CN105524176A - Bi-specific fusion proteins - Google Patents

Bi-specific fusion proteins Download PDF

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CN105524176A
CN105524176A CN201510960252.3A CN201510960252A CN105524176A CN 105524176 A CN105524176 A CN 105524176A CN 201510960252 A CN201510960252 A CN 201510960252A CN 105524176 A CN105524176 A CN 105524176A
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cell
mhsa
fusion rotein
target
sequence
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CN105524176B (en
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U·尼尔森
T·威克汉姆
B·舍贝尔
B·哈姆斯
B·林吉
M·昂桑姆
B·德拉巴瑞
S·M·利珀
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Silver Brook Pharmaceutical Ltd By Share Ltd
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Merrimack Pharmaceuticals Inc
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Abstract

Bi-specific fusion proteins with therapeutic uses are provided, as well as pharmaceutical compositions comprising such fusion proteins, and methods for using such fusion proteins to repair damaged tissue. The bi-specific fusion proteins generally comprise: (a) a targeting polypeptide domain that binds to an ischemia-associated molecule; and (b) an activator domain that that detectably modulates the activity of a cellular network.

Description

Bispecific fusion protein
Patent application of the present invention is international application no is PCT/US2011/037459, international filing date is on May 20th, 2011, enter the application number of National Phase in China for " 201180035656.1 ", denomination of invention is the divisional application of the application for a patent for invention of " bispecific fusion protein ".
Related application
This application claims the U.S.Provisional Serial 61/347,040 submitted on May 21st, 2010; The Application U.S. Serial No 13/112,907 that on May 20th, 2011 submits to; Be entitled as " Bi-specificproteins (dual specificity protein) ", attorney docket 132463-010104, the rights and interests of U. S. application that on May 20th, 2011 submits to and right of priority, described application is respectively included in herein by reference of text.
Technical field
The present invention relates generally to the fusion rotein of therepic use, particularly relate to bispecific fusion protein, pharmaceutical composition containing this fusion rotein, use this fusion rotein to repair the method for damaged tissue.
Background of invention
Tissue regeneration is a cross discipline, and target recovers biological function that is ill or damaged tissue.Tissue regeneration solves main clinical problem as myocardial infarction.Myocardial infarction is commonly referred to heart attack, occurs when coronary occlusion cuts off cardiac component blood supply.Because heart can not fully activate endogenous reproducer and self-regeneration, the anoxic produced causes irreversible tissue injury (downright bad and apoptosis).This tissue injury is the major cause of congestive heart failure, and described illness cardiac no longer can effective pump blood.In the U.S., have the heart attack more than 1,000,000 examples every year, the people of nearly 5,000,000 is subject to congestive heart failure impact.
The effective treatment regenerated not used for making damaged cardiac tissue.The treatment of current congestive heart failure focuses on prevention arrhythmia, arteriosclerosis development and Relapse myocardial infarction, but does not solve basic tissue injury.Diagnosis has in the patient of congestive heart failure over half dead in diagnosis 5 years.
Stem-cell therapy is the potential New Policy for cardiac repair.In laboratory, myocardial cell can be generated from stem cell.This shows that stem cell can be used for repairing the heart tissue of damaged tissue as patient; But, at present not based on the available treatment process of described method.The difficult point run in stem-cell therapy is significantly repaired clinically to produce by the stem cell target damaged tissue of enough numbers.
Therefore, this area needs method for repairing or regenerating damaged tissue, improves cell if stem cell target is to promote tissue repair.The present invention meets these demands, and provides other related advantages.
Summary of the invention
The invention provides bispecific fusion protein, encoding bispecific fusion rotein nucleic acid molecule and use the methods for the treatment of of this bispecific fusion protein.In some aspects, the invention provides containing following bispecific fusion protein: (a) has the target structural domain of binding specificity, wherein said molecule to be in viable cell in born of the same parents to tissue damaged's cell target molecule of being correlated with and be exposed to extracellular space in damaged cell; (b) the activation structure territory of binding specificity is had to cell surface relevant growth factors acceptor in tissue, after wherein said activation structure territory is exposed to described growth factor receptors, this activation structure territory in conjunction with described growth factor receptors to regulate tissue regeneration or survival.In some embodiments, described bispecific fusion protein also comprises peptide transformation period instrumentality.
Of the present invention in some, described bispecific fusion protein comprises (a) to be had the target structural domain of binding specificity, wherein said molecule to be in viable cell in born of the same parents to tissue damaged's cell target molecule of being correlated with and be exposed to extracellular space in damaged cell; B () has the activation structure territory of binding specificity to cell surface associated molecule in tissue, after wherein said activation structure territory is exposed to surface associated molecule, this activation structure territory in conjunction with described surface associated molecule to regulate tissue regeneration or survival; (c) transformation period instrumentality, wherein said transformation period instrumentality regulates and controls the transformation period of described bispecific fusion protein.
In other aspects of the present invention, described bispecific fusion protein comprises (a) has binding specificity target structural domain to the relevant target molecule of tissue; B () has the activation structure territory of binding specificity to cell surface associated molecule in tissue, after wherein said activation structure territory is exposed to described molecule, this activation structure territory in conjunction with described molecule to regulate tissue regeneration or survival; (c) transformation period instrumentality, wherein said transformation period instrumentality regulates and controls the transformation period of described bispecific fusion protein.
In other aspects of the present invention, described bispecific fusion protein comprises (a) has binding specificity target structural domain to the relevant target molecule of tissue; B () has the binding domain of binding specificity to cell surface associated molecule in tissue, after wherein said binding domain is exposed to described molecule, this binding domain in conjunction with described molecule to promote tissue regeneration or survival; (c) transformation period instrumentality, wherein said transformation period instrumentality regulates and controls the transformation period of described bispecific fusion protein.
Another aspect of the present invention relates to containing following fusion rotein: (a) has at least one target structural domain of binding specificity to the relevant target molecule of at least one tissue; B () has at least one activation structure territory of binding specificity to cell surface associated molecule at least one tissue, after wherein said activation structure territory is exposed to described molecule, this activation structure territory in conjunction with described molecule to promote tissue regeneration or survival; (c) transformation period instrumentality, wherein said transformation period instrumentality regulates and controls the transformation period of described fusion rotein.
In some embodiments, described activation structure territory or binding domain are combined with growth factor receptors, cytokine receptor or stem cell correlation antigen specific.In some embodiments, described target structural domain does not have biologic activity.Described target structural domain and described activation structure territory can maybe can in conjunction with the differing moleculars on different cell in conjunction with the differing molecular on same cell.
In some embodiments, described activation structure territory is selected from lower group: fibroblast growth factor (FGF), Urogastron (EGF), neuregulin/tune albumen (NRG/HRG), rhIGF-1 (IGF), pHGF (HGF), thymosin, granulocyte colony-stimulating factor (G-CSF), STEM CELL FACTOR (SCF)/mast cell growth factor (MGF), periostin (periostin), vascular endothelial growth factor (VEGF), stroma cell derivative factor (SDF), Thr6 PDGF BB (PDGF), teratoma derivative growth factor (TDGF), β-nerve growth factor (NGF), Brain Derived Neurotrophic Factor (BDNF), neurotrophic factor-3 (NT-3), thrombopoietin (TPO), Delicious peptide (BMP), activin A, second born of the same parents element, beta-catenin, dickkopf homologue 1 (DKK1), erythropoietin (EPO), tethelin (GH), heparin binding epidermal growth factor like growth factor (HBEGF), Regular Insulin, interleukin (IL) leukaemia inhibitory factor (LIF), MCP 1 (MCP1/CCL2), PTN (PTN), transforming growth factor (TGF), tumour necrosis factor (TNF), Wnt (one has specific antibody to activator receptor), its variant, its isotype, its fragment and its combination.In some embodiments, described activation structure territory comprises sequence according to any one of SEQIDNO:3-9,32-40 or 50-64.
Described target structural domain can at the N-terminal of described fusion rotein and described activation structure territory at C-terminal.In some embodiments, described target structural domain at the C-terminal of described fusion rotein and described activation structure territory at N-terminal.In some embodiments, described target structural domain at the C-terminal of described fusion rotein and described activation structure territory at N-terminal.
In some embodiments, described transformation period instrumentality is non-immunogenic protein.Described transformation period instrumentality can comprise from the single stranded form of the Domain III of human serum albumin, human serum albumin, alpha-fetoprotein, vitamin D binding protein, transthyretin antibody Fc domain, antibody Fc domain, proline(Pro)-, the sequence of L-Ala-and/or Serine enriched sequence, its variant, one of its fragment and its combination.Such as, described transformation period instrumentality comprises at least 100 continuous amino acids identical with serum albumin aminoacid sequence at least 80%.In some embodiments, described transformation period instrumentality have SEQIDNO:10,12, arbitrary described aminoacid sequence in 14-29,45-49,65-71 or 105.
