragment of scFv storehouse separation at the part of albumin fusion proteins of the present invention or albumin fusion proteins of the present invention.To contain reactive antibody fragment storehouse from the gene constructed one-tenth of the isolating natural V of existence of people PBL at the part of albumin fusion proteins of the present invention or albumin fusion proteins of the present invention, wherein donor can be once or not to be exposed to it (referring to as United States Patent (USP) 5,885,793, with its complete being incorporated herein by reference).

The recovery in storehouse (rescue).Described in international publication number WO 92/01047, from the RNA structure scFv storehouse of people PBL.In order to reclaim the phage of showing antibody fragment, with about 10 ⁹The individual intestinal bacteria that contain phagemid are used to inoculate the 2xTY (2xTY-AMP-GLU) that 50ml contains 1% glucose and 100 μ g/ml penbritins, and shaking culture reaches 0.8 to O.D..This culture of 5ml is used to inoculate 50ml 2xTY-AMP-GLU, adds 2 * 10 ⁸TU has deleted the helper phage (having deleted the M13 of delta gene 3, referring to international publication number WO 92/01047) of delta gene 3, culture is cultivated 45 minutes in 37 ℃ under non-oscillating condition, then in 37 ℃ of shaking culture 45 minutes.With culture centrifugal 10 minutes with 4000r.p.m., throw out is resuspended among 2 liters of 2xTY that contain 100 μ g/ml penbritins and 50 μ g/ml kantlex, and overnight incubation.Described in international publication number WO92/01047, prepare phage.

The M13 that has deleted delta gene 3 is prepared as follows: the M13 helper phage of having deleted delta gene 3 is encoding gene 3 protein not, and the phage (phagemid) of therefore showing antibody fragment has higher and antigen bonded avidity.Infective M13 particle of having deleted delta gene 3 prepares by cultivate helper phage in the cell that contains the pUC19 derivative, and described pUC19 derivative provides wild type gene 3 protein in morphology of phages generating process.With culture under non-oscillating condition in 37 ℃ of incubations 1 hour, and then in 37 ℃ of shaking culture 1 hour.With cell centrifugation precipitation (IEC-Centra 8,400r.p.m.10 minute), be resuspended in the 2xTY substratum (2xTY-AMP-KAN) that 300ml contains 100 μ g/ml penbritins and 25 μ g/ml kantlex, and spend the night in 37 ℃ of shaking culture.By twice PEG precipitation people such as (, 1990) Sambrook purifying and concentrated phage particle from substratum, it is resuspended in 2ml PBS, and filter (the Minisart NML by 0.45 μ m; Sartorius) to produce about 10 ¹³The final concentration of transduced unit/ml (amicillin resistance clone).

The elutriation in storehouse.Immunotubes (Nunc) is spent the night with the part bag of 4ml 100 μ g/ml or 10 μ g/ml albumin fusion proteins of the present invention or albumin fusion proteins of the present invention in PBS.Effective 2%Marvel-PBS in 37 ℃ of sealings 2 hours, is washed 3 times with PBS then.With about 10 ¹³The phage of TU is applied in the pipe, and goes up the upset incubation 30 minutes in room temperature at upset rotating disk (over and under turntable), and then leaves standstill 1.5 hours.Effective PBS, 0.1%Tween-20 are washed 10 times and wash 10 times with PBS.For wash-out bacteriophage, add 1ml 100mM triethylamine, and on the upset rotating disk, rotated 15 minutes, use 0.5ml 1.0M Tris-HCl pH7.4 neutralization solution thereafter at once.Then, by with the phage of wash-out and bacterium in 37 ℃ of incubations 30 minutes, phage is used to infect the e. coli tg1 of 10ml mid-log phase (mid-log).Then intestinal bacteria are coated on the TYE flat board that contains 1% glucose and 100 μ g/ml penbritins.Reclaim the bacterium storehouse that so produces with the helper phage of having deleted delta gene 3 as mentioned above then and be used for the phage that next round is selected with preparation.Then this process is repeated 4 affinity purifications of taking turns altogether, wherein take turns and the 4th take turns, wash pipe and be increased to PBS, 0.1%Tween-20 and wash 20 times and wash 20 times with PBS the 3rd.

Evaluation in conjunction with the person.Take turns and the 4th take turns that the phage of wash-out is used for ehec infection HB2151 in the selection the 3rd, and be used for measuring from single bacterium colony production solubility scFv people such as (, 1991) Marks.Adopt microtiter plate enforcement ELISA with the part bag quilt of 10pg/ml albumin fusion proteins of the present invention that is dissolved in 50mM supercarbonate pH9.6 or albumin fusion proteins of the present invention.The clone who is positive at ELISA and then by PCR fingerprinting (referring to for example international publication number WO 92/01047) with identify by order-checking subsequently.These ELISA positive colonies also can be by technology known in the art, further identifies such as for example epitope mapping, binding affinity, receptor signal transduction, blocking-up or competitive inhibition antibody/antigen bonded ability and competitive antagonism or antagonistic activity.

Embodiment 13:[ ³ H]-2-deoxyglucose picked-up test

Fat, skeletal muscle and liver are the insulin sensitivity tissues.Regular Insulin can promote glucose uptake/be transported in these tissues.In the fat and the situation of skeletal muscle, insulin promoter finally causes glucose transporter 4 molecules, GLUT4, and the signal that compartment is transferred to cell surface in the special born of the same parents conducts.In case on cell surface, GLUT4 allows the picked-up/transhipment of glucose.

[ ³ H]-picked-up of 2-deoxyglucose

Many fat and muscle relevant cell system can be used for testing the picked-up/transport activity of glucose when lacking or have the combination of any or multiple curative drug of listing for the treatment diabetes.Particularly, 3T3-L1 mouse fibroblast cell and L6 mouse Skeletal Muscle Cell can be divided into respectively 3T3-L1 adipocyte and myotube with as [ ³H]-the suitable external model of 2-deoxyglucose picked-up assay method (people such as Urso, J Biol Chem 274 (43): 30864-73,1999; People such as Wang, J Mol Endocrinol 19 (3): 241-8,1997; People such as HSApel, J Membr Biol 169 (1): 45-53,1999; People such as Tsakiridis, Endocrinology 136 (10): 4315-22,1995).Briefly, with 2 * 10 ⁵The adipocyte of individual cell/100 μ l or broken up the L6 cell transfer to handling through 50 μ g/ml poly-l-lysines after the i.e. differentiation in " TC " of 96 hole tissue culture wares promptly wrap quilt in the substratum (post-differentiationmedium), and in 37 ℃ at 5%CO ₂Middle overnight incubation.Cell is at first washed once with serum-free low dextrose DMEM substratum, then with the combination of the same medium in 100 μ l/ holes and or the multiple curative drug for treatment diabetes listed any with the damping fluid in 100 μ l/ holes, the cumulative concentration of therapeutical agent for example of the present invention (for example disclosed specific fusions of SEQ ID NO:Y and fragment and variant) is 1nM, 10nM and 100nM, hungryly when lacking or having 1nM Regular Insulin cultivates (starve) 16 hours in 37 ℃.Plate is given a baby a bath on the third day after its birth inferior with 100 μ l/ hole HEPES buffer saline.Exist 10 μ M through mark [ ³H]-(Amersham, #TRK672) (SIGMA, Regular Insulin added 30 minutes with 1nM in the HEPES buffer saline in 37 ℃ the 2-deoxyglucose in the time of D-3179) with the unmarked 2-deoxyglucose of 10 μ M.In contrast, carry out identical condition, but lack Regular Insulin.(SIGMA is C6762) to measure non-special picked-up to add podophyllotoxin (cytochalasin) B of final concentration 10 μ M with 100 μ l/ holes in different holes.Cell is given a baby a bath on the third day after its birth inferior with the HEPES buffer saline.I.e. 10 μ M[through mark ³H]-2-deoxyglucose and unlabelled i.e. 10 μ M 2-deoxyglucoses added 10 minutes in room temperature.Cell with cold phosphate buffered saline (PBS) promptly " PBS " give a baby a bath on the third day after its birth time.By adding 150 μ l/ hole 0.2N NaOH and coming lysing cell in 20 minutes in room temperature vibration insulation subsequently.Then with sample transfer in the scintillation vial that is added with the 5ml scintillation solution.Pipe is counted in β-scintillometer.Picked-up in the repeat condition, difference are to lack or exist Regular Insulin, measure by following equation: [(count per minute of Regular Insulin " the cpm "-special cpm of non-)/(the non-specific cpm of the cpm-of no Regular Insulin)].On average reply respectively in the limited field of about 5 times and 3 times of adipocyte and myotube contrast.

The differentiation of cell

At T-75cm ²Cell is converged fully.Remove substratum, and replaced 48 hours with substratum (pre-differentiation medium) before the 25ml differentiation.Cell in 37 ℃ at 5%CO ₂, cultivate in 85% humidity.After 48 hours, remove and dedifferente preceding substratum, and replaced 48 hours with the 25ml division culture medium.Cell once more in 37 ℃ at 5%CO ₂, cultivate in 85% humidity.After 48 hours, remove substratum, replace with 30ml differentiation back substratum.Differentiation back substratum kept 14-20 days, perhaps up to realizing differentiation completely.Changed substratum in every 2-3 days.People's adipocyte can be available from Zen-Bio, INC (#SA-1096).

Embodiment 14:[ ³ H]-thymidine mixes the external test method of pancreatic cell system

Proved GLP-1 induces pancreas in rat pipe epithelial cell line ARIP with time and dose-dependent mode differentiation recently, relevant (the people such as Hui of this rising with pancreas islet duodenum homology frame-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 be obtained by culture collecting center of U.S. typical organization (ATCC clone numbering CRL-2057).RIN-M clone is derived from radiation inductive portable Islet cells knurl.This is to be to set up from the bare mouse different species graft of tumour.This cell produces justacrine pancreas islet polypeptide hormone, and produces L-dopa decarboxylase (mark with amine precursor picked-up and decarboxylation or the active cell of APUD).

The ARLP cell: these are exocrine pancreas cells of the epithelium form that can be obtained by culture collecting center of U.S. typical organization (ATCC clone numbering CRL-1674).Also can be referring to reference: Jessop, N.W. and Hay, R.J., " Characteristics of two rat pancreatic exocrine cell linesderived from transplantable tumors ", In Vitro 16:212,1980; Cockell, M. wait the people, " Identification of a cell-specific DNA-binding activity that interacts with atranscriptional activator of genes expressed in the acinar pancreas ", Mol.Cell.Biol.9:2464-2476,1989; Roux, people such as E., " The cell-specific transcription facotrPTF1 contains two different subunits that interact with the DNA ", Genes Dev.3:1613-1624,1989; And Hui, H. wait the people, " Glucagon-like peptide 1 inducesdifferentiation of islet duodenal homcobox-1-positive pancreatic ductal cells intoinsulin-secreting cells ", Diabetes 50:785-796,2001.

The preparation of cell

RIN-M clone contain 10% foetal calf serum (HyClone, RPMI1640 substratum #SH30088.03) (HyClone cultivates in #SH300027.01), and every 6-8 days with the cultivation of going down to posterity of 1: 3 to 1: 6 ratio.Changed substratum in every 3-4 days.

ARIP (ATCC #CRL-1674) clone is containing the 2mM L-glutaminate and is transferring to HamShi F12K substratum (ATCC, #30-2004) the middle cultivation of containing 1.5g/L sodium bicarbonate and 10% foetal calf serum.ARIP clone goes down to posterity with 1: 3 to 1: 6 ratio per week and cultivates twice.Changed substratum in every 3-4 days.

The mensuration scheme

Cell is inoculated 96 hole plates with 4000 cells/well, and cultivates and to converge to 50% in 48-72 hour.Convert cell to serum free medium with 100 μ l/ holes.Cultivate after 48-72 hour, Xiang Kongzhong adds serum and/or therapeutical agent of the present invention (as albumin fusion proteins of the present invention and fragment and variant).Cultivate again and continue 36 hours.[ ³H]-(Amersham Pharmacia #TRK120) is diluted to 1 microcurie/5 microlitres to thymidine (5-20Ci/mmol).Cultivate after 36 hours, in each hole, add 5 microlitres, cultivated again 24 hours.By with cold phosphate buffered saline (PBS) promptly " PBS " cell of washing gently once come termination reaction.Cell is fixed 15 minutes with 100 microlitres, 10% ice-cold TCA in 4 ℃ subsequently.Remove PBS, and add 200 microlitre 0.2N NaOH.Plate was in room temperature vibration insulation 1 hour.Solution is transferred in the scintillation vial, and added the 5ml scintillation solution compatible, violent mixing with the aqueous solution.This pipe is counted in β-scintillometer.As negative control, only use damping fluid.As positive control, use foetal calf serum.

Embodiment 15: the mensuration of glycosuria

Glycosuria (promptly sugar is excessive in the urine) can be easy to measure so that the morbid state index of diabetes to be provided.Compare with the normal patient sample, excessive urine is the symptom of IDDM and NIDDM in patient's sample.The amount that the patient's of this IDDM of suffering from and NIDDM result of treatment shows as excessive glucose in the urine that causes thus reduces.In the preferred embodiment of IDDM and NIDDM monitoring, use technology known in the art patient's urine sample to be measured the existence of glucose.People's glycosuria is defined as the urine glucose concn and surpasses 100mg/100ml.By obtaining blood sample and measure serum glucose, even can measure over-drastic sugar level among those patients that show glycosuria more accurately.

Embodiment 16: detect B cell proliferation and the stimulation of differentiation or the assay method of inhibition

The generation of functional humoral immunoresponse(HI) needs the soluble and relevant signal between B-pedigree cell and its microenvironment.Signal can transmit and cause B-pedigree cell to continue the just stimulation that its sequencing is grown, or indicator cells stops the negative stimulation of current development pathway.So far, have been found that a large amount of stimulations and suppress effect of signals B cellular response degree, comprise IL-2, IL-4, IL-5, IL-6, IL-7, IL10, IL-13, IL-14 and IL-15.What is interesting is that these signals itself are the weak effect things, but can in the B cell mass, induce activation, breed, break up, go back to the nest, tolerate and death in conjunction with multiple stimulatory protein(SP) altogether.

A class B-cell co-stimulatory albumen of studying preferably is the TNF superfamily.In this family, have been found that CD40, CD27 and CD30 regulate panimmunity with their corresponding part CD154, CD70 and CD153 and reply.Can be used for detecting and observe the propagation of these B cell masses and precursor thereof and the assay method of differentiation is to measure range protein for the useful tool of these B cell masses in the influence that may have aspect propagation and the differentiation.Below listed be two kinds of assay methods that are designed for the differentiation, propagation or the inhibition that detect B cell mass and precursor thereof.

External test Fa-can assess it to albumin fusion proteins of the present invention (comprising the fragment that contains therapeutic protein or the fusion rotein of variant and/or white protein or albuminised fragment or variant) in B cell mass and precursor thereof, to induce activation, propagation, differentiation or suppress and/or dead ability.Albumin fusion proteins of the present invention stimulates the assay method at standard B-lymphocyte altogether in the human tonsil B cell activity to purifying of observational measurement from 0.1 to 10000ng/ml dosage range to be assessed, wherein the tonsilla B cell of culture purified when having the streptococcus aureus of formalin fixed (Staphylococcus aureus) Cowan 1 (SAC) or the anti-human IgM antibody of immobilization as initiator (priming agent).According to containing the measurement that the tritium thymidine mixes, the crosslinked synergy of second signal such as IL-2 and IL-15 and SAC and IgM is to cause B cell proliferation.Novel synergist can use this assay method to be easy to identify.This assay method comprises that exhausting the CD3-positive cell by magnetic bead (MACS) comes separation of human tonsilla B cell.According to the expression of CD45R (B220), assessment draws that to surpass 95% in the cell mass of generation like this be the B cell.

The multiple dilution of each sample is placed each holes of 96 hole plates, wherein add and be suspended in substratum (RPMI 1640, contain 10%FBS, 5X10 ^-5M 2ME, 100U/ml penicillin, 10 μ g/ml Streptomycin sulphates and 10 ^-5Dilution SAC) 10 ⁵Individual B cell, cumulative volume 150 μ l.Quantitative by the pulses in 20 hours (1uCi/ hole) that add the 3H-thymidine (6.7Ci/mM) that began in 72 hours after the factor to propagation or inhibition.Positive and negative contrast is respectively IL2 and substratum.

Assay method in the body-BALB/c mouse injection (i.p.) twice independent damping fluid every day or 2mg/Kg albumin fusion proteins of the present invention (comprising the fragment that contains therapeutic protein or the fusion rotein of variant and/or white protein or albuminised fragment or variant).Mouse was accepted this processing in continuous 4 days, at this moment put to death mouse, and collected various tissues and serum and be used for analyzing.The H﹠amp of the spleen of handling from normal spleen with albumin fusion proteins of the present invention; E section relatively determined the active result of fusion rotein to splenocyte, such as the diffusion of PALS and/or the remarkable increase of red pulp district karyocyte structure, this can indicate the differentiation of B cell mass and the activation of propagation.Whether any physiological change such as the splenolysis (disorganization) that will use the immunohistochemistry research of the promptly anti-CD45R of B cell marking (B220) to be used for measuring splenocyte increases owing to the B cell expressivity (representation) of the B cellular regions of infiltrating the loose definition of having set up the T cellular regions.

The flow cytometry analysis of the spleen of the mouse that in the future personal albumin fusion proteins is handled is used for indicating albumin fusion proteins, and whether the special ratio that increases ThB+, CD45R (B220) dual (dull) B cell surpasses viewed in control mice.

Same, the expected results that increases the mature B cell performance in vivo is the corresponding rising of serum I g titre.Therefore, comparison buffer and fusion rotein are handled serum IgM and the IgA level between the mouse.

Embodiment 17:T cell proliferating determining method

PBMC is carried out CD-3 induce proliferation assay, and pass through ³The picked-up of H-thymidine is measured.This assay method is following carries out.96 hole plates with 100 μ l/ vent needles to the mAb of CD3 (HIT3a, Pharmingen) or the contrast mAb (B33.1) of isotype coupling spent the night (1 μ g/ml is dissolved in 0.5M bicarbonate buffer pH9.5) in 4 ℃ of bags, give a baby a bath on the third day after its birth time with PBS then.Separate PBMC by the F/H gradient centrifugation from human peripheral, and be added to the mAb bag by four holes (5 * 10 of plate ⁴/ hole) contains 10%FCS and P/S in and exist among the RPMI of albumin fusion proteins of the present invention (comprising the fragment that contains therapeutic protein or the fusion rotein of variant and/or white protein or albuminised fragment or variant) of different concns (cumulative volume 200 μ l).With independent related protein damping fluid and substratum in contrast.After 48 hours, plate takes out 100 μ l supernatant liquors and is stored in-20 ℃ to measure IL-2 (or other cytokine), if observe cultivation effect with 1000rpm rotation 2 minutes in 37 ℃ of cultivations.Xiang Kongzhong adds 100 μ l and contains 0.5 μ Ci ³The substratum of H-thymidine, and in 37 ℃ of cultivations 18-24 hour.The results hole, and with ³The H-thymidine mixes the tolerance as propagation.With the positive control of independent anti-CD3 as propagation.Also use IL-2 (100U/ml) as the contrast that strengthens propagation.Use the negative control of the control antibodies of the propagation of inducing T cell not as fusion rotein effect of the present invention.

Embodiment 18: fusion rotein of the present invention is to people's dendritic cell of monocyte and monocyte derived The influence of II class MHC, costimulatory molecules and adhesion molecule expression and cytodifferentiation

Dendritic cell produce by expand the propagation precursor of finding in peripheral blood: the monocyte fraction of adhesivity PBMC or elutriation was cultivated 7-10 days with GM-CSF (50ng/ml) and IL-4 (20ng/ml).These dendritic cell have the characteristic phenotype (CD1, CD80, CD86, CD40 and the antigenic expression of II class MHC) of immature cell.The processing of carrying out such as TNF-α with incitant causes the rapid change (I class and II class MHC, stimulate altogether and the expression of adhesion molecule increases, FC γ RII downward modulation, CD83 raises) of surperficial phenotype.These change increases relevant with the function maturation of dendritic cell with the antigen presentation ability.

The facs analysis of surface antigen is following to carry out.Cell was handled 1-3 days with the albumin fusion proteins of the present invention or the LPS (positive control) of progressive concentration, clean with the PBS that contains 1%BSA and 0.02mM sodium azide, suitable FITC-or the PE-labeled monoclonal antibody with dilution in 1: 20 is incubated 30 minutes in 4 ℃ then.After cleaning once more, labeled cell is gone up at FACScan (Becton Dickinson) and is analyzed by flow cytometry.

The influence that the pair cell factor produces.The cytokine, particularly IL-12 that are produced by dendritic cell are important in T cell dependent immune response initial.IL-12 influences the generation of Th1 helper cell immunne response strongly, and inducing cytotoxic T and NK cell function.ELISA is used for the release of following measurement IL-12.Dendritic cell (10 ⁶/ ml) handled 24 hours with the albumin fusion proteins of the present invention of progressive concentration.LPS (100ng/ml) is joined in the cell culture as positive control.The supernatant liquor of collecting cell culture subsequently, and with commercialization ELISA test kit (R﹠amp for example; D Systems, Minneapolis MN) analyzes IL-12 content.The standard scheme that use provides with test kit.

Influence to II class MHC, its stimulation molecule and adhesion molecule expression.Can be on monocyte three major families of identification of cell surface antigen: adhesion molecule, participate in the molecule and the Fc acceptor of antigen presentation.The regulation and control of the expression of II class MHC antigen and other costimulatory molecules such as B7 and ICAM-1 can cause the change of monocyte antigen presentation ability and inducing T cell activation capability.The increase of Fc receptor expression may be relevant with monocyte cellular cytoxicity activity, release of cytokines and the phagolysis of improvement.

Use the following check surface antigen of facs analysis.Monocyte was handled 1-5 days with the albumin fusion proteins of the present invention or the LPS (positive control) of progressive concentration, clean with the PBS that contains 1%BSA and 0.02mM sodiumazide, suitable FITC-or the PE-labeled monoclonal antibody with dilution in 1: 20 is incubated 30 minutes in 4 ℃ then.After cleaning once more, labeled cell is gone up at FACScan (Becton Dickinson) and is analyzed by flow cytometry.

Monocyte activation and/or survival improve.Assay method at the molecule that activates (or deactivation) monocyte and/or raising monocyte survival (or reducing monocyte survival) is known in the art, and can conventional be used to measure the function whether molecule of the present invention has monocyte inhibition or activator.Albumin fusion proteins of the present invention can use following three kinds of assay methods to screen.For in these assay methods each, peripheral blood lymphocytes (PBMC) centrifugal by through Histopaque gradient (Sigma) from the packed white corpuscle of single donor (leukopacks) (American Red Cross, Baltimore, MD) purifying.Monocyte separates from PBMC by the centrifugal elutriation of adverse current.

The monocyte survival assay method.When cultivating under the condition that lacks serum or other stimulation, the human peripheral blood mononuclear cell is devitalization gradually.Their death results from the process (apoptosis) of inner regulation and control.Adding incitant such as TNF-α in culture obviously improves cell survival and prevents dna break.Use the following measurement apoptosis of propidium iodide (PI) dyeing.With monocyte in polypropylene tube in serum free medium (positive control) at (negative control) under the condition that has 100ng/ml TNF-α with exist under the condition of fusion rotein to be measured of different concns and cultivated 48 hours.Cell is with 2 * 10 ⁶The concentration of/ml is suspended in and contains among the PBS that final concentration is 5 μ g/ml PI, is incubated 5 minutes in room temperature then before FACScan analyzes.In this experiment example, proved that the PI picked-up is relevant with dna break.

The influence that the pair cell factor discharges.A critical function of monocyte/macrophage is that they are by the regulation activity of post-stimulatory release of cytokines to other cell mass of immunity system.Measure following the carrying out of ELISA of release of cytokines.The person monocytic cell is with 5 * 10 ⁵The albumin fusion proteins of the present invention of the density of cell/ml and progressive concentration and at similarity condition but lack under the situation of fusion rotein and cultivate.For the generation of IL-12, cell spends the night existing under the condition of fusion rotein to cause with IFN (100U/ml).Add LPS (10ng/ml) then.Collection condition substratum after 24 hours, and keep freezing up to use.Commodity in use ELISA test kit (R﹠amp for example then; D Systems, Minneapolis MN) carries out the measurement of TNF-α, IL-10, MCP-1 and IL-8, and the standard scheme that provides with test kit is provided.

Oxidation burst (oxidative burst).With the monocyte of purifying with 2-1 * 10 ⁵Cells/well places 96 hole plates.In the substratum (RPMI 1640+10%FCS, glutamine and microbiotic) of cumulative volume 0.2ml, Xiang Kongzhong adds the albumin fusion proteins of the present invention of progressive concentration.Cultivate after 3 days, plate is centrifugal, from the hole, remove substratum.Add every hole 0.2ml phenol red solution (140mM NaCl, 10mM potassium phosphate buffer pH7.0,5.5mM dextrose, 0.56mM is phenol red and 19U/ml HRPO) to the scavenger cell individual layer, and stimulator (200nM PMA).With plate in 37 ℃ of insulations 2 hours, by in each hole, adding 20 μ l 1N NaOH termination reactions.Read absorbancy in 610nm.In order to calculate the H that produces by scavenger cell ₂O ₂Amount, each experiment is carried out the H of known volumetric molar concentration ₂O ₂The typical curve of solution.

Embodiment 19: albumin fusion proteins of the present invention is to the influence of vascular endothelial cell growth

First day, with human umbilical vein's endotheliocyte (HUVEC) with 2-5 * 10 ⁴The density inoculation of cell/35mm plate contains 4% foetal calf serum (FBS), 16 units/ml heparin and 50 units/ml endothelial cell growth fill-in, and (ECGS, Biotechnique is in M199 substratum Inc.).The 2nd day, substratum is changed into the M199 that contains 10%FBS, 8 units/ml heparin.Add albumin fusion proteins of the present invention and positive control with different concns, such as VEGF and basic FGF (bFGF).The 4th day and the 6th day, change substratum.The 8th day, measure cell number with the Coulter counter.

The increase of HUVEC cell number shows that fusion rotein can make vascular endothelial cell proliferation, and the minimizing of HUVEC cell number shows that 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:

Make 1-1.5mm long from cornea central authorities to hypothallus otch.

Under notching edge, insert scraper, towards the exterior angle of eye.

Make bag (its bottom is apart from the edge 1-1.5mm of eye).

In bag, place the particle that contains 50ng-5 μ g albumin fusion proteins of the present invention.

The treatment of carrying out with albumin fusion proteins of the present invention also can be applied topically to corneal wound (treatment every day continues five days) with the dosage range of 20mg-500mg.

Embodiment 21: diabetic mice and glucocorticosteroid infringement wound healing model

Diabetes db+/db+ mouse model

In order to prove albumin fusion proteins healing acceleration process of the present invention, use the hereditary diabetic mice model of wound healing.Holostrome skin wound healing model in the db+/db+ mouse is abundant evaluation, clinical relevant and reproducible infringement wound healing model.The healing of diabetic wound depends on the formation of granulation tissue and epithelium and forms rather than shrink (Gartner, people such as M.H., J.Surg.Res.52:389,1992; Greenhalgh, people such as D.G., Am.J.Pathol.136:1235,1990).

Diabetic animal have many in type ii diabetes observed characteristic feature.Isozygoty (db+/db+) mouse than they normal heterozygosis (db+ /+m) littermate obesity.Diabetes (db+/db+) mouse of sudden change has single autosomal recessive sudden change (db+) people such as (, Proc.Natl.Acad.Sci.USA 77:283-293,1982) Coleman on No. 4 karyomit(e)s.Animal shows many foods, drinks and diuresis more.The diabetic mice (db+/db+) of sudden change have rising blood sugar, rising or normal insulin level and the cell-mediated immunity that suppresses (people such as Mandel, J.Immunol.120:1375,1978; Debray-Sachs, people such as M., Clin.Exp.Immunol.51 (1): 1-7,1983; People such as Leiter, Am.J.of Pathol.114:46-55,1985).Peripheral neuropathy, myocardium complication and microvascular lesions have been described, basement membrane thickening and glomerular filtration unusual (Norido, people such as F., Exp.Neurol.83 (2): 221-232,1984 in these animals; People such as Robertson, Diabetes 29 (1): 60-67,1980; People such as Giacomelli, Lab Invest.40 (4): 460-473,1979; Coleman, D.L., Diabetes 31 (Suppl): 1-6,1982).These diabetic mices that isozygoty form hyperglycemia people such as (, J.Immunol.120:1375-1377,1978) Mandel that similarly Regular Insulin is had resistance with human type ii diabetes.

Observed feature shows that the healing in this model may similar to observed healing in the human diabetes (people such as Greenhalgh, Am.J.of Pathol.136:1235-1246,1990) in these animals.

Used in this research the female C57BL/KsJ of hereditary diabetes (db+/db+) mouse and they non-diabetic (db+ /+m) heterozygosis littermate (Jackson Laboratories).Animal was buied when its age in 6 week, and was 8 ages in week when the research beginning.Animal is raised separately, and arbitrarily obtains food and water.All operations all adopts Aseptic technique to carry out.Test is according to Human Genome Sciences, the regulation of the Inc. scientific research the care of animal and the use council (Institutional Animal Care and Use Committee) and guilding principle and carry out about the guilding principle of management of laboratory animal and use.

The wound scheme was carried out (Tsuboi, R. and Rifkin, D.B., J.Exp.Med.172:245-251,1990) according to former reported method.Briefly, on wound same day, be dissolved in avertin (0.01mg/ml), tribromo-ethanol and the 2-methyl-2-butanols anesthetized animal of deionized water by peritoneal injection.With the dorsal area defeathering of animal, skin cleans with 70% ethanolic soln and the tincture of iodine.Operative region is dried up with sterile gauze before wound.Organize punch tool to create the holostrome skin wound of 8mm with Keyes subsequently.Follow closely after the wound, skin is to eliminate the wound expansion near stretching gently.Wound keeps open at experimental session.From wound same day, implemented Local treatment in continuous 5 days.Before processing, wound cleans gently with Sterile Saline and gauze.

The visual control wound, and on operation same day and after this taking pictures in fixed range every three days.At 1-5 days with measured the closed situation of wound on the 8th day by measurement every day.Use indicates graduated Jameson calipers level and vertical survey wound.Covered by the successive epithelium if no longer see granulation tissue and wound, then thought wound healing.

Albumin fusion proteins of the present invention adopts the scope various dose, uses in carrier 8 days every day to the every wound of 500mg from 4mg.The vehicle Control group obtains the 50ml carrier soln.

Animal passed through peritoneal injection vetanarcol (300mg/kg) euthanasia at the 8th day.Collect wound and near skin then to be used for histology and immunohistochemistry.Tissue sample places between the tissue cassette examination of living tissue sponge 10% neutral buffered formalin to be used for further processing.

Three groups of assessments of every group of 10 animals (5 diabetes and 5 non-diabetic contrasts): 1) carrier placebo, 2) untreated fish group and 3) treatment group.

By analyzing wound closure with level and Z-axis measured zone and the site area that obtains wound.Difference assessment contraction by definite initial wound area (the 0th day) and between the wound area (the 8th day) after handling subsequently.The 1st day wound area is 64mm ², i.e. the corresponding size of skin punch tool.Use following formula to calculate:

The 8th day open area of a.[]-[the 1st day open area]/[the 1st day open area]

Sample is fixed in 10% buffered formalin, and wax embedding block is cut into slices (5mm) perpendicular to wound surface, the Reichert-Jung slicing machine is adopted in cutting.The cross section of dividing wound equally is carried out conventional hematoxylin-eosin (H﹠amp; E) dyeing.Whether agglutination that uses the histological examination of wound to assess to repair skin and morphology outward appearance change because of the processing of albumin fusion proteins of the present invention.This assessment comprises checking cell aggregation, inflammatory cell, kapillary, inoblast, epithelium forms again and epidermis is sophisticated exists (Greenhalgh, people such as D.G., Am.J.Pathol.136:1235,1990).Lens micrometer through calibration is used by unwitting viewer (blinded observer).

Tissue slice also carries out immunohistochemical staining with the anti-human keratinous antibody of multi-clone rabbit, wherein uses ABC Elite detection system.End user's skin is as the contrast of positive tissue, and the not immune IgG of use is as negative control.Measure the growth of keratinocyte by using the degree that forms again through calibration lens micrometer assessment wound epithelium.

By using the proliferating cell nuclear antigen/cyclin (PCNA) in anti-PCNA antibody (1: 50) and the ABC Elite detection system displaying skin samples.End user's colorectal carcinoma is as the contrast of positive tissue, and end user's cerebral tissue is used as negative control.Each sample comprises and has omitted a section anti-and that replace with immune mouse IgG not.The classification of these sections is based on the grade that the propagation degree is divided into 0-8, and the low side grade of the slight propagation of reflection is to the high-end of propagation of having strong complaints.

Use non-paired t test to analyze experimental data.P value less than 0.05 is thought significantly.

Steroid infringement rat model

Steroid is to the inhibition of wound healing good proof (Wahl in the system in various external and bodies, " Glucocorticoids and Wound healing ", in " Anti-Inflammatory SteroidAction:Basic and Clinical Aspects ", 280-302,1989; People such as Wahl, J.Immunol.115:476-481,1975; People such as Werb, J.Exp.Med.147:1684-1694,1978).Glucocorticosteroid is by suppressing blood vessel and take place, reduce vascular permeability people such as (, An.Intern.Med.37:701-705,1952) Ebert, fibroblast proliferation and collagen synthetic (people such as Beck, Growth Factors5:295-304,1991; People such as Haynes, J.Clin.Invest.61:703-797,1978) and the instantaneous reduction (people such as Haynes, J.Clin.Invest.61:703-797,1978 that produce circulating monocytic cell; Wahl, " Glucocorticoids and wound healing " is in " Antiinflammatory SteroidAction:Basic and Clinical Aspects ", Academic Press, New York, pp.280-302,1989) postpone wound healing.Systemic application steroid infringement wound healing is the phenomenon that fully proves (people such as Beck, Growth Factors 5:295-304,1991 in rat; People such as Haynes, J.Clin.Invest.61:703-797,1978; Wahl, " Glucocorticoids and wound healing ", in " Antiinflammatory Steroid Action:Basic and Clinical Aspects ", AcademicPress, New York, pp.280-302,1989; People such as Pierce, Proc.Natl.Acad.Sci.USA86:2229-2233,1989).

In order to prove that albumin fusion proteins of the present invention can the healing acceleration process, assessed repeatedly the topical application fusion rotein to the effect of holostrome excision skin wound in the rat, wherein heal and damage because of the systemic application methyl meticortelone.

This embodiment uses the young bull Sprague Dawley rat (Charles River Laboratories) of body weight 200-300g.Animal was buied when 8 ages in week, was 9 ages in week when the research beginning.Systemic application methyl meticortelone when the healing of rat is replied because of wound (in the 17mg/kg/ rat muscle) is impaired.Animal is raised separately, and arbitrarily obtains food and water.All operations all adopts Aseptic technique to carry out.This research is according to Human Genome Sciences, the regulation of the Inc. scientific research the care of animal and the use council (Institutional Animal Care and Use Committee) and guilding principle and carry out about the guilding principle of management of laboratory animal and use.

Follow above-described wound scheme.On wound same day, draw piperazine (5mg/kg) anesthetized animal by peritoneal injection Ketamine (50mg/kg) and plug.With the dorsal area defeathering of animal, skin cleans with 70% ethanolic soln and iodine solution.Confined surgical areas is dried up with sterile gauze before wound.Organize punch tool to create the holostrome skin wound of 8mm with Keyes subsequently.Wound keeps open at experimental session.From wound and use methyl meticortelone same day subsequently, implement one time the topical application test material continuous 7 day every day.Before processing, wound cleans gently with Sterile Saline and gauze.

