CN1980687A - Albumin fusion proteins - Google Patents
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- CN1980687A CN1980687A CNA2005800122525A CN200580012252A CN1980687A CN 1980687 A CN1980687 A CN 1980687A CN A2005800122525 A CNA2005800122525 A CN A2005800122525A CN 200580012252 A CN200580012252 A CN 200580012252A CN 1980687 A CN1980687 A CN 1980687A
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Abstract
The present invention encompasses albumin fusion proteins. Nucleic acid molecules encoding the albumin fusion proteins of the invention are also encompassed by the invention, as are vectors containing these nucleic acids, host cells transformed with these nucleic acids vectors, and methods of making the albumin fusion proteins of the invention and using these nucleic acids, vectors, and/or host cells. Additionally the present invention encompasses pharmaceutical compositions comprising albumin fusion proteins and methods of treating, preventing, or ameliorating diseases, disorders or conditions using albumin fusion proteins of the invention.
Description
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 e-file.Each all contains file " PF612PCT SL.txt " (929,048 bytes are built on February 7th, 2005) these CDs, with its complete being incorporated herein by reference.
Background of invention
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 albumin or albuminised fragment or variant fusion.Polynucleotide, therapeutic albumin fusion proteins, compositions, 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 generate 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)), is responsible for the great ratio of serum osmotic pressure, 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 361 991 to produce reorganization HA (rHA) in microorganism.
The therapeutic protein that native state or reorganization generate such as interferon and growth hormone, normally presents the unstable molecule of of short duration storage life, when particularly preparing in aqueous solution.When preparation was used to use, the unstability of these molecules was pointed out necessary lyophilizing of a lot of molecules and cold preservation always when storage, thereby made 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 Min. storage back operation.
After using in the body, the therapeutic protein that native state or reorganization generate such as interferon and growth hormone, thereby presents of short duration plasma stability owing to removing rapidly from blood flow.Therefore, the therapeutic effect 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 dose administering therapeutic molecule more.Yet, increase reaction, side effect and toxicity increase that the proteinic scheme of taking medicine of administering therapeutic usually causes patient's injection site.Similarly, also usually cause patient's toxicity and side effect to increase with high dose administering therapeutic protein more.
The practical solution of the minority of the plasma stability of the raising therapeutic molecules of having proposed, comprise chemically conjugated, for the patient brings limited being benefited.Usually, in most cases, the therapeutic molecules of these chemical modifications is still used with the frequent scheme of taking medicine, and keeps 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 albumin or albuminised fragment (part) or variant is contained in the present invention.The present invention is also contained the nucleic acid molecules that comprises the therapeutic protein (as polypeptide, antibody or peptide or its fragment or variant) that coding and albumin or albuminised fragment (part) or variant merge or by its polynucleotide of forming.The present invention is also contained and is comprised coding following proteinic nucleic acid molecule or by its polynucleotide of forming, this protein comprises the therapeutic protein that merges with albumin or albuminised fragment (part) or variant (as polypeptide, antibody, or peptide or its fragment or variant), this albumin or albuminised fragment (part) or variant are enough to the proteinic storage life 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 the method for use these polynucleotide of the present invention and/or host cell and generation 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 description about the prolongation storage life 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 quantity 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 the table 1 or 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 the table 1 or 2.
The present invention also comprises the transgenic organism that contains nucleic acid molecules of the present invention (include but not limited to table 1 and 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 vehicle ATCC preservation thing PTA-3278.
Fig. 3 has shown the restriction map (people such as Sleep, BioTechnology 8:42,1990) of pSAC35 saccharomyces cerevisiae expression vector.
Fig. 4 shown by the various dilution factors of the coded IFNb albumin fusion proteins of the DNA that comprises in CID 2011 and 2053 in ISRE-SEAP/293F report cell to the active influence of SEAP (seeing embodiment 76).With protein serial dilution in DMEM/10%FBS, from 5e-7 to 1e-14g/ml, and be used to handle ISRE-SEAP/293F report cell.Take out supernatant and measure the SEAP activity by the report cell after 24 hours.From three stable clone: 293F/#2011, CHO/#2011 and NSO/#2053 purification IFNb albumin fusion proteins.The deutero-IFNb of mammal, Avonex are from Biogen and it is reported that the ratio of 2.0e5IU/ μ g is alive.
Fig. 5 has compared the antiproliferative activity to the Hs294T melanoma cells by the IFN albumin fusion proteins (CID 3165 protein) of CID 3165 coding and recombiant protein IFNa (rIFNa).Cell is cultivated in the culture medium that contains variable concentrations 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 inhibitory action, has reached 50% and suppress when about 200ng/ml.(■)=and CID 3165 protein, (◆)=rIFNa.
The various dilution factors that Fig. 6 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. 7 has shown by the influence (seeing embodiment 78) 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 excipient contrast, second is intravenous injection in first day 30 μ g/kgCID 2249 protein, article three, be first day subcutaneous injection 30 μ g/kg CID 2249 protein, article four, be first day subcutaneous injection 300 μ g/kg CID 2249 protein, the 5th is first and third, five day subcutaneous injection 40 μ g/kg reorganization IFNa.
Fig. 8 has shown the dosage-response relation (seeing embodiment 80 and 81) 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 reorganization BNP (■) and merged BNP in albuminised upstream.
Fig. 9 has shown the influence (seeing embodiment 80) to mean arterial pressure in spontaneous hypertensive rat of BNP albumin fusion proteins.Excipient (), reorganization BNP albumen (●) 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).
Figure 10 has shown the blood plasma cGMP level (see embodiment 80) of male C57/BL6 mice behind intravenous injection reorganization BNP protein (●) 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 11 shown in about 8 week age diabetes db/db mices of fasting single administration series connection GLP-1 (7-36A8G) 2x-HSA fusions (CID 3610) (◇), monomer GLP-1 (7-36A8G)-HSA fusions (Δ) or the independent back 24 hours blood sugar level of HSA (●).Blood sugar level is measured by the oral glucose tolerance method of testing.In about 8 week age diabetes db/db mices of fasting; after single administration 24 hours, series connection GLP-1 (7-36A8G) 2x-HAS fusions (CID 3610) (◇) had the beyond thought more effective activity (glucose-normalizing activity) that makes glucose normalization than monomer GLP-1 (7-36A8G)-HSA fusions (Δ).
Detailed Description Of The Invention
Definition
It is to run through employed some term of this description for the ease of understanding that following definition is provided.
When being used for this paper, " polynucleotide " refer to have the nucleic acid molecules of the nucleotide sequence of the following fusion rotein of coding, and this fusion rotein comprises 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 is made up of it; The nucleic acid molecules of nucleotide sequence with the following fusion rotein of coding, this fusion rotein comprise the aminoacid sequence of SEQ IDNO:Y (as described in table 2 the 6th row) or its fragment or variant or are made up of it; Have and comprise sequence shown in the SEQ ID NO:X or by the nucleic acid molecules of its nucleotide sequence of forming; Have the nucleic acid molecules of the nucleotide sequence of the following fusion rotein of coding, this fusion rotein comprises the aminoacid sequence of SEQ ID NO:Z or is made up of it; 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 nucleotide sequence that the albumin fusion construct described contained; The nucleic acid molecules that perhaps has the nucleotide sequence that is contained in the albumin fusion construct (as described in Table 3) of ATCC preservation.
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 polynucleotide of the therapeutic protein (or its fragment or variant) of at least one molecule of coding with identical reading frame albumin (or its fragment or variant) polynucleotide or by its nucleic acid molecules of forming; Comprise at least one molecule of coding that is connected with at least one section polynucleotide 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) polynucleotide or by its nucleic acid molecules of forming; Perhaps 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 albumin (or its fragment or variant) polynucleotide or by its nucleic acid molecule of forming, it also comprises for example one or more following elements: (1) functional autonomously replicationg vector (includes, but are 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 scalable or inducible promoter, constitutive promoter), (3) transcription termination region, (4) targeting sequencing and (5) selected marker.Coding therapeutic protein and albuminised polynucleotide, in case become the part of albumin fusion construct, each can be described as " part ", " zone " or " module " of this albumin fusion construct.
The present invention relates generally to the polynucleotide of coding albumin fusion proteins; Albumin fusion proteins; And the method for disease or disorder is treated, prevents or improved to the polynucleotide that use 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 by gene fusion link to each other (be that albumin fusion proteins produces by translating following nucleic acid, the polynucleotide of wherein encode complete or part therapeutic protein with identical reading frame with encode complete or the albuminised polynucleotide of part are connected).Therapeutic protein and albumin protein, in case become the part of albumin fusion proteins, each can be described as " part ", " zone " 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 processed justacrine in culture medium on every side by host cell.The processing to newborn albumin fusion proteins that takes place at the host's who is used for expressing secretory pathway can include but not limited to that signal peptide cutting, disulfide bond form, polymer protein is cut and is assembled in the interpolation of correct folding, carbohydrate and processing (glycosylation that is connected with O-such as for example N-), specific proteins hydrolysis.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 that this paper also is called " sophisticated albumin fusion proteins " through the albumin fusion proteins product of the terminal signal peptide cutting of N-.
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, might obtain specified albumin fusion construct again from the preservation thing by known in the art and other local technology of describing of this paper.ATCC_ be positioned at No. 10801, the big ways for education in 20110-2209 Manassas, Virginia, The United States state town (10801 University Boulevard, Manassas, Virginia 20110-2209, USA).ATCC_ preservation thing 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 preferred embodiments, 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 IDNO:Y in the table 2.In other embodiments, the invention provides the biologic activity that comprises therapeutic protein and/or therapeutic active 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 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 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 active 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 another preferred embodiment, the therapeutic protein of albumin fusion proteins partly is the outer solubility domain of born of the same parents of therapeutic protein.In 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 fragment or variant and serum albumin and/or therapeutic active 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.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 therapeutic protein or variant and human serum albumin's at least one fragment or variant or by its protein of forming, described fragment or variant are preferably to be 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 product.(term peptide, protein and polypeptide are used interchangeably in this article).Clearly imagine term " therapeutic protein " and contained 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 cancerous cell)) specific bond and therefore be can be used for protein with chemical compound (medicine or cytotoxic agents) this cell type of special target.
For example, can include, but are not limited to GLP-1, GLP-2, PACAP-27, PACAP-28, VIP, CD4M33, secretin, glucagon-like peptide, secrete sour regulin, PHM, IFN α, IFN β, ANP, BNP, NGF, BDNF, GDNF and somatostatin by " therapeutic protein " incomplete tabulation partly that albumin fusion proteins of the present invention comprises.
Interferon hybrid (hybrid) also can be blended in albuminised amino or carboxyl terminal to form interferon hybrid albumin fusion proteins.The interferon activity that interferon 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 hybrid albumin fusion proteins can be used for treatment, prevention or improves viral infection (as hepatitis (as HCV); Or HIV), multiple sclerosis or cancer.
In one embodiment, the interferon hybrid of interferon 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 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 is in total BglII restriction site place and interferon-ALPHA D fusion, and wherein the amino acid/11-62 of the N-end portion of A/D hybrid and interferon-ALPHA A is corresponding and the C-end portion is corresponding with the aminoacid 64-166 of interferon-ALPHA D.For example, this A/D hybrid will comprise aminoacid sequence:
CDLPQTHSLGSRRTLMLLAQMRX
1ISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFTTKDSSAAWDEDLLDKFCTELYQQLNDLEACVMQEERVGETPLMNX
2DSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIMRSLSLSTNLQERLRRKE(SEQ?ID?NO：99)，
X wherein
1Be R or K and X
2Be A or V (for example seeing construction ID#2875).In another embodiment, the A/D hybrid merges at total PvuIII restriction site place, and wherein the amino acid/11-91 of the N-end portion of A/D hybrid and interferon-ALPHA A is corresponding and the C-end portion is corresponding with interferon-ALPHA D aminoacid 93-166.For example, this A/D hybrid will comprise aminoacid sequence:
CDLPQTHSLGSRRTLMLLAQMRX
1ISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVMQEERVGETPLMNX
2DSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIMRSLSLSTNLQERLRRKE(SEQ?ID?NO：100)，
X wherein
1Be R or K and second X
2Be A or V (for example seeing construction ID#2872).These hybrids are at U.S. Patent number 4,414, further description are arranged in 510, with its complete being incorporated herein by reference.
In another embodiment, the α of interferon 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-end portion of A/F hybrid and interferon-ALPHA A is corresponding and the C-end portion is corresponding with the aminoacid 93-166 of interferon-ALPHA F.For example, this A/F hybrid will comprise aminoacid sequence:
CDLPQTHSLGSRRTLMLLAQMRXISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDMEACVIQEVGVEETPLMNVDSILAVKKYFQRITLYLTEKKYSPCAWEVVRAEIMRSFSLSKIFQERLRRKE(SEQ?ID?NO：101)，
Wherein X or R or K (for example seeing construction ID#2874).These hybrids are at U.S. Patent number 4,414, further description are arranged in 510, with its complete being incorporated herein by reference.In another embodiment, the α of interferon 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-end portion of A/B hybrid and interferon-ALPHA A is corresponding and the C-end portion is corresponding with the aminoacid 93-166 of interferon-ALPHA B.For example, this A/B hybrid will comprise aminoacid sequence:
CDLPQTHSLGSRRTLMLLAQMRX
1ISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEX
2X
3X
4X
5QEVGVIESPLMYEDSILAVRKYFQRITLYLTEKKYSSCAWEVVRAEIMRSFSLSINLQKRLKSKE(SEQ?ID?NO：102)，
X wherein
1Be R or K and X
2To X
5Be SCVM or VLCD (for example seeing construction ID#2873).These hybrids are at U.S. Patent number 4,414, further description are arranged in 510, with its complete being incorporated herein by reference.
In another embodiment, the interferon hybrid of interferon 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 hybrid albumin fusion proteins-α hybrid partly comprises the interferon beta-1 that merges with interferon-ALPHA D (being referred to herein 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-end portion and interferon beta-1 is corresponding and the C-end portion is corresponding with the aminoacid 74-167 of interferon-ALPHA D.For example, this β-1/ α D hybrid will comprise aminoacid sequence:
MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSAAWDEDLLDKFCTELYQQLNDLEACVMQEERVGETPLMNXDSILAVKKYFRRITLYLTEKKYSPCAWEVVRAEIMRSLSLSTNLQERLRRKE(SEQ?ID?NO：103)，
Wherein X is A or V.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 another embodiment, the interferon hybrid of interferon 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 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-end portion and interferon-ALPHA D is corresponding and the C-end portion is corresponding with the aminoacid 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 hybrid of interferon hybrid albumin fusion proteins part can comprise other compositions of α-interferon-alpha hybrid, alpha-beta interferon hybrid and β-interferon-alpha hybrid.In other embodiments, the interferon hybrid of interferon hybrid albumin fusion proteins part can modify with comprise sudden change, substitute interferon hybrid aminoacid sequence, deletion or add.These modifications to interferon 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 hybrid albumin fusion proteins mentioned above is contained in the present invention, and the host cell and the package carrier that contain the polynucleotide of these polypeptide of encoding.In one embodiment, the interferon hybrid albumin fusion proteins by above-mentioned polynucleotide encoding has increased shelf-life.In another embodiment, by the interferon 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 enterocyte propagation, reduce the enterocyte permeability, stimulate insulin secretion, induce bronchiectasis and vasodilation, suppress aldosterone and renin secretion, blood pressure regulation, promote nerve growth, enhance immunity is replied, strengthen inflammation, appetite-suppressing, described and/or the table 1 (secondary series) of perhaps following zhang " biologic activity " part is disclosed any or various biological is active to TA protein.
In one embodiment, IFN-β-HSA fusions is used to suppress Ebola virus (Ebolavirus) and SARS virus (Toronto-2 strain, Toronto-2 strain).For example, in the Vero cell, assessed the IFN-β (CID 2053 albumen) that is blended in ripe HSA upstream extracorporeal antivirus effect activity at Ebola virus and SARS virus.Based on the inhibitory action of cytopathic effect (CPE) and the dimethyl diaminophenazine chloride algoscopy of cell viability, these cells are used to assess CID 2053 proteinic protective effects.External signal transduction is assessed by gene expression analysis.In addition, CID 2053 proteinic pharmacokineticss and pharmacodynamics are assessed in Rhesus Macacus.Result's demonstration has realized strong extracorporeal antivirus effect activity with favourable safety index.CID 2053 protein are 0.4ng/ml and be 2ng/ml at the IC50 of SARS virus at the IC50 of Ebola virus.Array analysis (array analysis) demonstration CID 2053 protein are with the beta induced one group of similar expression of gene of IFN-and cause response element (ISRE) signal transduction pathway that IFN excites.Using in the CID 2053 proteinic Rhesus Macacus with the dosage of intravenous 50 μ g/kg or subcutaneous 300 μ g/kg, the terminal half-life (terminal half-life) is 36-40 hour.Use CID 2053 protein and cause the lasting rising of the expression of serum mopterin (neopterin) level and OAS1 mRNA.
In another embodiment, IFN-α-HSA fusions is used for suppress ranging category-A-Filovirus (Filo) (Ebola), arenavirus (Arena) (Pichende), category-B-togavirus (Toga) (VEE) or the virokine of C class-Bunyavirus (Bunya) (Pang Tatuolu (Punto toro)), banzi virus (Flavi) (yellow fever, Xi Niluo (West Nile)).For example, adopt CPE inhibition, dimethyl diaminophenazine chloride dyeing and virus yield algoscopy to assess the antiviral activity of the IFN-α (CID 3165 protein) that is blended in the HSA downstream.CID 3165 proteinic pharmacokineticss and pharmacodynamics are assessed in Rhesus Macacus and human experimenter.The result shows with favourable safety index and has realized antiviral activity at all RNA viruses.In the CPE algoscopy scope of IC50 value be＜0.1ng/ml (Pang Tatuolu A) is to 19ng/ml (VEE).In Rhesus Macacus, the 3165 proteinic half-life of CID are 90 hours and still can detect in back 14 days reaching to take medicine.In the human experimenter, CID 3165 protein be safe and toleration good.C after single injection is taken medicine
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 take medicine and have once obtained the support of pharmacokinetics.In single injection group (120-500 μ g), in most experimenters, observe antiviral response at hepatitis C.
In another embodiment, IFN-α-HSA fusions is used for the treatment of the patient of chronic hepatitis C infection (HCV).Interferon-ALPHA is also referred to as interferon alfa or LeIF, is treatment HCV infection patient's nursing standard.Term " interferon-ALPHA " refers to have the family of the homologous related polypeptide of height of antiviral activity.The interferon-ALPHA of IFN-α-HAS 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 that the present invention is contained includes but not limited to disclosed interferon-ALPHA protein in the table 1 therapeutic protein string.In specific embodiment, interferon-ALPHA partly comprises any commercialization form of Intederon Alpha-2a, Interferon Alpha-2b, interferon c, total interferon, interferon alfacon-1, interferon alfa-n1, Alferon N, interferon-ALPHA such as for example INTRON_A (Schering Corp., Kenilworth, N.J.), ROFERON_ (Hoffman-La Roche, Nutley, N.J.), Berofor interferon-alpha (Boehringer IngelheimPharmaceutical, Inc., Ridgefied, Conn.), OMNIFERON
TM(Viragen, Inc., Plantation, FL), MULTIFERON
TM(Viragen, Inc., Plantation, FL), WELLFERON_ (GlaxoSmithKline, London, Great Britian), INFERGEN_ (Amgen, Inc., Thousands Oaks, CA), SUMIFERON_ (Sumitomo, Japan), BELEROFON_ (Nautilus Biotech, France) or the interferon-ALPHA product of any purification or its fragment or form by it.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 Pegylation.Therefore, in other embodiments, the interferon-ALPHA of IFN-α-HSA fusion rotein partly comprises the Pegylation form of Intederon Alpha-2a, 2b or total interferon or is made up of it, and including but not limited to the commercialization glycol interferon alpha, such as for example PEG-INTRON_ (Schering Corp., Kenilworth, N.J.), PEGASYS_ (Hoffman-La 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 the HSA with any interferon-ALPHA protein known in the art or the fusion of its fragment.
Be used for the treatment of the interferon therapy scheme that HCV infects according to before whether accepting, subject suffering from HCV infection can be divided into two classes." patient who receives treatment first " refers to the patient of those never received interferon therapy scheme treatments." patient who once received treatment " refers to that those once accepted or accepting the patient of interferon therapy scheme treatment." not respondent " refers to such patient who once received treatment, and they had before accepted interferon therapy scheme treatment but main purpose such as the early stage virus load that do not reach treatment reduces (EVR) or treats whole end and reply (ETR).Yet, be used for this speech when literary composition, " HCV patient " is no matter referred to infect the patient of HCV and he is that receive treatment first, that once received treatment or respondent not.
In addition, hepatitis C virus can be divided into four kinds of genotype, 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.
The patient's that carries HCV standard care involved with the agent of interferon-ALPHA combination antiviral treat such as ribavirin (virazole).Usually, once a day, weekly twice, or use interferon-ALPHA once in a week and use ribavirin 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.
As mentioned above, CID 3165 proteinic pharmacokineticss are supported every 2-4 week or the longer time scheme of taking medicine once.Therefore, in another embodiment, antiviral agent once independent by every 2-4 week or the associating effective dose is used IFN-α-HSA fusions and is treated HCV patient.In preferred embodiments, use IFN-α-HSA fusions by the antiviral agent of once uniting effective dose 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 the antiviral agent of using effective dose.
In another embodiment, IFN-α-HSA fusions monotherapy of can be used as low dosage is used for the maintenance therapy of HCV.In another embodiment, IFN-α-HSA fusions can be united the treatment that ribavirin and one or more other antiviral agent are used for HCV.Perhaps, in another embodiment, IFN-α-HSA fusions can be united the treatment that one or more antiviral agent except that ribavirin are used for HCV.
In another embodiment, IFN-α-HSA fusions can be used for treating other viral 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, malignant melanoma, follicular lymphoma, chronic granulocytic leukemia, AIDS relevant Kaposi sarcoma, multiple myeloma or renal cell carcinoma.
In another embodiment, the GLP-1-HAS fusions is used to regulate the blood sugar level of diabetics.In another embodiment, the blood sugar level that the fused in tandem thing of wild type or saltant GLP-1 is used to regulate diabetics.For example, behind the diabetes db/db mouse subcutaneous injection GLP-1-HAS albumen of giving about 8 ages in week, assessed the ability of monomer GLP-1 (7-36A8G)-HAS and series connection GLP-1 (7-36A8G)-HSA (CID 3610) fusions blood sugar regulation level by oral glucose tolerance test (giving the 1g glucose) by the every kg body weight of oral gavage.This glucose tolerance test comprises subcutaneous injection GLP-1-HSA fusions, gives 1g glucose by the every kg body weight of oral gavage subsequently.Use for the diabetes db/db mice of fasting to wait the monomer or the series connection GLP-1-HSA fusion rotein of molar dose (100 and 171nmol/kg), and after the single dispenser, carried out oral glucose tolerance in 6 or 24 hours and test.Quite astonishing and unexpectedly, to compare with monomer GLP-1 (7-36A8G)-HSA fusions, series connection GLP-1 (7-36A8G)-HAS fusions (CID3610) is at back 6 hours remarkable blood sugar lowering of injection.In addition, when back 24 hours of injection is assessed diabetes db/db mice, the difference between monomer GLP-1 (7-36A8G)-HSA and series connection GLP-1 (7-36A8G)-HAS (CID 3610) fusions even more remarkable.As shown in figure 11; series connection GLP-1 (7-36A8G)-HAS fusions (CID 3610) (◇) has unexpectedly strong blood glucose normalization activity, however the fasting serum glucose level of single GLP-1 (7-36A8G)-HAS (Δ) fusions with obviously be that diabetes and the animal of using HSA (●) separately are similar in essence.
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, or 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 are external or the cell culture assays method is normally obtainable in this area for described therapeutic protein.The example of algoscopy includes but not limited to described in this paper embodiment part or table 1 " exemplary activation measurement " string (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 oligosaccharide groups usually such as cell surface and secreted protein.This modification is called glycosylation, but the proteinic physical characteristic of appreciable impact, and may be important for proteinic stability, secretion and location.Glycosylation betides along the ad-hoc location of polypeptide main chain.Two kinds of main type of glycosylation are arranged usually: the glycosylation that O-connects oligosaccharide that is characterized as that is attached to serine or threonine residues; Be attached to the glycosylation that is characterized as N-connection oligosaccharide of the asparagine residue in Asn-X-Ser or the Asn-X-Thr sequence, wherein X can be any aminoacid except that proline.N-n acetylneuraminic acid n (being also referred to as sialic acid) normally N-connects the terminal residue that is connected oligosaccharide with O-.Influence the quantity 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 analog 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 agedoite with glutamine, perhaps can by can not be with it in glycosylated host cell marking protein produce not glycosylated recombiant protein, for example in the yeast of escherichia coli 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 data base such as the GenSeq (as Derwent) of subscription to obtain from public database such as Chemical Abstracts Service data base (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 that the variant of described wild type polynucleotide sequence is (as the polynucleotide of encoding wild type therapeutic protein in some cases, the DNA sequence 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 SEQ ID 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 polypeptide or its fragment or variant in table 1 " therapeutic protein X " string (the 1st row).
Table 1 provides the therapeutic protein corresponding with the therapeutic protein of albumin fusion proteins of the present invention part or by the incomplete tabulation 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 round parentheses comprising this proteinaceous therapeutic molecule or its fragment or variant or by its proteinic formal name used at school and brand 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 list of references of the algoscopy of biologic activity.With complete being incorporated herein by reference of every piece of list of references of being quoted in " biologic activity " 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 list of references (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 albumin fusion construct of its albumin fusion proteins of forming.
Table 1
| Therapeutic protein: X | Biologic activity | Exemplary activation measurement | Preferred indication: Y | Construction ID | Therapeutic protein: Z |
| Human growth hormone (Pegvisamont; Somatrem; Somatropin; TROVERT; PROTROPIN; BIO-TROPIN; HUMATROPE; NUTROPIN; NUTROPIN AQ; NUTROPHIN; NORDITROPIN; GENOTROPIN; SAIZEN; SEROSTIM) | Transduce in conjunction with two GHR molecules and by the receptor dimerization inducement signal | The Ba/F3-hGHR proliferation assay is used for serum human growth hormone's novel specific bioassary method.J Clin Endocrinol Metab 2000 Nov; 85 (11): 4274-9 PGH (GH) immunoassay and tibia bioassary method.Appl Physiol 2000 Dec; 89 (6): the receptor-mediated cell-mediated propagation of 2174-8 growth hormone (hGH).Growth Horm IGF Res 2000 Oct; 10 (5): the international standard of 248-55 growth hormone.Horm Res 1999; 51 Suppll:7-12 | Acromegaly; Growth is not enough; Growth hormone is replaced (replacement); Growth hormone deficiency; Infant growth hormonoprivia; Adult's growth hormone deficiency; Retarded growth; Pu-Wei two syndromes; Pu-Wei two syndromes among 2 years old or the bigger child; Growth defect; The growth deficiency relevant with chronic renal insufficiency; Osteoporosis; Postmenopausal osteoporosis; Osteopenia; Osteoclast takes place; Burn; Cachexia; The cancer cachexia; Dwarfism; Metabolism disorder; Fat; Renal failure; Turner's syndrome; Fibromyalgia; Fracture is handled; Weak; The AIDS loss; Muscle loss; Short stature; Diagnostic agent; Infertility; Lipodystrophy. | 3468,3469,3470,3475. | Concrete construction sees Table 2 SEQ ID NO:Z |
| Interferon-ALPHA (Interferon Alpha-2b; Recombinant; Interferon alfa-n1; Alferon N; The PEG Interferon Alpha-2b; Ribavirin and Interferon Alpha-2b; Interferon alfacon-1; Total interferon; YM 643; CIFN; Total interferon-' alpha '; The total interferon of reorganization methionyl; Heavy | Give a series of cell responses, comprise antiviral, antiproliferative, antitumor and immunoregulation activity; Stimulate the generation of two kinds of enzymes: protein kinase and oligoadenylate synthetase. | Antiviral algoscopy: Rubinstein S, Familletti PC, Pestka S. (1981) Convenient assay for interferons. J. Virol. 37 (2): 755-8; Antiproliferative algoscopy: Gao Y, et al (1999) Sensitivity of an epstein-barr virus-positive tumor line, Daudi, to alpha interferon correlates with expression of a GC-rich viral transcript.Mol Cell | Viral infection comprises severe acute respiratory syndrome (SARS) and other coronavirus infection; Filovirus includes but not limited to Ebola virus and Marburg virus; Arenavirus includes but not limited to Pichende virus, Lassa virus, Junin virus, MAC, guanarito virus; And lymphocytic choriomeningitis virus (LCMV); Bunyavirus includes but not limited to Punta Toro virus, the Crimea peninsula-Congo hemorrhagic fever virus, phlebotomus fever virus, Rift valley fever virus, La Crosse virus and Hantaan virus; Banzi virus includes but not limited to yellow fever, BAN, west nile virus, dengue fever disease | 2249,2343,2366,2381,2382,2410,3165,3422,3423,3424,3476. | Concrete construction sees Table 2 SEQ ID NO:Z |
| Therapeutic raw egg white matter: X | Biologic activity | Exemplary activation measurement | Preferred indication: Y | Construction ID | Therapeutic protein: Z |
| The total interferon of group; CGP 35269; RO 253036; RO 258310; INTRON A; PEG-INTRON; OIF; OMNIFERON; PEG-OMNIFERO N; VELDONA; PEG-REBETRON; ROFERON A; WELLFERON; ALFERON N/LDO; REBETRON; ALTEMOL; VIRAFERONPE G; PEGASYS; VIRAFERON; VIRAFON; AMPLIGEN; INFERGEN; INFAREX; ORAGEN) | Biol.19 (11): 7305-13. | Poison, Japanese encephalitis virus, tick borne encephalitis, Omsk hemorrhagic fever and Kyasanur Forest disease virus; Togavirus includes but not limited to Venezuela, east and western equine encephalitis virus, ross river virus and rubella virus; Vaccinia subgroup virus includes but not limited to vaccine, cowpox, variola and monkeypox; Herpesvirus; Influenza A/B; Respiratory syncytial virus (RAV); Parainfluenza; Measles; Rhinovirus; Adenovirus; Semliki Forest virus; Viral hemorrhagic fever; Baculovirus; Paramyxovirus includes but not limited to Nipah virus and Heng Dela 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.2002; 20 (2): 311-30 and Darling et al., Emerg.Med.Clin.North Am. 2002; 20 (2): 273-309). | |||
| Interferon beta (interferon beta-1a; Interferon beta-1b; Interferon-beta-serine; SH 579; ZK 157046; BCDF; β-2 IF; Do | IFNg in regulation and control MHC antigen presentation, NK cytoactive and the mononuclear cell generates and IL12 generates. | Antiviral algoscopy: Rubinstein S, Familletti PC, Pestka S. (1981) Convenient assay for interferons.J.Virol. 37 (2): 755-8; Antiproliferative algoscopy: Gao Y, et al (1999) Sensitivity of an | Viral infection comprises severe acute respiratory syndrome (SARS) and other coronavirus infection; Filovirus includes but not limited to Ebola virus and Marburg virus; Arenavirus includes but not limited to Pichende virus, Lassa virus, Junin virus, MAC, guanarito virus; And lymphocytic choriomeningitis virus | 1778,1779,2011,2013,2053,2054,2492 | Concrete construction sees Table 2 SEQ ID NO:Z |
| Therapeutic protein: X | Biologic activity | Exemplary activation measurement | Preferred indication: Y | Construction ID | Therapeutic protein: Z |
| Disturb element-β-2; RhIL-6; SJ0031; DL 8234; FERON; Interferon beta; BETASERON; AVONEX; REBIF; BETAFERON; SIGOSIX) | epstein-barr virus-positive tumor line；Daudi,to alpha interferon correlates with expression of a GC-rich viral transcript. Mol Cell Biol.19 (11)： 7305-13. | (LCMV); Bunyavirus includes but not limited to Punta Toro virus, the Crimea peninsula-Congo hemorrhagic fever virus, phlebotomus fever virus, Rift valley fever virus, La Crosse virus and Hantaan virus; Banzi virus includes but not limited to yellow fever, BAN, west nile virus, dengue virus, Japanese encephalitis virus, tick borne encephalitis, Omsk hemorrhagic fever and Kyasanur Forest disease virus; Togavirus includes but not limited to Venezuela, east and western equine encephalitis virus, ross river virus and rubella virus; Vaccinia subgroup virus includes but not limited to vaccine, cowpox, variola and monkeypox; Herpesvirus; Influenza A/B; Respiratory syncytial virus (RAV); Parainfluenza; Measles; Rhinovirus; Adenovirus; Semliki Forest virus; Viral hemorrhagic fever; Baculovirus; Paramyxovirus includes but not limited to Nipah virus and Heng Dela 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.2002; 20 (2): 311-30 and Darling et al., Emerg.Med.Clin.North Am. 2002; 20 (2): 273-309). | 2580,2795,2796,2797. | ||
| Type B natriuretic peptide (BNP, brain natriuretic factor(peptide)) | Stimulate smooth muscle loosening and vasodilation, natriuresis and inhibition renin angiotensin and endothelin | The inhibitory action of angiotensin can be measured with algoscopy known in the art, for example uses Naunyn Schmiedebergs Arch Pharmacol 1999 May; 359 (5): the in-vitro multiplication algoscopy of describing among the 394-9 of carrying out with the rat heart fibroblast.Blood vessel relaxes | Congestive heart failure; The cardiac volume over loading; Cardiac decompensation; Heart failure; The left ventricle malfunction; Dyspnea.The glomerule hypertrophy; Glomerular injury; Glomerulopathy; Acute renal failure. | 3618,3690,3691,3715,3723,3724,3725,3736,3769 | Concrete construction sees Table 2 SEQ ID NO:Z |
| Therapeutic protein: X | Biologic activity | Exemplary activation measurement | Preferred indication: Y | Construction ID | Therapeutic protein: Z |
| Open and can be in animal to reply to measure and (see Am J Physiol Regul Integr Comp Physiol 2002 Aug by in isobaric arteriogram system, measuring little arteriorenal flesh source; 283 (2): R349-R355) .Natriuresis is measured by the sodium content of measuring in the urine. | 3778,3783,3795,3796,3809,3896,3897,3898,3899,3900. | ||||
| BDNF isoform c (neurotrophic factor derived from brain) | Promote nerve growth, differentiation and survival; Around in growth course, keeping and the survival of axoneuron subclass; In ripe nervous function, work; The neurotrophic factor that can help pain reaction. | BDNF can use neure growth and synaptic activity algoscopy to measure to the effect of neure growth, such as Bartrup et al (1997) Neuroreport 1:8 (17): described in the 3791-4; BDNF can measure by the behavior of measurement pain, hyperalgia and/or allodynia (allodynia) described in Shu et al Pain (1999) 80:463-470 and Zhou et al Eur.J. Neurosci. (2000) 12:100-105 the effect that pain receives. | Pain; Neuropathic pain; Complexity locality pain syndrome I; Reflex sympathetic dystrophy; Trigeminal neuralgia; Allodynia (allodynia); Constitutional and/or Secondary cases hyperpathia; Causalgia; Phantom pain; Postoperative pain; The burning feet syndrome; Ge-Ba two syndromes; Dejerine Roussy syndrome; Pain after the apoplexy; Vasculitis/angiopathy pain; Spontaneous pain; With following every relevant pain: entrapment neuropathy, nerve blocks, spinal cord injury, cicatrization, alcoholic neuropathy, pellagra, vitamin B1 deficiency, postherpetic neuralgia, HIV/AIDS pain, the vincristine neurotoxicity, the cisplatin neurotoxicity, the paclitaxel neurotoxicity, the thallium neurotoxicity, the arsenic neurotoxicity, radiotherapy, diabetes, malignant tumor, multiple sclerosis, the Fa Bulishi disease, Tangier, or amyloid. | 3549. | Concrete construction sees Table 2 SEQ ID NO:Z |
| Glucagon-like peptide 1 (GLP1; GLP-1; Pancreotropic hormone) | Stimulate the synthetic and release of insulin; Strengthen fat, muscle and liver organization sensitivity to insulin; Thorn | The GLP1 activity can use [3-H]-glucose uptake algoscopy at external test (J Biol Chem 1999 Oct 22; 274 (43): 30864-30873).GLP-1 is right | Hyperglycemia; Diabetes; Urine collapses disease; Diabetes; |
3610,3696. | Concrete construction sees Table 2 SEQ ID NO:Z |
| Therapeutic protein: X | Biologic activity | Exemplary activation measurement | Preferred indication: Y | Construction ID | Therapeutic protein: Z |
| Swash glucose uptake; Slow down digestion process; Appetite-suppressing; The secretion of blocking-up glucagon. | The influence of study can use passive avoidance and Mo Lisi water maze (MWM) model to study (Brain Res.1996 in rat, 716:29-38 and Nature 1982,297:681-683). | Disease, hyperglycemia, infection, retinopathy and/or ulcer; Metabolism disorder; Immunologic derangement; Fat; The blood vessel disorder; Body weight goes down; Loss of appetite; The X syndrome; Cognitive impaired; The loss of memory.Be used to strengthen study, memory, learning by association, space learning, Hippocampus plasticity, cognition and/or neuroprotective. | |||
| Lysosomal glucocerebrosidase; (Alglucerase; β-glucocerebrosidase; β-D-glucityl-N-acyl sphingosine glucose hydrolytic enzyme; Glucosylceramidase; Acid β-Pu Tangganmei; Imiglucerase; CEREDASE; CEREZYME) (GCB) | Catalysis D-glucityl-N-acyl sphingosine is hydrolyzed into D-glucose and N-acyl sphingosine, and the catalysis ceramide glucoside is hydrolyzed into the glucose ceramide. | Enzymatic activity can use means known in the art to assess, Poulos et al. for example, Clin.Chim.Acta.1976 Nov; 72 (3): the thin layer chromatography algoscopy of describing among the 327-335; Or Rudensky et al., Blood Cells Mol.Dis.2003 Jan-Feb; 30 (1): the FACS method of describing among the 97-99. | Gaucher disease, lysosomal storage disease | 3920,3921,3922,3923. | Concrete construction sees Table 2 SEQ ID NO:Z.In addition, for specific lysosomal glucocerebrosidase sequence or its variant, as seen GLCM HUMAN, Genbank registration number P04062; CAS-143003-46-7, all are incorporated herein by reference.U.S. Patent number 20040009165; 20030133924; Disclose other lysosomal glucocerebrosidase sequence in 20020127219, all be incorporated herein by reference.U.S. Patent number 6,696,272; 5,879,680; Open in 5,549,892 |
| Therapeutic protein: X | Biologic activity | Exemplary activation measurement | Preferred indication: Y | Construction ID | Therapeutic protein: Z |
| Other lysosomal glucocerebrosidase, all be incorporated herein by reference. | |||||
| Arginine deiminase (deiminase) (AD1-SS) | Irreversible citrulline and the ammonia of being hydrolyzed into of catalysis arginine; It is synthetic to suppress nitric oxide; Suppressing blood vessel takes place; Suppress the massive tumor cell proliferation. | Enzymatic activity can use method well-known in the art to measure, such as Weickmann et al., J.Biol. Chem.1977 Apr; 252 (8): the enzyme assay of describing among the 2615-2620.The inhibitory action that blood vessel takes place can use means known in the art to assess, Park et al. for example, Br.J. Cancer 2003 Sept; 89 (5): the Matrigel algoscopy of describing among the 907-914.The inhibition that nitric oxide generates can use means known in the art to assess, Noh et al. for example, Mol.Cells 2002 Feb; 13 (1): described in the 137-143.Tumor cell proliferation can use method well-known in the art to assess, Miyazaki et al. for example, Cancer Res. 1990 Aug; 50 (15): the proliferation assay described in the 4522-4527. | Cancer includes but not limited to melanoma and hepatocarcinoma.Other cancer includes but not limited to squamous cell carcinoma; Osteosarcoma; Glioma/astrocytoma; Glioblastoma; Promyelocytic leukemia; Lymphoblastic leukemia; And the cancer of cervix uteri, breast, ovary, prostate, colon or lung. | 3910,3915,3917,3918. | Concrete construction sees Table 2 SEQ ID NO:Z.In addition, for specific arginine deiminase sequence or its variant, see Baur et al., Eur.J.Biochem 1989 Jan; 179 (1): 53-60; Misawa et al., J.Biotechnol. 1994; 361:145-155; All be incorporated herein by reference.Disclose other arginine deiminase sequence in the U.S. Patent number 20040096437, be incorporated herein by reference.U.S. Patent number 6,180,387; 5,804,183; 5,474,928; Other arginine deiminase sequence is disclosed in 5,196,195, all |
| Therapeutic protein: X | Biologic activity | Exemplary activation measurement | Preferred indication: Y | Construction ID | Therapeutic protein: Z |
| Be incorporated herein by reference. | |||||
| Uricase (urate oxidase; Aspergillis flavus uricase; Candida utilis uricase; Rasburicase; FASTURTEC; ELITEK) | The catalysis uric acid is oxidized to allantoin. | Uric acid level can or use other method well-known in the art to measure by spectrophotography, colorimetry, such as Kageyama, Clin. Chim.Acta.1971; 31 (2): 421-426. | Gout; Hyperuricemia; Tumor dissolubility polyuria (hyperurecia); The polyuria of chemotherapy induction (hyperurecia); Cerebral infarction; Dyslipidemias; Cardiovascular disorder includes but not limited to hypertension, coronary artery disease, atherosclerosis. | 3903,3904,3936,3937,3938,3939,3940,3941,3942,3943,3944. | Concrete construction sees Table 2 SEQ ID NO:Z.In addition, for specific uricase sequence or its variant, see CAS-134774-45. 1; Wu et al., P.N. A.S.USA 1989 Dec; 86 (23): 9412-9416; All be incorporated herein by reference.U.S. Patent number 4,062,731; 4,273,874; 4,987,076; 5,376,545; 5,700,674; 5,728,562; 5,801,036; 5,834,273; 5,955, disclose other uricase sequence in 336, all be incorporated herein by reference. |
| The interferon hybrid, concrete preferred: IFN α A/D hybrid (BglII type); IFN α A/D | Give a series of cell responses, comprise antiviral, antiproliferative, antitumor and immunoregulation | Antiviral algoscopy: Rubinstein S, Familletti PC, Pestka S. (1981) Convenient assay for interferons.J.Virol. | Viral infection comprises severe acute respiratory syndrome (SARS) and other coronavirus infection; Filovirus includes but not limited to Ebola virus and Marburg virus; Viral infection; HIV infects; Hepatitis; Arenavirus, bag | 2872,2873,2874,2875,2876. | Concrete construction sees Table 2 SEQ ID NO:Z |
| Therapeutic protein: X | Biologic activity | Exemplary activation measurement | Preferred indication: Y | Construction ID | Therapeutic protein: Z |
| Hybrid (PvuII type); IFN α A/F hybrid; IFN α A/B hybrid; |
Active; Stimulate the generation of two kinds of enzymes: protein kinase and oligoadenylate synthetase.Also have, the IFNg that regulates in MHC antigen presentation, NK cytoactive and the mononuclear cell generates and the IL12 generation. | 37 (2): 755-8; Antiproliferative determination method: Gao Y; Et al (1999) Sensitivity of an epstein-barr virus-positive tumor line; Daudi, to alpha interferon correlates with expression of a GC-rich viral transcript. Mol Cell Biol.19 (11): 7305-13. | Draw together but be not limited to Pichende virus, Lassa virus, Junin virus, MAC, guanarito virus; And lymphocytic choriomeningitis virus (LCMV); Bunyavirus includes but not limited to Punta Toro virus, the Crimea peninsula-Congo hemorrhagic fever virus, phlebotomus fever virus, Rift valley fever virus, La Crosse virus and Hantaan virus; Banzi virus includes but not limited to yellow fever, BAN, west nile virus, dengue virus, Japanese encephalitis virus, tick borne encephalitis, Omsk hemorrhagic fever and Kyasanur Forest disease virus; Togavirus includes but not limited to Venezuela, east and western equine encephalitis virus, ross river virus and rubella virus; Vaccinia subgroup virus includes but not limited to vaccine, cowpox, variola and monkeypox; Herpesvirus; Influenza A/B; Respiratory syncytial virus (RSV); Parainfluenza; Measles; Rhinovirus; Adenovirus; Semliki Forest virus; Viral hemorrhagic fever; Baculovirus; Paramyxovirus includes but not limited to Nipah virus and Heng Dela 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. 2002; 20 (2): 311-30 and Darling et al., Emerg. Med.Clin.North Am.2002; 20 (2): 273-309). | ||
| IL-2 (Aldesleukin; The interleukin-2 fusion toxin; The T cell growth factor; PROLEUKIN; IMMUNACE; CELEUK; | Promote the growth of B and T cell and the killing activity of raising NK cell and CTL cell. | T cell proliferating determining method " Biological activity of recombinant human interleukin-2 produced in Escherichia coli. " Science 223:1412-1415,1984.Natural | Cancer; Solid tumor; The pancreas cancer; Colon cancer; Hepatocarcinoma; The T-lymphoma; Graft versus host disease (GVH); Autoimmune disease; The autoimmune sexual disorder; IL-2 receptor positive malignant tumor. | 1757,1758,1812,1813,1952,1954. | Concrete construction sees Table 2 SEQ ID NO:Z |
| Therapeutic protein: X | Biologic activity | Exemplary activation measurement | Preferred indication: Y | Construction ID | Therapeutic protein: Z |
| ONCOLIPIN2; MACROLIN) | Kill and wound (NK) cell and CTL cytotoxicity assay " Control of homeostasis of CD8+ memory T cells by opposing cytokines. " Science 288:675-678,2000.CTLL-2 propagation: Gillis et al (1978) J. Immunol.120:2027. | ||||
| Medulla renis quality (ADM) | Suppress kidney Na +-K +-ATP enzyme; Synthesizing of the PGE2 of enhancing adjusting smooth muscle contraction and diastole and vasodilation and contraction; Suppress the aldosterone secretion; Cause diuresis and natriuresis.Involve the salt/water/electrolyte balance of blood pressure regulating and body fluid; The protection kidney. | Kidney Na +-K +-atpase activity can use algoscopy known in the art to measure, such as Ku et al., 1987; Endocrinology 120:2166-2173.Vasodilation can use algoscopy known in the art to measure (Ashton et al. Pharmacology 2000; 61 (2): 101-105).The synthetic of PGE2 can use algoscopy known in the art to measure (Cheng et al., J Endocrinol.2004 Aug; 182 (2): 249-56).The aldosterone level can use means known in the art to measure, for example, Yamato et al., Circ J 2003 May; 67 (5): 384-90.Blood pressure can or use other method known in the art to measure with sphygomanometer, such as Reddy et al., Ultrasound Med Biol 2003 Mar; 29 (3): 379-85.Urine | The treatment congestive heart failure; Hypertension; Cardiovascular disease; Acute renal failure; Acute tubular necrosis; Nephropathy; Glomerulopathy; The cardiac volume over loading; Cardiac decompensation; The left ventricle malfunction; Dyspnea.The aldosterone level that treatment raises, it can cause vasoconstriction, cardiac output to weaken and/or hypertension; Heart failure; Reperfusion injury of cardiac muscle; Left ventricle is moulded again. | 3924,3925,3926,3927. | Concrete construction sees Table 2 SEQ ID NO:Z.Also visible Vesely Am J Physiol Renal Rhysiol 2003; 285:F167-177 is introduced into as a reference. |
| Therapeutic protein: X | Biologic activity | Exemplary activation measurement | Preferred indication: Y | Construction ID | Therapeutic protein: Z |
| Sodium increases by the sodium amount of measuring in the urine to be measured.Diuresis is measured by measuring excretory urine amount. | |||||
| Long-acting natriuretic peptide (LANP; ProANP-(31-67)) | Suppress kidney Na +-K +-ATP enzyme; Synthesizing of the PGE2 of enhancing adjusting smooth muscle contraction and diastole and vasodilation and contraction; Suppress plasma renin activity; Cause diuresis and natriuresis.Involve the salt/water/electrolyte balance of blood pressure regulating and body fluid; The protection kidney. | Kidney Na +-K +-atpase activity can use algoscopy known in the art to measure, such as Ku et al., 1987; Endocrinology 120:2166-2173.Vasodilation can use algoscopy known in the art to measure (Ashton et al. Pharmacology 2000; 61 (2): 101-105).The synthetic of PGE2 can use algoscopy known in the art to measure (Cheng et al., J Endocrinol.2004 Aug; 182 (2): 249-56).Blood pressure can or use other method known in the art to measure with sphygomanometer, such as Reddy et al., Ultrasound Med Biol 2003 Mar; 29 (3): 379-85.Natriuresis is measured by the sodium amount of measuring in the urine.Diuresis is measured by measuring excretory urine amount. | The treatment congestive heart failure; Hypertension; Cardiovascular disease; The cardiac volume over loading; Cardiac decompensation; The left ventricle malfunction; Dyspnea; Acute tubular necrosis; Acute renal failure; Renal tubules regeneration; Chronic renal failure; Nephropathy; Glomerulopathy. | 3886,3887. | Concrete construction sees Table 2 SEQ ID NO:Z.Also visible Vesely Am J Physiol Renal Rhysiol 2003; 285:F167-177 is introduced into as a reference. |
| Vasodilatin (VDP; ProANP-(79-98)) | Suppress kidney Na +-K +-ATP enzyme; Strengthen and regulate smooth muscle contraction and diastole and vasodilation and contraction | Kidney Na +-K +-atpase activity can use algoscopy known in the art to measure, such as Ku et al., 1987; Endocrinology 120:2166-2173.Vasodilation can use algoscopy known in the art to survey | The treatment congestive heart failure; Hypertension; Cardiovascular disease; The cardiac volume over loading; Cardiac decompensation; The left ventricle malfunction; Dyspnea; Acute tubular necrosis; Acute renal failure; Nephropathy; Glomerulopathy.The aldosterone level that treatment raises, it can cause vasoconstriction, cardiac output to weaken and/or high blood | 3888,3889 | Concrete construction sees Table 2 SEQ ID NO:Z.Also visible Vesely Am J Physiol Renal Rhysiol 2003; 285: |
| Therapeutic protein: X | Biologic activity | Exemplary activation measurement | Preferred indication: Y | Construction ID | Therapeutic protein: Z |
| PGE2 synthetic; Suppress the aldosterone secretion; In patients with congestive heart failure, cause natriuresis; Cause kaliuresis.Involve the salt/water/electrolyte balance of blood pressure regulating and body fluid; The protection kidney. | Amount (Ashton et al. Pharmacology 2000; 61 (2): 101-105).The synthetic of PGE2 can use algoscopy known in the art to measure (Cheng et al., J Endocrinol.2004 Aug; 182 (2): 249-56).The aldosterone level can use algoscopy known in the art to measure, Yamato et al. for example, Circ J 2003 May; 67 (5): 384-90.Blood pressure can or use other method known in the art to measure with sphygomanometer, such as Reddy et al., Ultrasound Med Biol 2003 Mar; 29 (3): 379-85.Natriuresis is measured by the sodium amount that detects in the urine.Kaliuresis is measured by the potassium amount of measuring in the urine. | Press; Heart failure; Reperfusion injury of cardiac muscle; Left ventricle is moulded again. | F167-177 is introduced into this paper as a reference. | ||
| Diuresis potassium peptide (KUP; ProANP-(99-126)) | Involve the salt/water/electrolyte balance of blood pressure regulating and body fluid. | Blood pressure can or use other method known in the art to measure with sphygomanometer, such as Reddy et al., Ultrasound Med Biol 2003 Mar; 29 (3): 379-85.Natriuresis is measured by the sodium amount of measuring in the urine.Diuresis is measured by measuring excretory urine amount. | The treatment congestive heart failure; Hypertension; Cardiovascular disease; Acute tubular necrosis; Acute renal failure; Nephropathy; Glomerulopathy. | 3890,3891. | Concrete construction sees Table 2 SEQ ID NO:Z.Also visible Vesely Am J Physiol Renal Rhysiol 2003; 285:F167-177 is introduced into this paper as a reference. |
| C type natriuretic peptide | Promote diuresis and urine sodium | Natriuresis is by measuring in the urine | The treatment congestive heart failure; Hypertension; Cardiovascular disease; | 3892, | Concrete construction sees Table |
| Therapeutic protein: X | Biologic activity | Exemplary activation measurement | Preferred indication: Y | Construction ID | Therapeutic protein: Z |
| (CNP) | Increase.Involve the salt/water/electrolyte balance of blood pressure regulating and body fluid. | The sodium amount is measured.Diuresis is measured by measuring excretory urine amount. | Acute tubular necrosis; Acute renal failure; Renal tubules regeneration; Chronic renal failure; Nephropathy; Glomerulopathy. | 3893. | 2?SEQ?ID?NO：Z。Also visible Vesely Am J Physiol Renal Rhysiol 2003; 285 F167-177 are introduced into this paper as a reference. |
| Mamba (Dendroaspis) natriuretic peptide (DNP) | Suppress kidney Na +-K +-ATP enzyme; Synthesizing of the PGE2 of enhancing adjusting smooth muscle contraction and diastole and vasodilation and contraction; Suppress the aldosterone secretion; Cause diuresis and natriuresis.Involve the salt/water/electrolyte balance of blood pressure regulating and body fluid; The protection kidney. | Kidney Na +-K +-atpase activity can use algoscopy known in the art to measure, such as Ku et al., 1987; Endocrinology 120:2166-2173.Vasodilation can use algoscopy known in the art and measure (Ashton et al. Pharmacology 2000; 61 (2): 101-105).The synthetic of PGE2 can use algoscopy known in the art to measure (Cheng et al., J Endocrinol.2004 Aug; 182 (2): 249-56).The aldosterone level can use means known in the art to measure, Yamato et al. for example, Circ J 2003 May; 67 (5): 384-90.Blood pressure can or use other method known in the art to measure with sphygomanometer, such as Reddy et al., Ultrasound Med Biol 2003 Mar; 29 (3): 379-85.Urine | The treatment congestive heart failure; Hypertension; Cardiovascular disease; Acute renal failure; Acute living renal tubular necrosis; Nephropathy; Glomerulopathy; The cardiac volume over loading; Cardiac decompensation; The left ventricle malfunction; Dyspnea.The aldosterone level that treatment raises, it can cause vasoconstriction, cardiac output to weaken and/or hypertension; Heart failure; Reperfusion injury of cardiac muscle; Left ventricle is moulded again. | 3894,3895. | Concrete construction sees Table 2 SEQ ID NO:Z.Also visible Vesely Am J Physiol Renal Rhysiol 2003; 285:F167-177 is introduced into this paper as a reference. |