In some embodiments, described target structural domain combines the target molecule being selected from lower group: myosin, cardiac myosin, DNA, phosphatidylserine, palatelet-selectin, ICAM-1, c-Met (HGF acceptor), its variant, its fragment and its combination.In some embodiments, described target structural domain is 10 in conjunction with dissociation constant Kd scope -6m-10 -12the target molecule of M.Described target structural domain can be selected from annexin, synaptotagmin, anti-phosphatidylserine antibody, PS4A7, lactadherin (lactadherin), antimyosin antibody, anti-DNA antibody, aDNASI1, aDNASI22, its variant, its fragment and its combination.In some embodiments, described target structural domain has arbitrary described sequence in SEQIDNO:1-2,30-31,72-73,76-83 or 85-86.In some embodiments, described antibody be have SEQIDNO:1,2,30,73, the scFv antibody of arbitrary described sequence in 76-80.In some embodiments, annexin is annexin V and has sequence described in SEQID.NO31,81,82 or 83.In some embodiments, described target structural domain have SEQIDNO:1,2,30,31 or 72-86 in arbitrary described sequence.
In some embodiments, described bispecific fusion protein also comprises the linker connecting described transformation period instrumentality and described fusion rotein.Described bispecific fusion protein can show 2 hours-6 hours, 6 hours-24 hours, be greater than 24 hours or be greater than the Half-life in vivo of 1 week.
In some embodiments, described fusion rotein promotes recruiting cells, apoptosis to suppress and/or cell proliferation induction.In some embodiments, described fusion rotein prevents cell injury, Promote cell's growth, the motion of promotion stem cell and/or promotes differentiation of stem cells.In some embodiments, described fusion rotein promotes tissue regeneration.Described tissue can be heart tissue, nephridial tissue, bone, cartilage, joint, skin, hepatic tissue, pancreatic tissue, hemocyte, lung tissue or neural system.
In some embodiments, described fusion rotein also comprises leading polypeptide.Described leading polypeptide can comprise SEQIDNO:41,42, arbitrary described sequence in 87-91 or 244.
In some embodiments, described fusion rotein also comprises polypeptide affinity tag.In some embodiments, described affinity tag is in the middle of the N-terminal of described fusion rotein, the C-terminal of described fusion rotein or described fusion rotein.In some embodiments, described fusion rotein comprises the polypeptide containing six Histidines.The described polypeptide containing six Histidines can have SEQIDNO:43,44 or 92-94 in arbitrary described sequence.
Bispecific binders provided herein need not fixed limit in 2 kinds of binding specificities.In some embodiments, described bispecific fusion protein comprises 2 or more activation structure territories, and described structural domain is directly or indirectly connected by peptide bond.In some embodiments, described bispecific fusion protein comprises beyond described activation structure territory 2 or more target structural domains, and described structural domain is directly or indirectly connected by peptide bond.
In other respects, the invention provides pharmaceutical composition, described composition comprises acceptable vehicle on above-mentioned bispecific fusion protein and physiology.
In other aspects, provide the method for the treatment of patient pathology tissue injury, described method comprises and gives pathological tissue injured patient by pharmaceutical composition, thus reduces the pathological tissue damage in this patient.
Many aspects of the present invention relate to the method promoting object tissue regeneration or survival, described method comprises (a) to be provided containing following bispecific fusion protein: (i) has the target structural domain of binding specificity, wherein said molecule to be in viable cell in born of the same parents to tissue damaged's cell target molecule of being correlated with and be exposed to extracellular space in damaged cell; (ii) growth factor receptors is had to the activation structure territory of binding specificity; (b) bispecific fusion protein for the treatment of significant quantity is given the patient needed, wherein said target structural domain specific binding tissue damaged cell is correlated with target molecule, thus by the first cell of described bispecific fusion protein target tissue and wherein after described activation structure territory is exposed to described growth factor receptors, the growth factor receptors of described activation structure territory specific activation second cell is to promote tissue regeneration.
In some embodiments, promote that the method for object tissue regeneration or survival comprises (a) and provides containing following bispecific fusion protein: (i) has the target structural domain of binding specificity to target molecule; (ii) acceptor is had to the activation structure territory of binding specificity; (iii) transformation period instrumentality, wherein said transformation period instrumentality regulates and controls the transformation period of described bispecific fusion protein; (b) bispecific fusion protein for the treatment of significant quantity is given the patient needed, wherein said target structural domain specific binding target molecule, thus by the first cell of described bispecific fusion protein target tissue and wherein after described activation structure territory is exposed to described growth factor receptors, the acceptor of described activation structure territory this tissue second cell of specific activation is to promote tissue regeneration.
In some embodiments, described first and second cells are identical.In other embodiments, described first and second cells are different.In some embodiments, described first cell is viable cell and described second cell is damaged cell.In other embodiments, described first cell is damaged cell and described second cell is viable cell.In some embodiments, described method also comprises and gives patient by stem cell.
In some embodiments, described pathological tissue damage is the damage to cardiac tissue relevant to myocardial infarction.In other embodiments, described pathological tissue damage is Renal tissues damage.In other embodiments, described pathological tissue damage is bone, cartilage, joint, skin, hepatic tissue, pancreatic tissue, hemocyte, lung tissue or neural system.In some embodiments, described method also comprises and gives patient by stem cell.
The nucleic acid molecule of above-mentioned bispecific fusion protein of encoding also is provided herein.In some embodiments, described nucleic acid molecule is DNA, described DNA also comprises operability and connects transcribing and translational regulation sequence of bispecific fusion protein encoding sequence, thus transcribing and translating of described encoding sequence occurs at least one eukaryotic cell type.
These and other aspects of the present invention are by becoming apparent with reference to following detailed description.
Sequence table describes
SEQIDNO:1 is the aminoacid sequence of anti-DNAscFvSI-1.
SEQIDNO:2 is the aminoacid sequence of anti-DNAscFvSI-22.
SEQIDNO:3 is the aminoacid sequence of the growth factor polypeptide corresponding to wild type human IGF-I (mature form).
SEQIDNO:4 is the aminoacid sequence of the growth factor polypeptide corresponding to the people IGF-1 having D12A to replace.
SEQIDNO:5 is the aminoacid sequence of the growth factor polypeptide corresponding to the people IGF-1 having E9A to replace.
SEQIDNO:6 is the aminoacid sequence of the growth factor polypeptide corresponding to people HGF α chain N-K1 structural domain.
SEQIDNO:7 is the aminoacid sequence of the growth factor polypeptide corresponding to people HGF α chain K1 structural domain.
SEQIDNO:8 is the aminoacid sequence corresponding to the growth factor polypeptide that people HGF α chain N-K2 merges.
SEQIDNO:9 is the aminoacid sequence of the growth factor polypeptide corresponding to people HGF α chain K2 structural domain.
SEQIDNO:10 is the aminoacid sequence of human serum albumin (HSA) joint having C34S and N503Q to replace.
SEQIDNO:11 is the aminoacid sequence of the HSA joint having C34S and N503Q to replace.
SEQIDNO:12 is the aminoacid sequence of HSA.
SEQIDNO:13 is the nucleotide sequence of HSA.
SEQIDNO:14 is the aminoacid sequence of HSA joint and the polypeptide linker having C34S and N503Q to replace.
SEQIDNO:15 is the aminoacid sequence of the HSA joint having C34S and N503Q replacement and polypeptide linker.
SEQIDNO:16 is the aminoacid sequence of the HSA joint having C34S and N503Q replacement and polypeptide linker.
SEQIDNO:17 is the aminoacid sequence of the HSA joint having C34S and N503Q replacement and polypeptide linker.
SEQIDNO:18 is the aminoacid sequence of the HSA joint having C34S and N503Q replacement and polypeptide linker.
SEQIDNO:19 is the aminoacid sequence of the HSA joint having polypeptide linker.
SEQIDNO:20 is the aminoacid sequence of the HSA joint having polypeptide linker.
SEQIDNO:21 is the aminoacid sequence of the HSA joint having polypeptide linker.
SEQIDNO:22 is the aminoacid sequence of the HSA joint having polypeptide linker.
SEQIDNO:23 is the aminoacid sequence of the HSA joint having polypeptide linker.
SEQIDNO:24 is the aminoacid sequence of the HSA joint having C34S to replace, structural domain I.
SEQIDNO:25 is the aminoacid sequence of HSA joint, domain II.
SEQIDNO:26 is the aminoacid sequence of the HSA joint having N503Q to replace, Domain III.
SEQIDNO:27 is the aminoacid sequence of HSA joint, structural domain I.
SEQIDNO:28 is the aminoacid sequence of HSA joint, Domain III.
SEQIDNO:29 is the aminoacid sequence of human a-fetoprotein.
SEQIDNO:30 is the aminoacid sequence of anti-phosphatidylserine scFvPS4A7.
SEQIDNO:31 is the aminoacid sequence of human annexin-V V (AnxV).