The visual control wound, and when the wound same day and processing end, take pictures in fixed range.At 1-5 days with measured the closed situation of wound on the 8th day by measurement every day.Use indicates graduated Jameson calipers level and vertical survey wound.Covered by the successive epithelium if no longer see granulation tissue and wound, then thought wound healing.

Albumin fusion proteins of the present invention adopts the scope various dose, uses in carrier 8 days every day to the every wound of 500mg from 4mg.The vehicle Control group obtains the 50ml carrier soln.

Animal passed through peritoneal injection vetanarcol (300mg/kg) euthanasia at the 8th day.Collect wound and near skin then to be used for histology.Tissue sample places between the tissue cassette examination of living tissue sponge 10% neutral buffered formalin to be used for further processing.

Three groups of assessments of every group of 10 animal (5 with methyl meticortelone and 5 no glucocorticosteroids): 1) untreated fish group, 2) carrier placebo, 3) treatment group.

By analyzing wound closure with level and Z-axis measured zone and the site area that obtains wound.Assess closure by the difference between the wound area (the 8th day) after definite initial wound area (the 0th day) and the processing subsequently.The 1st day wound area is 64mm ², i.e. the corresponding size of skin punch tool.Use following formula to calculate:

The 8th day open area of b.[]-[the 1st day open area]/[the 1st day open area]

Sample is fixed in 10% buffered formalin, and wax embedding block is cut into slices (5mm) perpendicular to wound surface, the Olympus slicing machine is adopted in cutting.The cross section of dividing wound equally is carried out conventional hematoxylin-eosin (H﹠amp; E) dyeing.Whether the histological examination of wound can be assessed the agglutination of repairing skin and morphology outward appearance and improve because of the processing of albumin fusion proteins of the present invention.Lens micrometer through calibration is used to measure the distance of wound breach by unwitting viewer.

Use non-paired t test to analyze experimental data.P value less than 0.05 is thought significantly.

Embodiment 22: the lymphedema animal model

The purpose of this experimental technique is to create suitable and reliable lymphedema model to be used for checking albumin fusion proteins of the present invention in the lymphatic vessel generation of the lymphokinesis system of rat hindlimb and the result of treatment of rebuilding.Come measurement effect by the swelling volume of ill limb, quantitative, the total plasma proteins and the histopathology of lymph vascular system quantity.The acute lymphoblastic oedema was observed 7-10 days.May more importantly be that the chronic process tracking of oedema reaches 3-4 week.

Begin before the operation, blood sample collection is used for the protein concn analysis.Male rat to the about 350g of body weight is taken Sodital.Subsequently, right leg from knee to the buttocks defeathering.Clean with the gauze that is immersed among the 70%EtOH in the defeathering zone.Blood sample collection is used for the total serum protein quality inspection and tests.Afterwards, carry out girth and cubing in 2 measurement levels of mark (the above 0.5cm of heel, the middle part of back of the body pawl), in claw, inject dyestuff then.The back intradermal injection 0.05ml 1%EvanShi indigo plant of right pawl and left pawl.After being expelled to dyestuff in the claw, carry out girth and cubing then.

Use knee endoprosthesis as boundary mark, produce the midleg groin incision along circumference, thereby can locate Femur blood vessel (femoral vessel).The use tweezers are dissected with mosquito forceps and are separated flap.Behind the Femur blood vessel of location, be positioned at below the blood vessel, along one sidle to lymphatic vessel.Main lymphatic vessel electricity with this zone condenses (electrically coagulated) or suture ligature (suture ligated) then.

Use microscope that the muscle of leg back (contiguous half tendon and adductor muscle) is directly cut.Locate popliteal lymphoglandula then.2 adjacent sides by suture ligature leg bending part lymphoglandula and 2 distally lymphatic vessels and distally blood supply then.Remove leg bending part lymphoglandula and any subsidiary fatty tissue by cutting off reticular tissue then.

Note controlling any slight bleeding that operation thus causes.After the lymph obturation, use hquid skin (Vetbond) (AJ Buck) sealing flap.The skin edge that separates is closed under the muscle tissue, and stays the breach of about 0.5cm around the leg.When needed, also can come grappling skin by being sewn onto the muscle below.

For fear of infection, animal is raised (not having the pad grass) separately with net.Just check every day,, generally occur in 5-7 days by best oedema peak the restorative animal.Observe subsequently and stablize the oedema peak.In order to assess the intensity of lymphedema, before operation and 7 days the girth of measuring 2 specified locationes on each claw every day and volume.Whether measure the influence of plasma proteins confrontation lymphedema, be effective scope of survey (testing perimeter) but also investigated protein analysis.Weight at 2 position assessment contrasts and oedema limbs.Analysis is carried out in the blindness mode.

Circumferential measurements: to prevent limb activity, use cloth tape rule to measure the limbs girth by simple gas anesthesia.Measure at anklebone and back of the body pawl place by 2 different people, and these two readings are averaged.Obtain reading from contrast and oedema limbs.

Cubing: performing the operation the same day, animal is anaesthetized with Sodital, and detects before operation.For measuring volume every day, animal is simply used halothane anesthesia (fast braking and fast quick-recovery), with two legs defeathering and leg is carried out same mark with the waterproof mark all.Leg is at first immersed in the water, immerse the level that arrives each mark in the instrument then, measure by Buxco oedema software (Chen/Victor) subsequently.Data are by a personal record, and another person is dipped into the mark zone with limbs.

Blood-plasma proteins is measured: gather blood, centrifugal and separation of serum before operation, when being end then, be used for comparison gross protein and Ca ²⁺

The limbs weight ratio is: after gathering blood, animal prepares for tissue collecting.With guillotine cutter (quillitine) excision limbs, then at ligation place cutting experiment and contrast leg and weigh.After shin-Gen joint breaks away from, carry out weighing the second time, and foot is weighed.

The histology prepared product: dissection is arranged in the transversus behind knee (leg bending part) zone and is arranged in metal die, fills up freezing gel (freezeGel), immerses cold methylbutane, and the tape label sample sack that places-80 ℃ is up to section.Be right after section, muscle is observed lymph under fluorescent microscope.

Embodiment 23: albumin fusion proteins of the present invention is to the inhibition of TNF α inductive adhesion molecule expression

The special receptor-ligand binding of lymphocyte between the cell surface adhesion molecule (CAM) that raising of inflammation and blood vessel generation area relates on lymphocyte and the blood vessel endothelium.Adhesion process is all followed the multistep cascade in normal and pathological conditions, it relates to the expression of adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and endothelial leukocyte adhesion molecule-1 (E-selects albumen) in endotheliocyte (EC) upper eye lid.These molecules and the expression of other molecule on blood vessel endothelium determined white corpuscle can during inflammatory reaction, adhere to the local vascular system and outside be seeped into efficient in the local organization.The concentration of local of cytokine and somatomedin participates in the expression regulation of these CAM.

Tumor necrosis factor alpha (TNF-a), a kind of effective pro-inflammatory cytokine is the stimulator of all three kinds of CAM on the endotheliocyte, and may relates to extremely various inflammatory reactions, causes pathological consequences usually.

Can check albumin fusion proteins of the present invention to mediate the potentiality of the inhibition that TNF-a inductive content-addressable memory is reached.Employing is measured a kind of member with the FGF protein families as the correction ELISA assay method of solid phase absorption agent with EC to stimulate altogether after the content-addressable memory amount of reaching on the EC that TNF-a handles.

In order to implement this experiment, obtain Human umbilical vein endothelial cells (HUVEC) culture from the umbilical cord gleanings (cord harvests) that merges, and containing 5%CO ₂37 ℃ of moist incubators in remain on the growth medium (EGM-2 that is supplemented with 10%FCS and 1% penicillin/streptomycin; Clonetics, San Diego, CA) in.With HUVEC with 1 * 10 ⁴The concentration of cells/well is inoculated in the 96 hole plates, and in 37 ℃ of incubation 18-24 hours or up to converging.Subsequently the serum-free solution of cell monolayer with the RPMI-1640 that is supplemented with 100U/ml penicillin and 100mg/ml Streptomycin sulphate is cleaned 3 times, and handled 24 hours in 37 ℃ with specified cytokine and/or somatomedin.After the cultivation, pair cell assessment content-addressable memory reaches.

Human umbilical vein endothelial cells (HUVEC) is cultured in standard 96 hole plates converges.Remove growth medium from cell, and replace with 90 μ l, 199 substratum (10%FBS).Sample that will be used for testing and positive or negative contrast are added to plate triplicate (10 μ l volume).With plate in 37 ℃ of insulations 5 hours (select albumen and integrin expression) or 24 hours (only integrin expression).Plate suction removing substratum, and is added 100 μ l, 0.1% paraformaldehyde-PBS (contains Ca in each hole ⁺⁺And Mg ⁺⁺).Plate was kept 30 minutes in 4 ℃.

From the hole, remove stationary liquid subsequently, the hole is cleaned 1 time with PBS (+Ca, Mg)+0.5%BSA, and drained.Do not allow orifice drying.Adding 10 μ l in test and control wells resists through one of dilution.Concentration (the 0.1mg/ml antibody liquid storage of dilution in 1: 10) with 10 μ g/ml uses anti-ICAM-1-vitamin H, anti-VCAM-1-vitamin H and anti-E-to select albumen-vitamin H.Cell is incubated 30 minutes in 37 ℃ in wet environment.The hole is cleaned 3 times with PBS (+Ca, Mg)+0.5%BSA.

In each hole, add the ExtrAvidin-alkaline phosphatase (1: 5,000 dilution) of 20 μ l then through dilution, and in 37 ℃ of insulations 30 minutes.The hole is cleaned 3 times with PBS (+Ca, Mg)+0.5%BSA.(p-Nitrophenol Phosphate pNPP) is dissolved in 5ml glycine buffer (pH 10.4) with 1 p-NP phosphoric acid ester.In each test hole, add the pNPP substrate that 100 μ l are dissolved in glycine buffer.The use diluent that is dissolved in glycine buffer by the ExtrAvidin-alkaline phosphatase prepares in triplicate standard orifice: 1: 5, and 000 (10 ⁰)＞10 ^-0.5＞10 ^-1＞10 ^-1.5Each extent of dilution is got 5 μ l and is joined in the in triplicate hole, and the AP content in each hole that so obtains is 5.50ng, 1.74ng, 0.55ng, 0.18ng.Must in each standard orifice, add 100 μ l pNNP reagent then.Plate must be in 37 ℃ of insulations 4 hours.To add the 3M NaOH of 50 μ l volumes in porose.Reading on the plate instrument in the 405nm quantized result.The blank well that glycine buffer only is housed is used the background deduction option.Template is set at the concentration [5.50ng that shows AP-conjugate in each standard orifice; 1.74ng; 0.55ng; 0.18ng].The result will be shown as the amount of bonded AP-conjugate in each sample.

The structure of embodiment 24:GAS reporter molecule construction

A kind of signal transduction pathway that will be referred to cytodifferentiation and propagation is called the Jaks-STAT approach.Activated protein bound γ activates site " GAS " element or interferon-sensitive response element (" ISRE ") in the Jaks-STAT approach, and they are arranged in the promotor of many genes.Protein changes Expression of Related Genes with combining of these elements.

GAS and ISRE element are subjected to being called the identification of the class transcription factor of signal transducer and transcriptional activator or " STAT ".There are six members in STAT family.Stat1 and Stat3 are present in many cell types, and Stat2 is (as being general to replying of IFN-α) too.Stat4 is more limited, and does not exist in many cell types, though found in the cell after it is handled at I class t helper cell, with IL-12.Stat5 is called the mammoplasia factor at first, but has been found that it is present in other cell with greater concn, comprises medullary cell.It can be subjected to the activation of many cytokines in tissue culture cells.

STAT is transferred to nuclear because of the one group of kinase whose tyrosine phosphorylation that is called Janus kinases (" Jaks ") family activates from tenuigenin.Jaks has represented a unique family of solubility Tyrosylprotein kinase, comprises Tyk2, Jak1, Jak2 and Jak3.These kinases are showed significant sequence similarity, and catalytically inactive in resting cell usually.

Jaks is subjected to the activation of the multiple acceptor that following table sums up.(reorganization is from the summary of Schidler and Darnell, Ann.Rev.Biochem.64:621-51,1995).The cytokine receptor family that can activate Jaks is divided into two groups: (a) 1 class comprises the acceptor of IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-11, IL-12, IL-IS, Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF and thrombopoietin; (b) 2 classes comprise IFN-a, IFN-g and IL-10.1 receptoroid has a conservative halfcystine motif (one group of 4 conservative halfcystine and 1 tryptophane) and WSXWS motif (the nearly central section of film of coding Trp-Ser-Xaa-Trp-Ser (SEQ ID NO:53))

Therefore, after part and receptors bind, Jaks is activated, and it activates STAT then, and it shifts and subsequently in conjunction with the GAS element.This whole process is included in the Jaks-STAT signal transduction pathway.Therefore, the activation by the Jaks-STAT approach that combination reflected of GAS or ISRE element can be used for indicating the protein that relates to cell proliferation and differentiation.For example, known somatomedin and cytokine activate Jaks-STAT approach (table 5 sees below).Therefore, by using the GAS element that is connected with acceptor molecule, can identify the incitant of Jaks-STAT approach.

Table 5

JAKs STAT GAS (element) or ISRE

Part Tyk2 Jak1 Jak2 Jak3

IFN family

IF-α/B?+ + - - 1，2，3ISRE

IFN-g + + - 1GAS(IRF1＞Lys6＞IFP)

Il-10 + ？ ？ - 1，3

Gp130 family

IL-6 (multi-purpose)+++? 1, and 3GAS (IRF1＞Lys6＞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)-+-+1,3,5GAS

IL-4 (lymph/marrow)-+-+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)--+-5 GAS (IRF1＞IFP＞＞Ly6)

IL-5 (marrow)--+-5 GAS

GM-CSF (marrow)--+-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++-(no IRF1)

In order to make up the synthetic GAS that contains promoter element, it is used for the biological assay that embodiment 27-29 describes, and adopts the strategy of PCR-based to produce the GAS-SV40 promoter sequence.5 ' primer contains 4 tandem copies of GAS binding site, to be proof that find in the IRF1 promotor and previous induce the back in conjunction with STAT (people such as Rothman what be subjected to the various kinds of cell factor for it, Immunity 1:457-468,1994), though can use other GAS or ISRE element as an alternative.5 ' primer also contains the sequence with SV40 early promoter sequence complementary 18bp, and flank is Xho I site.The sequence of 5 ' primer is:

5′-GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAATGATTTCCCCGAAATATCTGCCATCTCAATTAG-3′(SEQ?ID?NO：54)

Downstream primer and the complementation of SV40 promotor, and flank is Hind III site:

5′-GCGGCAAGCTTTTTGCAAAGCCTAGGC-3′(SEQ?ID?NO：55)

The B-gal that use obtains from Clontech: the SV40 promoter templates that exists the promoter plasmid carries out pcr amplification.The PCR fragment that so obtains is digested with Xho I/Hind III, and subclone is in BLSK2-(Stratagene).The order-checking of carrying out with forward and reverse primer has confirmed that inset contains following sequence:

5’- CTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAATGATTTCCCCGAAATATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAA AAGCTT-3′(SEQ?ID?NO：56)

By this GAS promoter element that is connected with the SV40 promotor, then transformed GAS:SEAP2 reporter molecule construction.Here, reporter molecule is the excretory alkaline phosphatase, or " SEAP ".Yet obvious any reporter molecule can substitute SEAP in this or any other embodiment.The well-known reporter molecule that can be used for alternative SEAP comprises that E.C. 2.3.1.28 (CAT), luciferase, alkaline phosphatase, beta-galactosidase, green fluorescent protein (GFP) maybe can be by any protein of antibody test.

Above-mentioned sequence has confirmed that synthetic GAS-SV40 promoter element uses HindIII and XhoI subclone to from the pSEAP-promoter vector that Clontech obtains, GAS:SV40 promoter element with amplification is effectively replaced the SV40 promotor, has created the GAS-SEAP carrier.Yet this carrier does not contain neomycin resistance gene, and therefore is not that mammalian expression system institute is preferred.

Therefore, in order to produce the stable mammal cell line of expressing the GAS-SEAP reporter, use SalI and NotI that the GAS-SEAP box is shifted out from the GAS-SEAP carrier, and these restriction sites in the use multiple clone site insert the main chain carrier that contains neomycin resistance gene, such as pGFP-1 (Clontech), created the GAS-SEAP/Neo carrier.In case this carrier is transformed in the mammalian cell, this carrier just can be used as GAS bonded reporter molecule as described in embodiment 27-29.

Can use description above and replace GAS and prepare other construction with the different promoters sequence.The structure of the reporter molecule that contains EGR and NF-KB promoter sequence for example, has been described among the embodiment 27-31.Yet, can use the scheme of describing among these embodiment to replace many other promotors.For example, can separately or unite (for example GAS/NF-KB/EGR, GAS/NF-KB, Il-2/NFAT or NF-KB/GAS) and replace SRE, IL-2, NFAT or osteocalcin promotor.Similarly, can use other clone to test reporter molecule construction activity, such as HELA (epithelium), HUVEC (endothelium), Reh (B-cell), Saos-2 (scleroblast), HUVAC (aorta) or myocardial cell.

The active assay method of embodiment 25:SEAP

As the reporter molecule that is used for the assay method that embodiment disclosed herein describes, measure the SEAP activity with Tropix Phospho-light test kit (Cat.BP-400) according to following general procedure.The TropixPhospho-light test kit provides following used dilution, mensuration and reaction buffer.

Divider is poured into 2.5x dilution buffer liquid, and 15 μ l 2.5x dilution buffer liquid are assigned to 35 μ l are housed contain among the Optiplate of albumin fusion proteins solution of the present invention.With plastic seal film phonograph seal plate, and in 65 ℃ of insulations 30 minutes.Separately Optiplate is with the inequality that keeps from heat.

Make sample be cooled to room temperature 15 minutes.Damping fluid is measured in emptying divider and perfusion.Adding 50ml measures damping fluid and is incubated 5 minutes in room temperature.The emptying divider also pours into reaction buffer (seeing the following form).Add 50 μ l reaction buffers and be incubated 20 minutes in room temperature.Because the intensity of chemiluminescence signal depends on the time, and on luminometer, read 5 plates and need about 10 minutes, handle 5 plates therefore at every turn, and second group of beginning after 10 minutes.

In luminometer, read relative light unit.It is blank setting H12, and print result.The chemoluminescence enhancing shows reporter activity.

Table 6

The plate numbering	Rxn buffer diluent (ml)	CSPD (ml)	The plate numbering	Rxn 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	7.75	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 assay method of identifying neuronal activity

When cell broke up and breed, one group of gene was activated by many different signal transduction pathways.One of these genes, EGR1 (early growth response gene 1) lures after activation in multiple tissue and cell type and is led.The promotor of EGR1 is responsible for this inducing.Use the EGR1 promotor that is connected with reporter molecule, can assess the ability of fusion rotein activating cells of the present invention.

Specifically, use following scheme in PC12 clone, to assess neuronal activity.Known PC12 cell (rat pheochromocyte oncocyte) is bred by the activation of multiple mitogen such as TPA (mnyristoyl phorbol acetic ester), NGF (nerve growth factor) and EGF (Urogastron) and/or is broken up.EGR1 genetic expression is activated in the reason process herein.Therefore, by with the construction stable transfection PC12 cell that contains the EGR promotor that is connected with the SEAP reporter gene, can assess the activation of albumin fusion proteins of the present invention to the PC12 cell.

EGR/SEAP reporter molecule construction can assemble by following scheme.EGR-1 promoter sequence (633 to+1) (people such as Sakamoto K, Oncogene 6:867-871,1991) can use following primer pcr amplification from the human gene group DNA:

First primer: 5 '-GCGCTCGAGGGATGACAGCGATAGAACCCCGG-3 ' (SEQ IDNO:57)

Second primer: 5 '-GCGAAGCTTCGCGACTCCCCGGATCCGCCTC-3 ' (SEQ IDNO:58)

Use the GAS:SEAP/Neo carrier that produces among the embodiment 24, the EGR1 amplified production can be inserted in this carrier then.Use restriction enzyme XhoI/HindIII with the linearizing of GAS:SEAP/Neo carrier, remove the GAS/SV40 weighting material.With identical enzyme with EGR1 amplified production restrictive diges-tion.Connection carrier and EGR1 promotor.

In order to prepare the 96 hole plates that cell cultures is used, add 2ml coating buffer (I class collagen (Upstate Biotech Inc. in each 10cm plate, products catalogue numbering 08-115) dilution (filtration sterilization) in 1: 30 in 30% ethanol) or each hole 50ml of 96 hole plates, and air drying 2 hours.

The PC12 cell routine is being supplemented with middle conventional cultivation of RPMI-1640 substratum (Bio Whittaker) of containing 10% horse serum (JRH BIOSCIENCES, products catalogue numbering 12449-78P), 5% heat-killed foetal calf serum (FBS) of 100U/ml penicillin and 100 μ g/ml Streptomycin sulphates on through the 10cm tissue culture ware of pre-bag quilt.One was divided into four in per 3 to 4 days.From plate, take out cell by scraping to get, and carry out resuspended above 15 times by sucking and blowing out.

Use technology known in the art with the transfection of EGR/SEAP/Neo construction in PC12.Obtain the EGR-SEAP/PC12 stabilized cell by culturing cell in 300 μ g/ml G418.Using the substratum that does not contain G418 to carry out routine and cultivate, is that per 1 to 2 months cells should be cultivated several generations again in 300 μ g/ml G418.

In order to measure neuronal activity, pair cell grows to the 10cm plate that about 70-80% converges and screens, and promptly removes old substratum.Clean cell once with PBS (phosphate buffered saline (PBS)).Then with cell hungry overnight incubation in low blood serum medium (containing 1% horse serum and 0.5%FBS and antibiotic RPMI-1640).

The next morning, remove substratum and clean cell with PBS.Scrape cell from plate, abundant suspension cell in the low blood serum medium of 2ml.The pair cell counting, and add how low blood serum medium, make final cell density reach 5 * 10 ⁵Cell/ml.

In each hole of 96 hole plates, add 200 μ l cell suspending liquids and (be equivalent to 1 * 10 ⁵Cells/well).The albumin fusion proteins of the present invention that adds a series of different concns was in 37 ℃ of insulations 48 to 72 hours.Can use and knownly activate the somatomedin of PC12 cell as positive control, such as the 50ng/ μ l neure growth factor (NGF) by EGR.Usually observing the SEAP that surpasses 50 times in the positive control hole induces.The SEAP assay method can use the technology routine of describing among known in the art and/or the embodiment 25 to carry out.

The assay method of embodiment 27:T cytoactive

Below scheme be used to be tested and appraised the factor and measure albumin fusion proteins of the present invention and whether breed and/or break up the T cell and assess the T cytoactive.Use the GAS/SEAP/Neo construction that produces among the embodiment 24 to assess the T cytoactive.Therefore, increase the ability that the active multiple indication of SEAP activates the Jaks-STAT signal transduction pathway.Employed T cell is JurkatT-cell (ATCC code T IB-152) in this assay method, though also can use Molt-3 cell (ATCC numbers CRL-1552) and Molt-4 cell (ATCC numbers CRL-1582).

Jurkat T-cell is a lymphocytoblast CD4+Th1 helper.In order to produce stable clone, use DMRIE-C (Life Technologies) (transfection method described below) the about 200 ten thousand Jurkat cells of GAS-SEAP/neo carrier transfection.Density inoculation transfectional cell and selection with about 20,000 cells in every hole have the transfection of resistance to give to the 1mg/ml gentamicin.Amplification resistance bacterium colony is tested their reactions to the interferon-gamma of concentration increase then.Demonstrated a selected clone's dose response.

Particularly, following scheme will produce enough cells for 75 holes of containing 200 μ l cells.Therefore,, perhaps amplify scale, perhaps repeatedly implement in order to be that a plurality of 96 orifice plates produce enough cells.In containing the RPMI+10% serum of 1% penicillin-Streptomycin sulphate, keep the Jurkat cell.Mixed 2.5ml OPTI-MEM (Life Technologies) and 10 μ g plasmid DNA in the T25 flask.Add the 2.5ml OPTI-MEM that contains 50 μ l DMRIE-C, and in room temperature insulation 15-45 minute.

Between soak, pair cell concentration counting, rotation precipitation requisite number purpose cell (10 ⁷And be resuspended in OPTI-MEM the each transfection of individual cell), to final concentration 10 ⁷Individual cell/ml.In the T25 flask, add 1 * 10 among the 1ml OPTI-MEM then ⁷Individual cell, and in 37 ℃ of insulations 6 hours.After the insulation, add 10ml RPMI+15% serum.

In RPMI+10% serum, 1mg/ml gentamicin and 1% penicillin-Streptomycin sulphate, keep Jurkat:GAS-SEAP and stablize reporting cell line.One or more fusion roteins of the present invention with different concns are handled these cells.

On the same day of handling with fusion rotein, cell should clean, and is resuspended in the density of fresh RPMI+10% serum to every milliliter 500,000 cell.The accurate number of required cell will depend on the quantity of the different concns of the quantity of fusion rotein and the fusion rotein that screens.For one 96 orifice plate, need about 1,000 ten thousand cells (, needing 100,000,000 cells) for 10 plates.

The porose vessel that will contain the Jurkat cell of handling through fusion rotein are placed 48 hours (noticing that this time can change between 48-72 hour) in incubator.Use then 12 road pipettors with the 35 μ l sample transfer in each hole in opaque 96 orifice plates.(using the sellophene coverture) covers opaque flat board, and is stored in-20 ℃ until carrying out the SEAP assay method according to embodiment 25.To contain the flat board that remains treated cell and place 4 ℃, and be used as the material source of replication on particular bore when needed.

Can use the 100U/ml interferon-gamma as positive colony, known its activation Jurkat T cell.In the positive control hole, observe usually and surpass 30 times induce.

Such scheme can be used for producing instantaneous and stable transfectional cell, and this is conspicuous to those skilled in the art.

The assay method of embodiment 28:T cytoactive

NK-KB (nf KB) is by the multiple factor, comprise inflammatory cytokine IL-1 and TNF, CD30 and CD40, lymphotoxin-α and lymphotoxin-β by being exposed to LPS or zymoplasm, and by the expression of some virogene product a kind of transcription factor of activated.As transcription factor, the NF-KB regulation and control relate to the expression of gene of activated immune cell, apoptosis control (NF-KB shows the protection cell and avoids apoptosis), B and T-cell development, antiviral and antimicrobial reaction and multiple stress reaction.

In excited state not, NF-KB and I-KB (inhibition KB) are retained in the tenuigenin.Yet after stimulation, phosphorylation and degraded take place in I-KB, cause NF-KB to shuttle back and forth in nucleus, thus the transcribing of activation target gene.Comprise IL-2, IL-6, GM-CSF, ICAM-1 and I class MHC by NF-KB activated target gene.

Because its vital role and the ability of replying multiple stimulation will utilize the reporter molecule construction of NF-KB promoter element to be used to screen fusion rotein.The activator of NF-KB or inhibition will be useful in treating, prevent and/or diagnosing the illness.For example, the inhibition of NF-KB can be used for treating the acute or chronic activated disease that those relate to NF-KB, such as rheumatoid arthritis.

In order to make up the carrier that contains the NF-KB promoter element, adopt the strategy of PCR-based.Upstream primer contain NF-KB binding site (GGGGACTTTCCC) (SEQ ID NO:59) 4 tandem copies, with 5 ' end complementary 18bp sequence of SV40 early promoter sequence, and flank is the XhoI site:

5′-GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTTCCATCCTGCCATCTCAATTAG-3′(SEQ?ID?NO：60)

3 ' end of downstream sequence and SV40 promotor is complementary, and flank is the HindIII site:

5′-GCGGCAAGCTTTTTGCAAAGCCTAGGC-3′(SEQ?ID?NO：55)

The pB-gal that use obtains from Clontech: the SV40 promoter templates that exists the promoter plasmid carries out pcr amplification.The PCR fragment that so obtains is digested with XhoI and HindIII, and subclone is in BLSK2-(Stratagene).The order-checking of carrying out with T7 and T3 primer has confirmed that inset contains following sequence:

5’-CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTTCCATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTT-3’(SEQ?ID?NO：61)

Then, replace the SV40 minimal promoter element of the existence in the pSEAP2-promoter plasmid (Clontech) with this NF-KB/SV40 fragment by XhoI and HindIII.Yet this carrier does not contain neomycin resistance gene, therefore is not that mammalian expression system institute is preferred.

In order to produce stable mammal cell line, use restriction enzyme SalI and NotI that the NF-KB/SV40/SEAP box is shifted out from above-mentioned NF-KB/SEAP carrier, and insert the carrier that contains neomycin resistance.Particularly, after using SalI and NotI, the NF-KB/SV40/SEAP box is inserted pGFP-1 (Clontech), replace the GFP gene the pGFP-1 restrictive diges-tion.

In case create the NF-KB/SV40/SEAP/Neo carrier, just create and keep stable Jurkat T-cell according to the scheme of describing among the embodiment 25.Similarly, embodiment 25 has also described the method for using these stable Jurkat T-cells to measure fusion rotein.As positive control, in hole H9, H10 and H11, add external source TNF α (0.1,1,10ng), wherein observe 5-10 activation doubly usually.

Embodiment 29: identify the active assay method of marrow

Below scheme be used for whether breeding and/or breaking up the marrow activity that medullary cell is assessed albumin fusion proteins of the present invention by measuring fusion rotein.Use the GAS/SEAP/Neo construction that produces among the embodiment 24 to assess the medullary cell activity.So, increase the ability that the active multiple indication of SEAP activates the Jaks-STAT signal transduction pathway.The medullary cell that uses in this assay method is U937, and a kind of premonocyte clone is though also can use TF-1, HL60 or KG1.

For with the GAS/SEAP/Neo construction transient transfection U937 cell that produces among the embodiment 24, use DEAE-Dextran method (people such as Kharbanda, 1994, Cell Growth ﹠amp; Differentiation5:259-265).At first, collect 2 * 10 ⁷Individual U937 cell also cleans with PBS.The U937 cell is cultivated in being supplemented with RPMI 1640 substratum that contain 10% heat-killed foetal calf serum (FBS) of 100U/ml penicillin and 100mg/ml Streptomycin sulphate usually.

Then, cell suspension is contained 0.5mg/ml DEAE-Dextran, 8 μ g GAS-SEAP2 plasmid DNA, 140mM NaCl, 5mM KCl, 375 μ M Na in 1ml ₂HPO ₄.7H ₂O, 1mM MgCl ₂With 675 μ M CaCl ₂20mM Tris-HCI (pH 7.4) damping fluid in.In 37 ℃ of insulations 45 minutes.Clean cell with RPMI 1640 substratum that contain 10%FBS, be resuspended in the 10ml perfect medium then, and in 37 ℃ of insulations 36 hours.

Obtain the GAS-SEAP/U937 stabilized cell by culturing cell in 400 μ g/ml G418.Using the substratum that does not contain G418 to carry out routine and cultivate, is that per 1 to 2 months described cells should be cultivated several generations again in 400 μ g/ml G418.

In order to test these cells, collect 1 * 10 ⁸Individual cell (this enough be used for 10 96 orifice plates measure) also cleans with PBS.In 200ml growth medium mentioned above, whole density is 5 * 10 with cell suspension ⁵Cell/ml.In each hole of 96 orifice plates, add 200 μ l cells (or 1 * 10 ⁵Cells/well).

The fusion rotein that adds different concns.In 37 ℃ of insulations 48 to 72 hours.Can use the 100U/ml interferon-gamma as positive control, known its activation U937 cell.In the positive control hole, observe usually and surpass 30 times induce.According to the scheme of describing among methods known in the art and/or the embodiment 25 supernatant liquor being carried out SEAP measures.

Embodiment 30: identify the assay method that small molecules concentration and membrane permeability change

Known ligand changes small molecules with combining of acceptor, such as level in the born of the same parents of calcium, potassium, sodium and pH, and changes membrane potential.These changes can be measured in the assay method of identifying with the fusion rotein of the receptors bind of specific cells.Though what following scheme was described is the assay method of calcium, this scheme can be easy to revise any other the micromolecular change to detect potassium, sodium, pH, membrane potential or can be detected by fluorescent probe.

Below assay method use fluorescence imaging to read plate instrument (" FLIPR ") to measure change in conjunction with micromolecular fluorescence molecule (Molecular Probes).Obviously, can use and detect micromolecular any fluorescence molecule, substitute calcium fluorescent molecule used herein, fluo-4 (Molecular Probes, Inc.; Products catalogue numbering F-14202).

For adherent cell, with cell with 10,000-20,000 cells/well inoculation has Co-star black 96 orifice plates of limpid bottom.With plate at CO ₂Insulation is 20 hours in the incubator.Adherent cell is cleaned twice with 200 μ l HBSS (HankShi balanced salt solution) in the Biotek washer, after final cleaning, stay 100 μ l damping fluids.

Preparation 1mg/ml fluo-4 liquid storage in 10%pluronic acid DMSO.In order to make cell loading fluo-4, in each hole, add 50 μ l, 12 μ g/ml fluo-4.With plate at CO ₂Be incubated 60 minutes in 37 ℃ in the incubator.Plate is cleaned 4 times with HBSS in the Biotek washer, and stay 100 μ l damping fluids.

For non-adherent cell, cell is rotated precipitation from substratum.With resuspended among the HBSS of cell in the 50ml conical tube to 2-5 * 10 ⁶Individual cell/ml.In every ml cells suspension, add the solution that 4 μ l1mg/ml fluo-4 are dissolved in 10%pluronic acid DMSO.Then pipe was placed 30-60 minute in 37 ℃ of water-baths.Cell cleans twice with HBSS, and is resuspended to 1 * 10 ⁶Cell/ml, and be assigned in the microtest plate 100 μ l/ holes.With plate centrifugal 5 minutes with 1000rpm.Then plate is cleaned once with 200 μ l in DenleyCell Wash, subsequently by drawing the final volume that step reaches 100 μ l.

For based on acellular assay method, fluorescence molecule is contained in each hole, such as fluo-4.Xiang Kongzhong adds fusion rotein of the present invention, and detects the change of fluorescence.

In order to measure the fluorescence of cellular calcium, FLIPR is set as follows parameter: (1) system-gain 300-800mW; (2) 0.4 second time shutter; (3) camera F/ stops F/2; (4) excite 488nm; (5) emission 530nm; (6) application of sample 50 μ l.The emission of 530nm increases indication and causes the extracellular signal incident by albumin fusion proteins of the present invention or albumin fusion proteins inductive molecule of the present invention, and it causes Ca in the born of the same parents ⁺⁺Concentration increases.

Embodiment 31: the assay method of identifying tyrosine kinase activity

Protein tyrosine kinase (PTK) is represented one group of various film and tenuigenin kinases of striding.The acceptor of multiple mitogenesis and metabolic somatomedin is arranged in receptor protein tyrosine kinases (RPTK) group, comprise PDGF, FGF, EGF, NGF, HGF and insulin receptor subfamily.In addition, have the RPTK extended familys, its corresponding part is unknown.The part of RPTK mainly comprises excretory small protein matter, but also comprises stromatin membrane-bound and that born of the same parents are outer.

Part relates to ligand-mediated receptor dimerizationization to the activation of RPTK, causes the activation of the transphosphorylation and the cytoplasmic tyrosine kinase of receptor subunit.Cytoplasmic tyrosine kinase comprises the cytosol protein tyrosine kinase that the acceptor relevant with src Family Tyrosine Kinases (as src, yes, Ick, lyn, fyn) is connected with no acceptor, such as Jak family, the signal transduction that its member's mediation is triggered by cytokine receptor superfamily (as interleukin, Interferon, rabbit, GM-CSF and Leptin).