| Therapeutic protein: X | Biologic activity | Exemplary activation measurement | Preferred indication: Y | Construction ID | Therapeutic protein: Z |
| Sodium increases by the sodium amount of measuring in the urine to be measured.Diuresis is measured by measuring excretory urine amount. | |||||
| (Fn 14 for TweakR; Fibroblast growth factor derivable 14) | Stimulate apoptotic cell death; Induction of vascular takes place; Promote endothelial cell migration and propagation; After sudden change, cause susceptibility to the Klippel-Trenaunay syndrome. | Apoptotic cell death can use algoscopy known in the art to measure, Nakayama et al. for example, J Immunol.2003 |
Treatment Klippel-Trenaunay syndrome; Cancer; Solid tumor; The pancreas cancer; Colon cancer; Hepatocarcinoma; The brain cancer; Gastric cancer; Pulmonary carcinoma; Breast carcinoma; Osteocarcinoma; Autoimmune disease; The autoimmune sexual disorder; Lupus; Rheumatoid arthritis; Multiple sclerosis; Blood vessel gets muddled; Cardiovascular disease. | 3919,3932. | Concrete construction sees Table 2 SEQ ID NO:Z |
| Kiss-1 (Metastin; Kisspeptin-54) | Suppress neoplasm metastasis; Activated G protein-coupled receptor GPR54 suppresses tumour progression; The secretion of control promoting sexual gland hormone. | The activation of GPR54 can use algoscopy known in the art to measure, Becker et al. for example, Biochem Biophys Res Commum. 2005 |
The treatment cancer; Gastric cancer; Breast carcinoma; The esophagus squamous cell carcinoma; Bladder cancer; Solid tumor; Thyroid carcinoma; The pancreas cancer; Colon cancer; Hepatocarcinoma; Pulmonary carcinoma; The brain cancer; Osteocarcinoma; Promoting sexual gland hormone lacks; Sterility and infertility; Hypopituitarism; Panhypopituitarism; Hypomenorrhea; Amenorrhea; Sexual anesthesia; Hectic fever; Dyspareunia; Sexual impotence; Osteopenia; Hypogonadotropic hypogonadism; Kalman's syndrome; Secondary hypogonadism. | 3928,3929. | Concrete construction sees Table 2 SEQ ID NO:Z.Also visible Dhar et al., Int J Cancer. 2004 |
| Ephrin B1 | Modulate tumor growth and | Tumor growth, blood vessel take place, migration | Cancer; Solid tumor; The pancreas cancer; Colon cancer; Hepatocarcinoma; The brain cancer; | 3930, | Concrete construction sees Table |
| Therapeutic raw egg white matter: X | Biologic activity | Exemplary activation measurement | Preferred indication: Y | Construction ID | Therapeutic protein: Z |
| (EFNB1) | Blood vessel takes place; The migration and the invasion of regulation and control cancerous cell; Strengthen platelet aggregation and grumeleuse maturation; Stimulate T cell proliferation, lymphokine to generate and the CTL activity; Stimulating endothelial cell migration and propagation; For normally cochlear function is required. | Can use algoscopy known in the art to measure (Nakada et al., Cancer Res.2004 May 1 with invasion; 64 (9): 3179-85 or Noren et al., Proc Natl Acad Sci USA. 2004 Apr 13; 101 (15): 5583-8).T cell proliferation, lymphokine generate and the CTL activity can use algoscopy known in the art to measure, Yu et al. for example, J Biol.Chem.2004; 279 (53): 55531-55539.Endothelial cell migration and propagation can use means known in the art to measure, Woods et al. for example, J Biol Chem.2002 Jan 18; 277 (3): 1924-7.Cochlear function can use algoscopy known in the art to measure (Howard et al., Hear Res. 2003 Apr; 178 (1-2): 118-30). | Colorectal carcinoma; Gastric cancer; Pulmonary carcinoma; Osteocarcinoma; Breast carcinoma; Cardiovascular disease; Hepatocarcinoma; The T lymphoma; Graft versus host disease (GVH); Autoimmune disease; The autoimmune sexual disorder; Hemophilia; Feng's von Willebrand disease; Hemorrhage disorder; By disorder afflicting connective tissue, leukemia, myeloma, the heart/pulmonary shunt art, kidney dialysis or drug-induced hemorrhage disorder; Blood vessel gets muddled; The cochlea malfunction; Hearing disability; Dizzy; Tinnitus; Bulge of the ear; Acute and/or chronic internal ear malfunction. | 3931,3934,3953. | 2 SEQ ID NO:Z |
| B7-H3 | Strengthen tumour immunity; Modulating T cell activates and immunne response. | Tumour immunity can be measured by means known in the art, Luo et al. for example, J Immunol.2004 |
Cancer; Solid tumor; The pancreas cancer; Colon cancer; Hepatocarcinoma; Pulmonary carcinoma; Osteocarcinoma; Breast carcinoma; The brain cancer; Autoimmune disease; The autoimmune sexual disorder; Molten bone osteopathia; The hypercalcemia of malignant tumor; The inductive osteodynia of malignant tumor. | 3933. | Concrete construction sees Table 2 SEQ ID NO:Z |
Table 2
| Fusions No. | Construction ID | The construction title | Describe | Expression vector | SEQ ID NO:Y | SEQ ID NO:X | SEQ ID NO:Z | SEQ ID NO:A | SEQ ID NO: | Targeting sequencing | |
| 1 | 3422 | PSAC35:APsp.HSA. IFNa | Ripe HSA and IFN α in succession after the acid phosphatase signal peptide. | PSAC35 | 149 | 117 | 181 | 213 | 214 | |
|
| 2 | 3423 | PSAC35:INVsp.HS A.IFNa | Ripe HSA and IFN α in succession after the invertase signal peptide. | |
150 | 118 | 182 | 215 | 216 | Invertase | |
| 3 | 3424 | PSAC35:KTsp.HSA. IFNa | Kill and wound in succession after the toxin signal peptide ripe HSA and IFN α. | PSAC35 | 151 | 119 | 183 | 217 | 218 | Kill and wound |
|
| 4 | 2249 | PSAC35:IFNa2-HS A is also referred to as pSAC23:IFNa2-HS A | |
PSAC35 | 152 | 120 | 184 | 219 | 220 | HSA/ |
|
| 5 | 2343 | PSAC35:INV-IFNA 2-HSA | |
PSAC35 | 153 | 121 | 185 | 221 | 222 | Invertase | |
| 6 | 2366 | PSAC35:MAF-IFNa 2-HSA | |
PSAC35 | 154 | 122 | 186 | 223 | 224 | MF α-1 | |
| 7 | 2381 | PC4:HSA-IFNa2 (C1 7-E181) | The aminoacid C17-E181 of IFN α 2 (aminoacid C1-E165 fragment shown in the SEQ ID NO:618) merges the downstream at HSA. | PC4 | 155 | 123 | 187 | 225 | 226 | HSA | |
| 8 | 2382 | PC4:IFNa2-HSA | The upstream that |
PC4 | 156 | 124 | 188 | 227 | 228 | |
|
| 9 | 2410 | PSAC35INV:IFNa-HSA | |
PSAC35 | 157 | 125 | 189 | 229 | 230 | Invertase | |
| 10 | 3165 | PSAC35:HAS.IFNa is also referred to as CID 3165, pSAC35:HAS.INFa | HSA merges in the upstream of IFN α and the downstream of HSA/kex2 targeting sequencing. | PSAC35 | 158 | 126 | 190 | HSA/kex2 | |||
| 11 | 1778 | PSAC35:IFNbeta.M | Residue M22-N187 (the SEQ ID of total length IFNb | PSAC35 | 159 | 127 | 191 | 231 | 232 | HSA/kex2 |
| Fusions No. | Construction ID | The construction title | Describe | Expression vector | SEQ ID NO:Y | SEQ ID NO:X | SEQ ID NO:Z | SEQ ID NO:A | SEQ ID NO:B | Targeting sequencing |
| 22-N187:HSA | M1-N166 shown in the NO:463) merges in the upstream of ripe HSA and the downstream of HSA/kex2 targeting sequencing. | |||||||||
| 12 | 1779 | PSAC35:HSA:IFNb eta.M22-N187 | The residue M22-N187 of total length IFNb (M1-N166 shown in the SEQ ID NO:464) merges the downstream at the HSA that has the HSA/kex2 targeting sequencing. | |
160 | 128 | 192 | HSA/kex2 | ||
| 13 | 2011 | PC4:IFNb-HSA | Total length IFNb merges the upstream at ripe HSA. | |
161 | 129 | 193 | 233 | 234 | Natural IFNb targeting sequencing |
| 14 | 2013 | PC4:HSA-IFNb.M2 2-N187 | The aminoacid M22-N187 of IFNb (aminoacid M1-N166 fragment shown in the SEQ ID NO:527) merges the downstream at HSA. | PC4 | 162 | 130 | 194 | HSA | ||
| 15 | 2053 | PEE12:IFNb-HSA is also referred to as pEE12.1:IFNbeta-H SA | Total length IFNb merges the upstream at ripe HSA. | PEE12.1 | 163 | 131 | 195 | Natural IFNb targeting sequencing | ||
| 16 | 2054 | PEE12:HSA-IFNb | Ripe IFNb merges the downstream at HSA. | PEE12.1 | 164 | 132 | 196 | HSA | ||
| 17 | 2492 | PC4:IFNb (deltaM22) .HSA | Mutant total length IFN β merges the upstream at ripe HSA.First residue (M22) deletion of natural ripe IFN β. | PC4 | 165 | 133 | 197 | Natural IFN β targeting sequencing | ||
| 18 | 2580 | PC4:IFNb (deltaM22, C38S) .HSA | IFNb merges the upstream at ripe HSA.Used IFNb lacks first residue of IFNb mature form in this fusions, and it is corresponding to the M22 of SEQ ID NO:1687.Simultaneously SEQ ID NO:1687 aminoacid 38 sports Ser by Cys. | PC4 | 166 | 134 | 198 | Natural IFN β | ||
| 19 | 2795 | PC4:HSA (A14)-IFN b.M22-N187 | The mature form of IFNb merges the C-terminal at HSA, and it contains modified signal peptide, is designed for to improve processing and homogeneity. | PC4 | 167 | 135 | 199 | Modified HSA (A14) | ||
| 20 | 2796 | PC4:HSA (S14)-IFN | The mature form of IFNb merges the C-terminal at HSA, | PC4 | 168 | 136 | 200 | Modified |
| Fusions No. | Construction ID | The construction title | Describe | Expression vector | SEQ ID NO:Y | SEQ ID NO:X | SEQ ID NO:Z | SEQ ID NO:A | SEQ ID NO:B | Targeting sequencing |
| B.M22-N187 | It contains modified signal peptide, is designed for to improve processing and homogeneity. | HSA (S14) | ||||||||
| 21 | 2797 | PC4:HSA (G14)-IFN b.M22-N187 | The mature form of IFNb merges the C-terminal at HSA, and it contains modified signal peptide. | PC4 | 169 | 137 | 201 | Modified HSA (G14) | ||
| 22 | 2872 | PSAC35:HAS.IFNa A (C1-Q91)/D (L93-E 166) | This construction contains IFN α A and IFN α D and merges hybrid form in ripe HSA downstream. | PSAC35 | 170 | 138 | 202 | 235 | 236 | HSA/kex2 |
| 23 | 2873 | PSAC35:HAS.IFNa A (C1-Q91)/B (L93-E 166) | This construction contains IFN α A and IFN α B and merges hybrid form in ripe HSA downstream. | PSAC35 | 171 | 139 | 203 | 237 | 238 | HSA/kex2 |
| 24 | 2874 | PSAC35:HAS.IFNa A (C1-Q91)/F (L93-E 166) | This construction contains IFN α A and IFN α F and merges hybrid form in ripe HSA downstream. | PSAC35 | 172 | 140 | 204 | 239 | 240 | HSA/kex2 |
| 25 | 2875 | PSAC35:HAS.IFNa A (C1-Q62)/D (Q64-E166) | This construction contains IFN α A and IFN α D and merges hybrid form in ripe HSA downstream. | PSAC35 | 173 | 141 | 205 | 241 | 242 | HSA/kex2 |
| 26 | 2876 | PSAC35:HAS.IFNa A (C1-Q91)/D (L93-E 166); R23K, A 113V | This construction contains IFN α A and IFN α D and merges hybrid form in ripe HSA downstream. | PSAC35 | 174 | 142 | 206 | 243 | 244 | HSA/kex2 |
| 27 | 1757 | PSAC35:IL2.A21-T 153.145C/S.HSA | The ripe human IL-2 of containing single amino acids sudden change (145 C sport S) is cloned in the downstream of HSA/kex2 targeting sequencing and the upstream of ripe HSA. | PSAC35 | 175 | 143 | 207 | HSA/kex2 | ||
| 28 | 1758 | PSAC35:HSA.IL2.A 21-T153.145C/S | The ripe human IL-2 of containing single amino acids sudden change (145 C sport S) is cloned in the downstream of the HSA of band HSA/kex2 targeting sequencing. | PSAC35 | 176 | 144 | 208 | HSA/kex2 | ||
| 29 | 1812 | PSAC35:IL2.A21-T 153.HSA | The aminoacid A21-T153 of IL-2 merges in the downstream of HSA/kex2 targeting sequencing and the upstream of ripe HSA. | pSAC35 | 177 | 145 | 209 | HSA/kex2 |
| Fusions No. | Construction ID | The construction title | Describe | Expression vector | SEQ ID NO:Y | SEQ ID NO:X | SEQ ID NO:Z | SEQ ID NO:A | SEQ ID NQ: | Targeting sequencing | |
| 30 | 1813 | PSAC35:HSA.IL2.A 21-T153 | The aminoacid A21-T1 53 of IL-2 merges the downstream at the HSA of band HSA/kex2 targeting sequencing. | PSAC35 | 178 | 146 | 210 | HSA/kex2 | |||
| 31 | 1952 | PcDNA3.1:IL2. HSA | Amino acid/11 45 is the upstream of total length human IL-2's fusion of serine at ripe HSA from cysteine mutation. | PCDNA 3.1 | 179 | 147 | 211 | Natural IL-2 targeting sequencing | |||
| 32 | 1954 | PC4:IL2.HSA | Amino acid/11 45 is the upstream of total length human IL-2's fusion of serine at ripe HSA from cysteine mutation. | |
180 | 148 | 212 | Natural IL-2 targeting sequencing | |||
| 33 | 3468 | PSAC35:KTsp.HSA. GH | Kill and wound the ripe HSA that ins succession after the toxin signal peptide growth hormone of ining succession again. | PSAC35 | 313 | 245 | 381 | 449 | 450 | Kill and wound toxin Qian Yuan district | |
| 34 | 3469 | PSAC35:INVsp.HS A.GH | The ripe HSA that ins succession after the invertase signal peptide growth hormone of ining succession again. | PSAC35 | 314 | 246 | 382 | 451 | 452 | Invertase | |
| 35 | 3470 | PSAC35:APsp.HSA. GH | The ripe HSA that ins succession after the acid phosphatase signal peptide growth hormone of ining succession again. | PSAC35 | 315 | 247 | 383 | 453 | 454 | Acid phosphatase | |
| 36 | 3475 | PSAC35:G19Rsp.H SA.GH | The ripe HSA that ins succession after the modified HSA/Kex2 signal sequence growth hormone of ining succession again. | PSAC35 | 316 | 248 | 384 | 455 | 456 | Modified HSA/Kex2 | |
| 37 | 3476 | PSAC35:G19Rsp.H SA.IFNa | The ripe HSA that ins succession after the modified HSA/kex2 signal sequence IFN α that ins succession again. | PSAC35 | 317 | 249 | 385 | 457 | 458 | Modified HSA/Kex2 | |
| 38 | 3549 | PSAC35:HSA/kex2. BDNFc.HSA | The ripe BDNFc (splice variant 6) that ins succession after the HSA/kex2 targeting sequencing ripe HSA that ins succession again. | PSAC35 | 318 | 250 | 386 | Do not have | Do not have | HSA/Kex2 | |
| 39 | 3610 | PSAC35:(HSA/KEX (R19G)) SP.GLP-1 (7-36A8G) x2.HSA | The codon optimized glp-1 dimer of yeast (wherein second dimer changed make that the swing position of each codon is all different with first dimer) of ining succession after the modified HSA/kex2 targeting sequencing merges at the N-of HSA end. | PSAC35 | 319 | 251 | 387 | 459 | 460 | Modified HSA/ |
|
| 40 | 3690 | PC4:MPIFSP.BNP/ HSA | The BNP that ins succession after bone marrow ancestors inhibitive factor-1 (MPIF) signal sequence merges at the N-of ripe HSA end. | |
320 | 252 | 388 | 461 | 462 | MPIF-1 | |
| 41 | 3691 | PC4:SPCON.BNP/H SA | The BNP that ins succession after the shared signal sequence merges at the N-of ripe HSA end. | PC4 | 321 | 253 | 389 | 463 | 464 | Consensus sequence |
| Fusions No. | Construction ID | The construction title | Describe | Expression vector | SEQ ID NO:Y | SEQ ID NO:X | SEQ ID NO:Z | SEQ ID NO:A | SEQ ID NO:B | Targeting sequencing |
| 42 | 3696 | PEE12.1:GLP-1 (7-3 6 (A8G) x2.HSA | The codon optimized glp-1 dimer of yeast (second binary changed makes that the swing position of each codon is all different with first dimer) of ining succession after bone marrow ancestors inhibitive factor-1 (MPIF) signal sequence merges at the N-of HSA end. | PEE12.1 | 322 | 254 | 390 | 465 | 466 | MPIF-1 |
| 43 | 3715 | PSAC35:BNP29/HS A.S65 | It is the N-end of the terminal truncate of N-(Δ 1-64) of HSA that the people BNP of single copy (amino acid/11-29) merges at HSA (S65-L585).This is in the downstream of HSA/Kex2 signal sequence. | PSAC35 | 323 | 255 | 391 | 467 | 468 | HSA/Kex2 |
| 44 | 3723 | PEE12.1:MPIFSP.B NP/HSA | The BNP that ins succession after bone marrow ancestors inhibitive factor-1 (MPIF) signal sequence merges at the N-of ripe HSA end. | PEE12.1 | 324 | 256 | 392 | Do not have | Do not have | MPIF-1 |
| 45 | 3724 | PEE12.1:SPCON.B NP/HSA | The BNP that ins succession after the shared signal sequence merges at the N-of ripe HSA end. | PEE12.1 | 325 | 257 | 393 | Do not have | Do not have | Consensus sequence |
| 46 | 3725 | PEE12.1:SPCON2.B NP/HSA | The BNP that ins succession after the shared signal sequence merges at the N-of ripe HSA end. | PEE12.1 | 326 | 258 | 394 | Do not have | Do not have | Shared |
| 47 | 3736 | PC4:SPCON2.BNP/ HSA | The BNP that ins succession after the shared signal sequence merges at the N-of ripe HSA end. | PC4 | 327 | 259 | 395 | 469 | 470 | Shared |
| 48 | 3769 | PC4:BNP (R13G)/HS A | The BNP mutant (R13G) of ining succession after bone marrow ancestors inhibitive factor-1 (MPIF) signal sequence merges at the N-of ripe HSA end. | PC4 | 328 | 260 | 396 | 471 | 472 | MPIF-1 |
| 49 | 3778 | PC4:SPCON.BNP29/HSA.S65 | It is the N-end of the terminal truncate of N-(Δ 1-64) of HSA that the people BNP of single copy (amino acid/11-29) merges at HSA (S65-L585).This is in the downstream of shared signal sequence. | PC4 | 329 | 261 | 397 | 473 | 474 | |
| 50 | 3783 | PC4:SPCON.BNP (R 13G)/HSA | The BNP mutant (R13G) of ining succession after the shared signal sequence merges at the N-of ripe HSA end. | PC4 | 330 | 262 | 398 | 475 | 476 | Consensus sequence |
| 51 | 3795 | PC4:SPCON.BNP (K | The BNP mutant of ining succession after the shared signal sequence | PC4 | 331 | 263 | 399 | 477 | 478 | Consensus sequence |
| Fusions No. | Construction ID | The construction title | Describe | Expression vector | SEQ ID NO:Y | SEQ ID NO:X | SEQ ID NO:Z | SEQ ID NO:A | SEQ ID NO:B | Targeting sequencing |
| 14G)/HSA | (K14G), merge at the N-of ripe HSA end. | |||||||||
| 52 | 3796 | PSAC35:HSA/BNP | The ripe HSA that ins succession afterwards merges at the N-of BNP end. | PSAC35 | 332 | 264 | 400 | 479 | 480 | HSA/Kex2 |
| 53 | 3809 | PSAC35:BNP30 (GG G)/HSA | The BNP (amino acid/11-30) that ins succession after the HSA/Kex2 signal sequence merges at the N-of ripe HSA end through three glycine. | PSAC35 | 333 | 265 | 401 | 481 | 482 | HSA/Kex2 |
| 54 | 3886 | PSAC35:HSA/KEX 2.LANP.HSA | The LANP that ins succession after the HSA/Kex2 signal sequence merges at the N-of ripe HSA end.LANP is corresponding to the aminoacid 26-55 (being referred to herein as LANP) of SEQ ID No:402. | PSAC35 | 334 | 266 | 402 | Do not have | Do not have | HSA/K |
| 55 | 3887 | PSAC35:HSA/KEX 2.HSA.LANP | The ripe HSA that ins succession after the HSA/Kex2 signal sequence merges at the N-of ripe LANP end. | PSAC35 | 335 | 267 | 403 | Do not have | Do not have | HSA/Kex2 |
| 56 | 3888 | PSAC35:HSA/KEX 2.VDP.HSA | The VDP that ins succession after the HSA/Kex2 signal sequence merges at the N-of ripe HSA end.VDP is corresponding to the aminoacid 56-92 (being referred to herein as VDP) of SEQ ID No:404. | PSAC35 | 336 | 268 | 404 | Do not have | Do not have | HSA/Kex2 |
| 57 | 3889 | PSAC35:HSA/KEX 2.HSA.VDP | The ripe HSA that ins succession after the HSA/Kex2 signal sequence merges at the N-of ripe VDP end. | PSAC35 | 337 | 269 | 405 | Do not have | Do not have | HSA/Kex2 |
| 58 | 3890 | PSAC35:HSA/KEX 2.KUP.HSA | The KUP that ins succession after the HSA/Kex2 signal sequence merges at the N-of ripe HSA end.KUP is corresponding to the amino acid/11 04-123 (being referred to herein as KUP) of SEQ ID No:406. | PSAC35 | 338 | 270 | 406 | Do not have | Do not have | HSA/Kex2 |
| 59 | 3891 | PSAC35:HSA/KEX 2.HSA.KUP | The ripe HSA that ins succession after the HSA/Kex2 signal sequence merges at the N-of ripe KUP end. | PSAC35 | 339 | 271 | 407 | Do not have | Do not have | HSA/ |
| 60 | 3892 | PSAC35:HSA/KEX 2.CNP.HSA | The CNP that ins succession after the HSAKex2 signal sequence merges at the N-of ripe HSA end.CNP corresponding to the amino acid/11 05-123 of SEQ ID No:408 (in this article | PSAC35 | 340 | 272 | 408 | Do not have | Do not have | HSA/Kex2 |
| Fusions No. | Construction ID | The construction title | Describe | Expression vector | SEQ ID NO:Y | SEQ ID NO:X | SEQ ID NO:Z | SEQ ID NO:A | SEQ ID NO:B | Targeting sequencing |
| Be called KUP). | ||||||||||
| 61 | 3893 | PSAC35:HSA/KEX 2.HSA.CNP | The ripe HSA that ins succession after the HSA/Kex2 signal sequence merges at the N-of ripe CNP end. | PSAC35 | 341 | 273 | 409 | Do not have | Do not have | HSA/Kex2 |
| 62 | 3894 | PSAC35:HSA/KEX 2.DNP.HSA | The DNP that ins succession after the HSA/Kex2 signal sequence merges at the N-of ripe HSA end. | PSAC35 | 342 | 274 | 410 | Do not have | Do not have | HSA/Kex2 |
| 63 | 3895 | PSAC35:HSA/KEX 2.HSA.DNP | The ripe HSA that ins succession after the HSA/Kex2 signal sequence merges at the N-of ripe DNP end. | PSAC35 | 343 | 275 | 411 | Do not have | Do not have | HSA/Kex2 |
| 64 | 3896 | PSAC35:HSA/KEX (R19G)-BNP30 (GGG) .HSA | The BNP (amino acid/11-30) that ins succession after the modified HSA/KEX-2 targeting sequencing merges at the N-of ripe HSA end through three glycine. | PSAC35 | 344 | 276 | 412 | Do not have | Do not have | Modified HSA/Kex2 |
| 65 | 3897 | PEE12:SPCON-BNP (K14G) .HSA | The mutant BNP (K14G) that ins succession after the shared signal sequence merges at the N-of ripe HSA end. | PEE12.1 | 345 | 277 | 413 | Do not have | Do not have | Consensus sequence |
| 66 | 3898 | PSAC35:HSA/Kex-2-BNP (K14G) .HSA | The mutant BNP (K14G) that ins succession after the HSA/KEX-2 signal sequence merges at the N-of ripe HSA end. | PSAC35 | 346 | 278 | 414 | Do not have | Do not have | HSA/Kex2 |
| 67 | 3899 | PSAC35:HSA/Kex-2 (R19G)-BNP (K14G) .HSA | The mutant BNP (K14G) that ins succession after the modified HSA/KEX-2 signal sequence merges at the N-of ripe HSA end. | PSAC35 | 347 | 279 | 415 | Do not have | Do not have | Modified HSA/Kex2 |
| 68 | 3900 | PSAC35:HSA/Kex-2-HSA.BNP (K14G) | The ripe HSA that ins succession after the HSA/KEX-2 signal sequence merges the N-end at mutant BNP (K14G). | PSAC35 | 348 | 280 | 416 | Do not have | Do not have | HSA/Kex2 |
| 69 | 3903 | PC4:SPCON-Uricas e.HSA | The rat uricase of ining succession after the shared signal sequence merges at the N-of ripe HSA end. | PC4 | 349 | 281 | 417 | Do not have | Do not have | |
| 70 | 3904 | PC4:HSA/Kex2-HS A.Uricase | The ripe HSA that ins succession after the HSA/KEX-2 signal sequence merges at the N-of rat uricase end. | |
350 | 282 | 418 | Do not have | Do not have | HSA/Kex2 |
| 71 | 3910 | PSAC35:HSA/Kex-2-ADI.HSA | The mycoplasma arginine deiminase of ining succession after the HSA/KEX-2 signal sequence merges the N-end at ripe HSA | PSAC35 | 351 | 283 | 419 | Do not have | Do not have | HSA/Kex2 |
| Fusions No. | Construction ID | The construction title | Describe | Expression vector | SEQ ID NO:Y | SEQ ID NO:X | SEQ ID NO:Z | SEQ ID NO:A | SEQ ID NO:B | Targeting sequencing |
| End. | ||||||||||
| 72 | 3915 | PC4:SPCON-ADI.H SA | The mycoplasma arginine deiminase of ining succession after the shared signal sequence merges at the N-of ripe HSA end. | PC4 | 352 | 284 | 420 | Do not have | Do not have | Consensus sequence |
| 73 | 3917 | PC4:HSA/Kex-2-HS A.ADI | The ripe HSA that ins succession after the HSA/KEX-2 signal sequence merges at the N-of mycoplasma arginine deiminase end. | PC4 | 353 | 285 | 421 | Do not have | Do not have | HSA/Kex2 |
| 74 | 3918 | PSAC35:HSA/Kex-2-HSA.ADI | The ripe HSA that ins succession after the HSA/KEX-2 signal sequence merges at the N-of mycoplasma arginine deiminase end. | PSAC35 | 354 | 286 | 422 | Do not have | Do not have | HSA/Kex2 |
| 75 | 3919 | PSAC35:KEX2.TW EAKR.E28-F75.HS A | The TweakR (aminoacid 28-75) that ins succession after the HSA/KEX-2 signal sequence merges at the N-of ripe HSA end.TweakR E28-F75 is corresponding to the aminoacid 28-75 (being referred to herein as TweakR E28-F75) of SEQ ID No:423. | PSAC35 | 355 | 287 | 423 | Do not have | Do not have | HSA/Kex2 |
| 76 | 3920 | PSAC35:HSA/Kex2-GCB.HSA | The lysosomal glucocerebrosidase of ining succession after the HSA/KEX-2 signal sequence merges at the N-of ripe HSA end. | PSAC35 | 356 | 288 | 424 | Do not have | Do not have | HSA/Kex2 |
| 77 | 3921 | PC4:SPCON-GCB. HSA | The lysosomal glucocerebrosidase of ining succession after the shared signal sequence merges at the N-of ripe HSA end. | PC4 | 357 | 289 | 425 | Do not have | Do not have | Consensus sequence |
| 78 | 3922 | PSAC35L:HSA/Kex2-HSA.GCB | The ripe HSA that ins succession after the HSA/KEX-2 signal sequence merges at the N-of lysosomal glucocerebrosidase end. | PSAC35 | 358 | 290 | 426 | Do not have | Do not have | HSA/Kex2 |
| 79 | 3923 | PC4:HSA/Kex2-HS A.GCB | The ripe HSA that ins succession after the HSA/KEX-2 signal sequence merges at the N-of lysosomal glucocerebrosidase end. | PC4 | 359 | 291 | 427 | Do not have | Do not have | HSA/ |
| 80 | 3924 | PSAC35:HSA/Kex2-ADM.HSA | The medulla renis quality of ining succession after the HSA/KEX-2 signal sequence merges at the N-of ripe HSA end. | |
360 | 292 | 428 | Do not have | Do not have | HSA/ |
| 81 | 3925 | PC4:SPCON-ADM. | The medulla renis quality of ining succession after the shared signal sequence merges and exists | |
361 | 293 | 429 | Do not have | Do not have | Consensus sequence |
| Fusions No. | Construction ID | The construction title | Describe | Expression vector | SEQ ID NO:Y | SEQ ID NO:X | SEQ ID NO:Z | SEQ ID NO:A | SEQ ID NO:B | Targeting sequencing |
| HSA | The N-end of ripe HSA. | |||||||||
| 82 | 3926 | PSAC35:HSA/Kex2-HSA.ADM | The ripe HSA that ins succession after the HSA/KEX-2 signal sequence merges at the N-of medulla renis quality end. | PSAC35 | 362 | 294 | 430 | Do not have | Do not have | HSA/Kex2 |
| 83 | 3927 | PC4:HSA/Kex2-HS A.ADM | The ripe HSA that ins succession after the HSA/KEX-2 signal sequence merges at the N-of medulla renis quality end. | PC4 | 363 | 295 | 431 | Do not have | Do not have | HSA/Kex2 |
| 84 | 3928 | Pc4:HSA.KISS.E20-Q145 | The ripe HSA that ins succession after the Qian Yuan district of HSA signal sequence merges the N-end at Kiss (aminoacid 20-145).Kiss E20-Q145 is corresponding to the aminoacid 20-145 (being referred to herein as Kiss E20-Q145) of SEQ ID No:432. | PC4 | 364 | 296 | 432 | Do not have | Do not have | HSA Qian Yuan district |
| 85 | 3929 | PSAC35:KEX2.HS A.KISS-1.E20-Q145 | The ripe HSA that ins succession after the HSA/KEX-2 signal sequence merges the N end at Kiss (aminoacid 20-145). | PSAC35 | 365 | 297 | 433 | Do not have | Do not have | HSA/Kex2 |
| 86 | 3930 | PSAC35:KEX2.Ephr inB1.K30-D229.HS A | The Ephrin B1 (aminoacid 30-229) that ins succession after the HSA/KEX-2 signal sequence merges at the N-of ripe HSA end.Ephrin B1 K30-D229 is corresponding to the aminoacid 30-229 (being referred to herein as Ephrin B1 K30-D229) of SEQ ID No:434. | PSAC35 | 366 | 298 | 434 | Do not have | Do not have | HSA/Kex2 |
| 87 | 3931 | Pc4:EphrinB1.M1-D 229.HSA | Ephrin B1 (amino acid/11-229) comprises natural Ephrin B1 signal sequence, merges at the N-of ripe HSA end. | PC4 | 367 | 299 | 435 | Do not have | Do not have | Natural Ephrin B1 |
| 88 | 3932 | Pc4:TWEAKR.M1-F75.HSA | TweakR (amino acid/11-75) comprises natural TweakR signal sequence, merges at the N-of ripe HSA end.TweakR M1-F75 is corresponding to the amino acid/11-75 (being referred to herein as TweakR M1-F75) of SEQ ID No:436. | PC4 | 368 | 300 | 436 | Do not have | Do not have | Natural TweakR |
| 89 | 3933 | Pc4:B7-H3.M1-E24 | B7-H3 (amino acid/11-247) comprises natural B 7-H3 | PC4 | 369 | 301 | 437 | Do not have | Do not have | Natural |
| Fusions No. | Construction ID | The construction title | Describe | Expression vector | SEQ ID NO:Y | SEQ ID NO:X | SEQ ID NO:Z | SEQ ID NO:A | SEQ ID NO:B | Targeting sequencing |
| 7.HSA | Signal sequence merges at the N-of ripe HSA end. | B7- |
||||||||
| 90 | 3934 | Pc4:HSA.EphrinB1. K30-D229 | The ripe HSA that ins succession after the Qian Yuan district of HSA signal sequence merges the N-terminal at Ephrin B1 (aminoacid 30-229). | PC4 | 370 | 302 | 438 | Do not have | Do not have | HSA Qian Yuan district |
| 91 | 3935 | PSAC35:HSA.Ephri nB1.K30-D229 | The ripe HSA that ins succession after the HSA/Kex2 signal sequence merges the N end at Ephrin B1 (aminoacid 30-229). | PSAC35 | 371 | 303 | 439 | Do not have | Do not have | HSA/Kex2 |
| 92 | 3936 | Pc4:SPCON.cUricas e.HSA | The chimpanzee uricase of ining succession after the shared signal sequence merges at the N-of ripe HSA end. | PC4 | 372 | 304 | 440 | Do not have | Do not have | Consensus sequence |
| 93 | 3937 | Pc4:HSA.cUricase | The ripe HSA that ins succession after the Qian Yuan district of HSA signal sequence merges at the N-of chimpanzee uricase end. | PC4 | 373 | 305 | 441 | Do not have | Do not have | HSA Qian Yuan district |
| 94 | 3938 | Pc4:SPCON.hUricas e.HSA | The uricase of ining succession after the shared signal sequence merges at the N-of ripe HSA end. | PC4 | 374 | 306 | 442 | Do not have | Do not have | Consensus sequence |
| 95 | 3939 | Pc4:HSA.hUricase | The ripe HSA that ins succession after the Qian Yuan district of HSA signal sequence merges at the N-of uricase end. | PC4 | 375 | 307 | 443 | Do not have | Do not have | HSA Qian Yuan district |
| 96 | 3940 | Pc4:SPCON .bUricas e.HSA | The baboon uricase of ining succession after the shared signal sequence merges at the N-of ripe HSA end. | PC4 | 376 | 308 | 444 | Do not have | Do not have | Consensus sequence |
| 97 | 3941 | Pc4:HSA.bUricase | The ripe HSA that ins succession after the Qian Yuan district of HSA signal sequence merges at the N-of baboon uricase end. | PC4 | 377 | 309 | 445 | Do not have | Do not have | HSA Qian Yuan district |
| 98 | 3942 | PSAC35:KEX2.hUri case.HSA | The uricase of ining succession after the HSA/Kex2 signal sequence merges at the N-of ripe HSA end. | PSAC35 | 378 | 310 | 446 | Do not have | Do not have | HSA/Kex2 |
| 99 | 3943 | PSAC35:KEX2.cUri case.HSA | The chimpanzee uricase of ining succession after the HSA/Kex2 signal sequence merges at the N-of ripe HSA end. | PSAC35 | 379 | 311 | 447 | Do not have | Do not have | HSA/ |
| 100 | 3944 | PSAC35:KEX2.bUri case.HSA | The baboon uricase of ining succession after the HSA/Kex2 signal sequence merges at the N-of ripe HSA end | PSAC35 | 380 | 312 | 448 | Do not have | Do not have | HSA/ |
| 101 | 3618 | PC4:SPCON.BNP1-32 (2x)/HSA | In succession after the shared signal sequence two tandem copies of ripe BNP merge at the N-of ripe HSA end. | PC4 | 483 | 484 | 485 | 463 | 464 | Consensus sequence |
Table 2 provides the nucleic acid molecules that comprises the albumin fusion proteins of encoding among the present invention or by the non-exhaustive inventory of its polynucleotide of forming.First row " fusion No. " have provided the fusion 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 raw egg white matter 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." construction title " row (the 3rd row) provide title for given albumin fusion construct or polynucleotide.
The 4th row " descriptions " of table 2 provide general description for given albumin fusion construct, and the 5th row " expression vector " have been listed the nucleic acid molecules 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 vehicle easily, (2) targeting sequencing, (3) promoter region, (4) translation termination is wherein one or multinomial or by its " expression cassette " of forming, then it is transferred in other carrier, such as the expression vector that for example comprises Yeast expression carrier for example or mammalian expression vector.In one embodiment, in order in saccharomyces cerevisiae, to express, will to comprise the nucleic acid molecules 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 molecules 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 molecules or be cloned among the pC4:HSA by the polynucleotide that it is formed.In another embodiment,, will comprise the nucleic acid molecules 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 NSO 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 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 cellular expression, 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 description 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 targeting sequencing 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 targeting sequencing listed.Comprising the compositions 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 description with different signal sequences such as back that help the excretory signal sequence substitution table 2 of albumin fusion proteins 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 derivant 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 the specific fragment of Protocols in Molecular Biology such as pcr amplification so and it is cloned in the appropriate carriers, this is within the technical scope of this area.
The 9th and 10 amplimers " SEQ ID NO:A " that provide 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 molecules or by the demonstration primer of its polynucleotide of forming.In one embodiment of the invention, have the oligonucleotide primers of sequence (SEQ ID NO:A and/or B) shown in the 9th 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 molecules 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, oligonucleotide 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 albumin fusion construct has been preserved in ATCC among the application.
Table 3
| Construction ID | The construction title | ATCC preserving number/date |
| 1812 | pSAC35：IL2.A21-T153.HSA | PTA-3759 2001-10-4 |
| 2053 | PEE12:IFNb-HSA is also referred to as pEE12.1:IFN β-HSA | PTA-3764 2001-10-4 |
| 2054 | pEE12：HSA-IFNb | PTA-3941 2001-12-19 |
| 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 |
| 2492 | pC4.IFNb(ΔM22).HSA | PTA-3943 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 albumin fusion construct by technology known in the art from depository, also is described (referring to embodiment 10) in other place of the application.ATCC be positioned at the Manassas of Virginia, US 20110-2209 No. 10801, the big ways for education (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 proprietary program.
In another embodiment of the present invention, the polynucleotide that comprise the given albumin fusion proteins of (1) coding, (2) targeting sequencing, (3) 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 " sub-clone " to another kind of carrier.By method well-known in the art,, can produce the fragment that is used for sub-clone such as for example pcr amplification (for example utilizing oligonucleotide primers) and/or restriction endonuclease digestion with sequence shown in SEQID 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 another preferred version, the therapeutic activity protein portion of albumin fusion proteins of the present invention is proteinic fragment or the variant by the sequential coding 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 aminoacid of the terminal deletion of proteinic N-, but still can keep other therapeutic activity and/or functional activity (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 conventional 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 biologys 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 amino 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 albumin 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 albumin 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 amino 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 albumin 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 amino 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 albumin 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 aminoacid 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 functional activity (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 usually and have the ability of antibody that the complete or mature form of this peptide species is discerned in C-terminal deletion polypeptid induction and/or combination.The conventional 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 carboxyl 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 albumin fusion construct).Particularly, the C-terminal deletion 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 albumin 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 carboxyl 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 albumin 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 carboxyl 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 albumin 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 albumin fusion construct coding, perhaps albumin fusion proteins, perhaps by the albumin fusion proteins of polynucleotide of the present invention or albumin 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 albumin 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 albumin fusion construct coding, perhaps albumin fusion proteins, perhaps by the albumin fusion proteins of polynucleotide of the present invention or albumin fusion construct coding) or its fragment have at least 80%, 85%, 90%, 95%, 96%, 97%, the protein of the polypeptide of 98% or 99% homogeneity.In preferred embodiments, the application relates to comprising and has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homogeneity with reference polypeptide and have N-mentioned above and the protein of the polypeptide of the aminoacid sequence of the terminal deletion of 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 functional activity (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 polynucleotide or the nucleic acid 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 " of table 1 row), albumin protein and/or albumin fusion proteins respectively but keep its herein 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 therapeutic protein partly, the albumin part or the albumin fusion proteins of the present invention of 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 albumin fusion construct coding; Or 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 albumin 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% homogeneity 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 the bonded DNA of filter membrane, 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 the bonded DNA of filter membrane, then at about 68 degrees centigrade, at 0.1X SSC, 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 in MolecularBiology ", Green publishing associates, Inc. and John Wiley ﹠amp; Sons Inc., NewYork, 6.3.1-6.3.6 and 2.10.3) hybridize with the complementary strand of the nucleic acid molecules of the albumin fusion proteins of the present invention of encoding down.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% " homogeneity " identical with the inquiry aminoacid sequence, just the desired polypeptides sequence can comprise nearly five amino acid change in per 100 aminoacid of inquiry aminoacid sequence.In other words, obtain to have the polypeptide that has the aminoacid sequence of at least 95% homogeneity 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 carboxyl terminal position of reference amino acid sequence, or between those terminal positions Anywhere, can individually be dispersed between the residue of reference sequences or be dispersed in one or more adjacent groups in the reference sequences.
In fact, can utilize known computer program to determine as usual whether the aminoacid sequence of any specific polypeptide and albumin fusion proteins of the present invention or its fragment (for example albumin part of the therapeutic protein of albumin fusion proteins part or albumin fusion proteins) has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homogeneity.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 method for optimizing 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 homogeneity percentage ratio.The preferred parameter that is used for the comparison of FASTDB aminoacid is: matrix=PAM0, 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 truncate of C-of aim sequence when calculating overall homogeneity percentage ratio.For with respect to the aim sequence of search sequence in the terminal truncate of N-and C-, following correction homogeneity percentage ratio, calculate the N-that is positioned at aim sequence in the search sequence and C-end, not with the residue number of corresponding aim sequence coupling/comparison, as the percentage ratio of search sequence base sum.Whether residue mates/compare is that result by the FASTDB sequence alignment determines.Deduct this percentage ratio from the homogeneity percentage ratio that utilizes special parameter to calculate by above-mentioned FASTDB program then, draw final homogeneity percentage ratio score.This final homogeneity percentage ratio score is used for score of the present invention exactly.Artificial N-that only considers to be positioned at aim sequence when adjusting homogeneity percentage ratio 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 homogeneity percentage ratio.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 homogeneity percentage ratio score that calculates by the FASTDB program.If remaining 90 residues mate fully, then final homogeneity percentage ratio 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 homogeneity percentage ratio that calculates by FASTDB need not to carry out manual correction.Statement once more, have only demonstration according to FASTDB comparison be positioned at beyond the N-and C-end of aim sequence, with the residue position needs craft rectification 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 homogeneity 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 by the homology or the homogeneity of BLAST (the promptly basic local comparison research tool of Basic LocalAlignment Search Tool) assay determination nucleotide or amino acid sequence level.
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 rectangular histogram, description, comparison, expected value (promptly reporting the significance,statistical threshold value at the paired sequence of database sequence), cutoff, matrix and filter 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 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 is (along search sequence at each wink
ThThe position produces the speech hits); Gapw=16 (set window width, wherein generation contains the breach comparison).Equal biastp 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 kit 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, noncoding 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 combination in any 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 aminoacid 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 bacterial host such as yeast or escherichia coli preference) at the specific host optimizing codon.
In a preferred version, 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 other embodiments, 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 chromosome, John Wiley ﹠amp for Genesll, 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 aminoacid 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 carboxyl 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 mutants, 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.Research worker is found " 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 aminoacid 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 conventional 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 functional activity 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 functional activity corresponding with one or more biologys 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 functional activity corresponding with one or more biologys 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 according to general rule known in the art select to the very little deletion of activity influence, insertion, inversion, repetition and 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 aliphatic 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, Tvr and Trp; And the replacement of small-sized aminoacid 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 the alternate guidance of phenotype silent amino acid, wherein the author points out to study aminoacid sequence has two kinds of main strategies to the toleration that changes.
First kind of strategy utilized the toleration 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 aminoacid variation at the ad-hoc location of clone gene, to identify the vital zone of protein function.For example, can use direct 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 aminoacid 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 aliphatic 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, Tvr and Trp; And the replacement of small-sized aminoacid Ala, Ser, Thr, Met and Gly.Except that conserved amino acid substitutes, variant of the present invention comprises that (i) contains the polypeptide of mentioning of one or more non-conservative amino acid residues, wherein 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 chemical compound (for example Polyethylene Glycol) that improves polypeptide stability and/or dissolubility, or (iv) contain other aminoacid such as for example polypeptide of IgG Fc district fusogenic peptide with another chemical 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 acid 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 aminoacid that the peptide isostere links together constitutes, and can comprise the aminoacid beyond the aminoacid of 20 kinds of gene codes.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 carboxyl 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 ramose, and for example because ubiquitin turns usefulness into, and they also can be to have or do not have 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 acetylation; acidylate; the ADP-ribosylation; amidatioon; the covalent attachment of flavin; 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 bond forms; demethylation; the formation of covalent cross-linking; the formation of cysteine; the formation of pyroglutamic acid; formylated; the γ carboxylation; glycosylation; the GPI anchor forms; hydroxylation; iodate; methylate; myristylization; oxidation; PEGization; Proteolytic enzyme processing; phosphorylation; prenylization; racemization; selenizing (selenoylation); sulphation; interpolation aminoacid 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-TRANSLATIONALCOVALENT MODIFICATION OF PROTEINS ", B.C.Johnson compiles, AcademicPress, New York, 1-12 page or leaf, 1983; People such as Seifer, Meth.Enzymol.182:626-646,1990; People such as Rattan, Ann.N.Y Acad.Sci.663:48-62,1992).
Functional activity
" polypeptide with functional activity " 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 functional activity include but not limited to biologic activity, antigenicity [in conjunction with the ability of (or combine with the polypeptide competition) anti-peptide antibody], immunogenicity (producing ability) with the bonded antibody of specific polypeptide of the present invention, and polypeptide of the present invention form polymeric ability and with the receptor or the bonded ability of part of polypeptide.
" polypeptide with biologic activity " refer to according to having or do not have the measurement of the particular biological algoscopy of dose dependent, 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 dependent really, compare with polypeptide of the present invention, given active dose dependent needn't be identical with polypeptide, only need similar substantially (promptly with respect to polypeptide of the present invention, candidate's 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 albumin.
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 merges state.Can utilize or conventional revise that algoscopy 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 functional activity (biological example is learned active) that algoscopy known in the art and algoscopy 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 algoscopy conventional determining and albumin fusion proteins of reference.In addition, but those skilled in the art utilize the segmental activity of albumin protein known in the art and/or that hereinafter the algoscopy 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-albumin 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 ", immunoradiometry, gel diffusion precipitation reaction, the immunodiffusion algoscopy, the original position immunoassay (is for example utilized gold colloidal, enzyme or radioisotopic tracer), Western blotting, precipitation, agglutination assay (example gel agglutination assay, the haemagglutination algoscopy), complement fixation assay, immunofluorescence assay, the competitiveness of technology such as a-protein algoscopy and immunoelectrophoresis algoscopy and noncompetitive are measured system.In one embodiment, detect antibodies by detecting a label 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 labelling.This area is known and is used for detecting bonded many methods in immunoassay, and they within the scope of the present invention.
In a preferred embodiment of the binding partners (for example receptor 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 reproducibility and irreducibility gel chromatography, protein affinity chromatograph 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 the multimerization ability of assessing albumin fusion proteins, for example can measure the association of polymer and other component by method well-known in the art such as for example reproducibility and irreducibility gel chromatography, protein affinity chromatograph 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 bond polypeptide or epi-position) relevant when not merging with albumin 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 antagonism) 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 bond 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 bond 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 algoscopy 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 antibacterial, 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 lists of references).Utilize or conventionally revise technology described herein or that other approach of this area is known, also can measure its specificity and affinity 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, 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 bond.
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 algoscopy, precipitin reaction, GDP reaction, immunodiffusion algoscopy, agglutination assay, complement fixation assay, immunoradiometry, fluorescence immunoassay and a-protein immunoassay, has only mentioned some here.This algoscopy 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., New York is with its complete being incorporated herein by reference).Hereinafter sketched exemplary immunoassay (but being not intended to restriction).
Exempt to survey the precipitation scheme and be usually included in the dissolving buffer such as being supplemented with protein phosphatase and/or protease inhibitor (EDTA for example, PMSF, press down the enzyme peptide, vanadic acid sodium) RIPA buffer (1%NP-40 or TFriton X-100,1% NaTDC, 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 a period of times (for example 1 to 4 hour), for example in the cytolysis thing, add sepharose pearl with anti-albumin antibody coupling, in about one hour of 40 degrees centigrade of incubations or longer time, in the dissolving buffer, 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 exempting to survey the precipitation 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 thin film such as celluloid from polyacrylamide gel, PVDF or nylon, closed film in confining liquid (PBS that for example contains 3%BSA or defatted milk), washing thin film in lavation buffer solution (for example PBS-polysorbas20), albumin fusion proteins of the present invention (diluting in the sealing buffer) is applied on the thin film, in lavation buffer solution, wash thin film, be applied to that sealing dilutes in the buffer with zymolyte (for example horseradish peroxidase or alkali phosphatase) or Geigers (for example
32P or
125Two anti-(it discerns albumin fusion proteins, for example AHS's albumin antibody) of I) puting together are washed thin film, and are detected antigenic existence in lavation buffer solution.Those skilled in the art understand can be increased the signal of detection and reduce background noise by revising parameter., compile referring to people such as for example Ausubel about more discussion of Western blotting scheme, 1994, " Current Protocols in Molecular Biology ", 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 microtitration plates, flush away not with the bonded antigen in hole, 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 a period of time of puting together with detection compound such as zymolyte (for example horseradish peroxidase or alkali phosphatase), the albumin fusion proteins of the unconjugated or non-specific bond of flush away, and detect and wrap by the existence of the bonded albumin fusion proteins of antigenic specificity 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 zymolyte (for example horseradish peroxidase or alkali phosphatase).Those skilled in the art understand can increase detection signal by revising parameter, and this area know other ELISA change., 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, and it is included under the condition that has the cumulative unlabelled antigen of quantity, will be through labelled antigen (for example
3H or
125I), and detect and through the bonded antibody of labelled antigen with albumin fusion proteins incubation of the present invention.The data determination albumin fusion proteins that can draw be analyzed by Scatchard is to the affinity 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, with protein, antigen or epi-position and (for example puted together labelled compound
3H or
125I) there is under the unmarked second cumulative proteinic condition of quantity incubation together in albumin fusion proteins, 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 bond of antigen at it.Preferred binding affinity comprises that dissociation constant or Kd are less than 5 * 10
-2M, 10
-2M, 5 * 10
-3M, 10
-3M, 5 * 10
-4M, 10
-4Binding affinity.Preferred binding affinity comprises that dissociation constant or Kd are less than 5 * 10
-5M, 10
-5M, 5 * 10
-6M, 10
-6M, 5 * 10
-7M, 10
7M, 5 * 10
-8M or 10
-8The binding affinity of M.Even preferred binding affinity comprises that dissociation constant or Kd are less than 5 * 10
-9M, 10
-9M, 5 * 10
-10M, 10
-10M, 5 * 10
-11M, 10
-11M, 5 * 10
-12M, 10
-12M, 5 * 10
-13M, 10
-13M, 5 * 10
-14M, 10
-14M, 5 * 10
-15M or 10
-15The binding affinity of M.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 affinity of the proteinic antibody of corresponding combined treatment (to the albumin fusion) to the affinity of given protein or epi-position.In addition, the algoscopy 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 derivant 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.
Albumin
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.
Other embodiments comprise 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 albumin (HA) are used interchangeably in this article.Term " albumin " and " serum albumin " scope are wideer, contain human serum albumin's (and fragment and variant) and from other the albumin (and fragment and variant) of species.
When being used for this paper, " albumin " refers to albumin protein or aminoacid sequence, perhaps has the overall of the albumin fragment of albuminised one or more functional activities (biological example learn active) or variant.Particularly, " albumin " refers to people's albumin or its fragment (for example referring to EP 201 239, EP 322094, 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 albumin or its fragment, perhaps these molecules or its segmental analog 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 is with reference to SEQ ID NO:1, and Leu-407 sports Ala, Leu-408 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 repellence of yeast Yap3p Proteolytic enzyme 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 albumin that is enough to the proteinic therapeutic activity of extended treatment, plasma stability or storage life 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 storage life or plasma stability partly compared the part albumin that has obtained prolongation or extended with the storage life 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 preferred embodiments, 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 infiltration (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.Albumin can be derived from any vertebrates, especially any mammal, for example people, cattle, sheep or pig.The nonmammalian albumin 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 aminoacid of the youthful and the elderly, preferably to 150 aminoacid of the youthful and the elderly.The HA variant can be formed or comprised it by at least one complete structure territory of HA, for example domain 1 (amino acid/11-194 of SEQ ID NO:1), domain 2 (the amino acid/11 95-387 of SEQ ID NO:1), domain 3 (the aminoacid 388-585 of SEQ ID NO:1), domain 1 and 2 (1-387 of SEQ ID NO:1), domain 2 and 3 (195-585 of SEQ ID NO:1) or domain 1 and 3 (the aminoacid 388-585 of the amino acid/11-194 of SEQ ID NO:1 and SEQ ID NO:1).Each domain 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 Val315 and G1u492 to Ala511.
Preferably, the albumin part of albumin fusion proteins of the present invention comprises minor structure territory or 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 bond therapeutic protein also is therapeutic protein
At least one fragment of the antibody that comprises disclosed therapeutic protein in the specific bond 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 amino 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 carboxyl 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 ", and the 3-5 chapter (Paul, W. compile, 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 overall structure, 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 specific epitopes.To the C-end, light chain and variable region of heavy chain all comprise domain FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from the N-end.The variable region links to each other with heavy chain or constant region of light chain.The aminoacid of each domain distributes and meets Kabat, " Sequences of Proteins of Immunological Interest ", National Institutes ofHealth, 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 bond.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 domain for example, the VL 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 bird and mammal from any animal origin.Preferably, antibody is the antibody of people, Mus (for example mice and rat), donkey, sheep, rabbit, goat, Cavia porcellus, 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 bond and comprises VL or the fragment of VH 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 domain.