SEQIDNO:32 is the aminoacid sequence of the growth factor polypeptide corresponding to people HGF α chain N-K1 structural domain.
SEQIDNO:33 is the aminoacid sequence of the growth factor polypeptide corresponding to people HGF α chain K1 structural domain.
SEQIDNO:34 is the aminoacid sequence of the growth factor polypeptide corresponding to people HGF α chain N-K2 structural domain.
SEQIDNO:35 is the aminoacid sequence of the growth factor polypeptide corresponding to people HGF α chain K2 structural domain.
SEQIDNO:36 is the aminoacid sequence of the growth factor polypeptide corresponding to people VEGF α monomer.
SEQIDNO:37 is the aminoacid sequence corresponding to the dimeric growth factor polypeptide of people VEGF α.
SEQIDNO:38 is the aminoacid sequence of the growth factor polypeptide corresponding to people FGF2.
SEQIDNO:39 is the aminoacid sequence of the growth factor polypeptide corresponding to people NRG1 α, EGF spline structure territory.
SEQIDNO:40 is the aminoacid sequence of the growth factor polypeptide corresponding to people NRG1 α, complete sequence.
SEQIDNO:41 is the aminoacid sequence of the leading polypeptide of bispecific fusion protein.
SEQIDNO:42 is the aminoacid sequence of the leading polypeptide of bispecific fusion protein.
SEQIDNO:43 is the aminoacid sequence of the polypeptide containing C-terminal six Histidine.
SEQIDNO:44 is the aminoacid sequence of the polypeptide containing C-terminal six Histidine.
SEQIDNO:45 is the aminoacid sequence of HSA joint.
SEQIDNO:46 is the aminoacid sequence of the HSA joint having N-terminal and the short linker polypeptide of C-terminal.
SEQIDNO:47 is the aminoacid sequence of the HSA joint having N-terminal and the short linker polypeptide of C-terminal.
SEQIDNO:48 is the aminoacid sequence of the HSA joint having N-terminal and the short linker polypeptide of C-terminal.
SEQIDNO:49 is the aminoacid sequence of the HSA joint having N-terminal and the short linker polypeptide of C-terminal.
SEQIDNO:50 is the aminoacid sequence of the growth factor polypeptide variant corresponding to people FGF2.
SEQIDNO:51 is the aminoacid sequence of the growth factor polypeptide corresponding to people HGF α chain.
SEQIDNO:52 is the aminoacid sequence of the growth factor polypeptide corresponding to people HGF α chain K1 structural domain.
SEQIDNO:53 is the aminoacid sequence of the growth factor polypeptide corresponding to people HGF α chain N-K2 structural domain.
SEQIDNO:54 is the aminoacid sequence of the growth factor polypeptide corresponding to people HGF α chain K2 structural domain.
SEQIDNO:55 is the aminoacid sequence of the growth factor polypeptide corresponding to people NRG1 β ectodomain.
SEQIDNO:56 is the aminoacid sequence of the growth factor polypeptide corresponding to people NRG1 β EGF spline structure territory.
SEQIDNO:57 is the aminoacid sequence of people's total length periostin.
SEQIDNO:58 is the aminoacid sequence in people's total length periostin region.
SEQIDNO:59 is the aminoacid sequence of the growth factor polypeptide corresponding to human bone morphogenesis protein-2.
SEQIDNO:60 is the aminoacid sequence of the growth factor polypeptide corresponding to strand human bone morphogenesis protein-2.
SEQIDNO:61 is the aminoacid sequence of the growth factor polypeptide corresponding to vascular endothelial growth factor B.
SEQIDNO62 is the aminoacid sequence of groups of people's vascular endothelial growth factor B.
SEQIDNO63 is the aminoacid sequence of groups of people's vascular endothelial growth factor B.
SEQIDNO64 is the aminoacid sequence of groups of people's vascular endothelial growth factor B.
SEQIDNO65 is the aminoacid sequence of human serum albumin (HSA) Domain III.
SEQIDNO66 is the aminoacid sequence of modification vitamin D binding protein (mVDBP).
SEQIDNO67 is the aminoacid sequence (aminosequence) of the Domain III of modification human serum albumin.
SEQIDNO68 is the aminoacid sequence (aminosequence) of people AFP.
SEQIDNO69 is the aminoacid sequence (aminosequence) of modification AFP.
SEQIDNO70 is the aminoacid sequence in albumin bound people from territory antibody (albudAb).
SEQIDNO71 is the aminoacid sequence of the mono-poly-variant form of Fc, is called scFc.
SEQIDNO72 is the aminoacid sequence of Synaptotagmin I.
SEQIDNO73 is the aminoacid sequence of anti-DNAscFv antibody.
SEQIDNO74 is the aminoacid sequence of non-binding Synaptotagmin I variant.
SEQIDNO75 is the aminoacid sequence of non-binding scFv variant (DAscFv).
SEQIDNO76 is the aminoacid sequence of B7scFv anti-myosin scFv antibody.
SEQIDNO77 is the aminoacid sequence of FD2 anti-myosin scFv antibody.
SEQIDNO78 is the aminoacid sequence of MCA1 anti-myosin scFv antibody.
SEQIDNO79 is the aminoacid sequence of MCB11 anti-myosin scFv antibody.
SEQIDNO80 is the aminoacid sequence of S3F5 anti-myosin scFv antibody.
SEQIDNO81 is the aminoacid sequence of human annexin-V V variant (AnxVmC317S).
SEQIDNO82 is the aminoacid sequence of human annexin-V V variant (AnxVm3).
SEQIDNO83 is the aminoacid sequence of human annexin-V V variant (AnxVm23).
SEQIDNO84 is the aminoacid sequence of the non-binding variant of human annexin-V V (AnxVm1234).
SEQIDNO85 is the aminoacid sequence of lactadherin variant.
SEQIDNO86 is the aminoacid sequence of lactadherin variant.
SEQIDNO87 is the aminoacid sequence of α conjugative element.
SEQIDNO88 is the aminoacid sequence of the leading polypeptide of app8.
SEQIDNO89 is the aminoacid sequence of aga2 signal peptide.
SEQIDNO90 is SUC2 signal peptide aminoacid sequence.
SEQIDNO91 is composite signal peptide amino acid sequence.
SEQIDNO92 is the aminoacid sequence of hexahistidine tag.
SEQIDNO93 is the aminoacid sequence of hexahistidine tag.
SEQIDNO94 is the aminoacid sequence of hexahistidine tag.
SEQIDNO95-104 and SEQIDNO182-184 corresponds to the aminoacid sequence of peptide linker.
SEQIDNO105 be proline(Pro)-, the aminoacid sequence of L-Ala-and/or Serine enriched sequence.
SEQIDNO106 is the aminoacid sequence of aDNASI1_mHSA_IGF1 fusion rotein.SEQIDNO107 is the nucleotide sequence of aDNASI1_mHSA_IGF1 fusion rotein.
SEQIDNO108 is the aminoacid sequence of aPS4A7_mHSA_IGF1 fusion rotein.SEQIDNO109 is the nucleotide sequence of aPS4A7_mHSA_IGF1 fusion rotein.
SEQIDNO110 is the aminoacid sequence of aDNASI1_mHSA_HGF (NK1) fusion rotein.SEQIDNO111 is the nucleotide sequence of aDNASI1_mHSA_HGF (NK1) fusion rotein.
The aminoacid sequence of SEQIDNO112aPS4A7_mHSA_HGF (NK1) fusion rotein.SEQIDNO113 is the nucleotide sequence of aPS4A7_mHSA_HGF (NK1) fusion rotein.
SEQIDNO114 is the aminoacid sequence of AnxVm1234_mHSA_IGF1 fusion rotein.SEQIDNO115 is the nucleotide sequence of AnxVm1234_mHSA_IGF1 fusion rotein.
SEQIDNO116 is the aminoacid sequence of AnxVm1234_mHSA_NRG1b (EGF) fusion rotein.SEQIDNO117 is the nucleotide sequence of AnxVm1234_mHSA_NRG1b (EGF) fusion rotein.
SEQIDNO118 is the aminoacid sequence of AnxV_mHSA_FGF2 fusion rotein.SEQIDNO119 is the nucleotide sequence of AnxV_mHSA_FGF2 fusion rotein.
SEQIDNO120 is the aminoacid sequence of AnxV_mHSA_NRG1b (EGF) fusion rotein.SEQIDNO121 is the nucleotide sequence of AnxV_mHSA_NRG1b (EGF) fusion rotein.
SEQIDNO122 is the aminoacid sequence of FGF2_mHSA_AnxVm1234 fusion rotein.SEQIDNO123 is the nucleotide sequence of FGF2_mHSA_AnxVm1234 fusion rotein.
SEQIDNO124 is the aminoacid sequence of aDNASI1_mHSA_FGF2 fusion rotein.SEQIDNO125 is the nucleotide sequence of aDNASI1_mHSA_FGF2 fusion rotein.