Because knownly can promote that the factor range of tyrosine kinase activity is wider, so be interested to identifying albumin fusion proteins of the present invention or whether can activating tyrosine kinase signal transduction pathway by albumin fusion proteins inductive molecule of the present invention.Therefore, following conceptual design is used to identify this molecule that can activate the tyrosine signal transduction pathway.

With the density of about 25,000 cells in every hole target cell (as former generation keratinocyte) is inoculated into that (Naperville is in 96 hole Loprodyne Silent Screen plates IL) available from Nalge Nunc.Plate is used water rinse by sterilizing in 30 minutes with two of 100% ethanol rinsings, and dried overnight.Some plates (all can be from Sigma Chemicals with 100ml cell cultures level I class collagen (50mg/ml), gelatin (2%) or poly-lysine (50mg/ml), St.Louis, MO buys) or 10%Matrigel (available from Becton Dickinson, Bedford, MA), or the bovine serum bag used the PBS rinsing by 2 hours, and was stored in 4 ℃.In order to be determined at the cell of growing on these plates, with on 5, the 000 cells/well inoculation growth medium and after 48 hours, use alamarBlue such as the AlamarBiosciences of manufacturers, Inc. (Sacramento, CA) quantity of described indirect measurement cell.Use BectonDickinson (Bedford, Falcon plate lid #3071 capping Loprodyne Silent Screen plate MA).Falcon Microtest III cell cultures plate also can be used for some proliferation experiments.

In order to prepare extract, with A431 cell inoculation (20, the 000/200ml/ hole) to the nylon membrane of Loprodyne plate, and in perfect medium overnight incubation.Made cell mourn in silence (quiesce) in 24 hours by insulation in the serum-free basic medium.After handling 5-20 minute with the albumin fusion proteins of the present invention of EGF (60ng/ml) or different concns, remove substratum, and in each hole, add 100ml extraction damping fluid (20mM HEPES pH7.5,0.15M NaCl, 1%Triton X-100,0.1%SDS, 2mM Na ₃VO ₄, 2mM Na ₄P ₂O ₇With from Boeheringer Mannheim (Indianapolis, IN) the protease inhibitor mixture (#1836170) of Huo Deing), and plate rocked 5 minutes in 4 ℃ on rotary shaker.Then plate is placed the vacuum transfer device, and use indoor vacuum to make extract pass through to filter at the bottom of the 0.45mm filter membrane in each hole.96 holes that extract is collected vacuum unit bottom are caught/are measured in the plate and also place on ice immediately.For by the clarifying extract of centrifugal acquisition, at detergent dissolution after 5 minutes, take out each hole content and in 4 ℃ with 16, centrifugal 15 minutes of 000xg.

To level through filtering extract test tyrosine kinase activity.Though know the many methods that detect tyrosine kinase activity, this paper has only described wherein a kind of method.

Usually, the tyrosine kinase activity of albumin fusion proteins of the present invention is to assess by the ability of measuring the tyrosine residues on its phosphorylation specific substrates (biotinylated peptide).The biotinylated peptide that can be used for this purpose comprises PSK1 (corresponding to the amino acid 6-20 of cell fission kinases cdc2-p34) and PSK2 (corresponding to the amino acid/11-17 of gastrin).Two kinds of peptides all are the substrates of multiple Tyrosylprotein kinase, and can obtain from Boehringer Mannheim.

Set up the tyrosine-kinase enzyme reaction by adding following component in order.At first, adding the biotinylated peptide of 10 μ l, 5 μ M, is 10 μ l ATP/Mg then ₂₊(5mM ATP/50mM MgCl ₂), be that 10 μ l5x measure damping fluid (40mM imidazole hydrochloride pH7.3,40mM β-Phosphoric acid glycerol esters, 1mM EGTA, 100mM MgCl then ₂, 5mM MnCl ₂, 0.5mg/ml BSA), be 5 μ l vanadic acid sodiums (1mM) then, be 5 μ l water then.Mixed gently each component, and with reaction mixture in 30 ℃ of pre-incubations 2 minutes.By adding 10 μ l control enzyme or beginning reaction through filtering supernatant liquor.

Add 10 μ l 120mm EDTA subsequently and stop the tyrosine kinase assay reaction, and reaction is placed on ice.

Measured tyrosine kinase activity in 20 minutes by 50 μ l reaction mixture samples being transferred in microtiter plate (MTP) assembly and in 37 ℃ of insulations.This makes 96 hole plates of Streptavidin bag quilt to combine with biotinylated peptide.Clean the MTP assembly 4 times with 300 μ l/ hole PBS.(anti-P-Tyr-POD 0.5u/ml), and is incubated 1 hour in 37 ℃ to add the anti-phosphotyrosine antibody that 75 μ l and horseradish peroxidase put together then in each hole.Hole flushing as mentioned above.

Add 100 μ l peroxidase substrate solution (Boehringer Mannheim) then, and at least 5 minutes (being 30 minutes) of room temperature insulation.Use ELISA to read the light absorption ratio of plate instrument measure sample at the 405nm place.Use ELISA to read the level that the plate instrument quantizes the bonded peroxidase activity, and the level of reflection tyrosine kinase activity.

Embodiment 32: the assay method of identifying phosphorylation activity

As the potential selection of the protein hydroxyphenylaminopropionic acid kinase activity assay method of describing among the embodiment 31 and/or replenish, also can use the assay method of the activation (phosphorylation) that detects main intracellular signal transduction intermediate.For example, as described below, a kind of concrete assay method can detect Erk-1 and the kinase whose tyrosine phosphorylation of Erk-2.Yet, other molecule can detect by Erk-1 or the Erk-2 that replaces in the following assay method with these molecules such as the phosphorylation of Raf, JNK, the kinase whose kinases of p38MAP, Map (MEK), MEK kinases, Src, muscle specific kinases (MuSK), IRAK, Tec and Janus and any other phosphoserine, Tyrosine O-phosphate or phosphothreonine molecule.

Particularly, by the hole of 96 hole ELISA plates was come the formation determination plate with 0.1ml Protein G (1 μ g/ml) in 2 hours in room temperature (RT) bag.Sealed 1 hour in room temperature then through plate PBS rinsing, and with 3%BSA/PBS.Then the Protein G plate is used at 2 kinds of commercialization monoclonal antibodies (100ng/ hole) of Erk-1 and Erk-2 and handled (room temperature 1 hour) (Santa CruzBiotechnology).(in order to detect other molecule, this step can be easy to revise by replacing the monoclonal antibody that detects any above-mentioned molecule) with after PBS rinsing 3-5 time, is stored in 4 ℃ until use with plate.

The A431 cell is inoculated 96 hole Loprodyne filter plates with 20,000/ holes, and in growth medium overnight incubation.With cell hungry cultivation 48 hours in basic medium (DMEM), use the fusion rotein of the present invention of EGF (6ng/ hole) or different concns to handle then 5-20 minute then.Dissolved cell then, and with the extract direct filtration to measuring in the plate.

Arise from the room temperature insulation after 1 hour, with extract one with hole rinsing once more.As positive control, use the commercialization prepared product (10ng/ hole) of map kinase to replace the A431 extract.Then plate is handled (room temperature 1 hour) with commercialization polyclone (rabbit) antibody (1 μ g/ml) of specific recognition Erk-1 and the kinase whose phosphorylation epi-position of Erk-2.This antibody has carried out biotinylation by standard program.Measure the bonded polyclonal antibody by in WallacDELFIA instrument (time resolved fluorescence), strengthening the continuous insulation of reagent then with europium-strepto-affinity element and europium fluorescence.The fluorescent signal that is higher than background increases the phosphorylation that indication is produced by fusion rotein of the present invention or albumin fusion proteins inductive molecule of the present invention.

Embodiment 33: the phosphorylation assay method

In order to measure the phosphorylation activity of albumin fusion proteins of the present invention, use the phosphorylation assay method of describing in the United States Patent (USP) 5,958,405 (being introduced into this paper as a reference).Briefly, can be by with the γ mark ³²P-ATP phosphorylated protein substrate is also measured the radioactivity of mixing with gamma activity isotropic substance counter and is measured phosphorylation activity.With fusion rotein of the present invention and protein substrate, ³²P-ATP and kinase buffer liquid are incubated together.To mix substrate by electrophoresis then ³²P and free ³²P-ATP separates, to what mix ³²P counts and compares with negative control.The phosphorylation activity that is higher than the radiocounting indication fusion rotein of negative control.

Embodiment 34: the phosphorylation activity that detects albumin fusion proteins of the present invention when having the peptide part (is lived Turn usefulness into)

Method known in the art or described herein can be used for measuring the phosphorylation activity of albumin fusion proteins of the present invention.The preferred method of measuring phosphorylation activity is to use US 5,817, the tyrosine phosphorylation assay method of describing in 471 (being incorporated herein by reference).

Embodiment 35: the assay method that stimulates marrow CD34+ cell proliferation

This assay method is based on the ability that people CD34+ breeds when having hemopoieticgrowth factor, and assessment fusion rotein of the present invention stimulates the ability of CD34+ cell proliferation.

Proved before that most of sophisticated precursors will only reply single signal.How immature precursor needs at least two kinds of signals to reply.Therefore, in order to test fusion rotein of the present invention to the active influence of the hematopoiesis of multiple progenitor cell, assay method contains specified fusion rotein of the present invention under the condition that has or do not exist hemopoieticgrowth factor.Exist STEM CELL FACTOR (SCF) to cultivate 5 days under together with the condition of specimen isolated cells.SCF has very limited effect to the propagation of marrow (BM) cell separately, it in this condition only as " existence " factor.Yet, with any factor (as IL-3) that these cells are shown effect of stimulation when combining, SCF will cause synergistic effect.Therefore, if the fusion rotein of test is to having effect of stimulation in the hemopoietic progenitor cell, this activity can be easy to detect so.Because normal BM cell has low-level circulating cells (cycling cells), thereby might can't detect any inhibition effect of specifying fusion rotein.Therefore, the assay method of the inhibition effect of progenitor cell is preferably tested in following cell, this cell at first carries out stimulated in vitro with SCF+IL+3, suppresses this and induces the compound of propagation to contact with will assessing then.

Briefly, use methods known in the art separation of C D34+ cell.With cell thawing and be resuspended in the substratum (QBSF 60 serum free mediums (500mI) that contain the 1%L-glutamine, QualityBiological, Inc., Gaithersburg, MD, products catalogue numbering 160-204-101).After the gentle centrifugation step of 200xg, make cell recuperation 1 hour several times.Cell count is transferred to 2.5 * 10 ⁵Cell/ml.During this period, in the outer perimeter holes of 96 hole plates, add 100 μ l sterilized waters.The cytokine of the enough albumin fusion proteins tests of the present invention of energy has independent 50ng/ml rhSCF (R﹠amp in this assay method; DSystems, Minneapolis, MN, products catalogue numbering 255-SC) and and rhSCF and 30ng/mlrhIL-3 (R﹠amp; D Systems, Minneapolis, MN, products catalogue numbering 203-ML) associating.After 1 hour, the albumin fusion proteins of the present invention of the ready cytokine of 10 μ l, different concns and 20 μ l diluting cells are joined in the substratum in the hole Already in, make that final cumulative volume is 100 μ l.Then with plate at 37 ℃/5%CO ₂Incubator in placed 5 days.

Assay method was gathered in the crops preceding 18 hours, added 0.5 μ Ci/ hole [3H] thymidine to measure multiplication rate in every hole with 10 μ l volumes.Test stops by using Tomtec Harvester 96 that cell is gathered in the crops filter bed (filtermat) from each 96 hole plate.After the results,, put in order and place and coil the OmniFilter assembly of forming by an OmniFilter plate and an OmniFilter with the filter bed drying.In each hole, add 60 μ l Microscint, and force sealing film phonograph seal plate with TopSeal-A.On first plate, paste barcode 15 pasters to be used for counting.Plate with sealing loads then, measures radioactive level by PackardTop Count, and the data that collection is printed are used for analyzing.Radioactive level has reflected the amount of cell proliferation.

The research test of describing in the present embodiment specifies fusion rotein to promote the activity of marrow CD34+ cell proliferation.Those skilled in the art can be easy to the activity of the research of modified example with test fusion rotein of the present invention and polynucleotide (as gene therapy) and exciting thing and antagonist.Albumin fusion proteins of the present invention promotes that the ability indication albumin fusion proteins of marrow CD34+ cell proliferation and/or the polynucleotide corresponding with fusion rotein are effective for diagnosing and treating the disease that influences immunity system and hemoposieis.Representational purposes is described in above " immunocompetence " and " transmissible disease " part, and other place of this paper.

Embodiment 36: the extracellular matrix enhanced cell is replied the assay method of (EMECR)

The purpose that the extracellular matrix enhanced cell is replied the assay method of (EMECR) is assessment fusion rotein of the present invention acts on hemopoietic stem cell under the background of extracellular matrix (ECM) inducement signal a ability.

Cell is replied regulatory factor under the background of received signal from microenvironment on every side.For example, inoblast and endothelium and epithelial stem cell can't duplicate when the signal that lacks from ECM.Hemopoietic stem cell can carry out self in marrow, but can't carry out in external suspension culture.Stem cell depends on the interaction of they and stroma cell and ECM protein fibronectin (fn) in external ability of carrying out self.The adhesion of cell and fn is by α ₅. β ₁And α ₄. β ₁The integrin receptor mediation, they are expressed by the hemopoietic stem cell of people and mouse.Do not identify the factor of integrating and being responsible for stimulating the stem cell self with the ECM environment as yet.The discovery of these factors will be extremely interested in gene therapy and bone marrow transplantation application.

Briefly, use the fn fragment with 0.2 μ g/cm ²Bag by the concentration bag by the 96 hole plates of polystyrene without tissue culture treated.In the 0.2ml serum free medium, distribute bone marrow cells in mice (1,000 cells/well).Cultured cells will be as positive control under the condition that has IL-3 (5ng/ml)+SCF (50ng/ml), estimates with this understanding stem cell seldom self but significantly differentiation.Test albumin fusion proteins of the present invention with suitable negative control under the condition that has and do not exist SCF (5.0ng/ml), the volume of using composition that wherein contains albumin fusion proteins of the present invention accounts for total volumetric 10%.Pass through at low-oxygen environment (5%CO then ₂, 7%O ₂And 88%N ₂) the tissue culture incubator in the cell growth of insulation 7 angels distribution.The thymidine that mixes cell DNA by measurement is measured the quantity of proliferative cell in the hole then.The confirmation of positive findings needs the phenotypic evaluation of cell in the mensuration, and this can carry out with the suitable antibodies reagent and the FACScan of use at cell-surface antigens by the scale of amplification culture system.

If finding specific fusion proteins of the present invention is the stimulator of hemopoietic progenitor cell, this fusion rotein and polynucleotide corresponding with this fusion rotein may be useful in the diagnosis of the disease that for example influences immunity system and hemoposieis and treatment so.Representative purposes is described in above " immunocompetence " and " infectious diseases " part, and other place of this paper.Fusion rotein may be in the amplification of the committed progenitor of stem cell and various hemocyte pedigrees, and also is useful in the differentiation of various cell types and/or the propagation.

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, and therefore are used in the effect that chemotherapeutic period protection bone marrow stem cell is avoided chemotherapeutics.This anti-proliferative effect tolerable is used the more chemotherapeutics of high dosage, and therefore carries out more effective chemotherapy and handle.

In addition, the polynucleotide of fusion rotein of the present invention and code book invention albumin fusion proteins also can be used for treatment and diagnosis hematopoiesis associated disorders, such as anaemia, pancytopenia, oligoleukocythemia, thrombopenia or leukemia, because stroma cell is important in the generation of hematopoietic lineage cell.Purposes comprises isolated culture, bone marrow transplantation, marrow reconstruct, excrescent radiotherapy or the chemotherapy of medullary cell.

Embodiment 37: human dermis inoblast and aortic smooth muscle cell propagation

Albumin fusion proteins of the present invention is added in normal people's dermal fibroblast (NHDF) and human aortic smooth muscle cell's (AoSMC) the culture, every duplicate samples is carried out two and is measured (co-assays) altogether.Measure for first and check the effect of fusion rotein normal people's dermal fibroblast (NHDF) or aortic smooth muscle cell (AoSMC) propagation.The misgrowth of inoblast or smooth muscle cell is the part of several pathologic processes, comprises fibrosis and restenosis.Measuring the IL6 that checks NHDF and SMC for second generates.IL6 generates and shows function activation.Activating cells will increase the generation of the various kinds of cell factor and other factor, can cause inflammation promoter action or immunomodulatory consequence.Having and measuring under the condition of TNFa stimulation altogether, to check common stimulation or to suppress active.

Briefly, at the 1st day, the black plate in 96 holes was set to 1000 cells/well (NHDF) or 2000 cells/well (AoSMC) in the 100 μ l substratum.The NHDF substratum contains: Clonetics FB basic medium, 1mg/ml hFGF, 5mg/ml Regular Insulin, 50mg/ml gentamicin, 2%FBS, and the AoSMC substratum contains Clonetics SM basic medium, 0.5g/ml hEGF, 5mg/ml Regular Insulin, 1 μ g/ml hFGF, 50mg/ml gentamicin, 50 μ g/ml amphotericin Bs, 5%FBS.In 37 ℃ be incubated at least 4-5 hour after, the sucking-off substratum replaces with cessation of growth cessation substratum (Growtharrest media).The cessation of growth cessation substratum of NHDF contains inoblast basic medium, 50mg/ml gentamicin, 2%FBS, and the cessation of growth cessation substratum of AoSMC contains SM basic medium, 50mg/ml gentamicin, 50 μ g/ml amphotericin Bs, 0.4%FBS.In 37 ℃ of insulations until the 2nd day.

At the 2nd day, the serial dilution thing and the template of design albumin fusion proteins of the present invention made them always comprise substratum contrast and known protein confrontation photograph.For stimulating and suppressing two kinds of experiments, protein all dilutes in the cessation of growth cessation substratum.For suppressing experiment, add TNFa to final concentration 2ng/ml (NHDF) or 5ng/ml (AoSMC).The substratum that contains contrast or albumin fusion proteins of the present invention that adds 1/3 volume, and in 37 ℃/5%CO ₂Insulation was until the 5th day.

Shift 60 μ l to another markd 96 hole plates from each hole, with the capping of plate sealer, and in 4 ℃ of preservations up to the 6th day (being used for IL6 ELISA).Remaining 100 μ l in the cell cultures plate, aseptic adding is equivalent to the Alamar Blue of culture volume 10% (10 μ l).Plate was put back to incubator 3-4 hour.Use CytoFluor to measure the 530nm place then and excite emitted fluorescence with the 590nm place.This has produced growth/inhibition data.

At the 5th day, the following IL6ELISA that carries out, the anti-people IL6 monoclonal antibody bag that dilutes in PBS pH7.4 with 50-100 μ l/ hole be by 96 orifice plates, and in the room temperature incubated overnight.

At the 6th day, the plate of in tank, turning, and on paper handkerchief, blot.The mensuration damping fluid that contains PBS with the 4%BSA preparation.With the sealing of the Pierce Super Block among the 200 μ l/ hole PBS damping fluid plate was sealed 1-2 hour, use cleaning buffer solution (PBS, 0.05%Tween-20) to clean plate then.Plate is blotted on paper handkerchief.Add anti-people IL-6 mono-clonal, the biotin labeled antibody of 50 μ l/ holes then in the 0.5mg/ml dilution.The IL-6 liquid storage is diluted in substratum (30,10,3,1,0.3,0ng/ml).Top row to plate adds the double sample.The capping plate is incubated 2 hours in room temperature on shaking table.

Plate is cleaned with cleaning buffer solution, and on paper handkerchief, blot.The strepto-affinity element of 1: 1000 dilution EU mark in measuring damping fluid, and add 100 μ l/ holes.Capping plate and in room temperature insulation 1 hour.Plate is cleaned with cleaning buffer solution once more, and on paper handkerchief, blot.

The enhancing liquid that adds 100 μ l/ holes.Shook 5 minutes.On Wallac DELFIA photofluorometer to the plate reading.The reading tabulation of three duplicate samples at every turn measuring is also average.

Positive findings explanation AoSMC cell proliferation and albumin fusion proteins during this measures may relate to dermal fibroblast propagation and/or smooth muscle cell proliferation.Positive findings also hints many potential uses of the polynucleotide of fusion rotein and coding albumin fusion proteins.For example, inflammation and immunne response, wound healing and blood vessel take place, as what this specification described in detail.Specifically, fusion rotein can be used for wound healing and skin regeneration, and promotes the two vascular of blood vessel and lymphatic vessel to take place.The growth of vascular can be used for treating for example cardiovascular disorder.In addition, the fusion rotein of demonstration antagonistic activity may relate to disease, disorder and/or the situation that blood vessel takes place by effectively being used for the treatment of as anti-angiogenic dose (for example angiogenesis inhibitor) in this assay method.These diseases, disorder and/or situation are known in the art and/or described herein, such as for example malignant tumour, solid tumor, innocent tumour, for example vascular tumor, acoustic tumor, neurofibroma, trachoma and botryomycosis hominis; Atherosclerotic plaque; The blood vessel generation disease of eye, for example uveitis and pteryium (the abnormal vascular growth) of diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, Terry's sign, rubescent, retinoblastoma, eye; Rheumatoid arthritis; Psoriatic; Wound healing is slow; Endometriosis; Angiogenesis (vasculogenesis); Granulation forms; Hypertrophic cicatrix (keloid); Disunited fracture; Scleroderma; Trachoma; Blood vessel adheres to; Myocardial vascular takes place; CC; The brain side shoot; Arteriovenous malformotion; (ischemic limbangiogenesis) takes place in the ischemia limb vessel; Ao-Wei two syndromes; Plaque neovessels forms; Telangiectasis; Bleeder's joint; Hemangiofibroma; Fibrillar muscle heteroplasia; The wound granulation forms; Crohn disease; And atherosclerosis.In addition, the albumin fusion proteins as antagonist can be used for treating anti-excess proliferative disease known in the art and/or described herein and/or anti-inflammatory in this assay method.

Embodiment 38: the cell adhesion molecule on the endotheliocyte (CAM) is expressed

The special receptor-ligand binding of lymphocyte between the cell surface adhesion molecule (CAM) that raising of inflammation and blood vessel generation area relates on lymphocyte and the blood vessel endothelium.Adhesion process is all followed the multistep cascade in normal and pathological conditions, it relates to the expression of adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and endothelial leukocyte adhesion molecule-1 (E-selects albumen) in endotheliocyte (EC) upper eye lid.These molecules and the expression of other molecule on blood vessel endothelium determined white corpuscle can during inflammatory reaction, adhere to the local vascular system and outside be seeped into efficient in the local organization.The concentration of local of cytokine and somatomedin participates in the expression regulation of these CAM.

Briefly, endotheliocyte (as Human umbilical vein endothelial cells (HUVEC)) is cultured in standard 96 hole plates converges, remove growth medium from cell, and replace with 100 μ l, 199 substratum (10% foetal calf serum (FBS)).Specimen (containing albumin fusion proteins of the present invention) and positive or negative contrast are added to (10 μ l volume) in the plate in triplicate.Then with plate in 37 ℃ of insulations 5 hours (select albumen and integrin expression) or 24 hours (only integrin expression).Plate suction removing substratum, and is added 100 μ l, 0.1% paraformaldehyde-PBS (having Ca++ and Mg++) in each hole.Plate was kept 30 minutes in 4 ℃.From the hole, remove stationary liquid, the hole is cleaned 1 time and drained with PBS (+Ca, Mg)+0.5%BSA.Adding 10 μ l in test and control wells resists through one of dilution.Anti-ICAM-1-vitamin H, anti-VCAM-1-vitamin H and anti-E-select albumen-vitamin H to use (1: 10 diluent of 0.1mg/ml antibody liquid storage) with the concentration of 10 μ g/ml.Cell is incubated 30 minutes in 37 ℃ in wet environment.Hole PBS (+Ca, Mg)+0.5%BSA is cleaned 3 times.In each hole, add the Extr affinity element-alkaline phosphatase (1: 5,000 dilution be referred to herein as use diluent) of 20 μ l through dilution, and in 37 ℃ of insulations 30 minutes.The hole is cleaned three times with PBS (+Ca, Mg)+0.5%BSA.1 p-NP phosphoric acid ester pNPP of dissolving in every 5ml glycine buffer (pH10.4).PNPP substrate in the 100 μ l Padil damping fluids will join each test hole.The use diluent that is dissolved in glycine buffer by Extr affinity element-alkaline phosphatase prepares in triplicate standard orifice: 1: 5, and 000 (10 ⁰)＞10 ^-0.5＞10 ^-1＞10 ^-1.5Each extent of dilution is got 5 μ l and is joined in the in triplicate hole, and the AP content in each hole that so obtains is 5.50ng, 1.74ng, 0.55ng, 0.18ng.In each standard orifice, add 100 μ l pNNP reagent then.Plate is incubated 4 hours in 37 ℃.To add the 3M NaOH of 50 μ l volumes in porose.Reading on the plate instrument that in 405nm the plate reading is used the background deduction option to the blank well that glycine buffer only is housed.In addition, template is set at the concentration [5.50ng that shows AP-conjugate in each standard orifice; 1.74ng; 0.55ng; 0.18ng].The result will be shown as the amount of bonded AP-conjugate in each sample.

Embodiment 39:Alamar Blue endothelial cell proliferation assay method

This assay method can be used for quantitative assay by protein mediation to bFGF inductive ox lymph endotheliocyte (LEC), bovine aortic endothelial cells (BAEC) or people's capillary blood vessel myometrium cell (UTMEC) inhibition of proliferation.This assay method is mixed the fluorescence growth indicator that detects based on metabolic activity.Pre-standard Alamar Blue proliferation assay in adding the 10ng/ml bFGF EGM-2MV that stimulation is originated as endotheliocyte.By the minor alteration of growth medium and cell concn, this assay method can will be used for various endotheliocytes.The diluent of protein to be tested batch is suitably diluted.Use the serum free medium that does not contain bEGF as non-stimulated contrast, and comprise that angiostatin (angiostatin) or TSP-1 contrast as known inhibition.

Briefly, LEC, BAEC or UTMEC density with 5000 to 2000 cells/well in growth medium are inoculated in the 96 hole plates, and spend the night in 37 ℃ of placements.After the cell incubated overnight, remove growth medium, and replace with GIBCO EC-SFM.Cell is handled at the suitable diluent (preparing in SFM) of triplicate Kong Zhongyong albumin fusion proteins of the present invention or reference protein quality sample, and added the concentration of bFGF to 10ng/ml.In case cell is handled with sample, plate was put back in 37 ℃ of incubators 3 days.After 3 days, in each hole, add 10ml alamar blue liquid storage (Biosource, products catalogue numbering DAL1100), and plate was put back in 37 ℃ of incubators 4 hours.Using CytoFluor fluorescence reader to excite with 590nm at 530nm then launches the plate reading.Directly export with the relative fluorescence unit record.

Alamar blue is an oxidation-reduction indicator, and fluorescence and color change all reflect the chemical reduction of the growth medium that is caused by the cell growth.When cell was grown in substratum, inborn metabolic activity caused the chemical reduction of immediate environment.The reduction reaction that relates to growth causes indicator to become reduction (redness that fluorescence is arranged) form from oxidation (non-blooming blueness) form (promptly being subjected to promoted propagation and will producing stronger signal, and the propagation that is suppressed will produce more weak signal, and the sum and their metabolic activity of resultant signal and cell are proportional).Observe active background level with independent hungry substratum.This and observed output from positive control sample (bFGF the growth medium) and protein diluent are compared.

Embodiment 40: the detection of the inhibition of mixed lymphocyte reacion

This assay method can be used for detection and assesses the inhibition of fusion rotein of the present invention to mixed lymphocyte reacion (MLR).The inhibition of MLR may be owing to the adjusting of sticking connection between adjusting, lymphocyte and the helper of costimulatory molecules on the direct influence of on cell proliferation and vigor, the interaction cell, the adjusting that the helper pair cell factor generates.Because the used single nuclear fraction of peripheral blood comprises T, B and nature killer cell and monocyte and dendritic cell in this assay method, thereby the albumin fusion proteins of inhibition MLR can be at various kinds of cell.

With find to suppress MLR albumin fusion proteins of the present invention can with lymphocyte and monocyte activation or propagation diseases associated in find application.These include but not limited to such as following disease, asthma, sacroiliitis, diabetes, inflammatory skin, psoriatic, eczema, systemic lupus erythematous, multiple sclerosis, glomerulonephritis, inflammatory bowel, Crohn disease, ulcerative colitis, arteriosclerosis, sclerosis, graft versus host disease, host versus graft disease, hepatitis, leukemia and lymphoma.Briefly, use LSM (

Density 1.0770g/ml, OrganonTeknika Corporation, West Chester is PA) by the PBMC of density gradient centrifugation purifying from people's donor.Will (Life Technologies, Grand Island transfer to 2 * 10 in NY) at the RPMI-1640 that is supplemented with 10%FCS and 2mM glutamine from the PBMS of two donors ⁶Cell/ml.To transfer to 2 * 10 from the PBMC of the 3rd donor ⁵Cell/ml.50 μ l are added in the hole of microtitration plate at the bottom of 96 hole circles from the PBMC of each donor.The diluent (50 μ l) of fusion rotein test material is added in the microtiter well in triplicate.Add specimen (target protein matter) to final 1: 4 extent of dilution; Add rhulL-2 (R﹠amp; D Systems, Minneapolis, MN, products catalogue numbering 202-IL) to final concentration 1 μ g/ml; Add anti-CD4mAb (R﹠amp; D Systems, clone 34930.11, products catalogue numbering MAB379) to final concentration 10 μ g/ml.With cell in 37 ℃ at 5%CO ₂The middle cultivation 7-8 days added 1 μ C[in the hole at last 16 hours that cultivate ³H] thymidine.Collecting cell, and measure thymidine with Packard TopCount and mix.Data are expressed as the mean value and the standard deviation of three parts of mensuration.

The sample of screening purpose fusion rotein in the experiment that separates, and handle with suppressing lymphopoietic negative control, anti-CD4mAb and strengthen lymphopoietic positive control and handle, IL-2 (recombined material or supernatant liquor) compares.

Embodiment 41: the assay method of protease activity

Following assay method can be used for assessing the protease activity of albumin fusion proteins of the present invention.

Gelatin and casease spectrometry (zymography) carry out (people such as Heusen, Anal.Biochem.102:196-202,1980 according to record in essence; People such as Wilson, Journal of Urology 149:653-658,1993).Sample is carried out electrophoresis containing on 1% gelatin or caseic 10% polyacrylamide/0.1%SDS gel, in 2.5%triton,, and in 0.1M glycine pH8.3, soaked 5-16 hour in 37 ℃ in soaking at room temperature 1 hour.After the dyeing, the proteolysis zone is shown as the limpid district in the blue-black background in amido black.Use trypsin Sigma T8642) as positive control.

Can also measure protease activity by the cutting of monitoring n-a-benzoyl-L-arginine ethyl ester (BAEE) (Sigma B-4500).At (25mM NaPO ₄, 1mM EDTA and 1mM BAEE), set up reaction among the pH 7.5.Add sample, and on Beckman DU-6 spectrophotometer, monitor the change of 260nm place absorbancy with the time drive pattern.Use trypsinase as positive control.

The absorbancy by measuring 280nm place or the colorimetric titration of use Folin method are based on people such as other assay method that is discharged solvable peptide by casein or oxyphorase such as Bergmeyer, Methods ofEnzymatic Analysis 5,1984) described in carry out.Other assay method relates to the dissolving (Ward, Applied Science 251-317,1983) of chromogenic substrate.

Embodiment 42: identify the serine protease substrate specificity

Method known in the art or described herein can be used for measuring the substrate specificity of the albumin fusion proteins of the present invention with serine protease.The preferred method of measuring substrate specificity is to use the synthetic combinatorial libraries (positional scanningsynthetic combinatorial libraries) of location scanning described in GB2324529 (complete being incorporated herein).

Embodiment 43: the part binding assay

Following assay method can be used for assessing the ligand-binding activity of albumin fusion proteins of the present invention.

The part binding assay provides confirms the pharmacological direct method of acceptor, and is suitable for high throughput format.Become height ratio to live (50-2000Ci/mmol) the part radio-labeling of the albumin fusion proteins of the present invention of purifying to be used in conjunction with research.Definite subsequently radiolabeled process is not subdued the activity of part to fusion rotein.Optimize the condition determination of damping fluid, ion, pH and other instrumentality such as Nucleotide, thereby the two all sets up feasible signal to noise ratio with cell polypeptide is originated entirely to film.For these assay methods, special polypeptide deducts the radioactivity that records when having excessive unmarked competitive part in conjunction with being defined as total relevant radioactivity.If possible, use defines remaining non-specific binding above a kind of competitive part.

Embodiment 44: the functional examination method in the xenopus leavis oocytes

Use RNA polymerase to add cap rna transcription thing according to standard program in the linearization plasmid template of external composite coding albumin fusion proteins of the present invention.The final concentration of in-vitro transcription thing with 0.2mg/ml suspended in water.Take out the ovary leaf from the female toad that grows up, what obtain the V stage removes folliculus (defolliculated) ovocyte, and uses the microinjection device to inject (bolus) injection rna transcription thing (10ng/ ovocyte) with 50nl.Use two electrode voltages to clamp and measure the electric current that indivedual xenopus leavis oocytes are replied fusion rotein and the exposure of polypeptide agonist.In no Ca2+BarthShi substratum, carry out record in room temperature.The tissue/cell extract that the Xenopus laevis system can be used for screening known ligand and is used to activate part.

Embodiment 45: little physiometry assay method (Microphysiometric Assay)

The activation of extremely multiple second messenger system causes small amount of acid to be extruded from cell.The acid that forms mainly is to promote the result that the required metabolic activity of intracellular signal process increases.The change of cell peripheral pH in culture medium is very little, but (Molecular DevicesLtd., Menlo Park Calif.) can detect the little physiometry device of CYTOSENSOR.Therefore CYTOSENSOR can detect albumin fusion proteins of the present invention and activate the ability of utilizing the second messenger that the intracellular signal approach is associated with energy.

Embodiment 46: extract/cell conditioned medium liquid screening

There is the multiple still also not relevant mammalian receptors that activates part (agonist).Thereby the active ligand of these acceptors may be not included in the part storehouse of identifying so far.Therefore, albumin fusion proteins of the present invention also can carry out therapeutic protein part and/or albumin protein partly the native ligand of functional screening (using functional screenings such as calcium, cAMP, little physiometry device, ovocyte electrophysiology) to identify albumin fusion proteins of the present invention at tissue extract.Produce extract that positive function replys and can continue inferior classification (subfractionate) until separating and evaluation obtains active ligand.

Embodiment 47:ATP is in conjunction with test

Following assay method can be used for assessing the ATP of fusion rotein of the present invention in conjunction with activity.