Combined treatment protein and can be antibody monospecific, bispecific, 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 to therapeutic protein and and allos epi-position such as heterologous polypeptide or solid support the two all have specificity.Referring to for example PCT publication WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; 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 bispecific 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 form.
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 derivant).Can utilize the standard technique that those skilled in the art will know that, comprise the direct 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 derivant) 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 aminoacid replacements with respect to reference VH domain, VHCDR1, VHCDR2, VHCDR3, VL 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 bond 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 bond therapeutic protein or therapeutic protein.Therefore, the antibody of specific bond 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.Not proteinic any other analog of combined treatment, directly in the antibody of congener (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 homogeneity (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 Mus 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.Not in conjunction with have less than 95% with therapeutic protein, 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 homogeneity (utilizing method calculating known in the art and described herein).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 bonded antibody of therapeutic protein part of albumin fusion proteins of the present invention.Preferred binding affinity comprises that dissociation constant or Kd are less than 5 * 10
-2M, 10
-2M, 5 * 10
-3M, 10
-3M, 5 * 10
-4M, 10
-4Binding affinity.Preferred binding affinity comprises that dissociation constant or Kd are less than 5 * 10
-5M, 10
-5M, 5 * 10
-6M, 10
-6M, 5 * 10
-7M, 10
7M, 5 * 10
-8M or 10
-8The binding affinity of M.Even preferred binding affinity comprises that dissociation constant or Kd are less than 5 * 10
-9M, 10
-9M, 5 * 10
-10M, 10
-10M, 5 * 10
-11M, 10
-11M, 5 * 10
-12M, 10
-12M, 5 * 10
-13M, 10
-13M, 5 * 10
-14M, 10
-14M, 5 * 10
-15M or 10
-15The binding affinity of M.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 affinity of the proteinic antibody of corresponding combined treatment (to the albumin fusion) to the affinity 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 competitive bonded any method, 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 the combination of the anti-epi-position to therapeutic protein of the albumin fusion proteins competitive inhibition two of at least one fragment of the proteinic antibody of combined treatment or variant.In other embodiment preferred, comprise at least one fragment of the proteinic antibody of combined treatment or 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% or at least 50% two combination that resists the epi-position of therapeutic protein of variant.
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 receptor 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 property aspect part combination and the/receptor activation have to the proteinic antibody of combined treatment do not merge fragment or variant is similar or similar substantially feature.
Characteristic of the present invention stops the two receptor specific antibody of part combination and receptor activation in addition, and the antibody of identification receptor-ligand complex, and preferably not unconjugated receptor 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 stimulating agent, promptly strengthen or activate the whole or subclass of the biologic activity of ligand-mediated receptor activation, for example by inducing the dimerization of receptor.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 purification, detection and targeted therapeutic protein, comprise external and intravital diagnosis and Therapeutic Method 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 measurement 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 purification, detection and targeted therapeutic protein, comprise external and intravital diagnosis and Therapeutic Method.
Combined treatment protein and can comprise derivant 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 derivatization by glycosylation, acetylation, PEGization, phosphorylation, amidatioon, known protection/blocking group, Proteolytic enzyme 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 cleavage, acetylation, formylated, tunicamycin is synthetic etc.In addition, derivant 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 suitable method 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, mice, 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 people's adjuvant such as the BCG (bacillus calmette-guerin vaccine) and the spillikin bacillus of Fu Shi (fully with incomplete) adjuvant, mineral coagulant agent such as aluminium hydroxide, surfactant such as LYSOLECITHIN SUNLECITHIN A, Pluronic polyhydric alcohol, polyanion, peptide, oil emulsion, key hole _ 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 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 list of references 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 limiting examples, can be with the cellular immunization mice 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 mice.Then by well-known technology with splenocyte and any suitable myeloma cell, for example, merge from the cell of the cell line SP20 that can obtain from ATCC.Select and the clone hybridization tumor 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 mice 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 monoclonal is the conversion that utilizes epstein-barr virus (EBV) to carry out.Being used to generate the scheme that EBV transforms B cell line 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 lymph node, tonsil, 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 cell line (ATCC numbers VR-1492).During approaching end of 3-4 week culture 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 culture after the time, EBV system can become monoclonal or polyclonal owing to the selective growth of particular B cell clone.Perhaps, can transform system to polyclonal EBV and carry out sub-clone (for example cultivating), perhaps be coated with to obtain monoclonal B cell line 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 myeloma (heteromyeloma) cell line (people * mice; 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 prepare the antibody fragment of discerning defined epitope by known technology.For example, enzyme be can utilize,, Fab of the present invention and F (ab ') 2 fragments generated by the Proteolytic enzyme cutting of immunoglobulin molecules such as papain (producing the Fab fragment) or pepsin (generation F (ab ') 2 fragments).F (ab ') 2 fragments contain the CH1 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 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 structural domain by complete or collected works or combinatorial antibody storehouse (for example people or Mus) expression.Can use antigen, for example labelled antigen or in conjunction with or be captured in antigen on the surface of solids or the pearl and select or identify the phage of expressing the antigenic antigen binding structural domain of binding purpose.The phage of using in these methods is typically the filobactivirus that comprises fd and M13, is wherein merged by the binding structural domain of the phage expression with the stable Fv antibody structure territory of Fab, Fv or disulfide bond and phage gene III or the reorganization of 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 list of references, 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, plant cell, yeast and antibacterial, 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 list of references 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 algoscopy 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's 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's 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 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 human immunoglobulin heavy chain and light chain gene complex are imported mouse embryo stem cell.Perhaps, except that people's heavy chain and light chain gene, also people variable region, constant region and multiformity zone can be imported in the mouse embryo stem cell.Import human immunoglobulin gene's seat by homologous recombination, can be respectively or make mouse immuning ball protein heavy chain and light chain 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 mode, 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.Human normal immunoglobulin's transgenic 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 PCT publication WO 98/24893; WO92/01047; WO 96/34096; WO 96/33735; European patent number 0 598 877; 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, 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 vehicle 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 of antibody, recombinant DNA technology for example, direct 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/or insert.
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 variable region of light chain 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 bond 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 Mus mAb and the antibody of human normal immunoglobulin's 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 aminoacid bridge, produce single chain polypeptide, form single-chain antibody thus the Fv district.Also can use the segmental technology of assembling function Fv in escherichia coli (people such as Skerra, Science 242:1038-1041,1988).
The recombinant 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, derivant or analog (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 variable region of light chain) 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 variable region of light chain with promoter.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 region 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 conventional 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 two 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 coded 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 antibacterial (for example escherichia coli, bacillus 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 promoter of mammalian cell genome (for example metallothionein promoter) or mammalian virus (for example gland virus stage starting, vaccinia virus 7.5K promoter).Preferably, use bacterial cell, such as escherichia coli, 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 from carriers such as early gene promoter sub-elements in the middle of people's cytomegalovirus main being effective antibody expression system (people such as Foecking, Gene 45:101,1986; People such as Cockett, Bio/Technology 8:2,1990).
In bacterial system,, can select multiple expression vector easily according to the desired use of expressed antibody molecule.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 purification 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 substrate glutathion-agarose pearl and combine and subsequently the eluting when having free glutathione can be easy to that purification comes out from dissolved cell.The pGEX carrier design is become to comprise thrombin or factor Xa protease cutting site, the target gene product of DCRP can partly be discharged from GST.
In the insecticide 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 promoter (for example polyhedrin promoter).
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 complex for example late promoter be connected with tripartite leader[.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 initial signal in order effectively to translate the antibody coding sequence that inserts.These signals comprise the sequence of ATG start codon and adjacency.And, start codon must with the expectation coded sequence the reading frame homophase to guarantee the translation of whole insert.These external sources translation control signals and start codon can have multiple source, natural and synthetic the two all can.Strengthen element, transcription terminator etc. and can strengthen expression efficiency (referring to people such as Bittner, Methods in Enzymol.153:51-544,1987) by comprising suitable transcribing.
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 outcome translation post-treatment and modified mechanism.Can select suitable cell line 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 outcome.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 cell line of stably express antibody molecule.Contain the expression vector that virus replication originates from its utilization, not as good as using DNA and the selected marker transformed host cell that is subjected to suitable expression control element (for example promoter, enhancer, 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 culture medium then into.Selected marker in the recombiant plasmid is given selection with resistance, and allows in the cell chromosome that is incorporated into them that plasmid is stable and growth forms focus (foci), can clone and increase then to be cell line.Can utilize this method to transform the cell line of expressed antibody molecule easily.This through the cell line transformed directly or indirectly and particularly useful aspect the interactional chemical 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), hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski, Proc.Natl.Acad.Sci.USA 48:202,1992) and adenine phosphoribosyl transferase (people such as Lowy, Cell 22:817,1980) gene.And the antimetabolite 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 glucosaminide G-41 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 TECH11 (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 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 labelling in the carrier system of expressing antibodies be can increase the time, the level that improves inhibitor in the host cell culture medium 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 sulfo-methionine (methionine sulphoximine) or methotrexate, increase respectively and utilize glutamine synthase (GS) or DHFR carrier as selected 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 endogenous gene performance function is provided.At PCT publication WO 87/04462; WO 86/05807; WO 89/01036; Among WO89/10404 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, can be from comprising that (Portsmouth NH) buys in interior suppliers in Lonza Biologics company according to the glutamine synthase expression vector that the present invention uses.People such as Bebbington, Bioltechnology 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 selected marker that makes that heavy chain and light chain polypeptide equivalent are expressed.Perhaps, also can use coding and can heavy chain and the two single carrier of 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 coded sequence of heavy chain and light chain can comprise cDNA or genomic DNA.
In case by animal, chemosynthesis or recombinant expressedly generated antibody molecule of the present invention, just can utilize any method that is used for the purification immunoglobulin molecules known in the art to carry out purification, for example by chromatography (for example ion exchange, affinity particularly after a-protein to the affinity and exclusion (sizing) 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 purification 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 labelled sequence such as the peptide of combined treatment protein or its fragment or variant can be merged to promote purification.In preferred embodiments, marker amino acid sequence is six polyhistidyl peptides, such as the label that provides in pQE carrier (9259 Eton 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 purification 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 purification.
Antibody or its fragment of puting together with diagnostic agent or therapeutic 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 tumor in diagnosis, thereby for example measures the effectiveness of given therapeutic scheme.By antibody and detectable substance coupling can be promoted to detect.The example of detectable substance comprises various enzymes, prothetic group, fluorescent material, luminescent material, bioluminescent material, radioactive substance, the positron emitting metal that utilizes various positron emission tomography arts and inactive paramagnetic metal ion.Utilize technology known in the art, can with detectable substance directly or by intermedium (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 agent 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, alkali phosphatase, beta galactosidase or acetylcholinesterase; The example of suitable prothetic group complex comprises Streptavidin/biotin and avidin/biotin; The example of suitable fluorescent material comprises umbelliferone, fluorescein, Fluorescein isothiocyanate, rhodamine, dichlorotriazine amine fluorescein (dichlorotriazinylamine fluorescein), dansyl Cl or phycoerythrin; The example of luminescent material comprises luminol; 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, therapeutic 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 paclitaxel, Cytochalasin B, Gramicidin D, ethidium bromide, ipecine, mitomycin, etoposide (etoposide, etoposide), teniposide (teniposide, tenoposide), vincristine, vincaleucoblastine, colchicine, amycin, daunorubicin, dihydroxy anthracin diketone, mitoxantrone, mithramycin, actinomycin D, 1-boldenone, glucocorticoid, procaine, tetracaine, lignocaine, Propranolol and puromycin and analog or homologue.Therapeutic agent includes but not limited to antimetabolite (methotrexate for example, Ismipur, 6-thioguanine, cytosine arabinoside, the 5-fluorouracil decarbazine), alkylating agent (dichloromethyldiethylamine (chlormethine) for example, plug is for group, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, mitobronitol, streptozotocin, ametycin and suitable dichloro diamino platinum (II) be cisplatin (DDP)), anthracycline antibiotics (for example daunorubicin (being called daunomycin in the past) and amycin), antibiotic (dactinomycin (being called D actinomycin D in the past) for example, bleomycin, mithramycin and anthramycin (AMC)) and antimitotic agent (for example vincristine and vincaleucoblastine).
Conjugate of the present invention can be used for modifying given biological answer-reply, does not think that therapeutic agent or medicine module are limited to classical chemotherapeutant.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, diphtherotoxin; Protein, such as tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet derived growth factor, 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 dressing agent, such as for example lymphokine, il-1 (" IL-1 "), interleukin-2 (" IL-2 "), interleukin-6 (" IL-6 "), granulocyte macrophage colony stimulating factor (" GM-CSF "), granulocyte colony-stimulating factor (" G-CSF ") or other somatomedin.
Antibody can also be attached to solid support, this immunoassay or purification to target antigen is useful especially.This solid support includes but not limited to glass, cellulose, polyacrylamide, nylon, polystyrene, polrvinyl chloride or polypropylene.
The technology that is used for 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 two can be resisted and put together described in 980 at U.S. Patent number 4,676 as Segal to form antibody allos conjugate, with its complete being incorporated herein by reference.
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-albumin 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 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 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 (Gly
4Ser)
3Therapeutic antibodies VL domain and therapeutic antibodies VH domain that peptide linkers such as (SEQ ID NO:4) connects.
Immunophenotyping
At least the fragment or the antibody of the present invention of variant or the immunophenotyping that albumin fusion proteins of the present invention can be used for cell line 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 maturation period differential expression in particular.To allow the cell mass of screening expression mark at the monoclonal antibody (or comprising the fragment of the proteinic antibody of combined treatment or the albumin fusion proteins of variant at least) of defined epitope or epi-position combination.Can utilize multiple technologies to use monoclonal antibody (or comprising the fragment of the proteinic antibody of combined treatment or the albumin fusion proteins of variant at least) 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 matrix (being plate), and flow cytometry (referring to for example United States Patent (USP) 5,985,660; Reach 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 (GVHD).Perhaps, these technology are allowed hematopoietic stem cell and the CFU-GM that screening can breed and/or break up, as finding in human cord blood.
Identify the proteinic antibody of combined treatment and comprise the fragment of the proteinic antibody of combined treatment or the albumin fusion proteins of variant
Antibody of the present invention or comprise the fragment of antibody of combined treatment protein (or its fragment or variant) at least 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 the fragment that comprises the proteinic antibody of combined treatment at least or variant is measured specificity in conjunction with the ability by the same antigen of following antibody specific bond, 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 the fragment of antibody of combined treatment protein (or its fragment or variant) at least 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 antibacterial (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 lists of references).Use or conventionally revise technology described herein or that other approach of this area is known, also can or comprise the fragment of antibody of combined treatment protein (or its fragment or variant) at least or the albumin fusion proteins of the present invention of variant is measured their specificity and affinitys to specified protein or epi-position antibody of the present invention.
Any method that can know by this area, to 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 by the bonded molecule of following antibody specificity, 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 at least.
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 algoscopy, agglutination assay, complement fixation assay, immunoradiometry, fluorescence immunoassay and protein A immunoassay and noncompetitive are measured system, only for several examples.Such algoscopy 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 protease inhibitor (as EDTA, PMSF, press down the enzyme peptide, vanadic acid sodium) RIPA buffer (1%NP-40 or TritonX-100,1% sodium deoxycholate, 0.1%SDS, 0.15M NaCl, 0.01 M sodium phosphate pH7.2,1%Trasylol) wait cell lysis group in the lysis buffer, in cell pyrolysis liquid, add antibody of the present invention or comprise the fragment of antibody of combined treatment protein (or its fragment or variant) or the albumin fusion proteins of the present invention of variant at least, in 40 ℃ of insulation a period of times (for example 1-4 hour), in cell pyrolysis liquid, add protein A and/or Protein G sepharose 4B (or in the situation of the albumin fusion proteins of fragment that comprises the proteinic antibody of combined treatment at least or variant, pearl through suitable anti-idiotype antibody or anti-albumin 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 4B pre cleaning cell pyrolysis liquid).Further discussion about the immunoprecipitation scheme 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, 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 the digestion fiber from polyacrylamide gel, on the film such as PVDF or nylon, closing membrane in confining liquid (PBS that for example contains 3%BSA or defatted milk), in cleaning buffer solution (for example PBS-Tween20), clean film, antibody of the present invention or albumin fusion proteins (diluting in the sealing buffer) are applied on the film, in cleaning buffer solution, clean film, be applied to that sealing dilutes in the buffer be conjugated with zymolyte (for example horseradish peroxidase or alkali phosphatase) or Geigers (for example
32P or
125I) two anti-(it discerns albumin fusion proteins, for example AHS's albumin 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 background noise.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., New York, 10.8.1.
ELISA comprises preparation antigen, hole with antigen coated 96 hole microtitration plates, flush away is not attached to the antigen on the hole, Xiang Kongzhong add be conjugated with such as zymolyte (for example horseradish peroxidase or alkali phosphatase) but etc. detection compound antibody of the present invention or albumin fusion proteins (fragment or the variant that comprise the proteinic antibody of combined treatment at least) and be incubated a period of 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) will 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 zymolyte (for example horseradish peroxidase or alkali 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) and antibody of the present invention or albumin fusion proteins be incubated together, and detect and through the bonded antibody of labelled antigen.Can measure the affinity 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 plot 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 labelled 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 the bonded speed that combines and dissociate of protein, antigen or epi-position.The BIAcore dynamic analysis comprises analyzes combining and dissociating of antibody, albumin fusion proteins or specific polypeptide, antigen or epi-position and chip, 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 the fragment of the proteinic antibody of combined treatment at least 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 nucleic acid of antibody of the present invention (comprising its fragment described herein, analog and derivant), code book invention antibody (comprising its fragment described herein, analog and derivant and anti-idiotype antibody), comprises fragment or the albumin fusion proteins of the present invention of variant and the nucleic acid of such albumin fusion proteins of encoding of the proteinic antibody of combined treatment at least.Antibody of the present invention or comprise the fragment of the proteinic antibody of combined treatment at least 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 the fragment of the proteinic antibody of combined treatment at least 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 compositions.
In the concrete and embodiment preferred, the present invention is devoted to the therapy based on antibody, it comprises antibody of the present invention or comprises the fragment of the proteinic antibody of combined treatment at least 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: neurological 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, analog and derivant 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 the fragment of the proteinic antibody of combined treatment at least 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 compositions.
Antibody wherein of the present invention or comprise the fragment of the proteinic antibody of combined treatment at least 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 lymphocyte (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 the fragment of the proteinic antibody of combined treatment at least or the albumin fusion proteins of the present invention of variant is used for diagnosis, monitoring or therapeutic purposes, and need not undo experimentation.
Antibody of the present invention or comprise the fragment of the proteinic antibody of combined treatment at least or the albumin fusion proteins of the present invention of variant can be favourable unites use with other monoclonal or chimeric antibody or with lymphokine or hemopoietic growth factor (such as IL-2, IL-3 and IL-7) for example helps to increase and the interactional effector lymphocyte's of antibody quantity or active.
Antibody of the present invention or comprise the fragment of the proteinic antibody of combined treatment at least 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 antitumor agent) of other type.Generally speaking, preferably use the product that belongs to the On 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, derivant, analog 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 affinity.Preferred binding affinity comprises less than 5 * 10
-2M, 10
-2M, 5 * 10
-3M, 10
-3M, 5 * 10
-4M, 10
-4The dissociation constant of M or Kd.Preferred binding affinity comprises that dissociation constant or Kd are less than 5 * 10
-5M, 10
-5M, 5 * 10
-6M, 10
-6M, 5 * 10
-7M, 10
-7M, 5 * 10
-8M or 10
-8Those of M.Even preferred binding affinity comprises that dissociation constant or Kd are less than 5 * 10
-9M, 10
-9M, 5 * 10
-10M, 10
-10M, 5 * 10
-11M, 10
-11M, 5 * 10
-12M, 10
-12M, 5 * 10
-13M, 10
-13M, 5 * 10
-14M, 10
-14M, 5 * 10
-15M or 10
-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 the fragment of the proteinic antibody of combined treatment at least 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 therapeutic effect.
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 excess syndrome of treatment or prophylactic activity
Before being used for the people, chemical compound of the present invention or pharmaceutical composition are preferred earlier at testing in vitro, test required treatment or prophylactic activity then in vivo.For example, the external test method that is used to confirm the treatment of chemical compound or pharmaceutical composition or prevention effectiveness comprises the effect of chemical 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 chemical compound or compositions pair cell system and/or tissue sample, include but not limited to that rosette forms algoscopy and cytolysis algoscopy.According to the present invention, sketched the external test method that can be used for determining whether to use specific compound, comprise that cell in vitro cultivates algoscopy, wherein cultivate patient's tissue sample and be exposed to or administered compound otherwise, and observe the effect of such chemical compound tissue sample.
Therapeutic/preventative using and compositions
The invention provides the The compounds of this invention by the experimenter being used effective dose or treatment, inhibition and the prevention method of pharmaceutical composition.In a preferred embodiment, chemical compound is (for example being substantially free of the material that limits its effect or produce undesired side effect) of purification basically.The preferred animal of experimenter includes but not limited to such as animals such as cattle, pig, horse, chicken, cat, dogs, and preferred mammal, and optimum is chosen.
When chemical compound comprised nucleic acid or immunoglobulin, 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 chemical compound of the present invention, for example be encapsulated in liposome, microgranule, the microcapsule, can express the reconstitution cell of this chemical 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 to Intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral route.Chemical compound or compositions 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 local application.In addition, may wish medical compounds of the present invention or compositions to be imported the central nervous system, comprise Intraventricular and intrathecal injection by any suitable way; Can be convenient to intracerebral ventricle injection by intraventricular catheter, for example connect storage, such as Ao Maye (Ommaya) storage.Also can adopt pulmonary's dispenser, for example by use inhaler or aerosol apparatus, and the dosage form that contains propellant.
In a specific embodiment, may wish medical compounds of the present invention or compositions local application to the zone that needs treatment; This can by such as but not limited to perioperative local infusion, topical application as in conjunction with postoperative wound dressing, by injection, via conduit, realize by suppository or by implant, described implant is porous, non-porous or spawn, comprise film, for example 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, chemical compound or compositions can be delivered in vesicle, 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, chemical compound or compositions 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 comment (Science 249:1527-1533,1990) of Langer is seen in the discussion of other controlled release system.
At chemical 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 compositions comprises the chemical compound and the pharmacopedics for the treatment of effective dose can accept carrier.In a specific embodiment, term " pharmacopedics is acceptable " refer to obtain the approval of federation or administrative organization 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 diluent, adjuvant, excipient or the media used with therapeutic 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 Oleum Arachidis hypogaeae semen, soybean oil, mineral oil, Oleum sesami or the like.When intravenous drug administration compositions, water is preferred carrier.Saline solution and moisture dextrose and glycerite also can be used as liquid-carrier, especially for injection.Suitable pharmacopedics excipient comprises starch, glucose, lactose, sucrose, gelatin, Fructus Hordei Germinatus, rice, flour, Chalk, silica gel, sodium stearate, glyceryl monostearate, Talcum, sodium chloride, defatted milk powder, glycerol, propylene, ethylene glycol, water, ethanol or the like.If desired, compositions also can contain a spot of wetting agent or emulsifying agent or pH buffer agent.These compositionss can be taked forms such as solution, suspension, emulsion, tablet, pill, capsule, powder, sustained release forms.Compositions can be mixed with suppository, contains traditional binding agent and such as carriers such as triglyceride.Peroral dosage form can comprise standard vector, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, saccharin sodium, cellulose, magnesium carbonate etc.The example of suitable pharmacopedics carrier be described in E.W.Martin's " Remington ' s Pharmaceutical Sciences ".Such compositions will contain the chemical compound for the treatment of effective dose, the form of preferred purification, and the carrier of appropriate amount is to provide the form that the patient is used of being suitable for.Dosage form should be fit to method of application.
In a preferred embodiment, according to old process compositions is mixed with and is suitable for the pharmaceutical composition that people's intravenous is used.Usually, being used for compositions that intravenous uses is solution at sterile isotonic aqueous buffer.When needing, compositions also can comprise solubilizing agent and such as local anesthetics such as lignocaine to ease the pain in the injection site.Generally speaking, composition is that separately provide or blended with unit dosage form, as the freeze-dried powder in the hermetically sealed container or there is not aqueous concentrate, the ampoule or the pouch (sachette) of activating agent quantity is arranged such as sign.When compositions will be used by infusion, available sterile pharmaceutical grade water or the brinish infusion bottle of being equipped with distributed.When compositions is used by injection, can provide ampoule Injectable sterile water or saline so that composition can used preceding mixing.
Chemical compound of the present invention can be mixed with neutrality or salt form.The acceptable salt of pharmacopedics comprises those that form with anion, such as derived from those of hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid etc., and form with cation those, such as derived from those of sodium hydroxide, potassium, ammonium, calcium, ferrum, 2-aminopropane., triethylamine, 3-ethylamino-ethanol, histidine, procaine 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 dose that will adopt in the preparation also depends on route of administration and the disease or the disorderly order of 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 immunoreation to external polypeptide, people's antibody has the longer half-life 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 labelling and derivant thereof and analog (comprising the fragment that comprises the proteinic antibody of combined treatment at least 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 that records thus is than the rising of standard expression or reduce the indication unconventionality expression.
The invention provides and be used to diagnose disorderly diagnostic algoscopy, comprise that (a) uses one or more to the special antibody of therapeutic protein or comprise the expression of the albumin fusion proteins of the fragment of the special antibody of therapeutic protein or variant being measured therapeutic protein in individual cells or the body fluid at least, and (b) gene expression dose and standard gene expression level are compared, the therapeutic protein expression that records thus is than the rising of standard expression 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 body constitution 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 to the fragment of the special antibody of therapeutic protein or variant at least 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 label is that this area is known, comprises the enzyme labelling thing, such as glucoseoxidase; Radiosiotope is such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (121In) and technetium (99Tc); Luminous marker is such as luminol; Fluorescent marker is such as fluorescein and rhodamine; And biotin.
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 purpose polypeptide that passes through labelling of (for example parenteral, subcutaneous or intraperitoneal) effective dose; B) waiting for a period of time after the dispenser to allow that labelled molecule is expressed therapeutic protein in the experimenter position preferentially concentrates (and unconjugated labelled molecule is removed to background level); C) measure background level; And d) the testing result indication experimenter that the labelled molecule among the detection experimenter, labelled 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 labelled molecule that detection is obtained and the standard value of before particular system being measured compare.
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 radiosiotope module,, inject radioactive amount usually in the scope of about 5 to 20 millicurie 99mTc for the human experimenter.Then through antibody, the antibody fragment of labelling or comprise the fragment of the proteinic antibody of combined treatment at least 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 label type and the dispenser pattern of use, allow after the dispenser that the position of labelled molecule in the experimenter preferentially concentrated and not the incorporation of markings molecule remove to the interval of background level be 6-48 hour or 6-24 hour or 6-12 hour.In another embodiment, the 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 behind the ID 1 month, at behind the ID 6 months, behind ID 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 label type.The technical staff 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 include but not limited to computed tomography (CT) (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 response 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 is used the fluorescent chemicals labelling and is used the scanning apparatus of fluorescence response to detect in the patient.In another embodiment, molecule uses positron emission tomography to detect with the positron emitting metal labelling 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 albumin or the antibody of therapeutic protein are embodiment preferred.These can be used for detecting the albumin fusion proteins of describing in the description full text.
Test kit
The invention provides the test kit that can be used for said 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 isolating basically polypeptide, 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 the bonded means of desired polypeptides (for example antibody can be conjugated with and can detect substrate, for example fluorescent chemicals, zymolyte, radioactive compound or luminophor, or discern one anti-two and anti-ly can be conjugated to and can 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 isolating basically polypeptide antigen, 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 the bonded means of antigen (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 anti-people's antibody that does not adhere to the reporter molecule labelling.In this embodiment, can detect the combination of antibody and polypeptide antigen by the antibody of described reporter molecule labelling.
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 labelling.Perhaps/in addition, detection means can comprise the competitive antigen through labelling.
In a kind of diagnostic form, test sera and the antigenic solid-phase reagent of surface combination 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 labelling react so that reporter molecule with solid support on the certain proportion binding reagents of bonded anti-antigen-antibody amount.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 protein material is attached to solid support material such as polymeric beads, dip rod, 96 orifice plates or filter material is prepared the solid surface reagent of said determination method.These adherence methods generally include the non-specific adsorption of protein and holder, or protein is covalently attached to chemical reaction 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.The carrier and being used to that test kit generally includes the recombinant antigen with surface combination detects anti-people's antibody of reporter molecule labelling of the anti-antigen-antibody of surface combination.
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 albumin (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 albumin in case become the part of albumin fusion proteins, can be described as " partly ", " 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 albumin 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 molecules 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 albumin (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 albumin (or its fragment or variant) polynucleotide or form by it; Perhaps described nucleic acid molecules 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 albumin (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 vector, expression vector, integration vector, and/or dubbing system), (2) transcription initiation region (promoter region for example, such as for example scalable or inducible promoter, constitutive promoter), (3) transcription termination region, (4) targeting sequencing and (5) selected 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 relevant 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 albumin 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 albumin 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 albumin 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 HAS molecule, joint is a particular importance.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 albumin 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, trimer, 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, trimer, the tetramer and Geng Gao polymer).In a specific embodiment, the therapeutic protein of albumin fusion proteins partly is polymeric form (being dimer, trimer, 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 zone 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 bond stable many rings or corner.According to the crystal 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 albumin molecule of particular biological to produce in essence.
Can insert peptide or polypeptide in people's albumin structure comprises with the ring that produces albumin fusion proteins of the present invention: Val54-Asn61, Thr76-Asp89, A1a92-Glu100, Gin170-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 albumin (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 aminoacid combination.
Such library can produce on HA or HA domain fragment by one of following method:
(a) aminoacid in HA or the segmental one or more peptide rings of HA domain is carried out random mutation.Intra-annular one, a plurality of or all residues by this way can suddenly change;
(b) carrying out length in one or more rings of HA or HA domain fragment (promptly inner the fusion) is X
nThe displacement or the insertion of the peptide at random of (wherein X is an aminoacid, 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 be transplanted in identical HA or the HA domain fragment, make HA or HA domain fragment become multi-functional by screening deutero-peptide at the difference that different targets carry out different rings.
In preferred embodiments, the peptide in insertion human serum albumin's the ring is the fragments of peptides 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 fragments of peptides 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 fragments of peptides 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 fragments of peptides or peptide variant.For example, the small peptide of describing in the table 1 and 2 (for example therapeutic 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 a plurality of zones of every kind of protein 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, half-life etc.
TMScFv fusions or other HA-fusions.
Also can prepare the therapeutic protein part targeting 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 other end.
Alternative method as the fusions of known treatment molecule, can obtain peptide as the library that HA or HA domain fragment N-terminal, C-terminal or N and C-terminal fusions make up by screening, (wherein X is aminoacid (aa) for their normally 6,8,12,20 or 25 or Xn, n is the residue number) random amino acid, wherein represented all possible aminoacid 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 aminoacid, makes its tool flexibility or has more rigidity.
Joint sequence can be by protease or chemical ablation to produce the growth hormone relevant portion.Preferably, protease is the natural generation of host, for example the protease of saccharomyces cerevisiae protease 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 albumin 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 interval between during removing.Half-life according to therapeutic protein in the blood flow is calculated plasma stability.The half-life of therapeutic protein can be easy to by the algoscopy 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 shelf-life of identical treatment that albumin merges the shelf-life of prolongation.Shelf-life 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 shelf-life after introducing albumin fusion proteins of the present invention.
Shelf-life " prolongation " albumin fusion proteins of the present invention shows bigger therapeutic activity with respect to the standard substance of accepting identical storage and treatment conditions.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 analog, 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 analog, 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 conditions therapeutic activity greater than about 100% or greater than about 105%, 110%, 120%, 130%, 150% or 200% of therapeutic activity.
Shelf-life 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 standardization.The albumin fusion proteins of the present invention that shelf-life 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 60%, 70%, 80% or 90% or more of about 50%, therapeutic activity.
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 antibacterial or the secretion of human or animal's cell line.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, the deutero-signal sequence of yeast (especially homologous) particularly with yeast host, with the fusion molecule of first aspect present invention, between signal and mature polypeptide, there is not yeast deutero-former (pro) sequence.
Saccharomyces cerevisiae invertase signal is the preferred example of the deutero-signal sequence of yeast.
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 chemical crosslinking.
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 Nutrient medium also contained in the present invention, preferred monoclonal (homogeneity on the clone) culture or by the deutero-culture of monoclonal culture.If polypeptide is excretory, then culture medium 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 antibacterial (for example colon bacillus (E.coli) and bacillus subtilis (Bacillus subtilis)), yeast (for example saccharomyces cerevisiae (Saccharomyces cerevisiae), Kluyveromyces lactis (Kluyveromyces lactis) and Pichia pastoris (Pichia pastoris)), filamentous fungi (for example aspergillus), plant 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 parent strain AH22his
+(be also referred to as DB1; Referring to people such as for example Sleep, Biotechnology 8:42-46,1990) derivant.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 promoter is subjected to the control of two kinds of checkpoints of monitoring glucose level and growth stage usually.In wild-type yeast, this promoter 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 resting stage.PRA1 gene code yeast vacuole protease, 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 targeting 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 derivant 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 protease in D88.This protease 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 HAS product (referring to for example people such as U.S. Patent number 5,965,386 and Kerry-Williams, Yast14:161-169 is 1998, with its complete being incorporated herein by reference).
BXP10 has following genotype: leu2-3, leu2-122, can1, pral, ubc4, ura3, yap3 ∷ URA3, ly2, 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-mannosylglycoprotein matter O-mannose transferase (Pmt) evolution conservative family.The Transmembrane Topology of Pmtlp 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 HAS 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 conventional method, for example by inserting in the host chromosome or the generation of the coded sequence on the free plasmid.With any common method for example electroporation with the coded 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 harvesting, 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.
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 selected 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:HAS, 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 saccharomyces cerevisiae terminator sequence of PRB1 saccharomyces cerevisiae promoter (PRB1p), fusion targeting sequencing (FL), coding HA.The sequence that merges targeting sequencing is made up of preceding 19 aminoacid of human serum albumin (SEQ ID NO:3) signal peptide and last 5 aminoacid of mating factor α 1 promoter (SLDKR is referring to EP-A-387319, with its complete being incorporated herein by reference).
Plasmid pPPC0005, pScCHSA, pScNHSA and pC4:Has 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 preserving number ATCC PTA-3278, PTA-3276, PTA-3279 and PTA-3277 respectively.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 promoter.Referring to for example Dominik Mumburg, RolfMuller and Martin Funk, Nucleic Acids Research 22 (25): 5767-5768,1994.The length of Met25 promoter 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 start codon and indicate underscore:
GCGGCCGCCGGATGCAAGGGTTCGAATCCCTTAGCTCTCATTATTTTTTGCTTTTTCTCTTGAGGTCACATGATCGCAAAATGGCAAATGGCACGTGAAGCTGTCGATATTGGGGAACTGTGGTGGTTGGCAAATGACTAATTAAGTTAGTCAAGGCGCCATCCTCATGAAAACTGTGTAACATAATAACCGAAGTGTCGAAAAGGTGGCACCTTGTCCAATTGAACACGCTCGATGAAAAAAATAAGATATATATAAGGTTAAGTAAAGCGTCTGTTAGAAAGGAAGTTTTTCCTTTTTCTTGCTCTCTTGTCTTTTCATCTACTATTTCCTTCGTGTAATACAGGGTCGTCAGATACATAGATACAATTCTATTACCCCCATCCATACA
A TG(SEQ?ID?NO：5)
Being used in other promoter of expressing albumin fusion proteins in the yeast comprises as follows:
A) cbh 1 promoter:
TCTAGAGTTGTGAAGTCGGTAATCCCGCTGTATAGTAATACGAGTCGCATCTAAATACTCCGAAGCTGCTGCGAACCCGGAGAATCGAGATGTGCTGGAAAGCTTCTAGCGAGCGGCTAAATTAGCATGAAAGGCTATGAGAAATTCTGGAGACGGCTTGTTGAATCATGGCGTTCCATTCTTCGACAAGCAAAGCGTTCCGTCGCAGTAGCAGGCACTCATTCCCGAAAAAACTCGGAGATTCCTAAGTAGCGATGGAACCGGAATAATATAATAGGCAATACATTGAGTTGCCTCGACGGTTGCAATGCAGGGGTACTGAGCTTGGACATAACTGTTCCGTACCCCACCTCTTCTCAACCTTTGGCGTTTCCCTGATTCAGCGTACCCGTACAAGTCGTAATCACTATTAACCCAGACTGACCGGACGTGTTTTGCCCTTCATTTGGAGAAATAATGTCATTGCGATGTGTAATTTGCCTGCTTGACCGACTGGGGCTGTTCGAAGCCCGAATGTAGGATTGTTATCCGAACTCTGCTCGTAGAGGCATGTTGTGAATCTGTGTCGGGCAGGACACGCCTCGAAGGTTCACGGCAAGGGAAACCACCGATAGCAGTGTCTAGTAGCAACCTGTAAAGCCGCAATGCAGCATCACTGGAAAATACAAACCAATGGCTAAAAGTACATAAGTTAATGCCTAAAGAAGTCATATACCAGCGGCTAATAATTGTACAATCAAGTGGCTAAACGTACCGTAATTTGCCAACGGCTTGTGGGGTTGCAGAAGCAACGGCAAAGCCCCACTTCCCCACGTTTGTTTCTTCACTCAGTCCAATCTCAGCTGGTGATCCCCCAATTGGGTCGCTTGTTTGTTCCGGTGAAGTGAAAGAAGACAGAGGTAAGAATGTCTGACTCGGAGCGTTTTGCATACAACCAAGGGCAGTGATGGAAGACAGTGAAATGTTGACATTCAAGGAGTATTTAGCCAGGGATGCTTGAGTGTATCGTGTAAGGAGGTTTGTCTGCCGATACGACGAATACTGTATAGTCACTTCTGGTGAAGTGGTCCATATTGAAATGTAAGTCGGCACTGAACAGGCAAAAGATTGAGTTGAAACTGCCTAAGATCTCGGGCCCTCGGGCCTTCGGCCTTTGGGTGTACATGTTTGTGCTCCGGGCAAATGCAAAGTGTGGTAGGATCGAACACACTGCTGCCTTTACCAAGCAGCTGAGGGTATGTGATAGGCAAATGTTCAGGGGCCACTGCATGGTTTCGAATAGAAAGAGAAGCTTAGCCAAGAACAATAGCCGATAAAGATAGCCTCATTAAACGGAATGAGCTAGTAGGCAAAGTCAGCGAATGTGTATATATAAAGGTTCGAGGTCCGTGCCTCCCTCATGCTCTCCCCATCTACTCATCAACTCAGATCCTCCAGGAGACTTGTACACCATCTTTTGAGGCACAGAAACCCAATAGTCAACCGCGGACTGGCATC(SEQ?ID?NO：113)
B) the cysD promoter of aspergillus nidulans (Aspergillus nidulans):
AGATCTGGTTCCTGAGTACATCTACCGATGCGCCTCGATCCCCCTCTTAGCCGCATGAGATTCCTACCATTTATGTCCTATCGTTCAGGGTCCTATTTGGACCGCTAGAAATAGACTCTGCTCGATTTGTTTCCATTATTCACGCAATTACGATAGTATTTGGCTCTTTTCGTTTGGCCCAGGTCAATTCGGGTAAGACGCGATCACGCCATTGTGGCCGCCGGCGTTGTGCTGCTGCTATTCCCCGCATATAAACAACCCCTCCACCAGTTCGTTGGGCTTTGCGAATGCTGTACTCTATTTCAAGTTGTCAAAAGAGAGGATTCAAAAAATTATACCCCAGATATCAAAGATATCAAAGCCATC(SEQ?ID?NO：114)
C) modified cbh1 promoter has sequence:
TCTAGAGTTGTGAAGTCGGTAATCCCGCTGTATAGTAATACGAGTCGCATCTAAATACTCCGAAGCTGCTGCGAACCCGGAGAATCGAGATGTGCTGGAAAGCTTCTAGCGAGCGGCTAAATTAGCATGAAAGGCTATGAGAAATTCTGGAGACGGCTTGTTGAATCATGGCGTTCCATTCTTCGACAAGCAAAGCGTTCCGTCGCAGTAGCAGGCACTCATTCCCGAAAAAACTCGGAGATTCCTAAGTAGCGATGGAACCGGAATAATATAATAGGCAATACATTGAGTTGCCTCGACGGTTGCAATGCAGGGGTACTGAGCTTGGACATAACTGTTCCGTACCCCACCTCTTCTCAACCTTTGGCGTTTCCCTGATTCAGCGTACCCGTACAAGTCGTAATCACTATTAACCCAGACTGACCGGACGTGTTTTGCCCTTCATTTGGAGAAATAATGTCATTGCGATGTGTAATTTGCCTGCTTGACCGACTGGGGCTGTTCGAAGCCCGAATGTAGGATTGTTATCCGAACTCTGCTCGTAGAGGCATGTTGTGAATCTGTGTCGGGCAGGACACGCCTCGAAGGTTCACGGCAAGGGAAACCACCGATAGCAGTGTCTAGTAGCAACCTGTAAAGCCGCAATGCAGCATCACTGGAAAATACAAACCAATGGCTAAAAGTACATAAGTTAATGCCTAAAGAAGTCATATACCAGCGGCTAATAATTGTACAATCAAGTGGCTAAACGTACCGTAATTTGCCAACGGCTTGTGGGGTTGCAGAAGCAACGGCAAAGCCCCACTTCCCCACGTTTGTTTCTTCACTCAGTCCAATCTCAGCTGGTGATCCCCCAATTGGGTCGCTTGTTTGTTCCGGTGAAGTGAAAGAAGACAGAGGTAAGAATGTCTGACTCGGAGCGTTTTGCATACAACCAAGGGCAGTGATGGAAGACAGTGAAATGTTGACATTCAAGGAGTATTTAGCCAGGGATGCTTGAGTGTATCGTGTAAGGAGGTTTGTCTGCCGATACGACGAATACTGTATAGTCACTTCTGGTGAAGTGGTCCATATTGAAATGTAAGTCGGCACTGAACAGGCAAAAGATTGAGTTGAAACTGCCTAAGATCTCGGGCCCTCGGGCCTTCGGCCTTTGGGTGTACATGTTTGTGCTCCGGGCAAATGCAAAGTGTGGTAGGATCGAACACACTGCTGCCTTTACCAAGCAGCTGAGGGTATGTGATAGGCAAATGTTCAGGGGCCACTGCATGGTTTCGAATAGAAAGAGAAGCTTAGCCTGCAGCCTCTTATCGAGAAAGAAATTACCGTCGCTCGTGATTTGTTTGCAAAAAGAACAAAACTGAAAAAACCCAGACACGCTCGACTTCCTGTCTTCCTATTGATTGCAGCTTCCAATTTCGTCACACAACAAGGTCCTAGCTTAGCCAAGAACAATAGCCGATAAAGATAGCCTCATTAAACGGAATGAGCTAGTAGGCAAAGTCAGCGAATGTGTATATAAAGGTTCGAGGTCCGTGCCTCCCTCATGCTCTCCCCATCTACTCATCAACTCAGATCCTCCAGGAGACTTGTACACCATCTTTTGAGGCACAGAAACCCAATAGTCAACCGCGGACTGGCATC(SEQ?ID?NO：115)
D) the cysD promoter 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 polymerase 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 hydrolysing 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 T4 dna ligase that can catalysis flush end dna molecular connects, the section of end-filling is incubated with the excessive greatly linkers of molal quantity.Thus, 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 action that produces the end compatible with the DNA segment ends with suitable restriction endonuclease.
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 the method, the flank of DNA that will enzymatic amplification is two kinds of specific oligonucleotide 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), Kluyveromyces (Kluyveromyces), mycocandida (Candida), Torulopsis (Torulopsis), spore torulopsis (Torulaspora) is arranged, fragmentation saccharomyces (Schizosaccharomyces), Citeromycesbaodingensis belongs to (Citeromyces), pipe capsule Saccharomyces (Pachysolen), Debaryomyces (Debaryomyces), the strange Saccharomyces of prunus mume (sieb.) sieb.et zucc. (Metschunikowia), red teliosporeae (Rhodosporidium), white winter spore yeast (Leucosporidium), Portugal's shape Saccharomyces (Botryoascus), lock is thrown Saccharomyces (Sporidiobolus), Endomycopsis (Endomycopsis) or the like.Is to be selected from down the genus of organizing preferred the genus: saccharomyces, fragmentation saccharomyces, 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 Kluyveromyces has the crisp kluyveromyces of wall (K.fragilis), Kluyveromyces lactis (K.lactis) and yeast Kluyveromyces marxianus (K.marxianus).The suitable spore torula species that have have Dell that spore torula T.delbrueckii is arranged).The example of Pichia sp. (Hansenula yeast) 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 saccharomyces cerevisiae transforms, all be incorporated herein by reference from EP 251 744, EP 258 067 and WO 90/01063.
Preferred saccharomyces cerevisiae example kind comprises that Saccharomyces cerevisiae (S.cerevisiae), Italian sugar yeast (S.italicus), saccharifying 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 Hansenula yeast example kind comprises multiple-shaped nuohan inferior yeast (Hpolymorpha) (being P.angusta now), unusual Hansenula yeast (H.anomala) (being unusual Pichia sp. P.anomala now) and broken capsule Pichia sp. (Pichia capsulata).Preferred in addition Pichia sp. example kind comprises Pichia pastoris (P.pastoris).Preferred aspergillosis 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 BY4743hsp150 mutant (ATCC accession number 4021266); Saccharomyces cerevisiae Hansen, teleomorph strain BY4743pmtl 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 promoter that is applicable to saccharomyces cerevisiae comprises the gene with PGK1, GAL1 or GAL10 gene, CYC1, PHO5, TRP1, ADH1, ADH2, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvic carboxylase, phosphofructokinase, phosphotriose isomerase, phosphogvlucoisomerase, the gene of glucokinase, α-mating factor pheromone, (pheromone of a mating factor) relevant promoter, the PRB1 promoter, the GUT2 promoter, the GPD1 promoter, and involve part 5 ' control region and other promoter part 5 ' control region or with the hybrid promoter (for example promoter of EP-A-258067) of the heterozygote of UAS.
Be used for foxtail millet wine fragmentation sugar yeast (Schizosaccharomyces pombe) then the startup of being convenient to regulate as Maundrell, J. Biol.Chem.265:10857-10864,1990 thiamines from the nmt gene of describing can be suppressed promoter, and as Hoffman and Winston, Genetics 124:807-816,1990 glucoses of describing can suppress the jbp1 gene promoter.
Transform Pichia sp. 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 promoter comprises AOX1 and AOX2.People such as Gleeson, J.Gen.Microbiol.132:3459-3465,1986 comprise the information about Hansenula yeast carrier and conversion, suitable promoter 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 promoter is PGK1.
3 ' flanking sequence of the preferred eukaryotic gene of transcription stop signals, it comprises the correct signal that is used for tanscription 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 promoter.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 targeting sequencing at first, and it can be effective any targeting sequencing in selected yeast.Useful targeting sequencing 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 calcium protein signal sequence (MLQNSAVLLLLVISASA, SEQ ID NO:7)
C) the pre-pro district of HAS signal sequence (for example MKWVTFISLLFLFSSAYSRGVFRR, SEQ ID NO:8)
D) the pre district of HAS signal sequence (for example MKWVTFISLLFLFSSAYS, SEQ IDNO:9) or its variant are such as for example MKWVSFISLLFLFSSAYS (SEQ ID NO:10)
E) invertase signal sequence (for example MLLQAFLFLLAGFAAKISA, SEQ ID NO:11)
F) yeast mating factor alpha 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 targeting sequencing
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 targeting sequencing (for example MNIFYIFLFLLSFVQGSLDKR, SEQ ID NO:15)
K) immunoglobulin 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 MMKTLLLFVGLLTWESGQVLG, SEQ ID NO:18)
N) IGFBP (insulin-like growth factor binding protein) 4 signal sequence (for example MLPLCLVAALLLAAGPGPSLG, SEQ ID NO:19)
O) variant in HAS 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 HAS (SEQ IDNO:24);
The leading HSA#66-MKWVTFISLLFLFGGVSG of modified HAS (SEQ IDNO:25);
Modified HAS (A14) is leading-MKWVTFISLLFLFAGVSG (SEQ ID NO:26);
Leading (being also referred to as modified the HAS#65)-MKWVTFISLLFLFSGVSG of modified HAS (S14) (SEQ ID NO:27),
Modified HAS (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 glucanase (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 aminoacid; 16 pre aminoacid and 13 pro aminoacid) MNIFYIFLFLLSFVQGLEHTHRRGSLDKR (SEQ ID NO:32)
W) saccharifying yeast (S.diastaticus) grape saccharogenic amylase II secretion targeting sequencing
X) saccharomyces carlsbergensis (S. carlsbergensis) alpha-galactosidase (MEL1) secretion targeting sequencing
Y) candida mycoderma glucoamylase targeting sequencing
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) saccharomyces cerevisiae invertase (SUC2) is leading, is disclosed in JP 62-096086 (grant number 911036516 is incorporated herein by reference); Or
Dd) multiple Flos Calystegiae sepii powder enzyme-MKLAYSLLLPLAGVSASVINYKR (SEQ ID NO:33)
Ee) the leading variant #1-of the former peptide of modified TA57
MKLKTVRSAVLSSLFASQVLGQPIDDTESQTTSVNLMADDTESAFATQTNSGGLDVVGLISMAKR(SEQ?ID?NO：34)
Ff) the leading variant #2-of the former peptide of modified TA57
MKLKTVRSAVLSSLFASQVLGQPIDDTESQTTSVNLMADDTESA
FATQTNSGGLDVVGLISMAEEGEPKR(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 technique.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 complementary host cell usually.
The polynucleotide of code book invention albumin fusion proteins can with contain the carrier that is useful on the selected marker of in the host, duplicating and be connected.Usually, plasmid vector is introduced precipitate, such as the calcium phosphate precipitation thing, or with the complex 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 insert should can be operatively connected with suitable promoter, early stage and late promoter and retrovirus LTR etc. such as bacteriophage lambda PL promoter, escherichia coli lac, trp, phoA and tac promoter, SV40.Other suitable promoter is known to those skilled in the art.Also will comprise the site that is used for transcription initiation and termination in the expression construct, and be used to the ribosome binding 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 termination codon (UAA, UGA or UAG) in the appropriate location of terminal point.
As mentioned above, expression vector preferably comprises at least a selected marker.Tetracycline, kanamycin or amicillin resistance that such labelling comprises dihydrofolate reductase, G418, glutamine synthase or is used for the neomycin resistance that eukaryotic cell cultivates and is used for cultivating on escherichia coli or other antibacterial.Suitably host's representative example includes but not limited to: bacterial cell belongs to and Salmonella typhimurium (Salmonella typhimurium) cell such as escherichia coli, strepto-; The fungal cell is such as yeast cells (for example 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 plant cell.The suitable culture medium and the condition of culture of above-mentioned host cell are known in this area.
The preferred vector that is used for antibacterial 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, pTEF 1/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 escherichia coli, 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 antibacterial 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 alkali 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 purification to improve in gram negative bacteria.Referring to U.S. Patent number 5,576,195 and 5,846,818, its content 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 calcium protein signal sequence (MLQNSAVLLLLVISASA, SEQ ID NO:7)
C) the pre-pro district of HAS signal sequence (for example MKWVTFISLLFLFSSAYSRGVFRR, SEQ ID NO:8)
D) the pre district of HAS signal sequence (for example MKWVTFISLLFLFSSAYS, SEQ IDNO:9) or its variant are such as for example MKWVSFISLLFLFSSAYS (SEQ ID NO:10)
E) invertase signal sequence (for example MLLQAFLFLLAGFAAKISA, SEQ ID NO:11)
F) yeast mating factor alpha signal sequence (for example
MRFPSIFTAVLAFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPF SNSTNNGLLFINTTIASIAAKEEGVSLEKR, SEQ ID NO:12 or MRFPSIFTAVLAFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPF SNSTNNGLLFINTTIASIAAKEEGVSLDKR, SEQ ID NO:12)
G) Kluyveromyces lactis (K.Lactis) kills and wounds the toxin targeting sequencing
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 MKWVSFISLLFLFS SAYSRSLDKR, SEQ ID NO:14)
J) Kluyveromyces lactis kill and wound/MF α-1 merges targeting sequencing (for example MNIFYIFLFLLSFVQGSLDKR, SEQ ID NO:15)
K) immunoglobulin 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 NQ:18)
N) IGFBP (insulin-like growth factor binding protein) 4 signal sequence (for example MLPLCLVAALLLAAGPGPSLG, SEQ ID NO:19)
O) variant in HAS 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 HAS (SEQ IDNO:24);
The leading HSA#66-MKWVTFISLLFLFGGVSG of modified HAS (SEQ IDNO:25);
Modified HAS (A14) is leading-MKWVTFISLLFLFAGVSG (SEQ ID NO:26);
Leading (being also referred to as modified HAS the #65)-MKWVTFISLLFLFSGVSG of modified HAS (S14) (SEQ ID NO:27),
Modified HAS (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 glucanase (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 aminoacid; 16 pre aminoacid and 13 pro aminoacid) MNIFYIFLFLLSFVQGLEHTHRRGSLDKR (SEQ ID NO:32)
W) saccharifying yeast (S.diastaticus) grape saccharogenic amylase II secretion targeting sequencing
X) saccharomyces carlsbergensis (S.carlsbergensis) alpha-galactosidase (MEL1) secretion targeting sequencing
Y) candida mycoderma glucoamylase targeting sequencing
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) saccharomyces cerevisiae invertase (SUC2) is leading, is disclosed in JP 62-096086 (grant number 911036516 is incorporated herein by reference); Or
Dd) multiple Flos Calystegiae sepii powder enzyme-MKLAYSLLLPLAGVSASVINYKR (SEQ ID NO:33)
Ee) the leading variant #1-of the former peptide of modified TA57
MKLKTVRSAVLSSLFASQVLGQPIDDTESQTTSVNLMADDTESAFATQTNSGGLDVVGLISMAKR(SEQ?ID?NO：34)
Ff) the leading variant #2-of the former peptide of modified TA57
MKLKTVRSAVLSSLFASQVLGQPIDDTESQTTSVNLMADDTESAFATQTNSGGLDVVGLISMAEEGEPKR(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 HAS/kex2 signal sequence (SEQ ID NO:112) is fused to the amino terminal of albumin fusion proteins, comprise the fusion rotein that comprises albumin 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 targeting sequencing 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 (methionine sulphoximine) or ammonia first dish purine, increase respectively as the carrier of selected 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 cell line of glutamine synthase feminine gender.The glutamine synthase expression system also can be by the extra inhibitor that stops endogenous 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 zones (for example promoter and/or enhancer).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 cells, perhaps host cell can be a prokaryotic cell, 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 inducer can improve the expression from some promoter; 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 cell line can guarantee modification and the processing that expressed exogenous proteins 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 vector.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 hereditary 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 mammal origin.Can operate the hereditary 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 zone (for example promoter and/or enhancer) 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 purification from the reconstitution cell culture by well-known method, comprises ammonium sulfate or ethanol precipitation, acid extraction, anion or cation-exchange chromatography, cellulose phosphate chromatography, hydrophobic interaction chromatography, affinity chromatograph, hydroxyapatite, dewatering electric charge interaction chromatography and agglutinin chromatography.Most preferably, adopt high performance liquid chroma-tography (" HPLC ") to carry out purification.