SEQIDNO126 is the aminoacid sequence of aDNASI1_mHSA_NRG1b (EGF) fusion rotein.SEQIDNO127 is the nucleotide sequence of aDNASI1_mHSA_NRG1b (EGF) fusion rotein.
SEQIDNO128 is the aminoacid sequence of AnxV_mHSA_VEGFB (111) fusion rotein.SEQIDNO129 is the nucleotide sequence of AnxV_mHSA_VEGFB (111) fusion rotein.
SEQIDNO130 is the aminoacid sequence of AnxV_mHSA_VEGFB (167) fusion rotein.SEQIDNO131 is the nucleotide sequence of AnxV_mHSA_VEGFB (167) fusion rotein.
SEQIDNO132 is the aminoacid sequence of AnxV_mHSA_HGF (NK1) fusion rotein.SEQIDNO133 is the nucleotide sequence of AnxV_mHSA_HGF (NK1) fusion rotein.
SEQIDNO134 is the aminoacid sequence of AnxV_mHSA_IGF1 fusion rotein.SEQIDNO135 is the nucleotide sequence of AnxV_mHSA_IGF1 fusion rotein.
SEQIDNO136 is the aminoacid sequence of IGF1_mHSA_AnxV fusion rotein.SEQIDNO137 is the nucleotide sequence of IGF1_mHSA_AnxV fusion rotein.
SEQIDNO138 is the aminoacid sequence of IGF1_mHSA_AnxVm1234 fusion rotein.SEQIDNO139 is the nucleotide sequence of IGF1_mHSA_AnxVm1234 fusion rotein.
SEQIDNO140 is the aminoacid sequence of HGF (NK1) _ mHSA_AnxV fusion rotein.SEQIDNO141 is the nucleotide sequence of HGF (NK1) _ mHSA_AnxV fusion rotein.
SEQIDNO142 is the aminoacid sequence of NRG1b (EGF) _ mHSA_AnxV fusion rotein.SEQIDNO143 is the nucleotide sequence of NRG1b (EGF) _ mHSA_AnxV fusion rotein.
SEQIDNO144 is the aminoacid sequence of FGF2_mHSA_AnxV fusion rotein.SEQIDNO145 is the nucleotide sequence of FGF2_mHSA_AnxV fusion rotein.
SEQIDNO146 is the aminoacid sequence of VEGFB (167) _ mHSA_AnxV fusion rotein.SEQIDNO147 is the nucleotide sequence of VEGFB (167) _ mHSA_AnxV fusion rotein.
SEQIDNO148 is the aminoacid sequence of VEGFB (111) _ mHSA_AnxV fusion rotein.SEQIDNO149 is the nucleotide sequence of VEGFB (111) _ mHSA_AnxV fusion rotein.
SEQIDNO150 is the aminoacid sequence of IGF1_mHSA_B7scFv fusion rotein.SEQIDNO151 is the nucleotide sequence of IGF1_mHSA_B7scFv fusion rotein.
SEQIDNO152 is the aminoacid sequence of IGF1_mHSA_Syt1 fusion rotein.SEQIDNO153 is the nucleotide sequence of IGF1_mHSA_Syt1 fusion rotein.
SEQIDNO154 is the aminoacid sequence of IGF1_mHSA_aDNASI1 fusion rotein.SEQIDNO155 is the nucleotide sequence of IGF1_mHSA_aDNASI1 fusion rotein.
SEQIDNO156 is the aminoacid sequence of NRG1b (EGF) _ mHSA_B7scFv fusion rotein.SEQIDNO157 is the nucleotide sequence of NRG1b (EGF) _ mHSA_B7scFv fusion rotein.
SEQIDNO158 is the aminoacid sequence of NRG1b (EGF) _ mHSA_Syt1 fusion rotein.SEQIDNO159 is the nucleotide sequence of NRG1b (EGF) _ mHSA_Syt1 fusion rotein.
SEQIDNO160 is the aminoacid sequence of NRG1b (EGF) _ mHSA_aDNASI1 fusion rotein.SEQIDNO161 is the nucleotide sequence of NRG1b (EGF) _ mHSA_aDNASI1 fusion rotein.
SEQIDNO162 is the aminoacid sequence of FGF2_mHSA_B7scFv fusion rotein.SEQIDNO163 is the nucleotide sequence of FGF2_mHSA_B7scFv fusion rotein.
SEQIDNO164 is the aminoacid sequence of FGF2_mHSA_Syt1 fusion rotein.SEQIDNO165 is the nucleotide sequence of FGF2_mHSA_Syt1 fusion rotein.
SEQIDNO166 is the aminoacid sequence of FGF2_mHSA_aDNASI1 fusion rotein.SEQIDNO167 is the nucleotide sequence of FGF2_mHSA_aDNASI1 fusion rotein.
SEQIDNO168 is the aminoacid sequence of B7scFv_mHSA_IGF1 fusion rotein.SEQIDNO169 is the nucleotide sequence of B7scFv_mHSA_IGF1 fusion rotein.
SEQIDNO170 is the aminoacid sequence of Syt1_mHSA_IGF1 fusion rotein.SEQIDNO171 is the nucleotide sequence of Syt1_mHSA_IGF1 fusion rotein.
SEQIDNO172 is the aminoacid sequence of aDNASI1_mHSA_IGF1 fusion rotein.SEQIDNO173 is the nucleotide sequence of aDNASI1_mHSA_IGF1 fusion rotein.
SEQIDNO174 is the aminoacid sequence of B7scFv_mHSA_NRG1b (EGF) fusion rotein.SEQIDNO175 is the nucleotide sequence of B7scFv_mHSA_NRG1b (EGF) fusion rotein.
SEQIDNO176 is the aminoacid sequence of Syt1_mHSA_NRG1b (EGF) fusion rotein.SEQIDNO177 is the nucleotide sequence of Syt1_mHSA_NRG1b (EGF) fusion rotein.
SEQIDNO178 is the aminoacid sequence of B7scFv_mHSA_FGF2 fusion rotein.SEQIDNO179 is the nucleotide sequence of B7scFv_mHSA_FGF2 fusion rotein.
SEQIDNO180 is the aminoacid sequence of Syt1_mHSA_FGF2 fusion rotein.SEQIDNO181 is the nucleotide sequence of Syt1_mHSA_FGF2 fusion rotein.
SEQIDNO185 is the aminoacid sequence of IGF1_mHSA_DAscFv fusion rotein.SEQIDNO186 is the nucleotide sequence of IGF1_mHSA_DAscFv fusion rotein.
SEQIDNOSEQIDNO:187-190 is the nucleotide sequence of the growth factor polypeptide corresponding to people FGF2 (SEQIDNO:38).
SEQIDNO191-94 is the nucleotide sequence of the growth factor polypeptide corresponding to HGF α chain N-K1 structural domain (SEQIDNO:6, SEQIDNO:32).
SEQIDNO195-197 is the nucleotide sequence of the growth factor polypeptide corresponding to wild type human IGF-I (SEQIDNO3).
SEQIDNO198 is the nucleotide sequence of the growth factor polypeptide corresponding to people NRG1 α full length sequence (SEQIDNO40).
SEQIDNO199 is the nucleotide sequence of the growth factor polypeptide corresponding to people NRG1 α EGF spline structure territory (SEQIDNO39).
SEQIDNO200-202 is the nucleotide sequence of the growth factor polypeptide corresponding to people NRG1 β EGF spline structure territory (SEQIDNO56).
SEQIDNO203 is the nucleotide sequence of the growth factor polypeptide corresponding to people's periostin region (SEQIDNO58).
SEQIDNO204 is the nucleotide sequence of the growth factor polypeptide corresponding to human bone morphogenesis protein-2 (SEQIDNO59).
SEQIDNO205 is the nucleotide sequence of the growth factor polypeptide corresponding to strand human bone morphogenesis protein-2 (SEQIDNO60).
SEQIDNO206 is the nucleotide sequence of the growth factor polypeptide corresponding to people VEGF α monomer (SEQIDNO36).
SEQIDNO207 is the nucleotide sequence of the growth factor polypeptide corresponding to people VEGF α dimer (SEQIDNO37).
SEQIDNO208-209 is the nucleotide sequence of the growth factor polypeptide corresponding to vascular endothelial growth factor B (SEQIDNO61).
SEQIDNO210-211 is the nucleotide sequence of the growth factor polypeptide corresponding to human vascular endothelial growth factor B.
SEQIDNO212-214 is the nucleotide sequence of the transformation period instrumentality corresponding to the human serum albumin having C34S and N503Q to replace (HSA) joint (SEQIDNO10).
SEQIDNO215 is the nucleotide sequence of the transformation period instrumentality corresponding to modification human serum albumin Domain III (SEQIDNO67).
SEQIDNO216 is the nucleotide sequence of the transformation period instrumentality corresponding to modification AFP (SEQIDNO69).
SEQIDNO217 is the nucleotide sequence of the transformation period instrumentality corresponding to albumin bound people from territory antibody (SEQIDNO70).