The ATP of albumin fusion proteins of the present invention can use United States Patent (USP) 5,858 in conjunction with activity, and the ATP binding assay of describing in 719 detects, with its complete being incorporated herein by reference.Briefly, the light avidity by 8-nitrine-ATP mark is measured and albumin fusion proteins bonded ATP of the present invention in competitive assays.The ATP that will contain proteic reaction mixture of 1mg/ml abc transport and different concns, or unhydrolyzable ATP analogue VITAMIN B4-5 '-imidodiphosphoric acid one arises from 4 ℃ of insulations 10 minutes.Add 8-nitrine-ATP (Sigma Chem.Corp., St.Louis, MO.) add 8-nitrine-ATP ( ³²P-ATP) (5mCi/ μ mol, ICN, Irvine, the final concentration of mixture to 100 μ M CA.), and the 0.5ml sample is placed the hole of porcelain system spot plate on ice.Plate is shining from the distance of plate 2.5cm two timed intervals of one minute with shortwave 254nm UV lamp, have therebetween one minute cooling station every.The dithiothreitol (DTT) termination reaction that adds final concentration 2mM.To be incubated liquid and carry out the SDS-PAGE electrophoresis, drying, and radioautograph.Downcut the protein band corresponding, and measure radioactivity with albumin fusion proteins of the present invention.Along with ATP or VITAMIN B4-5 '-imidodiphosphoric acid is cumulative and radioactivity that reduce provides the affine force measurement to the ATP of fusion rotein.

Embodiment 48: with the evaluation of the interactional signal transducer matter of albumin fusion proteins of the present invention

Albumin fusion proteins of the present invention can be used as research tool, is used for evaluation, sign and purifying signal transduction pathway protein or receptor protein.Briefly, the fusion rotein of the present invention of process mark can be used as reagent, is used for purifying molecule interactional with it.In an embodiment of affinity purification, with albumin fusion proteins of the present invention and chromatography column covalent coupling.Make the cell-free extract that is derived from supposition target cell such as cancerous tissue flow through pillar, and the molecule with suitable avidity combine with albumin fusion proteins.Reclaim protein complex by pillar, dissociate, and the molecule that reclaims is carried out the order-checking of N end protein matter.This aminoacid sequence is used for identifying the molecule that captures subsequently or is designed for from the degenerate oligonucleotide probe of suitable cDNA storehouse clone's corresponding gene.

Embodiment 49:IL-6 bioassay method

The multiple assay method that is used to test the cultivation effect of albumin fusion proteins of the present invention is known in this area.For example, a kind of like this assay method is described IL-6 bioassay methods such as Marz (Proc.Natl.Acad.Sci.U.S.A.95:3251-56,1998, be introduced into this paper as a reference).,, add tetrazolium salts tetrazolium bromide (MTT) and be incubated 4 hours again after 68 hours in 37 ℃ of insulations in 37 ℃ in order to measure the quantity of survivaling cell.With SDS dissolving B9 cell, and in 570nm measuring light density.Added the contrast that contains the IL-6 (positive) and the acellular factor (feminine gender).(briefly, IL-6 dependency B9 mouse cell is cleaned 3 times with no IL-6 substratum, and be assigned in the plate with 50 μ l, and add 50 μ l fusion rotein of the present invention with the concentration of every hole 5,000 cells.) show by fusion protein mediated cultivation effect with respect to negative control enhanced propagation in the specimen (containing albumin fusion proteins of the present invention).

Embodiment 50: the support of chicken embryo neuronal survival

Whether support the survival of sympathetic neuron cell in order to test albumin fusion proteins of the present invention, can utilize people's such as Senaldi chicken embryo neuronal survival assay method (Proc.Natl.Acad.Sci.U.S.A.96:11458-63,1998, be introduced into this paper as a reference).Briefly, from disengaging movement of chicken embryo and sympathetic neuron, be resuspended in the L15 substratum respectively and (contain 10%FCS, glucose, Sodium Selenite, progesterone, conalbumin, putrescine and Regular Insulin; Life Technologies, Rockville MD.) (contains 10%FCS, glucose, penicillin and 25mM Hepes damping fluid (pH 7.2) with the improved EaglesShi substratum of DulbeccoShi; Life Technologies, Rockville, MD.), and when the fusion rotein of the present invention of the purifying that has different concns in 37 ℃ at 5%CO ₂Middle insulation, and the negative control that lacks any cytokine.After 3 days, measure neuronal survival by the colorimetric method (Mosmann, T., J.Immunol.Methods 65:55-63,1983) of assessment morphocytology and use Mosmann.Compare the survival of enhanced neuronal cell with the contrast that lacks cytokine and show that albumin fusion proteins strengthens the ability of neuronal cell survival.

Embodiment 51: the assay method of phosphatase activity

Following assay method can be used for assessing serine/threonine Phosphoric acid esterase (PTPase) activity of albumin fusion proteins of the present invention.

In order to measure serine/threonine Phosphoric acid esterase (PTPase) activity, the assay method that can utilize those skilled in the art extensively to know.For example, serine/threonine Phosphoric acid esterase (PSPase) activity of albumin fusion proteins of the present invention can be used New England Biolabs, and the PSPase of Inc measures test kit and measures.Exist [ ³²P] during ATP with cAMP deopendent protein kinase phosphorylation myelin basic protein (MyBP) on Serine and threonine residues, the substrate of PSPase.Measure protein thread propylhomoserin/Threonine Phosphatases activity by the inorganic phosphate of measuring 32P-mark MyBP release subsequently.

Embodiment 52: serine/threonine Phosphoric acid esterase and other protein interactions

Fusion rotein of the present invention (for example according to embodiment 51 mensuration) with serine/threonine phosphatase activity for example can be used as research tool, be used for other interacting proteins of evaluation, sign and purifying or receptor protein, or other signal transduction pathway protein.Briefly, will can be used as reagent, be used for purifying molecule interactional with it through the fusion rotein of the present invention of mark.In an embodiment of affinity purification, with albumin fusion proteins of the present invention and chromatography column covalent coupling.Make from inferring target cell and flow through pillar, and the molecule with suitable avidity combines with fusion rotein such as neural or hepatocellular cell-free extract.Reclaim fusion rotein-mixture from post, dissociate, and the molecule that reclaims is carried out the order-checking of N end protein matter.This aminoacid sequence is used for identifying the molecule that captures subsequently or is designed for from the degenerate oligonucleotide probe of suitable cDNA storehouse clone's corresponding gene.

Embodiment 53: the active assay method of heparanase (heparanase)

The multiple assay method of the heparanase activity that can be used for measuring albumin fusion proteins of the present invention is known in this area.In an example, the heparanase activity of albumin fusion proteins of the present invention is measured (people such as Vlodavsky, Nat.Med.5:793-802,1999) as described in people such as Vlodavsky.Briefly, with cell lysates, conditioned medium, intact cell (each 35mm dish 1 * 10 ⁶Cell), the fusion rotein of cell culture supernatant or purifying in 37 ℃ of pH 6.2-6.6 with ³⁵The ESC of S mark or the solvable ECM that is derived from peak I proteoglycan are incubated 18 hours together.The heat insulating culture base is centrifugal, and at Sepharose CL-6B post (0.9 * 30em) goes up by gel-filtration clear liquid analytically.Use the PBS elutriated fraction, and measure their radioactivity.The degradation fragment of heparitin sulfate side chain is at 0.5＜K _AvDuring＜0.8 (peak II) from the Sepharose 6B wash-out.Each experiment is carried out three times at least.As described in people such as Vlodavsky, the degradation fragment corresponding with " peak II " shows the activity of albumin fusion proteins of the present invention in the cutting heparitin sulfate.

Embodiment 54: biomolecules fixing

This embodiment provides and has been used for stablizing the method for albumin fusion proteins of the present invention (referring to people such as for example Bieri at non-host cell lipid bilayer construction, Nature Biotech 17:1105-1108,1999, complete being incorporated herein by reference), it can adapt to the research of fusion rotein of the present invention in the above-mentioned various functional examination method.Briefly, will be used for biotinylated carbohydrate specific chemical method and be used for biotin label is limited to albumin fusion proteins of the present invention, thereby cause the unified orientation in fixing back.Solution and 20mM NaIO with 50 μ M albumin fusion proteins of the present invention in the film of washing ₄And 1.5mg/ml (4mM) BACH or 2mg/ml (7.5mM) vitamin H-hydrazides are in 1 hour (reaction volume, 150 μ l) of room temperature insulation.Then sample is at first dialysed in 4 ℃ (Pierce Slidealizer Cassett, 10kDa holds back; Pierce Chemical Co., Rockford, IL) 5 hours, each hour exchange buffering liquid was used 500ml damping fluid R (0.15M NaCl, 1mM MgCl at last ₂, 10mM sodium phosphate pH7) dialysis 12 hours.Only before adding cuvette, sample is diluted with damping fluid ROG50 (being supplemented with the damping fluid R of 50mM octyl glucoside) at 1: 5.

Embodiment 55: the assay method of metal proteinase activity

Metalloprotease is a peptidohydrolase, and it utilizes metal ion such as Zn ²⁺As catalyst mechanism.The metal proteinase activity of albumin fusion proteins of the present invention can be measured according to methods known in the art.The following illustrative method is provided:

The proteolysis of α-2-macroglobulin

In order to confirm protease activity, with the fusion rotein of the present invention and substrate α-2-macroglobulin (0.2 unit/ml of purifying; Boehringer Mannheim Germany) measures damping fluid (50mMHEPES pH 7.5,0.2M NaCl, 10mM CaCl at 1x ₂, 25 μ M ZnCl ₂And 0.05%Brij-35) mix in, and in 37 ℃ of insulations 1-5 days.Use trypsinase as positive control.Negative control only contains α-2-macroglobulin in measuring damping fluid.Collect sample, in containing the SDS-PAGE sample buffer of 5%2-mercaptoethanol, boiled 5 minutes, be loaded into then on the 8%SDS-polyacrylamide gel.Behind the electrophoresis, dye by silver protein is developed.The appearance of lower molecular weight band has proved proteolysis by compare more with negative control.

The inhibition of metalloprotease is to the proteoclastic inhibition of α-2-macroglobulin

Known metalloprotein enzyme inhibitor (metal chelator (EDTA, EGTA and HgCl ₂), peptide metalloprotein enzyme inhibitor (TIMP-1 and TIMP-2) and commercialization small molecules MMP inhibition) also can be used for characterizing the proteolytic activity of albumin fusion proteins of the present invention.Operable three kinds of synthetic MMP inhibitions are: MMP inhibition I, [IC ₅₀=1.0 μ M are to MMP-1 and MMP-8; IC ₅₀=30 μ M are to MMP-9; IC ₅₀=150 μ M are to MMP-3]; MMP-3 (molten stromatin enzyme-1) inhibition I, [IC ₅₀=5 μ M are to MMP-3] and MMP-3 inhibition II, [Ki=130nM is to MMP-3]; Inhibition can obtain from Calbiochem, and the products catalogue numbering is respectively 444250,444218 and 444225.Briefly, with the small molecules MMP inhibition of different concns and the fusion rotein of the present invention of purifying (50 μ g/ml) at 22.9 μ l 1x HEPES damping fluid (50mM HEPES pH 7.5,0.2M NaCl, 10mM CaCl ₂, 25 μ M ZnCl ₂And 0.05%Brii-35) mixes in, and in room temperature (24 ℃) insulation 2 hours, add 7.1 μ l substrate α-2-macroglobulin (0.2 unit/ml), and then in 37 ℃ of insulations 20 hours.Add the 4x sample buffer and come termination reaction, and boiled at once 5 minutes.Behind the SDS-PAGE, dye by silver and to manifest protein band.

Synthetic fluorescence peptide substrates cutting assay method

Substrate specificity with the fusion rotein of the present invention that confirms metal proteinase activity can use technology known in the art to measure, such as using synthetic fluorescence peptide substrates (available from BACHEMBioscience Inc).The test substrate comprises M-1985, M-2225, M-2105, M-2110 and M-2255.Preceding 4 kinds is the MMP substrate, and at last a kind of is the substrate of tumor necrosis factor-alpha (TNF-α) saccharase (TACE).These substrates preferably prepare in 1: 1 dimethyl sulfoxide (DMSO) (DMSO) and water.Liquid storage is 50-500 μ M.The luminous spectrometer of Perkin Elmer LS 50B that use is furnished with water bath with thermostatic control carries out fluorometric assay.Exciting λ is 328nm, and emission λ is 393nm.Briefly, following mensuration is with 176 μ l1x HEPES damping fluid (0.2M NaCl, 10mM CaCl ₂, 0.05%Brij-35 and 50mM HEPESpH 7.5) and 20 μ l substrate solutions (50 μ M) in 25 ℃ of insulations 15 minutes, in measuring cuvette, add the fusion rotein of the present invention of 20 μ l purifying then.The final concentration of substrate is 1 μ M.To initial hydrolysis rate monitoring 30 minutes.

The generation of diabetes in the embodiment 56:NOD mouse

The feature of female NOD (no obese diabetes) mouse is to show IDDM with the similar course of disease of being found in the mankind, though disease ratio in female is more obvious male NOD mouse.Hereinafter, except as otherwise noted, the female NOD mouse of term " NOD mouse " expression.The NOD mouse has the β cell that chronic autoimmune disease causes and destroys gradually.Have euglycemia or normal glucose level when therefore, NOD mouse life begins.Yet during age in week, the NOD mouse begins to become hyperglycemia to about 15-16, and this destruction and corresponding pancreas incompetence that shows their most of pancreatic beta cells produces enough Regular Insulin.Therefore, the cause of disease and progress are all similar with people IDDM patient.

Can be in the body of assessment immunization protocol effect in the female NOD/LtJ mouse (can be, Bar Harbor, Me. buys) from The Jackson Laboratory assay method.The female mice of report 80% forms diabetes in the document when 24 ages in week, and the outbreak of insulitis starts from 6-8 between age in week.The NOD mouse is an inbred lines, and high response panimmunity regulation and control strategy.The NOD mouse (6-8 age in week) that grows up has the weight in average of 20-25g.

These mouse can be undressed (contrasts), handle separately or in conjunction with other therapeutic compound mentioned above with therapeutical agent of the present invention (as albumin fusion proteins of the present invention and fragment and variant).These different therapies can followingly be measured the effect of diabetes progress:

When 14 ages in week, can determine the phenotype of female NOD mouse according to the glucose tolerance.The glucose tolerance can be measured by intraperitoneal glucose tolerance method of testing (IPGTT).Briefly, blood is got from the other plexus vasculosus of socket of the eye in the 0th minute behind peritoneal injection glucose (1g/kg body weight) and the 60th minute.The normal plasma glucose that is defined as the 0th minute that tolerates is lower than 144mg%, or is lower than 160mg% on the 60th minute.Glucose level is measured with Glucometer Elite device.

Based on this phenotype analytical, animal can be assigned to different test group.Specifically, the animal with glucose level of rising can be assigned to glucose and tolerate impaired group.Mouse can ad libitum access, and supply acidified water (pH2.3).

Tolerance can further be subdivided into control group, albumin fusion proteins group of the present invention and albumin fusion proteins/therapeutic compound combination group with the mouse that does not tolerate glucose.But the mouse in the control group is accepted the peritoneal injection of carrier every day, 6 times weekly.But the mouse in the white protein fusion group is accepted the peritoneal injection of the therapeutical agent of the present invention (as albumin fusion proteins of the present invention and fragment and variant) in the carrier, 6 times weekly every day.Mouse can be accepted albumin fusion proteins and therapeutic compound makes up the two as mentioned above in albumin fusion proteins/therapeutic compound combination group.

The level of sugar of NOD mouse can be used Labstix, and (England) two weeks were measured once for Bayer Diagnostics, Hampshire.Body weight and liquid are taken in also two all mensuration once.Glycosuria in double mensuration, occurs and determine the outbreak of diabetes afterwards.After handling for 10 weeks, can carry out additional IPGTT, and put to death animal at second day.

After 10 courses of treatment in week, tolerance and the control animal that does not tolerate in the glucose group form diabetes (seeing U.S. Patent number 5,866,546, people such as Gross) with 60% and 86% ratio respectively.Therefore, if do not intervene, even in the NOD mouse of initial tolerance glucose, a high proportion of diabetes occur yet.

Can come validate result by the glucose level of measuring in the NOD mouse of treatment front and back.The glucose level of tolerance and two kinds of mouse that do not tolerate glucose is all measured as mentioned above in all groups of describing.

In optional embodiment, therapeutical agent of the present invention (for example as the disclosed specific fusion proteins of SEQ ID NO:Y and fragment and variant) can use analysis of spectral method to measure, and before injection the protein of proper amt is resuspended in every dosage 50 μ l phosphate buffered saline (PBS)s (PBS).But the double injection subcutaneous administration in the week of being separated by is under the skin of back of every mouse.Monitoring can be carried out in immunity preceding two periods of separating, and can carry out weekly during treating and continue thereafter.Can test glucose in urine (Keto-Diastix.RTM. weekly; Miles Inc., Kankakee, Ill.), and can to the glycosuria mouse check serum glucose (ExacTech.RTM., MediSense, Inc., Waltham, Mass.).When being higher than 2.5g/L, the fasting glycemia is diagnosed as diabetes.

The histological examination of embodiment 57:NOD mouse

The histological examination of NOD mouse tissue sample can prove that the combination of the present composition and/or the present composition and other Remedies for diabetes increases the ability of β cell relative concentration in the pancreas.Experimental technique is as follows:

Mouse from embodiment 56 can be put to death when treatment stage finishes, and takes out tissue sample from pancreas.Can sample is fixing in being dissolved in 0.9% brinish, 10% formaldehyde, and bag wax.Two groups 5 continuous 5 μ m sections can be immune labeled to be used for the cutting cut-space of 150 μ m.Section can be Regular Insulin (1: 1000 diluent of cavy synalbumin antiserum(antisera), ICN Thames U.K.) and glucagon (1: 2000 diluent of the anti-hyperglycemic-glycogenolytic factor antiserum(antisera) of rabbit) carry out immune labeled, and with the anti-cavy (Dako that has puted together peroxidase, High Wycombe, U.K.) or puted together the anti-rabbit anteserum of peroxidase (1: 50 diluent Dako) detects.

Composition of the present invention for the visual quality (visible mass) of β cell can have or not have with its to tolerance with do not tolerate the same strong effect of diabetes clinical manifestation in the animal of glucose.

Mouse model in the body of embodiment 58:NIDDM

From Jackson Laboratory (Bar Harbor, male C 57 BL/6 J mouse ME) can obtain during ages in 3 weeks, and the conventional food or be rich in fat (35.5%wt/wt of feeding; Bioserv.Frenchtown, NJ) or fructose (60%wt/wt; Harlan Teklad, Madison, food Wl).Conventional food is fibrous by 4.5%wt/wt fat, 23%wt/wt protein, 31.9%wt/wt starch, 3.7%wt/wt fructose and 5.3%wt/wt.Higher fatty acid (lard) food is fibrous by 35.5%wt/wt fat, 20%wt/wt protein, 36.4%wt/wt starch, 0.0%wt/wt fructose and 0.1%wt/wt.High fructose food is fibrous by 5%wt/wt fat, 20%wt/wt protein, 0.0%wt/wt starch, 60%wt/wt fructose and 9.4%wt/wt.Mouse is raised in the watch-keeping cubicle of illumination in 12 hours of 22 ± 3 ℃ of temperature, 50 ± 20% humidity at 6 (in the morning to 6 pm)/dark cycle, every cage is no more than 5 (people such as Luo, 1998, Metabolism 47 (6): 663-8, " Nongenetic mouse models ofnon-insulin-dependent diabetes mellitus "; People such as Larsen, Diabetes 50 (11): 2530-9,2001, " Systemic administration of the long-acting GLP-1 derivative NN2211induces lasting and reversible weight loss in both normal and obese rats ").Give corresponding food after 3 weeks, but the streptozocin of mouse peritoneal injection 100mg/kg body weight, and " STZ " (Sigma, St.Louis MO) or carrier (0.05mol/L citric acid pH4.5), and keep same food in ensuing 4 weeks.Under the condition of non-fasting, get blood by cutting off tail end 1,2 and 4 weeks behind STZ.The glucose and the insulin concentration that sample are used to measure non-fasting blood plasma.Write down body weight and ingestion of food weekly.

In order directly to measure food rich in fat promotes the glucose controllability to Regular Insulin influence, can begin experiment to three groups of mouse when finish mentioned above 7 all periods, promptly fat is fed mouse, is fed mouse with the food nursing mouse of vector injection with the fat of STZ injection.Mouse can be before experiment fasting 4 hours.In first group of experiment, can be by sucking Methoxyflurane (Pitman-Moor, Mundelein, IL) anesthetized mice.Insulin regular (Sigma) can carry out intravenous injection ([IV] 0.1U/kg body weight) by tail vein, and 3,6,9,12 gets blood with 15 minutes from different tail vein after injection.Can be to these sample determination plasma glucose concentrations, and (NC), a kind of pharmacokinetics/pharmacodynamics software program calculates the transformation period (t1/2) that glucose disappears in the blood plasma for Scientific Consulting, Apex to use WinNonlin.

In second group of experiment, mouse can be by intraperitoneal vetanarcol (Sigma) anesthesia.Open the abdominal cavity, expose main epigastric vein, and insert No. 24 IV conduits (Johnson-Johnson Medical, Arlington, TX).Conduit is fixed near the epigastric vein the muscle tissue, cuts on the bottom that syringe connects, hang up the PE50 plastics tubing of filling with in advance, it connects the syringe that perfusion liquid is housed then.Sew up the abdominal cavity then.By this method, the obstacle that the lower position that does not exist blood from health is refluxed.Give mouse with 10 μ l/min perfusion volume continous pouring glucose (24.1mg/kg/min) and Regular Insulin (10mU/kg/min).In order to measure plasma glucose and insulin concentration, can gather socket of the eye in 90,105,120 and 135 minutes in perfusion beginning back after blood sample (each 70 μ l).The steady state plasma glucose (SSPG) and Regular Insulin (SSPI) concentration that the mean value of these 4 samples are used to estimate each animal.

At last, be used to assess the application's albumin fusion proteins, therapeutic composition, reducing the experiment of plasma glucose ability when using separately or unite any or multiple curative drug of listing for the treatment diabetes can carry out in following two groups of " NIDDM " mouse models with the STZ injection: the C57BL/6J that C57BL/6J that (1) fat is fed and (2) fructose are fed.The plasma glucose concentration that is used for the mouse of these researchs arrives in the scope of 555mg/dL 255.Mouse is specified at random with carrier or with white protein of the present invention and merges therapeutical agent separately or unite any or multiple curative drug of listing for the treatment diabetes and treat.Can use three dosage altogether.Can be used to measure plasma glucose concentration by 3 hours collection tail vein blood samples before first administration and after the last administration.

Plasma glucose concentration can use the glucose diagnostic kit (Sigma No.315) from Sigma, and a kind of enzyme colorimetric method is measured.Plasma insulin level can use the plain RIA test kit of the mouse islets (#RI-13K from Linco Research; St.Charles MO) measures.

Embodiment 59: the external H4IIe-SEAP reporter assay method of determining to relate to insulin action Various H4IIe reporter genes

H4IIe/rMEP-SEAP: separate malic enzyme (rMEP) from rat and contain PPAR-γ element in the Regular Insulin approach.This reporter molecule construction is stably transfected in the liver H4IIe clone.

H4IIe/SREBP-SEAP: sterol controlling element conjugated protein (SREBP-1c) is a kind of transcription factor, it acts on multiple insulin replies gene, the for example promotor of fatty acid synthetase (FAS), and the expression of key gene in the adjusting fatty acid metabolism in inoblast, lipocyte and liver cell.SREBP-1c is also referred to as adipocyte decision and differentiation factor 1 (ADD-1), thinks that Regular Insulin in the adipocyte influences the main intermediary of genetic expression.Its activity is subjected to the adjusting of Regular Insulin, sterol and glucose level.This reporter molecule construction is stably transfected in the liver H4IIe clone.

H4IIe/FAS-SEAP: fatty acid synthetase reporter molecule construction contains minimum SREBP responsiveness FAS promotor.This reporter molecule construction is stably transfected in the liver H4IIe clone.

H4IIe/PEPCK-SEAP: phosphoenolpyruvate carboxykinase (PEPCK) promotor is to regulate the major hormone regulatory site of the active PEPCK genetic transcription of PECK.Key and rate-limiting step in the PEPCK catalysis liver glyconeogenesis maintain glucose level in the NL thereby therefore must carefully control.This reporter molecule construction is stably transfected in the liver H4IIe clone.

Also these reporter molecule constructions can be stably transfected in 3T3-L1 inoblast and the L6 sarcoplast.Make these stable cell lines be divided into 3T3-L1 adipocyte and L6 myotube then as described in the previous embodiment 13.The clone of differentiation can be used for SEAP assay method hereinafter described then.

Growth and mensuration substratum

Growth medium contains 10% foetal calf serum (FBS), 10% calf serum, 1%NEAA, 1x 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).For H4IIe/PEPCK-SEAP, growth medium is made up of 10%FBS, 1% penicillin/streptomycin, 15mMHEPES buffer saline and 0.50mg/ml G418.

For H4IIe/rFAS-SEAP, H4IIe/SREBP-SEAP, H4IIe/rMEP-SEAP reporter gene, measure substratum and form by low dextrose DMEM substratum (Life Technologies), 1%NEAA, 1x penicillin/streptomycin.The assay method substratum of H4IIe/PEPCK-SEAP reporter gene is made up of 0.1%FBS, 1% penicillin/streptomycin and 15mM HEPES buffer saline.

Method

96 hole plates are inoculated in 100 μ l/ hole growth mediums with 75,000 cells/well, become adherent up to the cell of logarithmic phase.Measure substratum by growth medium being replaced with 200 μ l/ holes, with the hungry cultivation of cell 48 hours.(, contain the mensuration substratum of 0.5 μ M dexamethasone with the adding of 100 μ l/ holes, and be incubated about 20 hours) for the H4IIe/PEPCK-SEAP cell.To measure substratum subsequently and replace with the fresh mensuration substratum in 100 μ l/ holes, and the Xiang Kongzhong adding is from 50 μ l samples of the cell conditioned medium liquid of the transfectional cell series acquisition of expression therapeutical agent of the present invention (as albumin fusion proteins of the present invention and fragment and variant).Use from the supernatant liquor of empty carrier transfectional cell series as negative control.Xiang Kongzhong interpolation 10nM and/or 100nM Regular Insulin are as positive control.Be incubated after 48 hours, the results conditioned medium, and measure SEAP activity (Phospha-Light System protocol, Tropix #BP2500).Briefly, with sample dilution in 1: 4 in dilution buffer liquid, and in 30 minutes SEAP with the endogenous non-placentation of deactivation of 65 ℃ of insulations.50 μ l samples of dilute sample are measured damping fluid with 50 μ l SEAP mix, the latter is contained the activated inhibition mixture of non-placenta SEAP isozyme, and is incubated 5 minutes again.In mixed solution, be added in 50 μ l samples of the CSPD chemical luminous substrate of dilution in 1: 20 in the Emerald luminescence enhancer, and be incubated 15-20 minute.Plate is carried out reading in Dynex plate photometer.

Embodiment 60: transgenic animal

Albumin fusion proteins of the present invention also can be expressed in transgenic animal.The animal of any species includes but not limited to mouse, rat, rabbit, hamster, cavy, pig, miniature pig, goat, sheep, ox and non-human primates, all can be used for producing transgenic animal as baboon, monkey and orangutan.In specific embodiment, use technology described herein or that other approach of this area is known in the people, to express fusion rotein of the present invention, as the part of gene therapy scheme.

Any technology known in the art all can be used for the polynucleotide of code book invention albumin fusion proteins are imported animal to produce the person of foundation system (founder lines) of transgenic animal.Such technology includes but not limited to pronucleus microinjection (people such as Paterson, Appl.Microbiol.Biotechnol.40:691-698,1994; People such as Carver, Biotechnology (NY) 11:1263-1270,1993; People such as Wright, Biotechnology (NY) 9:830-834,1991; Reach people such as Hoppe, U.S. Patent number 4,873,191,1989); The transgenosis of retrovirus-mediated method is to reproductive tract (people such as Van derPutten, Proc.Natl.Acad.Sci.USA 82:6148-6152,1985), blastocyst or embryo; Gene target in the embryonic stem cell (people such as Thompson, Cell 56:313-321,1989); Cell or embryo's electroporation (Lo, 1983, Mol.Cell.Biol.3:1803-1814,1983); Use the importing (referring to people such as for example Ulmer, Science 259:1745,1993) of the polynucleotide of the present invention of particle gun; The nucleic acid construct thing is imported embryonic pleuripotent stem cell and stem cell is shifted back blastocyst; And the transgenosis of sperm mediation people such as (, Cell 57:717-723,1989) Lavitrano; Deng.The summary of these technology is referring to Gordon, " Transgenic Animals ", Intl.Rev.Cytol.115:171-229,1989), with its complete being incorporated herein by reference.

Can use any technology known in the art to produce the transgene clone of the polynucleotide that contain code book invention albumin fusion proteins, for example will or induce into immobilized from embryo, the fetus cultivated becomes somatic nuclear consideration convey to move on in the non-nucleus egg mother cell (people such as Campell, Nature 380:64-66,1996; People such as Wilmut, Nature 385:810-813,1997).

The invention provides the transgenic animal of the polynucleotide that in its all cells, carry code book invention albumin fusion proteins, and at it some but the animal of carrying these Nucleotide in the non-all cells, i.e. chimaeric animals or mosaic.Transgenosis can be used as single transgenosis or as multiple copied concatermer for example, as series connection head to head or head to the tail series connection and integrate.According to people's such as for example Lasko instruction, also the transgenosis selectivity can be imported particular cell types and activate people such as (, Proc.Natl.Acad.Sci.USA 89:6232-6236,1992) Lasko therein.This cell type specificity activates required regulating and controlling sequence will depend on concrete purpose cell type, and it will be apparent to those skilled in the art that.When the polynucleotide of expectation code book invention fusion rotein were incorporated into the chromosome position of the native gene corresponding with the therapeutic protein part of fusion rotein of the present invention or albumin part, gene targeting was preferred.Briefly, when using such technology, the carrier design that will contain some and native gene homologous nucleotide sequence is used for the function that is incorporated into the nucleotide sequence of native gene and destroys it by the homologous recombination with chromosome sequence.According to people's such as for example Gu instruction, also the transgenosis selectivity can be imported particular cell types, thus deactivation native gene people such as (, Science265:103-106,1994) Gu in this cell type only.The required regulating and controlling sequence of this cell type specificity deactivation will depend on concrete purpose cell type, and it will be apparent to those skilled in the art that.

In case produced transgenic animal, can utilize standard technique to measure the expression of recombination.Confirm to take place the integration of the polynucleotide of code book invention fusion rotein by Southern engram analysis or round pcr analyze animal tissue, finished preliminary screening thus.Also can use following technology to assess the mRNA expression level of the polynucleotide of code book invention fusion rotein in the tissue of transgenic animal, include but not limited to Northern engram analysis, in situ hybridization analysis and the reverse transcription PCR (rt-PCR) of the tissue sample that from animal, obtains.The sample of the tissue of expressed fusion protein can also use the special antibody of fusion rotein is carried out immunocytochemistry or immunohistochemistry assessment.

In case produced the person of foundation animal, can make their mating, inbreeding, outbreeding or hybridization to produce the colony of special animal.The example of such mating strategy includes but not limited to: have the outbreeding of the person of foundation animal that surpasses an integration site, isolate system (separate lines) to set up; Isolate the inbreeding of system, to produce owing to the effect of each genetically modified additional expression with the compound transgenic animal of higher level express transgenic; The needs that screen animal by DNA analysis are expressed and eliminated to the hybridization of heterozygosis transgenic animal to be created in the animal of specifying integration site to isozygoty, thereby increase; Isolate the hybridization of isozygotying and being, be to produce compound heterozygosis or to isozygoty; And mating, so that transgenosis (being the polynucleotide of code book invention albumin fusion proteins) is placed the unique background that is suitable for the purpose experimental model.The purposes of transgenic animal of the present invention includes but not limited to be used in the animal model system that biological function, the research of the therapeutic protein that describes fusion rotein of the present invention and fusion rotein of the present invention in detail and/or white protein component situation relevant with unconventionality expression and/or disease and screening are effectively improved the compound of these situations and/or disorder.

Embodiment 61: the methods of treatment-ex vivo (ex vivo) that uses gene therapy

A kind of method of gene therapy can be expressed the inoblast of albumin fusion proteins of the present invention and be transplanted on one's body the patient.Usually, obtain inoblast by Skin biopsy from the experimenter.The tissue that so obtains is placed tissue culture medium (TCM), and be divided into fritter.Little block organization is placed on the wet surface of tissue culture flasks, place about ten in each flask.Flask is turned upside down, airtight, and place in room temperature and to spend the night.After room temperature was placed 24 hours, with the flask reversing, and tissue block still was fixed on drag, added the fresh culture HamShi F12 substratum of 10%FBS, penicillin and Streptomycin sulphate (as contain).Then flask is incubated about weeks in 37 ℃.

At this moment, add fresh culture, changed in every subsequently several days.After extra 2 weeks of cultivation, the inoblast individual layer appears.With the cell monolayer tryptic digestion, and the amplification scale is to bigger flask.

With flank is pMV-7 (Kirschmeier, people such as P.T., DNA 7:219-25,1988) EcoRI and the HindIII digestion of the long terminal repeat of Moloney murine sarcoma virus, handles with the calf intestinal phosphatase enzyme then.With linear carrier classification on sepharose, and use the granulated glass sphere purifying.

The polynucleotide of code book invention albumin fusion proteins can use technology known in the art to produce, and adopt the PCR primer corresponding with its 5 ' and 3 ' terminal sequence to increase, and choose wantonly if desired to have suitable restriction site and initial/terminator codon.Preferably, 5 ' primer contains the EcoRI site, and 3 ' primer contains the HindIII site.The Moloney murine sarcoma virus linear backbone of equivalent and EcoRI and the HindIII fragment that obtains that increase are added to together when having the T4 dna ligase.The mixture that maintenance so obtains under the condition that is suitable for two kinds of fragments connections.To connect mixture then and be used for transform bacteria HB101, be coated on subsequently on the agar that contains kantlex to confirm correctly to have inserted goal gene in the carrier.

Two preferendum pA317 or GP+aml2 packing cell be cultured in tissue culture in the EaglesShi substratum (DMEM) of the DulbeccoShi improvement that contains 10% calf serum (CS), penicillin and Streptomycin sulphate converge density.In substratum, add the MSV carrier that contains gene then, use the carrier transduction packing cell.Packing cell produces the infectious viral particle (packing cell is called the production cell now) that contains gene now.

Produce in the cell to transduction and to add fresh culture, subsequently from the 10cm plate results substratum of the production cell that converges.The exhausted substratum that contains infectious viral particle is filtered to remove the production cell that comes off by millipore filter, then this substratum is used to infect inoblast.Converge plate from fibroblastic Asia and take out substratum, and be replaced with from the substratum of producing cell.Take out this substratum, and be replaced with fresh culture.If the titre height of virus, so in fact all inoblasts all will be infected, and not need to select.If titre is very low, must use retroviral vector so with selective marker such as neo or his.In case effectively infected inoblast, be parsed into fibrocyte and whether produce albumin fusion proteins to measure.

To be transplanted on the host after converging separately or growing on cytodex 3 microcarrier beads through the inoblast of transforming then.

Embodiment 62: use in the methods of treatment-body of gene therapy

Another aspect of the present invention is to use the vivo gene therapy to treat disorder, disease and situation.Gene therapy method relates to the exposed nucleic acid of code book invention albumin fusion proteins (DNA, RNA and antisense DNA or RNA) sequence importing animal.The polynucleotide of code book invention albumin fusion proteins can be operatively connected (promptly linking to each other) with promotor or by necessary any other gene element of target tissue express polypeptide.Such gene therapy and delivery technology and method are known in the art, see for example WO 90/11092, WO 98/11779; U. S. application numbers 5693622,5705151,5580859; People such as Tabata, Cardiovasc.Res.35 (3): 470-479,1997; People such as Chao, Pharmacol.Res.35 (6): 517-522,1997; Wolff, Neuromuscul.Disord.7 (5): 314-318,1997; People such as Schwartz, Gene Ther.3 (5): 405-411,1996; People such as Tsurumi, Circulation94 (12): 3281-3290,1996 (being incorporated herein by reference).