In preferred embodiments, albumin fusion proteins of the present invention carries out purification 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 purification 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 purification 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 purification 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 purification with affinity chromatograph, includes but not limited to HAS 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 purification with above-mentioned one or more chromatography methods.In other embodiment preferred, albumin fusion proteins of the present invention carries out purification with following one or more chromatographic columns: Q Sepharose FF post, SP Sepharose FF post, QSepharose high-efficiency 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 purification 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 purification 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 technique from protokaryon or eucaryon host generation, and described host comprises for example antibacterial, yeast, higher plant, insecticide 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 of the N-of translation initiation codon coding.Though the terminal methionine of most proteinic N-is also effectively removed in most prokaryotic cells, 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 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 methanol 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 methanol as sole carbon source, pichia pastoris phaff must generate high-caliber Alcohol oxidase, and partly cause is that Alcohol oxidase is to O
2Affinity relatively low.So in relying on the growth medium of methanol as main carbon source, the promoter region height of one of two kinds of Alcohol oxidase genes (AOX1) has activity.When having methanol, 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 sp. of when having methanol, 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 promoter that is connected with pichia pastoris phaff alkali 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 culture yeasts culture among for example pGAPZ or the pGAPZalpha and when not having methanol 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, hydroxyproline, sarcosine, citrulline, Homocitrulline, cysteic acid, tert-butyl group glycine, tert-butyl group alanine, phenylglycine, Cyclohexylalanine, the b-alanine, fluoroamino acid, tape label aminoacid is such as the b-methylamino acid, the Ca-methylamino acid, the Na-methylamino acid, and general amino acid analogue.In addition, aminoacid 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, acetylation, phosphorylation, amidatioon, by known protection/blocking groups derive, Proteolytic enzyme cuts, with being connected of antibody molecule or other cell ligand etc.Can carry out any numerous chemical modification by known technology, include but not limited to Bromine cyanide., trypsin, chymase, papain, V8 protease, NaBH
4Special chemical cleavage; Acetylation, formylated, oxidation, reduction; Metabolism when having tunicamycin is synthetic; Or the like.
Other post translational 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 chemical modification 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, isotope or affinity tag, thereby can detect and isolated protein.
The example of suitable enzyme comprises horseradish peroxidase, alkali phosphatase, beta galactosidase or acetylcholine esterase; The example of suitable prothetic group complex comprises strepto-affinity element/biotin and affinity element/biotin; The example of suitable fluorescent material comprises umbelliferone, fluorescein, Fluorescein isothiocyanate, rhodamine, dichlorotriazine amine fluorescein, dansyl Cl or phycoerythrin; The example of luminescent substance comprises luminol; The bioluminescence examples of substances comprises luciferase, fluorescein and aequorin; And the example of suitable radioactive substance comprise iodine (
121I,
123I,
125I,
131I), carbon (
14C), sulfur (
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 also can be cyclic, 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 acetylation, acidylate, the ADP-ribosylation, amidatioon, the covalent attachment of flavin, the covalent attachment of haemachrome 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 bond forms, demethylation, the formation of covalent cross-linking, the formation of cysteine, the formation of pyroglutamic acid, formylated, gamma-carboxylation, glycosylation, the formation of GPI anchor, hydroxylation, iodate, methylate, myristylization, oxidation, PEGization, Proteolytic enzyme processing, phosphorylation, isoprenylation, raceme, selenonylization, sulphation, interpolation aminoacid 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 purification.In preferred embodiments, the mark aminoacid sequence is six histidine peptides, such as label that provides in the pQE carrier (QIAGEN company, 9259 Eton Avenue, 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 histidine are that the purification of fusion rotein facilitates.Other peptide tag that can be used for purification 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, therapeutic 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 paclitaxel, Cytochalasin B, Gramicidin D, ethidium bromide, ipecine, mitomycin, etoposide, teniposide, vincristine, vincaleucoblastine, colchicine, amycin, daunorubicin, dihydroxy anthracin diketone, mitoxantrone, mithramycin, actinomycin D, 1-boldenone, glucocorticoid, procaine, tetracaine, lignocaine, Propranolol and puromycin and analog or homologue.Therapeutic agent includes but not limited to antimetabolite (methotrexate for example, Ismipur, 6-thioguanine, cytosine arabinoside, the 5-fluorouracil decarbazine), alkylating agent (dichloromethyldiethylamine (chlormethine) for example, plug is for group, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, mitobronitol, streptozotocin, ametycin and suitable dichloro diamino platinum (II) be cisplatin (DDP)), anthracene nucleus class (for example daunorubicin (being called daunomycin in the past) and amycin), antibiotic (dactinomycin (being called D actinomycin D in the past) for example, bleomycin, mithramycin and anthramycin (AMC)) and antimitotic agent (for example vincristine and vincaleucoblastine).
Conjugate of the present invention can be used for modifying given biological answer-reply, does not think that therapeutic agent or medicine module are limited to classical chemotherapeutant.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, diphtherotoxin; Protein, such as tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet derived growth factor, 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 dressing agent, such as for example lymphokine, il-1 (" IL-1 "), interleukin-2 (" IL-2 "), interleukin-6 (" IL-6 "), granulocyte macrophage colony stimulating factor (" GM-CSF "), granulocyte colony-stimulating factor (" G-CSF ") or other somatomedin.The technology that is used for 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 to albumin fusion proteins of the present invention immunoassay bonded, bonded or associating polypeptide with it or purification with it be useful especially.This solid support includes but not limited to glass, cellulose, polyacrylamide, nylon, polystyrene, polrvinyl 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 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 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 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 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 albumin 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 albumin C-terminal.
The present invention also provides the chemical modification derivant of albumin fusion proteins of the present invention, and it can provide extra advantage, such as solubility, stability and the circulation time increase or the immunogenicity reduction (referring to U.S. Patent number 4,179,337) of polypeptide.The chemical module that is used for derivant can be selected from water-soluble polymer, such as Polyethylene 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 precalculated position, and can comprise one, two, the three or more chemical module that adheres to.
Polymer can be any molecular weight, and can be branched or not branched.For Polyethylene Glycol, for easy operating and manufacturing, preferred molecular weight is (term " about " refers in the Polyethylene 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 Polyethylene Glycol to other known effect of therapeutic protein or analog).For example, the mean molecule quantity of Polyethylene Glycol can be about 200,500,1000,1500,2000,2500,3000,3500,4000,4500,5000,5500,6000,6500,7000,7500,8000,8500,9000,9500,10,000,10,500,11,000,11,500,12,000,12,500,13,000,13,500,14,000,14,500,15,000,15,500,16,000,16,500,17,000,17,500,18,000,18,500,19,000,19,500,20,000,25,000,30,000,35,000,40,000,45,000,50,000,55,000,60,000,65,000,70,000,75,000,80,000,85,000,90,000,95,000 or 100,000 kDa.
As mentioned above, Polyethylene Glycol can have apparatus derivatorius.Branched Polyethylene 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, Bioeonjug.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 adherence method, such as disclosed method (PEG is coupled on the G-CSF) among the EP 0 401 384 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 HFC-143a sulfonic acid (tresyl) chloride GM-GSF carry out PEGization.For example, Polyethylene 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; Aminoacid 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 purposes, such as being attached on N-terminal or the lysine group.
As mentioned above, Polyethylene Glycol can by with being connected of any several amino acids residue attached on the protein.For example, Polyethylene Glycol can be by being connected with protein with the covalent bond of lysine, histidine, aspartic acid, glutamic acid or cysteine residues.Can adopt one or more reactive chemistries that Polyethylene Glycol is attached to proteinic particular amino acid residue (for example lysine, histidine, aspartic acid, glutamic acid or cysteine) or the proteinic amino acid residue (for example lysine, histidine, aspartic acid, glutamic acid, cysteine and combination thereof) that surpasses a type.
May wish especially in N-terminal chemically modified protein matter.With the example of Polyethylene Glycol as compositions, can multiple peg molecule (according to molecular weight, branch situation, etc.), the type of the ratio in the reactant 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 purification N-terminal PEGization material from PEGization protein molecule colony.Can realize that at N-terminal selective chemical modification protein the reproducibility alkanisation utilizes specified protein to can be used for the difference reaction of deutero-dissimilar primary amino radical (lysine is to N-terminal) by the reproducibility 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, Polyethylene Glycol directly or by transition joint can be attached to albumin fusion proteins.Be used for Polyethylene 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.ofHematol.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 Polyethylene Glycol directly being attached to proteinic amino acid residue and not having a kind of system of transition joint to adopt the sulfonated MPEG of HFC-143a, it is the chloride (ClSO that uses HFC-143a sulfonic acid
2CH
2CF
3) single methoxy Polyethylene Glycol (MPEG) modified generate.After protein and the sulfonated MPEG reaction of HFC-143a, Polyethylene 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-HFC-143a reacts and protein-Polyethylene Glycol conjugate of generating.
Also available multiple different transition joints are attached to protein with Polyethylene Glycol.For example, U.S. Patent number 5,612,460 disclose and are used for Polyethylene Glycol is connected to urethane joint on the protein, and its complete open book is incorporated herein by reference.Wherein Polyethylene Glycol is attached to proteinic protein-Polyethylene Glycol conjugate by joint and also can generates by protein and following compounds are reacted, such as MPEG-succinimido succinate/ester, with 1,1 '-the activatory MPEG of carbonyl dimidazoles, MPEG-2,4,5-trichlorine amyl group carbonate/ester, MPEG-paranitrophenol carbonate/ester, and various MPEG-succinate/ester derivants.Described among the international publication number WO 98/32466 and be used for Polyethylene 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 Polyethylene 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 Polyethylene 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 purification from chemosynthesis and reconstitution cell culture by standard method, includes but not limited to ammonium sulfate or ethanol precipitation, acid extraction, anion or cation-exchange chromatography, cellulose phosphate chromatography, hydrophobic interaction chromatography, affinity chromatograph, hydroxyapatite and agglutinin chromatography.Most preferably, adopt high performance liquid chroma-tography (" HPLC ") to carry out purification.When polypeptide separating and/or purge process in during degeneration, 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 albumin antibody sandwich elisa plate, plate is sealed to stop non-specific binding, clean elisa plate, (with one or more variable concentrations) 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 labelling two can be anti-people's albumin 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, cell line or the tissue of expressing by the coded albumin fusion proteins of the polynucleotide of code book invention albumin fusion proteins by genetic engineering 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 genetic defect.Polynucleotide disclosed by the invention provide the method with these genetic defectes of height accurate way targeting.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 algoscopy, 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 label is road known in the art, comprises the enzyme labelling thing, such as glucoseoxidase; Radiosiotope, such as iodine (
131I,
125I,
123I,
121I), carbon (
14C), sulfur (
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; Fluorescent marker is such as fluorescein and rhodamine; And biotin.
Albumin fusion proteins of the present invention also can detect in vivo by imaging.Being used for the label of protein in-vivo imaging or mark comprises and can pass through X-radiography, nuclear magnetic resonance, NMR (NMR) or electron spin resonance (electron spin relaxtion, ESR) material of Jian Ceing.For the X-radiography, suitable label comprises radiosiotope, 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 nutrient that offers the cell line of expressing albumin fusion proteins of the present invention by labelling.
Will be through suitably detecting image-forming module, (for example such as radiosiotope
131I,
112In,
99mTc, iodine (
131I,
125I,
123I,
121I), carbon (
14C), sulfur (
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 labelling import the mammal 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 radiosiotope module,, inject radioactive amount usually in about 5 to 20 millicuries for the human experimenter
99mIn the scope of Tc.Then the labelling albumin fusion proteins will be preferentially existed in vivo position (for example organ, cell, extracellular space or the substrate) 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, through the labelling albumin fusion proteins will preferentially in health, exist with (be used to generate albumin fusion proteins of the present invention) therapeutic antibodies the position (for example organ, cell, extracellular space or substrate) of the corresponding polypeptide/epi-position of bonded polypeptide/epi-position accumulate.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 molecules 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 destroys the method for cell (for example destroying tumor 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 chemical compounds of activation of endogenous cellulotoxic effect system, radiosiotope, holotoxin, improvement toxin, toxin or cell in or non-existent usually on the surface, cause any molecule or the enzyme of cell death under given conditions.Can include but not limited to radiosiotope known in the art according to the toxin that method of the present invention is used, chemical compound is such as for example in conjunction with the antibody (or it contains the complement fixation part) of inherent or inductive endogenous cell toxic effect system, thymidine kinase, endonuclease, alpha-toxin, ricin, Agglutinin, Pseudomonas exotoxin A, diphtheria toxin, diphtherotoxin, the Saponaria officinalis toxalbumin, the Fructus Momordicae charantiae toxalbumin, spend more the Rhizoma Melaleuca Viridiflora toxalbumin, pokeweed antiviral protein, α-broom aspergillin and cholera toxin." toxin " also comprise and suppress cell agent or cytocide, therapeutic agent or radioactive metal ion, and alpha emitter for example is such as for example
213Bi, or other radiosiotope are such as for example
103Pd,
133Xe,
131I,
68Ge,
57Co,
65Zn,
85Sr,
32P,
35S,
90Y,
153Sm,
153Gd,
169Yb,
51Cr,
54Mn,
75Se,
113Sn,
90Yttrium,
117Stannum,
186Rhenium,
166Holmium and
188Rhenium; Luminous marker is such as luminol; Fluorescent marker is such as fluorescein and rhodamine; And biotin.In a specific embodiment, the invention provides by using and radiosiotope
90Associating polypeptide of the present invention of Y or antibody and specificity destroy the method for cell (for example destroying tumor cell).In another specific embodiment, the invention provides by using and radiosiotope
111Associating polypeptide of the present invention of In or antibody and specificity destroy the method for cell (for example destroying tumor cell).In another concrete embodiment, the invention provides by using and radiosiotope
131Associating polypeptide of the present invention of I or antibody and specificity destroy the method for cell (for example destroying tumor cell).
Can use technology known in the art to come labelling 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 content 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 mammal 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 or reduce as disorderly index.For cancer, the tendency that a large amount of relatively transcripies 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 measure 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 neurological 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 insulin) that disappearance or level descend, (for example Hb-S is to replenish hemoglobin 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 receptor), 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 bond of albumin fusion proteins, and/or reduce the excessive generation of the polypeptide of the therapeutic antibodies institute specific bond 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 bond that is used to generate albumin fusion proteins, and it is gone up bonded polypeptide by binding film (receptor) 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 algoscopy
Chemical compound of the present invention can be used for diagnosing, treat, preventing and/or predicts particularly people's various disorders of mammal.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 ", " disordered breathing ", " angiogenesis inhibitor activity ", " cellular level disease ", " wound healing and epithelial cell proliferation ", " neural activity and neurological disorder ", " endocrine regulation ", " reproductive system disorder ", " infectious 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, synovial fluid 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 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 algoscopys 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 gene expression strengthens or the patient of reduction will suffer bad luck thus.
Qualitative or the quantitative measurement 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 average level 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, cell line, 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, synovial fluid 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 mammal 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 suitable method 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 (RT-LCR).
The invention still further relates to and be used for the imitate diagnostic methods method of level of product (for example cell and tissue) or by it bonded or with it associating polypeptide bonded of detection of biological with albumin fusion proteins of the present invention, such as quantitative and diagnostic algoscopy, comprise the normal and abnormal level of measuring polypeptide.So, for example, according to of the present invention be used to detect bonded or can be used for detecting the existence of tumor with respect to the diagnostic algoscopy of the abnormal level of normal control tissue sample by its bonded or associating polypeptide with albumin fusion proteins with it.Can be used for measuring derived from the determination techniques of the level of bonded in host's the sample or or with it associating polypeptide bonded those skilled in the art are known by it with albumin fusion proteins of the present invention.Such assay method comprises radioimmunoassay, competitive binding assay method, Western engram analysis and ELISA algoscopy.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 label is road known in the art, comprises the enzyme labelling thing, such as glucoseoxidase, with radiosiotope, such as iodine (
125I,
121I), carbon (
14C), sulfur (
35S), tritium (
3H), indium (
112In) and technetium (
99mTc), and fluorescent marker, such as fluorescein and rhodamine, and biotin.
Tissue to be analyzed or cell type generally include those known or those (such as for example cancers) suspection expression genes of interest.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 gene expression.
For example, albumin fusion proteins can be used for quantitatively or qualitative detection and albumin fusion proteins of the present invention is bonded or the existence of or with it associating polypeptide bonded by 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 fluorimetry.
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 bond or the albumin fusion proteins of variant at least can be used for quantitatively or the existence of qualitative detection gene outcome or its conservative variant or fragments of peptides.This can realize that it adopts fluorescently-labeled antibody by for example immunofluorescence technique, and detects coupling mutually with light microscopy, flow cytometry or fluorimetry.
Albumin fusion proteins of the present invention also can be used for picture and be used in situ detection and albumin fusion proteins of the present invention is bonded or by its bonded or associating with it polypeptide in immunofluorescence, immunoelectron microscope or non-immunologic assay method carefully knitting on.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 labelling realize.Preferably use albumin fusion proteins on the biological sample by covering through the albumin fusion proteins of labelling.By a kind of like this program, not only might determine the existence of or by it bonded or with it associating polypeptide bonded with albumin fusion proteins, also may determine its distribution in being examined tissue.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 albumin fusion proteins immunoassay and non-immunoassay bonded or or with it associating polypeptide bonded and generally include sample by it, lysate such as biological fluid, tissue extract, the new cell of collecting or the cell that in cell culture, had been incubated, exist can in conjunction with gene outcome or its conservative variant or fragments of peptides detect through traget antibody the time be incubated, and detect bonded antibody by any 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 celluloid 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 holder then through the albumin fusion proteins of the present invention of labelling.Available then buffer cleans solid support once more to remove unconjugated antibody or polypeptide.Optional traget antibody subsequently.Can detect the quantity of bonded label on the solid support then by conventional method.
" solid support or carrier " mean can in conjunction with polypeptide (albumin fusion proteins for example, or with albumin fusion proteins of the present invention bonded or by its bonded or associating polypeptide with it) any holder.Well-known holder or carrier comprise glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and cellulose, polyacrylamide, gabbro and magnetic iron ore improvement.For the purposes of the present invention, the character of carrier can be to a certain degree soluble or insoluble.In fact holder can have any possible construction profile, as long as link coupled molecule can be in conjunction with polypeptide.Therefore, the profile of holder can be spheric, as pearl, or columniform, as the inwall of test tube, or the outer surface of bar.Perhaps, the surface can be flat, such as thin slice, test strips etc.Preferred holder 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 label or the mark that are used for albumin fusion proteins in-vivo imaging of the present invention comprise the material that available X-radiography, NMR, MRI, CAT-scanning or ESR detect.For the X-radiography, suitable label comprises radiosiotope, 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 nutrient of engineered cells system (perhaps antibacterial or yeast strain) by labelling.
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 chemical compound or other suitable chemical compound labelling.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 radiosiotope
131I,
112In,
99mTc), radio-opaque substance, maybe can be by magnetic resonance detection the polypeptid specificity antibody or the antibody fragment of material labelling import the mammal 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 radiosiotope module,, inject radioisotopic amount usually in about 5 to 20 millicuries for the human experimenter
99mIn the scope of Tc.To preferentially contain bonded or by the position accumulation of its bonded or associating polypeptide or other material then in vivo with it with albumin fusion proteins of the present invention through the labelling 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.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 spectrophotography, fluorometry or range estimation mode.The report enzyme that can be used for detectable label antibody includes but not limited to malic dehydrogenase, staphylococcal nuclease, δ-5-steroid isomerase, yeast alcohol dehydrogenase, α-Gan Youlinsuantuoqingmei, phosphotriose isomerase, horseradish peroxidase, alkali phosphatase, asparaginase, glucoseoxidase, beta galactosidase, ribonuclease, 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 radioactive label and be used for multiple other immunoassay.For example, by the radioactive label albumin fusion proteins, albumin fusion proteins might be used for radioimmunoassay (RIA) (referring to for example Weintraub, B., Principles of radioimmunoassay, Seventh training Course on Radioligand Assay Techniques, The EndocrineSociety, in March, 1986, be incorporated herein by reference).Can come the detection of radioactive isotope by following means, include but not limited to gamma counter, scintillation counter or autoradiography.
In addition, this area knows that chelating molecule and they can be used for the labelling 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 labelling, and metal ion comprises radionuclide or fluorescent marker.For example, referring to Subramanian, R. and Meares, C.F., " Bifunctional Chelating Agentsb 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 chelating agen with described albumin and all can be used for the present invention.Chelating agen also can comprise and connects 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 chelating agen, such as diethylenetriamines-N, N, N ', N ", N "-analog of pentaacetic acid (DPTA), DPTA, and the derivant of DPTA.As nonrestrictive example, chelating agen can be 2-(right-the isothiocyanato benzyl)-6-methyl diethylene-triamine pentaacetic acid (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-triamine pentaacetic acid (CHX-DTPA), or N-[2-amino-3-(p-nitrobenzophenone) propyl group]-trans-thiacyclohexane-1,2-diamidogen-N, N ', N "-pentaacetic acid (nitro-CHX-A-DTPA).
In another embodiment, albumin fusion proteins of the present invention is attached to acyclic terpyridyl chelating agen, such as 6,6 " two [[N, N, N ", N " four (carboxymethyl) amino] methyl]-4 '-(3-amino-4-anisyl)-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, chelating agen 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 chelating agen is conjugated on the antibody, thereby those skilled in the art can be easy to disclosed method wherein made amendment chelating agen 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 ", _-tetraacethyl); People such as S.VDeshpande, 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 chelating agen.In another embodiment, chelating agen is the chelating agen that is selected from DOTA, DOTA analog and DOTA derivant.
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 triacetic acid), NOTA (1,4,7-7-triazacyclononane triacetic acid), 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 analog and derivant.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 chelating agen comprises U.S. Patent number 4,647,447; 4,687,659; 4,885,363; EP-A-71564; WO 89/00557; And disclosed chelating agen among the EP-A-232751.
In another embodiment, can be used for suitable macro ring carboxylic acid chelating agen 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 " triacetic acid (9N3); 1,5,9-triazododecane-N, N ', N " triacetic acid (12N3); And 6-acetyl bromide 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-anisyl)-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-anisyl)-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 chelating agen can (by the coordination site of chelating agen) 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,
99mTe,
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 chelating agen can be with being selected from
90Y,
111In,
177Lu,
166Ho,
215Bi and
225The metal ion of Ac carries out labelling.
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 chelating agen can carry out labelling with above-mentioned radionuclide.
In another embodiment, covalent attachment the albumin fusion proteins of the present invention of chelating agen can carry out labelling with paramagnetic metal ion, the ion that comprises transition metal and lanthanide series metal, such as atomic 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 compositions 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 chelating agen can carry out labelling 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 chelating agen can carry out labelling with the reporter that contains heavy metal, can comprise the atom of Mo, Bi, Si and W.
Might use fluorescent chemicals labelling 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 labeling chemical compound has Fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, phthaladehyde (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-triamine pentaacetic acid (DTPA) or ethylenediaminetetraacetic acid 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 chemical compound has luminol, different luminol, theromatic acridinium ester, imidazoles, acridinium salt and oxalate.
Equally, bioluminescent compound also can be used for labelling albumin fusion proteins of the present invention.Bioluminescence is a class chemiluminescence of finding in the system in biology, 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 labelling 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 shift the genetically modified organism, GMO of reorganization, external source or clone's hereditary material.Such hereditary material often is called transgenic.Genetically modified nucleotide sequence can comprise one or more transcription regulating nucleotide sequences and other nucleotide sequence such as intron, and they may be that coded proteinic optimum expression and secretion are necessary.Transgenic 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.Transgenic can be by forming from the deutero-nucleotide sequence of genome of species identical with the target animals species or different species.Transgenic 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 hereditary information is imported germ line cell, thereby gives the ability that transgenic organism passes to hereditary information the offspring.If in fact such offspring has some or all of these changes or hereditary information, they also are transgenic organisms so.This change or hereditary information can be external sources for the living species under the receiver, and external source with regard to the particular individual receiver, perhaps can be the hereditary information that the receiver 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 distinct 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 molecules 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 SV40T-antigenic (U.S. Patent number 5,728,915); Do not express (U.S. Patent number 5,731,490) of interferon 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) mice.
Though most of transgenic experiments are still selected mice and rat, preferred or even necessary other animal species that uses in some situation.The transgenic process has been successfully applied to multiple non-Mus animal, comprises that sheep, goat, pig, dog, cat, monkey, chimpanzee, hamster, rabbit, cattle and Cavia porcellus 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.AnimaiScience 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 promoter, this promoter preferentially activates in galactophore epithelial cell.The promoter of the gene of preferred control coding milk protein matter, for example the promoter of casein, beta lactoglobulin, whey acid protein or lactalbumin (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, cattle, camel or sheep.
Albumin fusion proteins of the present invention can also be expressed in transgenic plant, for example the DNA transgenic is inserted into the plant in nucleus or the plastom.Be used for the Plant Transformation flow process of external nucleic acid importing plant cell or protoplast 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 conventional method, comprise that parenteral (for example subcutaneous or intramuscular) is injected or intravenous is inculcated.Treatment can be taken medicine or repeatedly taking medicine in one period constitutes by single.
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 receiver harmless with regard to, carrier must be " acceptable ".Usually, carrier will be aseptic and pyrogen-free water or saline.Albumin fusion proteins of the present invention is particularly suitable for preparing in such as aseptic pyrogen-free water, saline or other isotonic solution at aqueous carrier, because their shelf-lifves 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 shelf-life of albumin fusion proteins in aqueous formulation to prolong.Just as discussed above, the many shelf-lifves in these therapeutic proteins significantly increase or have prolonged after merging with HA.
In being suitable for situation about using, can use characterization program that albumin fusion proteins of the present invention is mixed with aerosol with aerosol.Term " aerosol " comprises any gas matchmaker suspended phase of the albumin fusion proteins of the present invention that can be drawn in bronchioles or the nasal meatus.Particularly, aerosol comprises the gas matchmaker float of the droplet of albumin fusion proteins of the present invention, and it can generate in metered dose inhaler upon actuation or nebulizer or in aerosol apparatus.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 Raeburn, 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 pharmaceutical field.Such method comprises the step that albumin fusion proteins is combined with the carrier that constitutes one or more supplementary elements.Usually,, make product shaping then as required, so prepare described preparation by with active component and liquid carrier or finely-divided solid carrier or both homogeneous and closely combine.
The preparation that is suitable for parenteral administration comprises aqueous or non-aseptic parenteral solution under water, can contain antioxidant, buffer agent, antibacterial and make described preparation be suitable for the solute of intended recipinent; And aqueous or non-aqueous sterile suspensions, can contain suspending agent and thickening agent.Preparation can present in unit dose or multi-dose container, for example Mi Feng ampoule bottle, phial or syringe, and can be stored under freeze-dried (lyophilizing) condition, only need face add before using aseptic liquid carrier for example water promptly can be used for injecting.Now joining injection 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 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 I " 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 shelf-life 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 12.0 IU/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 DE in the unit will present more heavy weight reagent, but the frequency of taking medicine for example can be reduced to twice weekly, once in a week or still less.
Preparation of the present invention or compositions can be packed or be included in the test kit with the description or the package insert that prolong about the albumin fusion proteins composition shelf-life.For example, such description or package insert can provide the shelf-life 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 description 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 health product (nutraceuticals).For example, some albumin fusion proteins of the present invention can be used in natural prodcuts, comprises the breast or the milk product that are obtained by the transgene mammal of expressing albumin fusion proteins.Such compositions can also comprise plant or the plant product that is obtained by the transgenic plant of expressing albumin fusion proteins.Can also provide albumin fusion proteins with medicated powder or the tablet form that contains or do not contain other additives known, carrier, filler and diluent.Health product are described in Scott Hegenhart, " Food Product Design ", in December, 1993.
The present invention also provides by accepting at pharmacopedics in the carrier that the experimenter is used the method that the albumin fusion proteins of the present invention of effective dose or the polynucleotide of code book invention albumin fusion proteins (" albumin 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 " effective dose " of the present invention will be determined by this class Consideration.
As general recommendation, total the pharmacopedics effective dose 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 when taking medicine at every turn.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 take medicine continuously, use 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 intravenous.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.Determining to take medicine to consider the increase of this plasma stability when using the effective dose of albumin fusion proteins and taking medicine application program at every turn.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-HAS fusion rotein is supported every 2-4 week or the longer time scheme of taking medicine once, even takes medicine with 4 weeks or the interval more than 4 weeks.
Albumin fusion proteins and/or polynucleotide can by in oral, rectum, parenteral, the brain pond, intravaginal, intraperitoneal, part (by powder, ointment, gel, drop 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, diluent, coating material or pharmaceutical adjunct.Term " parenteral " refers to comprise when being used for this paper in intravenous, 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 slow release albumin fusion proteins and/or polynucleotide has is oral, in the rectum, parenteral, brain pond, intravaginal, intraperitoneal, part (by powder, ointment, gel, drop 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, diluent, coating material or the pharmaceutical adjunct of any kind.Term " parenteral " refers to comprise when being used for this paper in intravenous, intramuscular, intraperitoneal, the breastbone, subcutaneous and intra-articular injection and be poured in mode of administration.Other example of slow release albumin fusion proteins and/or polynucleotide comprises suitable polymeric material (such as the semi permeability polymeric matrices of for example formed product form, for example thin film or microcapsule), suitable hydrophobic material (for example can accept the emulsion 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 polyactide (U.S. Patent number 3,773,919, EP 58,481), the copolymer of L-glutamic acid and 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), ethylene vinyl acetate people such as (, the same) Langer or poly--D-(-)-3-hydroxybutyric acid (EP 133,988).
Slow release 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 components 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 receiver in the dosage that is adopted and concentration by accepting carrier with pharmacopedics with the purity of required degree in unit dose injectable forms (solution, suspension or emulsion) for albumin fusion proteins and/or polynucleotide.For example, the preferred oxygen-free agent of preparation and known to deleterious other chemical 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 dosage form.Preferably, carrier is the parenteral carrier, more preferably with the isoosmotic solution of receiver's blood.The example of such carrier medium comprises water, saline, 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 receiver at dosage that is adopted and concentration, comprises buffer agent such as phosphate, citrate, succinate, acetic acid and other organic acid or its salt; Antioxidant is such as ascorbic acid; Low-molecular-weight (being less than about 10 residues) polypeptide, for example pR60 or tripeptides; Protein is such as serum albumin, gelatin or immunoglobulin; Hydrophilic polymer is such as polyvinylpyrrolidone; Aminoacid is such as glycine, glutamic acid, aspartic acid or arginine; Monosaccharide, disaccharide and other carbohydrate comprise cellulose or derivatives thereof, glucose, mannose or dextrin; Chelating agen is such as EDTA; Sugar alcohol is such as mannitol or sorbitol; Counter ion counterionsl gegenions are such as sodium; And/or non-ionic surface active agent, 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 stabilizing agent 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 unit dose or multi-dose container as aqueous solution or the lyophilized formulations that is used for rehydration usually, for example in Mi Feng ampoule bottle or the phial.As the example of lyophilized formulations, 5ml is packed in the phial through 1% (w/v) albumin fusion proteins of aseptic filtration and/or polynucleotide aqueous solution, and with the lyophilizing of gained mixture.Make freeze dried albumin fusion proteins and/or polynucleotide rehydration with the preparation infusion 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 chloride, 0.16 micromole sodium caprylate/milligram fusion rotein, 15 mcg/ml polysorbate80 pH7.2.In the concrete and embodiment preferred, the albumin fusion proteins preparation is made up of 0.01M sodium phosphate, 0.15mM sodium chloride, 0.16 micromole sodium caprylate/milligram fusion rotein, 15 mcg/ml polysorbate80 pH7.2 at another.The pH and the buffer agent of selection and physiological conditions coupling, and add salt as permeation promoter (tonicfier).Select sodium caprylate to be because its that reported increases the ability of the heat stability 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 pharmacopedics packing or the test kit that comprises one or more containers, and one or more compositions that one or more comprise albumin fusion proteins of the present invention and/or polynucleotide are housed in the described container.Can provide with such container by the standard medicine of administrative organization of government promulgation or biology product the announcement of production, use or sale, this announcement has reflected the approval of administrative organization to production, use or the sale of human body dispenser.In addition, 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 Alumen, Alumen add dexycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21 (Genentech, Inc.), the non-living body goods of BCG (for example THERACYS_), MPL and spillikin bacillus (Corynebacterium parvum).In a specific embodiment, albumin fusion proteins of the present invention and/or polynucleotide and Alumen 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, Afiu Vax 100a, QS-21, QS-18, CRL1005, aluminum salt, MF-59 and virion (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); chickenpox; tetanus/diphtheria; hepatitis A; hepatitis B; hemophilus influenza (Haemophilus influenzae) B; pertussis (whooping cough); pneumonia; influenza; the Lai Mushi disease; rotavirus; cholera; yellow fever; Japanese encephalitis; poliomyelitis (poliomyelitis); rabies; typhoid fever; and pertussis (pertussis) provides the vaccine of protection.Co-administered both can be concomitant administration, for example as mixture, separately but simultaneously or parallel using; Also can be that order is used.This comprises wherein unites medicament as presenting that the therapeutic mixture is used together, and also comprises wherein uniting medicament separately but the program of using simultaneously for example enters same individuality via the intravenous circuit that separates." associating " used and also comprised such separate administration, promptly gives a kind of in chemical 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 therapeutic agent separately and be used.Can include but not limited to chemotherapeutics, antibiotic, steroid and nonsteroidal anti-inflammatory, routine immunization therapeutic 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, separately but simultaneously or parallel using; Also can be that order is used.This comprises wherein unites medicament as treatment following mixture presenting of using together, and also comprises wherein uniting medicament separately but the program of using simultaneously for example enters same individuality via the intravenous circuit that separates." associating " used and also comprised such separate administration, promptly gives a kind of in chemical compound or the medicament earlier, gives second kind then.
In one embodiment, albumin fusion proteins of the present invention and/or polynucleotide and anticoagulant are co-administered.Can include but not limited to heparin, low molecular weight heparin, warfarin (warfarin) sodium (for example COUMADIN_), dicoumarol, 4 hydroxy coumarin, anisindione (MIRADON for example with the co-administered anticoagulant of compositions of the present invention
TM), acenocoumarol (acenocoumarol) (acenocoumarol (nicoumalone) for example, SINTHROME
TM), 1,3-indenes diketone, phenprocoumon (MARCUMAR for example
TM), dicoumarol ethyl acetate (TROMEXA for example
TM), and aspirin.In a specific embodiment, compositions of the present invention and heparin and/or warfarin are co-administered.In another specific embodiment, compositions of the present invention and warfarin are co-administered.In another specific embodiment, compositions of the present invention and warfarin and aspirin are co-administered.In another specific embodiment, compositions of the present invention and combination with heparin are used.In another specific embodiment, compositions of the present invention and heparin and aspirin are co-administered.
In another embodiment, albumin fusion proteins of the present invention and/or polynucleotide and thrombolytic medicine are co-administered.Can include but not limited to plasminogen, lys-plasminogen, α 2-antiplasmin, streptokinase (KABIKINASE for example with the thrombolytic medicine that compositions 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, (prourokinase, single chain urokinase type plasminogen activator), and aminocaproic acid (AMICAR for example
TM).In a specific embodiment, compositions of the present invention and tissue-type plasminogen activator and aspirin 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 aspirin, dipyridamole (dipyridamole) (PERSANTINE for example with the antiplatelet drug that compositions 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 anticoagulant, thrombolytic 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 anticoagulant, thrombolytic medicine and/or antiplatelet drug and uniting of albumin fusion proteins of the present invention and/or polynucleotide, reduce the peri-operation period thrombosis risk that may follow with angioplasty, stroke risk, reduction and Cardiac valve prosthesis and/or the relevant thromboembolism risk of mitral valve disease that reduction atrial fibrillation comprises non-rheumatic atrial fibrillation patient.Therapeutic 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, anticoagulant 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 protease 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 protease 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 combination in any of antiretroviral agent, nucleoside reverse transcriptase inhibitor, non-nucleoside reverse transcriptase inhibitor and/or protease 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 absolute acid stability 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 essence ratio of lamivudine resistance separator; Biochem Pharma); (approval of FDA refusal is used for anti-HIV treatment to adefovirdipivoxil (Adefovir); Gilead Sciences); PREVEON_ (adefovir dipivoxil, the active parent drug of adefovirdipivoxil; 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); ZIAGENE
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 (second filial generation derivant 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 protease 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 analog; Merck); DMP-450 (cyclisation urea compounds; Avid ﹠amp; DuPont); AG-1776 is (at the external active peptide mimics that has at protease inhibitor resistance virus; Agouron); VX-175/GW-433908 (An Ruinawei, the former medicine of the phosphate of amprenavir; Vertex ﹠amp; Glaxo Welcome); CGP61755 (Ciba); And AGENERASE
TM(An Ruinawei; Glaxo Welcome Inc.).
Other antiretroviral agent comprises fusion inhibitor/gp41 binding agent.Fusion inhibitor/gp41 binding 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 resting state T-20; Trimeris) and T-1249 (second filial generation fusion inhibitor; Trimeris).
Other antiretroviral agent comprises fusion inhibitor/chemokine receptor anagonists.Fusion inhibitor/chemokine receptor anagonists comprises the CXCR4 antagonist, such as AMD 3100 (bicyclam), SDF-1 and analog thereof and ALX40-4C (cationic peptide), T22 (18 amino acid peptides; Trimeris) and T22 analog 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 analog).Also comprise CCR2B, CCR3 and CCR6 antagonist.Chemokine receptor agonists such as RANTES, SDF-1, MIP-1 α, MIP-1 β etc. also can suppress to merge.
Other antiretroviral agent comprises integrase inhibitor.Integrase inhibitor comprises two caffeoyl quinine (DFQA) acid; L-chicoric acid (two caffeoyl winestone (DFQA) acid); Alizarin bordeaux (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 chemical 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)) chemical compound; The inhibitor that HIV invades is such as the soluble complex of AOP-RANTES, NNY-RANTES, RANTES-IgG fusion rotein, RANTES and glycosaminoglycans (GAG), and AMD-3100; Nucleocapsid zinc refers to inhibitor, such as the dithiane chemical compound; The target of HIV Tat and Rev; And drug effect reinforcing agent (pharmacoenhancer), such as ABT-378.
Other antiretroviral therapy and complementary therapy comprise cytokine and lymphokine, such as MIP-1 α, MIP-1 β, SDF-1 α, IL-2, PROLEUKIN
TM(aldesleukin, aldesleukin/L2-7001; Chiron), IL-4, IL-10, IL-12 and IL-13; Interferon is such as IFN-α 2a, IFN-α 2b or IFN-β; The antagonist of TNF, NF κ B, GM-CSF, M-CSF and IL-10; Regulate the medicament of immune activation, such as ciclosporin and prednisone; Vaccine is such as Remune
TM(HIV Immunogen), APL 400-003 (Apollon), reorganization gp120 and fragment, bivalence (B/E) recombinant envelope glycoprotein, rgp120CM235, MN rgp120, SF-2 rgp120, gp120/ solubility CD4 complex, Delta JR-FL albumen, derived from the synthetic peptide of the branch of discontinuous gp120 C3/C4 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 (ntrakine) (through the CC chemotactic factor targeting 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 stimulating agent 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 antiviral agents are co-administered.Can include but not limited to acyclovir (acyclovir), ribavirin (ribavirin), amantadine (amantadine), remantidine, maxamine or thymalfasin (thymalfasin) with the co-administered antiviral agents of albumin fusion proteins of the present invention and/or polynucleotide.Particularly, the interferon 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 albumin fusion proteins and ribavirin are co-administered.In another preferred embodiment, interferon-ALPHA albumin fusion proteins and ribavirin are co-administered.In another preferred embodiment, interferon-ALPHA 2a albumin fusion proteins and ribavirin are co-administered.In another preferred embodiment, interferon alpha 2 b albumin fusion proteins and ribavirin are co-administered.In another preferred embodiment, interferon beta albumin fusion proteins and ribavirin are co-administered.In another preferred embodiment, hydridization interferon albumin fusion proteins and ribavirin are co-administered.
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, RIFAB UTIN
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 combination in any 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) MOI so that combination in any 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 combination in any 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 the opportunistic cytomegalovirus to infect so that combination in any 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 combination in any 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 combination in any 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 Mus toxoplasma (Toxoplasma gondii) to infect so that combination in any 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 bacterial infection so that combination in any 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 amoxicillin with the co-administered antibiotics of albumin fusion proteins of the present invention and/or polynucleotide, beta-lactamase, aminoglycoside, beta-lactam (glycopeptide), beta-lactamase, clindamycin, chloromycetin, cephalosporin, ciprofloxacin, erythromycin, fluoroquinolone, macrolide, metronidazole, penicillin, quinolinones, rapamycin, rifampicin, streptomycin, sulfanilamide, tetracycline, trimethoprim, trimethoprim-sulfalene _ azoles, 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 (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 (for example interferon-ALPHA) and interleukin (for example IL-2).
In other embodiments, albumin fusion proteins of the present invention and/or polynucleotide and immunosuppressant are co-administered.Can include but not limited to steroid, cyclosporin, cyclosporin analog, cyclophosphamide methyl prednisone, prednisone, imuran, FK-506,15-deoxyspergualin and other immunosuppressant that works by the function that suppresses response T cell with the co-administered immunosuppressant of albumin fusion proteins of the present invention and/or polynucleotide.Can include but not limited to meticortelone, methotrexate, thalidomide (thalidomide), methoxsalen (methoxsalen), rapamycin, leflunomide (leflunomide), mizoribine (mizoribine) (BREDININ with co-administered other immunosuppressant of albumin fusion proteins of the present invention and/or polynucleotide
TM), brequinar (brequinar), deoxyspergualin and aza spiro alkane (azaspirane) (SKF 105685), ORTHOCLONE OKT_3 (muromonab-CD3), SANDIMMUNE
TM, NEORAL
TM, SANGDYA
TM(cyclosporin), PROGRAF_ (FK506, tacrolimus), CELLCEPT_ (mycophenolic acid ethyl ester, active metabolite wherein is a mycophenolic acid), IMURAN
TM(imuran), glucocorticoid, adrenocortical steroid are such as DELTASONE
TM(prednisone) and HYDELTRASOL
TM(meticortelone), FOLEX
TMAnd MEXATE
TM(methotrexate), OXSORALEN-ULTRA
TM(methoxsalen) and RAPAMUNE
TM(sirolimus).
In another embodiment, albumin fusion proteins of the present invention and/or polynucleotide are used separately or are co-administered with one or more intravenous immunoglobulin products.Can include but not limited to GAMMAR with the co-administered intravenous immunoglobulin product of albumin fusion proteins of the present invention and/or polynucleotide
TM, IVEEGAM
TM, SANDOGLOBULIN
TM, GAMMAGARDS/D
TM, ATGAM
TM(antithymocyte globulin) and GAMIMUNE
TMIn a specific embodiment, albumin fusion proteins of the present invention and/or polynucleotide and intravenous 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 JIUYUE 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 anti-inflammatory agent.Can include but not limited to corticosteroid (betamethasone for example with the co-administered anti-inflammatory agent of albumin fusion proteins of the present invention and/or polynucleotide, budesonide, cortisone, dexamethasone, hydrocortisone, methyl meticortelone, meticortelone, prednisone, and triamcinolone), non-steroid anti-inflammatory agent (diclofenac for example, diflunisal, etodolac, fenoprofen, floctafenine, flurbiprofen, ibuprofen, indomethacin, ketoprofen, the meclofenamic acid ester, mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin, Phenylbutazone (BUTE), piroxicam, sulindac, tenoxicam, tiaprofenic acid, and antihistaminic and tolmetin),, the aminoaryl carboxylic acid derivates, the Arylacetic acids derivant, arylbutyric acid derivatives, aryl carboxylic acid, aryl propionic acid derivatives, pyrazoles, pyrazolone, salicyclic acid derivatives, thiazine Methanamide (thiazinecarboxamide), e-acetylamino caproic acid (acetamidocaproic acid), S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide, ditazole, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime, general Lu Kuizong, proxazole, and tenidap.
In another embodiment, compositions 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 compositions of the present invention, Rockville, MD), troponin-1 (Boston Life Sciences, Boston, MA), the anti-invasion factor, tretinoin and derivant thereof, paclitaxel (Taxol), suramin, metalloproteases-1 organize mortifier, metalloproteases-2 to organize mortifier, 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 complex comprises oxo vanadium complex, such as vanadate and vanadyl complex.Suitable vanadate complex comprises metavanadate and orthovanadate complex, such as for example ammonium metavanadate, sodium metavanadate and sodium orthovanadate.Suitable vanadyl complex comprises for example acetylacetone,2,4-pentanedione vanadyl and vanadium oxysulfate, comprises the vanadium oxysulfate hydrate, as one and three hydration vanadium oxysulfates.
The representative example of tungsten and molybdenum complex also comprises oxo complex.Suitable oxo tungsten complex comprises tungstates and tungsten oxide complex.Suitable tungstates complex comprises ammonium tungstate, artificial schellite, Disodium tungstate (Na2WO4) dihydrate and wolframic acid.Suitable tungsten oxide comprises tungsten oxide (IV) and tungsten oxide (VI).Suitable oxo molybdenum complex comprises molybdate, molybdenum oxide and molybdenyl complex.Suitable molybdate complex comprises ammonium molybdate and hydrate, sodium molybdate and hydrate thereof and potassium molybdate and hydrate thereof.Suitable molybdenum oxide comprises molybdenum oxide (VI), molybdenum oxide (VI) and molybdic acid.Suitable molybdenyl complex comprises for example acetylacetone,2,4-pentanedione oxygen molybdenum.Tungsten that other is suitable and molybdenum complex comprise derived from for example hydroxyl derivant of glycerol, tartaric acid 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; Sulphation chitin derivative (making) (people such as Murata, Cancer Res.51:22-26,1991) by the snowflake Carapax Eriocheir sinensis; Sulphation polysaccharide Peptidoglycan complex (SP-PG) (existence of steroid such as estrogen and citric acid tamoxifen can strengthen this compound functions); D-82041 DEISENHOFEN; The matrix metabolism regulator comprises for example proline analogs, cis hydroxyproline, d, L-3,4-dehydroproline, Thiaproline, α, α-two pyridine, Fumaric acid aminopropionitrile; 4-propyl group-5-(4-pyridine radicals)-2 (3H)-_ oxazolone; Methotrexate; Mitoxantrone; Heparin; Interferon; 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); Myristyl sulphuric acid cyclodextrin; Eponemycin; Camptothecine; Amebacilin (people such as Ingber, Nature 348:555-557,1990); Kidon (Ono) (" 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)-carboxy phenyl-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, Bethesda, MD); Conbretastatin A-4 (CA4P) (OXiGENE, Boston, MA); Squalamine (Magainin Pharmaceuticals, Plymouth Meeting, PA); TNP-470 (Tap Pharmaceuticals, Deerfield, IL); ZD-0101 AstraZeneca (London, UK); APRA (CT2584); Benefin, Byrostatin-1 (SC339555); CGP-41251 (PKC412); CM101; Dexrazoxane (ICRF187); DMXAA; Endostatin; Flavopridiol; Genestein; GTE; ImmTher; Iressa (ZD1839); Octreotide (Somat); Panretin; Penacillamine; Photopoint; PI-88; Prinomastat (AG-3340); Purlytin; Suradista (FCE26644); Tamoxifen (Nolvadex); Tazarotene; Tetrathiomolybdate; Xeloda (capecitabine); And 5-fluorouracil.
Can work by number of mechanisms with the co-administered angiogenesis inhibitor medicine of compositions of the present invention, include but not limited to suppress Proteolytic enzyme, the blocking-up endotheliocyte-extracellular matrix adhesion molecule of extracellular matrix function, antagonizing vessel generation inducer 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 compositions 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 compositions of the present invention, Germany) and Vitaxin (Ixsys, La Jolla, CA/Medimmune, Gaithersburg, MD).By direct antagonism or suppress that blood vessel generation inducer works and can include but not limited to Angiozyme (Ribozyme with the example of the co-administered antiangiogenic agent of compositions 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 compositions of the present invention, Kirkland, WA), interferon-ALPHA, IL-12 (Roche, Nutley, NJ) and many sulphuric acid pentosan (Georgetown University, Washington, DC).
In specific embodiment, imagination is used for treating, prevents by making uniting of compositions 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 arthritis by making uniting of compositions 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 compositions 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 basic fibroblast growth factor with the co-administered proteinic example of blood vessel generation of compositions of the present invention, VEGF-1, VEGF-2, VEGF-3, epidermal growth factor α and β, platelet-derived endothelial cell growth factor (ECGF), platelet derived growth factor, tumor necrosis factor, hepatocyte growth factor, insulin like growth factor, colony stimulating factor, M-CSF, granulocyte/M-CSF, with the nitric oxide synthase.
In other embodiments, compositions 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 chlormethine (chlormethine for example, cyclophosphamide, the cyclophosphamide ifosfamide, melphalan (Phenylalanin-Lost), and chlorambucil), Ethylenimine and methylmelamine (for example altretamine and thio-tepa), alkylsulfonate (for example busulfan), nitroso ureas (carmustine (BCNU) for example, lomustine (CCNU), semustine (Semustine), and streptozocin (streptozotocin)), triazenes (dacarbazine (DTIC for example; Dacarbazine)) folacin (for example methotrexate (methotrexate)), pyrimidine analogue (fluorouracil (5-fluorouracil for example; 5-FU), floxuridine (fluorodeoxyuridine; FudR), and cytosine arabinoside (cytarabin)), purine analogue and relevant inhibitor (mercaptopurine (Ismipur for example; 6-MP), thioguanine (6-thioguanine; TG), and pentostatin (2 '-deoxycoformycin)), vinca alkaloids (for example vinblastine (VLB, vinblastine sulfate)) and vincristine (vincristine sulfate)), epipodophyllotoxin (for example etoposide and teniposide), antibiotic (for example dactinomycin (actinomycin D), daunoblastin (daunomycin; Daunorubicin), amycin, 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 cisplatin (cis-DDP) and carboplatin), amerantrone (anthracenedione) (mitoxantrone), replace urea (for example hydroxyl urea), methylhydrazine derivant (procarbazine (N-methylhydrazine for example; MIH)), adrenocortical steroid (for example prednisone), progesterone (caproic acid hydroxyl progestogen for example, medroxyprogesterone, medroxyprogesterone acetate, and megestrol acetate), estrogen (diethylstilbestrol (DES) for example, stilphostrol, estradiol, and ethinylestradiol), estrogen antagonist (for example tamoxifen), androgen (Testosterone Propionate and fluoxymesterone), androgen antagonist (for example flutamide), gonadotropin releasing hormone analogues (for example leuprorelin), other hormone and hormone analogs (methyltestosterone for example, estramustine, estramustine phosphate sodium, chlorotrianisene, and testolactone), and other medicament (dacarbazine for example, glutamic acid, and mitotane).
In one embodiment, compositions 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 Roussel), Kineret
TM(the IL-1 receptor antagonist is also referred to as Anakinra, from Amgen, Inc.).
In a specific embodiment, compositions of the present invention and CHOP (cyclophosphamide, amycin, vincristine and prednisone) or use with the combinatorial association of one or more compositions of CHOP.In one embodiment, compositions of the present invention and anti-CD 20 antibodies, human monoclonal anti-CD 20 antibodies are co-administered.In another embodiment, particularly the combination in any of cyclophosphamide and/or prednisone is co-administered for one or more compositions of compositions of the present invention and anti-CD 20 antibodies and CHOP or anti-CD 20 antibodies and CHOP.In a specific embodiment, compositions of the present invention and Rituximab are co-administered.In another embodiment, compositions of the present invention and Rituximab and CHOP, or with Rituximab and one or more compositions of CHOP particularly the combination in any of cyclophosphamide and/or prednisone use.In another embodiment, compositions of the present invention with tositumomab and CHOP or tositumomab and one or more compositions of CHOP particularly the combination in any of cyclophosphamide and/or prednisone use.Anti-CD 20 antibodies can be chosen wantonly with the former medicine of radiosiotope, toxin or cellular toxicity and be associated.
In another specific embodiment, compositions of the present invention and Zevalin
TMCo-administered.In another embodiment, compositions of the present invention and Zevalin
TMWith CHOP or Zevalin
TMWith one or more compositions of CHOP particularly the combination in any of cyclophosphamide and/or prednisone use together.Zevalin
TMCan be associated with one or more radiosiotope.Particularly preferred isotope 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, IL15, 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 correlation 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; Platelet derived growth factor (PDGF-A) is disclosed in european patent number EP-682110; Platelet derived growth factor-B (PDGF-B) is disclosed in european patent number EP-282317; Placental growth factor (PIGF) is disclosed in international publication number WO 92/06194; Placental growth factor-2 (PIGF-2) is disclosed in people such as Hauser, Growth Factors 4:259-268,1993); VEGF (VEGF) is disclosed in international publication number WO 90/13649; VEGF-A (VEGF-A) is disclosed in european patent number EP-506477; VEGF-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; VEGF-D (VEGF-D) is disclosed in international publication number WO98/02543; VEGF-D (VEGF-D) is disclosed in international publication number WO98/07832; And VEGF-E (VEGF-E), be disclosed in German patent DE19639601.Be incorporated herein by reference above-mentioned list of references 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 hemopoietic growth factor are co-administered.Can include but not limited to granulocyte macrophage colony stimulating factor (GM-CSF) (Sargramostim, sargramostim, LEUKINE with the co-administered hemopoietic growth 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), M-CSF (M-CSF, CSF-1), erythropoietin (Epoetin Alfa, epoetin alfa, EPOGEN
TM, PROSCRIT
TM), stem cell factor (SCF, c-kit part, 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 (atenolol), betaxolol (betaxolol), bisoprolol (bisoprolol), carteolol (carteolol), labetalol (labetalol), metoprolol (metoprolol), nadolol (nadolol), oxprenolol (oxprenolol), penbutolol (penbutolol), pindolol (pindolol), Propranolol (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), Folium Digitalis Purpureae (digitalis), digoxin (digoxin), Digitoxin (digitoxin), diliazem, norpace (disopyramide), esmolol (esmolol), flecainide (flecainide), lignocaine (lidocaine), mexiletine (mexiletine), moracizine (moricizine), phenytoin (phenytoin), procainamide (procainamide), N-acetyl procainamide, Propafenone (propafenone), Propranolol, quinidine (quinidine), sotalol, tocainide (tocainide), and verapamil (verapamil)).