SEQIDNO218 is the nucleotide sequence corresponding to the transformation period instrumentality being called the mono-poly-variant form (SEQIDNO71) of the Fc of scFc.
SEQIDNO219 is the nucleotide sequence of the transformation period instrumentality corresponding to modification vitamin D binding protein mVDBP (SEQIDNO66).
SEQIDNO220-221 is the nucleotide sequence corresponding to anti-DNAscFv antibody (SEQIDNO73).
SEQIDNO222 is the nucleotide sequence corresponding to anti-DNAscFvSI-1 (SEQIDNO1).
SEQIDNO223 is the nucleotide sequence corresponding to B7scFv anti-myosin scFv antibody (SEQIDNO76).
SEQIDNO224 is the nucleotide sequence corresponding to anti-phosphatidylserine scFvPS4A7 (SEQIDNO30).
SEQIDNO225-227 is the nucleotide sequence corresponding to human annexin-V V (SEQIDNO31).
SEQIDNO228 is the nucleotide sequence corresponding to human annexin-V V variant (C317S, SEQIDNO81).
SEQIDNO229-230 is the nucleotide sequence corresponding to human annexin-V V variant (AnxVm3, SEQIDNO82).
SEQIDNO231-232 is the nucleotide sequence corresponding to the non-internalization variant (AnxVm23, SEQIDNO83) of annexin V.
SEQIDNO233-234 is the nucleotide sequence corresponding to the non-binding variant (AnxVm1234, SEQIDNO84) of annexin V.
SEQIDNO235 is the nucleotide sequence corresponding to Synaptotagmin I (SEQIDNO72).
SEQIDNO236-237 corresponds to non-binding scFv variant (DAscFv; SEQIDNo75) nucleotide sequence.
SEQIDNO238 is the nucleotide sequence corresponding to leading polypeptide.
SEQIDNO239 is the nucleotide sequence corresponding to α conjugative element.
SEQIDNO240 is the nucleotide sequence corresponding to the leading polypeptide of app8.
SEQIDNO241 is the nucleotide sequence corresponding to aga2 signal peptide.
SEQIDNO242 is the nucleotide sequence corresponding to SUC2 signal peptide.
SEQIDNO243 is the nucleotide sequence corresponding to composite signal peptide.
SEQIDNO:244 is the aminoacid sequence corresponding to alpha factor signal sequence.SEQIDNO245 is the nucleotide sequence corresponding to alpha factor signal sequence.
SEQIDNO246 is the aminoacid sequence of DAscFv_mHSA_IGF1 fusion rotein.SEQIDNO247 is the nucleotide sequence corresponding to DAscFv_mHSA_IGF1 fusion rotein.
SEQIDNO248 is the aminoacid sequence of DAscFv_mHSA_HGF (NK1) fusion rotein.SEQIDNO249 is the nucleotide sequence corresponding to DAscFv_mHSA_HGF (NK1) fusion rotein.
SEQIDNO250 is the aminoacid sequence of AnxVm1234_mHSA fusion rotein.SEQIDNO251 is the nucleotide sequence corresponding to AnxVm1234_mHSA fusion rotein.
SEQIDNO252 is the aminoacid sequence of AnxV_mHSA fusion rotein.SEQIDNO253 is the nucleotide sequence corresponding to AnxV_mHSA fusion rotein.
SEQIDNO254 is the aminoacid sequence of NRG1b (EGF) _ mHSA_AnxVm1234 fusion rotein.SEQIDNO255 is the nucleotide sequence corresponding to NRG1b (EGF) _ mHSA_AnxVm1234 fusion rotein.
SEQIDNO256 is the aminoacid sequence of AnxVm23_mHSA fusion rotein.SEQIDNO257 is the nucleotide sequence corresponding to AnxVm23_mHSA fusion rotein.
SEQIDNO258 is the aminoacid sequence of AnxVm1234_mHSA_VEGFB (111) fusion rotein.SEQIDNO259 is the nucleotide sequence corresponding to AnxVm1234_mHSA_VEGFB (111) fusion rotein.
SEQIDNO260 is the aminoacid sequence of AnxVm1234_mHSA_VEGFB (167) fusion rotein.SEQIDNO261 is the nucleotide sequence corresponding to AnxVm1234_mHSA_VEGFB (167) fusion rotein.
SEQIDNO262 is the aminoacid sequence of AnxVm1234_mHSA_HGF (NK1) fusion rotein.SEQIDNO263 is the nucleotide sequence corresponding to AnxVm1234_mHSA_HGF (NK1) fusion rotein.
SEQIDNO264 is the aminoacid sequence of AnxVm1234_mHSA_FGF2 fusion rotein.SEQIDNO265 is the nucleotide sequence corresponding to AnxVm1234_mHSA_FGF2 fusion rotein.
SEQIDNO266 is the aminoacid sequence of mHSA_AnxV fusion rotein.SEQIDNO267 is the nucleotide sequence corresponding to mHSA_AnxV fusion rotein.
SEQIDNO268 is the aminoacid sequence of mHSA_AnxVm23 fusion rotein.SEQIDNO269 is the nucleotide sequence corresponding to mHSA_AnxVm23 fusion rotein.
SEQIDNO270 is the aminoacid sequence of mHSA_AnxVm1234 fusion rotein.SEQIDNO271 is the nucleotide sequence corresponding to mHSA_AnxVm1234 fusion rotein.
SEQIDNO272 is the aminoacid sequence of HGF (NK1) _ mHSA_AnxVm1234 fusion rotein.SEQIDNO273 is the nucleotide sequence corresponding to HGF (NK1) _ mHSA_AnxVm1234 fusion rotein.
SEQIDNO274 is the aminoacid sequence of VEGFB (167) _ mHSA_AnxVm1234 fusion rotein.SEQIDNO275 is the nucleotide sequence corresponding to VEGFB (167) _ mHSA_AnxVm1234 fusion rotein.
SEQIDNO276 is the aminoacid sequence of VEGFB (111) _ mHSA_AnxVm1234 fusion rotein.SEQIDNO277 is the nucleotide sequence corresponding to VEGFB (111) _ mHSA_AnxVm1234 fusion rotein.
Brief Description Of Drawings
Fig. 1 is the SDS-PAGE of purified IGF1_mHSA_AnxV (136), IGF1_mHSA_AnxVm1234 (138), NRG1b (EGF) _ mHSA_AnxV (142) and NRG1b (EGF) _ mHSA_AnxVm1234 fusion rotein.
Fig. 2 is the flow cytometry that in apoptosis heart cell, annexin V-FITC and propidium iodide (PI) apoptosis detect positive control.
Fig. 3 is the flow cytometric histograms of the passage of FITC and PI shown in Fig. 2.
Fig. 4 is the flow cytometry of IGF1_mHSA_AnxV and propidium iodide in apoptosis heart cell.
Fig. 5 is the flow cytometric histograms of the passage of FITC and PI shown in Fig. 4.
Fig. 6 is the flow cytometry of IGF1_mHSA_AnxVm1234 and propidium iodide in apoptosis heart cell.
Fig. 7 is the flow cytometric histograms of the passage of FITC and PI shown in Fig. 6.
Fig. 8 is the flow cytometry that in apoptosis heart cell, annexin V-FITC and propidium iodide (PI) apoptosis detect positive control.
Fig. 9 is the flow cytometric histograms of the passage of FITC and PI shown in Fig. 8.
Figure 10 is the flow cytometry of IGF1_mHSA_AnxV and propidium iodide in apoptosis heart cell, does not block in advance with IGF1.
Figure 11 is the flow cytometric histograms of the passage of FITC and PI shown in Figure 10.
Figure 12 is the flow cytometry of IGF1_mHSA_AnxVm1234 and propidium iodide in apoptosis heart cell, does not block in advance with IGF1.
Figure 13 is the flow cytometric histograms of the passage of FITC and PI shown in Figure 12.
Figure 14 is the flow cytometry of IGF1_mHSA_AnxV and propidium iodide in apoptosis heart cell, blocks 10 minutes in advance with 800nMIGF1.
Figure 15 is the flow cytometric histograms of the passage of FITC and PI shown in Figure 14.
The ESC that Figure 16 display presents apoptosis group derives heart cell, is with or without Zorubicin process.Ann-HSA＝AnxV_mHSA.M1234-Ann-HSA＝AnxVm1234_mHSA。
The flow cytometry that Figure 17 is AnxV_mHSA instead of AnxVm1234_mHSA specific binding apoptosis, ESC derives heart cell.
The flow cytometry that Figure 18 is AnxV_mHSA_NRG1b (EGF) specific binding apoptosis, ESC derives heart cell.
Figure 19 shows IGF1_mHSA_Syt1 and IGF1_mHSA_AnxV specific binding phosphatidylserine.
Figure 20 shows aDNASI1_mHSA_FGF2, aDNASI1_mHSA_NRG1b (EGF) and IGF1_mHSA_aDNASI1 specific binding DNA.