The polynucleotide construction can be delivered by any method that injectable substance is delivered in the zooblast, such as being injected to tissue (heart, muscle, skin, lung, liver, intestines etc.) intercellular space.The polynucleotide construction can be delivered in pharmacopedics acceptable liquid or aqueous carrier.

Term " exposed " polynucleotide, DNA or RNA refer to that sequence is free on any delivery carrier auxiliary, that promote or quicken to enter cell, comprises virus sequence, virion, Liposomal formulation, fat transfection or precipitation reagent etc.Yet, the polynucleotide of code book invention albumin fusion proteins also can be able to delivered (such as people such as Felgner P.L. in the Liposomal formulation by method preparation well known to those skilled in the art, 1995, people such as Ann.NY Acad.Sci.772:126-139 and Abdallah B., 1995, Biol.Cell 85 (1): instructed among the 1-7).

The polynucleotide carrier construction that is used for gene therapy method is preferably and neither can be incorporated into host genome and also contain the construction that allows the sequence of duplicating.Can use any strong promoter that those skilled in the art will know that to drive the expression of DNA.Different with other gene therapy technology, a major advantage that exposed nucleotide sequence is imported target cell is the of short duration character of polynucleotide synthetic in the cell.Studies show that can be with non-repetition DNA sequence transfered cell, thus be 6 months during the generation of expectation polypeptide is provided.

The polynucleotide construction can be delivered to the intercellular space of organizing of animal, comprise muscle, skin, brain, lung, liver, spleen, marrow, thymus gland, the heart, lymph, blood, bone, cartilage, pancreas, kidney, gall-bladder, stomach, intestines, testis, ovary, uterus, rectum, neural system, eye, gland and reticular tissue.Organize the intercellular space to comprise the collegen filament of spandex fiber in mucopolysaccharide layer, tube wall or the locular wall between the reticulin fiber of intercellular fluid, organ-tissue, fibrous tissue or include myocyte's reticular tissue or the same matrix in the bone crack in.Also similar by the space that the lymph liquid of circulating plasma and lymph pipeline occupies.Owing to the reason of discussing below preferably is delivered to the muscle tissue intercellular space.They can be delivered to the tissue that comprises these cells easily by injection.They preferably are delivered to persistent, nondividing noble cells and express therein, can be although deliver and express at not differentiation or the more incomplete cell of differentiation, and such as finishing in the stem cell of for example blood or the skin flbroblast.In vivo, muscle cell is competent especially aspect the ability of their picked-ups and expression polynucleotide.

For exposed polynucleotide injection, the effective dose of DNA or RNA will be in about 0.05g/kg body weight to the scope of about 50mg/kg body weight.Preferably, dosage will be about 0.005mg/kg to about 20mg/kg and 0.05mg/kg about 5mg/kg extremely more preferably from about.Certainly, those of ordinary skills will understand, and this dosage will change along with the tissue site of injection.Suitable and the effective dosage of nucleotide sequence can be easy to determine, and may depend on the situation and the drug delivery route of treatment by those of ordinary skills.Preferred drug delivery route is to organizing in the intercellular space by the parenteral injection approach.Yet, also can use other parenteral route, suck such as aerosol, be specially adapted to be delivered to lung or bronchial tissue, larynx or nasal mucosa.In addition, the conduit that can be in the angioplasty process uses in by program of exposed polynucleotide construction is delivered to artery.

The following mensuration of administration reaction effect of intramuscular injection polynucleotide in the body.The suitable template DNA that is used to generate the mRNA of code book invention polypeptide according to the preparation of conventional recombinant DNA method.Template DNA may be annular or linear, or uses or compound with liposome as naked DNA.Give the template DNA of the different amounts of musculus quadriceps injection of mouse subsequently.

Female and the male Balb/C mouse in five to six ages in week is anaesthetized by peritoneal injection 0.3ml 2.5% avertin.On preceding thigh, do the otch of a 1.5cm, directly expose musculus quadriceps.Template DNA was injected with one minute by No. 27 pins with the 1cc syringe in the 0.1ml carrier, and is dark to the about 0.5cm of knee and about 0.2cm from muscle tip insertion position.For location in the future, on the injection site, sew up, and skin is closed with the stainless steel folder.

After suitable soaking time (for example 7 days), prepare the muscle extract by downcutting whole musculus quadriceps.To indivedual quricipital per five 15 μ m slices across is that protein expression carries out histological stain.The time course of expressing fusion protein can be finished by similar form, and the musculus quadriceps that is different mouse is in the different time collection.The persistence of DNA can be measured by the Southern engram analysis for preparing total cell dna and HIRT supernatant liquor from injection mouse and control mice in the muscle of injection back.Other treatment parameter that above-mentioned result of experiment can be used for extrapolating proper dosage and use naked DNA in people and other animal in the mouse.

Embodiment 63: the biology effect of fusion rotein of the present invention

Stellate cell and neurone assay method

Can to albumin fusion proteins of the present invention test the survival, the spinous process that promote the cortical neuron cell outwards grow or phenotypic differentiation in active and induce glial fibrillary acidic protein immuning positive cell, the ability of stellate cell propagation.Select tegumental cell for bioassay method and be based on FGF-1 and generally expressing of FGF-2 and strengthening of previous report in the cortex construction because of FGF-2 handles the cortical neuron survival that causes.For example, thymidine mixes assay method and can be used for illustrating albumin fusion proteins of the present invention to these cell activity.

In addition, the previous FGF-2 (basic FGF) that describes has confirmed that at the report of external biology effect to cortex or hippocampal neuron neuronal survival and spinous process outwards grow the increase of the two (people such as Walicke, " Fibroblast growth factor promotes survival of dissociated hippocampalneurons and enhances neurite extension ", Proc.Nntl.Acad.Sci.USA 83:3012-3016,1986, be incorporated herein by reference assay method is complete).Yet, show to reply for these two kinds from report to need not to be synonym, but what may not only depend on test is which kind of FGF depends on that also which kind of acceptor what express on the target cell is to PC-12 experiment that cell is done.Use former generation cortical neuron to cultivate calligraphy or painting model, albumin fusion proteins of the present invention induce ability that spinous process outwards grows can with for example use thymidine to mix assay method to compare with replying of realizing of FGF-2.

Inoblast and endotheliocyte assay method

(San Diego CA) obtains people's lung fibroblast, and keeps the growth medium that obtains from Clonetics from Clonetics.(SanDiego CA) obtains dermal microvascular endothelial cell from Cell Applications.For proliferation assay, people's lung fibroblast and dermal microvascular endothelial cell can be cultivated 1 day with 5,000 cells/well in growth medium in 96 hole plates.Then cell is incubated 1 day in the 0.1%BSA basic medium.After changing substratum, cell is incubated 3 days with the proteinic tester fusion protein of the present invention with fresh 0.1%BSA substratum.(Alamar Biosciences, Sacramento is CA) to final concentration 10% to add Alamar Blue in each hole.With cell insulation 4 hours.Measure cell viability by the reading in the CytoFluor fluorescence reader.For PGE ₂Assay method is cultivated people's lung fibroblast 1 day with 5,000 cells/well in 96 hole plates.After substratum is replaced by the 0.1%BSA basic medium, cell is incubated 24 hours with FGF-2 or fusion rotein of the present invention under the condition that contains or do not contain IL-1 α.Collect supernatant liquor and use EIA test kit (Cayman, Ann Arbor, MI) mensuration PGE ₂For the IL-6 assay method, people's lung fibroblast was cultivated 1 day with 5,000 cells/well in 96 hole plates.After substratum is replaced by the 0.1%BSA basic medium, with cell and FGF-2 or insulation 24 hours under the condition that contains or do not contain albumin fusion proteins of the present invention and/or IL-1 α.Collect supernatant liquor and use ELISA test kit (Endogen, Cambridge, MA) mensuration IL-6.

Adding Alamar Blue, people's lung fibroblast was cultivated in basic medium 3 days with FGF-2 or albumin fusion proteins of the present invention with before assessing effect to fibroblastic growth.FGF-2 should show effect of stimulation at 10-2500ng/ml, and this can be used for and fusion rotein comparison stimulus effect of the present invention.

The cell proliferation of mixing based on [3H] thymidine

Below [3H] thymidine mix assay method and can be used for measuring therapeutic protein, for example growth factor protein is to such as inoblast, epithelial cell or the isocellular cultivation effect of prematurity muscle cell.

By in the substratum that does not contain serum, cultivating 18 hours, culture is converged in the Asia be arrested in the G1 stage.Add therapeutic protein then and reach 24 hours, and last 4 hours with [3H] thymidine mark culture, 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.Subsequently, cell is used the icy water rinsing subsequently with 10% ice-cold trichoroacetic acid(TCA) continuously.In 0.5M NaOH, after the dissolving, merge lysate and PBS rinsing liquid (500ml), and measure the amount of radioactivity.

Parkinson's model

Motor function forfeiture in the Parkinson's disease will be attributed to the striatum Dopamine HCL shortage of degenerating and causing because of nigrostriatum dopaminergic projection neuron.The animal model for parkinsonism of extensively identifying relates to systemic application 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).In CNS, MPTP is absorbed by stellate cell, and is 1-methyl-4-phenylpyridinium (MPP by monoamine oxidase B katabolism ⁺) and discharge.Subsequently, MPP ⁺Initiatively accumulation in dopaminergic neuron by the high-affinity re-uptake translocator of Dopamine HCL.MPP then ⁺In plastosome, concentrate and selectivity inhibition nicotinamide-adenine bisphosphate by electrochemical gradient: ubiquinone oxide-reductase enzyme (mixture 1), disturb electron transport and the final oxyradical that produces thus.

In the tissue culture example, confirmed FGF-2 (basic FGF) have nutritional activities at substantia nigra dopaminergic neuron (people such as Ferrari, Dev.Biol.1989).Recently; doctor's Unsicker team is verified uses FGF-2 with the gel foam implant and causes being close to and protect substantia nigra dopaminergic neuron to avoid exposing relevant toxicity (Otto and Unsicker with MPTP fully in striatum; J.Neuroscience, 1990).

Data according to FGF-2; albumin fusion proteins of the present invention can be assessed determining whether it has the similar effect in external enhancing dopaminergic neuron survival to FGF-2, but also the effect that dopaminergic neuron in the protection striatum avoids handling with MPTP relevant damage can be tested in vivo.At first at the external latent effect of in dopaminergic neuron cell cultures example, checking albumin fusion proteins of the present invention.In order to prepare culture, dissect the midbrain base plate from 14 days Wistar rat embryos of gestation.Use trypsinase disintegrated tissue, and with 200,000 cells/cm ²Be inoculated on the glass cover slide of polyorthinine-ln bag quilt.In EagleShi substratum that DulbeccoShi improves and the F12 substratum that contains hormonal supplementation thing (N1), keep cell.At external use paraformaldehyde fixed culture, and process for the immunohistochemical staining of tyrosine hydroxylase (special marking of dopaminergic neuron) after 8 days.Prepare dissociated cell culture from fetal rat.Substratum changed in per three days one, and added the described factor at that time.

Because dopaminergic neuron is isolating from 14 days animal of gestation, development time has at that time passed through the stage of dopaminergic precursor cell propagation, so the increase quantitatively of tyrosine hydroxylase immuning positive neuron will be represented the increase of the dopaminergic neuron quantity of external survival.Therefore, if therapeutic protein of the present invention plays the effect that prolongs the dopaminergic neuron survival, show that so this fusion rotein may relate to Parkinson's disease.

Embodiment 64: the pancreas beta cell is transplanted combination therapy

Transplanting is the common treatment form of autoimmune disease, especially when target autologous tissue has been badly damaged.For example, but not be limited to, transplantation of pancreas and islet cell transplantation are that the common treatment of IDDM is selected (for example to see people such as Stewart, Journal of Clinical Endocrinology ﹠amp; Metabolism 86 (3): 984-988,2001; Brunicardi, Transplant.Proc.28:2138-40,1996; Kendall and Robertson, Diabetes Metab.22:157-163,1996; People such as Hamano, Kobe J.Med.Sci.42:93-104,1996; Larsen and Stratta, Diabetes Metab.22:139-146,1996; Reach people such as Kinkhabwala, Am.J.Surg.171:516-520,1996).As any implantation method, autoimmunization patient's transplantation treatment comprises that the risk that the host is repelled transplanted tissue drops to minimum processing.Yet autoimmune disease comprises that relating to the host's autoimmune response that is pre-existing in that destroys initial autologous tissue will produce additional, the risk independently of identical damage effect to transplanted tissue.Therefore, the present invention is encompassed in and uses albumin fusion proteins combined immunization conditioning agent/immunosuppressor of the present invention to treat the method and composition of autoimmunity pancreatic disease in the individuality of the transplantation treatment of accepting autoimmune disease.

According to the present invention, use above-described composition and preparation and prevent and treat the individual initial infringement that transplant organ, tissue or cell is produced at the autoimmune response of former autologous tissue by the host based on the white protein fusions.Can and transplant the back before transplanting and carry out dispenser with each 2 to 4 dosage at a distance of a week.

Following immunomodulator/immunosuppressor includes but not limited to AI-401, CDP-571 (anti-TNF monoclonal antibody), CG-1088, Diamyd (vaccine for diabetes), ICM3 (anti-ICAM-3 monoclonal antibody), linomide (Roquinimex), NBI-6024 (the peptide part of change), TM-27, VX-740 (HMR-3480), Caspase 8 proteinase inhibitor, Thalidomide, hOKT3gammal (Ala-ala) (CD 3-resisting monoclonal antibody), oraferon-α, orally administering lactobacillus and LymphoStat-B ^TM, can in islet cells and transplantation of pancreas, use with white protein fusions therapeutical agent of the present invention.

The evaluation of embodiment 65:VH and VL structural domain and clone

A kind of method of identifying and cloning VH and VL structural domain from the clone of expressing specific antibodies is with VH and VL special primer the cDNA from the preparation of antibody expression clone to be carried out PCR.Briefly, from the clone isolation of RNA, and in the RT-PCR of antibody VH that is designed for amplification EBV expression of cell lines and VL structural domain, be used as template.Can use

Reagent (Life Technologies, Rockville.MD) dissolved cell, and with the chloroform extracting of 1/5th volumes.After adding chloroform, make solution in room temperature insulation 10 minutes, and in 4 ℃ in desk centrifuge 14, centrifugal 15 minutes of 000rpm.Collect supernatant liquor, and with isopyknic isopropanol precipitating RNA.By in desk centrifuge in 4 ℃ with 14, the centrifugal RNA that came settle precipitates in 15 minutes of 000rpm.After centrifugal, abandon supernatant liquor and also clean with 75% ethanol.After the cleaning, with RNA once more in 4 ℃ with 800rpm centrifugal 5 minutes.Abandon supernatant liquor and make the throw out dry air.Be dissolved in RNA in the DEPC water and be heated to 60 ℃ and reach 10 minutes.Can measure the amount of RNA by photodensitometry.

Can according to method well-known in the art from 1.5-2.5 microgram RNA with reversed transcriptive enzyme and at random the sexamer primer synthesize cDNA.Subsequently with the template of cDNA as pcr amplification VH and VL structural domain.The primer of VH and VL gene of being used to increase is shown in table 7.Be typically, single 5 ' primer and single 3 ' primer are used in the PCR reaction.Sometimes, when the limited amount of obtainable RNA template,, can use 5 ' and/or 3 ' primer in groups perhaps for higher efficient.For example, in single PCR reaction, use all five kinds of VH-5 ' primers and all JH3 ' primers sometimes.PCR is reflected in the 50 microlitre volumes of the high-fidelity Taq polysaccharase, 5 ' primer mixture, 3 ' primer mixture and the 7.5 microlitre cDNA that contain 1XPCR damping fluid, every kind of dNTP of 2mM, 0.7 unit and carries out.VH and VL the two 5 ' and 3 ' primer mixture can prepare by every kind of indivedual primers that merge 22pmole and 28pmole respectively.The PCR condition is: 96 ℃ 5 minutes; 94 ℃ 1 minute, 50 ℃ of 25 round-robin 1 minute and 72 ℃ are 1 minute subsequently; One was extended 72 ℃ of round-robin 10 minutes subsequently.After reaction is finished, sample hose is stored in 4 ℃.

Table 7: the primer sequence of be used to increase VH and VL structural domain

The primer title SEQ ID NO Primer sequence [5 '-3 ']

The VH primer

Hu?VH?1-5′ 62 CAGGTGCAGCTGGTGCAGTCTGG

Hu?VH?2-5′ 63 CAGGTCAACTTAAGGGAGTCTGG

Hu?VH?3-5′ 64 GAGGTGCAGCTGGTGGAGTCTGG

Hu?VH?4-5′ 65 CAGGTGCAGCTGCAGGAGTCGGG

Hu?VH?5-5′ 66 GAGGTGCAGCTGTTGCAGTCTGC

Hu?VH?6-5′ 67 CAGGTACAGCTGCAGCAGTCAGG

Hu?JH?1，2-5′ 68 TGAGGAGACGGTGACCAGGGTGCC

Hu?JH?3-5′ 69 TGAAGAGACGGTGACCATTGTCCC

Hu?JH?4，5-5′ 70 TGAGGAGACGGTGACCAGGGTTCC

Hu?JH?6-5′ 71 TGAGGAGACGGTGACCGTGGTCCC

The VL primer

Hu?Vkappa1-5′ 72 GACATCCAGATGACCCAGTCTCC

Hu?Vkappa2a-5′ 73 GATGTTGTGATGACTCAGTCTCC

Hu?Vkappa2b-5′ 74 GATATTGTGATGACTCAGTCTCC

Hu?Vkappa3-5′ 75 GAAATTGTGTTGACGCAGTCTCC

Hu?Vkappa4-5′ 76 GACATCGTGATGACCCAGTCTCC

Hu?Vkappa5-5′ 77 GAAACGACACTCACGCAGTCTCC

Hu?Vkappa6-5′ 78 GAAATTGTGCTGACTCAGTCTCC

Hu?Vlambda1-5′ 79 CAGTCTGTGTTGACGCAGCCGCC

Hu?Vlambda2-5′ 80 CAGTCTGCCCTGACTCAGCCTGC

Hu?Vlambda3-5′ 81 TCCTATGTGCTGACTCAGCCACC

Hu?Vlambda3b-5′ 82 TCTTCTGAGCTGACTCAGGACCC

Hu?Vlambda4-5′ 83 CACGTTATACTGACTCAACCGCC

Hu?Vlambda5-5′ 84 CAGGCTGTGCTCACTCAGCCGTC

Hu?Vlambda6-5′ 85 AATTTTATGCTGACTCAGCCCCA

Hu?Jkappa1-3′ 86 ACGTTTGATTTCCACCTTGGTCCC

Hu?Jkappa2-3′ 87 ACGTTTGATCTCCAGCTTGGTCCC

Hu?Jkappa3-3′ 88 ACGTTTGATATCCACTTTGGTCCC

Hu?Jkappa4-3′ 89 ACGTTTGATCTCCACCTTGGTCCC

Hu?Jkappa5-3′ 90 ACGTTTAATCTCCAGTCGTGTCCC

Hu?Jlambda?1-3′ 91 CAGTCTGTGTTGACGCAGCCGCC

Hu?Jlambda2-3′ 92 CAGTCTGCCCTGACTCAGCCTGC

Hu?Jlambda3-3′ 93 TCCTATGTGCTGACTCAGCCACC

Hu?Jlambda3b-3′ 94 TCTTCTGAGCTGACTCAGGACCC

Hu?Jlambda4-3′ 95 CACGTTATACTGACTCAACCGCC

Hu?Jlambda5-3′ 96 CAGGCTGTGCTCACTCAGCCGTC

Hu?Jlambda6-3′ 97 AATTTTATGCTGACTCAGCCCCA

Then with PCR sample electrophoresis on 1.3% sepharose.Can downcut the DNA band (the VH structural domain is about 506 base pairs, and the VL structural domain is 344 base pairs) of expection size from gel, and with method purifying well-known in the art.The PCR product of purifying can be connected to (from Invitrogen Inc., Carlsbad, the TA carrier of CA) in the PCR cloning vector.Can select the back to separate indivedual clones' PCR product at transfection Escherichia coli and indigo plant/white colour.The method order-checking that clone's PCR product generally can be known with this area then.

The PCR band that contains VH structural domain and VL structural domain also can be used for creating total length Ig expression vector.VH and VL structural domain can be cloned in the carrier of the nucleotide sequence that contains heavy chain (for example human IgG1 or human IgG 4) or light chain (people κ or people λ) constant region, make after transfection is in the appropriate host cell, can be from these vector expressions complete heavy chain or light chain molecule.In addition, when clone's heavy chain and light chain when all expressing in a clone (from a kind of or two kinds of carriers), they can be assembled into complete functional antibodies molecule, are secreted in the cell culture medium.The method that produces the expression vector of coding complete antibody molecule with the polynucleotide in coding VH and VL antibody structure territory is well-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 purpose cytokine or somatomedin such as NGF can separate by several different methods, comprises from the cDNA storehouse, and by RT-PCR with by adopting the PCR of a series of overlapping synthetic oligonucleotide primer things, all use standard method.All these proteinic nucleotide sequences are known and obtainable.CDNA can be 5 ' and 3 ' end prune to produce restriction site, to make and can use oligonucleotide joint that cDNA is cloned in the carrier of the cDNA that contains HA.This can be at N or C-end, uses or without the spacer sequence.NGF (or other cytokine) cDNA is cloned into such as in the carriers such as pPPC0005 (Fig. 2), pScCHSA, pScNHSA or pC4:HSA, then from wherein downcutting the expressed intact box and be inserted into the plasmid pSAC35, thereby can in yeast, express albumin fusion proteins.Can collect and purifying yeast excretory albumin fusion proteins from substratum then, and test its biologic activity.For the expression in mammal cell line, adopt similar program, be that used expression cassette adopts mammalian promoter, leader sequence and terminator (seeing embodiment 1).Downcut this expression cassette then and be inserted in the plasmid that is suitable for transfection mammalian cell system.

The preparation of embodiment 67:HA-IFN fusion rotein (such as IFNa)

The cDNA of purpose Interferon, rabbit such as IFNa can separate by several different methods, includes but not limited to from the cDNA storehouse, and by RT-PCR with by adopting the PCR of a series of overlapping synthetic oligonucleotide primer things, all use standard method.The nucleotide sequence of Interferon, rabbit such as IFNa is known and is obtainable, for example at United States Patent (USP) 5,326, and 859 and 4,588,585, EP 32 134 and such as in the public databases such as GenBank.CDNA can be 5 ' and 3 ' end prune to produce restriction site, to make and can use oligonucleotide joint that cDNA is cloned in the carrier of the cDNA that contains HA.This can be at N or C-end, uses or without the spacer sequence.IFNa (or other Interferon, rabbit) cDNA is cloned into such as in the carriers such as pPPC0005 (Fig. 2), pScCHSA, pScNHSA or pC4:HSA, then from wherein downcutting the expressed intact box and be inserted into the plasmid pSAC35, thereby can in yeast, express albumin fusion proteins.Can collect and purifying yeast excretory albumin fusion proteins from substratum then, and test its biologic activity.For the expression in mammal cell line, adopt similar program, be that used expression cassette adopts mammalian promoter, leader sequence and terminator (seeing embodiment 1).Downcut this expression cassette then and be inserted in the plasmid that is suitable for transfection mammalian cell system.

Maximum protein recovery from pipe

Albumin fusion proteins of the present invention has high stability, even when they are packed with lower concentration.In addition, although protein concn is low, the fusion rotein rate of recovery of still observing is reduced to minimum with combining of tube wall and during other protein of adding even do not contain at the aqueous solution in order to make.To compare with the recovery and the liquid storage of managing the HA-IFN solution of storing.6 or 30 μ g/ml HA-IFN solution are placed pipe and are stored in 4 ℃.After 48 or 72 hours, take out initial suitable volume and use the IFN sandwich ELISA to measure with the 10ng sample.Estimated value and high density liquid storage are compared.As what proved, the sample in these pipes is not loss in essence, illustrates in order to prevent sample optional such as white protein because of the loss of tube wall adds allogenic material.

The body internal stability and the bioavailability of HA-α-IFN fusions

In order to measure the body internal stability and the bioavailability of HA-α-IFN fusion molecule, monkey is used the fusion molecule (from yeast) of purifying.Pharmaceutical composition by HA-α-IFN fusions preparation can cause serum half-life and bioavailability to improve.Therefore, can prepare to compare and contain than the active pharmaceutical composition of low dosage alpha-interferon with natural alpha-interferon molecule.

The pharmaceutical composition that contains HA-α-IFN fusions is used in that suffer from can be by treatment or preventing disease among the patient who uses any disease that α-IFN regulates or morbid state.Such disease includes but not limited to hairy cell (hairy cell leukemia), Kaposi sarcoma (kaposi ' s sarcoma), sexual organ and anus wart, chronic hepatitis B, chronic non-A non-B hepatitis, third liver particularly, the fourth liver, chronic granulocytic leukemia, renal cell carcinoma, bladder cancer, ovary and cervical cancer, skin carcinoma, recurrent respirator papillomatosis (recurrent respirator papillomatosis), Fei Huoqijinshi (non-Hodgkin ' s) and skin T-cell lymphoma, melanoma, multiple myeloma, AIDS, multiple sclerosis, glioblastomas etc. (are seen Interferon Alpha, in " AHFS Drug Information ", 1997).

Therefore, the present invention includes the pharmaceutical composition that contains HA-α-IFN fusion rotein, polypeptide or peptide and be used for human administration with the correct dose preparation.The present invention also comprises the patient's of this treatment of treatment needs method, and it comprises at least one step of using the pharmaceutical composition that contains at least a HA-α-IFN fusion rotein, polypeptide or peptide.

Difunctional HA-α-IFN fusions

HA-α-IFN expression vector can be modified into and comprise the inset that is used to express difunctional HA-α-IFN fusion rotein.For example, can be after removing dual terminator codon or moving to the encoding sequence downstream, the downstream of the cDNA of second target protein being inserted " rHA-IFN " sequence in reading frame.

In a version of difunctional HA-α-IFN fusion rotein, can be with an end that merges at the antibody of B-LS thing protein (GenBank numbering 4455139) or polypeptide or fragment at the HA of fusion molecule composition.This bifunctional protein is useful to any immunne response that α-IFN composition of regulating by fusions produces.

The preparation of embodiment 68:HA-hormone fusion rotein

The cDNA of purpose hormone can separate by several different methods, includes but not limited to from the cDNA storehouse, and by RT-PCR with by adopting the PCR of a series of overlapping synthetic oligonucleotide primer things, all use standard method.All these proteinic nucleotide sequences are known and are obtainable, for example in such as public databases such as GenBank.CDNA can be 5 ' and 3 ' end prune to produce restriction site, to make and can use oligonucleotide joint that cDNA is cloned in the carrier of the cDNA that contains HA.This can be at N or C-end, uses or without the spacer sequence.Hormone cDNA is cloned into such as in the carriers such as pPPC0005 (Fig. 2), pScCHSA, pScNHSA or pC4:HSA,, thereby can in yeast, expresses albumin fusion proteins then from wherein downcutting the expressed intact box and be inserted into the plasmid pSAC35.Can collect and purifying yeast excretory albumin fusion proteins from substratum then, and test its biologic activity.For the expression in mammal cell line, adopt similar program, be that used expression cassette adopts mammalian promoter, leader sequence and terminator (seeing embodiment 1).Downcut this expression cassette then subsequently and be inserted in the plasmid that is suitable for transfection mammalian cell system.

The preparation of embodiment 69:HA-soluble receptors or the conjugated protein fusion rotein of HA-

Purpose soluble receptors or protein-bonded cDNA can separate by several different methods, include but not limited to from the cDNA storehouse, and by RT-PCR with by adopting the PCR of a series of overlapping synthetic oligonucleotide primer things, all use standard method.All these proteinic nucleotide sequences are known and are obtainable, for example in GenBank.CDNA can be 5 ' and 3 ' end prune to produce restriction site, to make and can use oligonucleotide joint that cDNA is cloned in the carrier of the cDNA that contains HA.This can be at N or C-end, uses or without the spacer sequence.Receptor cdna clone to such as in the carriers such as pPPC0005 (Fig. 2), pScCHSA, pScNHSA or pC4:HSA, then from wherein downcutting the expressed intact box and be inserted into the plasmid pSAC35, thereby can be expressed albumin fusion proteins in yeast.Can collect and purifying yeast excretory albumin fusion proteins from substratum then, and test its biologic activity.For the expression in mammal cell line, adopt similar program, be that used expression cassette adopts mammalian promoter, leader sequence and terminator (seeing embodiment 1).Downcutting this expression cassette then also inserts in the plasmid that is suitable for transfection mammalian cell system.

The preparation of embodiment 70:HA-somatomedin

The cDNA of purpose somatomedin can separate by several different methods, includes but not limited to from the cDNA storehouse, and by RT-PCR with by adopting the PCR of a series of overlapping synthetic oligonucleotide primer things, all use standard method (seeing GenBank numbering NP_000609).CDNA can be 5 ' and 3 ' end prune to produce restriction site, to make and can use oligonucleotide joint that cDNA is cloned in the carrier of the cDNA that contains HA.This can be at N or C-end, uses or without the spacer sequence.Somatomedin cDNA is cloned into such as in the carriers such as pPPC0005 (Fig. 2), pScCHSA, pScNHSA or pC4:HSA,, thereby can in yeast, expresses albumin fusion proteins then from wherein downcutting the expressed intact box and be inserted into the plasmid pSAC35.Collect and purifying yeast excretory albumin fusion proteins from substratum then, and test its biologic activity.For the expression in mammal cell line, adopt similar program, be that used expression cassette adopts mammalian promoter, leader sequence and terminator (seeing embodiment 1).Downcut this expression cassette then and be inserted in the plasmid that is suitable for transfection mammalian cell system.

The preparation of embodiment 71:HA-scfv fusion protein

Single-chain antibody prepares by several method, include but not limited to: from phage library, select, cDNA by clonal antibody also uses the flank constant region to clone the variable region of specific antibodies as primer clone variable region, perhaps by the synthetic oligonucleotide corresponding with the variable region of any specific antibodies.CDNA can be 5 ' and 3 ' end prune to produce restriction site, to make and can use oligonucleotide joint that cDNA is cloned in the carrier of the cDNA that contains HA.This can be at N or C-end, uses or without the spacer sequence.Cell cDNA is cloned into such as in the carriers such as pPPC0005 (Fig. 2), pScCHSA, pScNHSA or pC4:HSA,, thereby can in yeast, expresses albumin fusion proteins then from wherein downcutting the expressed intact box and be inserted into the plasmid pSAC35.

In fusion molecule of the present invention, VH and VL can make up by following a kind of method or its and be connected: V _HC-end and V _LThe N-end between peptide linker; V _HAnd V _LBetween Kex2p proteolytic enzyme cutting site, make the two can when secretion, cut and subsequently the oneself associate; And cystine residue, its position makes V _HAnd V _LCan between them, form disulfide linkage so that they are linked together.Selectable option is with V _HPlace the segmental N-of HA or HA structural domain terminal and with V _LPlace the segmental C-end of HA or HA structural domain.

Can collect and purifying yeast excretory albumin fusion proteins from substratum then, and test its biologic activity.For the expression in mammal cell line, adopt similar program, be that used expression cassette adopts mammalian promoter, leader sequence and terminator (seeing embodiment 1).Downcut this expression cassette then and be inserted in the plasmid that is suitable for transfection mammalian cell system.Zhi Bei antibody can be from the substratum purifying in this way, and tests it with the standard immunoassay chemical process and combine with antigenic.

The preparation of embodiment 72:HA-cell adhesion molecule fusion rotein

The cDNA of purpose cell adhesion molecule can separate by several different methods, includes but not limited to from the cDNA storehouse, and by RT-PCR with by adopting the PCR of a series of overlapping synthetic oligonucleotide primer things, all use standard method.The nucleotide sequence of known cell adhesion molecule is known and is obtainable, for example in GenBank.CDNA can be 5 ' and 3 ' end prune to produce restriction site, to make and can use oligonucleotide joint that cDNA is cloned in the carrier of the cDNA that contains HA.This can be at N or C-end, uses or without the spacer sequence.Cell adhesion molecule cDNA is cloned into such as in the carriers such as pPPC0005 (Fig. 2), pScCHSA, pScNHSA or pC4:HSA,, thereby can in yeast, expresses albumin fusion proteins then from wherein downcutting the expressed intact box and be inserted into the plasmid pSAC35.Collect and purifying yeast excretory albumin fusion proteins from substratum then, and test its biologic activity.For the expression in mammal cell line, adopt similar program, be that used expression cassette adopts mammalian promoter, leader sequence and terminator (seeing embodiment 1).Downcut this expression cassette then and be inserted in the plasmid that is suitable for transfection mammalian cell system.

Embodiment 73: supressor and peptide are (antibiotic such as HA-antiviral drug, HA-as the HA fusion rotein Medicine (antibiotic), HA-enzyme inhibitors and HA-resistance are answered albumen) preparation

The cDNA of purpose peptide such as antibacterial peptide can separate by several different methods, includes but not limited to from the cDNA storehouse, and by RT-PCR with by adopting the PCR of a series of overlapping synthetic oligonucleotide primer things, all use standard method.CDNA can be 5 ' and 3 ' end prune to produce restriction site, to make and can use oligonucleotide joint that cDNA is cloned in the carrier of the cDNA that contains HA.This can be at N or C-end, uses or without the spacer sequence.Peptide cDNA is cloned into such as in the carriers such as pPPC0005 (Fig. 2), pScCHSA, pScNHSA or pC4:HSA,, thereby can in yeast, expresses albumin fusion proteins then from wherein downcutting the expressed intact box and be inserted into the plasmid pSAC35.Collect and purifying yeast excretory albumin fusion proteins from substratum then, and test its biologic activity.For the expression in mammal cell line, adopt similar program, be that used expression cassette adopts mammalian promoter, leader sequence and terminator (seeing embodiment 1).Downcut this expression cassette then and be inserted in the plasmid that is suitable for transfection mammalian cell system.

Embodiment 74: the preparation of target HA fusion rotein

The cDNA of target protein matter can use the standard molecular biology method to separate from the cDNA storehouse or can prepare with several overlapping oligonucleotide are synthetic.The suitable Nucleotide that can transform among the cDNA is attached to white protein cDNA to form easily restriction site and to allow with protein cDNA.Equally, can with can be in cell targeting proteins matter or peptide cDNA such as the single-chain antibody or the peptide of pilot protein matter, merge at the albuminised the other end such as nuclear localization signal.Target protein matter and target peptide are cloned into such as in the carriers such as pPPC0005 (Fig. 2), pScCHSA, pScNHSA or pC4:HSA, thereby cause fusion with white protein cDNA.In this mode, two ends of albuminised N-and C-all merge other protein.From pPPC0005, cut out the cDNA of fusion then and be inserted into, thereby can in yeast, express albumin fusion proteins such as in the plasmids such as pSAC35.All said procedures can use molecular biological standard method to carry out.Can collect and purifying yeast excretory albumin fusion proteins from substratum, and use suitable biochemistry and biology method of testing to test its biologic activity and its target activity.