In another embodiment, albumin fusion proteins of the present invention and/or polynucleotide and diuretic are co-administered, such as carbonic anhydrase inhibitors (for example acetazolamide, daranide (dichlorphenamide) and methazolamide), osmotic diuretic (for example glycerol, isosorbide, mannitol and carbamide), inhibition Na
+-K
+-2Cl
-The diuretic of symport (furosemide for example, bumetanide, azosemide, piretanide, tripamide, etacrynic acid, muzolimine, and torsemide), thiazine and thiazine sample diuretic (bendroflumethiazide for example, benzthiazide, chlorothiazide, hydrochlorothiazide, hydroflumethiazide, methyclothiazide, many thiazines, trichlormethiazide, chlortalidone, indapamide, metolazone, and quinethazone), Potassium-sparing diuretic (for example amiloride and triamterene), with mineralocorticoid receptor antagonist (spironolactone for example, canrenone, and canrenoate potassium).
In one embodiment, the treatment of albumin fusion proteins of the present invention and/or polynucleotide and endocrine and/or hormone imbalances disorder is co-administered.The treatment of endocrine and/or hormone imbalances disorder includes but not limited to
127The radiosiotope of I, iodine such as
131I and
123I; Recombinant human growth hormone is such as HUMATROPE
TM(reorganization growth hormone); Growth hormone analogs is such as PROTROPIN
TM(somatrem); Dopamine agonist is such as PARLODEL
TM(bromocriptine); The Somat analog is such as SANDOSTATIN
TM(octreotide); The promoting sexual gland 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 promoting sexual gland hormone agonist is such as LUPRON
TM(leuprorelin acetate), SUPPRELIN
TM(Supprelin (Roberts) .), SYNAREL
TM(Nafarelin Monoacetate) and ZOLADEX
TM(goserelin acetate); The throtropin releasing hormone synthetic products is such as RELEFACT TRH
TMAnd THYPINONE
TM(Protirelin); Recombined human TSH is such as THYROGEN
TMThyroxin 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 chemical 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 Propranolol (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 iopodate).
Other treatment of endocrine and/or hormone imbalances disorder includes but not limited to estrogen or puts together estrogen, such as ESTRACE
TM(estradiol), ESTINYL
TM(ethinylestradiol), PREMARIN
TM, ESTRATAB
TM, ORTHO-ES
TM, OGEN
TMAnd estropipate (estrone), ESTROVIS
TM(quinestrol), ESTRADERM
TM(estradiol), DELESTROGEN
TMAnd VALERGEN
TM(estradiol valerate), DEPO-E STRADIOL CYPIONATE
TMAnd ESTROJECTLA
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(megestrol acetate), NORLUTIN
TM(norethindrone), and NORLUTATE
TMAnd AYGESTIN
TM(norethindrone acetate); The progesterone implant is such as NORPLANT SYSTEM
TM(hypodermic implant of norgestrel); Progesterone antagonist 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/norethindrone), LEVLEN
TM, NORDETTE
TM, TRI-LEVLEN
TMAnd TRIPHASIL-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(norethindrone/mestranol), DESOGEN
TMAnd ORTHO-CEPT
TM(ethinylestradiol/desogestrel), ORTHO-CYCLEN
TMAnd ORTHO-TRICYCLEN
TM(ethinylestradiol/norgestimate), MICRONOR
TMAnd NOR-QD
TM(norethindrone) and OVRETTE
TM(norgestrel).
Other treatment of endocrine 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(testosterone enanthatas), DEPO-TESTOSTERONE
TM(depo-testosterone), DAQNOCRINE
TM(danazol), HALOTETIN
TM(fluoxymesterone), ORETON METHYL
TM, TESTRED
TMAnd VIRILON
TM(methyltestosterone) and OXANDRIN
TM(oxandrolone); 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(alclometasone diproionate), CYCLOCORT
TM(amcinonide), BECLOVEN
TMAnd VANCERIL
TM(beclomethasone), CELESTONE
TM(betamethasone), BENISONE
TMAnd UTICOR
TM(betamethasone benzoate), DDIPROSONE
TM(betamethasone dipropionate), CELESTONEPHOSPHATE
TM(betamethasone sodium phosphate), CELESTONE SOLUSPAN
TM(betamethasone phosphoric acid and sodium acetate), BETA-VAL
TMAnd VALISONE
TM(betamethasone valerate), TEMOVATE
TM(clobetasol propionate), CLODERM
TM(trimethylace tonitric clocortolone), CORTEF
TMAnd HYDROCORTONE
TM(hydrocortisone (hydrocortisone)), HYDROCORTONE ACETATE
TM(acetic acid hydrocortisone (hydrocortisone)), LOCOID
TM(butanoic acid hydrocortisone (hydrocortisone)), HYDROCORTONE PHOSPHATE
TM(hydrocortisone (hydrocortisone) sodium phosphate), A-HYDROCORT
TMWith SOLU CORTEF
TM(hydrocortisone (hydrocortisone) sodium succinate), WESTCORT
TM(valeric acid hydrocortisone (hydrocortisone)), CORTISONE ACETATE
TM(cortisone acetate), DESOWEN
TMAnd TRIDESILON
TM(desonide), 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(flurandrenolide), HALOG
TM(halcinonide), HMS LIZUIFILM
TM(medrysone), MEDROL
TM(methylprednisolone), DEPO-MEDROL
TMAnd MEDROLACETATE
TM(methylprednisolone acetate), A-METHAPRED
TMAnd SOLUMEDROL
TM(Urbason Solubile), ELOCON
TM(momestasone furoate), HALDRONE
TM(paramethasone acetate), DELTA-COITEF
TM(prednisolone), ECONOPRED
TM(prednisolone acetate), HYDELTRASOL
TM(Inflamase), HYDELTRA-T.B.A
TM(tert-butyl group 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 biosynthesis and effect is such as CYTADREN
TM(aminoglutethimide), NIZORAL
TM(ketoconazole), MODRASTANE
TM(trilostane) and METOPIRONE
TM(metyrapone); Cattle, 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(chlorpropamide), TOLAMIDE
TMAnd TOLINASE
TM(tolazamide), DYMELOR
TM(acetohexamide), glibenclamide, MICRONASE
TM, DIBETA
TMAnd GLYNASE
TM(glibenclamide), GLUCOTROL
TM(glipizide) and DIAMICRON
TM(gliclazide), GLUCOPHAGE
TM(metformin), ciglitazone, pioglitazone and alpha-glucosidase inhibitor; Cattle or Pancreas Sus domestica glucagon; Somatostatin is such as SANDOSTATIN
TM(octreotide); 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 puting together estrogen (PREMARIN for example
_And ESTRATAB
_), estradiol (CLIMARA for example
_And ALORA
_), estropipate and chlorotrianisene; Progesterone medicine (AMEN for example
_(medroxyprogesterone), MICRONOR
_(norethindrone acetate), PROMETRIUM
_Progesterone and megestrol acetate); And estrogen/progesterone conjoint therapy, such as for example puting together estrogen/medroxyprogesterone (PREMPRO for example
TMAnd PREMPHASE
_) and norethindrone 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), ferrum dextran injection (INFED for example
TM), blue vitriol, pyridoxol (pyroxidine), riboflavin, vitamin B
12, cobalamin injection (REDISOL for example
TM, RUBRAMIN PC
TM), hydroxocobalamine, folic acid (FOLVITE for example
TM), formyl tetrahydrofolic acid (folinic acid, 5-CHOH4PteGlu, calcium folinate) or WELLCOVORIN (calcium salt of formyl tetrahydrofolic acid), transferrins 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, clozapine, fluphenazine, haloperidol, loxapine, mesoridazine, molindone, olanzapine, perphenazine, pimozide, Kui sulfur is flat, risperidone, thioridazine, for thioxanthene difficult to understand, trifluoperazine, and triflupromazine), anti-manic medicine (carbamazepine for example, divalproex sodium, lithium carbonate, and Lithium Citrate de), antidepressants (amitriptyline for example, amoxapine, BUP, citalopram, clomipramine, desipramine, doxepin, fluvoxamine, fluoxetine, imipramine, isocarboxazid, maprotiline, mirtazapine, nefazodone, nortriptyline, paroxetine, phenelzine, protriptyline, Sertraline, tranylcypromine, trazodone, trimeprimine, with a zhang traction therapy suffering), antianxiety drugs (alprazolam for example, buspirone, Chlordiazepoxidum, chlordiazepoxide, diazepam, halazepam, lorazepam, oxazepam, and prazepam), and analeptic (d-amfetamine for example, methylphenidate, 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 neurological disorder co-administered.Can include but not limited to antuepileptic (carbamazepine for example with the co-administered neurological medicament of albumin fusion proteins of the present invention and/or polynucleotide, clonazepam, ethosuximide, phenobarbital, phenytoin, primidone, valproic acid, divalproex sodium, 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; Benzhexol, tolcapone) and ALS therapeutic agent (for example riluzole).
In another embodiment, albumin fusion proteins of the present invention and/or polynucleotide and vasodilation and/or calcium channel blocker are co-administered.Can include but not limited to Angiotensin-Converting (ACE) inhibitor (papaverine for example with the co-administered vasodilation of albumin fusion proteins of the present invention and/or polynucleotide, isoxsuprine, benazepril, captopril, cilazapril, enalapril, enalaprilat, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, spirapril, trandolapril, and buphenine), and nitrate (Dilatrate-SR for example, isosorbide mononitrate, and nitroglycerin).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, isradipine, nicardipine, nifedipine, nimodipine and verapamil.
In certain embodiments, the therapeutic agent of albumin fusion proteins of the present invention and/or polynucleotide and gastrointestinal dysfunction is co-administered.Can include but not limited to H with the therapeutic agent of the co-administered gastrointestinal dysfunction of albumin fusion proteins of the present invention and/or polynucleotide
2Histamine receptor antagonists (TAGAMET for example
TM(cimetidine), ZANTAC
TM(ranitidine), 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(basic bismuth salicylate) 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 surfactant laxative, zest laxative, saline and permeability laxative); Diarrhea (LOMOTIL for example
TM(diphenoxylate), MOTOFEN
TM(diphenoxin) and IMODIUM
TM(loperamide hydrochloride)), the synthetic analogues of somatostatin, such as SANDOSTATIN
TM(octreotide), Bendectin (ZOFRAN for example
TM(ondansetron), KYTRIL
TM(Granisetron Hydrochloride), tropisetron, dolasetron, metoclopramide, chlorpromazine, perphenazine, prochlorperazine, promethazine, thiethylperazine, triflupromazine, domperidone, haloperidol, droperidol, trimethobenzamide, dexamethasone, methylprednisolone, dronabinol and Fructus Cannabis dragon); D2 antagonist (for example metoclopramide, trimethobenzamide and chlorpromazine); Gallbladder salt; Chenodeoxycholic acid; Ursodesoxycholic acid; With the pancreas enzyme preparation, such as pancreatin and pancreatic lipase.
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 pharmacopedics packing 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, with such container provide by the standard medicine of administrative organization of government promulgation or biology product the announcement of production, use or sale, 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 method for optimizing that is used in vivo the nucleic acid transfered cell is the viral 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 viral vector nucleic acid of for example encoding in the viral vector by the contained cDNA of viral 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 cell line of replication defect type (being called " incasing cells ") has increased the effectiveness of retrovirus in gene therapy, and the deficiency retrovirus of being identified be used for gene therapy purposes gene transfer (summary referring to Miller, A.D., Blood 76:271,1990).By standard technique, the replication defect type retrovirus can be packaged into virion, 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 viral gene delivery system of the present invention and use the deutero-carrier of adenovirus.The genome that can operate adenovirus like this makes it coding and expresses the genes of interest product, but its ability of in normal molten born of the same parents' sexually transmitted disease (STD) 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 vector 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 purification 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) unconformity 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 mutation 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 lysine conjugate, reaches artificial viral envelope.In a representative embodiment, the nucleic acid molecules 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 Iipofectin) in the tagged liposome of the antibody of target tissue cell surface antigen, 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 diluent 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 the method requirement code book invention albumin fusion proteins and promoter 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 promoter 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 tremulous pulse, in the tissue around the tremulous pulse 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 promoter comprises adenovirus promoter, such as adenovirus major late promoter; Or allogeneic promoter, such as cytomegalovirus (CMV) promoter; Respiratory syncytial virus (RSV) promoter; Inducible promoter is such as MMT promoter, metallothionein promoter; The heat shock promoter; The albumin promoter; The ApoAI promoter; The human immunoglobulin promoter; Viral thymidine kinase promoter is such as herpes simplex thymidine kinase promoter; Retrovirus LTR; The b-actin promoter; And human growth hormone's promoter.Promoter also can be natural promoter 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 synthetic of short duration character of polynucleotide 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, bone marrow, thymus, the heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, intestinal, testis, ovary, uterus, rectum, nervous system, eye, gland and connective tissue.Organize the intercellular space to comprise the collagen fiber of elastic fiber in mucopolysaccharide layer, tube wall or the locular wall between the reticular fiber of iuntercellular, liquid, organ-tissue, fibrous tissue or include myocyte's connective tissue or the same matrix in the bone crack in.Also similar by the space that the lymph fluid of circulating plasma and lymph pipeline occupies.Owing to the reason of discussing below preferably is delivered to the muscular 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 0.05g/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 naked DNA construction is delivered to tremulous pulse.
Deliver exposed polynucleotide by any method that this area is known, comprise that the direct pin injection, intravenous injection, local 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 cation (positively charged), anion (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 purification (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-(trimethylammonium) 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, anion 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, PHOSPHATIDYL ETHANOLAMINE, dioleoyl phospholipid phatidylcholine (DOPC), DOPG (DOPG), DOPE (DOPE) etc.These materials also can mix with proper proportion with DOTMA and DOTAP parent material.Method with these material preparation liposomees is well-known in the art.
For example, can be used to prepare conventional liposome by various combination, can add or not add cholesterol at commercial dioleoyl phospholipid phatidylcholine (DOPC), DOPG (DOPG) and DOPE (DOPE).Therefore, for example, can by with each 50mg DOPG and DOPC under the nitrogen current 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 Ultrasound Instrument 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 vesicle 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 of Immunology 101:512-527,1983, be introduced into this paper as a reference.For example, can be by phospholipid membrane being deposited on the teat glass 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 homogenizing.The material of desiring to catch is added in the treated MLV suspension supersound process then.When using the liposome of cation lipid, exsiccant lipid film is resuspended in suitable solution such as sterilized water or waits buffer 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 complex.SUV can be used for the nucleic acid small fragment.Many method preparations that LUV knows by this area.Method commonly used comprises Ca
2+-EDTA chelating (people such as Papahadiopoulos, 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 the compound genetic stocks of cationic-liposome carrier.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 mammal.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 complex 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 by the retrovirus of its retroviral plasmid vector of deriving 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.
Use retroviral plasmid vector transduction package cell line to form Producer cell line.But the example of the incasing cells 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 cell line described in 1990 are with its complete being incorporated herein by reference.Any method carrier transduction incasing cells 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 cell line 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 vector.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' sexually transmitted disease (STD) 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 mutation.In addition, adenovirus has fabulous security feature people such as (, Am.Rev.Respir.Dis.109:233-238,1974) Schwartz as live body intestinal vaccine for many years.At last, demonstrate many examples of adenovirus mediated gene transfer, 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 vector 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 vector 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 infection cell, and can express the polynucleotide of interest that can be operatively connected with promoter, 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 spatial constraints 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 herpesvirus.In case incasing cells 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 zone 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 of JIUYUE in 1996 publication on the 26th; 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).The method relate to exist in the target cell but in cell, do not express under the normal condition 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 promoter and this promoter flank is the targeting sequence.This paper has described suitable promoter.Targeting sequence and endogenous sequence are fully complementary to allow the homologous recombination of promoter-targeting sequence and endogenous sequence.The targeting sequence will make promoter can be operatively connected by homologous recombination and endogenous sequence fully near 5 ' end of the endogenous polynucleotide sequence of expectation.
Can pcr amplification promoter and targeting sequence.Preferably, the promoter of amplification contains distinct restriction enzyme sites at 5 ' and 3 ' end.Preferably, 3 ' end of the first targeting sequence contains the identical restriction enzyme sites of 5 ' end with the amplification promoter, and 5 ' end of the second targeting sequence contains with 3 ' of the promoter that increases and holds identical restriction enzyme sites.The promoter of digest amplification is with the targeting sequence and connect together.
Promoter-targeting 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, precipitant.Can deliver P promoter-targeting sequence by any method, comprise that direct pin injection, intravenous injection, local application, conduit are inculcated, particle accelerator etc.Hereinafter more detailed description these methods.
Promoter-targeting 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 promoter.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 homologous or allogenic for polynucleotide of interest, and for treating that transfectional cell can be 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 therapeutic effect 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 pumps (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 gene expression of exogenous gene in rats'liver (people such as Kaneda in the portal vein, Science 243:375,1989).
A kind of method for optimizing of local application is to pass through direct injection.Preferably, will with deliver the compound albumin fusion proteins of the present invention of media and be administered in the tremulous pulse by direct injection or locally apply in the arteriosomes.Compositions locally applied in the arteriosomes refer to compositions is expelled to several centimetres preferred several millimeters of intra-arterial.
The another kind of method of local application is to place polynucleotide constructs of the present invention among the surgical 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 targeting of the present invention delivers the compound fusion rotein of the present invention of media.The delivery media that is applicable to systemic application comprises the liposome that comprises the part of media targeting specific part.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 targeting specific part.
The method for optimizing of systemic application comprises intravenous injection, aerosol, oral and percutaneous (part) delivery.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 enzyme 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 effective dose that the decision material is delivered can be depending on multiple factor, comprise the age of the chemical constitution of these materials for example and biologic activity, animal and body weight, needs treatment the definite situation and the order of severity thereof, reach drug delivery route.The frequency of treatment depends on multiple factor, the quantity of the polynucleotide construction of using such as take medicine at every turn and experimenter's health status and medical history.Attending doctor or veterinary will determine definite consumption, medicining times and medicine time.
Albumin fusion proteins of the present invention can be applied to any animal, preferred mammal and birds.Preferred mammal comprises people, dog, cat, mice, rat, rabbit, sheep, cattle, 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 algoscopy 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 relevant 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 quantity 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 quantity 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 cellular expression, 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 description.So, the most preferred embodiment of the present invention comprises the albumin fusion proteins (it lacks table 2 the 4th and/or the listed targeting sequencing 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 targeting sequencing of 11 row.Comprising the compositions 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 hormonal system (referring to " endocrine regulation " part for example hereinafter), nervous system (referring to " neurological disorder " part for example hereinafter), immune system (referring to " immunocompetence " part for example hereinafter), respiratory system (referring to " disordered breathing " part for example hereinafter), cardiovascular system (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 system (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, cell differentiation 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 activeness (chemotaxis) of for example activation or inhibition immunocyte.Immunocyte is grown in being called the process of hemoposieis, produces bone marrow (platelet, erythrocyte, neutrophil cell and macrophage) and lymph (B and T lymphocyte) cell from pluripotent 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 infectious.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 immunodeficiency, comprise two kinds of geneogenous and acquired immunodeficiency.Wherein the example of the B cellular immunity deficiency of immunoglobulin level B cell function and/or B cell number reduction comprises: the agammaglobulinemia (Bu Ludunshi disease) that X-is chain; the infantile agammaglobulinemia that X-is chain; the high IgM immunodeficiency that X-is chain; the high IgM immunodeficiency that non-X-is chain; the lymphopoiesis syndrome (XLP) that X-is chain; agammaglobulinemia comprises congenital and acquired agammaglobulinemia disease; the agammaglobulinemia of adult age outbreak; tardy property agammaglobulinemia; dysgammaglobulinemia; hypogammag lobulinemia; not qualitative hypogammag lobulinemia; 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: the enlightening George is unusual, Reconstruction in Sever Combined Immunodeciency (SCID) (includes but not limited to the SCID that X-is chain, autosomal recessive SCID, the ADA Adenosine deaminase defective, purine nucleoside phosphorylase (PNP) defective, II class MHC defective (bare lymphocyte syndrome), Wei-Ao two syndromes, ataxia-telangiectasia), thymic dysplasia, 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 immunodeficiency of dominance T cell defect (not qualitative), immunodeficiency 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 immunodeficiency of diagnosis and/or prediction includes but not limited to chronic granulo matosis, Qie-Xi two syndromes, myeloperoxidase deficiency, leukocyte glucose-6-phosphate dehydrogenase (G6PD) defective, the lymphopoiesis syndrome (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, immunodeficiency with thymoma, the severe congenital leukopenia, abnormal development with immunodeficiency, the neonate neutrophil reduces, micromelic dwarf disease, and Nei Ziluofu syndrome-merging Ig immunodeficiency.
In a preferred embodiment, with polynucleotide are treated, prevent, diagnosed and/or prediction is relevant with above-mentioned immunodeficiency the immunodeficiency 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 enhance immunity response in the immunodeficiency 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 enhance immunity response 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 sexual disorder.Many autoimmune sexual disorders all be since the immunocyte mistake self is identified as foreign substance.This wrong identification causes and causes host tissue to suffer 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 Therapeutic Method of prevention autoimmune sexual disorder.
Can treat with the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins, prevention, the autoimmune disease of diagnosis and/or prediction and disorderly include but not limited to following one or more: systemic lupus erythematosus (sle), rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis, autoimmune thyroiditis, struma lymphomatosa, autoimmune hemolytic anemia, hemolytic anemia, thrombocytopenia, idiopathic thrombocytopenic purpura, autoimmunity neonate thrombocytopenia, idiopathic thrombocytopenic purpura, purpura (for example prosperous-permitted Er Shi purpura), autoimmunity cytopenia, the Goodpasture syndrome, pemphigus vulgaris, myasthenia gravis, Graves' disease (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 autoimmune factor, other disorder of prevention and/or diagnosis includes but not limited to the inductive arthritis of II Collagen Type VI, anti-phospholipid syndrome, dermatitis, allergic encephalitis, myocarditis, relapsing polychondritis, rheumatic heart disease, the neuritis, the uveitis ophthalmia, many endocrinopathys, Lay Te Shi disease, stiff people's syndrome, 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 autoimmune 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 antiglomerular basement membrane antibody or immune complex usually), bulla pemphigoid (being characterized as IgG and complement in the basement 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 insular cellular antibody), 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 autoimmune 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), constitutional gallbladder 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 SC in the tube wall for example usually), back MI (being characterized as for example heart antibody usually), cardiotomy syndrome (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 disease 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 diagnostic 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 nephropathy.
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 disease 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 immunosuppressant.
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 leukopenia, neutrophil cell minimizing, anemia and the thrombocytopenic effort, strengthen differentiation and propagation that hematopoietic cell comprises pluripotent 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 pluripotent stem cell.
Allergy 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 allergic asthma) or other respiratory problems.In addition, these molecules can be used for treating, prevent, predicting and/or diagnose anaphylaxis, 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 allergy of IgE mediation.Such allergy 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 inductive pulmonary lesion of hyperacute rejection, nephritis, cytokine or chemotactic factor that infects relevant inflammation (for example septic shock, septicemia or systemic inflammatory response syndrome), ischemia-reperfusion injury, endotoxin lethal, complement-mediated, inflammatory bowel, Crohn disease, cytokine (for example TNF or IL-1), disordered breathing (for example asthma and allergy); 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, neurodegenerative diseases (for example parkinson 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 erythematosus (sle), 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 adrenalitis, alveolitis (alveolitis), angiocholecystitis, appendicitis, balanitis, blepharitis, bronchitis, bursitis, carditis, cellulitis, cervicitis, cholecystitis, chorditis, cochlitis, colitis, conjunctivitis, cystitis, dermatitis, diverticulitis, encephalitis, endocarditis, esophagitis, salpingitis, fibrositis, folliculitis, gastritis, gastroenteritis, gingivitis, glossitis, hepatosplenitis, keratitis, labyrinthitis, laryngitis, lymphangitis, mastitis, otitis media, meningitis, metritis, mucositis, myocarditis, myositis, myringitis, nephritis, the neuritis, orchitis, osteochondritis, otitis, pericarditis, tenosynovitis, peritonitis, pharyngitis, phlebitis, poliomyelitis, prostatitis, pulpitis, retinitis, rhinitis, salpingitis, scleritis, sclerochoroiditis, scrotitis, sinusitis, spondylitis, pimelitis, stomatitis, synovitis, salpingitis, tendinitis, 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.The organ rejection destroys transplanted tissue by the host immune cell through immunoreation and takes place.Similarly, GVHD also relates to immunoreation, but in the case, external transplantation immunity cytoclasis host tissue.Suppress effective Therapeutic Method that immunoreation, 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 immunoreation, 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 Therapeutic Method that prophylactic tria allergic and super acute xenograft 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, joint knot property polyarteritis and the inductive vasculitis of immune complex.
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 infection prevention 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 enhance immunity reaction.Immunoreation can be by strengthening existing immunoreation or strengthening by starting new immunoreation.Perhaps, the polynucleotide of fusion rotein of the present invention and/or code book invention albumin fusion proteins also can directly suppress infectious agent (listing the part of infectious agent etc. in the application reference book), and need not to cause immunoreation.
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 immunoreation as adjuvant.
In another specific embodiment, albumin of the present invention merge egg from and/or the polynucleotide of code book invention albumin fusion proteins be used to strengthen the antiviral immunity reaction as adjuvant.Can use compositions of the present invention as adjuvant and enhanced antiviral immunity reaction comprises virus and viral relevant disease or symptom described herein or that other approach of this area is known.In specific embodiment, compositions of the present invention is used to strengthen immunoreation at the virus, disease or the symptom that are selected from down group as adjuvant: AIDS, meningitis, dengue fever, EBV and hepatitis (for example hepatitis B).In another specific embodiment, compositions of the present invention is used to strengthen immunoreation at the virus, disease or the symptom that are selected from down group as adjuvant: HIV/AIDS, respiratory syncytial virus, dengue virus, rotavirus, Type B Japanese encephalitis, A and Type B influenza, parainfluenza, measles, cytomegalovirus, rabies, Junin virus, chikungunya disease, Rift valley fever, herpes simplex and yellow fever.
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 compositions of the present invention is as adjuvant and the reaction of enhanced antibacterium or antifungal immunity comprises antibacterial or fungus or antibacterial and fungus antibacterial or fungus relevant disease or symptom described herein or that other approach of this area is known.In specific embodiment, compositions of the present invention is used to strengthen at the antibacterial that is selected from down group or the immunoreation of fungus, disease or symptom as adjuvant: tetanus, diphtheria, botulism and Type B meningitis.
In another specific embodiment, compositions of the present invention is used to strengthen antibacterial or the fungus of organizing at being selected from down as adjuvant, the immunoreation of disease or symptom: vibrio cholera (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, shigella (Shigella spp.), enterotoxigenic dust Xi Shi escherichia coli (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 compositions of the present invention is as adjuvant and enhanced antiparasitic immunity reaction comprises parasite and parasite relevant disease or symptom described herein or that other approach of this area is known.In specific embodiment, compositions of the present invention is used for strengthening at parasitic immunoreation as adjuvant.In another specific embodiment, compositions of the present invention is used for the immunoreation 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 infectious 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 (mice for example, rat, rabbit, hamster, Cavia porcellus, pig, miniature pig, chicken, camel, goat, horse, cattle, sheep, dog, cat, non-human primates, and people, optimum is chosen) produce one or more antibody (IgG for example of greater number with the enhance 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 enhance 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 stimulus object at the B cellular response of pathogen.
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, compositions of the present invention can be before transplanting, use simultaneously and/or afterwards.In a specific embodiment, compositions of the present invention after transplanting, the T cell colony uses before beginning to recover.In another specific embodiment, compositions 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 enhance immunity response 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 chronic 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 enhance immunity response in having the individuality of temporary immunodeficiency.Cause the situation of the temporary immunodeficiency 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 viral infection (for example influenza) recover, the situation relevant with malnutrition, from infectious mononucleosis recoverys, pressure-dependent situation, from measles recover, from the blood transfusion recovery, reach from surgery recovery.
In another specific embodiment, the regulator that the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins are presented as mononuclear cell, 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 immune 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 effect 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 therapeutic schemes 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 regenerated Therapeutic Method after wound or the genetic 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 bone 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 Therapeutic Method based on gene that causes such as the congenital heredity sexual disorder of viewed immune incapability/immunodeficiency 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 parasitic disease of influence such as leishmanial means as activated mononuclear cell/macrophage.
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 secreted cytokine 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 immunoreation different aspect of self.The example that may wish to block the disease of some aspect of immunoreation wherein or situation comprise autoimmune sexual disorder such as lupus and arthritis and to the immune responsiveness of skin allergic reaction, inflammation, enteropathy, damage and with pathogen 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 Therapeutic Method relevant with autoimmune disease such as idiopathic thrombocytopenic purpura, systemic lupus erythematosus (sle) 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 immunoreation 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 are as the Therapeutic Method such as tangible chronic hypergammaglobulinemia in the diseases such as not qualitative MG (MGUS), waldenstrom's disease, the relevant special property sent out MG and plasmocytoma.
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 polypeptide chemotaxis and macrophage and precursor, neutrophil cell, basophilic granulocyte, bone-marrow-derived lymphocyte and some T cell subclass such as activation activatory and cd8 cell toxicity T cell and natural killer cell in some autoimmunity and chronic inflammatory and infectious 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 syndrome 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 mononuclear cell 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 constitutional 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 tumor, and/or with these infection, disease, any disease that disorder and/or malignant tumor 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, herpes zoster (for example serious herpes 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 anemia, thrombocytopenia 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 myelogenous leukemia, chronic lymphocytic leukemia, lymphogranulomatosis, the non-Hodgkin lymphomas, acute lymphocytic anemia (ALL), chronic lymphocytic leukemia, plasmocytoma, multiple myeloma, 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 Therapeutic Method 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 lymphocytic leukemia and the means that involve of Ig.
In specific embodiment, compositions 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 enhance immunity response.
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 thrombolytic (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 thrombocytopenia) 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 (infarction), 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 Cardiac valve prosthesis and/or the relevant thromboembolism 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 hemopoietic 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 hemocytees or hemocyte subclass such as for example erythrocyte, lymphocyte (B or T cell), medullary cell (for example basophil, eosinocyte, neutrophil(e) cell, mastocyte, macrophage) and hematoblastic quantity.The ability that reduces hemocyte or hemocyte subclass quantity can be used for preventing, detect, diagnose and/or treating anemia described below and leukopenia.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 hemocytees or hemocyte subclass such as for example erythrocyte, lymphocyte (B or T cell), medullary cell (for example basophil, eosinocyte, neutrophil(e) cell, mastocyte, macrophage) 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 dyscrasia.
Anemia is that erythrocyte number or hemoglobin wherein (carrying the protein of oxygen) quantity are lower than the situation of normal level.Anemia 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 anemia.The polynucleotide treatment of available albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins, the anemia of prevention or diagnosis comprises iron deficiency 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 anemia 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 erythematosus (sle), cancer, lymphoma, chronic nephropathy and splenauxe relevant anemia.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 anemia that is caused by Drug therapy, such as the anemia relevant with methyldopa, dapsone and/or sulphonamides.In addition, the anemia 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 heritability sphaerocyst increases, the heritability 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 thrombocytopenia (idiopathic thrombocytopenic purpura for example, with the thrombosis 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 heritability thrombopathia, the TXA2. dysfunction, Thrombasthenia, and Bai-Su two syndromes), the hemolytic uremic syndrome syndrome, 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, condense in anaphylactoid purpura (prosperous-permitted Er Shi 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, from disorders of blood, multiple myeloma, liver cirrhosis, with the dysfunction of platelet and the dysfunction of platelet relevant of systemic lupus erythematosus (sle), comprise aspirin with high dose with Drug therapy, ticlopidine, non-steroid anti-inflammatory agent (is used for arthritis, 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 leukocyte number increases or reduce or diseases associated and disorder with it.Leukopenia takes place when being lower than normal level when the leukocyte number is reduced to.Leukopenia includes but not limited to that neutrophil reduces and lymphopenia.The leukocyte number is called leukocytosis with respect to the increase of normal level.Body produces the leukocyte that number increases between infection period.Therefore, leukocytosis may only be the normal physiological mathematic(al) parameter that reflection is infected.Perhaps, leukocytosis may be the index of damage or other diseases such as cancer.Leukocytosis includes but not limited to eosinophilia's disease and macrophage 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 leukopenia.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.
Leukopenia 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 neutrophil cell decreased 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 heritability 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 Drug therapy (regimen of antibiotics for example, treat such as penicillin, the sulfonamide treatment, the anticoagulant treatment, anticonvulsant drug, antithyroid drug and cancer chemotherapy) or relevant with it neutrophil minimizing, with come from the neutropenia that neutrophil cell destroy to increase, neutrophil cell destroy increase may with some antibacterials or viral infection, allergic disorder, autoimmune disease, splenomegaly (Fil base of a fruit 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, Drug therapy (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 erythematosus (sle), chronic infection, some viral infection and/or heritability disorder (enlightening George syndrome for example, one-tenth-Ao two syndromes, serious compressibility immunodeficiency, 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 macrophage number and/or macrophage function diseases associated and disorder, include but not limited to familial splenic anemia, niemann-Pick disease, letterer-Siwe disease and Hand Sch.
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 syndrome (idiopathic hypereosinophilic syndrome), eosinophile granulocyte-myalgia syndrome and Hand Sch.
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 (lymphoblast property) leukemia (ALL), acute myeloid sample (myeloid, bone marrow, myeloblast property, or bone marrow mononuclear cell) leukemia, chronic lymphocytic leukemia (B cell leukemia for example, the T chronic myeloid leukemia, the Sai Zhali syndrome, and hairy cell), chronic myeloid (myelocyte sample, bone 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 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, thrombocytosis (comprising constitutional and Secondary cases thrombocytosis) 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, phagocytosis, the superoxides that strengthen neutrophil cell, eosinophile granulocyte and macrophage 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 platelet is extracted.
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 constitutional hemopoietic 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, abdominal part, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine gland (adrenal gland, parathyroid gland, hypophysis, testis, ovary, thymus, thyroid), 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 period of acute, acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, adult's (constitutional) hepatocarcinoma, adult's (constitutional) hepatocarcinoma, 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's soft tissue sarcoma, the AIDS lymphoma of being correlated with, the AIDS associated malignancies, anus cancer, astrocytoma, cancer of biliary duct, bladder cancer, osteocarcinoma, the brain stem glioma, cerebroma, breast carcinoma, renal pelvis and carcinoma of ureter, central nervous system's (constitutional) lymphoma, central nervous system lymphoma, cerebellar astrocytoma, big cerebral astrocytoma, cervical cancer, the childhood period (constitutional) hepatocarcinoma, the childhood period (constitutional) hepatocarcinoma, the childhood period acute lymphoblastic leukemia, the childhood period acute myeloid leukemia, the childhood period brain stem glioma, the childhood period cerebellar astrocytoma, the childhood period big cerebral astrocytoma, the childhood period extracranial germ cell tumor, the childhood period lymphogranulomatosis, the childhood period hodgkin's lymphomas, the childhood period hypothalamus and look the road glioma, the childhood period lymphoblast leukemia, the childhood period medulloblastoma, the childhood period non-Hodgkin lymphomas, the childhood period pinus and curtain go up an original neuroectodermal tumor, the childhood period primary hepatocarcinoma, the childhood period rhabdomyosarcoma, the childhood period soft tissue sarcoma, the childhood period look road and hypothalamus glioma, chronic lymphocytic leukemia, chronic granulocytic leukemia, colon cancer, cutaneous T cell lymphoma, the endocrine islet-cell carcinoma, carcinoma of endometrium, ependymoma, epithelial cancer, the esophageal carcinoma, ewing's sarcoma and related neoplasms, the exocrine pancreas cancer, the extracranial germ cell tumor, the outer sexual cell tumor of gonad, cholangiocarcinoma, cancer eye, women's breast carcinoma, familial splenic anemia, carcinoma of gallbladder, gastric cancer, the gastrointestinal carcinoid tumor, the gastrointestinal tumor, germinoma, gestational trophoblastic neoplasms, hairy cell, head and neck cancer, hepatocarcinoma, lymphogranulomatosis, hodgkin's lymphomas, hypergammaglobulinemia, hypopharyngeal cancer, intestinal cancer, intraocular melanoma, islet-cell carcinoma, the islet cells cancer of pancreas, Kaposi sarcoma, renal carcinoma, laryngeal carcinoma, lip and oral cancer, hepatocarcinoma, pulmonary carcinoma, the lymphocytic hyperplasia sexual disorder, macroglobulinemia, women's breast carcinoma, malignant mesothe, malignant thymoma, medulloblastoma, melanoma, mesothelioma, the disguised constitutional squamous cell of transitivity neck cancer, transitivity constitutional squamous cell neck cancer, transitivity squamous cell neck cancer, multiple myeloma, multiple myeloma/plasma cell vegetation, myelodysplastic syndrome, myelocytic leukemia, myelocyte sample leukemia, the bone marrow proliferation sexual disorder, nasal cavity and paranasal sinuses, nasopharyngeal carcinoma, neuroblastoma, trimester of pregnancy the non-Hodgkin lymphomas, non-melanoma skin cancer, nonsmall-cell lung cancer, disguised constitutional transitivity squamous cell neck cancer, the oropharynx cancer, bone-/the malignant fibrous sarcoma, osteosarcoma/malignant fibrohistiocytoma, the malignant fibrohistiocytoma of osteosarcoma/bone, epithelial ovarian cancer, the ovarian germ cell tumor, the rudimentary pernicious potential tumor of ovary, cancer of pancreas, paraproteinemia, purpura, the parathyroid gland cancer, carcinoma of penis, pheochromocytoma, pituitary tumor, plasma cell vegetation/multiple myeloma, primary central nervous system lymphoma, primary hepatocarcinoma, carcinoma of prostate, rectal cancer, renal cell carcinoma, renal pelvis and carcinoma of ureter, retinoblastoma, rhabdomyosarcoma, salivary-gland carcinoma, the sarcoidosis sarcoma, the Sai Zhali syndrome, skin carcinoma, small cell lung cancer, carcinoma of small intestine, soft tissue sarcoma, the squamous cell neck cancer, gastric cancer, original neuroderm and pinealoma on the curtain, t cell lymphoma, carcinoma of testis, thymoma, thyroid carcinoma, renal pelvis and ureteral transitional cell carcinoma, transitional type renal pelvis and carcinoma of ureter, trophoblastic tumor, ureter and renal pelvis cell carcinoma, carcinoma of urethra, uterus carcinoma, sarcoma of uterus, cancer of vagina, look road and hypothalamus glioma, the vaginal orifice cancer, Walden Si Telunshi macroglobulinemia, the Wei Ermusishi tumor, 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 hypertrophy, the change that is made of non-neoplastic cell growth given birth to, or most particularly abnormal development (summary about these misgrowth situations can be consulted Robbins and Angell, 1976, Basic Pathology ", the 2nd edition, W.B.Saunders company, Philadelphia, pp.68-79).
Hypertrophy is a kind of form of controlled cell propagation, involve 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 hypertrophy sexual disorder of treatment includes but not limited to blood vessel folliculus mediastinal lymph nodes hypertrophy, blood vessel lymphocytic hyperplasia with the eosinophilia, atypical melanocytic hyperplasia, basal cell hyperplasia, optimum giant lymph node hyperplasia, hypercementosis, adrenal,congenital hyperplasia, congenital sebaceous hyperplasia, 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 hypertrophy, 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), connective tissueization is given birth to, epithelial metaplasia, intestinalization is given birth to, metaplastic anemia, metaplastic ossification, metaplastic polyp, myeloid metaplasia, the constitutional myeloid metaplasia, the Secondary cases myeloid metaplasia, squamous metaplasia, the squamous metaplasia of amniotic membrane, with Symptomatic myeloid metaplasia.
Abnormal development usually is the omen of cancer, and mainly finds in epithelium; It is the most of disorderly form of non-neoplastic cell growth, involves the forfeiture of cell individual concordance and cell structure orientation.The abnormal development cell usually has unusual large-scale, dense painted nuclear, and shows pleomorphism.Abnormal development 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 abnormal development sexual disorder of treatment includes but not limited to anhidrotic ectodermal dysplasia, anterofacialdysplasia, asphyxiating thoracic dysplasia, atrium finger abnormal development, bronchopulmonary dysplasia, encephalodysplasia, dysplasia of cervix, chondroectodermal dysplasia, clavicle skull abnormal development, congenital ectodermal dysplasia, skull is done abnormal development, cranium wrist instep abnormal development, cranium metaphysis abnormal development, dentinal dysplasia, diaphyseal sclerosis, ectodermal dysplasia, enamel abnormal development, the brain eyeball development is unusual, inclined to one side side seam epiphysis abnormal development, dysplasia epiphysealis multiplex, stippled epiphyses abnormal development, epithelial development is unusual, face refers to that (toe) genital development is unusual, the familial fiber abnormal development of jaw (jaw), familial white fold abnormal development, fiber flesh sexual abnormality, the fiber abnormal development of bone, vigorous bone sexual abnormality, heritability kidney retinal development is unusual, the antiperspirant ectodermal dysplasia is arranged, the hypohidrosis ectodermal dysplasia, the lymphopenia thymus development is unusual, development of breast is unusual, lower jaw face abnormal development, metaphysis abnormal development, Mondini abnormal development, single bone fiber abnormal development, mucous epithelium abnormal development, dysplasia epiphysealis multiplex, oculoauricular dysplasia, eye vertebra abnormal development, tooth source sexual abnormality, eye ramus of mandible abnormal development, the all dentinal dysplasias of point, boniness fiber abnormal development, false cartilage forms spinal column epiphyseal dysplasia (pseudoachondroplasticspondyloepiphysial dysplasia), retinal development is unusual, every dysplasia of eye, the spinal column epiphyseal dysplasia, with ventricles of the brain radius abnormal development.
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 benign tumor, fibrous capsule character condition, tissue hypertrophy, polyp intestinal, polyp of colon and esophagus abnormal development), leukoplakia, 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 radiosiotope 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 radiosiotope 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 colon cancer, cardiac tumor, cancer of pancreas, melanoma, retinoblastoma, Glioblastoma, pulmonary carcinoma, intestinal cancer, carcinoma of testis, gastric cancer, neuroblastoma, myxoma, muscular tumor, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast carcinoma, carcinoma of prostate, Kaposi sarcoma, and ovarian cancer), the autoimmune sexual disorder is (such as multiple sclerosis, the Si Yegelun syndrome, struma lymphomatosa, biliary cirrhosis, Behcet, Crohn disease, polymyositis, systemic lupus erythematosus (sle) and immunity relevant glomerulonephritis and rheumatoid arthritis) and viral infection (such as herpesvirus, poxvirus, and adenovirus), inflammation, graft versus host 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 tumor and associated disorders with cell survival, (comprise that acute leukemia is (as acute lymphoblastic leukemia such as leukemia, acute myeloid leukemia (comprises myeloblast, promyelocyte (promyelocyte), myelomonocyte, mononuclear cell and erythroleukemia)) and chronic leukemia (as chronic myeloid (granulocytic) leukemia and chronic 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 tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, cancer of pancreas, breast carcinoma, ovarian cancer, carcinoma of prostate, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, syringocarcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocarcinoma, cancer of biliary duct, choriocarcinoma, spermocytoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, pulmonary carcinoma, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, 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, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration and cerebroma or aforementioned relevant disease); Autoimmune sexual disorder (such as multiple sclerosis, Si Yegelun syndrome, struma lymphomatosa, biliary cirrhosis, Behcet, Crohn disease, polymyositis, systemic lupus erythematosus (sle) and relevant glomerulonephritis of immunity and rheumatoid arthritis); Development of bone marrow abnormal syndrome (such as aplastic anemia), graft versus host disease, ischemia injury (such as causing), hepatic injury (as the relevant hepatic injury of hepatitis, ischemic/reperfusion injury, cholestasis (bile duct injury) and hepatocarcinoma) by myocardial infarction, apoplexy and reperfusion injury; The hepatopathy of toxin-induced (such as what cause), septic shock, cachexia and anorexia by ethanol.
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, abdominal part, bone, breast, digestive system, pancreas, peritoneum, endocrine gland (adrenal gland, parathyroid gland, hypophysis, testis, ovary, thymus, thyroid), eye, head and neck, nervous 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 syndrome, 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 carrier, the encode DNA sequence of described polynucleotide of this recombinant expression carrier effective expression.In another preferred embodiment of the present invention, utilize retrovirus or preferred adenovirus vector 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, viral vector is a deficiency, 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 micromolecule, chemicals or medicament administration etc.) of inducing coded protein to express via the promoter 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 therapeutic effect 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, stops the protein translation post-treatment, destroys protein or Profilin 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 liposomees, fat transfection or any other method of describing in full as exposed polynucleotide or description.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 lists of references 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 is the needed reverse transcription virus gene of its biocycle and can not self replication for want of, therefore adopt this retrovirus delivery system will make the cell of described gene and construction targeting 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 organs, 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 part or its combination in any, 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, suppress effect biology as long as it has the propagation of treatment cell.In addition, might be applied to same area simultaneously with surpassing a kind of polynucleotide of the present invention." inhibition biology " mean part or growth inhibited completely, and the reduction of cell proliferation or growth rate.Suppress biology dosage can by assessment polynucleotide of the present invention to target in the tissue culture effect of tumor growth and cell culture 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 hemopoietic, 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 rotein 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 tumor necrosis factor (TNF) receptor-1, CD95 (Fas/APO-1), the mediation of TNF receptor related apoptosis, and TNF apoptosis induction ligand related (TRAIL) receptor-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 rotein 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 agglutinin, thioredoxin, antiinflammatory 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 therapeutic effect of the present invention can separately or associating small-molecule drug or adjuvant realize.
In another embodiment, the invention provides the compositions that will contain the polynucleotide of albumin fusion proteins of the present invention and/or code book invention albumin fusion proteins be delivered to express that albumin fusion proteins of the present invention institute is bonded, the method for the target cell of its combination or associating polypeptide.Albumin fusion proteins of the present invention can associate via hydrophobicity, hydrophilic, ion and/or covalent interaction and heterologous polypeptide, heterologous nucleic acids, toxin or prodrug.
Albumin fusion proteins of the present invention can be used for strengthening 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 chemotactic factor) of described antigen and immunogenic immunne response.
The kidney disorder
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 compositions diagnosis of the present invention, prediction, prevention and/or treatment includes but not limited to urinary disturbance, autoimmune sexual disorder, sclerosis and necrosis, electrolyte imbalance and the renal carcinoma of the blood vessel disorder of renal failure, nephritis, kidney, metabolic and congenital kidney disorder, kidney.
Available compositions diagnosis of the present invention, prediction, prevention, and/or the nephropathy of treatment includes but not limited to acute renal failure, chronic renal failure, medicated porridge sample embolic renal failure, end-stage renal disease, the inflammatory diseases of kidney is (as acute glomerulonephritis, infect the back glomerulonephritis, fast-developing glomerulonephritis, the 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, lupus nephritis, chronic nephritis, interstitial nephritis, and post-streptococcal glomerulonephritis), the blood vessel disorder of kidney is (as renal infarction, medicated porridge sample embolic nephropathy, 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 disease (as pyelonephritis, hydronephrosis, urolithiasis (renal calculus, nephrolithiasis), reflux nephropathy, urinary tract infection, urinary retention, with acute or chronic one-sided obstructive uropathy).
In addition, compositions 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 glucosuria, 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, fingernail patella syndrome, the congenital nephrotic syndrome, the extruding syndrome, horseshoe kidney, diabetic nephropathy, diabetes insipidus,nephrogenic, the analgesic nephropathy, renal calculus, and membranous nephropathy), with the autoimmune sexual disorder of kidney (as systemic lupus erythematosus (sle) (SLE), the Goodpasture syndrome, IgA nephropathy, with IgM mesentery proliferative glomerulonephritis).
Compositions 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), renal carcinoma is (as nephroncus, hypernephroma, nephroblastoma, renal cell carcinoma, transitional cell carcinoma, renal adenocarcinoma, squamous cell carcinoma, and Wilms' tumor), and electrolyte imbalance is (as nephrocalcinosis, pyuria, edema, hydronephrosis, albuminuria, hyponatremia, hypernatremia, hypokalemia, hyperpotassemia, hypocalcemia, hypercalcemia, hypophosphatemia, and hyperphosphatemia).
Any method that compositions of the present invention can use this area to know is used, include but not limited to that direct pin injection, intravenous injection, local application, the conduit of delivering the position are inculcated, in the solid-state pharmaceutical formulations of biological projectile ejector, particle accelerator, gelfoam stock, other commercialization storage material, osmotic pumps, oral or suppository, operation process topple over or topical application, aerosol are delivered.These methods are roads known in the art.Compositions of the present invention can be used as the part of therapeutic 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 disorder, include but not limited to the peripheral arterial disease, such as limb ischemia.
Cardiovascular disorder includes but not limited to Cardiovascular abnormality, such as tremulous pulse-fistula of artery, artery-vein fistula, cerebral arteries deformity, congenital heart defect, pulmonary atresia and tulwar syndrome.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 syndrome, 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 syndrome is risen in the Shandong, method Rockwell triad, the ventricle heart septum defect.
Cardiovascular disorder also includes but not limited to heart disease, such as arrhythmia, 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, rupture of heart after the infarction, RIVS, valvular heart disease, cardiomyopathy, myocardial ischemia, pericardial effusion, pericarditis (comprising constriction and tuberculosis), pneumopericardium, pericardial incision postoperative syndrome, pulmonary heart disease, rheumatic heart disease, the ventricle malfunction, congested, the cardiovascular pregnancy complications, the tulwar syndrome, cardiovascular syphilis, with the cardiovascular tuberculosis disease.
Arrhythmia includes but not limited to arrhythmia, atrial fibrillation, atrial flutter, bradycardia, premature contraction, Adam Mu-Stokes syndrome, bundle branch block, sinoatrial block, long QT syndrome, parasystole, Lang-Gan-Lai three syndromes, Ma Haimu type pre-excitation syndrome, Wo-Pa-Huai syndrome, 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, subvalvular aortic stenosis, subvalvular pulmonary stenosis, restrictive cardiomyopathy, looks into Ge Sishi cardiomyopathy, 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 angina pectoris, coronary aneurysm, coronary atherosclerosis, coronary artery thrombosis, coronary vasospasm, myocardial infarction and myocardial stunning.
Cardiovascular disease also comprises angiopathy, such as aneurysm, angiodysplasia, hemangioma, the bacilus hemangioma, hippel-Lindau disease, Ke-Te-Wei three syndromes, Sturge-Weber syndrome, vasodilation, arotic disease, high iS-One arteritis, aortitis, Amur syndrome in reining in, arteriosclerosis obliterans, arteritis, enarteritis, polyarteritis nodosa, cerebrovascular disorder, diabetic angiopathy, diabetic retinopathy, thromboembolism, thrombosis, erythromelalgia, hemorrhoid, hepatic veno-occulusive disease, hypertension, hypotension, ischemia, peripheral vascular disease, phlebitis, pulmonary veno-occlusive disease, Raynaud disease, the CREST syndrome, retinal vein occlusion, the tulwar syndrome, superior vena cava syndrome, telangiectasis, ataxia telangiectasis, hereditary hemorrhagic telangiectasia, varicocele, varicosis, varicose ulcer, vasculitis, and venous insufficiency.
Aneurysm includes but not limited to dissecting aneurysm, false aneurysm, infectious aneurysm, ruptured aneurysm, aortic aneurysm, cerebral aneurysm, coronary artery aneurysm, arterio-cardiac aneurysm and iliac artery tumor.
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, carotid artery thrombosis, thrombosis of venous sinus, Wahlen Burger syndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma, subarachnoid hemorrhage, cerebral infarction, cerebral ischemia (comprising temporary), subclavian artery is stolen the blood syndrome, periventricular leukomalacia, vascular headache, cluster headache, migraine, and VBI.
Thromboembolism includes but not limited to air embolism, amniotic embolism, cholesterol thromboembolism, blue toe syndrome, fat embolism, pulmonary infarction and thromboembolism.Thrombosis includes but not limited to coronary artery thrombosis, hepatic vein thrombosis formation, retinal vein occlusion, carotid artery thrombosis, thrombosis of venous sinus, Wahlen Burger syndrome and thrombophlebitis.
The ischemia sexual disorder includes but not limited to cerebral ischemia, ischemic colitis, interval syndrome (compartment syndrome), tibialis anterior syndrome (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 lymph node syndrome, 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, local application, the conduit of delivering the position are inculcated, in the solid-state pharmaceutical formulations of biological projectile ejector, particle accelerator, gelfoam stock, other commercialization storage material, osmotic pumps, oral or suppository, operation process 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.
Disordered breathing
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, the atrophic rhinitis, vasomotor rhinitis), nasal polyp and sinusitis, the juvenile form fibrohemangioma, rhinocarcinoma and juvenile form papilloma, polyp of vocal cord, brief summary (singer's brief summary), contact ulcer, vocal cord paralysis, the larynx bulging, pharyngitis (as viral and bacillary), tonsillitis, the tonsil cellulitis, parapharyngeal abscess, laryngitis, the larynx bulging, and laryngocarcinoma is (as nasopharyngeal carcinoma, carcinoma of tonsil, laryngeal carcinoma), pulmonary carcinoma is (as squamous cell carcinoma, minicell (oat cells) cancer, large cell carcinoma, and adenocarcinoma), allergic disorder (eosinocyte pneumonia, the hypersensitivity pneumonia is (as extrinsic allergic alveolitis, allergic interstitial pneumonitis, the organic dust pneumoconiosis, allergic bronchopulmonary aspergillosis, asthma, Wa Genashi granuloma (granuloma vasculitis), the Goodpasture syndrome)), pneumonia is (as bacterial pneumonia (as streptococcus pneumoniae Streptococcus pneumoniae (pneumococcal pneumonia), staphylococcus aureus Staphylococcus aureus (staphylococcal pneumonia), gram negative bacteria 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 viral pneumonia is (as influenza, fowl pox (chickenpox)).