Figure 21 shows fusion rotein NRG1b (EGF) _ mHSA_AnxV and positive control NRG1b (EGF) to the stimulation of pAkt in DU145 cell.
Figure 22 shows fusion rotein AnxV_mHSA_NRG1b (EGF) and positive control NRG1b (EGF) to the stimulation of pAkt in DU145 cell.
Figure 23 shows fusion rotein IGF1_mHSA_AnxV, fusion rotein IGF1_mHSA_AnxVm1234 and the positive control IGF1 stimulation to pAkt in DU145 cell.
Figure 24 shows the stimulation of fusion rotein IGF1_mHSA_B7scFv and positive control IGF1 to pAkt in DU145 cell.
Figure 25 shows IGF1 and IGF1_mHSA_AnxV to be stimulated the dose response of pAkt in heart cell.
Figure 26 shows FGF2 and AnxV_mHSA_FGF2 derives pErk in myocardial cell stimulation to ESC.
The pAkt level that Figure 27 display is induced by albumen and apoptosis HL-1 cell (secret note) and untreated HL-1 cell (lath) pre-mixing.
Figure 28 display measures preparation 2 hearts/organize anatomical cardiac used with regard to ELISA.The cross section of the illustration display section B1 in the upper right corner.Section B1-infraction and B2 comprise most of or all infractions and neighbouring fringe region, and distal sections A and B1 mainly comprises health tissues.LV: left ventricle.
After Figure 29 shows three administrations, in heart, IGF1_mHSA_AnxV and non-binding IGF1_mHSA_AnxVm1234 fusion rotein detect.Secret note representative infraction is with the protein concentration that finds in fringe region and lath represents the protein concentration found in non-infarct area.Display often organizes 2 mouse.Group 2 corresponds to the mouse with targeting proteins IGF1_mHSA_AnxV administration, and group 3 corresponds to the mouse with non-binding variant IGF1_mHSA_AnxVm1234 administration.
Figure 30 is the representative Photomicrograph of the immunohistochemical staining of IGF1_mHSA_AnxV process mouse heart sections after 24 hours.
The representative Photomicrograph display of the heart sections immunohistochemical staining of Figure 31 blocking tissue of the mouse of IGF1_mHSA_AnxVm1234 process and fringe region.Time point is after administration 24 hours.
The contrast Photomicrograph of Figure 32 is for showing the dyeing specificity of mouse experiment anti-HSA primary antibodie used to the tissue containing HSA albumen or generation HSA.
Detailed Description Of The Invention
Many aspects of the present invention are for bispecific fusion protein, described albumen comprises 2 binding domain, and namely one has the target structural domain of binding specificity and one to the activation structure territory regulating the acceptor of tissue regeneration to have binding specificity to certain target molecules or target cell.In some embodiments, described target structural domain be used for by described bispecific fusion protein target target cell or tissue, activation structure territory is used for activating cells thus promote target tissue regeneration." bispecific fusion protein " used herein refers to the fusion rotein of energy specific binding 2 or more special molecular.
In some embodiments, described bispecific fusion protein comprises (1) has binding specificity target structural domain to tissue damaged's cell associated molecules, and wherein said molecule to be in born of the same parents and to be exposed to extracellular space in damaged cell in viable cell; (2) the activation structure territory of binding specificity is had to growth factor receptors or cytokine receptor in tissue, after wherein said activation structure territory is exposed to described growth factor receptors or cytokine receptor, this activation structure territory in conjunction with described growth factor receptors or cytokine receptor to regulate tissue regeneration or survival.
In some embodiments, described bispecific fusion protein comprises (1) has binding specificity target structural domain to tissue damaged's cell associated molecules, and wherein said molecule to be in born of the same parents and to be exposed to extracellular space in damaged cell in viable cell; (2) the activation structure territory of binding specificity is had to cell surface associated molecule in tissue, after wherein said activation structure territory is exposed to film associated molecule, this activation structure territory is in conjunction with described film associated molecule to regulate tissue regeneration and (3) transformation period instrumentality, and wherein said transformation period instrumentality regulates and controls the transformation period of described bispecific fusion protein.
In some embodiments, described bispecific fusion protein comprises: (1) is in conjunction with the target polypeptide structural domain of ischemic associated molecule; (2) activation structure territory, if growth factor polypeptide or cytokine antagonist polypeptide are to promote tissue regeneration or survival.
In some embodiments, described bispecific fusion protein comprises (1) has binding specificity target structural domain to the relevant target molecule of tissue; (2) have the binding domain (as activation structure territory) of binding specificity to cell surface associated molecule in tissue, after wherein said binding domain is exposed to described molecule, this binding domain in conjunction with described molecule to promote tissue regeneration or survival; (3) transformation period instrumentality, wherein said transformation period instrumentality regulates and controls the transformation period of described bispecific fusion protein.
In some embodiments, described bispecific fusion protein is transformation period instrumentality (HLM).In some embodiments, described HLM is polypeptide.Described HLM can have a 2 ends i.e. N-terminal and a C-terminal, to be connected and to be connected with described activation structure territory through peptide bond at another end at an end through peptide bond with described target polypeptide structural domain.In other embodiments, described transformation period instrumentality connects described activation structure territory or described target structural domain at an end (N-terminal or C-terminal).Therefore, described transformation period instrumentality can at the N-terminal of described bispecific fusion protein or C-terminal.Described transformation period instrumentality can connect described target structural domain or described activation structure territory through peptide bond.
The fusion rotein that other aspects of the present invention relate to comprises (1) has binding specificity at least one target structural domain to the relevant target molecule of at least one tissue; (2) cell surface associated molecule at least one tissue is had at least one binding domain (as activation structure territory) of binding specificity, after wherein said binding domain is exposed to described molecule, this binding domain in conjunction with described molecule to promote tissue regeneration or survival; (3) optional transformation period instrumentality, wherein said transformation period instrumentality regulates and controls the transformation period of described fusion rotein.In some embodiments, described fusion rotein comprises 2 or more target structural domains, and each target structural domain has the binding affinity to the relevant target molecule of tissue.Each target structural domain can have identical combination specificity (binding specificity as to same target molecule) or different binding specificity (binding specificity as to different target molecule).In some embodiments, described fusion rotein comprises 2 or more activation structure territories.Each activation structure territory can have identical combination specificity (binding specificity as to target molecule same on cell) or different binding specificity (binding specificity as to target molecule different on cell).
It will be understood by those skilled in the art that this bispecific fusion protein can be applicable to tissue regeneration.In some embodiments, after tissue or organ damage or histocyte may after impaired event, bispecific fusion protein can be used for diseased cells.In some embodiments, described bispecific fusion protein can activate the cell of expressing one or more somatomedins and/or cytokine (as chemokine) and/or integrin.In other embodiments, described bispecific fusion protein obtains application, such as, after such as damage or histocyte impairedly maybe may may become the event of functional disorder (the β cell dysfunctions as in diabetes), for the recruiting cells of one or more somatomedins and/or cytokine (as chemokine) acceptor and/or integrin (as stem cell, precursor cell or immune system cell) will be expressed to tissue.Reparation or regeneration that this bispecific fusion protein can be used for promoting damaged tissue or organ is given in body.
In some embodiments, bispecific fusion protein described herein can be used for regulating tissue survival.Such as, described bispecific fusion protein can strengthen or maintain cell viability.In some embodiments, described bispecific fusion protein can activate short survival or cell survival path.In some embodiments, the adjustable apoptosis of described dual specificity protein.
In some embodiments, described dual specificity protein can have (a) target polypeptide structural domain, wherein said target structural domain binding target molecule thus the activation structure territory the first cell of this bispecific fusion protein target tissue and (b) had receptor binding specificities.After described activation structure territory is exposed to described acceptor, the second cell receptor can be activated to promote that recruiting cells, apoptosis suppress, cell proliferation is induced, to urge survival pathway activation, regeneration, tissue survival in this activation structure territory.It will be understood by those skilled in the art that described bispecific fusion protein can act on same cell mass (as autocrine mode) or different cell mass (as paracrine mode) in conjunction with the first cell mass.In some embodiments, impaired first cell mass of described target structural domain specific binding is correlated with the second cell mass acceptor of target molecule and described activation structure territory specific binding viable cell.In some embodiments, the organizing specific target molecule on described target structural domain specific binding first cell mass surface and described activation structure territory specific effect are in the second cell mass.Described first cell can be viable cell, or " risky " cell." risky " used herein cell refers to not yet to experience apoptosis or not impaired but have the viable cell of damaged risk.
In some embodiments, described dual specificity protein has 2 different binding domain (described target structural domain and activation structure territory) of differing molecular on different cell in conjunctive tissue or organ.In other embodiments, described dual specificity protein has 2 different binding domain of different cell on same target cell in conjunctive tissue, and described target structural domain is chosen to specific binding target cell and described activation structure territory is chosen to promote tissue regeneration.