The preparation of embodiment 75:HA-enzyme fusions

The cDNA of purpose enzyme can separate by several different methods, includes but not limited to from the cDNA storehouse, by RT-PCR with by using the PCR of a series of overlapping synthetic oligonucleotide primer things, all uses standard method.CDNA can be 5 ' and 3 ' end prune to produce restriction site, to make and can use oligonucleotide joint that cDNA is cloned in the carrier of the cDNA that contains HA.This can be at N or C-end, uses or without the spacer sequence.Enzyme cDNA is cloned into such as in the carriers such as pPPC0005 (Fig. 2), pScCHSA, pScNHSA or pC4:HSA,, thereby can in yeast, expresses albumin fusion proteins then from wherein downcutting the expressed intact box and be inserted into the plasmid pSAC35.Can and test its biologic activity from substratum collection and purifying yeast excretory albumin fusion proteins then.For the expression in mammal cell line, adopt similar program, be that used expression cassette adopts mammalian promoter, leader sequence and terminator (seeing embodiment 1).Downcut this expression cassette then and be inserted in the plasmid that is suitable for transfection mammalian cell system.

Embodiment 76: construction ID 2249, the generation of IFNa2-HSA

Construction ID 2249, pSAC35:IFNa2.HSA comprises the DNA of coding IFNa2-albumin fusion proteins in yeast saccharomyces cerevisiae expression vector pSAC35, this fusion rotein has the chimeric leader sequence of HSA, then be that the proteinic mature form of IFNa2 is C1-E165, merge N-terminal at the HSA mature form.

The clone of IFNa2cDN4

Use the polynucleotide of primer I FNa2-1 and IFNa2-2PCR amplification coding IFNa2 hereinafter described.With SalI/Cla I cutting pcr amplification thing, and be connected in the pScCHSA of Xho I/Cla I cutting.The construction ID #2249 albumin fusion proteins of encoding, it contains the mature form of chimeric HSA leader sequence, IFNa2, and the back is ripe HSA protein.

Synthetic two oligonucleotide that are suitable for the polynucleotide of pcr amplification coding IFNa2 mature form, IFNa2-1 and IFNa2-2:

IFNa2-1：5′-CGCGCGC GTCGACAAAAGATGTGATCTGCCTCAAACC

CACA-3’(SEQ?ID?NO：348)

IFNa2-2：5′-GCGCGC ATCGATGAGCAACCTCACTCTTGTGTGCATCT

TCCTTACTTCTTAAACTTTCT-3′(SEQ?ID?NO：349)

The IFNa2-1 primer mixes Nucleotide, and 22 Nucleotide (runic demonstration) of 7 amino-acid residues of coding IFNa2 mature form of last three amino-acid residues of Sal I cloning site (underscore demonstration), the chimeric HSA leader sequence of coding.In IFNa2-2, Cla I site (underscore demonstration) and subsequent DNA are the reverse complementary sequence of proteinic 10 amino acid whose DNA of encoding mature HSA, and 22 last Nucleotide (runic demonstration) are the reverse complementary sequence (seeing embodiment 2) of the DNA of last 7 amino-acid residues of coding IFNa2.With the pcr amplification thing of these primers generations IFNa2-HSA, purifying,, and be cloned among the Xho I and Cla I site of pScCHSA carrier with Sal I and the digestion of Cla I restriction enzyme.After confirming sequence, with the expression cassette subclone of this IFNa2-albumin fusion proteins of coding in the pSAC35 of NotI digestion.

In addition, can confirm exist (the seeing below) of the IFNa2 sequence of expecting to the analysis of expressed albumin fusion proteins N-end by amino acid sequencing.

Made up other IFNa2-albumin fusion proteins (seeing embodiment 2) that adopts different leader sequences by methods known in the art.The example of various leader sequences includes but not limited to saccharase " INV " (construction 2343 and 2410) and mating alpha factor " MAF " (construction 2366).Can (seeing embodiment 5) as discussed previously these IFNa2-albumin fusion proteins subclones be arrived such as in the mammalian expression vectors such as pC4 (construction 2382) and pEE12.1.Can also make up the IFNa2-albumin fusion proteins (construction 2381) of therapeutic meromixis in HSA C-end.

The mature form that IFNa2-albumin fusion proteins of the present invention preferably comprises HSA is Asp-25 to Leu-609, and the mature form that it merges at IFNa2 is N-or the C-end of Cys-1 to Glu-165.In one embodiment of the invention, IFNa2-albumin fusion proteins of the present invention also is included in the signal sequence of the host's who is used for expressing the initial fusion polypeptide of Secretory Pathway guiding.In another preferred embodiment, excised by the signal peptide of signal sequence encoding, and sophisticated IFNa2-albumin fusion proteins direct secretion is in substratum.IFNa2-albumin fusion proteins of the present invention can comprise the allos signal sequence, includes but not limited to that MAF, INV, Ig, fine protein B, bunch albumen (clusterin), IGFBP4, HSA leader sequence variant include but not limited to chimeric HSA/MAF leader sequence or other allos signal sequence known in the art.In a preferred embodiment, IFNa2-albumin fusion proteins of the present invention comprises natural IFNa2.In another preferred embodiment, IFNa2-albumin fusion proteins of the present invention also comprises the terminal methionine residues of N-.The polynucleotide of these polypeptide of encoding are also contained in the present invention, comprise fragment and/or variant.

The expression of construction ID 2249 and purifying

In Expression in Saccharomyces Cerevisiae

By methods known in the art construction 2249 is transformed among the Wine brewing yeast strain BXP10 and (sees embodiment 3).Can collect the cell of steady stage in growth after 72 hours.By cell was collected supernatant liquor in centrifugal 10 minutes with 3000g.Use anti-HSA serum (Kent Laboratories) or check expression level as anti-a detection by immunoblotting.Can obtain the IFNa2-albumin fusion proteins of the about 88.5kDa of molecular weight.

From brewing yeast cell supernatant liquor purifying

The cell conditioned medium liquid that contains the IFNa2-albumin fusion proteins of being expressed by construction ID#2249 in brewing yeast cell can use Dyax peptide affinity column to carry out (seeing embodiment 4) on a small scale, perhaps carries out large scale purification by following 5 steps: diafiltration, the anion-exchange chromatography that carries out with DEAE-Sepharose Fast Flow post, the hydrophobic interaction chromatography (HIC) that carries out with Butyl 650S post, the cation-exchange chromatography or the Blue-Sepharose chromatography that carry out with SP-Sepharose Fast Flow, and the efficient chromatography (seeing embodiment 4) that carries out with the Q-Sepharose high performance column.The IFNa2-albumin fusion proteins can from DEAE-Sepharose Fast Flow post with 100-250mM NaCl, from SP-SepharoseFast Flow post with 150-250mM NaCl with at 5-7.5mS/em wash-out from the Q-Sepharose high performance column.The N-end sequencing should obtain the sequence C DLPQ consistent with the IFNa2 mature form (SEQ IDNO:98).

The activity of IFNa2 can be measured with external ISRE-SEAP assay method

Method

Can as described in previous embodiment 76, in the ISRE-SEAP assay method, test activity to the IFNa2-albumin fusion proteins of encoding by construction ID #2249.Briefly, condition yeast supernatant liquor is tested it instructs the ISRE signal transduction on the ISRE-SEAP/293F reporting cell line ability with 1: 1000 extent of dilution.ISRE-SEAP/293F is reported that cell handling the day before yesterday with 3 * 10 ⁴Cells/well is assigned in 96 hole plates of poly--D-Methionin bag quilt.To report cell insulation 18 to 24 hours then, take out 40 μ l subsequently, be used for SEAP reporter gene chemiluminescence assay (Roche products catalogue numbering 1779842).Use recombinant human interferon beta, " rhIFNb " (Biogen), as positive control.

The result

The purifying prepared product of IFNa2-HSA in the ISRE-SEAP assay method in concentration range 10 ^-1To 10 ¹Ng/ml (see figure 4) or 10 ^-10To 10 ^-8Show linear relatively increase on the ng/ml (see figure 5).

Induce in the body of interferon alpha fusions by construction ID 2249 codings OAS

Method

The OAS enzyme, 2 '-5 '-oligoadenylate synthetase activates at transcriptional level by Interferon, rabbit in replying anti-virus infection.The effect of Interferon, rabbit construction can obtain blood sample by the monkey of handling from acceptance and to two kinds of OAS mRNA of these sample analysis, the transcriptional activation of p41 and p69 is measured.From 7 different time points of every group of 4 animals each animal, the 0th day, the 1st day, the 2nd day, the 4th day, the 8th day, the 10th day and the 14th day obtain the whole blood of 0.5ml volume.Different groups comprises vehicle contrast, intravenous injection in the 1st day 30 μ g/kg HSA-IFN, subcutaneous injection in the 1st day 30 μ g/kg HSA-IFN, subcutaneous injection in the 1st day 300 μ g/kg HSA-IFN, reaches subcutaneous injection in the 1st, 3 and 5 day 40 μ g/kg interferon alphas (Schering-Plough) as positive control.The level of p41 and p69mRNA transcript is used p41-OAS and the special probe of p69-OAS is measured by real-time quantitative PCR (Taqman).OAS mRNA level is carried out quantitatively with respect to the endogenous contrast of 18S ribosome-RNA(rRNA).White protein by construction 2249 codings merges and can similarly test.

The result

In the monkey of handling, observe the remarkable increase of the mRNA transcript degree of p41 and two kinds of OAS of p69, form contrast (the p41 data are seen Fig. 6) with the monkey of handling with IFNa with the HSA-Interferon, rabbit.This effect continues nearly 10 days.

The indication of embodiment 77:IFNa2-albumin fusion proteins

IFN α-albumin fusion proteins (include but not limited to by construction 2249,2343,2410,2366,2382 and 2381 codings those) can be used for treatment, prevention, improves and/or detects multiple sclerosis.Other indication includes but not limited to virus infection, comprises severe acute respiratory syndrome (SARS) and other coronavirus infection; Filovirus (filovirus) includes but not limited to Ebola virus (Ebolavirus) and Marburg virus (Marburg virus); Arenavirus (Arenavirus) includes but not limited to 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 (Puntatoro virus), the Crimea peninsula-Congo hemorrhagic fever virus (Crimean-Congo hemorrhagic fevervirus), phlebotomus fever virus (sandfly fever virus), Rift valley fever virus (Rift Valley fevervirus), La Crosse virus (La Crosse virus) and Hantaan virus (hantavirus); Flavivirus (Flavivirus) includes but not limited to yellow jack, BAN (Banzi virus), west nile virus (WestNile virus), dengue fever virus, japanese encephalitis virus, tick encephalitis, Omsk hemorrhagic fever (OmskHemorrhagic fever) and Kyasanur Forest disease virus (Kyasanur Forest Disease virus); Togavirus (Togavirus) includes but not limited to Venezuela, east and western equine encephalitis virus, ross river virus (Ross River virus) and rubella virus; Vaccinia subgroup virus includes but not limited to bovine vaccine, cowpox, smallpox and monkeypox; Simplexvirus; Influenza A/B; Respiratory syncytial virus (RAV); Parainfluenza; Measles; Rhinovirus; Adenovirus; Semliki Forest virus (Semliki Forest virus); Viral hemorrhagic fever; Baculovirus; Paramyxovirus includes but not limited to Nipah virus (Nipah virus) and Heng Dela virus (Hendra virus); And (be A, B and the C class factor by other virokine that U.S. CDC is defined as the disease factor of high-priority; See for example Moran, Emerg.Med.Clin.North.Am.20 (2): 311-30,2002 and people such as Darling, Emerg.Med.Clin.NorthAm.20 (2): 273-309,2002).

Preferably, IFNa-albumin fusion proteins or IFN hybrid fusion rotein and CCR5 antagonist combination are used, also can or unite the inverase therapy again with at least a associating in ribavirin (ribavirin), IL-2, IL-12, the pentafuside (pentafuside), for example HAART is used for preparing and was not accepting treatment and accepting the adult of treatment and the medicine of pediatric patient treatment HIV-1 infection, HCV or HIV-1 and HCV coinfection.

Embodiment 78: construction #3691, the generation of BNP-HSA

Construction ID#3691, pC4:SPCON.BNP1-32/HSA comprises the DNA of coding BNP-albumin fusion proteins in mammalian expression vector pC4, it has total leader sequence, secrecon, the back is fusion is processed in the aminoterminal process of HSA mature form, activated BNP peptide (amino acid/11-32).

The clone who is used for the BNP cDNA of construction 3691

Use the polynucleotide of primer BNP-1 and BNP-2PCR amplification coding BNP hereinafter described,, and be connected in the pC4:HSA of Bam HI/Cla I cutting, produce construction ID #3691 with Bam HI/Cla I cutting.The construction ID #3691 albumin fusion proteins of encoding, it contains total leader sequence (SEQ ID NO:111) and through the BNP of the activity form of processing, the back is sophisticated HSA protein (seeing Table the SEQ ID NO:204 of construction 3691 in 2).

Synthetic be suitable for the pcr amplification coding activated, through two Nucleotide of the polynucleotide of the BNP of form processing, BNP-1 and BNP-2:

BNP-1：5′-GAGCGC GGATCCAAGCTTCCGCCATCATGTGGTGGCGC

CTGTGGTGGCTGCTGCTGCTGCTGCTGCTGCTGTG

GCCCATGGTGTGGGCCAGCCCCAAGCTGGTGCAAGG -3′

(SEQ?ID?NO：364)

BNP-2：5′-AGTCCC ATCGATGAGCAACCTCACTCTTGTGTGCATCA

TGCCGCCTCAGCACTTTGC-3′(SEQ?ID?NO：365)

BNP-1 mixes the polynucleotide (runic) of 7 aminoacid sequences of the polynucleotide (italic) of Bam HI cloning site (underscore), the total leader sequence (SEQ ID NO:111) of coding and the BNP that encodes.In BNP-2, the underscore sequence is Cla I site, and the polynucleotide of its back contain encode last 6 amino acid (runic) of BNP and the reverse complementary sequence of proteinic 10 amino acid whose DNA of ripe HSA.Use this two kinds of primer PCR amplification BNP protein.Must rule of thumb be that every pair of special primer and template are determined annealing and elongating temperature and time.

Purified pcr product (for example uses Wizard PCR Preps dna purification system; Promega company), then with Bam HI and Cla I digestion.After being further purified the BamHI-ClaI fragment, product cloning in the pC4:HSA of Bam HI/Cla I digestion, is produced construction ID#3691 by gel electrophoresis.The sequence of checking expression constructs.

The expression of construction ID 3691 and purifying

Expression in the 293F cell

With construction ID #3691, the pC4:SPCON.BNP1-32/HSA transfection (is seen embodiment 6) in the 293F cell by methods known in the art.

From 293F cell conditioned medium liquid purifying

Collected 2 in 3 days after the transfection and go up clear liquid.(NJ USA) catches and with 2M NaCl wash-out recombinant protein for Amersham Biosciences, Piscataway with 5ml Blue Sepharose CL-6B post.Material is attached on HiPrep 16/10 Phenyl FF (high sub) post also with 20mM MES pH6.7 wash-out.Be further purified BNP-HSA at pH6.8 with sodium phosphate buffer gradient (0-20mS/cm among the 200ml) by the hydroxylapatite column chromatography.By HiPrep 26/10 desalting column (AmershamBiosciences) final product is transformed among the PBS pH7.2.

The activity of BNP-HSA can be measured with external NPR-A/cGMP assay method

Natriuratic peptide receptor-A (NPR-A) is the frizzled receptor of BNP, thereby most of biological actions of responsible BNP.The biological activity of BNP is to be mediated by the NPR-A guanylate cyclase structural domain that GTP is transformed into cGMP when activating.The active assay method that makes things convenient for of BNP is to measure the BNP hormesis of stablizing the 293F clone of expressing NPR-A.Can be exposed to that the cGMP in the cell generates behind the BNP by cGMP ELISA measurement.

The screening method of NPR-A 293F stable clone

The opening code-reading frame of people NPR-A is building up in the pcDNA3.1 expression vector (Invitrogen)., and select with plasmid DNA stable transfection 293F cell by fat transfection amine reagent (Lipofectamine) method with 0.8 μ g/ml G418.To the preferably response of 293F/NPR-A stable clone screening to reorganization BNP.

The measurement of cGMP activation

BNP carries out in the 293F/NPR-A cell the activation of cGMP, and (CA USA) measures for Molecular Devices, Sunnyvale to use CatchPoint cGMP fluorometric assay test kit.Briefly, the 293F/NPR-A cell cultivated with 50,000 cells/well in the 96 hole plates is stimulated damping fluid (contain 10mM glucose pH7.4,15nM sodium bicarbonate, and the Krebs-Ringe bicarbonate buffer of 0.75mM 3-isobutyl-1-methylxanthine) cleaning in advance with 80 μ l.Stimulate the BNP-HSA in the damping fluid or the BNP that recombinates to join in advance 40 μ l and reach 10 minutes in the cell in 37 ℃.Cell is rocked dissolving 10 minutes with 40 μ l dissolving damping fluid.Measure the amount of cGMP in the lysate according to the indication of manufacturers.

The result

Measured dosage-response relation (see figure 7) of BNP-HSA and reorganization BNP.The maximum activity of construction ID#3691 and reorganization BNP is that similar (be respectively 1.63 ± 0.016 and 1.80 ± 0.016pm), the EC50 value is respectively 28.4 ± 1.2 and 0.46 ± 1.1nM.

BNP-HSA brings high blood pressure down in vivo

Method

BNP brings high blood pressure down by direct vasorelaxation with by suppressing feritin/Angiotensin/endothelin/aldosterone system.In that (test b NP-HSA reduces the ability of arteriotony in the male spontaneous hypertensive rat of 3 monthly ages USA) for Germantown, NY available from Taconic.Spontaneous hypertensive rat is an essential hypertension, and hypertension was shown effect after 3 monthly ages.Reconstruct in 0.3cc PBS gives every rat with BNP-HSA or reorganization BNP.Medicine is by the tail vein injected delivery.(CT is USA) by cover tail method record heart contraction and diastolic blood pressure for Kent Scientific, Torrington to use the XBP-1000 system.For each blood pressure data point, gather 4-5 continuous-reading and average.Press with 1/3 systolic pressure+2/3 diastole and to calculate mean arterial pressure (MAP).For dosage-reply mensuration, with 0.5,2,6 and the dosage of 18nmol/kg measured blood pressure in 20 hours after using pC4:SPCON.BNP1-32/HSA.

The result

The typical heart systolic pressure of spontaneous hypertensive rat is 180-200mmHg before administration.The single 6nmol/kg BNP-HSA that injects that delivers through the tail vein injection reduces heart contraction and two kinds of blood pressures of diastole, reaches mean arterial pressure (MAP) and reduces above 30mmHg.The blood pressure stabilization that reduces also continues 1 day, returns to the baseline (see figure 8) then in several days gradually.On the contrary, because its moment removes, the single 6nmol/kg of reorganization BNP injects the very of short duration MAP that only produces about 15mmHg and reduces.

In addition, in 4 spontaneous hypertensive rats, measured bolus infusion BNP-HSA after 20 hours dosage-reply.0.5nmol/kg BNP-HSA has the MAP of average 7mmHg to reduce, and 6nmol/kg BNP-HSA has the MAP of average 30mmHg to reduce, the amplitude that the BNP-HSA of 18nmol/kg high dosage brings high blood pressure down is only slightly more than 6nmol/kg.

Induce in the body of BNP-HSA to blood plasma cGMP

Method

The intracellular cGMP activation of BNP causes it to be discharged into the circulation from cell.Blood plasma cGMP level is cardiovascular relevant with kidney physiology with the BNP inductive.Blood plasma cGMP has been used as the biomarker of BNP effect in the body.For test b NP-HSA in vivo to the inducing of blood plasma cGMP, to 11-12 age in week male C57/BL6 mouse deliver reorganization BNP or BNP-HSA through tail vein by single injecting with the dosage of 6nmol/kg.Prepare blood plasma 5,10,20,40 and 80 minutes time points of reorganization BNP administration group and BNP-HSA group extra 640,1440,2880 and 5760 minutes from tail blood.The plasma sample of collecting the mouse of handling with PBS at the zero-time point contrasts as vehicle.Use CatchPoint cGMP fluorometric assay kit measurement cGMP level according to the indication of manufacturers.

The result

Inject 6nmol/kg reorganization BNP or BNP-HSA in azygos vein after, the peak value blood plasma cGMP level that surpasses baseline has increased by 3.9 times or 5.6 times of (see figure 9)s respectively.In addition, in the single-phase exponential decay transformation period (one-pHSAe exponential decayhalf-life) of handling back cGMP with reorganization BNP is 16 minutes (10 to 42 minutes, 95%CI), and after using BNP-HSA the single-phase exponential decay transformation period of cGMP be 1538 minutes (1017 to 3153 minutes, 95%CI).

By Pharmacokinetic analysis in the body of the BNP-white protein fusions of construction ID 3691 codings

Method

Male C57/BL6 mouse in 11-12 age in week (from Ace Animals, Boyertown, PA, USA acquisition) is 25.1 ± 0.12g in the search time body weight.All animals are with the volume administration of 10ml/kg body weight.Give pre-administration animal injection PBS.Reorganization BNP peritoneal injection is to afterbody or be subcutaneously injected into the omoplate region intermediate.

Following group is carried out pharmacokinetic analysis:

Table 8

Group	Medicine	Dosage (mg/kg)	Approach	N/ time	Time (hour)
						1	BNP-HSA	2.19	Subcutaneous	3	0.5、2、6、16、24、32、48、 72、96
2	BNP-HSA	2.19	Intravenously	3	0.083、2、6、16、24、32、 48、72、96
						3	Vehicle	0	Subcutaneous	3	Pre-administration (predose)
4	Vehicle	0	Intravenously	3	Pre-administration

From the postcava blood sample collection, place miniature vessel, and be stored in the ice through EDTA bag quilt.With sample in Eppendorf centrifuge in room temperature with 14,000rpm (16,000xg) centrifugal 10 minutes.Transfer to bunch blood plasma in the pipe (cluster tube) and be stored in-80 ℃.

Use BNP EIA test kit (Phoenix Pharmaceutical, Belmont, CA, USA) the BNP-HSA concentration in the mensuration plasma sample.On the plate identical, produce typical curve in the identical time with specimen.The limit of detection of reorganization BNP is 0.11ng/ml.This assay method detect reorganization BNP and not with mouse BNP cross reaction.

By non-compartment method (WinNonlin; 4.1 version; Pharsight Corp., Mountain View, CA USA) analyzes.Use the mean plasma concentration of each time in the analysis.Use linear rising/logarithm decline trapezoidal method to calculate AUC _0-tCarry out infinitely great AUC with last observed concentration divided by the terminal elimination rate constant _0-∞Extrapolation.Data in these analyses are unified weighting.

The result

Before administration in the sample in the detected blood plasma BNP-HSA average baselining concentration be about 0.081-0.095 μ g/ml.In the azygos vein or after the subcutaneous injection, BNP-HSA has the terminal of 11.2 (intravenously deliveries) or 19.3 hours (subcutaneous delivery) to eliminate the transformation period (terminal elimination half-life), and the reorganization transformation period of BNP in mouse is 3.1 minutes.The non-compartment analysis (noncompartmental analysis) of BNP-HSA has disclosed BNP-HSA and has had following feature:

Table 9

4 points that are chosen in 5 points of intravenously curve terminal stage and subcutaneous curve terminal stage are used for the calculating of final transformation period.The AUC of this terminal stage that so obtains is respectively about 10% of intravenously and the total AUC of subcutaneous curve.By comparison, when selecting last 3 points to be used for the calculating of final transformation period, 2% and 4% of intravenously and the total AUC of subcutaneous curve only arranged respectively.

Embodiment 79: construction ID #3618, the generation of BNP (2X)-HSA

Construction ID #3618, pC4:SPCON.BNP1-32 (2x)/HSA comprises the DNA of coding BNP-albumin fusion proteins in mammalian expression vector pC4, it has total leader sequence, secrecon, the back is the polyphone fusion is processed in aminoterminal two processes of HSA mature form, activated BNP peptide (amino acid/11-32).

The clone who is used for the BNP cDNA of construction 3618

At first use hereinafter described four kinds of primer BNP-1, BNP-2, BNP-3 and BNP-4 polynucleotide, to produce two Segment A and B from the dual BNP module of encoding through activity form BNP (amino acid/11-32) pcr amplification of processing.After the amplification, two purifying fragments (A and B) are mixed with equimolar amount, and as pcr template, with primer BNP-5 and BNP-6 amplification hereinafter described.Digest BNP (2x) inset with BamHI and Cla I then, and be connected in BamHI and the predigested pC4:HSA carrier of ClaI, produce construction ID # 3618.Construction ID # 3618 albumin fusion proteins of encoding, it contains total leader sequence, secrecon, (SEQ ID NO:111), with two tandem copies of the activity form BNP that passes through processing, the back is ripe HSA protein (seeing Table the SEQ ID NO:226 of construction 3618 in 2).

At first synthetic four oligonucleotide that are suitable for two segmental polynucleotide of pcr amplification coding BNP protein:

BNP-1 5′AGCCCCAAGATGGTGCAAGGGTCTGGCTGCTTTGGGAG

GAAGATGGACCGGATCAGCTCCTCCAGTGGCTGGGCTGC

AAAGTGCTGAGGCGGCAT-3′(SEQ?ID?NO：460)

BNP-2 5′-CCTTGCACCATCTTGGGGCTATGCCGCCTCAGCACTTT

GC-3′(SEQ?ID?NO：461)

BNP-3 5′-GCAAAGTGCTGAGGCGGCATAGCCCCAAGATGGTGCA

AGG-3′(SEQ?ID?NO：462)

BNP-4 5′-AGTCCCATCGATGAGCAACCTCACTCTTGTGTGCATCA

TGCCGCCTCAGCACTTTGC-3′(SEQ?ID?NO：463)

Use primer sets BNP-I/BNP-2 and BNP-3/BNP-4, two BNP protein fragments of pcr amplification (being respectively A and B).Must rule of thumb be that every pair of special primer and template are determined annealing and elongating temperature and time.Purifying Segment A and B (for example use Wizard PCR Preps dna purification system; Promega Corp), mix, and be used to use two the oligonucleotide BNP-5 in addition that are suitable for pcr amplification and the pcr amplification of BNP-6 as template with equimolar amount:

BNP-5 5′-GAGCGC GGATCCAAGCTTCCGCCATCA?TGTGGTGGCGC

CTGTGGTGGCTGCTGCTGCTGCTGCTGCTGCTGTGGCCCAT

GGTGTGGGCCAGCCCCAAGCTGGTGCAAGG-3′(SEQ ID

NO：382)

BNP-6 5′-AGTCCC ATCGATGAGCAACCTCACTCTTGTGTGCATCA

TGCCGCCTCAGCACTTTGC-3′(SEQ?ID?NO：383)

BNP-5 mixes the polynucleotide (runic) of preceding 7 aminoacid sequences of the polynucleotide (italics) of Bam HI cloning site (underscore), the total leader sequence (SEQ ID NO:111) of coding and the BNP that encodes.In BNP-6, the underscore sequence is Cla I site, and the polynucleotide of its back contain encode last 6 amino acid (runic) of BNP and the reverse complementary sequence of proteinic 10 amino acid whose DNA of ripe HSA.Use this two kinds of primers, two tandem copies of total leader sequence of pcr amplification and active BNP peptide.Must rule of thumb be that every pair of special primer and template are determined annealing and elongating temperature and time.

Purified pcr product (for example uses Wizard PCR Preps dna purification system; Promega company), then with Bam HI and Cla I digestion.After being further purified the BamHI-ClaI fragment, product cloning in the pC4:HSA of Bam HI/Cla I digestion, is produced construction ID#3618 by gel electrophoresis.The sequence of checking expression constructs.

The expression of construction ID 3618 and purifying

Expression in the 293F cell

With construction ID #3618, pC4:SPCON.BNP1-32 (2x)/HSA transfection (is seen embodiment 6) in the 293F cell by methods known in the art.

From 293F cell conditioned medium liquid purifying

As before above among the embodiment 78 subtitle " from 203F cell conditioned medium liquid purifying " down as described in purifying by pC4:SPCON.BNP1-32 (the 2x)/HSA of construction ID#3618 coding.

The activity of BNP (2X)-HSA can be measured with external NPR-A/cGMP assay method

Can be by the activity of BNP (the 2X)-HSA of construction ID#3618 coding as passing through the NPR-A/cGMP assay method at external test at subtitle " activity of BNP-HSA can be measured with external NPR-A/cGMP assay method " and " screening method of NPR-A 293F stable clone " as described in down among the previous embodiment 78.

The result

Measured dosage-response relation (see figure 7) of BNP (2X)-HSA and reorganization BNP.By the maximum activity of BNP (the 2X)-HSA of construction ID#3618 coding and the BNP that recombinates is that similar (be respectively 1.68 ± 0.021 and 1.80 ± 0.016pm), the EC50 value is respectively 9.8 ± 1.1 and 0.46 ± 1.1nM.

The external NPR-A/cGMP assay method of embodiment 80:BNP

Background or method

Natriuretic peptide receptor-A (NPR-A) is the frizzled receptor of BNP, therefore, is responsible for most of biological effects of BNP.The BNP biological activity is by the mediation of NPR-A amidino groups/guanylate cyclase (guanylyl cyclase) structural domain, and it is transformed into cGMP with GTP after activation.The active a kind of convenient assay method of BNP is to measure the BNP that stablized the 293F clone of expressing NPR-A to stimulate.The cGMP of cell after being exposed to BNP generates and can measure by cGMP ELISA.

The method of screening NPR-A 293F stable clone

The open reading-frame (ORF) of people NPR-A is built into pcDNA3.1 expression vector (Invitrogen).Select with plasmid DNA stable transfection 293F cell and by 0.8 μ g/ml G418 by the Lipofectamine method.To 293F/NPR-A stable clone screening preferably replying to reorganization BNP.

The cGMP activatory is measured

The cGMP that carries out being caused by BNP in the 293F/NPR-A cell activates and (CA USA) measures for Molecular Devices, Sunnyvale by CatchPoint cyclo GMP fluorometry test kit.In brief, 50, the 000 cells/well 293F/NPR-A cells of cultivating in 96 orifice plates are cleaned into 80 μ l stimulate damping fluid (Krebs-Ringer bicarbonate buffer in advance, contain 10mM glucose, pH 7.4,15nM sodium bicarbonate and 0.75mM 3-isobutyl-1-methylxanthine).Stimulate BNP-HSA or reorganization BNP in the damping fluid to add to cell in advance 40 μ l, in 37C insulation 10 minutes.Cell was shaken cracking 10 minutes with 40 μ l lysis buffers.According to the indication of manufacturers quantitatively and measure the EC50 value to the amount of cGMP in the lysate.In this assay method, high cGMP level causes low signal (relative fluorescence unit or RFU).

The generation of construction ID #3 796

Construction ID # 3796, pSAC35:HSA.BNP (1-32), the DNA that comprises coding BNP albumin fusion proteins, described fusion rotein has the HSAsp/KEX2 leader sequence, then be that it merges to the N-end through BNP peptide (amino acid/11-32) processing, mature form in Yeast expression carrier pSAC35 (construction 3796 in the table 2 is seen SEQ ID NO:214) through HSA processing, mature form.

The clone who is used for the BNP cDNA of construction 3796

Use the hereinafter polynucleotide of primer BNP-102689 and BNP-102692PCR amplification coding BNP,, and connect into pSAC-NEC, produce construction ID # 3796 through the Bsu36I/AscI cutting with the Bsu36I/AscI cutting.The template that is used for pcr amplification is the polynucleotide of the whole ripe BNP of coding (1-32 sequence).

Synthesized be suitable for pcr amplification coding activated, through two oligonucleotide of the polynucleotide of the BNP of form processing, BNP-102689 and BNP-102692:

BNP-102689：5′-AAGCTGCCTTAGGCTTAAGCCCCAAGATGGTGCAAGGGTC-3′(SEQ?ID?NO：378)

BNP-102692：5′-GCACCGGGCGCGCCTTAATGCCGCCTCAGCACTTTGCAGC-3′(SEQ?ID?NO：379)

BNP-102689 mixes the polynucleotide (italic) of last five amino acid of Bsu36I cloning site (underscore), coding HSA and the polynucleotide (runic) of the first eight aminoacid sequence of coding BNP.In BNP-102692, the underscore sequence is Asc I site, and polynucleotide thereafter comprise reverse complementary sequence (runic) and the terminator codon (italic) of last 8 the amino acid whose DNA of coding BNP.Use this two primers, pcr amplification HSA/BNP corresponding circle of sensation.Be necessary for every pair of concrete primer and each concrete template and determine annealing and the temperature and time that extends by rule of thumb.

Purified pcr product (for example use Wizard PCR to prepare dna purification system, PromegaCorp), then with Bsu36I and Asc I digestion.By gel electrophoresis to Bsu36I/Asc I is segmental be further purified after, product cloning is gone into pSAC35-NEC through Bsu36I/Asc I digestion, produce construction ID # 3796.Expression constructs has been carried out the sequence checking.

The expression of construction ID 3796 and purifying

In Expression in Saccharomyces Cerevisiae

With construction ID#3796, pSAC35:HSA.BNP (1-32) is transfected into BXP10 cell (seeing embodiment 4) by methods known in the art.

From BXP10 cell conditioned medium liquid purifying

After about 84 hours, the results culture also filters by centrifugal and 0.2 μ m and to clarify cell.Catch recombinant protein and the combination by sodium-chlor and Sodium octoate comes wash-out by 5ml Blue Sepharose fast flow post (GE Healthcare).Some prepared products are by also finishing protein bound to ceramic hydroxyl appetite post (BioRad) with the phosphoric acid salt wash-out of cumulative concentration.Other prepared product is bonded to DEAESepharose fast flow post (GE Heathcare) and passes through linear sodium-chlor gradient elution.Then the wash-out set is bonded to Q sepharose high performance column (GE Healthcare) and uses the sodium-chlor gradient elution.End product is concentrated and in the preparation damping fluid, exchange by tangential flow filtration.

The generation of construction ID # 3959 and clone

Construction ID # 3959, pSAC35:HSA.BNP (1-29), the DNA that comprises coding BNP albumin fusion proteins, described fusion rotein has the HSAsp/KEX2 leader sequence, then be through processing, the HSA of mature form, it merges in Yeast expression carrier pSAC35 (in the table 2 construction 3959 see SEQ ID NO:501) to through processing, N-end mature form, that lack last three amino acid whose BNP peptides (S1-L29).

Use the polynucleotide of primer BNP-103801 and BNP-104315PCR amplification coding BNP hereinafter described,, and connect into pSAC-NEC, produce construction ID # 3959 through the Bsu36I/AscI cutting with the Bsu36I/AscI cutting.The template that is used for pcr amplification is primer construction ID 3796 (seeing below).

Synthesized be suitable for pcr amplification coding activated, through two oligonucleotide of the polynucleotide of the BNP of form processing, BNP-103801 and BNP-104315:

BNP-103801：5′-CAGGAGCCCCTTAGGCTTAAGCCCCAAGATGGTGCAAGGGTCT-3′(SEQ?ID?NO：578)

BNP-104315：5′-CCTCACTCGGCGCGCCTTACAGCACTTTGCAGCCCAGGCCACTGGA-3′(SEQ?ID?NO：579)

BNP-103801 mixes the polynucleotide (italic) of last five amino acid of Bsu36I cloning site (underscore), coding HSA and the polynucleotide (runic) of the first eight aminoacid sequence of coding BNP.In BNP-104315, the underscore sequence is Asc I site, and polynucleotide thereafter comprise reverse complementary sequence (runic) and the terminator codon (italic) of the amino acid whose DNA of S21-L29 of the BNP that encodes.Use this two primers, pcr amplification HSA/BNP corresponding circle of sensation.Be necessary for every pair of concrete primer and each concrete template and determine annealing and the temperature and time that extends by rule of thumb.

Purified pcr product (for example use Wizard PCR to prepare dna purification system, PromegaCorp), then with Bsu36I and Asc I digestion.By gel electrophoresis to Bsu36I/Asc I is segmental be further purified after, product cloning is gone into pSAC35-NEC through Bsu36I/Asc I digestion, produce construction ID # 3959.Expression constructs has been carried out the sequence checking.