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, German measles (rubella), and subacute sclerosing panencephalitis, fungal pneumonia is (as histoplasmosis, coccidioidomycosis, blastomycosis, immune system is subjected to the fungal infection of serious philtrum of containing (as the cryptococcosis 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), emphysema, chronic or acute bronchitis), occupational pneumonopathy is (as silicatosis, black lung (miner lung coniosis), asbestosis, berylliosis, occupational asthma, byssinosis, with optimum pneumoconiosis), wellability pneumonopathy 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), edema, pulmonary infarction, bronchitis is (as viral, bacillary), bronchiectasis, pulmonary atelectasis, pulmonary abscess (by for example staphylococcus aureus or invade lung legionella Legionella pneumophila cause), and cystic fibrosis.
The angiogenesis inhibitor activity
Natural between endogenous stimulus object that blood vessel takes place and mortifier exists the influence that suppresses in the balance to preponderate.People such as Rastinejad, Cell, 56:345-355,1989.Issue in the angiopoietic rare situation of tissue regeneration promoting in the normal physiologic situation, 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 psoriasis of solid tumor growth and transfer, arthritis, 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 tumor, 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 relevant disease and/or disorderly method, comprise the albumin fusion proteins of the present invention that needed individual administering therapeutic effective dose 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, lung, breast, ovary, stomach, pancreas, larynx, esophagus, testis, liver, the parotid gland, bile duct, colon, rectum, cervix uteri, uterus, endometrium, kidney, bladder, thyroid carcinoma; Primary tumor and transfer; Melanoma; Glioblastoma; Kaposi sarcoma; Leiomyosarcoma; The non-cancer of non-small cell; Colorectal carcinoma; Senior malignant tumor; With the blood propagation tumor, 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 neoplasms, 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 tumor 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 involves except that cancer.These disorders include but not limited to: benign tumor, for example hemangioma, acoustic neuroma, neurofibroma, trachoma and botryomycosis hominis; Atheromatous plaque; The blood vessel generation disease of eye, for example uveitis and the pterygium (abnormal vascular growth) of diabetic retinopathy, retinopathy of prematurity, degeneration of macula, corneal graft rejection, neovascular glaucoma, Terry's sign, rubescent, retinoblastoma, eye; Rheumatoid arthritis; Psoriasis; 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 ischemic limb vessel; Ao-Wei two syndromes; Plaque neovessels forms; Telangiectasis; Bleeder's joint; Fibrohemangioma; Fibrillar muscle abnormal development; 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 degeneration of macula.
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, degeneration of macula, corneal transplantation neovascularization and with choroid or relevant ophthalmology inflammatory diseases, tumor of the eye 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 chemical 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 effective dose.In brief, cornea is the tissue that under normal circumstances lacks blood vessel.Yet in some pathological condition, capillary tube can extend into cornea by the cornea peripheral vessels 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 vision.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 malnutrition state, and as the complication of wearing contact lens.
In particularly preferred embodiment of the present invention, preparation is used for local application can (to unite any antiseptic and the antimicrobial that are usually used in the ophthalmology prepared product) in saline, 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 compositions of preparation also can directly be applied to cornea as mentioned above.In preferred embodiments, the angiogenesis inhibitor compositions prepares with the mucoadhesive polymers in conjunction with cornea.In other embodiments, the angiogenesis inhibitor factor or angiogenesis inhibitor compositions can be used as the complementary 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, chemical compound mentioned above can be injected directly in the corneal stroma under microscope instructs by ophthalmologists.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 compositions is placed blood vessel structure.In most applications, this cornea injection that will involve 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 capillary tube invasion of corneal transplant.In sustained release form, may only need 2-3 injection every year.Also can in injection, 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 effective dose and/or code book invention albumin fusion proteins, make the formation of blood vessel be suppressed the patient.In one embodiment, but the chemical compound local application to the eye with the treatment old model a neovascular glaucoma.In other embodiments, chemical compound can beat in the angle of anterior chamber zone by injection.In other embodiments, chemical compound also can place any position, makes chemical 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 effective dose 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 local concentration of agonist in retina by being expelled in aqueous humor or the vitreous body.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 effective dose and/or code book invention albumin fusion proteins, make the formation of blood vessel be suppressed the patient.Chemical compound can be implanted local application by intravitreal injection and/or ophthalmic.
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 hemangioma, arthritis, psoriasis, fibrohemangioma, atheromatous 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 tumor is such as leukemia, neoplasm metastasis, Kaposi sarcoma, benign tumor is hemangioma for example, acoustic neuroma, neurofibroma, trachoma, and botryomycosis hominis, rheumatoid arthritis, psoriasis, ophthalmology blood vessel generation disease is diabetic retinopathy for example, retinopathy of prematurity, degeneration of macula, 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 ischemic limb vessel takes place, Ao-Wei two syndromes, plaque neovessels forms, telangiectasis, bleeder's joint, fibrohemangioma, fibrillar muscle abnormal development, the wound granulation forms, Crohn disease, atherosclerosis, by preventing that the embryo from implanting the needed angiopoietic contraceptive 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, using quantity before or after sexual intercourse and fertilization generation is enough to block the chemical 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 ectopic treatment of film 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, compositions (for example form of spray or thin 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 prevent disease diffuses to surrounding tissue.In others of the present invention, compositions (for example form of spray) can be delivered by endoscopic procedure, thus the blood vessel generation that covers tumor 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 compositions bag quilt of the present invention.For example, in one embodiment of the invention, the surgery reticulated that has loaded angiogenesis inhibitor compositions 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 tumor being used after the excision 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 compositions is applied directly to tumor resection position (for example using by the margins of excision of smearing, brushing with the angiogenesis inhibitor chemical compound or alternate manner covers tumor).Perhaps, the angiogenesis inhibitor chemical compound can mix known surgery patch before using.In particularly preferred embodiment of the present invention, the angiogenesis inhibitor chemical compound is used after the hepatectomy of malignant tumor 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 chemical compound can be applied to the neurological tumor locus after excision, 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, tretinoin and derivant thereof, paclitaxel (Paclitaxel), suramin, metalloproteases-1 organize mortifier, metalloproteases-2 organize mortifier, 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 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 complex comprises oxo vanadium complex, such as vanadate and vanadyl complex.Suitable vanadate complex comprises metavanadate and positive vanadate complex, such as for example ammonium metavanadate, sodium metavanadate and sodium vanadate.Suitable vanadyl complex comprises that for example acetylacetone,2,4-pentanedione vanadyl and vanadium oxysulfate comprise that the vanadium oxysulfate hydrate is such as one and three hydration vanadium oxysulfates.
The representative example of tungsten and molybdenum complex also comprises oxo complex.Suitable oxo tungsten complex comprises tungstates and tungsten oxide complex.Suitable tungstates complex comprises ammonium tungstate, artificial schellite, Disodium tungstate (Na2WO4) dihydrate and wolframic acid.Suitable tungsten oxide comprises tungsten oxide (IV) and tungsten oxide (VI).Suitable oxo molybdenum complex comprises molybdate, molybdenum oxide and molybdyl complex.Suitable molybdate complex comprises ammonium molybdate and hydrate, sodium molybdate and hydrate thereof and potassium molybdate and hydrate thereof.Suitable molybdenum oxide comprises molybdenum oxide (VI), molybdenum oxide (VI) and molybdic acid.Suitable molybdyl complex copies for example acetylacetone,2,4-pentanedione oxygen molybdenum.Tungsten that other is suitable and molybdenum complex comprise by for example deutero-coordination hydroxyl of glycerol, tartaric acid and saccharide derivant.
Extremely multiple other angiogenesis inhibitor factor also can be used for content of the present invention.Representational example comprises platelet factor 4; Protamine sulfate; Sulphuric acid chitin derivatives (by snowflake Carapax Eriocheir sinensis preparation) people such as (, Cancer Res., 51:22-26,1991) Murata; Sulfated polysaccharide Peptidoglycan complex (SP-PG) (function of this complex can strengthen by the existence of steroid, such as estrogen and citric acid tamoxifen); Staurosporine; The modulator of matrix metabolism comprises for example proline analogs, cis hydroxyl groups proline, d, L-3,4-dehydroproline, thioproline, α, α-bipyridyl aminopropionitrile fumarate, 4-propyl group-5-(4-pyridine radicals)-2 (3H)-azolactones; Methotrexate; Mitoxantrone; Heparin; Interferon; 2 macroglobulin-albumin; 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; Amebacilin (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)-carboxy phenyl-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 cellular level
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 colon cancer, cardiac tumor, cancer of pancreas, melanoma, retinoblastoma, Glioblastoma, pulmonary carcinoma, intestinal cancer, carcinoma of testis, gastric cancer, neuroblastoma, myxoma, muscular tumor, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast carcinoma, carcinoma of prostate, Kaposi sarcoma, and ovarian cancer); Autoimmune sexual disorder (such as multiple sclerosis, Si Yegelun syndrome, struma lymphomatosa, biliary cirrhosis, Behcet, Crohn disease, polymyositis, systemic lupus erythematosus (sle) and relevant glomerulonephritis of immunity and rheumatoid arthritis) and viral infection (such as herpesvirus, poxvirus and adenovirus), inflammation, graft versus host disease, acute grafing 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 tumor and associated disorders, (comprise that acute leukemia is (as acute lymphoblastic leukemia such as leukemia, acute myeloid leukemia (comprises myeloblast, promyelocyte (promyelocyte), myelomonocyte, mononuclear cell and erythroleukemia)) and chronic leukemia (as chronic myeloid (granulocytic) leukemia and chronic 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 tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, cancer of pancreas, breast carcinoma, ovarian cancer, carcinoma of prostate, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, syringocarcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocarcinoma, cancer of biliary duct, choriocarcinoma, spermocytoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, pulmonary carcinoma, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, 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, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration and cerebroma or aforementioned relevant disease); Autoimmune sexual disorder (such as multiple sclerosis, Si Yegelun syndrome, struma lymphomatosa, biliary cirrhosis, Behcet, Crohn disease, polymyositis, systemic lupus erythematosus (sle) and relevant glomerulonephritis of immunity and rheumatoid arthritis); Development of bone marrow abnormal syndrome (such as aplastic anemia), graft versus host disease, ischemia injury (such as causing), hepatic injury (as the relevant hepatic injury of hepatitis, ischemic/reperfusion injury, cholestasis (bile duct injury) and hepatocarcinoma) 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 ethanol by ethanol.
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 excision wound, the gash that involves 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 chemical drugs causes, and other unusual wound healing situation, such as uremia, malnutrition, vitamin deficiency, and and steroid, radiotherapy, systemic treatment complications associated with arterial system with anti-superfluous crude drug and antimetabolite.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, xenograft (heterologous graft), xenograft (xenograft), allograft (homologous graft), activated graft, cornea thin layer graft, mesh graft, mucosal graft, ollier-Thiersch graft, omental grafts, patch graft, double-end graft, the 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 intestinal 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, hepatocyte, II type pneumonocyte, the goblet cell that generates mucoitin and skin, lung, liver and the gastrointestinal 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 organs (gut) toxic side effects that is caused by radiotherapy, chemotherapy or viral 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 (oral ulcer) that is caused by chemotherapy and viral 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 sebaceous 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 blister 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 mucosa lining and the regeneration of gland mucosa and duodenal mucosa lining.Inflammatory bowel such as Crohn disease and ulcerative colitis, refers to cause respectively small intestinal or the destructive disease of large intestine mucomembranous surface.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 gastrointestinal mucus generated and is had remarkable result, and can be used for protecting intestinal mucosa avoid the harmful substance that absorbs 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 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 hepatic injury that causes such as the explosive liver failure that causes by liver cirrhosis, by viral hepatitis and noxious substance (being acetaminophen, carbon tetrachloride (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 complementary 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 nerve problems of available compositions 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, neuron is subdued or degeneration 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 anoxia causes neuronal damage or death, comprises cerebral infarction or ischemia or spinal infarction or ischemia; (2) traumatic injury comprises damage that caused by physical injury or relevant with operation, and for example neural damage of cut-off parts perhaps weighs wounded; (3) malignant lesion, wherein the partial nerve system suffers the destruction or the damage of malignant tissue, this malignant tissue or nervous system associated malignancies or by the deutero-malignant tumor of non-neural system tissue; (4) infectious damage, wherein the partial nerve system is destroyed or is damaged because infect, for example by trachoma or with human immunodeficiency virus, herpes zoster or herpes simplex infections or relevant with Lyme disease, tuberculosis or syphilis; (5) degeneration damage, wherein the partial nerve system is destroyed or is damaged because of degenerative process, includes but not limited to and the relevant degeneration of parkinson, Alzheimer, hungtington's chorea or amyotrophic lateral sclerosis (ALS); (6) with nutritional 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 nutritional 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 erythematosus (sle), cancer or sarcoidosis; (8) damage that is caused by noxious substance comprises ethanol, 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 etiology, 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 hypoxia.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 compositions 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 nerve problems to select by the biologic activity of test in promoting neuronal survival or differentiation.For example, but not as restriction, cause the compositions of any following effect and can use according to the present invention: (1) increases the time-to-live of neuron in cultivation existing or lack in anoxia or the hypoxia condition; (2) in cultivation or increase neuronic germination in vivo; (3) in cultivation or increase the generation of neuron correlation molecule in vivo, as be choline acetyltransterase 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 general measure, 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 neuron correlation molecule can be used that this area is known and depend on that the technology of molecule to be measured waits and measures by bioassary method, enzymatic algoscopy, antibodies, Northern trace algoscopy; And the motor neuron dysfunction can be measured by the physical manifestations (physicalmanifestation) of assessment motor neuron disorder, for example weakness, motor neuron conduction 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 tumor of motor neuron and other composition of nervous 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 period PBP (Fa Qiao-Longde two syndromes), syndrome 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, synapse formation, conduction, Neural Differentiation etc.Thus, compositions 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.Compositions 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, Huntington's disease, the many syndromes 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, compositions 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 carotid artery thrombosis; carotid artery stenosis; or moyamoya); cerebral amyloid angiopathy; cerebral aneurysm; cerebral anoxia; cerebral arteriosclerosis; arteriovenous malformation of brain; cerebral arterial disease; cerebral embolism and thrombosis are (as carotid artery thrombosis; thrombosis of venous sinus; or Wahlen Burger syndrome); cerebral hemorrhage 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 syndrome; or VBI); vascular dementia (as multiple infarct); 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 purposes 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 disease 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 neurological 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, the 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 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 carotid artery thrombosis, thrombosis of venous sinus, with Wahlen Burger syndrome, cerebral hemorrhage 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 syndrome, 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 progressive supranuclear plasy, 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 syndrome, parkinson 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 syndrome, tetanic clonic epilepsy, the localized epilepsy of localized epilepsy such as complexity, frontal epilepsy and temporal-lobe epilepsy, post-traumatic epilepsy, status epilepticus 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, intracranial tuberculoma and Ze Weige syndrome, the dull-witted compound disease of central nervous system infection such as AIDS, brain 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 syndrome, myelitis is such as transverse myelitis, neurosyphilis is such as tabes dorsalis, poliomyelitis comprises syndrome 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's 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, meninges 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, meningism, 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, tabes dorsalis, stiff body syndrome, the mental retardation is such as angel's syndrome, cri du chat 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 syndrome, auspicious special syndrome, Rubinstein Taybi Syndrome, tuberous sclerosis, the WAGR syndrome, nervous system abnormality is such as holoprosencephaly, neural tube defect comprises hydranencephaly such as anencephaly, A-Cha Er Shi deformity, the brain bulging, the meninges bulging, the bulging of spinal cord spinal meninges, spinal dysraphism such as spina bifida cystica and spina bifida occulta.
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 heritability motion and esthesioneurosis, comprise Xia Ke-Malian Er Shi disease, hereditary optic atrophy, Refsum's disease, hereditary spastic paraplegia, Werdnig Hoffmann, heritability sensation and autonomic neuropathy such as congenital absence of pain and dysautonomia, neural performance (such as agnosia, comprises the special Man syndrome 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 logagraphia such as aphasia, anomia, Broca's aphasia and Wernicke's aphasia, dyslexia is such as the posteriority dyslexia, 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, echolalia, mutism and stutter, get muddled such as crying out and hoarseness, decerebrate state, delirium, fasciculation, hallucination, meningism, the dyskinesia is such as angel's syndrome, ataxia, athetosis, chorea, dystonia, hypokinesia, muscle tone is low excessively, myoclonus, twitch, torticollis and trembling, muscle tone is too high to ossify such as stiff body syndrome 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 syndrome, chronic progressive external ophthalmoplegia is such as the Ji Ensi syndrome, bulbar paralysis, torrid zone spastic paraplegia, paraplegia such as Blang-fork clip that syndrome, quadriplegia, respiratory paralysis and vocal cord paralysis, 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, sleep disorder such as hypersomnia comprises the Kleine-Levin syndrome, insomnia and somnambulism, spasm is such as gnathospasma, 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 neuron disease, amyotrophy is such as Duchenne-Arandisease, Xia-Ma Er Shi disease and Werdnig Hoffmann, syndrome 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 syndrome, 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 syndrome, reflex sympathetic dystrophy and Xia Yi-De Leige syndrome, cranial nerve diseases comprises neurofibromatosis 2 such as acoustic nerve disease such as acoustic neuroma, the nervus facialis disease is such as opsialgia, Mel Ke Song-Rosenthal syndrome, moving eye disorderly (ocular motility disorder) comprises amblyopia, nystagmus, oculomotor paralysis, ophthalmoplegia is such as the Du An syndrome, the Horner syndrome, chronic progressive external ophthalmoplegia comprises the Ji Ensi syndrome, stravismus such as esotropia and exotropia, 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, vocal cord paralysis, 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 nerve compression syndrome is such as complication of wrist, instep pipe syndrome, the thoracic outlet syndrome is such as scalenussyndrome, ulnar nerve compressing syndrome, neuralgia is such as causalgia, cervico-brachial neuralgia, opsialgia and trigeminal neuralgia, the neuritis is such as the EAN, optic neuritis, polyneuritis, polyradiculoneuropathy and radiculitis are such as polyradiculitis, heritability motion and esthesioneurosis such as Xia Ke-Malian Er Shi disease, hereditary optic atrophy, Refsum's disease, hereditary spastic paraplegia and Werdnig Hoffmann, heritability sensation and autonomic neuropathy comprise congenital absence of pain and dysautonomia, the POEMS syndrome, 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 hormonal system.
Hormone by the glandular secretion of hormonal 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 hormonal system and/or hormone imbalances or disorderly mark or detection thing.
Hormonal 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 the disorder and/or the disease (as dysmenorrhea and endometriosis) of menstrual cycle.
Hormonal 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 hypoplasia, pheochromocytoma-islet cell tumor syndrome; Adrenal disorder and/or disease are such as for example Addison's disease, corticosteroid deficiency disease, manlike disease, hirsutism, Cushing syndrome, hyperaldosteronism, pheochromocytoma; The disorder of pituitary gland and/or disease are such as for example hyperpituitarism, hypopituitarism, growth hormone deficiency dwarfism, pituitary adenoma, panhypopituitarism, acromegaly, gigantism; Thyroid disorder and/or disease include but not limited to hyperthyroidism, hypothyroidism, general Lu's Mo's disease, Graves' disease (graves), 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 thyroid 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, hormonal 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 syndrome (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, hormonal 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 endocrine 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 defect and causes sterile disease or the complication and the difficulty in puerperal (postpartum difficulties) of disorder, gestation, childbirth or production.
Reproductive system disorder and/or disease comprise the disease and/or the disorder of testis, comprise atrophy of testis, testicular feminization, cryptorchidism (one-sided and bilateral), anorchia, ectopic testis, epididymitis and orchitis (are caused by infection, usually such as for example gonorrhea, parotitis, tuberculosis, and syphilis), testicular torsion, joint knot shape deferentitis, germ cell tumor is (as spermocytoma, the cells,primordial cancer, teratocarcinoma, choriocarcinoma, yolk sac tumor, and teratoma), stromal tumors (as the interstitial cells tumor), hydrocele of tunica vaginalis, hematocele, varicocele, seminal cyst, 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 dystonia, prostatosis, granulomatous prostatitis, malakoplakia, benign prostatauxe or regeneration, and the superfluous natural disposition disorder of prostate, comprise adenocarcinoma, transitional cell carcinoma, duct carcinoma and squamous cell carcinoma.
In addition, compositions of the present invention can be used for diagnosis, 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, herpes simplex virus, gonorrhea, non gonococcal urethritis, chlamydia, mycoplasmas, trichomonacide, HIV, AIDS, Lai Teer syndrome, condyloma acuminatum, flat condyloma and pearly penile papules; Urethra is unusual, such as hypospadias, epispadias and phimosis; Damage before worsening comprises erythroplasia of Queyrat, bowen's disease, rich warm bowenoid papulosis, Bu-huge condyloma latum of Le Er Shi and verrucous carcinoma; Carcinoma of penis comprises squamous cell carcinoma, cancer in situ, verrucous carcinoma cancer and dispersivity carcinoma of penis; The urethra natural disposition disorder of going to live in the household of one's in-laws on getting married comprises penis carcinoma of urethra, bulb of urethra film cancer (bulbomembranous urethralcarcinoma) and prostate-urethra cancer; And it is disorderly to erect, such as priapism, Perun Nie Shi disease, erection disturbance and sexual impotence.
In addition, deferential disease and/or disorder comprise vasculitis and CBAVD (deferent duct 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 syndrome, premature ejaculation, diabetes, cystic fibrosis, Ka Tagena syndrome, hyperpyrexia, 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 candida mycoderma vaginitis, herpes simplex virus, chancroid, granuloma inguinale, lymphogranuloma venereum, scabies, the human papillomavirus, the vagina wound, the vaginal orifice wound, adenopathy, the chlamydia vaginitis, gonorrhea, the trichomonacide vaginitis, condyloma acuminatum, syphilis, molluscum contagiosum, atrophic vaginitis, Paget, lichen sclerosus, lichen planus, vulvodynia, toxic shock syndrome, vulvismus, vulvovaginitis, vulvar vestibulitis, with superfluous natural disposition disorder, such as the squamous cell hypertrophy, clear cell carcinoma, basal cell carcinoma, melanoma, Ba Tuolin adenocarcinoma, form with Intradermal vegetation on the pudendum.
The disorder in uterus and/or disease comprise dysmenorrhea, retroversion, endometriosis, fibroma, gland myopathy (adenomyosis), anovulatory bleeding, amenorrhea, Cushing syndrome, hydatidiform mole (hydatidiform mole), Ah thank'sing Man syndrome, premature menopause, sexual precosity, metropolypus, anovulatory dysfunctional uterine hemorrhage (as because unusual hormone signal) and superfluous natural disposition disorder, such as adenocarcinoma, 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 uterus bicornis, uterus septus, simple unicornuate uterus, unicornuate uterus, unicornuate uterus, unicornuate uterus, uterus arcuatus, double 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 cyst, hypo ovaria, ovary is insensitive to promoting sexual gland hormone, ovary excessively generates androgen, the right ovarian vein syndrome, amenorrhea, hirutism, and ovarian cancer (includes but not limited to the carcinous growth of constitutional and Secondary cases, Sai Ertuoli-Lai Dixi Er Shi tumor, the ovary endometrioid carcinoma, the ovary papillary serous adenocarcinoma, the ovary mucinous adenocarcinoma, with the ovary krukenberg's tumor).
The disease of cervix uteri and/or disorder comprise cervicitis, chronic cervicitis, sticking purulence cervicitis, dysplasia of cervix, cervical polyp, naboths cysts, cervical erosion, incompetence,cervical and cervix uteri vegetation (comprising that for example cervical cancer, squamous metaplasia, squamous cell carcinoma, glandular scale shape cell vegetation form and cylindrical cell 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, TA, threatened abortion, missed abortion, incomplete abortion, artificial abortion, habitual abortion, missed abortion and septic abortion; Ectopic pregnancy, anemia, Rh incompatibility, pregnancy duration vaginal hemorrhage, gestational diabetes, intrauterine growth retardation, polyhydramnios, HELLP syndrome, placental abruption, placenta previa, hypermesis, preeclampsia, eclamposia, 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 disease, heart failure, rheumatic heart disease, congenital heart disease, mitral valve prolapse, hypertension, anemia, nephropathy, infectious disease is (as rubella, cytomegalovirus, toxoplasmosis, infectious hepatitis, chlamydia, HIV, AIDS, and genital herpes), diabetes, Graves' disease, thyroiditis, hypothyroidism, struma lymphomatosa, chronic active hepatitis, liver cirrhosis, primary biliary cirrhosis, asthma, systemic lupus erythematosus (sle), rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, appendicitis, ovarian cyst, the gallbladder 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 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, mastitis, cystitis, 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 syndrome, pseudohermaphroditism, premenstrual syndrome, pelvic inflammatory disease, pelvic congestion (congestion of blood vessel), frigidity, ahedonia, dyspareunia, salpingorrhexis and middle pain.
Infectious disease
The polynucleotide of 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, infectious 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: arbovirus (Arbovirus), Adenoviridae (Adenoviridae), Arenaviridae (Arenaviridae), Arteriviridae (Arterivirus), birnavirus section (Bimaviridae), Bunyaviridae (Bunyaviridae), Caliciviridae (Caliciviridae), porcine circovirus section (Circoviridae), coronaviridae (Coronaviridae), dengue virus, EBV, HIV, flaviviridae (Flaviviridae), Hepadnaviridae (Hepadnaviridae) (hepatitis virus), herpetoviridae (Herpesviridae) is (such as cytomegalovirus, herpes simplex virus, 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), human papillomavirus, papovaviridae (Papovaviridae), Parvoviridae (Parvoviridae), Picornaviridae (Picornaviridae), Poxviridae (Poxviridae) (such as variola 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: arthritis, 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 fever, 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), dermatosis 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, dengue 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 antibacterial and the fungus media of symptom include but not limited to following Gram-negative and gram-positive bacterium, antibacterial section, and fungus: 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), dermatomycosis (Dermatocycoses), bacillus coli (E.coli) (as enterotoxigenic escherichia coli and enterohemorrhagic Escherichia coli), Enterobacter (Enterobacter) (as clostridium perfringen (Enterobacteraerogenes)), enterobacteriaceae (Enterobacteriaceae) (Klebsiella (Klebsiella), Salmonella (Salmonella) is (as salmonella typhi (Salmonella typhi), Salmonella enteritidis (Salmonella enteritidis), salmonella typhi (Salmonella typhi)), Serratia (Serratia), Yersinia (Yersinia), Shigella (Shigella)), erysipelothrix (Erysipelothrix), haemophilus (Haemophilus) (as Type B hemophilus influenza (Haemophilus influenzae)), Helicobacterium (Helicobacter), Legionnella (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), spirillum (Spirochetes) is (as treponema (Treponema spp.), Leptospira (Leptospiraspp.), Borrelia (Borrelia spp.)), Shigella (Shigella spp.), staphylococcus (Staphylococcus) (as staphylococcus 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 antibacterials, parasite, can cause disease or symptom with fungus section, include but not limited to: antibiotic resistance infects, bacteremia, endocarditis, septicemia, ocular infection (as conjunctivitis), uveitis, tuberculosis, gingivitis, 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 pertussis or empyema, sepsis, Lyme disease, cat scratch disease, dysentery, paratyphoid fever, alimentary toxicosis, legionnaires disease, chronic and acute inflammation, erythema, yeast infection, typhoid fever, pneumonia, lymph, meningitis (as A type and Type B meningitis), chlamydia, syphilis, diphtheria, leprosy, brucellosis, peptic ulcer, anthrax, spontaneous abortion, birth defect, pneumonia, pulmonary infection, ear infection, deaf, blind, drowsiness, uncomfortable, vomiting, chronic diarrhea, Crohn disease, colitis, vaginitis, sterile, pelvic inflammatory disease, candidiasis, paratuberculosis, tuberculosis, lupus, botulism, gangrene, tetanus, impetigo, rheumatic fever, scarlet fever, sexually transmitted disease (STD), dermatosis is (as cellulitis, dermatomycosis), toxemia, urinary tract infection, wound infection, 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, botulism 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: ameba worm (disease), crust is this worm (disease) doubly, coccidiosis (disease), Cryptosporidium (disease), double-core ameba worm (disease), covering diseases such as horse, epizoa (disease), giardia lamblia (disease), anthelmintic (disease), Leishmania (disease), schistosomicide (schistosomicide) disease, Taylor's that piroplasm (disease), toxoplasma (disease), taper worm (disease), and infusorian (disease) and sporozoon 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, pneumonopathy, 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 effective dose, 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 infectious 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 defect, wound (wound, burn, otch or ulcer), aging, disease (as osteoporosis, osteoarthritis, periodontal disease, liver failure), performing the operation comprises face-lifting plastic operation, fibrosis, reperfusion injury or the impaired tissue of systemic cytokine damage.
Can use the regenerated tissue of the present invention to comprise organ (as pancreas, liver, intestinal, kidney, skin, endothelium), muscle (smooth muscle, skeletal muscle or cardiac muscle), vascular system (comprising blood vessel and lymphatic vessel), nerve, hemopoietic and skeleton (bone, cartilage, tendon and ligament) tissue.Preferably, outgrowth do not have cicatrix or cicatrix 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 recovery time 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 tendinitis, 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, with peripheral nerve injury, peripheral neuropathy (as causing), localized neuropathy and central nervous system disease (as Alzheimer, parkinson, Huntington disease, amyotrophic lateral sclerosis and Xia Yi-De Leige syndrome) diseases associated by chemotherapy or other medical therapy all can use albumin of the present invention merge egg from and/or the polynucleotide of code book invention albumin fusion proteins treat.
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 intestinal vegetation (enteric carcinoid tumor, small intestinal non-Hodgkin lymphomas, enteric lymphoma)) and ulcer, such as peptic ulcer.
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, adenocarcinoma, the gastric retention disorder, gastroenteritis, gastratrophia, gastric cancer, polyp of stomach, the autoimmune sexual disorder is such as pernicious anemia, pyloric stenosis, gastritis is (bacillary, viral, eosinocyte, pressure inducement, chronic aggressivity, atrophic, plasma cell, with the Menetrey Erichsen), and disease of peritoneum is (as chyloperitoneum (chyloperioneum), hemoperitoneum, mesenteric cyst, adenomesenteritis, the mesentery angiemphraxis, panniculitis, vegetation, peritonitis, pneumoperitoneum, bubphrenic abscess).
Gastrointestinal dysfunction also comprises the disorder relevant with small intestinal, 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, appendicitis, ileocleisis, Meckel's diverticulum, multiple diverticulum, the complete operational failure of small intestinal and large intestine, lymphoma, with antibacterial and parasitic disease (such as traveler's diarrhea, typhoid fever and paratyphoid fever, cholera, ascarid (ascariasis Ascariasislumbricoides), ancylostome (Ancylostoma duodenale Ancylostoma duodenale), nematicide (shape worm Enterobius vermicularis of wriggling) in the gutstring, cestode (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, biliary cirrhosis), fatty liver (alcoholic fatty liver, the Lay syndrome), hepatic vein thrombosis forms, the degeneration of liver bean shape grain, hepatomegaly, liver lung syndrome, hepatorenal syndrome, portal hypertension (esophagus stomach function regulating varix), liver abscess (amebic liver abscess), liver cirrhosis (ethanol, bile, with experimental), alcoholic liver disease (fatty liver, hepatitis, sclerosis), parasite (hepatic echinococcosis, fascioliasis, amebic liver abscess), jaundice (hemolytic, hepatocyte property, with cholestasis), cholestasis, portal hypertension, liver enlarges, ascites, hepatitis (alcoholic hepatitis, animal hepatitis, chronic hepatitis (autoimmune, hepatitis B, hepatitis C, hepatitis D, drug-induced), toxic hepatitis, viral people's hepatitis (hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E), Wilson's disease, granulomatous hepatitis, the Secondary cases biliary cirrhosis, hepatic encephalopathy, portal hypertension, varix, hepatic encephalopathy, primary biliary cirrhosis, primary sclerosing cholangitis, adenoma, hemangioma, cholelithiasis, liver failure (hepatic encephalopathy, acute hepatic failure), regulating liver-QI vegetation (angiomyoliopma, calcified liver metastasis, the capsule metastatic liver cancer, epithelial tumor, fibre plate hepatocarcinoma, kitchen range joint knot property hypertrophy, liver adenocarcinoma, hepatobiliary cystadenoma, hepatoblastoma, hepatocarcinoma, hepatoma, hepatocarcinoma, angioendothelioma of liver, mesenchymal hamartoma, the mesenchymal cell tumor of liver, joint knot regenerative proliferation, optimum liver tumor (hepatic cyst [simple cyst, polycystic liver disease, hepatobiliary cystadenoma, choledochal cyst], mesenchymal cell tumor [mesenchymal hamartoma, child's hemangioendothelioma, hemangioma, peliosis hepatis, lipoma, inflammatory pseudotumor, mix type], epithelioma [epithelial duct (bile duct hamartoma, cholangioadenoma), hepatocyte (adenoma, kitchen range joint knot property hypertrophy, joint knot regenerative proliferation)], malignancy hepatic tumor [hepatocyte, hepatoblastoma, hepatocarcinoma, bile duct cell, cancer of biliary duct, cystadenocarcinoma, vascular tumor, angiosarcoma, Kaposi sarcoma, hemangioendothelioma, other tumor, embryonal sarcoma, fibrosarcoma, leiomyosarcoma, rhabdomyosarcoma, carcinosarcoma, teratoma, carcinoid, squamous cell carcinoma, the constitutional lymphoma]), peliosis hepatis, the erythropoiesis hepatic porphyria, hepatic porphyria (acute intermittent porphyria, porphyria cutanea tarda), the Ze Weige syndrome).
The disease of pancreas and/or disorder comprise acute pancreatitis, chronic pancreatitis (acute necrotizing pancreatitis, alcoholic pancreatitis), vegetation (adenocarcinoma of pancreas, cystadenocarcinoma, insulinoma, gastrinoma and glucagonoma, capsule vegetation, islet cells tumor, pancreas blastoma) and other pancreatic diseases (as cystic fibrosis, cyst (pancreatic pseudocyst, pancreas fistula, insufficiency)).
Gallbladder disease comprises cholelithiasis (cholelithiasis and choledocholithiasis), postcholecystectomy syndrome, diverticulum of gallbladder disease, acute cholecystitis, chronic cholecystitis, tumor of bile duct and mucous cyst.
The disease of large intestine and/or disorder comprise antibiotic resistance colitis, diverticulitis, ulcerative colitis, acquired megacolon, abscess, fungus and bacterial infection, anal orifice and rectal intestine disorder (as anal fissure, hemorrhoid), colonic diseases (colitis, colon vegetation [colon cancer, adenoma shape polyp of colon (as villous adenoma), colon cancer, colorectal carcinoma], diverticulitis of colon, diverticulosis of colon, megacolon [Hirschsprung disease, toxic megacolon]; Sigmoid diseases [proctocolitis, 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 atresia, parasitic disease (anisakiasis, balantidiasis, yeast infection (blastocystis infection), cryptosporidiosis, double-core ameba parasitosis, amebic dysentery, giardiasis), intestinal fistula (rectal fistula), intestinal vegetation (caecum vegetation, colon vegetation, duodenum vegetation, ileum vegetation, polyp intestinal, jejunum vegetation rectum vegetation), intestinal obstruction (input loop syndrome, duodenal obstruction, feces blocks, the pseudoileus proctoptosis), peptic ulcer (duodenal ulcer, peptic esophagitis, hemorrhage, perforation, gastric ulcer, Zollinger Ellison syndrome), syndrome behind the gastrectomy (dumping syndrome), gastropathy is (as achlorhydria, duodenogastric reflux (bile reflux), gastric antrum vasodilation, gastric fistula, gastric outlet obstruction, gastritis (atrophic or plumpness), gastroparesis, flatulence, gastric diverticulum, stomach vegetation (gastric cancer, polyp of stomach, adenocarcinoma of stomach, the hypertrophy polyp of stomach), gastric rupture, gastric ulcer, gastric volvulus), tuberculosis, visceroptosis, vomiting is (as hematemesis, the hyperemesis gravidarum, postoperative nausea and vomiting), and hemorrhagic colitis.
Other disease and/or the disorder of gastronintestinal 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, gastrointestinal vegetation, the adenocarcinoma of pancreas vegetation such as pancreas, the mucin capsule vegetation of pancreas, pancreatic capsule vegetation, the pancreas blastoma, with peritoneum vegetation), esophagus disease is (as big kitchen disease, candidiasis, glycogenesis property acanthosis, ulcer, Barrett esophagus varix, locking, cyst, diverticulum (as divicine), fistula (as tracheo esophageal fistula), the motility disorder is (as the CREST syndrome, dysphagia, achalasia, spasm, gastroesophageal reflux), vegetation, perforation (is breathed out not syndrome as boolean, Mallory-Weiss syndrome), narrow, esophagitis, diaphragmatocele (as hiatal hernia); Gastrointestinal disease is such as gastroenteritis (infecting as eholera morbus, Norwalk virus), hemorrhage (as hematemesis, melena, digestive ulcerative bleeding), stomach vegetation (gastric cancer, polyp of stomach, adenocarcinoma of stomach, gastric cancer)), hernia (as congenital diaphragmatic hernia, femoral hernia, inguinal hernia, obturator hernia, umbilical hernia, abdominal hernia) and intestinal diseases (as caecum disease (appendicitis, 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 mononuclear cell, fibroblast, neutrophil cell, T cell, mastocyte, eosinocyte, epithelial cell and/or endotheliocyte) or mobilizes specific part 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 specific part in the targeting 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 fibroblast, 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 bonded molecule of therapeutic protein part 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 receptor) or micromolecule.
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 simulation 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 bonded natural receptor of therapeutic protein part of albumin fusion proteins of the present invention, perhaps is can be by the bonded receptor fragments of therapeutic protein part (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 involves the suitable cell that generates expression albumin fusion proteins of the present invention.Preferred cell comprises from mammal, yeast, fruit bat or colibacillary cell.
Algoscopy can only be tested combining of candidate compound and albumin fusion proteins of the present invention, wherein in conjunction with being to detect by label or involving in the algoscopy of the competition of labelling competition thing.In addition, algoscopy can be tested candidate compound by producing signal with combining of fusion rotein.
Perhaps, algoscopy 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.Algoscopy 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 algoscopy can be used fusion rotein level or the activity in monoclonal or the polyclonal antibody measuring samples (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 bonded receptor of therapeutic protein part 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 expression cloning, 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 receptor 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 labelling.Can come the labelling 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 receptor evaluation, can will be connected by light is affine with the cell membrane 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 labelling 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 receptor through labeled complex, resolve to fragments of peptides, 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 albumin 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 involves 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 domain, fragment etc. and one or more heterologous molecule, motif, parts, partly, reorganization such as domain, fragment.In preferred embodiments, heterologous molecule is the family member.In embodiment preferred more, heterologous molecule is a somatomedin, such as for example platelet derived growth factor (PDGF), insulin like growth factor (IGF-1), transforming growth factor (TGF)-α, epidermal growth factor (EGF), fibroblast growth factor (FGF), TGF-β, bone morphogenetic protein (BMP)-2, BMP-4, BMP-5, BMP-6, BMP-7, activator protein A and B, decapentaplegic (dpp), 60A, OP-2, back of the body albumen, growth and differentiation factor (GDF), nodal, MIS, inhibin-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 albumin 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 albumin 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 chemical compound of evaluation regulation and control albumin fusion proteins effect of the present invention.The example of this algoscopy comprise with mammal fibroblast, albumin fusion proteins of the present invention and chemical compound to be screened and
3[H] thymidine is mixed under with normal proliferating cells condition of culture fibroblast.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 chemical compound compares to determine whether this chemical 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 chemical compounds of agonist and antagonist all can be identified by this program.
In another approach, there is incubation under the condition of chemical compound in the mammalian cell or the film preparation thing of receptor of expressing the therapeutic protein composition of fusion rotein of the present invention with the fusion rotein of the present invention through labelling.Can measure the chemical 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 chemical compound bind receptor and cause whether the ability that the second message,second messenger replys is potential fusion rotein with definite this chemical compound.This second messenger system includes but not limited to cAMP guanylate cyclase, ion 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 algoscopys to find can be used for treating disease or produce particular result (as angiogenic growth) in the patient.In addition, these algoscopys 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 chemical 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.
Targeting is delivered
In another embodiment, the invention provides the method that compositions is delivered to the target cell of the receptor 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, hydrophilic, 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 compositions 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 tumor 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 poisonous effect system system chemical compound, radiosiotope, 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 cell death.Can include but not limited to radiosiotope known in the art according to the toxin that the present invention uses, such as the antibody chemical compounds such as (or it contain the complement fixation part) of for example uniting in conjunction with intrinsic or inductive endogenous cell poisonous effect system, thymidine kinase, endonuclease, the RNA enzyme, alpha-toxin, ricin, Agglutinin, Pseudomonas exotoxin A, diphtheria toxin, diphtherotoxin, the Saponaria officinalis toxalbumin, the Fructus Momordicae charantiae toxalbumin, spend more the Rhizoma Melaleuca Viridiflora toxalbumin, pokeweed antiviral protein, α-broom aspergillin, and cholera toxin." 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 benzoic acid Semen Sinapis 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 derivant of daunoblastin and amycin.
Drug screening
The polynucleotide of also having imagined albumin fusion proteins of the present invention or these fusion rotein 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 chemical 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 that this cell obtains medicine is screened at this.Can measure for example formation of complex 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 complex between reagent and albumin fusion proteins or its fragment.In this competitive binding assay method, reagent to be screened has carried out labelling usually.Behind the incubation, free reagent is separated with the reagent that exists with combining form, and quantity free or the not label of complexation is 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 chemical compound of appropriate combination affinity, and in the european patent application 84/03564 of JIUYUE in 1984 announcement on the 13rd, 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 bonded peptide by method well-known in the art then.Can with the albumin fusion proteins direct coated of purification 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 algoscopy, 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 technique 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 chromatograph process, and wherein affinity substrate is made of immobilized albumin fusion proteins of the present invention.Can come purification 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 that separates 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 outer 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 inducer, 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 celluloid 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 coded sequence of the DNA of coded polypeptide more easily by direct mensuration of conventional 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 spectrography.
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 bond albumin fusion proteins of the present invention at random or combined peptide or non-peptide library.Many available libraries are 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, 91:11422-1 1426,1994; 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, hydantoin, piperazinedione, biphenyl, sugar analogue, β-sulfydryl ketone, Arylacetic acids, acylpiperidine .alpha.-5:6-benzopyran, 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 oligomer.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 the benzodiazepine grass structure.
Non-peptide oligomer 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-amido but not the peptide sample oligomer of alpha-carbon, has constituted the basis in the non-peptide oligomer library of another kind of form.Initial non-peptide oligomer library utilizes the monomer of single type, thereby contains multiple main chain.Nearest library has utilized and has surpassed a kind of monomer, makes the motility (flexibility) in library increase.
The screening library can be undertaken by the several different methods of generally knowing.Consult for example following list of references, 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 bonded those libraries of albumin fusion proteins 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 described in the list of references of quoting and be called " elutriation "
The example of these screening techniques of technology
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 bond 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 multiformity 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 assigned address 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 aminoacid lysine 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 aminoacid.In another embodiment, has 15-100 aminoacid or 20-50 aminoacid 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 mitosis of the various kinds of cell of Different Origin, such as fibroblast 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 neuron disorder or neurodegenerative conditions, to take place, such as Alzheimer, parkinson 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 the chondrocyte 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 primary tissue.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 catabolism, 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 condition by influencing biorhythm, the rhythm of the heart, depression (comprising depression), violent tenet, pain tolerance level, reproductive performance (preferably by activin or inhibin sample activity), hormone or endocrine level, appetite, libido, 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 food additive or antiseptic, such as being used for improving or reducing storage capacity, fat content, lipid, protein, carbohydrate, microorganism, mineral, the spoke factor or other nutritional labeling.
Above-mentioned application can be used for host extremely widely.These hosts include but not limited to people, Mus, rabbit, goat, Cavia porcellus, camel, horse, mice, rat, hamster, pig, miniature pig, chicken, goat, cattle, sheep, dog, cat, non-human primates and people.In specific embodiment, the host is mice, rabbit, goat, Cavia porcellus, chicken, rat, hamster, pig, sheep, dog or cat.In preferred embodiments, the host is a mammal.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 remainder 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 derivants 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 vehicle, 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 albumin 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 XhoI and ClaI restriction site prepare.
The first step by remove the inherent XhoI of pPPC0005 and ClaI site (being positioned at 3 ' end of ADH1 terminator sequence) with XhoI 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 XhoI and ClaI restriction site are added in the nucleotide sequence (HSA targeting sequencing with from the kex2 site of mating factor α " MAF " chimera) 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 coded portion HSA signal peptide sequence, from the kex2 site of mating factor α targeting sequencing and the aminoterminal nucleotide sequence of section H SA mature form.Introduce four point mutation in the sequence, be created in the XhoI and the ClaI 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
ATT
TAAAGATTTGGG-3 ' (SEQ ID NO:36) and
5′-AATCGATGAGCAACCTCACTCTTGTGTGCATCTCTTTTCTCGAGGCTC
CTGGAATAAGC-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 purification then with AflII and XbaI digestion, and is connected in the same loci of pPPC0006 to produce pScCHSA.So the plasmid that produces has XhoI and the ClaI site that is incorporated in the signal sequence.Having produced single amino acids in the end that exists in signal sequence in XhoI site changes promptly from LDKR to LEKR.After the nucleotide sequence that will have 5 ' SalI site (it is compatible with the XhoI site) and 3 ' ClaI site, comprise the polynucleotide of coding albumin fusion proteins therapeutic part is connected in the XhoI of pScCHSA and the ClaI site, D will can not be present in the final albumin fusion proteins expression plasmid to the change of E.SalI has recovered the original amino acid of signal peptide sequence to the connection of XhoI.The DNA of coding albumin fusion proteins therapeutic part can be inserted into after the Kex2 site before (Kex2 cuts after two basic amino acid sequence KR of signal peptide end) and ClaI site.
The generation of pScNHSA: albumin part is positioned at the albumin 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.AscI, FseI and PmeI restriction site are added between the Bsu36I and Hind III site of encoding mature HSA nucleic acid sequences to proteins end.This is to contain AscI, FseI that underscore indicates and two complementary synthetic primers (SEQ ID NO:40 and SEQ ID NO:41) of PmeI restriction site are realized by use.
5′-AAGCTGCCTTAGGCTTATAATAA
GGCGCGCCGGCCGGCCGTTTAAAC
TAAGCTTAATTCT-3 ' (SEQ ID NO:40) and
5′-AGAATTAAGCTTA
GTTTAAACGGCCGGCCGGCGCGCCTTATTATAAG
CCTAAGGCAGCTT-3′(SEQ?ID?NO：41)。With these primer annealings, with Bsu36I and
Hind III digestion, and be connected in the same loci of pScCHSA to produce pScNHSA.
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 vehicle 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 albumin 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 can design the HSA mature form of will encoding last four aminoacid (with containing the Bsu36I site) polynucleotide to add to the coding therapeutic protein DNA 5 ' end 5 ' primer; And the 3 ' primer that termination codon and suitable cloning site is added to 3 ' end of therapeutic protein coded 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 aminoacid 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 termination codoies, 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 (AscI, FseI or PmeI), and is connected among the pScNHSA.
In the chimeric targeting sequencing of HSA the XhoI 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 albumin 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 targeting sequencing last three aminoacid 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 '-aggagcg
TcGACAAAAGA (N)
15-3 ' (SEQ ID NO:46), wherein the underscore sequence is the SalI site, last three aminoacid (DKR) of capitalization nucleotide coding HSA targeting sequencing, 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 the ClaI site, and underscore nucleotide is the reverse complementary sequence of DNA of initial 9 aminoacid (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 SalI and ClaI, and is connected in the pScCHSA of XhoI and ClaI digestion.May need different signals or targeting sequencing, for example can be by standard method known in the art with invertase " 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) sub-clone of IGFBP (insulin-like growth factor binding protein) 4 (Swiss-Prot AccessionP22692) and HSA targeting sequencing is in suitable carrier.
The generation of the albumin fusion construct that is adapted at expressing in the saccharomyces cerevisiae
Contain the NotI 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 NotI site of the pSAC35 with LEU2 selected marker.Then the carrier that will so produce is used for the conversion of saccharomyces cerevisiae expression system.
Embodiment 3: the general expression in the 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 saccharomyces cerevisiae.Expression vector imports among Wine brewing yeast strain DXY1, D88 or the BXP10 by transforming, indivedual transformants 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 the stable phase collecting cell.By cell was collected supernatant in centrifugal 10 minutes with 3000g.
Except that the LEU2 selected marker, pSAC35 (people such as Sleep, 1990, Biotechnology8:42 and referring to Fig. 3) comprise whole yeast 2 μ m plasmids, PRB1 promoter and ADH1 termination signal that copy function is provided.
Embodiment 4: the general purification of the albumin fusion proteins of being expressed by the albumin fusions in the 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 fused 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 culture medium.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, IGFBP (insulin-like growth factor binding protein) 4, the variant HSA targeting sequencing that includes but not limited to chimeric HSA/MAF targeting sequencing 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 description " 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.Ooze rate to reduce volume and to remove pigment from the zymic supernatant of expressing albumin fusion proteins with 3mM phosphate buffer pH 6.2,20mM NaCl and 0.01%Tween20.Making solution pass through 0.22 μ m device then filters.Filter liquor is loaded on the Dyax peptide affinity column.Carry out eluting with 100mM Tris/HCl pH8.2 buffer coupled columns.Collection contains proteinic peak fraction, and analyzes on SDS-PAGE after concentrating 5 times.
For large scale purification, can use following method.Supernatant above 2 liters is oozed rate and is concentrated into 500ml in 20mMTris/HCl pH8.0.Spissated protein solution is loaded into through on the 50ml DEAE-Sepharose Fast Flow post of pre-equilibration, washes post, and with among the 20mM Tris/HCl pH8.0 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 solution, adds final concentration 0.9M
4)
2SO
4, and whole solution is loaded into through on the 50ml Butyl650S post of pre-equilibration.Ammonium sulfate (0.9 to 0M (NH with linear gradient
4)
2SO
4) elute protein.Merge the fraction that contains the albumin fusions once more, use 10mM Na
2HPO
4/ citrate buffer solution pH5.75 oozes rate, and is loaded into 50ml through on the SP-Sepharose Fast Flow post of pre-equilibration.NaCl linear gradient elution protein with 0 to 0.5M.Merge the fraction that contains destination protein matter, change buffer into 10mM Na with the Amicon concentrator
2HPO
4/ citric acid pH6.25, electrical conductivity＜2.5mS/cm.This protein solution is loaded into 15ml through on the Q-Sepharose high-efficiency column of pre-equilibration, 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 buffer is changed purification then.
Embodiment 5: be used for the generation of the general construction of mammalian cell transfection
The generation of the albumin fusion construct that is adapted at expressing in the mammal cell line
Can produce the albumin 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 systems.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 mammal culture systems, produces plasmid pC4:HSA (ATCC preserving number PTA-3277).This carrier has dihydrofolate reductase, 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 sub-clone of forming in candidate's expression vector.For example, can with comprise the DNA of encoding mature albumin fusion proteins or by its fragment sub-clone 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 sub-clone of albumin 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 albumin fusion construct of HSA-therapeutic protein fusion product
Use pC4:HSA (ATCC preserving number PTA-3277), can produce the albumin fusion construct that therapeutic protein wherein partly is positioned at ripe albumin 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 AscI, 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 albumin fusion construct of gene-HSA fusion product
Use pC4:HSA (ATCC preserving number PTA-3277), can produce the albumin fusion construct that therapeutic protein wherein partly is cloned into ripe albumin 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 ClaI 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 XhoI and ClaI site of pC4:HSA.When using the XhoI 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?ID
NO：51)
In 5 ' primer (SEQ ID NO:50), the underscore sequence is the XhoI site; And the last seven amino acid of the dna encoding natural human serum albumin targeting sequencing behind XhoI site and the XhoI 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 the ClaI site; And ClaI 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 XhoI and ClaI restriction endonuclease, and it is cloned among the XhoI and ClaI site of pC4:HSA carrier.
Candidate's targeting sequencing can replace to chimeric albumin targeting sequencing with the native albumin targeting sequencing by standard method known in the art so if desired, i.e. HSA-kex2 signal peptide, or other optional targeting sequencing.(for example, those skilled in the art can be conventional pcr amplification replace with targeting sequencing and with PCR product sub-clone in the albumin fusion construct to replace the albumin targeting sequencing, 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 albumin 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 cell line.Then by selecting transfectional cell by the existence of the selective agent of the selected marker in expression vector decision.
PC4 expression vector (ATCC Accession No.209646) is the derivant 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 " enhancer (people such as Boshart, 1985, Cell41:521-530).This carrier also contains 3 ' intron, polyadenylic acidization and the termination signal of rat proinsulin protogene, and the mice DHFR gene under the control of SV40 early promoter.Chinese hamster ovary " CHO " cell or other cell line of lacking active DHFR gene are used for transfection.By methods known in the art the albumin fusion construct transfection among the pC4 will be allowed and in Chinese hamster ovary celI, to express albumin fusion proteins that excise targeting sequencing subsequently, justacrine is in supernatant in the Chinese hamster ovary celI.Be further purified albumin fusion proteins by supernatant then.
The pEE12.1 expression vector is by Lonza Biologics, and (Portsmouth NH) provides Inc., and it is the derivant of pEE6 (Stephens and Cockett, 1989, Nucl.Acids Res.17:7110).This carrier comprises promoter, enhancer and the complete 5 ' untranslated region (international publication number WO89/01036) of the main immediate early gene of people's 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 culture medium of sulfur-bearing acute pyogenic infection of nails methyllanthionine (methionine sulphoximine) optionally and select transfectional cell.The albumin 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 targeting sequencing subsequently, justacrine is in supernatant.Can use technology described herein or that other approach of this area is known to be further purified albumin fusion proteins then by supernatant.
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 cell line through the transfection of albumin fusion construct, the cultivation when for example selecting to have dihydrofolate reductase " DHFR " gene by methods known in the art (for example fat transfection amine reagent transfection) as the carrier of selected marker or by the shortage glutamine with the 100nM methotrexate.Expression 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 by immunoblotting.Concrete productivity ratio is measured by ELISA, wherein capture antibody can be the monoclonal antibody at the therapeutic protein part of albumin fusions, and detect antibody can be monoclonal anti HSA biotinylated antibody (or vice versa), subsequently according to the scheme of manufacturer 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 tanscription termination and the needed signal of transcript polyadenylic acidization of mediation mRNA transcription initiation.Other element comprises that enhancer, 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 promoter).