Term used herein " polypeptide " refers to the molecule that the multiple amino-acid residues be connected by peptide bond form.This term does not imply connected amino acid residue numbers.
Term used herein " dual specific " refers to fusion rotein and 2 interactional abilities of different ligands: target molecule (being combined by target polypeptide structural domain) and activation structure domain receptor.The binding characteristic in described target polypeptide structural domain and activation structure territory more discusses in detail hereinafter.
Term used herein " target molecule " refers to any molecule relevant to tissue (as ill or damaged tissue)." target molecule " refers to the cell of dual specificity protein or its target structural domain energy specific binding.Preferred target molecule is exposed to target molecule outside or enrichment thereon.In some embodiments, described target molecule is relevant to damaged cell, and described target molecule is living or to be in born of the same parents in non-damaged cell and to be exposed to extracellular space in damaged cell.This quasi-molecule comprises the molecule, myosin (comprising its tissue type specificity hypotype), ICAM-1 or the palatelet-selectin that such as expose in the cell experiencing downright bad (as DNA) or apoptosis (as phosphatidylserine).In other embodiments, described target molecule is the level comparing health or functioning cell or detection in organizing, at ill or the appearance of functional disorder cell or tissue surface or enrichment molecule.
Cell is surrounded by the plasma membrane (or cytolemma) containing lipid bilayer.Described cytolemma can regard as the surface of surface (tenuigenin side or cell interior) and the Cell-oriented outside with Cell-oriented matter, or extracellular space.Anionic phospholipid occurring apoptosis across bilayer movement from plasma membrane internal lobe to siphonal lobe.Described anionic phospholipid associated proteins such as annexin V, Synaptotagmin I or lactadherin can for detecting the existence of phosphatidylserine on cytolemma siphonal lobe.Phosphatidylserine is usually limited to live or the tenuigenin side form of non-damaged cell, and become in apoptosis and be exposed on external cell surface or the phosphatide of extracellular space.Phosphatidylserine has been used as the mark (see table 2) of in-vivo imaging research.
In some embodiments, described target molecule is " ischemic associated molecule "." ischemic associated molecule " is with any molecule of remarkable higher (as at least high 2 times) level detection after ischemic or anoxic.The test of any appropriate combination can be used for qualification ischemic associated molecule, comprise provided herein those.Survey molecule level increase may be the result that turnover is raised or reduced, may be maybe because accessibility increases (as cell injury causes).In some embodiments, detect that the described ischemic associated molecule level in post-ischemic tissue cell is significantly higher than the same histocyte (molecular specific is namely in post-ischemic tissue or enrichment wherein) that (as at least high 2 times) does not experience ischemic event.In other embodiments, described ischemic associated molecule relevant to cell injury (namely detecting that the level of molecule described in damaged cell is significantly higher than int same type cell).Some ischemic associated molecule enrichment in heart after ischemic event (2 times or higher) (or for simulating in the model system of heart ischemia).This quasi-molecule comprises the molecule of molecule or the enrichment in scar heart tissue such as exposed on the myocyte or other heart cells of experience downright bad (as DNA) or apoptosis (as phosphatidylserine), as the extracellular matrix protein of collagen (collagen I, III), myosin (comprising its cell type specificity hypotype) or enrichment in post ischemic cardiac.Identify that this molecular energy is according to enrichment after ischemia-reperfusion in body or in-vitro simulated ischemia-reperfusion, or being exposed to such as anoxic, ATP reduces, active oxygen (ROS) or nitric oxide synthetase (NOS) generate increase or cultured cell in vitro the condition such as serum starvation after.
Target polypeptide structural domain
Regulated by target polypeptide structural domain to the combination of the relevant target molecule of tissue (as ischemic associated molecule).This structural domain can be any peptide sequence for this function.Preferably, the combination of described target structural domain and described target molecule does not have biologic activity." biologic activity " used herein refers to the known clear and definite activity completed by molecule being exposed to fusion rotein structural domain.
In some embodiments, described target structural domain has the natural generation polypeptide of the non-antibody of binding affinity to described target molecule, its fragment or its variant.In other embodiments, described target structural domain comprises one or more antibody variable region.It will be understood by those skilled in the art that can consider can directly or indirectly in conjunction with any target structural domain of described target molecule.
In some embodiments, described target structural domain is annexin V (SEQIDNO:31), its fragment or its variant (SEQIDNO:81-83).Annexin V is in conjunction with phosphatidylserine (PS).In some embodiments, annexin V is modified into and replaces halfcystine 316 to reduce dimer formation with Serine or L-Ala.In some embodiments, annexin V is modified into and reduces annexin V internalization and maintain phosphatidylserine binding affinity simultaneously.In some embodiments, one or more annexin V residue can be changed combine to modify thus obtain more favourable target molecule combination rate or more favourable target molecule in conjunction with dissociation yield.In some embodiments, can human annexin V variant be used, wherein D144 be replaced to N and/or E228 A replace (see Mira, 1997; Kenis, 2004; Kenis2010 and Ungthum, 2010).
In other embodiments, described target structural domain is Synaptotagmin I, its fragment or its variant.Synaptotagmin I (SytI) display is in Ca (2+) dependency mode in conjunction with phosphatidylserine, and binding affinity is about 5-40nM.In some embodiments, one of 2 C2 structural domains (as C2B) of synaptotagmin can be used as target structural domain.In some embodiments, described target structural domain is the C2 structural domain (as protein kinase C, factor V and VIII) of the Ca2+ dependency film targeting proteins participating in signal transduction or film transport.In some embodiments, described target structural domain has sequence described in SEQID.NO:72.Lactadherin, also referred to as butterfat ball-EGF8, is in conjunction with glycoprotein by the 45kDa phosphatidylserine of macrophages secrete.Lactadherin comprises 2 C-structure territories of aminoterminal EGF spline structure territory and C-terminal.Therefore, in some embodiments, described target structural domain comprises the C-structure territory of lactadherin, its fragment or its variant.In some embodiments, one or more C2 domain residues can be changed combine to modify thus obtain more favourable target molecule combination rate or obtain more favourable target molecule in conjunction with dissociation yield.In some embodiments, described target structural domain has sequence described in SEQID.NO:85 or 86.In some embodiments, described target polypeptide structural domain comprises T cell immunoglobulin (Ig) MUC-1 and 4 (TIM albumen).In other embodiments, comprise can through blood plasma 2-glycoprotein 1 indirectly in conjunction with 3G4 antibody or the antibody domain of phosphatidylserine for described target polypeptide structural domain.In other embodiments, comprise can in conjunction with the anti-phosphatidylserine antibody (as PS4A7, SEQIDNO.30) of phosphatidylserine for described target polypeptide structural domain.
In some embodiments, described target polypeptide structural domain comprises the polypeptide of binding target molecule.Representational described polypeptide comprise or have be provided as herein SEQIDNO:31,72, the sequence of 81-83 or 85-86.Representational described polypeptide-nucleic acid sequence comprises or has the sequence being provided as SEQIDNO:225-232 or 235 herein.
Natural polypeptides can be used as target structural domain.But obviously also can use described natural polypeptides and the polypeptide of the sequence that changes, prerequisite is that described polypeptide retains with the ability of appropriate combination affinity (Kd) binding target molecule, as described in more detail below.
The albumen that " antibody " used herein is made up of one or more polypeptide of substantially being encoded by immunoglobulin gene.Classical antibody is the tetramer formed identical polypeptide chain by 2, and often pair has " gently " chain (about 25kD) and " weight " chain (about 50-70kD)." V l" and " V h" refer to these light and heavy chains respectively." antibody variable region " is antibody variable chain (V lor V h) N-terminal region, comprise the amino-acid residue of primary responsibility antigen recognition.Those of ordinary skill in the art easily can identify antibody variable region and measure the minimum size of giving needed for antigen recognition.Usually, antibody variable region comprises at least 70 amino-acid residues, is more often at least 100 amino-acid residues.The polypeptide comprising antibody variable region (but not must) can comprise other light and/or sequence of heavy chain further, and (but not must) can comprise non-antibody derived sequence further.Obvious antibody variable sequences can be natural generation, or using standard techniques is modified, and prerequisite is reservation function (antigen recognition).Some polypeptide comprising antibody variable region is single-chain antibody (antibody as Single polypeptide chain exists), more preferably single-chain Fv antibody (scFv), wherein Weight variable sequence and variable light district link together (directly or through peptide linker) form continuous polypeptide.Described scFv antibody can chemosynthesis or can from comprising the V directly connecting or connect through peptide encoding linker h-and V l-encoding sequence is at interior expression of nucleic acid.