The expression of construction ID 3959 and purifying

In Expression in Saccharomyces Cerevisiae

With construction ID # 3959, pSAC35:HSA.BNP (S1-L29) is transfected into BXP10 cell (seeing embodiment 4) by methods known in the art.

From BXP10 cell conditioned medium liquid purifying

After about 84 hours, the results culture also filters by centrifugal and 0.2 μ m and to clarify cell.Catch recombinant protein and the combination by sodium-chlor and Sodium octoate comes wash-out by 5ml Blue Sepharose fast flow post (GE Healthcare).Some prepared products are by also finishing protein bound to ceramic hydroxyl appetite post (BioRad) with the phosphoric acid salt wash-out of cumulative concentration.Other prepared product is bonded to DEAESepharose fast flow post (GE Heathcare) and passes through linear sodium-chlor gradient elution.Then the wash-out set is bonded to Q sepharose high performance column (GE Healthcare) and uses the sodium-chlor gradient elution.End product is concentrated and in the preparation damping fluid, exchange by tangential flow filtration.

The result

Measured dosage-response relation (see figure 10) of HSA-BNP (1-29) and HSA-BNP (1-32) and reorganization BNP.The EC50 value of HSA-BNP (1-29) and HSA-BNP (1-32) is than the big several times of EC50 value (being respectively 467.9,45.06 and 0.2227) of reorganization BNP.In addition, the EC50 value of HSA-BNP (1-29) fusion rotein is bigger 10 times than HSA-BNP (1-32) fusion rotein.

The external test method of embodiment 81:ANP and BNP degraded

Background and method:

Neutral lyase (Neprilysin), be also referred to as MME, CALLA, CD10, common acute lymphoblastic leukemia antigen, enkephalinase, EPN, NEP, neutral endopeptidase or neutral endopeptidase 24.11, be that (MW 90 for 743 amino acid, 000-110, cell surface metallopeptidase 000kD), by multiple tissue expression, include but not limited to prostate gland, kidney, intestines, uterine endometrium, suprarenal gland and lung.Neutral lyase includes but not limited to ANP, BNP, CNP, Substance P, bradykinin, pitocin, Leu-and Met-enkephalin, neurotensin, bombesin, endothelium (blood vessel contracts) peptide-1 and bombesin-like peptide by the multiple physiologically active peptide of amino side deactivation of cutting water transport property residue.

After measured to the relative susceptibility of neutral lyase hydrolysis, approximately be that CNP is 4-5 minute, ANP is 8 minutes, and BNP is that (Kenny, A.J.et al., Biochem be (1) J.291: 83-8 (1993)) in 2 hours.

Neutral lyase is to the influence of ANP and BNP peptide

ANP and BNP peptide have been measured activity in CatchPoint cGMP assay method (Molecular Devises), wherein comprised or do not contain and be exposed to the neutral lyase of proteolytic enzyme.Particularly, 5 μ M ANP or BNP are being contained or are not being contained the neutral lyase (R﹠amp of 10nM; D Systems) in the MES damping fluid (0.1M2-(N-morpholino) ethyl sulfonic acid (Sigma)) in 37 ℃ of incubations 24 hours.Then will be described in embodiment 80 stable transfection the 293F cell of NPR-A stimulate with the ANP or the BNP through protease treatment of various concentration.The pair cell lysate is analyzed the cGMP activation then, uses CatchPoint cGMP assay method (Molecular Devices) described in embodiment 80.

The result

For BNP and ANP have calculated dosage-response curve, wherein there is or do not have neutral lyase incubation (seeing Figure 11 A).Have or when not having neutral lyase incubation, BNP shows similar BNP activity, the EC50 value is respectively 0.2966 and 0.2702.Yet, cause the ANP activity to compare remarkable reduction with neutral lyase incubation ANP with untreated ANP, the EC50 value is respectively 0.2965 and 60.47.Use technology known in the art, selected sample is further analyzed by reversed-phase HPLC.Carry out per-cent relatively (seeing Figure 11 D) with the sample of identical time of incubation under the condition that does not have neutral lyase.Although handling generation ANP proteolysis in 20 minutes with neutral lyase, yet even also do not observing significant BNP proteolysis after 24 hours with neutral lyase incubation.

Neutral lyase is to the influence of ANP-HSA fusion rotein

ANP and ANP-HSA (CID 3484) are being contained or are not being contained the neutral lyase (R﹠amp of 10nM; DSystems) in the MES buffering (0.1M 2-(N-morpholino) ethyl sulfonic acid (Sigma)) in 37 ℃ of incubations 20 minutes, 1 hour or 24 hours.Then will be described in embodiment 80 stable transfection the 293F cell of NPR-A stimulate with the ANP or the ANP-HSA through protease treatment of various concentration.The pair cell lysate is analyzed the cGMP activation then, uses CatchPoint cGMP assay method (Molecular Devices) described in embodiment 80.

The result

For ANP and ANP-HSA have calculated dosage-response curve, wherein there is or do not have neutral lyase incubation (seeing Figure 11 B and 11C respectively).The ANP peptide shows significant active the reduction handling with neutral lyase in 1 hour.Yet, ANP-HSA (CID 3484) even after handling 24 hours, also do not showing significant active the reduction with neutral lyase.Use technology known in the art, selected sample is further analyzed by reversed-phase HPLC.Carry out per-cent relatively (seeing Figure 11 D) with the sample of identical time of incubation under the condition that does not have neutral lyase.Although handling generation ANP proteolysis in 20 minutes with neutral lyase, yet even also do not observing ANP-HSA (CID 3484) proteolysis after 24 hours with neutral lyase incubation.

Embodiment 82:HSA-IFN α 2b associating ribavirin in genotype 1, do not accept interferon therapy Antiviral activity among chronic hepatitis C (HCV) patient

Background

The conventional processing of accepting the HCV patient (interferon-naiveHCV patient) of Interferon, rabbit (IFN) treatment utilizes interferon alpha associating ribavirin (RBV) to reach for 48 weeks to genotype 1, first.Yet this processing has great practical limits.Because the well-known side effect of current interferon therapy, patient's quality of life have substantive the reduction after using Interferon, rabbit at every turn.Current scheme requires weekly at least dosed administration, the time lengthening that causes quality of life to reduce.The result is that a large amount of patients ends treatment, and some research report termination rate surpasses 50%.In addition, the great hematologic parameter that current interferon therapy also has considerable ratio reduces, and this low RBV dosage of begging to surrender is possibly perhaps more requiring the temporary transient interferon therapy scheme that stops to recover normal until hematology numerical value in the serious situation.So, obviously, the treatment plan that need improve is used for the treatment of the genotype 1HCV among the patient who accepts the IFN treatment first.

Ultimate principle

By the albuminised C-terminal gene of maturation being merged N-end, produce HSA-IFN α 2b to ripe Interferon Alpha-2b.In the clinical study of positive contrast (active controlled clinical study) (in genotype 1, do not accept to carry out in the HCV human patients of IFN treatment) is arranged, assessed the security and the effect of the treatment of HSA-IFN α 2b associating RBV, and with compare as the conventional treatment that uses PEG-IFN α-2a (PEG-IFN) associating RBV described in the embodiment 82 of actively contrast (active control).

Method

With genotype 1, do not accept IFN treatment human HCV patient with HSA-IFN α 2b or actively contrast PEG-IFN and unite ribavirin (RBV) and treat.In particular, 458 human experimenters are randomized into 4 subcutaneous (sc) treatment groups: (a) PEG-IFN dosed administration 180 μ g, weekly (Q1w); (b) HSA-IFN α 2B dosed administration 900 μ g, whenever biweekly (Q2w); (c) HSA-IFN α 2b dosed administration 1200 μ g Q2w; Or (d) HSA-IFN α 2b dosed administration 1200 μ g, every around once (Q4w).

According to body weight, every experimenter in each treatment group also accepts 1000-1200mg/ days RBV.The foundation of grouping comprises weight index (body mass index, BMI) (＜25kg/m ²Or 〉=25kg/m ²) and HCV RNA titre (＜800,000IU/ml or 〉=800,000IU/ml).

The treatment time length of this research was 48 weeks, followed up a case by regular visits in 24 weeks in addition.The main effect terminal point of this research (primary efficacy endpoint) is to continue virusology to reply (SVR).

Use the PCR in real time assay method, sensitivity range (quantitatively restriction (LOQ)) is 43IU to 69 * 10 ⁶IU/mL and detection level (LOD) are the Quantasure of 10IU/mL ^TM(Labcorp) measure the HCVRNA titre.Use standard technique known in the art to measure alanine transaminase (ALT) and hematology effect, comprise absolute neutrophil count (ANC), oxyphorase and platelet count.

Be absorbed in treatment (Intent to treat, ITT) the patient experimenter that is defined as all random packet in each treatment group and receives treatment is no matter whether the patient lacks any data point or withdraw from research.Revise be absorbed in treatment (Modified intent to treat, MITT) patient is defined as the believable patient with the visit of the 24th week of those research date of registration according to them.

Result and discussion

Having summed up experimenter's demographics, antiviral response and the hematology in the 4th week and the 12nd week in table 10 (preliminary interim analysis (preliminary interim analysis)) and the table 11 (final interim analysis (finalinterim analysis)) reduces.In a word, all four treatment plans have all obtained good tolerance, and do not have significant difference in 3-4 level laboratory values or aspect the termination of adverse events between each treatment group.

Table 10: the preliminary interim analysis of demographics, antiviral response and hematology effect

^*P value＜0.05HSA-IFN α 2b 1200 μ g Q2w are than Q4w; P value 0.068 HSA-IFN α 2b 1200 μ g Q2w are than PEG-IFN;

#p value＜0.05 HSA-IFNA2B, 1200 μ g Q4w are than PEG-IFN; ^*P value＜0.05HSA-IFN α 2b 1200 μ g Q4w are than all other groups

Table 11: the final interim analysis of demographics, antiviral response and hematology effect

^*P value＜0.05HSA-IFN α 2b 1200 μ g Q2w are than Q4w;

P value 0.15HSA-IFN α 2b 1200 μ g Q2w are than PEG-IFN; #p value＜0.05HSA-IFNA2B 1200 μ g Q4w are than PEG-IFN; ^*P value＜0.05HSA-IFN α 2b 1200 μ g Q4w are than all other groups

The antiviral response predictor of SVR

The antiviral response predictor (predictor) of SVR is defined as treatment the 12nd when week HCV RNA titre negative (HCV RNA titre＜LOQ), its subordinate phase slope＞0.6log/wk (2 phase slope).Two kinds of activity of each stage antiviral response rate of curve indication.Fs demonstrates the direct antiviral activity of replying.Subordinate phase indication treatment compound is to the destruction of HCV cells infected.So, the numerical value of subordinate phase slope＞0.6log/wk is the good predictor (positive predicted value (PPV)＞90%) of SVR.

The antiviral response predictor of SVR during the 12nd all ITT is maximum in HSA-IFN α 2b 1200 μ gQ2w treatment groups, wherein 82/110 experimenter or 74.5% (final interim analysis (finalinterim analysis)) (77/104 experimenter or 74.0% (preliminary interim analysis (preliminaryinterim analysis))) to have HCV RNA negative levels (be that level is lower than LOQ (＜43IU/mL)) and 58% and shows subordinate phase slope＞0.6log/wk, comparatively speaking, PEG-IFN contrast treatment group is 75/114 experimenter or 65.8% (final interim analysis) (70/112 experimenter or 62.5% (preliminary interim analysis)) and 49%.These data show that in the 12nd week, HSA-IFN α 2b 1200 μ g Q2w treatment plans have suitable with the PEG-IFN conventional treatment at least antiviral activity.Because in the 12nd when week HSA-IFN α 2b 1200 μ g Q2w treatment plans patient's number of RNA feminine gender (HCV RNA titre level is lower than LOQ) than PEG-IFN contrast treatment larger about 9% (final interim analysis) and 12% (tentatively interim analysis), and the subordinate phase slope is larger about 9% (the preliminary interim analysis of final sum the two), so the possible HSA-IFN of being α 2b 1200 μ g Q2w treatment plans can cause remarkable effect, it surpasses conventional PEG-IFN treatment.The negative patient's number of HCV RNA is similar to the PEG-IFN conventional treatment in HSA-IFN α 2b 900 μ g Q2w and the HSA-IFN α 2b 1200 μ gQ4w treatment groups.

The 20th week and the 24th when all the antiviral response predictor of SVR be expressed as HCV RNA titre and be lower than detection level (LOD) (＜10IU/mL) experimenter.The antiviral response predictor of SVR is maximum in HSA-IFN α 2b 1200 μ g Q2w treatment groups during the 20th week, wherein 82/110 experimenter or 74.5% (final interim analysis) have detection less than the HCV rna level (promptly＜10IU/mL), comparatively speaking, PEG-IFN contrast treatment is 77/114 experimenter or 67.5% (final interim analysis).Similarly, the antiviral response predictor of SVR is maximum in IFN α 2b 1200 μ gQ2w treatment groups during the 24th week, 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 experimenter or 63.3% have detection less than the HCV rna level.The data in the 20th week and the 24th week show that all HSA-IFN α 2b 1200 μ g Q2w treatment plans have antiviral activity and security, and use the conventional treatment of PEG-IFN suitable at least with the improvement dosage regimen.

The normalizing of ALT level

The measurement commonly used of liver function is alanine transaminase (ALT) level among the patient.One of characteristics of HCV infected patient are the high Serum ALT levels of indication liver injury.So, the normalizing of ALT level is corresponding to the improvement of liver function, and to the favourable prognosis of treatment response.Although all representing the ALT level of sening as an envoy to, all treatment plans recover more normal abilities; yet in HSA-IFN α 2b 1200 μ g Q4w treatment plans, observe the most significant effect, wherein compare the ALT level that patient's (preliminary interim analysis of final sum) above twice has realized normalizing with the PEG-IFN conventional treatment.So, with the HSA-IFN α 2b of 1200 μ g Q4w dosed administrations make genotype 1, not accept HCV patient's the liver function recovery of IFN treatment more effective than conventional PEG-IFN treatment unexpectedly aspect normal.

The hematology effect

The full dosage IFN and the RBV that guarantee to obey and be exposed in the combined treatment are vital for making the maximization of SVR ratio.

Hematology is common during being reduced in the combination therapy of IFN and RBV.Oxyphorase (Hb) is owing to the reduction that haemolysis that RBV brought out causes need reduce RBV dosage.Particularly, Hb＜12g/dL requires RBV was reduced to 800mg/ days from 1000-1200mg/ days.Recurrence is vital to RBV dosage for prevention HCV.HSA-IFN α 2b 1200 μ g Q4W treatment plans make the amplitude of Hb reduction＜12g/dL significantly reduce (52% 65% (final interim analysis) than PEG-IFN unexpectedly; 49.1% 64% (preliminary interim analysis)) than PEG-IFN.This recurrence rate that can be interpreted as HSA-IFN α 2b 1200 μ g Q4W treatment plans is lower, causes SVR to improve.

ANC reduction＜750/mm ³Require to reduce IFN dose of components in the combination therapy.Unexpectedly, HSA-IFN α 2b 1200 μ g Q4W treatment plans make ANC＜750/mm ³The reduction amplitude significantly reduces, and this compares with PEG-IFN, and (6% than 20.2% (final interim analysis); 4.3% than 17.5% (preliminary interim analysis)).This is interpreted as reducing under the less condition of amplitude at the desired dosage of HSA-IFN α 2b 1200 μ g Q4W treatment plans once more, and the SVE ratio is higher.

Similar hematology takes place and reduces in the 12nd when week between HSA-IFN α 2b Q2w and PEG-IFN treatment group.Unexpectedly, the 12nd week in HSA-IFN α 2b 1200 μ g Q4w treatment groups viewed hematological indices reduction amplitude than viewed little by about 75% in PEG-IFN treatment group.These results show, compare with conventional PEG-IFN treatment, and HSA-IFN α 2b Q4w can provide the safety features of brilliance and the recurrence rate of improvement.

Conclusion

In the 12nd week, in HSA-IFN α 2b 1200 μ g Q2w treatment groups, observe genotype 1, do not accept the maximum antiviral activity of the HCV of IFN treatment.In HSA-IFN α 2b 1200 μ g Q2w and PEG-IFN treatment group, also observe the similar effect that hematology is reduced.In addition, in the 20th week and the 24th week, in HSA-IFN α 2b 1200 μ g Q2w treatment groups, also observe maximum antiviral activity.The 20th the week and the 24th week in HSA-IFN α 2b 900 μ g Q2w treatment groups, continued to observe suitable antiviral activity.Correspondingly, HSA-IFN α 2b 900 μ g Q2w can provide suitable with current standard treatment PEG-IFN at least effect and safety features, but it has the dosage regimen of improvement, has made things convenient for the patient widely.In addition, 1200 μ g Q2w can provide suitable with current standard treatment PEG-IFN at least safety features, but the remarkable effect and the dosage regimen of improvement can be arranged, thereby greatly facilitate the patient.

Use the treatment of HSA-IFN α 2b 1200 μ g Q4w schemes demonstrating unexpectedly in the 12nd week and the suitable effect of conventional PEG-IFN treatment, even the experimenter who accepts the PEG-IFN conventional treatment is owing to dosage regimen has been accepted extra 3 doses of PEG-IFN.The effect that HSA-IFN α 2b 1200 μ g Q4w are suitable with conventional PEG-IFN treatment continued through the 20th week and the 24th week.In HSA-IFN α 2b 1200 μ g Q4w treatment groups, also observing the ability of stablizing liver function and significantly alleviating the improvement that the hematology factor reduces the 12nd week, show the improvement of liver injury among these patients and dosage reduces or suspensory incidence and treatment after the reduction of the incidence that may recur.So, these presentation of results, the treatment of HSA-IFN α 2b 1200 μ g Q4w can provide with the PEG-IFN combination therapy compares suitable effect, and the dosage regimen that the former advantage is to improve, make the normal ability of liver function recovery bigger, and hematological indices to reduce amplitude less, cause the patient that bigger conformability and bigger facility are arranged, and achievement after the more favourable treatment.In a word, consider the latest developments in HCV therapeutics field, HSA-IFN α 2bQ4W treatment plan will have Ideal Characteristics (for example suitable effect, remarkable tolerance, remarkable convenience cause bigger answering property of clothes), and the Interferon, rabbit trunk that will become the interferon anti-reflecting virus conjoint therapy is selected.

In a word, these presentation of results, to genotype 1, do not accept IFN treatment HCV patient's HSA-IFN α 2b effective as conventional PEG-IFN and RBV combination therapy at least with the RBV combination therapy, advantage is the dosage regimen improved.Particularly, this presentation of results, the treatment of HSA-IFN α 2b associating RBV can have compares remarkable effect and similar safety characteristic, similarly effect and remarkable safety features or remarkable effect and safety features with conventional PEG-IFN associating RBV treatment, but has improvement and highly favourable dosage regimen.

Embodiment 83: chronic hepatitis C (HCV) does not have the response patient to HSA-IFN α 2b associating ribavirin Reply

Background

Have in the U.S. to surpass 4,000,000 people and infected hepatitis C virus (HCV), make this virus become the common cause of U.S.'s hepatopathy.Think that in history the interferon alpha (IFN α) that has or do not have antiviral molecule ribavirin (RBV) concurrent treatment is to the most effective treatment of patient.Recently, the interferon alpha of PEGization form has been ratified associating RBV and has been used for HCV treatment.These PEGization Interferon, rabbit have demonstrated in treatment HCV more effective than standard Interferon, rabbit or Interferon, rabbit associating ribavirin therapy, and have become the conventional treatment of HCV.

Yet this treatment has great practical limits.Outside the substantive side effect that reduces patients ' life quality during well-known treatment the and considerable ratio that the great hematology relevant with routine treatment reduces, routine treatment is invalid to the patient of the vast scale of accepting current HCV treatment.The clinical studies show of not accepting the HCV patient treatment of IFN α-RBV therapy in the past goes out about 45% patient who starts conventional treatment and fails to remove HCV and keep chronic infection (for example not answering the person).Socially, there is patient's the ratio of response quite little to treatment.

The clinical investigation personnel have made response to the needs of not answering person colony of failing the treatment of previous use IFN α or associating RBV is made the HCV patient of response, promptly control these patients again with the routine treatment of PEGization IFN α and RBV.Referring to Shiffman et al., " Peginterferon alfa-2a andribavirin in patients with chronic hepatitis C who have failed prior treatment, " Gastroenterology 126 (4): 1015-23 (2004).Although the patient of 35% registration in this test does not have the evidence of HCV RNA after treating for 20 weeks with routine treatment, many among these patients have recurred after treatment is ended.So, have only 18% patient in fact realized lasting virus reply (Sustained Viral Response, SVR) and cured HCV.Similarly, when the kaput nothing response of the treatment of controlling previous conventional PEGization IFN α therapy with another kind of PEGization IFN α again patient, according to evidence (anecdotal evidence), have only these patients of about 5-10% can realize SVR according to unmatchful.So, not only a kind of interferon therapy has been failed but also all kaput patient of all current interferon therapies colony continues significantly to increase.Therefore, obviously the treatment plan that needs improvement, not only be used for general HCV treatment, but also those are previous with patients of interferon therapy treatment (for example accepting IFN α treatment) with do not answer the person as treatment, particularly those nothings that are difficult to treat most response patients' (treatment of the current routine treatment of for example previous use or control kaput patient again) alternative therapy.

Ultimate principle

By the albuminised C-terminal gene of maturation being merged N-end, produce HSA-IFN α 2b to ripe Interferon Alpha-2b.Security, tolerance and the effect of the treatment of HSA-IFN α 2b associating RBV in clinical study random packet, open-label (accepting not answering in the person HCV human patients of IFN α treatment carries out), have been assessed.For this research, do not answer the person to be defined as those and reduce (for example the 12nd week early stage virus was replied or EVR12) failing to realize SVR after the HCV patient who stops previous treatment the 12nd week or those are finished treatment plan patient owing to fail to realize the 2-log of HCV rna level.Get rid of outside research the patient who ends the recurrence of treatment back.In addition, at least 50% the previous PEGization IFN of patient α treatment plan has been failed in this research.

Method

The person HCV patient that do not answer that the kaput people IFN of previous at least a IFN α treatment plan α was once received treatment is divided into 3 subcutaneous (sc) HSA-IFN α 2b treatment groups at random: (a) 900 μ g whenever biweekly (Q2w); (b) 1200 μ g Q2w and (c) 1200 μ g every around once (Q4w).Every experimenter in each treatment group also accepts 1000-1200mg/ days RBV.After the data of safety of assessment from these three initial groups, the dosage of HSA-IFN α 2b is amplified in two extra groups of follow-up increase, they accept HSA-IFN α 2b and unite 1000-1200mg/ days RBV with 1500 μ g Q2w or 1800 μ g Q2w.

The treatment time length of this research was 48 weeks, followed up a case by regular visits in 24 weeks in addition.The main effect terminal point of this research is to continue virusology to reply (SVR).

Use the PCR in real time assay method, sensitivity range is 10IU to 100 * 10 ⁶The Quantasure of IU/mL ^TM(Labcorp) measure HCV RNA titre.Use standard technique known in the art to measure alanine transaminase (ALT) and hematology effect, comprise absolute neutrophil count (ANC), oxyphorase and platelet count.

Result and discussion

Demography

Identify many Demographics, served as the individual index that those tend to not respond the patient of treatment.Predictor comprises before the crucial treatment of these of non-responsiveness: (1) genotype 1, and (2) high baseline intermediate value HCV rna level, (3) had not before responded the PEG+RBV therapy, (4) African American, the horizontal F3-F4 of (5) fibrosis in late period (uses

Staging) and (6) high BMI (for example 〉=25mg/kg).Perhaps, the best overall index of responsiveness is not the number based on the therapy of IFN of previous PEG-RBV treatment failure and previous failure.

Experimenter's demography is summarized in table 12.In a word, all experimenter's demographys are similar in all treatment groups.Most of experimenters have been exposed to above a kind of IFN of containing α scheme and previous PEG+RBV therapy and have failed.In addition, though 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 most a high proportion of previous PEG+RBV failure.So, the experimenter in the 1800 μ g Q2w treatment groups has presented maximum treatment refractoriness patient group.

Table 12: demography and baseline characteristic

	900μg?Q2w N＝23	1200μg?Q2w N＝24	?1200μg?Q4w?N＝24	?1500μg?Q2w?N＝22	?1800μg?Q2w?N＝22	The P value
							Genotype
1	20(87.0％)	24(100％)	22(91.7％)	21(95.5％)	21(95.5％)	0.4531
							Intermediate value HCV RNA (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
							The African 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

Effect and biologic activity

Table 13 has shown HCVRNA that genotype 1, PEG+RBV do not answer person, the refractoriness HCV patient group reduction with respect to level before the treatment during treating.In 2-12 week, the degree that HCV RNA reduces is suitable between 900-1500 μ g treatment group.Yet, in 1800 μ g treatment groups, observe maximum virus load and reduce.Consider HCV RNA and the most a high proportion of PEG+RBV failure before the higher levels of treatment in this treatment group, this is unexpected.The degree of treatment 12 all antiviral response has reflected the subordinate phase slope of dynamics of virus and has been the positive predictor (positivepredictor) of SVR.

As shown in figure 12, not answer the HCVRNA of person's 900-1500 μ g treatment group to reduce slope be suitable in the 12nd week for genotype 1, PEG+RBV.Unexpectedly, the HCV RNA reduction degree of 1800 μ g treatment groups is maximum.It is suitable that the HCV RNA of 1500 μ g of this patient subgroups and 1800 μ g treatment groups was reduced in for the 24th week.

In the 24th week, experimenter's ratio of HCV RNA feminine gender is suitable between 900-1500 μ g treatment group.According to investigator's judgement and consider from cumulative data (showing the high negative predicted value that SVR is lacked EVR12 and the 24th all RNA feminine genders) based on the therapy of Interferon, rabbit, allow the experimenter end in default of effect in the 24th week.(end-of-treatment response, ETR, the 48th all HCV feminine genders) replied at the whole end of overall treatment of 900-1200 μ g treatment group is 30% (22/73).So, realized ETR in the 12nd week (for example EVR12) or the experimenter at high proportion who becomes HCV RNA feminine gender the 24th week.In addition, continuing during most of experimenters (13/22) following up a case by regular visits in the 12nd week after 48 week treatments is HCV RNA feminine gender.This shows that the potential SVR after the HSA-IFN α 2b/RBV treatment is 18%.

In a word, these data show, with 900-1200 μ g HSA-IFN α 2b associating RBV treat PEG+RBV failure at high proportion, once accept IFN α treatment, refractoriness HCV patient causes strong and suitable antiviral activity.This treatment refractoriness do not answer also observe in the person colony low virus break through (breakthrough) rate (for example HCV RNA detect less than but are male at two or more time points subsequently) and low recurrence rate.In addition, in 1800 μ g treatment groups, also observe significant reduction by a larger margin in 12 week, show that the SVR that unites the patient of ribavirin therapy with HSA-IFN α 2b with this dosage leads remarkable rising can be arranged in treatment.

The antiviral response that table 13:GT1 and PEG+RBV do not answer the person

	900μg?Q2w N＝12	1200μg?Q2w N＝16	1200μg?Q4w N＝13	1500μg?Q2w N＝15	1800μgQ2w 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％)
Detected in the 24th week 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％)

^aBe defined as detection less than HCV RNA or 〉=the HCV RNA of 2-log reduces

^bAccurate 95% fiducial interval

The hematology effect

The full dosage IFN and the RBV that guarantee the conformability for the treatment of and be exposed in the combined treatment are vital for making SVR lead maximization.

Hematology is common during being reduced in the combination therapy of IFN and RBV.Recurrence is vital to RBV dosage for prevention HCV.Yet oxyphorase (Hb) and thrombocyte (PLT) counting is because the reduction RBV dosage that lowers the requirement that the haemolysis that RBV brought out causes.Absolute neutrophil count (ANC) reduction＜750/mm ³Require to reduce IFN dose of components in the combination therapy.

Reduce although observe some ANC and PLT, these are reduced between all Q2w treatment groups is suitable, and reaches platform (plateau) in about 4-8 week.Same, it is suitable that Hb (comprises 1800 μ g treatment groups) with respect to being reduced in of baseline between all Q2w treatment groups, after the 12nd week reached.Being reduced in the Q4w treatment group of hematology numerical value is less.Generally, there is 12/115 experimenter to reduce dosage in order to control adverse events.Most of HSA-IFN α 2b dosage reduce the reduction that is derived from ANC, as what summarized in the treatment plan.Between Q2w treatment group, do not observe dose response.So, not observing needing that dosage reduces in high-dose therapy group more increases.

In a word, though hematology numerical value is observed in the treatment of HSA-IFN α 2b associating RBV some reductions are arranged, these are reduced between each treatment group is suitable, shows with 900-1800 μ gHSA-IFN α 2b associating RBV treatment once to accept do not have significant difference between IFN α security treatment, that do not answer person HCV patient.

Conclusion

In a word, the treatment of these presentation of results HSA-IFN α 2b and RBV is realizing that aspect the SVR be effective, and so remarkable ratio accept not the answering among the person HCV patient of IFN α treatment, comprise among those kaput patients of previous PEG+RBV treatment plan and eradicated HCV.Particularly, these presentation of results, the treatment of HSA-IFN α 2b 900-1200 μ g associating RBV can cause 18% patient to realize SVR, even also is like this in the situation of PEG+RBV failure formerly.In addition, 1800 μ g treatment groups demonstrate the 24th maximum all HCV RNA negative rate in most of refractoriness patient groups, show that this treatment can cause these patients even bigger SVR leads.In addition, the safety features between all treatment groups of HSA-IFN α 2b is similar.In addition, these results show, per two to use HSA-IFN α 2b around thoughtful be useful, thereby the dosage regimen that improves is provided.Therefore, these results show, the treatment of HSA-IFN α 2b associating RBV is the kaput patient of those therapies based on Interferon, rabbit, those conventional PEGization Interferon, rabbit-kaput patients of RBV therapy effective and safe treatment alternatives of providing this area to lack at present particularly, it has dosage regimen favourable and that improve.

Embodiment 84:HSA-IFN α 2b associating ribavirin in genotype 2 or 3, do not accept Interferon, rabbit and control Antiviral activity among chronic hepatitis C (HCV) patient who treats

Background

The whole world has and surpasses 1.7 hundred million people and infected hepatitis C virus (HCV), and this virus has become great public health problem, and has become the common cause of whole world hepatopathy rapidly.Acute HCV infection does not have symptom usually, makes early diagnosis be a problem.In fact, HCV infects and trends towards chronic condition, and wherein about 70% acute infection becomes persistent.So, although the new incidence that infects is descending, however popular will the remaining unchanged in the recent period of prediction HCV infection.

Think that in history the interferon alpha (IFN α) that has or do not have antiviral molecule ribavirin (RBV) concurrent treatment is to the most effective treatment of chronic hepatitis C (CHC) patient.Recently, the interferon alpha of PEGization form has been ratified associating RBV and has been used for HCV treatment.These PEGization Interferon, rabbit have demonstrated in treatment HCV more effective than standard Interferon, rabbit or Interferon, rabbit associating ribavirin therapy, and have become the conventional treatment of HCV.

The overall antiviral response (SVR) that continues to current recommended therapy changes in CHC patient greatly, depends on virus and host's characteristic, particularly virogene type.For example, the scope of SVR ratio is about 42-46% among more common genotype 1 patient.On the other hand, the SVR ratio is 76-80% among more uncommon genotype 2 or 3 patients.In addition, 1 patient compares with genotype, and genotype 2 that treatment difficulty reduces greatly or 3 patients can treat with shorter treatment time with the ribavirin of low dosage more.

Although to the current recommended therapy of genotype 2 or 3, promptly PEGization Interferon, rabbit associating RBV reached for 24 weeks, then was 24 all follow-up period, caused the patient of great ratio to realize SVR, and this treatment plan has still kept and the great practical limits of therapy common based on IFN.Particularly, current recommended therapy still is subjected to the puzzlement of untoward reaction, promptly in the quality of life of using the substantive patient of reduction in back at every turn.So, continue to need effectively and the patient that infected genotype 2 or 3HCV tolerates better new treatment plan.

Ultimate principle

By the albuminised C-terminal gene of maturation being merged N-end, produce HSA-IFN α 2b to ripe Interferon Alpha-2b.The security and the effect of the treatment of HSA-IFN α 2b associating RBV in the clinical study of random packet, polycentric, open-label (in genotype 2 or 3, do not accept to carry out in the CHC human patients of IFN treatment), have been assessed.

Method

43 genotype 2 or 3, the human HCV patient that do not accept IFN treatment are randomized into two subcutaneous (sc) HSA-IFN α 2b treatment groups: (a) 1500 μ g dosed administrations, whenever biweekly (Q2w) or (2) 1500 μ g dosed administrations, every around once (Q4w).Every experimenter in each treatment group also accepts 800mg/ days RBV.The main foundation of grouping comprise genotype (2 or 3) and HCV RNA (＜800,000IU/mL or 〉=800,000IU/mL).

The treatment time length of this research was 24 weeks, followed up a case by regular visits in 24 weeks in addition.Main effect terminal point (primary efficacy end-point) is to continue virusology to reply (SVR).

Use PCR in real time, sensitivity range (quantitatively restriction (LOQ)) is 43IU to 69 * 10 ⁶The Quantasure of IU/mL ^TM(Labcorp) measure HCV RNA titre.Use stable state assessment models (Homeostasis Assessment Model, HOMA) assessment insulin resistant.

Be absorbed in treatment (Intent to treat, ITT) the patient experimenter that is defined as all random packet in each treatment group and receives treatment is no matter whether the patient lacks any data point or withdraw from research.Revise be absorbed in treatment (Modified intent to treat, MITT) patient is defined as the patient that those research date of registration according to them can have the visit of the 12nd week credibly.

Result and discussion

The experimenter's demographics and the antiviral response in the 4th week and the 12nd week have been summed up in the table 14.In a word, HSA-IFN α 2b has obtained good tolerance in two treatment groups.

Table 14: demographics and antiviral response

ITT=is absorbed in treatment; (experimenter meets the condition of the 12nd week visit) of being absorbed in treatment that MITT=revises

Degree that HCV RNA reduces and the genotype 2 of HCV RNA＜LOQ or 3 patients' ratio are suitable between HSA-IFN α 2b Q2w and two treatment groups of HSA-IFN α 2b Q4w.In the 4th week, the genotype 2 of HCV RNA＜LOQ or 3 patients' ratio, 1500 μ g Q2w treatment groups are 76.2% and 1500 μ g Q4w treatment groups are 68.2%.In the 12nd week, a high proportion of genotype 2 or its HCV RNA＜LOQ of 3 patients (1500Q2w be 82.4% and 1500Q4w is 88.9%) are arranged all in two treatment groups.

So, these results show, cause powerful antiviral response rate with 1500 μ g HSA-IFN α 2b Q2w or Q4w therapeutic gene type 2 or 3HCV patient.In addition, in genotype 2 or 3 patients, the treatment of HSA-IFN α 2b 1500 μ g Q4w schemes demonstrates the suitable effect of treatment with HSA-IFN α 2b 1500 μ g Q2w schemes.So, these presentation of results, the treatment of HSA-IFN α 2b 1500 μ g Q2w or the Q4w current recommended therapy as the patient who has infected HCV genotype 2 or 3 at least is effective, and advantage is the dosage regimen improved greatly, thereby causes remarkable tolerance and made things convenient for the patient.