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, Cos1, Cos7 and CV1, Carnis Coturnicis japonicae 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 chromosome, express albumin fusion proteins.Allow evaluation and separate transfectional cell with the cotransfection of selected marker such as DHFR, gpt, neomycin or hygromycin.
Transfection polynucleotide that can also the amplification coding fusion rotein are to express a large amount of coded fusion rotein.DHFR (dihydrofolate reductase) labelling can be used for setting up carry hundreds of or even the cell line of the genes of interest 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 selected 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 labellings, in selective medium, cultivate mammalian cell and select to have the cell of high resistance.These cell lines contain the amplification gene that is incorporated in the chromosome.Chinese hamster ovary (CHO) and NSO cell are usually used in proteinic production.
The derivant 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 genes of interest.Carrier also contains 3 ' intron, polyadenylic acidization and the termination signal of rat proinsulin protogene, and the mice DHFR gene under the control of SV40 early promoter.
Specifically, for example use suitable restriction endonuclease digested plasmid pC6, use calf intestinal phosphatase enzyme dephosphorylation by program known in the art then.Then from 1% agarose gel 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% agarose gel for " Geneclean ", BIO 101 Inc..Digest this fragment with suitable restriction endonuclease then, and purification on 1% agarose gel once more.
Then with the amplified fragments of identical restriction endonuclease digestion code book invention albumin fusion proteins, and on 1% agarose gel purification.Then connect isolating fragment and dephosphorylized carrier with the T4 dna ligase.Transformed into escherichia coli HB101 or XL-1 Blue cell subsequently, and identify by for example restriction enzyme analysis and to contain the segmental antibacterial 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 trypsinization 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 trypsinization, be inoculated into then in the 6 hole petri disses or 10ml flask that use variable concentrations 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 higher concentration 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 general purification of the albumin fusion proteins of expressing from the albumin fusion construct in the mammal cell line
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 fused 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 culture medium.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, IGFBP (insulin-like growth factor binding protein) 4, the variant HSA targeting sequencing that includes but not limited to chimeric HSA/MAF targeting sequencing 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 description " 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 purification albumin fusion proteins according to employed expression system.
From CHO and 293T cell line purification
Can comprise by the 293T cell conditioned medium liquid purification albumin fusion proteins of Chinese hamster ovary celI supernatant or transient transfection and at first to use sodium phosphate buffer to catch with anion HQ resin, and use the phosphate gradient elution, on Blue Sepharose FF post, carry out affinity chromatograph subsequently, use the salt gradient eluting.BlueSepharose FF removes main BSA/ myosin pollutant.Being further purified that employing phosphate gradient is carried out on Poros PI50 resin can remove and reduce endotoxin contaminants and concentrate albumin fusion proteins.
From NSO cell line purification
Can comprise the Q-Sepharose anion-exchange chromatography by NSO cell conditioned medium liquid purification albumin fusion proteins, follow SP-sepharose purification by stepwise elution, be the Phenyl-650M purification of stepwise elution and the last rate of oozing then.
Then can be by the protein of buffer conversion preparation purification.
Embodiment 9: the bacterial expression of albumin fusion proteins
Use the PCR oligonucleotide primers amplification corresponding to comprise the polynucleotide of antibacterial signal sequence, code book invention albumin fusion proteins, insert fragment to synthesize with 5 ' and 3 ' end of DNA sequence.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 antibiotic resistance (Amp
r), antibacterial origin of replication (ori), the adjustable promoter/operon of IPTG (P/O), ribosome binding site (RBS), hexahistidine tag (6-His) and restriction endonuclease 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 antibacterial RBS.To connect then mixed liquor be used for transformed into escherichia coli bacterial strain M15/rep4 (Qiagen, Inc.), it contains the plasmid pREP4 of multicopy, 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 ampicillin/kalamycin resistance bacterium colony.Isolated plasmid dna is also confirmed by restriction analysis.
To contain being cloned in the LB culture medium of replenishing 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 culture is arrived optical density 600 (O.D.
600) between 0.4 and 0.6.Add the final concentration of IPTG (isopropyl-B-D-thiogalactoside) subsequently to 1mM.IPTG suppresses the removing that son is induced P/O by deactivation lacI, causes gene 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 carbamide 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 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 purification (details are referring to The QIAexpressionist, 1995, QIAGEN, Inc., the same).
Briefly, supernatant 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 eluting polypeptide at last.
Dialyse and make the protein renaturation of purification by it is added 200mM NaCl to phosphate buffered saline (PBS) (PBS) or 50mM sodium acetate pH6 buffer 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 carbamide gradient among 500mM NaCl, 20% glycerol, 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 buffer is added 200mMNaCl.The protein of purification 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 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 selected marker, 2) escherichia coli origin of replication, 3) T5 bacteriophage promoter sequences, 4) two lac operator sequences, 5) Shine-Delgamo sequence and 6) lactose operon repressor thing gene (lacIq).Origin of replication (oriC) derived from pUC19 (LTI, Gaithersburg, MD).Promoter 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 insert 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 insert gel-purified, and carry out restrictive diges-tion with compatible enzyme.The secundum legem scheme connects insert 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 albumin fusion construct of the present invention are by the ATCC preservation.The albumin fusion construct can comprise any one following expression vector: 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 ampicillin, and in 37 ℃ of overnight incubation, these carriers and/or the albumin 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 albumin fusion construct, every kind of albumin fusion proteins that coding is different.Therefore, the preservation thing of shared same ATCC preserving number contains the albumin fusion construct of determining at least a table 3 corresponding line.
There are two kinds of methods to can be used for this albumin fusion construct of preservation sample separation of the plasmid DNA from table 3, quoted for specific albumin fusion construct.
Method 1: screening
At first, can directly separate the albumin 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 conventional method
32P-γ-ATP carries out labelling and carries out purification (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 albumin 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 transformants of each plate (bacterium colony) is applied on 1.5% agar plate (containing suitable selective agent, for example ampicillin).According to the conventional method of bacterial clump 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 albumin fusion construct of preservation with SEQ ID NO:X, for example uses in 5 of the albumin 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 polymerase of 0.25 unit.Carry out 35 PCR circulation (94 ℃ of degeneration 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 purification with expection molecular weight.Confirm that by sub-clone 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-coded portion.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 genes of interest 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 said 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 phosphatase subsequently, they may influence the RNA ligase step of back.Then should the deactivation phosphatase, and handle RNA is positioned at messenger RNA 5 ' end with removal cap with the acid pyrophosphatase of Nicotiana tabacum L..This 5 ' end that is reflected at the RNA of excision medicated cap stays 5 ' phosphate group, and it can use T4 RNA ligase 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 genes of interest 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 albumin (or its fragment or variant)) can merge to produce " many fusion rotein " with other protein is extra.These many fusion rotein can be used for using widely.For example, the fusion of albumin fusion proteins of the present invention and His label, HA label, protein A, IgG domain and maltose-binding protein will help purification (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 targeting, 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 dissolubility 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 termination 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:
GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGT
GCCCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCC
CAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACAT
GCGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAAC
TGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCG
GGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCG
TCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTC
TCCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCC
AAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCG
GGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAG
GCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGC
CGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGC
TCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAG
CAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAAC
CACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGA
CGGCCGCGACTCTAGAGGAT (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 the bonded antibody of part (as the therapeutic protein part or the albumin part of fusion rotein) 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 purification 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 mice).Extract the splenocyte of these mices 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 culture medium, 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 anti-idiotype 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 the method, protein-specific antibody is used for immune animal, preferred mice.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 anti-idiotype 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 deutero-gene constructs of hybridoma 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 escherichia coli that contain phasmid are used to inoculate the 2xTY (2xTY-AMP-GLU) that 50ml contains 1% glucose and 100 μ g/ml ampicillin, and shaken cultivation 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 gene 3, referring to international publication number WO 92/01047) of gene 3, culture is cultivated 45 minutes in 37 ℃ under non-oscillating condition, then in 37 ℃ of shaken cultivation 45 minutes.With culture centrifugal 10 minutes with 4000r.p.m., precipitate is resuspended among 2 liters of 2xTY that contain 100 μ g/ml ampicillin and 50 μ g/ml kanamycin, and overnight incubation.Described in international publication number WO 92/01047, prepare phage.
The M13 that has deleted gene 3 is prepared as follows: the M13 helper phage of having deleted gene 3 is encoding gene 3 protein not, and the phage (phasmid) of therefore showing antibody fragment has higher and the bonded affinity of antigen.The communicable M13 granule of having deleted gene 3 prepares by cultivate helper phage in the cell that contains the pUC19 derivant, and described pUC19 derivant provides the wild type gene group 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 shaken cultivation 1 hour.With cell centrifugation precipitation (IEC-Centra 8,400r.p.m.10 minute), be resuspended in the 2xTY culture medium (2xTY-AMP-KAN) that 300ml contains 100 μ g/ml ampicillin and 25 μ g/ml kanamycin, and spend the night in 37 ℃ of shaken cultivation.By twice PEG precipitation people such as (, 1990) Sambrook purification and concentrated phage particle from culture medium, 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 eluting and antibacterial in 37 ℃ of incubations 30 minutes, phage is used to infect the e. coli tg1 of 10ml mid-log phase (mid-log).Then escherichia coli are coated on the TYE flat board that contains 1% glucose and 100 μ g/ml ampicillin.Reclaim the antibacterial storehouse that so produces with the helper phage of having deleted 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 eluting 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 microtitration plate enforcement ELISA with the part bag quilt of 10pg/ml albumin fusion proteins of the present invention that is dissolved in 50mM bicarbonate 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 the bonded ability of competitive inhibition antibody/antigen and competitive antagonism or antagonistic activity.
Embodiment 13:[
3H]-2-deoxyglucose picked-up test
Fat, skeletal muscle and liver are the insulin sensitivity tissues.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.
[
3H]-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 Os Mus bone myocyte can be divided into respectively 3T3-L1 adipose cell and myotube with as [
3H]-the suitable external model of 2-deoxyglucose picked-up algoscopy (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 Haspel, J Membr Biol 169 (1): 45-53,1999; People such as Tsakiridis, Endocrinology 136 (10): 4315-22,1995).Briefly, with 2 * 10
5The adipose cell 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 culture medium (post-differentiationmedium), and in 37 ℃ at 5%CO
2Middle overnight incubation.Cell is at first washed once with serum-free LG DMEM culture medium, 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 buffer in 100 μ l/ holes, the cumulative concentration of therapeutic 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 the 1nM 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 labelling [
3H]-(Amersham, #TRK672) (SIGMA, 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 insulin.(SIGMA is C6762) to measure non-special picked-up to add cytochalasin (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 labelling
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 cell lysis 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-scintillation counter.Picked-up in the repeat condition, difference are to lack or exist insulin, measure by following equation: [(count per minute of insulin " the cpm "-special cpm of non-)/(the non-specific cpm of the cpm-of no insulin)].On average reply respectively in the limited field of about 5 times and 3 times of adipose cell and myotube contrast.
The differentiation of cell
At T-75cm
2Cell is converged fully.Remove culture medium, and replaced 48 hours with culture medium (pre-differentiation medium) before the 25ml differentiation.Cell is cultivated in 5%CO2,85% humidity in 37 ℃.After 48 hours, remove and dedifferente preceding culture medium, 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 culture medium, replace with 30ml differentiation back culture medium.Differentiation back culture medium kept 14-20 days, perhaps up to realizing differentiation completely.Changed culture medium in every 2-3 days.People's adipose cell can be available from Zen-Bio, INC (#SA-1096).
Embodiment 14:[
3H]-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 islets of langerhans 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 cell line numbering CRL-2057).RIN-M cell line is derived from the inductive transplantation Islet cells tumor of radiation.This is to be to set up from the bare mouse different species graft of tumor.This cell produces justacrine islets of langerhans polypeptide hormone, and produces L-DOPA decarboxylase (labelling 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 cell line numbering CRL-1674).Also can be referring to list of references: 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 difderent 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 cell line contain 10% hyclone (HyClone, RPMI1640 culture medium #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 culture medium in every 3-4 days.
ARIP (ATCC#CRL-1674) cell line is containing the 2mM L-glutaminate and is transferring to HamShi F12K culture medium (ATCC, #30-2004) the middle cultivation of containing 1.5g/L sodium bicarbonate and 10% hyclone.ARIP cell line goes down to posterity with 1: 3 to 1: 6 ratio jede Woche and cultivates twice.Changed culture medium 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 therapeutic 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 cessation 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 aqueous solution.This pipe is counted in β-scintillation counter.As negative control, only use buffer.As positive control, use hyclone.
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 therapeutic effect 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 concentration of glucose 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 algoscopy 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 algoscopy 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 algoscopys 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 albumin 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 algoscopy at standard B-lymphocyte altogether in the human tonsil B cell activity to purification of observation measurements from 0.1 to 10000ng/ml dosage range to be assessed, wherein the tonsil B cell of culture purified when having the staphylococcus 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 synergism 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 algoscopy to be easy to identify.This algoscopy comprises that exhausting the CD3-positive cell by magnetic bead (MACS) comes separation of human tonsil 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 culture medium (RPMI 1640, contain 10%FBS, 5 X10
-5M 2ME, 100U/ml penicillin, 10 μ g/ml streptomycins and the dilution SAC of 10-5) 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 culture medium.
Algoscopy in the body-BALB/c mouse injection (i.p.) twice independent buffer 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 albumin or albuminised fragment or variant).Mice was accepted this processing in continuous 4 days, at this moment put to death mice, 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 nucleated cell 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 mice 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 mice.
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 algoscopy 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 albumin or albuminised fragment or variant) of variable concentrations (cumulative volume 200 μ l).With independent related protein buffer and culture medium in contrast.After 48 hours, plate takes out 100 μ l supernatant 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 culture medium 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 the influence of II class MHC, costimulatory molecules and the adhesion molecule expression and the cell differentiation of people's dendritic cell of mononuclear cell and monocyte derived
Dendritic cell produce by expand the propagation precursor of finding in peripheral blood: the monocyte fraction of adhesiveness 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 activity factor 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 Thl helper T 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 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, costimulatory molecules and adhesion molecule expression.Can be on mononuclear cell three major families of identification of cell surface antigen: adhesion molecule, participate in the molecule and the Fc receptor 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 mononuclear cell antigen presentation ability and inducing T cell activation capability.The increase of Fc receptor expression may be relevant with mononuclear cell cellular cytoxicity activity, release of cytokines and the phagocytosis of improvement.
Use the following check surface antigen of facs analysis.Mononuclear cell 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 Hydrazoic acid,sodium salt, 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.Algoscopy at the molecule that activates (or deactivation) mononuclear cell 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 mononuclear cell mortifier or activator.Albumin fusion proteins of the present invention can use following three kinds of algoscopys to screen.For in these algoscopys each, peripheral blood lymphocytes (PBMC) centrifugal by through Histopaque gradient (Sigma) from the packed leukocyte of single donor (leukopacks) (American Red Cross, Baltimore, MD) purification.Mononuclear cell separates from PBMC by the centrifugal elutriation of adverse current.
The monocyte survival algoscopy.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 activity factor 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 mononuclear cell 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 variable concentrations 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 immune 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 culture medium 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 is burst.With the mononuclear cell of purification with 2-1 * 10
5Cells/well places 96 hole plates.In the culture medium (RPMI 1640+10%FCS, glutamine and antibiotic) 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 culture medium.Add every hole 0.2ml phenol red solution (140mM NaCl, 10mM kaliumphosphate buffer pH7.0,5.5mM dextrose, 0.56mM is phenol red and 19U/mlHRPO) to the macrophage monolayer, and stimulus object (200nM PMA).With plate in 37 ℃ of insulations 2 hours, by in each hole, adding 20 μ l 1N NaOH cessation reactions.Read absorbance in 610nm.In order to calculate the H that produces by macrophage
2O
2Amount, each experiment is carried out the H of known molar concentration
2O
2The standard 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% hyclone (FBS), 16 units/ml heparin and 50 units/ml endothelial cell growth fill-in, and (ECGS, Biotechnique is in M199 culture medium Inc.).The 2nd day, culture medium 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 variable concentrations, such as VEGF and basic FGF (bFGF).The 4th day and the 6th day, change culture medium.The 8th day, measure cell number with the Coulter enumerator.
The increase 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 granule 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 glucocorticoid 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+ mice 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+) mice than they normal heterozygosis (db+ /+m) littermate obesity.Diabetes (db+/db+) mice 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 chromosomes.Animal shows polyphagia, polydipsia and polyuria.The diabetic mice (db+/db+) of sudden change have rising blood glucose, 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 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+) mice 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, Inc. scientific research the care of animal and use the regulation of committee (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 Rifkn, 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% alcoholic solution and iodine tincture.Operative region is dried up with sterile gauze before wound.Organize card punch to create the holostrome skin trauma 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 macroscopy 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 caliper level and vertical survey wound.Covered by 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 solution.
Animal passed through peritoneal injection pentobarbital sodium (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 biopsy 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) processed group.
By analyzing wound closure with level and vertical 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 card punch.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 microtome 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, capillary tube, fibroblast, 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 observer (blinded observer).
Tissue slice also carries out immunohistochemical staining with the anti-human keratin 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 colon cancer 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).Glucocorticoid 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 trauma 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, Inc. scientific research the care of animal and use the regulation of committee (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% alcoholic solution and iodine solution.Confined surgical areas is dried up with sterile gauze before wound.Organize card punch to create the holostrome skin trauma of 8mm with Keyes subsequently.Wound keeps open at experimental session.From wound and use methyl meticortelone same day subsequently, implement one time the local application test material continuous 7 day every day.Before processing, wound cleans gently with Sterile Saline and gauze.
The macroscopy 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 caliper level and vertical survey wound.Covered by 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 solution.
Animal passed through peritoneal injection pentobarbital sodium (300mg/kg) euthanasia at the 8th day.Collect wound and near skin then to be used for the histology.Tissue sample places between the tissue cassette biopsy sponge 10% neutral buffered mole Malaysia to be used for further processing.
Three groups of assessments of every group of 10 animals (5 diabetes and 5 non-diabetic contrasts): 1) untreated fish group, 2) carrier placebo, 3) processed group.
By analyzing wound closure with level and vertical 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 card punch.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% buffering mole Malaysia, and wax embedding block is cut into slices (5mm) perpendicular to wound surface, the Olympus microtome 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 observer.
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 at the lymphatic vessel generation of the lymph circulation system of rat hindlimb and the therapeutic effect of rebuilding.Come measurement effect by the swelling volume of ill limb, quantitative, the total plasma protein and the histopathology of lymph vascular system quantity.The acute lymphoblastic edema was observed 7-10 days.May more importantly be that the chronic process tracking of edema reaches 3-4 week.
Begin before the operation, blood sample collection is used for the protein concentration analysis.Male rat to the about 350g of body weight is taken pentobarbital.Subsequently, right lower limb 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 labelling (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 lower limb back (contiguous half tendon and adductor muscle) is directly cut.Locate popliteal lymph node then.2 adjacent sides by suture ligature leg bending part lymph node and 2 distally lymphatic vessels and distally blood supply then.Remove leg bending part lymph node and any subsidiary fatty tissue by cutting off connective 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 muscular tissue, and stays the breach of about 0.5cm around the lower limb.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 edema peak restorative animal.Observe subsequently and stablize the edema peak.In order to assess the intensity of lymphedema, before operation and 7 days the girth of measuring 2 assigned addresses on each claw every day and volume.Whether measure the influence of plasma protein confrontation lymphedema, be effective test range (testing perimeter) but also investigated protein analysis.Weight at 2 position assessment contrasts and edema 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 edema limbs.
Cubing: performing the operation the same day, animal is anaesthetized with pentobarbital, and detects before operation.For measuring volume every day, animal is simply used halothane anesthesia (fast braking and fast quick-recovery), with two lower limbs defeathering and lower limb is carried out same labelling with the waterproof labelling all.Lower limb is at first immersed in the water, immerse the level that arrives each labelling in the instrument then, measure by Buxco edema 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 cutting machine (quillitine) excision limbs, then at ligation place cutting experiment and contrast lower limb and weigh.After shin-Gen joint breaks away from, carry out weighing the second time, and foot is weighed.
Histology's 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 methybutane, and the tape label sample sack that places-80 ℃ is up to section.Be right after section, muscle is observed lymph under fluorescence microscope.
Embodiment 23: albumin fusion proteins of the present invention is to the inhibition of the inductive adhesion molecule expression of TNF α
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 leukocyte 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 (TNF-a), a kind of effective proinflammatory cytokine is the stimulus object 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 the inductive content-addressable memory of TNF-a is reached.Employing is measured a kind of member with the FGF protein families as the correction ELISA algoscopy of solid phase absorbent 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 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 culture medium (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 culture medium, 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 fixative 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-biotin, anti-VCAM-1-biotin and anti-E-to select albumen-biotin.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-alkali 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 (pH10.4) with 1 paranitrophenol phosphate ester.In each instrument connection, 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-alkali phosphatase prepares in triplicate gauge orifice: 1: 5, and 000 (10
0)＞10
-0.5＞10
-1＞10
-1.5Each dilution factor 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 gauge 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 gauge 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 cell differentiation 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 promoter 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 higher concentration, 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 Cytoplasm.Jaks has represented a unique family of solubility tyrosine 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 receptor 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 receptor 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 cysteine motif (one group of 4 conservative cysteine and 1 tryptophan) and WSXWS motif (the nearly center 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 activity factor of Jaks-STAT approach.
Table 5
| JAKs Part | STAT | GAS (element) or ISRE | |||
| tyk2 | ? Jak1 | ? Jak2 | ? Jak3 | ||
| IFN family IFN-α/B IFN-g Il-10 | + + | + + ？ | - + ？ | - - - | 1，2，3? |
| Gp130 familyIL-6 (multi-purpose) I1-11 (multi-purpose) OnM (multi-purpose) LIF (multi-purpose) | + ？ ？ ？ | + + + + | + ？ + + | ？ ？ ？ ？ | 1，3 GAS(IRF1＞Lys6＞IFP) 1，3 1，3 1，3 |
| CNTF (multi-purpose) G-CSF (multi-purpose) IL-12 (multi-purpose) | -/+ ？ + | + + - | + ？ + | ？ ？ + | 1，3 1，3 1，3 | |
| G-C familyIL-2, (lymphocyte) IL-4, (lymph/bone marrow) IL-7, (lymphocyte) IL-9, (lymphocyte) IL-13, (lymphocyte) IL-15 | - - - - - ？ | + + + + + + | - - - - ？ ？ | + + + + ？ + | 1，3，5 6 5 5 6 5 | GAS GAS(IRF1＝IFP＞＞Ly6)(IgH) GAS GAS GAS GAS |
| Gp140 familyIL-3 (bone marrow) IL-5 (bone marrow) GM-CSF (bone marrow) | - - - | - - - | + + + | - - - | 5 5 5 | GAS(IRF1＞IFP＞＞Ly6) GAS GAS |
| Somatotropin family GH PRL EPO | ？ ？ ？ | - +/- - | + + + | - - - | 5 1，3，5 5 | GAS(B-CAS＞IRF1＝IFP＞＞Ly6) |
| Receptor tyrosine kinase EGF PDGF CSF-1 | ？ ？ ？ | + + + | + + + | - - - | 1，3 1，3 1，3 | GAS (IRF1) GAS (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 promoter 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 and the complementary 1 8bp sequence of SV40 early promoter sequence, and flank is the XhoI site.The sequence of 5 ' primer is:
5′-GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCC
CGAAATGATTTCCCCGAAATATCTGCCATCTCAATTAG-3′(SEQ?ID?NO：
54)
Downstream primer and the complementation of SV40 promoter, 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 XhoI/Hind III, and sub-clone is in BLSK2-(Stratagene).The order-checking of carrying out with forward and reverse primer has confirmed that insert contains following sequence:
5’-
CTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGA
AATGATTTCCCCGAAATATCTGCCATCTCAATTAGTCAGCAACCATAGTC
CCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCC
ATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAG
GCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTT
GGAGGCCTAGGCTTTTGCAAA
AAGCTT-3′(SEQ?ID?NO：56)
By this GAS promoter element that is connected with the SV40 promoter, then transformed GAS:SEAP2 reporter molecule construction.Here, reporter molecule is excretory alkali 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 chloramphenicol acetyltransferase (CAT), luciferase, alkali 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 sub-clone to from the pSEAP-promoter vector that Clontech obtains, GAS:SV40 promoter element with amplification is effectively replaced the SV40 promoter, has created the GAS-SEAP carrier.Yet this carrier does not contain neomycin resistance gene, and therefore is not that mammalian expression systems 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 the bonded reporter molecule of GAS 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 promoteres.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 promoter.Similarly, can use other cell line to test reporter molecule construction activity, such as HELA (epithelium), HUVEC (endothelium), Reh (B-cell), Saos-2 (osteoblast), HUVAC (aorta) or myocardial cell.
The active algoscopy of embodiment 25:SEAP
As the reporter molecule that is used for the algoscopy 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.
Allotter 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.Buffer is measured in emptying allotter and perfusion.Adding 50ml measures buffer and is incubated 5 minutes in room temperature.The emptying allotter 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 chemiluminescence 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 algoscopy 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 promoter of EGR1 is responsible for this inducing.Use the EGR1 promoter that is connected with reporter molecule, can assess the ability of fusion rotein active cell of the present invention.
Specifically, use following scheme in PC12 cell line, to assess neuronal activity.Known PC12 cell (rat pheochromocyte oncocyte) is bred by the activation of multiple mitogen such as TPA (myristoyl phorbol acetate), NGF (nerve growth factor) and EGF (epidermal growth factor) and/or is broken up.EGR1 gene expression is activated in the reason process herein.Therefore, by with the construction stable transfection PC12 cell that contains the EGR promoter 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 ID
NO：57)
Second primer: 5 '-GCGAAGCTTCGCGACTCCCCGGATCCGCCTC-3 ' (SEQ ID
NO：58)
Use the GAS:SEAP/Neo carrier that produces among the embodiment 24, the EGR1 amplified production can be inserted in this carrier then.Use restriction endonuclease XhoI/HindIII with the linearisation of GAS:SEAP/Neo carrier, remove the GAS/SV40 implant.With identical enzyme with EGR1 amplified production restrictive diges-tion.Connection carrier and EGR1 promoter.
In order to prepare the 96 hole plates that cell culture is used, add 2ml coating buffer (I class collagen (Upstate Biotech Inc. in each 10cm plate, 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 PCI2 cell routine is being supplemented with middle conventional cultivation of RPMI-1640 culture medium (Bio Whittaker) of containing 10% horse serum (JRH BIOSCIENCES, catalogue numbering 12449-78P), 5% heat-killed hyclone (FBS) of 100U/ml penicillin and 100 μ g/ml streptomycins 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 cultured cell in 300 μ g/ml G418.Using the culture medium 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 culture medium.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 culture medium 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 variable concentrations 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 algoscopy can use the technology routine of describing among known in the art and/or the embodiment 25 to carry out.
The algoscopy 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 algoscopy, though also can use Molt-3 cell (ATCC numbers CRL-1552) and Molt-4 cell (ATCC numbers CRL-1582).
Jurkat T-cell is a lymphoblast CD4+Th1 accessory cell.In order to produce stable cell line, use DMRIE-C (Life Technologies) (transfection method described below) the about 200 ten thousand Jurkat cells of GAS-SEAP/neo carrier transfection.Also select the 1mg/ml gentamycin is had the transfectant of resistance with the density inoculation transfectional cell of about 20,000 cells in every hole.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, 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 gentamycin and 1% penicillin-streptomycin, keep the stable report of Jurkat:GAS-SEAP system.One or more fusion rotein of the present invention with variable concentrations 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 variable concentrations 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 covering) covers opaque flat board, and is stored in-20 ℃ until carrying out the SEAP algoscopy 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 algoscopy of embodiment 28:T cytoactive
NK-KB (nuclear factor 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 thrombin, and by the expression of some viral gene product activated a kind of transcription factor.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.
In excited state not, NF-KB and I-KB (mortifier KB) are retained in the Cytoplasm.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 the activated target gene of NF-KB.
Because its important function 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 mortifier will be useful in treating, prevent and/or diagnosing the illness.For example, the mortifier 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.Forward primer contain NF-KB binding site (GGGGACTTTCCC) (SEQ ID NO:59) 4 tandem copies, with the complementary 18bp sequence of 5 ' end of SV40 early promoter sequence, and flank is the XhoI site:
5′-GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGG
GACTTTCCATCCTGCCATCTCAATTAG-3′(SEQ?ID?NO：60)
3 ' end of downstream sequence and SV40 promoter 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 sub-clone is in BLSK2-(Stratagene).The order-checking of carrying out with T7 and T3 primer has confirmed that insert contains following sequence:
5’-CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTT
TCCATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTC
CGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCA
TGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCC
TCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGC
TTTTGCAAAAAGCTT-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 systems institute is preferred.
In order to produce stable mammal cell line, use restriction endonuclease 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 algoscopy of bone marrow
Below scheme be used for whether breeding and/or breaking up the bone 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 algoscopy is U937, and a kind of premonocyte cell line 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 culture medium that contain 10% heat-killed hyclone (FBS) of 100U/ml penicillin and 100mg/ml streptomycin 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 (pH7.4) buffer in.In 37 ℃ of insulations 45 minutes.Clean cell with RPMI 1640 culture medium that contain 10%FBS, be resuspended in the 10ml complete medium then, and in 37 ℃ of insulations 36 hours.
Obtain the GAS-SEAP/U937 stabilized cell by cultured cell in 400 μ g/ml G418.Using the culture medium 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 400 μ g/mlG418.
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 variable concentrations.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 being carried out SEAP measures.
Embodiment 30: identify the algoscopy that micromolecule concentration and membrane permeability change
Known ligand changes micromolecule with combining of receptor, such as level in the born of the same parents of calcium, potassium, sodium and pH, and changes transmembrane potential.These changes can be measured in the algoscopy of identifying with the fusion rotein of the receptors bind of specific cells.Though what following scheme was described is the algoscopy of calcium, this scheme can be easy to revise any other the micromolecular change to detect potassium, sodium, pH, transmembrane potential or can be detected by fluorescent probe.
Below algoscopy 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.; 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 scrubber, after final cleaning, stay 100 μ l buffer.
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 scrubber, and stay 100 μ l buffer.
For non-adherent cell, cell is rotated precipitation from culture medium.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 algoscopy, 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) time of exposure is 0.4 second; (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 the inductive molecule of albumin fusion proteins of the present invention, and it causes Ca in the born of the same parents
++Concentration increases.
Embodiment 31: the algoscopy of identifying tyrosine kinase activity
Protein tyrosine kinase (PTK) is represented one group of various film and Cytoplasm kinases of striding.The receptor of multiple mitogenesis and metabolic somatomedin is arranged in receptor protein tyrosine kinases (RPTK) group, comprise PDGF, FGF, EGF, NGF, HGF and Insulin receptor INSR 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 involves 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 receptor relevant with src Family Tyrosine Kinases (as src, yes, Ick, lyn, fyn) is connected with no receptor, such as Jak family, the signal transduction that its member's mediation is triggered by cytokine receptor superfamily (as interleukin, interferon, 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 the inductive molecule of albumin fusion proteins 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 culture level I class collagen (50mg/ml), gelatin (2%) or poly-D-lysine (50mg/ml), St.Louis, MO buys) or 10%Matrigel (available from Becton Dickinson, Bedford, MA), or the Ox blood 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 manufacturer, Inc. (Sacramento, CA) quantity of described indirect determination cell.Use BectonDickinson (Bedford, Falcon plate lid #3071 capping Loprodyne Silent Screen plate MA).Falcon Microtest III cell culture 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 complete medium overnight incubation.Made cell mourn in silence (quiesce) in 24 hours by insulation in the serum-free basal medium.After handling 5-20 minute with the albumin fusion proteins of the present invention of EGF (60ng/ml) or variable concentrations, remove culture medium, and in each hole, add 100ml extraction buffer (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 depurator 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 equipment 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 residue on its phosphorylation specific substrates (biotinylated peptide).The biotinylated peptide that can be used for this purpose comprises PSK1 (corresponding to the aminoacid 6-20 of cell division kinases cdc2-p34) and PSK2 (corresponding to the amino acid/11-17 of gastrin).Two kinds of peptides all are the substrates of multiple tyrosine 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 buffer (40mM imidazole hydrochloride pH7.3,40mM β-phosphoglycerol, 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 reactant mixture in 30 ℃ of pre-incubations 2 minutes.By adding 10 μ l control enzyme or beginning reaction through filtering supernatant.
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 reactant mixture samples being transferred in microtitration 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 absorptance of plate instrument measuring samples at the 405nm place.Use ELISA to read the level that the plate instrument quantizes bonded peroxidase activity, and the level of reflection tyrosine kinase activity.
Embodiment 32: the algoscopy of identifying phosphorylation activity
As the potential selection of the protein hydroxyphenylaminopropionic acid kinase activity algoscopy of describing among the embodiment 31 and/or replenish, also can use the algoscopy of the activation (phosphorylation) that detects main intracellular signal transduction intermedium.For example, as described below, a kind of concrete algoscopy 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 algoscopy with these molecules such as the phosphorylation of Raf, JNK, the kinase whose kinases of p38 MAP, Map (MEK), MEK kinases, Src, muscle specific kinases (MuSK), IRAK, Tec and Janus and any other phosphoserine, phosphotyrosine 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 basal medium (DMEM), use the fusion rotein of the present invention of EGF (6ng/ hole) or variable concentrations 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 standardization program.Measure 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 fluorescence signal that is higher than background increases the phosphorylation that indication is produced by fusion rotein of the present invention or the inductive molecule of albumin fusion proteins 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 labelling
32P-ATP phosphorylating protein substrate is also measured the radioactivity of mixing with gamma activity isotope enumerator 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 (activation) that detects albumin fusion proteins of the present invention when having the peptide part
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 method for optimizing of measuring phosphorylation activity is to use US 5,817, the tyrosine phosphorylation algoscopy of describing in 471 (being incorporated herein by reference).
Embodiment 35: the algoscopy that stimulates bone marrow CD34+ cell proliferation
This algoscopy is based on the ability that people CD34+ breeds when having hemopoietic growth 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 hemopoietic of multiple CFU-GM, algoscopy contains specified fusion rotein of the present invention under the condition that has or do not exist hemopoietic growth 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 bone 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 cooperative 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 algoscopy of the inhibition effect of CFU-GM is preferably tested in following cell, this cell at first carries out stimulated in vitro with SCF+IL+3, suppresses this and induces the chemical 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 culture medium (QBSF 60 serum-free mediums (500mI) that contain the 1%L-glutamine, QualityBiological, Inc., Gaithersburg, MD, catalogue numbering 160-204-101).After the gentle centrifugation step of 200xg, make cell recuperation 1 hour several times.Cell number 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 water.The cytokine of the enough albumin fusion proteins tests of the present invention of energy has independent 50ng/ml rhSCF (R﹠amp in this algoscopy; DSystems, Minneapolis, MN, catalogue numbering 255-SC) and and rhSCF and 30ng/mlrhIL-3 (R﹠amp; D Systems, Minneapolis, MN, catalogue numbering 203-ML) associating.After 1 hour, the albumin fusion proteins of the present invention of the ready cytokine of 10 μ l, variable concentrations and 20 μ l diluting cells are joined in the culture medium 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.
Algoscopy 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 bar code 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 bone 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 bone marrow CD34+ cell proliferation and/or the polynucleotide corresponding with fusion rotein are effective for diagnosing and treating the disease that influences immune system and hemoposieis.Representational purposes is described in above " immunocompetence " and " infectious disease " part, and other place of this paper.
Embodiment 36: the extracellular matrix enhanced cell is replied the algoscopy of (EMECR)
The purpose that the extracellular matrix enhanced cell is replied the algoscopy of (EMECR) is assessment fusion rotein of the present invention acts on hematopoietic 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, fibroblast and endothelium and epithelial stem cell can't duplicate when the signal that lacks from ECM.Hematopoietic stem cell can carry out self renewal in bone marrow, but can't carry out in external suspension culture.Stem cell depends on the interaction of they and stromal cell and ECM protein fibronectin (fn) in external ability of carrying out self renewal.The adhesion of cell and fn is by α
5.β
1And α
4.β
1The integrin receptor mediation, they are expressed by the hematopoietic stem cell of people and mice.Do not identify the factor of integrating and being responsible for stimulating the stem cell self renewal 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 renewal 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 compositions 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%O2 and 88%N
2) tissue culture's 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 antigen by the scale of amplification culture system.
If finding specific fusion proteins of the present invention is the stimulus object 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 immune system and hemoposieis and treatment so.Representative purposes is described in above " immunocompetence " and " infectious disease " 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 dose, 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 hemopoietic associated disorders, such as anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia, because stromal cell is important in the generation of hematopoietic lineage cell.Purposes comprises isolated culture, bone marrow transplantation, bone marrow reconstruct, excrescent radiotherapy or the chemotherapy of medullary cell.
Embodiment 37: human dermis fibroblast and aortic smooth muscle cell propagation
Albumin fusion proteins of the present invention is added in normal person'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 person's dermal fibroblast (NHDF) or aortic smooth muscle cell (AoSMC) propagation.The misgrowth of fibroblast or smooth muscle cell is the part of several pathological processes, comprises fibrosis and restenosis.Measuring the IL6 that checks NHDF and SMC for second generates.IL6 generates and shows function activation.Active cell will increase the generation of the various kinds of cell factor and other factor, can cause short inflammation or immunomodulating 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 culture medium.The NHDF culture medium contains: Clonetics FB basal medium, 1mg/ml hFGF, 5mg/ml insulin, 50mg/ml gentamycin, 2%FBS, and the AoSMC culture medium contains Clonetics SM basal medium, 0.5g/ml hEGF, 5mg/ml insulin, 1 μ g/ml hFGF, 50mg/ml gentamycin, 50 μ g/ml amphotericin Bs, 5%FBS.In 37 ℃ be incubated at least 4-5 hour after, the sucking-off culture medium replaces with growth retardation culture medium (Growtharrest media).The growth retardation culture medium of NHDF contains fibroblast basal medium, 50mg/ml gentamycin, 2%FBS, and the growth retardation culture medium of AoSMC contains SM basal medium, 50mg/ml gentamycin, 50 μ g/ml amphotericin Bs, 0.4%FBS.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 culture medium contrast and known protein confrontation photograph.For stimulating and suppressing two kinds of experiments, protein all dilutes in the growth retardation culture medium.For suppressing experiment, add TNFa to final concentration 2ng/ml (NHDF) or 5ng/ml (AoSMC).The culture medium 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.
At the 5th day, the following IL6 ELISA 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 napkin, blot.The mensuration buffer that contains PBS with the 4%BSA preparation.With the sealing of the Pierce Super Block among the 200 μ l/ hole PBS buffer plate was sealed 1-2 hour, use cleaning buffer solution (PBS, 0.05%Tween-20) to clean plate then.Plate is blotted on napkin.Add anti-people IL-6 monoclonal, the biotin labeled antibody of 50 μ l/ holes then in the 0.5mg/ml dilution.The IL-6 liquid storage is diluted in culture medium (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 napkin, blot.The strepto-affinity element of 1: 1000 dilution EU labelling in measuring buffer, 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 napkin, blot.
The enhancing liquid that adds 100 μ l/ holes.Shook 5 minutes.In Wallac DELFIA exometer 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 disease.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 anti-angiogenic becomes) in this algoscopy.These diseases, disorder and/or situation are known in the art and/or described herein, such as for example malignant tumor, solid tumor, benign tumor, for example hemangioma, acoustic neuroma, neurofibroma, trachoma and botryomycosis hominis; Atheromatous plaque; The blood vessel generation disease of eye, for example uveitis and the pterygium (abnormal vascular growth) of diabetic retinopathy, retinopathy of prematurity, degeneration of macula, corneal graft rejection, neovascular glaucoma, Terry's sign, rubescent, retinoblastoma, eye; Rheumatoid arthritis; Psoriasis; 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 ischemic limb vessel; Ao-Wei two syndromes; Plaque neovessels forms; Telangiectasis; Bleeder's joint; Fibrohemangioma; Fibrillar muscle abnormal development; The wound granulation forms; Crohn disease; And atherosclerosis.In addition, the albumin fusion proteins as antagonist can be used for treating anti-excess proliferative disease known in the art and/or described herein and/or antiinflammatory in this algoscopy.
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 leukocyte 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 culture medium (10% hyclone (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 culture medium, 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 fixative, 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-biotin, anti-VCAM-1-biotin and anti-E-select albumen-biotin 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-alkali 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 paranitrophenol phosphate ester pNPP of dissolving in every 5ml glycine buffer (pH10.4).PNPP substrate in the 100 μ l glycine buffer will join each inspection hole.The use diluent that is dissolved in glycine buffer by Extr affinity element-alkali phosphatase prepares in triplicate gauge orifice: 1: 5, and 000 (10
0)＞10
-05＞10
-1＞10
-1.5Each dilution factor 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 gauge 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 gauge 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 algoscopy
This algoscopy can be used for quantitative assay by protein mediation to the inductive cattle lymph of bFGF endotheliocyte (LEC), bovine aortic endothelial cells (BAEC) or people's blood capillary myometrium cell (UTMEC) inhibition of proliferation.This algoscopy 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 concentration, this algoscopy 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 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, 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 electronation of the growth medium that is caused by the cell growth.When cell was grown in culture medium, inborn metabolic activity caused the electronation 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, and (propagation that is promptly promoted will produce 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 culture medium.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 reaction
This algoscopy can be used for detection and assesses the inhibition of fusion rotein of the present invention to mixed lymphocyte reaction (MLR).The inhibition of MLR may be owing to the adjusting of sticking connection between adjusting, lymphocyte and the attached cell of costimulatory molecules on the direct influence of on cell proliferation and vigor, the interaction cell, the adjusting that the accessory cell pair cell factor generates.Because used peripheral blood mononuclear fraction comprises T, B and natural killer lymphocyte and mononuclear cell and dendritic cell in this algoscopy, 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, arthritis, diabetes, inflammatory skin, psoriasis, eczema, systemic lupus erythematosus (sle), 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 (LSM
_, density 1.0770g/ml, OrganonTeknika Corporation, West Chester is PA) by the PBMC of density gradient centrifugation purification 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 (destination protein matter) to final 1: 4 dilution factor; Add rhulL-2 (R﹠amp; D Systems, Minneapolis, MN, catalogue numbering 202-IL) to final concentration 1 μ g/ml; Add anti-CD4 mAb (R﹠amp; D Systems, clone 34930.11, 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 meansigma methods 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-CD4 mAb and strengthen lymphopoietic positive control and handle, IL-2 (recombined material or supernatant) compares.
Embodiment 41: the algoscopy of proteinase activity
Following algoscopy can be used for assessing the proteinase 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 proteinase 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 absorbance with the time drive pattern.Use trypsin as positive control.
The absorbance by measuring 280nm place or the colorimetric titration of use Folin method are based on people such as other algoscopy that is discharged solvable peptide by casein or hemoglobin such as Bergmeyer, Methods ofEnzymatic Analysis 5,1984) described in carry out.Other algoscopy involves 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 method for optimizing 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 algoscopy 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 receptor, and is suitable for high throughput format.Become height ratio to live (50-2000Ci/mmol) the part radioactive label of the albumin fusion proteins of the present invention of purification 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 buffer, 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 algoscopys, 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 medicated cap rna transcription thing according to standardization 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 Bufo siccus that grows up, what obtain the V stage removes folliculus (defolliculated) oocyte, and uses the microinjection device to inject (bolus) injection rna transcription thing (10ng/ oocyte) 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 culture medium, 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 algoscopy (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 message,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 receptors 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, oocyte 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 algoscopy 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 among the 7l9 detects, with its complete being incorporated herein by reference.Briefly, the light affinity by 8-nitrine-ATP labelling is measured and the bonded ATP of albumin fusion proteins of the present invention in competitive assays.The ATP that will contain proteic reaction mixture of 1mg/ml abc transport and variable concentrations, or unhydrolyzable ATP analog adenine-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 two intervals of one minute with shortwave 254nm UV lamp from the distance of plate 2.5cm, have therebetween one minute chilling room every.The dithiothreitol, DTT cessation reaction that adds final concentration 2mM.To be incubated liquid and carry out the SDS-PAGE electrophoresis, drying, and autoradiography.Downcut the protein band corresponding, and measure radioactivity with albumin fusion proteins of the present invention.Along with ATP or adenine-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 purification signal transduction pathway protein or receptor protein.Briefly, the fusion rotein of the present invention of process labelling can be used as reagent, is used for purification molecule interactional with it.In an embodiment of affinity purification, with albumin fusion proteins of the present invention and chromatographic 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 affinity 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 bioassary method
The multiple algoscopy 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 algoscopy is described IL-6 bioassary 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 Mus cell is cleaned 3 times with no IL-6 culture medium, 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.) specimen shows by fusion protein mediated cultivation effect with respect to the enhanced propagation of negative control in (containing albumin fusion proteins of the present invention).
Embodiment 50: the support of Embryo Gallus domesticus 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 Embryo Gallus domesticus neuronal survival algoscopy (Proc.Natl.Acad.Sci.U.S.A.96:11458-63,1998, be introduced into this paper as a reference).Briefly, from Embryo Gallus domesticus isolated movement and sympathetic neuron, be resuspended in the L15 culture medium respectively and (contain 10%FCS, glucose, sodium selenite, progesterone, conalbumin, putrescine and insulin; Life Technologies, Rockville MD.) (contains 10%FCS, glucose, penicillin and 25mM Hepes buffer (pH7.2) with the improved EaglesShi culture medium of DulbeccoShi; Life Technologies, Rockville, MD.), and when the fusion rotein of the present invention of the purification that has variable concentrations 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 enhanced neuronal cell survival with the contrast that lacks cytokine and show that albumin fusion proteins strengthens the ability of neuronal cell survival.
Embodiment 51: the algoscopy of phosphatase activity
Following algoscopy can be used for assessing serine/threonine phosphatase (PTPase) activity of albumin fusion proteins of the present invention.
In order to measure serine/threonine phosphatase (PTPase) activity, the algoscopy that can utilize those skilled in the art extensively to know.For example, serine/threonine phosphatase (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-labelling MyBP release subsequently.
Embodiment 52: serine/threonine phosphatase 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 purification or receptor protein, or other signal transduction pathway protein.Briefly, will can be used as reagent, be used for purification molecule interactional with it through the fusion rotein of the present invention of labelling.In an embodiment of affinity purification, with albumin fusion proteins of the present invention and chromatographic column covalent coupling.Make from inferring target cell and flow through pillar, and the molecule with suitable affinity combines with fusion rotein such as neural or hepatocellular cell-free extract.Reclaim fusion rotein-complex 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 algoscopy of heparanase (heparanase)
The multiple algoscopy 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 purification in 37 ℃ of pH 6.2-6.6 with
35The ESC of S labelling or the solvable ECM that is derived from peak I Dan Baijutang are incubated 18 hours together.The heat insulating culture base is centrifugal, and at Sepharose CL-6B post (0.9 * 30cm) the last analytically clear liquid of gel filtration that passes through.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 eluting.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: biomolecule 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) biotin-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 buffer R (0.15M NaCl, 1mM MgCl at last
2, 10mM sodium phosphate pH7) dialysis 12 hours.Only before adding cuvette, sample is diluted with buffer ROG50 (being supplemented with the buffer R of 50mM octyl glucoside) at 1: 5.
Embodiment 55: the algoscopy of metal proteinase activity
Metalloproteases 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 Proteolytic enzyme of α-2-macroglobulin
In order to confirm proteinase activity, with the fusion rotein of the present invention and substrate α-2-macroglobulin (0.2 unit/ml of purification; Boehringer Mannheim Germany) measures buffer (50mMHEPES pH7.5,0.2M 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 trypsin as positive control.Negative control only contains α-2-macroglobulin in measuring buffer.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 low-molecular-weight band has proved Proteolytic enzyme by compare more with negative control.
The mortifier of metalloproteases 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 micromolecule MMP mortifier) also can be used for characterizing the proteolytic activity of albumin fusion proteins of the present invention.Operable three kinds of synthetic MMP mortifiers are: MMP mortifier 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) mortifier I, [IC
50=5 μ M are to MMP-3] and MMP-3 mortifier II, [Ki=130nM is to MMP-3]; Mortifier can obtain from Calbiochem, and the catalogue numbering is respectively 444250,444218 and 444225.Briefly, with the micromolecule MMP mortifier of variable concentrations and the fusion rotein of the present invention of purification (50 μ g/ml) at 22.9 μ l 1x HEPES buffer (50mM HEPES pH7.5,0.2M NaCl, 10mM CaCl
2, 25 μ M ZnCl
2And 0.05%Brij-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 cessation reaction, and boiled at once 5 minutes.Behind the SDS-PAGE, dye by silver and to manifest protein band.
Synthetic fluorescence peptide substrates cutting algoscopy
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) invertase (TACE).These substrates preferably prepare in 1: 1 dimethyl sulfoxide (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 fluoremetry.Exciting λ is 328nm, and emission λ is 393nm.Briefly, following mensuration is with 176 μ l1x HEPES buffer (0.2M NaCl, 10mM CaCl
2, 0.05%Brij-35 and 50mM HEPESpH7.5) 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 purification 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 mice
The feature of female NOD (no obese diabetes) mice 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 mice.Hereinafter, except as otherwise noted, the female NOD mice of term " NOD mice " expression.The NOD mice has the β cell that chronic autoimmune disease causes and destroys gradually.Have euglycemia or normal blood sugar level when therefore, NOD mice life begins.Yet during age in week, the NOD mice begins to become hyperglycemia to about 15-16, and this destruction and corresponding pancreas impotentia that shows their most of pancreatic beta cells produces enough insulins.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 mice (can be, Bar Harbor, Me. buys) from The Jackson Laboratory algoscopy.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 mice is an inbred line, and high response panimmunity regulation and control strategy.The NOD mice (6-8 age in week) that grows up has the average weight of 20-25g.
These mices can be undressed (contrasts), handle separately or in conjunction with other therapeutic compound mentioned above with therapeutic 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 mice 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 vascular plexus 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.Blood sugar level is measured with Glucometer Elite device.
Based on this phenotype analytical, animal can be assigned to different test group.Specifically, the animal with blood sugar level of rising can be assigned to glucose and tolerate impaired group.Mice can ad libitum access, and supply acidifying water (pH2.3).
Tolerance can further be subdivided into matched group, albumin fusion proteins group of the present invention and albumin fusion proteins/therapeutic compound combination group with the mice that does not tolerate glucose.But the mice in the matched group is accepted the peritoneal injection of carrier every day, 6 times weekly.But the mice in the albumin fusion group is accepted the peritoneal injection of the therapeutic 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.Mice 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 mice 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 mice of initial tolerance glucose, a high proportion of diabetes occur yet.
Can come validate result by the blood sugar level of measuring in the NOD mice of treatment front and back.The blood sugar level of tolerance and two kinds of mices that do not tolerate glucose is all measured as mentioned above in all groups of describing.
In optional embodiment, therapeutic 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 right quantity 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 mice.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 mice check serum glucose (ExacTech.RTM., MediSense, Inc., Waltham, Mass.).When being higher than 2.5g/L, the fasting glucohemia is diagnosed as diabetes.
The histological examination of embodiment 57:NOD mice
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:
Mice 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 insulin (1: 1000 diluent of Cavia porcellus glucagon antiserum, ICN Thames U.K.) and glucagon (1: 2000 diluent of the anti-glucagon antiserum of rabbit) carry out immune labeled, and with the anti-Cavia porcellus (Dako that has puted together peroxidase, High Wycombe, U.K.) or puted together the anti-rabbit anteserum of peroxidase (1: 50 diluent Dako) detects.
Compositions 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 the clinical proof of diabetes 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 W1).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 (Adeps Sus domestica) 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.Mice is raised in the control room 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 mice 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 food intake weekly.
In order directly to measure food rich in fat promotes the glucose control ability to insulin influence, can begin experiment to three groups of mices when finish mentioned above 7 all periods, promptly fat is fed mice, is fed mice with the food nursing mice of vector injection with the fat of STZ injection.Mice can be before experiment fasting 4 hours.In first group of experiment, can be by sucking methoxiflurane (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 half-life (t1/2) that glucose disappears in the blood plasma for Scientific Consulting, Apex to use Win Nonlin.
In second group of experiment, mice can be by intraperitoneal pentobarbital sodium (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 muscular tissue, cuts on the bottom that syringe connects, hang up the PE50 plastic tube of filling with in advance, it connects the syringe that infusion 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 mice with 10 μ l/min perfusion volume continous pouring glucose (24.1mg/kg/min) and 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 statue plasma glucose (SSPG) and insulin (SSPI) concentration that the meansigma methods 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 mice of these researchs arrives in the scope of 555mg/dL 255.Mice is specified at random with carrier or with albumin of the present invention and merges therapeutic agent separately or unite any or multiple curative drug of listing for the treatment diabetes and treat.Can use three dosage altogether.Can be before taking medicine first and take medicine for the last time after gathered the tail vein blood sample in 3 hours and be used to measure plasma glucose concentration.
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 malate dehydrogenase (rMEP) from rat and contain PPAR-γ element in the insulin approach.This reporter molecule construction is stably transfected in the liver H4IIe cell line.
H4IIe/SREBP-SEAP: sterin controlling element conjugated protein (SREBP-1c) is a kind of transcription factor, it acts on multiple insulin replies gene, the for example promoter of fatty acid synthetase (FAS), and the expression of key gene in the adjusting fatty acid metabolism in fibroblast, lipocyte and hepatocyte.SREBP-1c is also referred to as adipose cell decision and differentiation factor 1 (ADD-1), thinks that insulin in the adipose cell influences the main mediator of gene expression.Its activity is subjected to the adjusting of insulin, sterin and glucose level.This reporter molecule construction is stably transfected in the liver H4IIe cell line.
H4IIe/FAS-SEAP: fatty acid synthetase reporter molecule construction contains minimum SREBP response FAS promoter.This reporter molecule construction is stably transfected in the liver H4IIe cell line.
H4IIe/PEPCK-SEAP: phosphoenolpy ruvate carboxy kinase (PEPCK) promoter 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 blood sugar level in the normal limit thereby therefore must carefully control.This reporter molecule construction is stably transfected in the liver H4IIe cell line.
Also these reporter molecule constructions can be stably transfected in 3T3-L1 fibroblast and the L6 sarcoplast.Make these stable cell lines be divided into 3T3-L1 adipose cell and L6 myotube then as described in the previous embodiment 13.The cell line of differentiation can be used for SEAP algoscopy hereinafter described then.