Embodiment 85: with the genotype 1 of HSA-IFN α 2b associating ribavirin therapy, do not accept interference The chronic hepatitis C of extract for treating (HCV) patient's quality of life (QOL)

Background

As previously mentioned, reach the conventional treatment in 48 weeks with PEGization interferon alpha associating ribavirin (RBV) therapeutic gene type 1, the HCV patient that do not accept Interferon, rabbit (IFN) treatment and have great practical limits.Because the well-known side effect of interferon therapy of current recommendation, patient's quality of life have substantive the reduction after using Interferon, rabbit at every turn.Current scheme requires weekly at least dosed administration, and the time lengthening that quality of life reduces due to causing treating and the fate of anergy increase.The result is that a large amount of patients ends treatment, and some research report termination rate surpasses 50%.So, obviously the treatment plan that need improve is used for the treatment of the genotype 1HCV among the patient who does not accept the IFN treatment, and requirement is compared with current standard treatment the influence of quality of life and made moderate progress.

Ultimate principle

In the clinical study of positive contrast (active controlled clinical study) (in genotype 1, do not accept to carry out in the HCV human patients of IFN treatment) is arranged, assessed the security and the effect of the treatment of HSA-IFN α 2b associating RBV, and with compare as the conventional treatment that uses PEG-IFN α-2a (PEG-IFN) associating RBV described in the embodiment 82 of actively contrast (active control).In 12 week of treatment, the treatment of using HSA-IFN α 2b associating RBV is united RBV to the influence of QOL and anergy fate (disability days) (for example inoperable fate) with PEG-IFN compare.

Method

458 genotype, 1 human HCV patient's random packet is also treated described in embodiment 82.SF-is passed through in the 4th week and the assessment of the 12nd week that reach treatment before treatment

Measurement model (QualityMetric, Lincoln, RI) QOL of Ce Dinging and anergy fate.Particularly, eight SF-36v2 fields have been assessed: physical function (physical functioning, PF), role-physical (RP), human body pain (bodily pain, BP), general health (general health, GH), vigor (vitality, VT), social function (social functioning, SF), role-emotional (RE) and Mental Health (mental health, MH).Preceding four fields (PF, RP, BP and GH) are corresponding to the physical health key element (Physical Health Component) of QOL model, and remain four fields (VT, SF, RE and MH) corresponding to Mental Health key element (Mental Health Component).

Assessed conversion (original) score in eight SF-36v2 fields, and gather (physical component summary based on the physique key element of standard (norm-based), PCS) score and spiritual key element gather (mental component summary, MCS) score until the treatment the 12nd week.

Result and discussion

Table 15: quality of life is measured

^*P value＜0.05 is than PEG-IFN; ^#Surpass (MCID) minimum clinical significant differences

During the 12nd week, the QOL that accepts the patient of 900 μ g HSA-IFN α 2b Q2w is having improvement with respect to PEG-IFN aspect each measurement, realized significance,statistical in MCS and PCS and 5/8 field.In 1200 μ g Q2w and 1200 μ g Q4w HSA-IFN α 2b treatment groups, the deterioration of QOL alleviates all having aspect each measurement almost with respect to PEG-IFN, all observes clinical significant difference in human body pain and Mental Health.

In a word, the genotype 1HCV patient in any HSA-IFN α 2b treatment group is because their HCV infection and successive treatment and inoperable fate (MDW) is less than the genotype 1HCV patient in the PEG-IFN treatment group.Particularly, the MDW that accepts the patient of 900 μ g HSA-IFN α 2b Q2w lacks 75% than the patient who accepts PEG-IFN, and the MDW that accepts the patient of 1200 μ g HSA-IFN α 2b Q2w or 1200 μ gHSA-IFN α 2b Q4w lacks 25% than the patient who accepts PEG-IFN.

Add the antiviral activity of the 2b of HSA-IFN α shown in the embodiment 82, these presentation of results, use HSA-IFN α 2b and RBV to genotype 1, do not accept the IFN treatment HCV patient's combination therapy effective as the treatment of using conventional PEG-IFN and RBV combination therapy at least, advantage is that dosage regimen improves to some extent and QOL makes moderate progress.Particularly, these presentation of results, use the treatment of HSA-IFN α 2b associating RBV that improvement and highly favourable treatment plan can be provided, surpass conventional PEG-IFN associating RBV treatment, be that the treatment institute that surpasses PEG-IFN associating RBV is getable for the patient provides the deterioration degree of QOL index to alleviate and the minimizing of inoperable fate.

Embodiment 86:HSA-BNP (construction ID # 3959) in the normal pig model to the body of cGMP In induce

Ultimate principle

The ability that brain (Type B) natriuretic peptide (BNP) is regulated cytological effect is well-known in the art such as the ability that changes renal function and vascular tone.The activity of BNP depends on its combination and follow-up activation to natriuretic peptide receptor A (NPR-A) (a kind of amidino groups/guanylate cyclase).BNP causes cGMP level rising in the born of the same parents to the activation of NPR-A, and wherein the cGMP level can be measured by assay method known in the art in the born of the same parents, such as for example ELISA.In the normal pig model, tested the ability that HSA-BNP (CID 3959) induces cGMP to generate in vivo.

Method

By the albuminised C-terminal gene of maturation being merged, produce HSA-BNP (CID 3959) to the N-end of the BNP (amino acid/11-29) of the terminal clipped form of C-.

Use single bolus 5mg/kgHSA-BNP (CID 3959) or vehicle preparaton only for normotensive, healthy pig (n=4-6 head/group) in time 0.After injection, gathered blood plasma and urine in 1,8,16,24,48 and 72 hour.By commercially available ELISA (Molecular Dynamics) measure the cGMP level in the blood plasma of gathering and the urine.

The result

Single IV injects 5mg/kg HSA-BNP (CID 3959) and causes IV to use the two the remarkable rising of back 1 hour blood plasma (Figure 13 A) and urine (Figure 13 B) cGMP level.In blood plasma 24 hours and in urine 48-72 hour cGMP level drop to baseline values gradually.

The natruresis in the pace-making model in body in heart failure of embodiment 87:BNP-HSA fusion construct is lived The property

Background

Using external source BNP has been used for promoting natruresis in congestive heart failure (CHF).Yet nearest studies show that used BNP and can be had a negative impact to renal function.Assessed the influence of HSA-BNP (CID 3959) in vivo in the severe CHF pig model to kidney or left ventricle (LV) function.

Method

By the albuminised C-terminal gene of maturation being merged, produce HSA-BNP (CID 3959) to the N-end of the BNP (amino acid/11-29) of the terminal clipped form of C-.

By reaching for 3 weeks, in 18 pigs, bring out CHF with the chronic pace-making of 240bpm.8 pigs serve as the reference contrast.Compare the following baseline characteristic of suffering from CHF with the reference contrast remarkable reduction is arranged.Base measurement is to use technology known in the art to carry out.

Table 16: the baseline measures of the pig of having brought out CHF in the reference contrast pig

	Brought out the pig of CHF	The contrast pig
			Left ventricle (the penetrating blood) mark that records by ultrasonic cardiography shortens (LVFS) (%)	21±2	32±2
The kidney vascular flow (RenFlow) that the use microsphere records (mL/min/g)	1.73±0.09	2.23±0.26
			Sodium is removed (Na CL)(mL/min)	0.43±0.11	4.35±2.45
Fractional excretion of sodium (FE Na)(％)	0.25±0.07	0.80±0.32

After baseline measures, for this research will show animal in heart failure (the LV expansion for example takes place, and mark shortens decline (to penetrate blood) subsequently) random packet.With Animal Anesthesia (n=10 head/group), use vehicle, 2mg/kg HSA-BNP (CID 3959) or 6mg/kg HSA-BNP (CID3959) IV, and monitored 4 hours.Measure the diastasis diameter by ultrasonic cardiography.

Result and conclusion

The HSA-BNP of two kinds of dosage (CID 3959) has no significant effect (data not shown) to heart rate, mean arterial pressure, ventricular end diastolic pressure, mean pulmonary arterial pressure, left ventricle peak pressure, peak positivity dp/dt (peak positive dp/dt) or cardiac output.Do not see that in the animal of the arbitrary dosage HSA-BNP of infusion (CID 3959) coronary flow changes (data not shown).In addition, although 30 minutes creatinines are removed and fractional excretion of sodium (fractional sodium excretion) rising after causing infusion for animal infusion 6mg/kg HSA-BNP (CID 3959), yet the two does not all reach the statistics that is higher than baseline restricted (data not shown).Similarly, infusion 6mg/kg HSA-BNP (CID 3959) causes the non-remarkable reduction (data not shown) that plasma renin activity and endothelium (blood vessel contracts) peptide (endothelin) blood plasma level are compared with vehicle.

In the animal of infusion 6mg/kg HSA-BNP (CID 3959), seen sodium remove remarkable rising than the vehicle group (behind the infusion 30 minutes 492 ± 281% and infusion after 60 minutes 950 ± 483%).Exception, the HSA-BNP (CID 3959) of a diastasis vary in diameter dosage in office that is caused by pace-making compares remarkable reduction (Figure 14 A) afterwards with the vehicle group.In addition, left ventricle (the penetrating blood) mark that is caused by pace-making shortens HSA-BNP (CID 3959) that (left ventricular fractional shortening) change a dosage in office to be compared with the vehicle group afterwards and has reduced, and wherein the reduction that is caused by 2mg/kg dosage HSA-BNP (CID 3959) is significant (Figure 14 B) than the vehicle group.

So, in a word, these results have proved, acute infusion HSA-BNP (CID 3959) can bring out natruresis but left ventricle or renal function are not had disadvantageous effect in the CHF model in vivo.

Embodiment 88:HSA-BNP (construction ID #3959) is to the shadow of cardiorenal function in the normal dogs of anesthesia Ring

Ultimate principle

The ability that brain (Type B) natriuretic peptide (BNP) is regulated renal function and vascular tone is well-known in the art.Dog is that research BNP influences useful especially model to cardiorenal function.Therefore, in the normal dogs model of anesthesia, carry out the extensive evaluation of HSA-BNP (CID 3959) to heart kidney influence (comprising Hemodynamics, kidney and hormone effect).

Method

By the albuminised C-terminal gene of maturation being merged, produce HSA-BNP (CID 3959) to the N-end of the BNP (amino acid/11-29) of the terminal clipped form of C-.

Hemodynamics, kidney and the hormone parameter of cardiorenal function in this research, have been assessed.Particularly, hemodynamic parameter comprises measurement cardiac output, mean arterial pressure, pulmonary capillary wedge pressure and mean pulmonary arterial pressure.The kidney parameter comprises measures urine stream, sodium excretion, glomerular filtration rate(GFR (GFR) and kidney blood flow.The hormone parameter comprises measures blood plasma cGMP, feritin, Angiotensin II, aldosterone and urine cGMP.

Reach 5 days before the research beginning, for normal, healthy hybrid dog (n=8/group) feeding fixed sodium meal.In acute experiment eve, give the animal fasting and give the 300mg Quilonum Retard to be used to assess renal tubular function.On acute experiment same day, with dog by IV vetanarcol (15mg/kg) anesthesia, intubate, and with supplemental oxygen power ventilation.

(flow-directed) end that fluid is guided is that spheric (balloon-tipped) thermodilution catheter inserts pulmonary artery to measure the Hemodynamics of the heart through external jugular vein.To femoral arteriography with monitoring blood pressure, blood sample collection, and infusion inulin (inulin) and physiological saline.To left kidney intra-ureteral cannula to collect urine.To place the Renal artery on every side to measure kidney blood flow (RBF) through the Electromagnetic Flow detector of calibration.

On acute experiment same day, implement single IV to dog and inject HSA-BNP (CID 3959) 0.5mg/kg or 5mg/kg (n=8 only/group).Influence to cardiorenal function was monitored 4.5 hours.

Measured cardio-vascular parameters comprises mean arterial pressure (MAP), RAP (RAP), Ppa pulmonary artery pressure (PAP), cardiac output (CO) and pulmonary capillary wedge pressure (PCWP).CO measures by thermodilution.MAP assesses via the direct measurement from femoral catheter.GFR removes by inulin (inulin) and measures.

Measure cardiovascular Hemodynamics when beginning each the removing.Each removing is collected arterial blood in the pipe that heparin and EDTA are housed and is placed on ice immediately.In 4 ℃ with 2, after 500rpm is centrifugal, pours out blood plasma and be stored in-20 ℃ until analysis.During each remove whole, collect urine with assessment urine amount, ionogen and inulin.For analyzing the urine of collecting, cGMP before preservation, is heated to above 90 ℃.

Result and discussion

Haemodynamic effect

Figure 15 A-H has shown that the HSA-BNP (CID 3959) that uses with 0.5mg/kg (Figure 15 A, C, E and G) or 5mg/kg (Figure 15 B, D, F and H) compared the influence of hemodynamic performance with the baseline reading during behind the infusion 4.5 hours.Hemodynamic parameter is to measure in 30,60,90,150,210 and 270 minutes after IV injects preceding baseline of HSA-BNP (CID3959) and infusion.

The haemodynamic effect of HSA-BNP (CID 3959) is kept at 4.5 hours viewing durations.All cause the significant and lasting pulmonary capillary wedge pressure of statistics (PCWP) to reduce (Figure 15 E and F) 0.5 inject HSA-BNP (CID 3959) with 5mg/kg IV.The amplitude that these two kinds of dosage reduce PCWP does not have significant difference.

HSA-BNP (CID 3959) is relevant with dosage with the influence of mean arterial pressure (MAP) (Figure 15 C and D) to Ppa pulmonary artery pressure (PAP) (Figure 15 G and H).When giving animal dosed administration 5mg/kg, observe the remarkable reduction (Figure 15 H) of PAP.Same, the remarkably influenced of in the 5mg/kg treatment group, behind infusion, observing MAP in 270 minutes (Figure 15 D).

The kidney effect

Figure 16 A-H has shown that the HSA-BNP (CID 3959) that uses with 0.5mg/kg (Figure 16 A, C, E and G) or 5mg/kg (Figure 16 B, D, F and H) compared the influence of renal output and blood flow with the baseline reading during behind the infusion 4.5 hours.The kidney performance perameter is to measure in 30,60,90,150,210 and 270 minutes after IV injects preceding baseline of HSA-BNP (CID 3959) and infusion.

HSA-BNP (CID 3959) uses the remarkably influenced that causes renal function.0.5 and 5mg/kgHSA-BNP (CID 3959) all observe kidney blood flow (Figure 16 E and F), diuretic properties (Figure 16 A and B), the natruresis (Figure 16 C and d) of remarkable rising.The GFR rising trend that dosage is relevant also is tangible (Figure 16 G and H).

HSA-BNP (CID 3959) reaches the maximum time to the influence of kidney parameter and compares with haemodynamic effect and trend towards delayed slightly.In addition, the degree that natruresis and diuretic properties raise in the 5mg/kg treatment group is significantly higher than the 0.5mg/kg treatment group.

Hormone effect

Figure 17 A-F has shown that the HSA-BNP (CID 3959) that uses with 0.5mg/kg (Figure 17 A, C and E) or 5mg/kg (Figure 17 B, D and F) compared the influence of RAAS hormone with the baseline reading during behind the infusion 4.5 hours.Plasma aldosterone, feritin and Angiotensin II level are to measure in 30,60,90,150,210 and 270 minutes after IV injects preceding baseline of HSA-BNP (CID 3959) and infusion.

0.5 and 5mg/kg IV injects the reduction that using of HSA-BNP (CID 3959) all causes viewing duration feritin behind 4.5 hours infusions, Angiotensin and aldosterone level.Influence to the aldosterone level in research in whole 270 minutes is significant and lasting.Influence to feritin and Angiotensin II all is significant in the HSA-BNP that uses two kinds of dosage (CID 3959) back between 30-90 minute, but bounce-back when the observation period finishes.

Conclusion

This studies have shown that HSA-BNP (CID 3959) turns round in the pharmacology mode similar to not merging BNP.With 0.5 and 5mg/kg single IV inject and use variation dose-dependently, significant and lasting that HSA-BNP (CID 3959) causes multinomial heart kidney parameter, consistent with the effect of its long-acting form BNP.Particularly, use HSA-BNP (CID 3959) with 5mg/kg dosage and cause blood plasma and urine cGMP level to raise, PCWP and PAP reduce, and natruresis, diuresis, kidney blood flow and glomerular filtration rate(GFR raise, plasma aldosterone, feritin and Angiotensin II reduce, and MAP reduces slightly.Differentia influence to each composition of renin-angiotensin-aldosterone system is some unpredictable consequence, and may be the beneficial characteristics of HSA-BNP (CID 3959).

In a word, these presentation of results can be to improve cardiorenal function but do not cause that dosage essence, that undesired systemic blood pressure reduces uses HSA-BNP (CID 3959).

Embodiment 89:HSA-BNP (construction ID #3959) is to blood pressure in the Bi Erge sleuth of Electronic Testing Influence

Ultimate principle

The ability that brain (Type B) natriuretic peptide (BNP) is regulated vascular tone is well-known in the art.As previously mentioned, dog is that research BNP influences useful especially model to cardiovascular function (comprising blood pressure).Accordingly, in Bi Erge (beagle) sleuth that regain consciousness, normal, assessed intravenously (IV) and used HSA-BNP (CID 3959) the cardiovascular function influence of (comprising systolic pressure, mean arterial pressure and heart rate).In addition, also having assessed subcutaneous (SC) uses the effectiveness of HSA-BNP (CID 3959) and replys the time length.

Method

Implant Data Sciences International radiotelemetry projector for healthy Bi Erge sleuth (n=4/group) operation, it has the ability of collecting systemic arterial blood pressure, heart rate and ECG data.After the implantation, dog is accepted single IV and injects 0.1,0.5 or 5mg/kg HSA-BNP (CID 3959) or vehicle (vehicle).

The continuous recording of ECG parameter and systemic blood pressure in 48 hours behind the monitoring infusion.By monitoring intermittently dog is followed the trail of 9 days (total time=11 day) again.

Inject and do not merge BNP (0.02mg/kg) and implement SC and use HSA-BNP (CID 3959) effect (10mg/kg) and assess subcutaneous administration HSA-BNP (CID 3959) to the effectiveness of systemic blood pressure with reply the time length by relatively implementing IV.

Result and discussion

HSA-BNP (CID 3959) is to the influence of systolic pressure and mean arterial pressure

Figure 18 A-C has shown that single IV injects the influence of HSA-BNP (CID 3959) to systolic pressure and mean arterial pressure in the clear-headed dog.Use 5mg/kg HSA-BNP (CID 3959) and cause systolic pressure to reduce gradually, wherein peak value was positioned at about 16 hours and got back to baseline in the time of 48 hour.8 hours beginning systolic pressures obviously have the lasting reduction of about 15mmHg and last till and use back 20 hours behind drug administration.

Use HSA-BNP (CID 3959) at identical viewing duration to diastolic pressure or heart rate without any obviously influence (data not shown).

Than low dosage (for example 0.5 and 0.1mg/kg) HSA-BNP (CID 3959) to blood pressure or not obviously influence of heart rate.In addition, do not observe processing associated change for the HSA-BNP (CID 3959) of any dosage to the ECG parameter.

IV uses and does not merge the comparison that BNP peptide and SC use HSA-BNP (CID 3959)

Figure 19 A and B have shown that IV injects and have not merged the influence that BNP and SC injection HSA-BNP (CID 3959) compares systemic blood pressure in normal, the healthy Bi Erge sleuth.HSA-BNP (CID 3959) and do not merge BNP the two all reduces systolic pressure in the healthy Bi Erge sleuth.The effect that does not merge BNP reached maximum and get back to baseline in a few hours in the time of about 30 minutes.In contrast, the effect of HSA-BNP (CID3959) is used the back at SC and was become in about 10 hours obviously, reached maximum in the time of about 40 hours, and got back to baseline between 48-72 after the injection hour.HSA-BNP (CID 3959) to the effect of blood pressure slowly performance absorb (Tmax in dog is about 36 hours) slowly with it consistent.The effect longer duration of HSA-BNP (CID3959) with it the long half-lift (being 72 hours in dog) consistent.

In a word, these presentation of results can be applied to heart failure patient with low the dosage that is enough to improve cardiorenal function but does not influence systemic blood pressure with HSA-BNP (CID 3959).

With whole disclosures of every piece of document (comprising patent, patent application, patent publications, journal article, summary, experiment guide, books or other open form) of being quoted among the application and can be collected herein by reference by the information completely of being mentioned among the application to obtaining such as the special identifier of databases such as GenBank, GeneSeq or CAS Registry.

In addition, with the specification sheets of following international application and each part of U. S. application with sequence table is complete is collected herein by reference: International Application PCT/US02/40891 that on December 23rd, 2002 submitted to; International Application PCT/US2004/001369 that on January 20th, 2004 submitted to; International Application PCT/US2005/004041 that on February 9th, 2005 submitted to; The U. S. application 10/775,204 that on February 11st, 2004 submitted to; The U. S. application 11/175,690 that on July 7th, 2005 submitted to; The U. S. application 11/429,373 that on May 8th, 2006 submitted to; The U. S. application 11/429,276 that on May 8th, 2006 submitted to; The U. S. application 11/429,374 that on May 8th, 2006 submitted to; The U.S. Provisional Application 60/707,521 that on August 12nd, 2005 submitted to; Ask 60/712,386 during the U.S. of submitting on August 31st, 2005 is interim; The U.S. Provisional Application 60/732,724 that on November 3rd, 2005 submitted to; The U.S. Provisional Application 60/776,914 that on February 28th, 2006 submitted to; The U.S. Provisional Application 60/781,361 that on March 13rd, 2006 submitted to; With the U.S. Provisional Application of submitting on June 2nd, 2,006 60/810,182; With the U.S. Provisional Application of submitting on June 6th, 2,006 60/813,682.

Explanation about the preservation biomaterial

(PCT detailed rules and regulations 13bis)

A. Xia Mian explanation relates to the preservation biomaterial of mentioning among the specification sheets table 1A.

B. The evaluation of preservation thing

Depositary institution's title: American type culture collection

Depositary institution address: 10801 University Boulevard

Manassas，Virginia?20110-2209

United?States?of?America

	Preserving number	Preservation date
			?1	PTA-3763	October 4 calendar year 2001
?2	PTA-3940	December 19 calendar year 2001
			?3	PTA-3942	December 19 calendar year 2001
?4	PTA-3939	December 19 calendar year 2001
			?5	PTA-4670	September 16 calendar year 2001

Canada: applicant's requirement, until on the application basis, authorizing Canadian Patent or apply for out of court, perhaps abandon and no longer recover, perhaps recall, the commissioner can only be provided by the sample of the preservation biologic material that provides among the application to be mentioned to the specified Independent Expert of chief, so the applicant must be with written statement notice international office before the technology of announcing international application be ready to complete.

Norway: the applicant is in this requirement, until application (by Norwegian Patent office) open to the public, perhaps finally make decision under the disclosed situation not having by Norwegian Patent office, sample should only be provided the expert who gives this area.According to Norwegian Patent method 22 and 33 (3) joints, should be earlier than to open the proposing to Norwegian Patent office day of the public to the requirement of this effectiveness by the applicant.If the applicant has proposed this generic request, any requirement of the sampling of third party's proposition should be understood that the expert that will use so.This expert can be anyone of applicant's approval in anyone or the case on generally acknowledged expert's table of drawing up of Norwegian Patent office.

Australia: the applicant notifies at this, microbiological specimens should before the granted patent or stopping, rejecting or withdrawal of an application before only provide give as and the present invention do not have personnel's (regulations 3.25 (3) of Australian Patent regulations) of the skilled practitioner of stake (interest).

Finland: the applicant is in this requirement, until applying for that (by national patent and management committee) is open to the public, perhaps finally do not made decision by national patent and management committee under the disclosed situation of the public, sample should only be provided the expert who gives this area.

Britain: applicant's requirement, the sample of microorganism can only offer the expert.This application must be submitted to international office by the applicant and just can come into force before this application performs the technology preparation of international publication.

Denmark: applicant's requirement, until this application international open (by Danish Patent office), or under the disclosed situation of the public, do not made final decision by Danish Patent office, biological sample only can be provided the expert to this area.According to Danish Patent bill the 22nd and 33 (3) joints, this requirements is being submitted to Danish Patent office day and just can come into force earlier than what disclose to the public by the applicant.If the applicant has submitted such requirement to, so the requirement of the sampling that any third party proposed all will be satisfied the regulation that the expert uses.Described expert can be any expert in the list of expert approved of Danish Patent office, or anyone of applicant's case approval.

Sweden: applicant's requirement, until this application international open (by Swedish patent office), or under the disclosed situation of the public, do not made final decision by Swedish patent office, biological sample only can be provided the expert to this area.This requirement must be from right of priority be submitted to the international office form of PCT/RO/134 table among the I twisting cohesion spare Z of PCT applicant's guide (preferably with) in 16 months before at the expiration and just can be come into force.If the applicant has submitted such requirement to, the requirement of so any third party institute requirement sampling all will be satisfied the regulation that the expert uses.Described expert can be any expert in the list of expert approved of Danish Patent office, or anyone of applicant's case approval.

Holland: applicant's requirement, until the day or the day out of court until this application or that recall or abandon of HOII P mandate, according to the regulation of patent law 31F (1), microorganism only can be provided to the expert.This requirement must precedingly be submitted to Dutch industrial property office and just can come into force to open day (two dates are formerly whichever) of the public according to the regulation of the 22C of HOII P bill joint or the 25th joint in this application by the applicant.

Claims (30)

1. the albumin fusion proteins that comprises the member who is selected from down group:

(a) fragment of therapeutic protein X or therapeutic protein X or variant and white protein or albuminised fragment or variant;

(b) fragment of therapeutic protein X or therapeutic protein X or variant and white protein or its fragment or variant, wherein said white protein or its fragment or variant comprise the aminoacid sequence of SEQ ID NO:1;

(c) (a) or the fragment of therapeutic protein X (b) or therapeutic protein X or variant and white protein or albuminised fragment or variant, the fragment of wherein said therapeutic protein X or variant have the biologic activity of therapeutic protein X, and wherein said albuminised fragment or variant have albuminised activity;

(d) fragment of therapeutic protein X (c) or therapeutic protein X or variant and white protein or albuminised fragment or variant, wherein said white protein activity are the abilities of comparing the preservation period of extended treatment protein X with the preservation period of the therapeutic protein X that does not merge state;

(e) fragment of therapeutic protein X (c) or therapeutic protein X or variant and white protein or albuminised fragment or variant, wherein said white protein activity are the abilities of comparing the serum half-life of extended treatment protein X with the serum half-life of the therapeutic protein X that does not merge state;

(f) (a) therapeutic protein X-(e) or the fragment of therapeutic protein X or variant and white protein or albuminised fragment or variant, wherein said albuminised fragment or variant comprise the aminoacid sequence of the amino acid/11-387 of SEQID NO:1;

(g) (a) therapeutic protein X-(f) or the fragment of therapeutic protein X or variant and white protein or albuminised fragment or variant, the fragment of wherein said therapeutic protein X or therapeutic protein X or variant merge the N-terminal at described white protein or albuminised fragment or variant;

(h) (a) therapeutic protein X-(f) or the fragment of therapeutic protein X or variant and white protein or albuminised fragment or variant, the fragment of wherein said therapeutic protein X or therapeutic protein X or variant merge the C-terminal at described white protein or albuminised fragment or variant;

(i) (a) therapeutic protein X-(f) or the fragment of therapeutic protein X or variant and white protein or albuminised fragment or variant, the fragment of wherein said therapeutic protein X or therapeutic protein X or variant merge N-terminal and the C-terminal at described white protein or albuminised fragment or variant;

(j) (a) therapeutic protein X-(f) or the fragment of therapeutic protein X or variant and white protein or albuminised fragment or variant, it comprises the first therapeutic protein X or its fragment or variant and the second therapeutic protein X or its fragment or variant, and the wherein said first therapeutic protein X or its fragment or variant are different from the described second therapeutic protein X or its fragment or variant;

(k) (a) therapeutic protein X-(j) or the fragment of therapeutic protein X or variant and white protein or albuminised fragment or variant, the fragment of wherein said therapeutic protein X or therapeutic protein X or variant separate by joint and described white protein or albuminised fragment or variant;

(l) (a) therapeutic protein X-(k) or the fragment of therapeutic protein X or variant and white protein or albuminised fragment or variant, wherein said albumin fusion proteins has following general formula: R1-L-R2; R2-L-R1; Or R1-L-R2-L-R1, and wherein R1 is fragment or the variant of therapeutic protein X or therapeutic protein X, L is a peptide linker, and R2 is white protein or albuminised fragment or the variant that comprises the aminoacid sequence of SEQID NO:1;

(m) (a) therapeutic protein X-(l) or the fragment of therapeutic protein X or variant and white protein or albuminised fragment or variant, the preservation period of wherein said albumin fusion proteins is greater than the fragment of therapeutic protein X that does not merge state or therapeutic protein X or the preservation period of variant;

(n) (a) therapeutic protein X-(l) or the fragment of therapeutic protein X or variant and white protein or albuminised fragment or variant, the serum half-life of wherein said albumin fusion proteins is greater than the fragment of therapeutic protein X that does not merge state or therapeutic protein X or the serum half-life of variant;

(o) (a) therapeutic protein X-(l) or the fragment of therapeutic protein X or variant and white protein or albuminised fragment or variant, the extracorporeal biology activity of the therapeutic protein X of wherein said albumin fusion proteins or the fragment of therapeutic protein X or variant is greater than the fragment of therapeutic protein X that does not merge state or therapeutic protein X or the extracorporeal biology activity of variant; With

(p) (a) therapeutic protein X-(l) or the fragment of therapeutic protein X or variant and white protein or albuminised fragment or variant, biologic activity is greater than biologic activity in the body of the fragment of therapeutic protein X that does not merge state or therapeutic protein X or variant in the body of the therapeutic protein X of wherein said albumin fusion proteins or the fragment of therapeutic protein X or variant.

2. the albumin fusion proteins of claim 1, it is expressed in host cell, and wherein said host cell is yeast cell, mammalian cell or bacterial cell.

3. the albumin fusion proteins of claim 1, wherein said albumin fusion proteins also comprises the secretion leader sequence.

4. comprise the albumin fusion proteins of claim 1 and the composition that pharmacopedics can be accepted carrier.

5. the test kit that comprises the composition of claim 4.

6. treat disease of patient or disorderly method, comprise the step of the albumin fusion proteins of using claim 1.

7. the method for claim 6, wherein said disease or disorder comprise indication: Y.

8. the method for claim 7, wherein said disease or disorder are infection with hepatitis C virus.

9. the method for claim 8, wherein said albumin fusion proteins are by the host cell expression that comprises the white protein fusion construct that is selected from down group:

(a) construction ID 2249;

(b) construction ID 2343;

(c) construction ID 2366;

(d) construction ID 2381;

(e) construction ID 2382;

(f) construction ID 2410;

(g) construction ID 3165;

(h) construction ID 3422;

(i) construction ID 3423;

(j) construction ID 3424;

(k) construction ID 3476;

(l) construction ID 3960;

(m) construction ID 4290;

(n) construction ID 4291;

(o) construction ID 4292;

(p) construction ID 4295; With

(q) construction ID 4296.

10. the method for claim 9, wherein said white protein fusion construct is (d).

11. the method for claim 9, wherein said white protein fusion construct is (g).

12. the method for claim 9, wherein said white protein fusion construct is (l).

13. the method for claim 11, the wherein said patient who suffers from infection with hepatitis C virus is patient who had not received treatment or the patient who once received treatment.

14. the method for claim 13, the wherein said patient who once received treatment treats not answer the person.

15. the method for claim 14 does not wherein saidly answer the person before at least a combined treatment that comprises PEGization interferon alpha and ribavirin to be failed.

16. the method for claim 13, wherein said infection with hepatitis C virus are genotype 1 or genotype 2/3.

17. the method for claim 16, the treatment significant quantity of wherein said albumin fusion proteins is selected from down group:

(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; With

(f) about 2000 μ g/ agent.

18. the method for claim 17, wherein said albumin fusion proteins carries out dosed administration according to the dosage regimen that is selected from down group:

(a) weekly;

(b) whenever biweekly;

(b) per three weeks once;

(c) whenever all around once; With

(d) per five weeks once.

19. suffer from the patient's of infection with hepatitis C virus method with the albumin fusion proteins treatment that comprises the ripe Interferon Alpha-2b that merges with ripe white protein of treatment significant quantity, wherein said ripe Interferon Alpha-2b merges at the albuminised C-terminal of maturation, and wherein (1) described patient is the patient who had not received treatment, (2) described infection with hepatitis C virus is a genotype 1, described treatment significant quantity is about 900 μ g/ agent to about 1800 μ g/ agent, and described albumin fusion proteins is with every dosed administration biweekly.

20. the method for claim 19, wherein said treatment significant quantity is selected from down group:

(a) about 900 μ g/ agent;

(b) about 1200 μ g/ agent; With

(c) about 1800 μ g/ agent.

21. suffer from the patient's of infection with hepatitis C virus method with the albumin fusion proteins treatment that comprises the ripe Interferon Alpha-2b that merges with ripe white protein of treatment significant quantity, wherein said ripe Interferon Alpha-2b merges at the albuminised C-terminal of maturation, and wherein (1) described patient is the patient who had not received treatment, (2) described infection with hepatitis C virus is a genotype 1, described treatment significant quantity is extremely about 1800 μ g/ agent of about 900 μ g/ agent, and described albumin fusion proteins is with dose administration around every.

22. the method for claim 21, wherein said treatment significant quantity is selected from down group:

(a) about 900 μ g/ agent;

(b) about 1200 μ g/ agent; With

(c) about 1800 μ g/ agent.

23. suffer from the patient's of infection with hepatitis C virus method with the albumin fusion proteins treatment that comprises the ripe Interferon Alpha-2b that merges with ripe white protein of treatment significant quantity, wherein said ripe Interferon Alpha-2b merges at the albuminised C-terminal of maturation, and wherein (1) described patient is the patient who once received treatment, (2) described infection with hepatitis C virus is a genotype 1, described treatment significant quantity is about 1200 μ g/ agent to about 1800 μ g/ agent, and described albumin fusion proteins is with every dosed administration biweekly.

24. the method for claim 23, wherein said treatment significant quantity is selected from down group:

(a) about 1200 μ g/ agent;

(b) about 1500 μ g/ agent; With

(c) about 1800 μ g/ agent.

25. suffer from the patient's of infection with hepatitis C virus method with the albumin fusion proteins treatment that comprises the ripe Interferon Alpha-2b that merges with ripe white protein of treatment significant quantity, wherein said ripe Interferon Alpha-2b merges at the albuminised C-terminal of maturation, and wherein (1) described patient is the patient who once received treatment, (2) described infection with hepatitis C virus is a genotype 1, described treatment significant quantity is extremely about 1800 μ g/ agent of about 1200 μ g/ agent, and described albumin fusion proteins is with dose administration around every.

26. the method for claim 25, wherein said treatment significant quantity is selected from down group:

(a) about 1200 μ g/ agent;

(b) about 1500 μ g/ agent; With

(c) about 1800 μ g/ agent.

27. the method for the fragment of extended treatment protein X or therapeutic protein X or the preservation period of variant or serum half-life, comprise the fragment of therapeutic protein X or therapeutic protein X or the step of variant and white protein or albuminised fragment or variant fusion, compare with the fragment of therapeutic protein X that does not merge state or therapeutic protein X or the preservation period or the serum half-life of variant, described white protein or albuminised fragment or variant are enough to prolong the fragment of described therapeutic protein X or therapeutic protein X or the preservation period or the serum stability of variant.

28. nucleic acid molecule, it comprises the polynucleotide sequence of the albumin fusion proteins of the claim 1 of encoding.

29. comprise the carrier of the nucleic acid molecule of claim 28.

30. comprise the host cell of the nucleic acid molecule of claim 28.

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