Growth and mensuration culture medium
Growth medium contains 10% hyclone (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 culture medium and form by LG DMEM culture medium (Life Technologies), 1%NEAA, 1x penicillin/streptomycin.The algoscopy culture medium 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 exponential phase.Measure culture medium by growth medium being replaced with 200 μ l/ holes, with the hungry cultivation of cell 48 hours.(, contain the mensuration culture medium 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 culture medium subsequently and replace with the fresh mensuration culture medium 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 therapeutic agent of the present invention (as albumin fusion proteins of the present invention and fragment and variant).Use from the supernatant of empty carrier transfectional cell series as negative control.Xiang Kongzhong interpolation 10nM and/or 100nM insulin are as positive control.Be incubated after 48 hours, the results conditioned medium, and measurement 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 buffer with 50 μ l SEAP mix, the latter is contained the activated mortifier mixture of non-Placenta Hominis SEAP isozyme, and is incubated 5 minutes again.In mixed liquor, 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 mice, rat, rabbit, hamster, Cavia porcellus, pig, miniature pig, goat, sheep, cattle 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 gene transfer of retrovirus-mediated method is to system genitale (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 gene transfer 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 the somatic nuclear consideration convey of immobilized one-tenth and move on in the non-nucleus egg mother cell (people such as Campell from embryo, the fetus cultivated, 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 chimera.Transgenic can be used as single transgenic or as multicopy concatemer 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 transgenic 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 endogenous 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 the homologous nucleotide sequence of endogenous gene is used for the function that is incorporated into the nucleotide sequence of endogenous gene and destroys it by the homologous recombination with chromosome sequence.According to people's such as for example Gu instruction, also the transgenic selectivity can be imported particular cell types, thus deactivation endogenous 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 to reduce the expression of measuring 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 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 copulation, inbreeding, outbreeding or hybridization to produce the colony of special animal.The example of such copulation 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 copulation, so that transgenic (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 albumin component situation relevant with unconventionality expression and/or disease and screening are effectively improved the chemical compound of these situations and/or disorder.
Embodiment 61: the Therapeutic Method-ex vivo (ex vivo) that uses gene therapy
A kind of method of gene therapy can be expressed the fibroblast of albumin fusion proteins of the present invention and be transplanted on one's body the patient.Usually, obtain fibroblast 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 piece of tissue still was fixed on drag, added the fresh culture HamShi F12 culture medium of 10%FBS, penicillin and streptomycin (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 fibroblast monolayer appears.With the cell monolayer trypsinization, and the amplification scale is to bigger flask.
With flank is pMV-7 (Kirschmeier, people such as P.T., DNA7:219-25,1988) 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 agarose gel, and use the bead purification.
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/termination 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 HB 101, be coated on subsequently on the agar that contains kanamycin to confirm correctly to have inserted genes of interest in the carrier.
Two preferendum pA317 or GP+am12 incasing cells be cultured in tissue culture in the EaglesShi culture medium (DMEM) of the DulbeccoShi improvement that contains 10% calf serum (CS), penicillin and streptomycin converge density.In culture medium, add the MSV carrier that contains gene then, use the carrier transduction incasing cells.Incasing cells produces the infectious viral particle (incasing cells 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 culture medium of the production cell that converges.The exhausted culture medium that contains infectious viral particle is filtered to remove the production cell that comes off by microfilter, then this culture medium is used to infect fibroblast.Converge plate from fibroblastic Asia and take out culture medium, and be replaced with from the culture medium of producing cell.Take out this culture medium, and be replaced with fresh culture.If the titre height of virus, so in fact all fibroblasts all will be infected, and not need to select.If titre is very low, must use retroviral vector so with selected marker such as neo or his.In case effectively infected fibroblast, 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 fibroblast of transforming then.
Embodiment 62: use in the Therapeutic Method-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 promoter 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, intestinal 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, the major advantage that the naked nucleotide sequence that falls is imported target cell is the synthetic of short duration character of polynucleotide 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, bone marrow, thymus, the heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, intestinal, testis, ovary, uterus, rectum, nervous system, eye, gland and connective tissue.Organize the intercellular space to comprise the collagen fiber of elastic fiber in mucopolysaccharide layer, tube wall or the locular wall between the reticular fiber of intercellular fluid, organ-tissue, fibrous tissue or include myocyte's connective tissue or the same matrix in the bone crack in.Also similar by the space that the lymph fluid of circulating plasma and lymph pipeline occupies.Owing to the reason of discussing below preferably is delivered to the muscular 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 competent especially aspect the ability of their picked-ups and expression polynucleotide in vivo.
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 tremulous pulse.
The following mensuration of medication 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 for annular or linear, or use or compound with liposome as naked DNA.Give the not commensurability template DNA of musculus quadriceps injection of mice 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.Do the otch of a 1.5cm on the pro-thigh, 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 rustless steel folder.
After suitable temperature retention 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 mices is in the different time collection.The persistency of DNA can be measured by the Southern engram analysis for preparing total cell dna and HIRT supernatant from injection mice 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 mice.
Embodiment 63: the biology effect of fusion rotein of the present invention
Spider cell and neuron algoscopy
Can to albumin fusion proteins of the present invention test the survival, the neurite 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 spider cell propagation.Select cortical cell for bioassary 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 algoscopy 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 neurite 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 algoscopy 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 receptor 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 neurite outwards grows can with for example use thymidine to mix algoscopy to compare with replying of realizing of FGF-2.
Fibroblast and endotheliocyte algoscopy
(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 basal medium.After changing culture medium, cell is incubated 3 days with the proteinic tester fusion protein of the present invention with fresh 0.1%BSA culture medium.(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
2Algoscopy is cultivated people's lung fibroblast 1 day with 5,000 cells/well in 96 hole plates.After culture medium is replaced by the 0.1%BSA basal 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 and use EIA test kit (Cayman, Ann Arbor, MI) mensuration PGE
2For the IL-6 algoscopy, people's lung fibroblast was cultivated 1 day with 5,000 cells/well in 96 hole plates.After culture medium is replaced by the 0.1%BSA basal 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 and use ELISA test kit (Endogen, Cambridge, MA) mensuration IL-6.
Adding Alamar Blue, people's lung fibroblast was cultivated in basal 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 that mixes based on [3H] thymidine
Below [3H] thymidine mix algoscopy and can be used for measuring therapeutic protein, for example growth factor protein is to such as fibroblast, epithelial cell or the isocellular cultivation effect of immaturity muscle cell.
By in the culture medium that does not contain serum, cultivating 18 hours, culture is converged in the Asia be arrested in the Gl stage.Add therapeutic protein then and reach 24 hours, and last 4 hours with [3H] thymidine labelling 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 trichloroacetic acid continuously.In 0.5M NaOH, after the dissolving, merge lysate and PBS rinsing liquid (500ml), and measure radioactive quantity.
The parkinson model
Motor function forfeiture in the parkinson disease will be attributed to the striatum dopamine shortage of degenerating and causing because of nigrostriatum dopaminergic projection neuron.The animal model for parkinsonism of extensively identifying involves systemic application 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).In CNS, MPTP is absorbed by spider cell, and is 1-methyl-4-phenylpyridinium (MPP by monoamine oxidase B catabolism
+) and discharge.Subsequently, MPP
+High-affinity reuptake transport protein by dopamine in dopaminergic neuron actively accumulates.MPP then
+In mitochondrion, concentrate and selectivity inhibition nicotinamide adenine diphosphonic acid by electrochemical gradient: ubiquinone oxide-reductase enzyme (complex 1), disturb electron transport and the final oxygen-derived free radicals that produces thus.
In tissue culture's 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 culture 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 trypsin disintegrated tissue, and with 200,000 cells/cm
2Be inoculated on the glass cover slide of polyorthinine-laminin bag quilt.In EagleShi culture medium that DulbeccoShi improves and the F12 culture medium 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.Culture medium was changed in per three days one, and added the 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 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 disease.
Embodiment 64: the pancreas beta cell is transplanted therapeutic alliance
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, pancreas transplantation 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, autoimmune patient's transplantation treatment comprises that the risk that the host is repelled transplanted tissue drops to minimum processing.Yet autoimmune disease comprises that involving 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 regulator/immunosuppressant of the present invention to treat the method and composition of autoimmunity pancreatic diseases in the individuality of the transplantation treatment of accepting autoimmune disease.
According to the present invention, use above-described compositions and preparation and prevent and treat the individual initial infringement that transplant organ, tissue or cell is produced at the autoimmune response of initial autologous tissue by the host based on the albumin 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/immunosuppressant 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 protease inhibitor, Thalidomide, hOKT3gammal (Ala-ala) (CD 3-resisting monoclonal antibody), oraferon-α, orally administering lactobacillus and LymphoStat-B
TM, can in islet cells and pancreas transplantation, use with albumin fusions therapeutic agent of the present invention.
The evaluation of embodiment 65:VH and VL domain and clone
A kind of method of identifying and cloning VH and VL domain from the cell line of expressing specific antibodies is with VH and VL special primer the cDNA from the preparation of antibody expression cell line to be carried out PCR.Briefly, from the cell line isolation of RNA, and in the RT-PCR of antibody VH that is designed for amplification EBV expression of cell lines and VL domain, be used as template.Can use TRlzol_ 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, 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 and also clean with 75% ethanol.After the cleaning, with RNA once more in 4 ℃ with 800rpm centrifugal 5 minutes.Abandon supernatant and make the precipitate air drying.Be dissolved in RNA in the DEPC water and be heated to 60 ℃ and reach 10 minutes.Can measure the quantity of RNA by photodensitometry.
Can according to method well-known in the art from 1.5-2.5 microgram RNA with reverse transcriptase and at random the hexamer primer synthesize cDNA.Subsequently with the template of cDNA as pcr amplification VH and VL 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 polymerase, 5 ' primer mixture, 3 ' primer mixture and the 7.5 microlitre cDNA that contain 1XPCR buffer, 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 circulation 1 minute and 72 ℃ are 1 minute subsequently; One was extended 72 ℃ of circulation 10 minutes subsequently.After reaction is finished, sample cell is stored in 4 ℃
Table 7: the primer sequence of be used to increase VH and VL domain
| ? The primer title | SEQ?ID?NO | ? Primer sequence [5 '-3 '] |
| VH primer Hu VH 1-5 ' Hu VH 2-5 ' Hu VH 3-5 ' Hu VH 4-5 ' Hu VH 5-5 ' Hu VH 6-5 ' |
62 63 64 65 66 67 68 69 70 71 | ?CAGGTGCAGCTGGTGCAGTCTGG ?CAGGTCAACTTAAGGGAGTCTGG ?GAGGTGCAGCTGGTGGAGTCTGG ?CAGGTGCAGCTGCAGGAGTCGGG ?GAGGTGCAGCTGTTGCAGTCTGC ?CAGGTACAGCTGCAGCAGTCAGG ?TGAGGAGACGGTGACCAGGGTGCC ?TGAAGAGACGGTGACCATTGTCCC ?TGAGGAGACGGTGACCAGGGTTCC ?TGAGGAGACGGTGACCGTGGTCCC |
| VL primer Hu Vkappal-5 ' | 72 | GACATCCAGATGACCCAGTCTCC |
| Hu?Vkappa2a-5′ Hu?Vkappa2b-5′ Hu?Vkappa3-5′ Hu?Vkappa4-5′ Hu?Vkappa5-5′ Hu?Vkappa6-5′ Hu?Vlambda1-5′ Hu?Vlambda2-5′ Hu?Vlambda3-5′ Hu?Vlambda3b-5′ Hu?Vlambda4-5′ Hu?Vlambda5-5′ Hu?Vlambda6-5′ Hu?Jkappa1-3′ Hu?Jkappa2-3′ Hu?Jkappa3-3′ Hu?Jkappa4-3′ Hu?Jkappa5-3′ Hu?Jlambda1-3′ Hu?Jlambda2-3′ Hu?Jlambda3-3′ Hu?Jlambda3b-3′ Hu?Jlambda4-3′ Hu?Jlambda5-3′ Hu?Jlambda6-3′ | ?73 ?74 ?75 ?76 ?77 ?78 ?79 ?80 ?81 ?82 ?83 ?84 ?85 ?86 ?87 ?88 ?89 ?90 ?91 ?92 ?93 ?94 ?95 ?96 ?97 | GATGTTGTGATGACTCAGTCTCC GATATTGTGATGACTCAGTCTCC GAAATTGTGTTGACGCAGTCTCC GACATCGTGATGACCCAGTCTCC GAAACGACACTCACGCAGTCTCC GAAATTGTGCTGACTCAGTCTCC CAGTCTGTGTTGACGCAGCCGCC CAGTCTGCCCTGACTCAGCCTGC TCCTATGTGCTGACTCAGCCACC TCTTCTGAGCTGACTCAGGACCC CACGTTATACTGACTCAACCGCC CAGGCTGTGCTCACTCAGCCGTC AATTTTATGCTGACTCAGCCCCA ACGTTTGATTTCCACCTTGGTCCC ACGTTTGATCTCCAGCTTGGTCCC ACGTTTGATATCCACTTTGGTCCC ACGTTTGATCTCCACCTTGGTCCC ACGTTTAATCTCCAGTCGTGTCCC CAGTCTGTGTTGACGCAGCCGCC CAGTCTGCCCTGACTCAGCCTGC TCCTATGTGCTGACTCAGCCACC TCTTCTGAGCTGACTCAGGACCC CACGTTATACTGACTCAACCGCC CAGGCTGTGCTCACTCAGCCGTC AATTTTATGCTGACTCAGCCCCA |
Then with PCR sample electrophoresis on 1.3% agarose gel.Can downcut the DNA band (the VH domain is about 506 base pairs, and the VL domain is 344 base pairs) of expection size from gel, and with method purification well-known in the art.The PCR product of purification can be connected to (from Invitrogen Inc., Carlsbad, the TA carrier of CA) in the PCR cloning vehicle.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 domain and VL domain also can be used for creating total length Ig expression vector.VH and VL domain can be cloned in the carrier of the nucleotide sequence that contains heavy chain (for example human IgG l 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 cell line (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.
The preparation of embodiment 66:HA-cytokine or HA-growth factor fusion protein (such as NGF, BDNFa, BDNFb and BDNFc)
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 sept 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 the excretory albumin fusion proteins of purifying yeast from culture medium then, and test its biologic activity.For the expression in mammal cell line, adopt similar program, be that used expression cassette adopts mammalian promoter, targeting sequencing 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 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 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 sept sequence.IFNa (or other interferon) 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 the excretory albumin fusion proteins of purifying yeast from culture medium then, and test its biologic activity.For the expression in mammal cell line, adopt similar program, be that used expression cassette adopts mammalian promoter, targeting sequencing 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 low concentration.In addition, although protein concentration is low, the fusion rotein response rate of still observing is reduced to minimum with combining of tube wall and during other protein of adding even do not contain at 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 concentration 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 albumin 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 purification.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 prevent disease among the patient who uses any disease that α-IFN regulates or morbid state.Such disease includes but not limited to hairy cell, Kaposi sarcoma, reproduction and anus wart, chronic hepatitis B, chronic non-A non-B hepatitis, hepatitis C particularly, hepatitis D, chronic granulocytic leukemia, renal cell carcinoma, bladder cancer, ovary and cervical cancer, skin carcinoma, recurrent respirator papillomatosis (recurrent respiratorpapillomatosis), Fei Huoqijinshi and skin T-cell lymphoma, melanoma, multiple myeloma, AIDS, multiple sclerosis, glioblastomas etc. (are seen Interferon Alpha, in " AHFS DrugInformation ", 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 insert that is used to express difunctional HA-α-IFN fusion rotein.For example, can be after removing dual termination codon or moving to the coded sequence downstream, the downstream of the cDNA of second destination protein being inserted " rHA-IFN " sequence with identical 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 sept 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 the excretory albumin fusion proteins of purifying yeast from culture medium then, and test its biologic activity.For the expression in mammal cell line, adopt similar program, be that used expression cassette adopts mammalian promoter, targeting sequencing 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 recepter or the conjugated protein fusion rotein of HA-
Purpose soluble recepter 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 sept 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 the excretory albumin fusion proteins of purifying yeast from culture medium then, and test its biologic activity.For the expression in mammal cell line, adopt similar program, be that used expression cassette adopts mammalian promoter, targeting sequencing 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 sept 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 the excretory albumin fusion proteins of purifying yeast from culture medium then, and test its biologic activity.For the expression in mammal cell line, adopt similar program, be that used expression cassette adopts mammalian promoter, targeting sequencing 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 sept 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 protease cutting site, make the two can after secretion, cut and oneself's association subsequently; And cystine residue, its position makes V
HAnd V
LCan between them, form disulfide bond so that they are linked together.Selectable option is with V
HPlace the segmental N-of HA or HA domain terminal and with V
LPlace the segmental C-end of HA or HA domain.
Can collect and the excretory albumin fusion proteins of purifying yeast from culture medium then, and test its biologic activity.For the expression in mammal cell line, adopt similar program, be that used expression cassette adopts mammalian promoter, targeting sequencing 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 culture medium purification in this way, and tests it with the standard immunoassay chemical method 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 sept 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 the excretory albumin fusion proteins of purifying yeast from culture medium then, and test its biologic activity.For the expression in mammal cell line, adopt similar program, be that used expression cassette adopts mammalian promoter, targeting sequencing 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: inhibitive factor and peptide are as the preparation of HA fusion rotein (answering albumen such as HA-antiviral agents, HA-antibiotics (antibiotic), HA-enzyme inhibitor and HA-resistance)
The cDNA of purpose peptide such as antibiosis 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 sept 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 the excretory albumin fusion proteins of purifying yeast from culture medium then, and test its biologic activity.For the expression in mammal cell line, adopt similar program, be that used expression cassette adopts mammalian promoter, targeting sequencing 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 targeting HA fusion rotein
The cDNA of destination 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 albumin 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 other end such as nuclear localization signal.Destination protein matter and targeting peptide are cloned into such as in the carriers such as pPPC0005 (Fig. 2), pScCHSA, pScNHSA or pC4:HSA, thereby cause fusion with albumin 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 the excretory albumin fusion proteins of purifying yeast from culture medium, and use suitable biochemistry and biology method of testing test its biologic activity and its targeting 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 sept 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 culture medium collection and the excretory albumin fusion proteins of purifying yeast then.For the expression in mammal cell line, adopt similar program, be that used expression cassette adopts mammalian promoter, targeting sequencing 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 2053, the generation of IFNb-HSA
Construction ID 2053, pEE12.1:IFNb.HSA comprises coding and has the DNA of the proteinic IFNb-albumin fusion proteins of total length IFNb in NSO expression vector pEE12.1, the natural IFNb targeting sequencing that merges comprising the amino terminal with the HSA mature form.
The clone of IFNb cDNA
Use the polynucleotide of primer I FNb-1 and IFNb-2 pcr amplification coding IFNb hereinafter described,, and be connected in the pC4:HSA of Bam HI/ClaI cutting, produce construction 2011 with Bam HI/ClaI cutting.With the Eco RI/Eco RI fragment sub-clone of construction ID#2011 in the EcoRI site of pEE12.1, produce construction ID#2053, it comprises the DNA of the albumin fusion proteins of encoding, and this fusion rotein contains targeting sequencing and IFNb mature form, and the back is ripe HSA protein.
Synthetic two oligonucleotide that are suitable for the polynucleotide of pcr amplification coding total length IFNb, IFNb-1 and IFNb-2:
IFNb-1：5′-GCGC
GGATCCGAATTCCGCCGCCATGACCAACAAGTGT
CTCCTCCAAATTGCTCTCCTGTTGTGCTTCTCCACTACAG
CTCTTTCCATGAGCTACAACTTGCTTGG-3’(SEQ?ID?NO：
107)
IFNb-2：5′-GCGCGC
ATCGATGAGCAACCTCACTCTTGTGTGCATCG
TTTCGGAGGTAACCTGT-3′(SEQ?ID?NO：108)
The IFNb-1 primer has mixed Bam HI cloning site (underscore demonstration), Eco RI cloning site and Kozak sequence (italic demonstration), and the back is 27 amino acid whose 80 nucleotide of coding total length form IFNb.In IFNb-2, ClaI site (underscore demonstration) and subsequent DNA are the reverse complementary sequence of 10 amino acid whose DNA of encoding mature HSA protein (SEQ ID NO:1), and 18 last nucleotide are the reverse complementary sequence (seeing embodiment 2) of the DNA of last 6 amino acid residues of coding IFNb.Produce the pcr amplification thing with these primers, purification with Bam HI and the digestion of ClaI restriction endonuclease, and is cloned among the Bam HI and ClaI site of pC4:HSA carrier.After confirming sequence, the Eco RI fragment sub-clone that will contain IFNb-albumin fusion proteins expression cassette is in the pEE12.1 of Eco RI digestion.
In addition, can confirm to expect exist (the seeing below) of IFNb sequence by amino acid sequencing to the analysis of expressed albumin fusion proteins N-end.
The mature form that IFNb-albumin fusion proteins of the present invention preferably comprises HSA is Asp-25 to Leu-609, and the mature form that it merges at IFNb is N-or the C-end of Met-22 to Asn-187.In one embodiment of the invention, IFNb-albumin fusion proteins of the present invention also comprises the signal sequence that is used in the host's who is used to express the nascent fused polypeptide of secretory pathway guiding.In another preferred embodiment, excised by the signal peptide of signal sequence encoding, and sophisticated IFNb-albumin fusion proteins direct secretion is in culture medium.IFNb-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, IGFBP (insulin-like growth factor binding protein) 4, HSA targeting sequencing variant include but not limited to chimeric HSA/MAF targeting sequencing or other allos signal sequence known in the art.In a preferred embodiment, IFNb-albumin fusion proteins of the present invention comprises natural IFNb.In another preferred embodiment, IFNb-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 2053 and purification
Expression in rat bone marrow tumour NSO cell line
With construction ID#2053, the pEE12.1:IFNb-HSA electroporation (is seen embodiment 6) in the NSO cell by methods known in the art.
From NSO cell conditioned medium liquid purification
Can be included in the Q-Sepharose anion-exchange chromatography that pH7.4 carries out with 0 to 1M NaCl gradient the 20mM Tris-HCl from NSO cell conditioned medium liquid purification IFNb-HSA, be Poros PI 50 anion-exchange chromatographies that carry out with 5 to 40mM sodium citrate gradient at pH6.5 subsequently, and during 6 DV diafiltrations are last buffer compositions to 10mM citrate pH6.5 and 140mM NaCl.The N-end sequencing should obtain sequence MSYNLL, and it is the amino terminal of IFNb mature form.Protein has the molecular weight of about 88.5kDa.
For more massive purification, for example, 50 liters of NSO cell conditioned medium liquid can be concentrated into about 8-10 liter.(10 * 19cm 1.5L), adopts the distribution eluting of being made up of 50mM NaOAc pH6.0 and 150mM NaCl to make concentrating sample flow through the Q-Sepharose anion-exchange column of pH7.5 then.Then can be with the sample of eluting with 0.75%Triton-X 100 in room temperature inactivation of viruses 60 minutes.Can adopt the SDR-reversed phase chromatography (10cm * 10cm that uses 50mM NaOAc and 150mM NaCl at pH6.0 then, 0.8L), perhaps can make sample flow cross the SP-Sepharose post of pH4.8, adopt the distribution eluting of 50mMNaOAc pH6.0 and 150mM NaCl.Filter to remove any viral content by DV 50 subsequently.Can be that (20cm * 12cm 3.8L), adopts by 350mM (NH4) for the Phenyl-650M chromatography of pH6.0 subsequently
2Distribution eluting that SO4 and 50mM NaOAc form or that form by 50mM NaOAcpH6.0.The diafiltration of 6-8 DV can become buffer exchange 10mM Na2HPO4+58mM sucrose+120mM NaCl pH7.2 or 10mM citrate pH6.5 and 140mMNaCl or 25mM Na
2Buffer is finally prepared in the expection of HPO4,100mM NaCl pH7.2.
The activity of IFNb can be measured with external ISRE-SEAP algoscopy
All I type ifn proteins signal by public receptor complex and similar Jak/STAT signal path, and the latter reaches peak value in by interferon sequence response element " ISRE " activation interferon " IFN " response gene.The active mensuration system that makes things convenient for algoscopy to be based on promoter-reporter gene of I type IFN, it contains a plurality of copy ISRE elements that merge with the downstream reporter gene.Can produce stable HEK293 cell line, and contain extremely responsive and surpassing the ISRE-SEAP reporter gene that demonstrates linear stable integration copy on 5 logarithm concentration I type IFN.
The screening technique of IFNb-HSA NSO stable clone
As described in the embodiment 6 with construction 2053 electroporations in the NSO cell.By test condition growth medium in the ISRE-SEAP algoscopy to NSO cell screening activity through construction ID#2053 transfection.ISRE-SEAP/293F is reported that cell handling the previous day with 3 * 10
4Cells/well is assigned in 96 hole plates of poly--D-lysine bag quilt.Handle the report cell with the condition supernatant of various dilution factors (including but not limited to 1: 500 and 1: 5000) or by the purification prepared product or the rhIFNb in contrast of the IFNb-albumin fusion proteins of construction ID 2053 codings.To report cell insulation 24 hours then, take out 40 μ l subsequently, be used for SEAP reporter gene chemiluminescence assay (Roche catalogue numbering 1779842).Use recombinant human interferon beta, " rhIFNb " (Biogen), as positive control.
The result
It is 1.8 * 10 that the purification prepared product of the IFNb-HSA that NSO expresses has than the EC50 value
-10The bigger EC50 value of rhIFNb (Biogen) of g/ml (seeing embodiment 4) is 9.3 * 10
-9G/ml.
Induce in the body of interferon to OAS
Method
The OAS enzyme, 2 '-5 '-oligoadenylate synthetase activates at transcriptional level by interferon in replying viral infection resisting.The effect of interferon 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.Can be 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 can comprise injection of vehicle contrast, first day intravenous and/or subcutaneous injection 30 μ g/kg and/or 300 μ g/kg IFN-albumin fusion proteins, and the 1st, 3 and 5 day subcutaneous injection 40 μ g/kg interferon-ALPHA (Schering-Plough) as positive control.The level of p41 and p69mRNA transcript can be used p41-OAS and the special probe of p69-OAS are measured by real-time quantitative PCR (Taqman).OAS mRNA level can be carried out quantitatively with respect to the endogenous contrast of 18S ribosomal RNA.
Induce in the interferon beta-body of albumin fusions by construction ID 2053 codings OAS
Method
Activity by the HSA-IFNb fusion rotein of construction 2053 coding can be measured by OAS algoscopy in the body, as before above described in subtitle " is induced in the body of interferon to OAS ".
The indication of embodiment 77:IFNb-albumin fusion proteins
IFN β-albumin fusion proteins (include but not limited to by construction 2053 coding those) can be used for treatment, prevention, improves and/or detects multiple sclerosis.Other indication includes but not limited to viral infection, comprises severe acute respiratory syndrome (SARS) and other coronavirus infection; Filovirus includes but not limited to Ebola virus and Marburg virus; Arenavirus includes but not limited to Pichende virus, Lassa virus, Junin virus, MAC, guanarito virus; And lymphocytic choriomeningitis virus (LCMV); Bunyavirus includes but not limited to Punta Toro virus, the Crimea peninsula-Congo hemorrhagic fever virus, phlebotomus fever virus, Rift valley fever virus, La Crosse virus and Hantaan virus; Banzi virus includes but not limited to yellow fever, BAN, west nile virus, dengue virus, Japanese encephalitis virus, tick borne encephalitis, Omsk hemorrhagic fever and Kyasanur Forest disease virus; Togavirus includes but not limited to Venezuela, east and western equine encephalitis virus, ross river virus and rubella virus; Vaccinia subgroup virus includes but not limited to vaccine, cowpox, variola and monkeypox; Herpesvirus; Influenza A/B; Respiratory syncytial virus (RAV); Parainfluenza; Measles; Rhinovirus; Adenovirus; Semliki Forest virus; Viral hemorrhagic fever; Baculovirus; Paramyxovirus includes but not limited to Nipah virus and Heng Dela 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.North Am.20 (2): 273-309,2002).
Embodiment 78: construction ID 2249, the generation of IFNa2-HSA
Construction ID 2249, pSAC35:IFNa2.HSA comprises the DNA of coding IFNa2-albumin fusion proteins in saccharomyces cerevisiae expression vector pSAC35, this fusion rotein has the chimeric targeting sequencing of HSA, the back is that the proteinic mature form of IFNa2 is Cl-E165, merges the amino terminal at the HSA mature form.
The clone of IFNa2 cDNA
Use the polynucleotide of primer I FNa2-1 and IFNa2-2PCR amplification coding IFNa2 hereinafter described.
With Sal I/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 targeting sequencing, 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：109)
IFNa2-2：5′-GCGCGC
ATCGATGAGCAACCTCACTCTTGTGTGCATCT
TCCTTACTTCTTAAACTTTCT-3′(SEQ?ID?NO：110)
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 targeting sequencing 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, purification,, and be cloned among the Xho I and Cla I site of pScCHSA carrier with Sal I and the digestion of Cla I restriction endonuclease.After confirming sequence, with the expression cassette sub-clone of this IFNa2-albumin fusion proteins of coding in the pSAC35 of Not I 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 targeting sequencings by methods known in the art.The example of various targeting sequencings includes but not limited to invertase " INV " (construction 2343 and 2410) and copulation alpha factor " MAF " (construction 2366).Can (seeing embodiment 5) as discussed previously these IFNa2-albumin fusion proteins sub-clones 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 partial fusion 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 fused 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 culture medium.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, IGFBP (insulin-like growth factor binding protein) 4, HSA targeting sequencing variant include but not limited to chimeric HSA/MAF targeting sequencing 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 purification
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 stabilization sub stage in growth after 72 hours.By cell was collected supernatant in centrifugal 10 minutes with 3000g.Use anti-HSA serum (Kent Laboratories) or check expression 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 purification
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-efficiency 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/cm eluting from the Q-Sepharose high-efficiency column.The N-end sequence 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 algoscopy
Method
Can as described in previous embodiment 76, in the ISRE-SEAP algoscopy, test activity to the IFNa2-albumin fusion proteins of encoding by construction ID#2249.Briefly, condition yeast supernatant is tested it instructs the ISRE signal transduction on the ISRE-SEAP/293F reporting cell line ability with 1: 1000 dilution factor.ISRE-SEAP/293F is reported that cell handling the previous day with 3 * 10
4Cells/well is assigned in 96 hole plates of poly--D-lysine 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 catalogue numbering 1779842).Use recombinant human interferon beta, " rhIFNb " (Biogen), as the positive.
The result
The purification prepared product of IFNa2-HSA in the ISRE-SEAP algoscopy in concentration range 10
-1To 10
1Ng/ml (see figure 5) or 10
-10To 10
-8Show linear relatively increase on the ng/ml (see figure 6).
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 in replying viral infection resisting.The effect of interferon 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 comprise excipient contrast, intravenous injection in the 1st day 30 μ g/kg HSA-IFN, the 1st day subcutaneous injection 30 μ g/kg HSA-IFN, the 1st day subcutaneous injection 300 μ g/kg HSA-IFN, and the 1st, 3 and 5 day subcutaneous injection 40 μ g/kg interferon-ALPHA (Schering-Plough) as positive control.The level of p41 and p69 mRNA 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 ribosomal RNA.Albumin 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. 7) with the monkey of handling with IFNa with the HSA-interferon.Effect continues nearly 10 days.
The indication of embodiment 79: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 viral 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); Banzi virus (Flavivirus) includes but not limited to yellow fever, BAN (Banzi virus), west nile virus (WestNile virus), dengue virus, Japanese encephalitis virus, tick borne 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 vaccine, cowpox, variola and monkeypox; Herpesvirus; 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, and then with ribavirin (ribavirin), IL-2, IL-12, pentafuside (pentafuside) at least a independent associating or associating inverase treatment, 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 80: construction # 3691, the generation of BNP-HSA
The clone who is used for the BNP cDNA of construction 3691
Use the polynucleotide of primer BNP-1 and BNP-2 pcr 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 targeting sequencing (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:321 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
GCCCA?TGGTGTGGGCCAGCCCCAAGCTGGTGCAAGG?-3′
(SEQ?ID?NO：463)
BNP-2：5′-AGTCCC
ATCGATGAGCAACCTCACTCTTGTGTGCATCA
TGCCGCCTCAGCACTTTGC-3′(SEQ?ID?NO：464)
BNP-1 mixes the polynucleotide (runic) of 7 aminoacid sequences of the polynucleotide (italic) of Bam HI cloning site (underscore), the total targeting sequencing (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 aminoacid (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.Be further purified BamHI-Cla I fragment by gel electrophoresis after, product cloning in the pC4:HSA of Bam HI/Cla I digestion, is produced construction ID#3691.The sequence of checking expression constructs.
The expression of construction ID 3691 and purification
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 purification
Collected 2 in 3 days after the transfection and go up clear liquid.(NJ USA) catches and with 2M NaCl eluting recombiant 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 eluting.Be further purified BNP-HSA at pH6.8 with sodium phosphate buffer gradient (0-20mS/cm among the 200ml) by the hydroxyapatite column chromatography.By HiPrep 26/10 desalting column (AmershamBiosciences) end product is transformed among the PBS pH7.2.
The activity of BNP-HSA can be measured with external NPR-A/cGMP algoscopy
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 domain that GTP is transformed into cGMP after activation.The active algoscopy that makes things convenient for of BNP is to measure the BNP stimulation of stablizing the 293F cell line of expressing NPR-A.Can be exposed to that the cGMP in the cell generates behind the BNP by cGMP ELISA measurement.
The screening technique of NPR-A293F 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 0.8ug/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 fluoremetry test kit.Briefly, the 293F/NPR-A cell cultivated with 50,000 cells/well in the 96 hole plates is stimulated buffer (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 buffer 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 buffer.Measure the quantity of cGMP in the lysate according to the indication of manufacturer.
The result
Measured dosage-response relation (see figure 8) 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 vasodilation 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 delivered by the tail vein injection.(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 taking medicine.The single 6nmol/kgBNP-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 9) then in several days gradually.Opposite, 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 dose 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 relevant with the kidney physiology with the inductive cardiovascular of BNP.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 mice deliver reorganization BNP or BNP-HSA through tail vein by single injecting with the dosage of 6nmol/kg.Prepare blood plasma in take medicine 5,10,20,40 and 80 minutes time points of group and BNP-HSA group extra 640,1440,2880 and 5760 minutes of reorganization BNP from tail blood.The plasma sample of collecting the mice of handling with PBS at the zero-time point contrasts as excipient.Use CatchPoint cGMP fluoremetry kit measurement cGMP level according to the indication of manufacturer.
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 10)s respectively.In addition, in the single-phase exponential decay half-life (one-phase 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 half-life of cGMP be 1538 minutes (1017 to 3153 minutes, 95%CI).
By pharmacokinetic analysis in the body of the BNP-albumin fusions of construction ID 3691 coding
Method
Male C57/BL6 mice 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 taken medicine with the volume of 10ml/kg body weight.Give the animal injection PBS that takes medicine in advance.Reorganization BNP peritoneal injection is to afterbody or be subcutaneously injected into the omoplate zone line.
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 | Intravenous | 3 | 0.083、2、6、16、24、32、 48、72、96 |
| 3 | Excipient | ?0 | Subcutaneous | 3 | (predose) in advance takes |
| 4 | Excipient | ?0 | Intravenous | 3 | Take medicine in advance |
From the postcava blood sample collection, place miniature vessel, and be stored in the ice through EDTA bag quilt.With sample in microcentrifuge 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 standard curve in the identical time with specimen.The detectable limit of reorganization BNP is 0.11ng/ml.This algoscopy detect reorganization BNP and not with mice 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 taking medicine 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 behind the subcutaneous injection, BNP-HSA has the terminal of 11.2 (intravenous deliveries) or 19.3 hours (subcutaneous delivery) to eliminate the half-life (terminal elimination half-life), and the reorganization half-life of BNP in mice is 3.1 minutes.The non-compartment analysis (noncompartmental analysis) of BNP-HSA has disclosed BNP-HSA and has had following feature:
Table 9
| Unit | Intravenous | Subcutaneous | |
| t max | ?h | ?NA | ?16 |
| C Max | μ g/ml | NA | 11.2 |
| t _, term | h | 11.2 | 19.3 |
| AUC 0-∞ | (h μ g/ml)/(mg/kg) | 658.9 | 227.9 |
| V Ss | Ml/kg | 37 | NA |
| V zOr V z/ F | Ml/kg | 53.5 | 268 |
| CL or CL/F | Ml/h/kg | 3.3 | 9.6 |
| MRT | h | 11.2 | 19.8 |
| Bioavailability | % | 34.6 | |
| C
Max, the peak plasma concentrations of medicine; t
Max, the time of maximal plasma concentration; AUC
0-∞, from the |
|||
4 points that are chosen in 5 points of intravenous curve terminal stage and subcutaneous curve terminal stage are used for the calculating of final half-life.The AUC of this terminal stage that so obtains is respectively about 10% of intravenous and the total AUC of subcutaneous curve.By comparison, when selecting last 3 points to be used for the calculating of final half-life, 2% and 4% of intravenous and the total AUC of subcutaneous curve only arranged respectively.
Embodiment 81: 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 targeting sequencing, secrecon, the back is 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 purification fragments (A and B) are mixed with equimolar amounts, and as pcr template, with primer BNP-5 and BNP-6 amplification hereinafter described.Digest BNP (2x) insert with BamHI and ClaI then, and be connected in BamHI and the predigested pC4:HSA carrier of ClaI, produce construction ID#3618.The construction ID#3618 albumin fusion proteins of encoding, it contains total targeting sequencing, 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:483 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：486)
BNP-2 5′-CCTTGCACCATCTTGGGGCTATGCCGCCTCAGCACTTT
GC-3′?(SEQ?ID?NO：487)
BNP-3 5′-GCAAAGTGCTGAGGCGGCATAGCCCCAAGATGGTGCA
AGG-3′(SEQ?ID?NO：488)
BNP-4 5′-AGTCCCATCGATGAGCAACCTCACTCTTGTGTGCATCA
TGCCGCCTCAGCACTTTGC-3′(SEQ?ID?NO：489)
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.Purification 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 amounts:
BNP-5 5′-GAGCGC
GGATCCAAGCTTCCGCCATCATGTGGTGGCGC
CTGTGGTGGCTGCTGCTGCTGCTGCTGCTGCTGTGGCCCAT
GGTGTGGGCCAGCCCCAAGCTGGTGCAAGG-3′(SEQ?ID
NO：463)
BNP-6 5′-AGTCCC
ATCGATGAGCAACCTCACTCTTGTGTGCATCA
TGCCGCCTCAGCACTTTGC-3′(SEQ?ID?NO：464)
BNP-5 mixes the polynucleotide (runic) of preceding 7 aminoacid sequences of the polynucleotide (italics) of Bam HI cloning site (underscore), the total targeting sequencing (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 aminoacid (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 targeting sequencing 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.Be further purified BamHI-Cla I fragment by gel electrophoresis after, product cloning in the pC4:HSA of Bam HI/Cla I digestion, is produced construction ID#3618.The sequence of checking expression constructs.
The expression of construction ID 3618 and purification
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 purification
As before above among the embodiment 80 subtitle " from 203F cell conditioned medium liquid purification " down as described in purification by pC4:SPCON.BNPl-32 (the 2x)/HSA of construction ID#3618 coding.
The activity of BNP (2X)-HSA can be measured with external NPR-A/cGMP algoscopy
Can be by the activity of BNP (the 2X)-HSA of construction ID#3618 coding as passing through the NPR-A/cGMP algoscopy at external test at subtitle " activity of BNP-HSA can be measured with external NPR-A/cGMP algoscopy " and " screening technique of NPR-A 293F stable clone " as described in down among the previous embodiment 80.
The result
Measured dosage-response relation (see figure 8) 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.
With the whole open of every piece of document quoting among the application (comprise patent, patent application, patent announcement, magazine article, summary, experiment guide, books or other open) and can be incorporated herein by reference by the GenBank, the GeneSeq that mention among the application or the information completely of the special identifier acquisition of CAS Registry.
In addition, with the description of following every U. S. application with sequence table is complete is incorporated herein by reference: the U. S. application of submitting on February 4th, 2005 number 60/542,274; The U. S. application of submitting on March 5th, 2004 number 60/549,901; The U. S. application of submitting on March 29th, 2004 number 60/556,906; And the U. S. application submitted in 17th of December in 2004 number 60/636,603.
Explanation about the preservation biomaterial
A. Xia Mian explanation relates to the preservation biomaterial that description the 44th page table 3 is mentioned.
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 | Preserving | Preservation date | |||
| 1 | ?PTA-3759 | October 4 calendar year 2001 | 2 | ?PTA-3764 | October 4 calendar year 2001 | |
| 3 | ?PTA-3941 | Calendar year 2001 December 19 |
4 | ?PTA-3763 | October 4 calendar year 2001 | |
| 5 | ?PTA-3940 | Calendar year 2001 December 19 days | 6 | ?PTA-3942 | Calendar year 2001 December 19 days | |
| 7 | ?PTA-3939 | Calendar year 2001 December 19 days | 8 | ?PTA-3943 | Calendar year 2001 December 19 days | |
| 9 | ?PTA-4670 | Calendar year 2001 JIUYUE 16 |
10 |
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 Norw P office) open to the public, perhaps finally make decision under the disclosed situation not having by Norw P office, sample should only be provided the expert who gives this area.According to Norw P method 22 and 33 (3) joints, should be earlier than to open the proposing to Norw P 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 Norw P 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 administration committee) is open to the public, perhaps finally do not made decision by national patent and administration 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 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 (29)
1. the albumin fusion proteins that comprises the member who is selected from down group:
(a) therapeutic protein: X and the albumin that comprises the aminoacid sequence of SEQ ID NO:1;
The fragment or the variant of the aminoacid sequence of (b) therapeutic protein: X and SEQ ID NO:1, wherein said fragment or variant have the albumin activity;
The fragment or the variant of the aminoacid sequence of (c) therapeutic protein: X and SEQ ID NO:1, wherein said fragment or variant have the albumin activity, and wherein said albumin activity is to compare the ability of the storage life of extended treatment protein: X with the storage life of the therapeutic protein that does not merge state: X;
The fragment or the variant of the aminoacid sequence of (d) therapeutic protein: X and SEQ ID NO:1, wherein said fragment or variant have the albumin activity, and wherein said fragment or variant comprise the aminoacid sequence of the amino acid/11-387 of SEQ IDNO:1;
(e) fragment of therapeutic protein: X or variant and the albumin that comprises the aminoacid sequence of SEQ ID NO:1, wherein said fragment or variant have therapeutic protein: the biologic activity of X;
(f) (a) therapeutic protein-(e): X or its fragment or variant and albumin or its fragment or variant, wherein said therapeutic protein: X or its fragment or variant merge not to be held at albuminised N-, or the N-end of albumin fragment or variant;
(g) (a) therapeutic protein-(e): X or its fragment or variant and albumin or its fragment or variant, wherein said therapeutic protein: X or its fragment or variant merge at albuminised C-end, or the C-end of albumin fragment or variant;
(h) (a) therapeutic protein-(e): X or its fragment or variant and albumin or its fragment or variant, wherein said therapeutic protein: X or its fragment or variant merge the terminal and C-end at albuminised N-, or the N-of albumin fragment or variant is terminal and the C-end;
(i) (a) therapeutic protein-(e): X or its fragment or variant and albumin or its fragment or variant, it comprises first therapeutic protein: X or its fragment or variant and second therapeutic protein: X or its fragment or variant, and wherein said first therapeutic protein: X or its fragment or variant are different from described second therapeutic protein: X or its fragment or variant;
(j) (a) therapeutic protein-(i): X or its fragment or variant and albumin or its fragment or variant, wherein said therapeutic protein: X or its fragment or variant separate by joint and described albumin or albumin fragment or variant; With
(k) (a) therapeutic protein-(j): X or its fragment or variant and albumin or its 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 a therapeutic protein: X or its fragment or variant, and L is a peptide linker, and R2 is albumin or albuminised fragment or the variant that comprises the aminoacid sequence of SEQ ID NO:1.
2. the albumin fusion proteins of claim 1, the storage life of wherein said albumin fusion proteins or plasma stability are greater than the storage life or the plasma stability of the therapeutic protein that does not merge state: X or its fragment or variant.
3. the albumin fusion proteins of claim 1, the wherein therapeutic protein that merges with albumin or its fragment or variant: the extracorporeal biology activity of X or its fragment or variant is greater than the extracorporeal biology activity of the therapeutic protein that does not merge state: X or its fragment or variant.
4. the albumin fusion proteins of claim 1, the wherein therapeutic protein that merges with albumin or its fragment or variant: biologic activity is greater than biologic activity in the body of the therapeutic protein that does not merge state: X or its fragment or variant in the body of X or its fragment or variant.
5. the albumin fusion proteins that comprises the therapeutic protein that is inserted in albumin or its fragment or the variant: X or its fragment or variant comprises aminoacid sequence or its fragment or the variant of SEQ ID NO:1.
6. the albumin fusion proteins that comprises the therapeutic protein that is inserted in albumin or its fragment or the variant: X or its fragment or variant comprises the one or more aminoacid sequences that are selected from down group:
(a) the aminoacid 54-61 of SEQ ID NO:1;
(b) the aminoacid 76-89 of SEQ ID NO:1;
(c) the aminoacid 92-100 of SEQ ID NO:1;
(d) the amino acid/11 70-176 of SEQ ID NO:1;
(e) the aminoacid 247-252 of SEQ ID NO:1;
(f) the aminoacid 266-277 of SEQ ID NO:1;
(g) the aminoacid 280-288 of SEQ ID NO:1;
(h) the aminoacid 362-368 of SEQ ID NO:1;
(i) the aminoacid 439-447 of SEQ ID NO:1;
(j) the aminoacid 462-475 of SEQ ID NO:1;
(k) the aminoacid 478-486 of SEQ ID NO:1; With
(l) the aminoacid 560-566 of SEQ ID NO:1.
7. the albumin fusion proteins of claim 5, wherein said albumin fusion proteins comprise compares the storage life that is enough to extended treatment protein: X or its fragment or variant or the part albumin of plasma stability with the storage life of the therapeutic protein that does not merge state: X or its fragment or variant or plasma stability.
8. the albumin fusion proteins of claim 6, wherein said albumin fusion proteins comprise compares the storage life that is enough to extended treatment protein: X or its fragment or variant or the part albumin of plasma stability with the storage life of the therapeutic protein that does not merge state: X or its fragment or variant or plasma stability.
9. the albumin fusion proteins of claim 5, wherein said albumin fusion proteins comprise to compare with the extracorporeal biology activity of the therapeutic protein that does not merge state: X or its fragment or variant is enough to prolong the therapeutic protein that merges with albumin: the active part albumin of the extracorporeal biology of X or its fragment or variant.
10. the albumin fusion proteins of claim 6, wherein said albumin fusion proteins comprise to compare with the extracorporeal biology activity of the therapeutic protein that does not merge state: X or its fragment or variant is enough to prolong the therapeutic protein that merges with albumin: the active part albumin of the extracorporeal biology of X or its fragment or variant.
Compare and be enough to prolong the therapeutic protein that merges with albumin 11. the albumin fusion proteins of claim 5, wherein said albumin fusion proteins comprise in the body with the therapeutic protein that does not merge state: X or its fragment or variant biologic activity: the part albumin of biologic activity in the body of X or its fragment or variant.
Compare and be enough to prolong the therapeutic protein that merges with albumin 12. the albumin fusion proteins of claim 6, wherein said albumin fusion proteins comprise in the body with the therapeutic protein that does not merge state: X or its fragment or variant biologic activity: the part albumin of biologic activity in the body of X or its fragment or variant.
13. each albumin fusion proteins of claim 1-12, it is nonglycosylated.
14. each albumin fusion proteins of claim 1-12, it is expressed in yeast.
15. the albumin fusion proteins of claim 14, wherein said yeast is a glycosylation defect.
16. the albumin fusion proteins of claim 14, wherein said yeast are glycosylation and protease defective.
17. each albumin fusion proteins of claim 1-12, it is by mammalian cell expression.
18. each albumin fusion proteins of claim 1-12, wherein said albumin fusion proteins are by the mammalian cell expression in cultivating.
19. each albumin fusion proteins of claim 1-12, wherein said albumin fusion proteins also comprises the secretion targeting sequencing.
20. comprise each albumin fusion proteins and the pharmacopedics compositions that can accept carrier of claim 1-12.
21. comprise the test kit of the compositions of claim 20.
22. treatment disease of patient or disorderly method comprise and use each the step of albumin fusion proteins of claim 1-12.
23. the method for claim 22, wherein said disease or disorder comprise indication: Y.
24. treatment suffers from and is subjected to therapeutic protein: disease that X or its fragment or variant are regulated or disorderly patient's method comprises each the step of albumin fusion proteins of claim 1-12 of using effective dose.
25. the method for claim 24, wherein said disease or disorder are indications: Y.
26. the storage life of extended treatment protein: X or its fragment or variant or the method for plasma stability, comprise therapeutic protein: the step that X or its fragment or variant and albumin or its fragment or variant merge, compare with the storage life of the therapeutic protein that does not merge state: X or its fragment or variant, described albumin or its fragment or variant are enough to the storage life or the plasma stability of extended treatment protein: X or its fragment or variant.
27. nucleic acid molecules comprises each the polynucleotide sequence of albumin fusion proteins of coding claim 1-12.
28. comprise the carrier of the nucleic acid molecules of claim 27.
29. comprise the host cell of the nucleic acid molecules of claim 28.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210139189.3A CN102816241B (en) | 2004-02-09 | 2005-02-09 | Albumin fusion proteins |
Applications Claiming Priority (13)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US54227404P | 2004-02-09 | 2004-02-09 | |
| US60/542,274 | 2004-02-09 | ||
| US60/542274 | 2004-02-09 | ||
| US54990104P | 2004-03-05 | 2004-03-05 | |
| US60/549901 | 2004-03-05 | ||
| US60/549,901 | 2004-03-05 | ||
| US55690604P | 2004-03-29 | 2004-03-29 | |
| US60/556906 | 2004-03-29 | ||
| US60/556,906 | 2004-03-29 | ||
| US63660304P | 2004-12-17 | 2004-12-17 | |
| US60/636,603 | 2004-12-17 | ||
| US60/636603 | 2004-12-17 | ||
| PCT/US2005/004041 WO2005077042A2 (en) | 2004-02-09 | 2005-02-09 | Albumin fusion proteins |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210139189.3A Division CN102816241B (en) | 2004-02-09 | 2005-02-09 | Albumin fusion proteins |
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| Publication Number | Publication Date |
|---|---|
| CN1980687A true CN1980687A (en) | 2007-06-13 |
| CN1980687B CN1980687B (en) | 2015-05-13 |
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ID=38131514
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200580012252.5A Expired - Fee Related CN1980687B (en) | 2004-02-09 | 2005-02-09 | Albumin fusion proteins |
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| ZA (1) | ZA200605871B (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102453094A (en) * | 2010-11-01 | 2012-05-16 | 天津药物研究院 | Fusion protein containing Exendin-4 and preparation method and application thereof |
| CN104302772A (en) * | 2012-05-18 | 2015-01-21 | 爱德迪安（北京）生物技术有限公司 | Protein and protein conjugate for diabetes treatment, and applications thereof |
| CN105254763A (en) * | 2015-06-30 | 2016-01-20 | 成都谨信恒生物技术有限公司 | Recombinant insulin secretion promoter fusion protein and its preparation method and use |
| CN107478637A (en) * | 2017-07-07 | 2017-12-15 | 复旦大学 | The quick unmarked imaging method for distinguishing Hb A hemoglobin adult and hemosiderin |
| CN107949638A (en) * | 2015-09-08 | 2018-04-20 | Jcr制药股份有限公司 | New human serum albumins mutant |
| CN109627343A (en) * | 2018-12-27 | 2019-04-16 | 北京美福源生物医药科技有限公司 | Long-acting cytokine gene derivative fusion protein |
| CN111373034A (en) * | 2017-07-07 | 2020-07-03 | 艾乐娜制药有限公司 | Recombinant uricase |
| CN112839587A (en) * | 2018-08-21 | 2021-05-25 | 加州理工学院 | Wireless ultrasonic monitoring equipment |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB9526733D0 (en) * | 1995-12-30 | 1996-02-28 | Delta Biotechnology Ltd | Fusion proteins |
| EA005584B1 (en) * | 2000-12-07 | 2005-04-28 | Эли Лилли Энд Компани | Glp-1 fusion proteins |
-
2005
- 2005-02-09 CN CN200580012252.5A patent/CN1980687B/en not_active Expired - Fee Related
-
2006
- 2006-07-17 ZA ZA200605871A patent/ZA200605871B/en unknown
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102453094A (en) * | 2010-11-01 | 2012-05-16 | 天津药物研究院 | Fusion protein containing Exendin-4 and preparation method and application thereof |
| CN104302772A (en) * | 2012-05-18 | 2015-01-21 | 爱德迪安（北京）生物技术有限公司 | Protein and protein conjugate for diabetes treatment, and applications thereof |
| CN105254763A (en) * | 2015-06-30 | 2016-01-20 | 成都谨信恒生物技术有限公司 | Recombinant insulin secretion promoter fusion protein and its preparation method and use |
| CN107949638A (en) * | 2015-09-08 | 2018-04-20 | Jcr制药股份有限公司 | New human serum albumins mutant |
| CN107949638B (en) * | 2015-09-08 | 2022-05-13 | Jcr制药股份有限公司 | Novel human serum albumin mutants |
| CN107478637A (en) * | 2017-07-07 | 2017-12-15 | 复旦大学 | The quick unmarked imaging method for distinguishing Hb A hemoglobin adult and hemosiderin |
| CN111373034A (en) * | 2017-07-07 | 2020-07-03 | 艾乐娜制药有限公司 | Recombinant uricase |
| CN107478637B (en) * | 2017-07-07 | 2021-04-30 | 复旦大学 | Rapid label-free imaging method for distinguishing normal hemoglobin from ferrihemoglobin |
| CN112839587A (en) * | 2018-08-21 | 2021-05-25 | 加州理工学院 | Wireless ultrasonic monitoring equipment |
| CN109627343A (en) * | 2018-12-27 | 2019-04-16 | 北京美福源生物医药科技有限公司 | Long-acting cytokine gene derivative fusion protein |
Also Published As
| Publication number | Publication date |
|---|---|
| ZA200605871B (en) | 2008-02-27 |
| CN1980687B (en) | 2015-05-13 |
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