" combination " or " specific binding " is exchanged in this article and is used and show dual specificity protein display to specific molecular (such as, the significant affinity of target structural domain display to target molecule, or the display of activation structure territory is to the significant affinity of cell surface associated molecule as acceptor) or the cell or tissue that carries this molecule have significant affinity, and think that it to have during significant affinity specific molecular at described fusion rotein (or its target polypeptide structural domain or its activation structure territory) and occur, its selectivity is not show the remarkable cross reactivity with other molecules.Preferred significantly combination comprises dissociation constant (K d) be 10 -6, 10 -7, 10 -8, 10 -9, 10 -10, 10 -11, 10 -12the combination of M or better.Such as, the interactional K of antibody-antigene dinstruction antibody concentration (being expressed as volumetric molar concentration), wherein 50% antibody and be combineding with each other of antigen molecule are in thermodynamic(al)equilibrium.Therefore, in suitable immobilized antigen concentration, higher (namely stronger) affinity antibodies of 50% is to obtain the antibody concentration conjugated antigen molecule needed for identical combination per-cent lower than with more low-affinity antibody.K dor kinetics combines and dissociation rate (k in conjunction withand k dissociate) ratio; I.e. K d=k dissociate/ k in conjunction with.Therefore, lower K dvalue instruction higher (stronger) avidity." better " used herein avidity is stronger avidity, identifies to have 10 of lower numerical value by the dissociation constant comparing the lower numerical value of its comparative -10mK dand therefore representative is better than 10 -9mK davidity.Generally preferably be better than 10 -7m, is preferably better than 10 -8the avidity of M (namely has lower K dvalue is also therefore stronger).Also consider the value in the middle of listed by herein, preferred binding affinity can be expressed as the dissociation constant of certain limit, and the preferred combination affinity of such as antibody disclosed herein is 10 by scope -6-10 -12the K of M (namely micromole is to picomole) dvalue represents, preferably 10 -7-10 -12m, more preferably 10 -8-10 -12m or better.The antibody " not showing remarkable cross reactivity " is that not obvious combination is missed the target the antibody of antigen.Such as, in one embodiment, the antibody of specificity and selective binding cardiac myosin can show to the binding affinity of cardiac myosin than the mhc molecule beyond cardiac myosin or non-myosin or peptide height at least 2 and preferably 3 or 4 an or more order of magnitude (namely combine that display is low by 2, the K of 3 or 4 an or more order of magnitude dvalue).Binding affinity and the selectivity art-recognized method of the described feature of any mensuration detect, and comprise and such as use Scatchard (Scatchard) to analyze and/or competitive (competition) binding tests.
Can with in multiple technologies well known in the art any one evaluate combine and measure K dvalue.Such as, DNA molecular is correlated with usually by being evaluated by appropriate solid upholder (as pearl, elisa plate or BIACORE chip) with target dna fragment bag in conjunction with ischemic.With regard to the target polypeptide structural domain in conjunction with any DNA sequence dna, 10 base pairs or larger DNA fragmentation (list or double-strand) are fixing on solid substrate.With regard to the target polypeptide structural domain of binding specificity sequence or DNA complex (as DNA-histone complex), fixing suitable corresponding target.Before adding ischemic associated molecule, the nonspecific binding site BSA of albumen, milk or any other suitable blocker are closed.Do not wrap and be used as Specificity control by hole or with the hole of non-target molecules bag quilt.The substrate or the contrast substrate that increase bispecific fusion protein (or target polypeptide structural domain) the target bag quilt of concentration are hatched.Also do not test as Specificity control in conjunction with the fusion rotein of target or structural domain.Then, by measuring with the standard scheme corresponding to solid support used and combination technology along with dosage increases the bispecific fusion protein (or target polypeptide structural domain) of change in conjunction with target and the amount contrasted, the target evaluating described bispecific fusion protein (or target polypeptide structural domain) is special, dose-dependently combination.Representational described scheme comprises Wassaf etc., Anal.Biochem.351 (2): 241-53 (2006); Epub2006 February 10 (BIACORE); With those described in Murray and Brown, J.Immunol.Methods.127 (1): 25-8 (1990) (ELISA).In addition, also carry out change fixed target molecule quantifier elimination or comprise level increase target molecule as competitor research with monitor combine and specificity.
Binding affinity with regard to binding target molecule and kinetics combine and dissociation rate standard technique is measured and with other negative control molecule (if any irrelevant target polypeptide fusion rotein or lack the fusion rotein of target polypeptide or have the fusion rotein of non-binding target polypeptide) and positive control molecule (as the parental generation antibody of target target molecule, or other antibody of known binding target molecule or antibody fragment) make comparisons.Such as, described non-binding target polypeptide can be non-in conjunction with human annexin V variant (SEQIDNO:84, nucleic acid sequence SEQ ID NO 233-234), non-binding synaptotagmin variant (SEQIDNO:74) or non-binding scFv (SEQIDNO:75; Nucleic acid sequence SEQ ID NO 236-237).
In some embodiments, K dmeasure with biosensor (such as, by surface plasma body resonant vibration (as BIAcore) or resonance image-forming analysis (resonance image-forming)).This mensuration can as Hefta etc., MeasuringAffinityUsingBiosensors (measuring avidity with biosensor), include in " AntibodyEngineering:APracticalApproach (antibody engineering: hands-on approach) ", McCafferty etc. (volume), 99-116 page (Oxford University Press (OxfordUniversityPress), 1996) and wherein carrying out described in institute's incorporated by reference document.In brief, kinetics combines and dissociation rate (k in conjunction withand k dissociate) measure with the sensor chip of coupling ischemic associated molecule.(k is combined for evaluating in conjunction with), the bispecific fusion protein (or target polypeptide structural domain) of different concns flows through chip, combines with Mass sen-sitivity test and monitoring simultaneously.Use BIAcore system (GE Medical Group (GEHealthcare); New Jersey Piscataway), k in conjunction withbe the slope that dR/dt compares R mapping, wherein R is the signal observed.In conjunction with after, to be observed by chip by making buffered soln and dissociate, measure k in a similar manner dissociate.Then following formula calculating K is used d:
K d=k dissociate/ k in conjunction with
In the context of the present invention, if bispecific fusion protein is to be less than 10 -8the K of M din conjunction with, be preferably less than 10 -7m, 10 -8m, 10 -9m or 10 -10m, then binding target molecule.In addition, described in this test, the combination of bispecific fusion protein and described target molecule is significantly higher than the combination of (as at least high 2,10 or 100 times) this bispecific fusion protein and negative control.Preferably, be combined also available excess soluble target to compete with fixed target target.
As mentioned above, some target molecule is specific to damaged cell (or enrichment).Representative target molecule includes but not limited to phosphatidylserine, DNA, myosin, cardiac myosin, c-Met (HGF acceptor), phosphatidylserine, palatelet-selectin and ICAM-1.With damaged cell in conjunction with the external confirmation of easy-to-use culturing cell, described cell is exposed to necrosis induced or apoptotic condition.Such as, necrosis is induced in cultured myocardial by simulated ischemia/Reperfu-sion, and monitor with LDH release test or Trypanblau test, then the apoptotic cell number of experience is deducted, basic as described in Shan etc., Am.J.Physiol.Cell.Physiol.294:833-841 (2008).The quantitatively total dead cell of this test and overall and apoptosis cell object difference owing to necrosis, as described in more detail below.The condition of cell death inducing comprises and is exposed to H 2o 2apoptosis can any one be monitored with in multiple technologies known in the art, described technology comprises such as annexin V and combines, the known caspase activated by apoptosis cuts target peptide sequence, or DNA ladder is (by TUNEL experimental measurement, basic as Kuramochi, J.Biol.Chem.279 (49): 51141 – 47 (2004) are described).By fluorescently-labeled bispecific fusion protein (or target polypeptide structural domain) or appropriate control albumen being added cell to evaluate the combination with the cell experiencing necrosis or apoptosis after cell death inducing or necrosis.Albumen and cell incubation number minute, to after one day, wash described cell and measure Cell binding fluorescence by immunofluorescence, flow cytometry or similar techniques.Or, detection can be used in conjunction with the additive method of bispecific fusion protein (or target polypeptide structural domain), comprise radio-labeling or use enzyme, bispecific fusion protein (or target polypeptide structural domain) or the antibody in conjunction with this fusion rotein (or target polypeptide structural domain) described in described enzyme coupling, this is the convention in ELISA operation scheme.If detect that the Cell binding after ischemic (as downright bad in cell experience or apoptosis) is significantly higher than the cell (as cell does not experience apoptosis or necrosis) that (as high 2 times) does not experience damage, then described bispecific fusion protein (or target polypeptide structural domain) is in conjunction with target cell.
By induce the ischemic in such as animal model and to compare before and after ischemic in target tissue give the level of bispecific fusion protein (or target polypeptide structural domain) to prove target in body.By the tissue injury in induced animal model, give bispecific fusion protein (or target polypeptide structural domain) and the level of bispecific fusion protein (or target polypeptide structural domain) in more impaired and non-damaged cell, prove target damaged cell in body.In one embodiment, described bispecific fusion protein is designed to the tissue injury region after target ischemical reperfusion injury.In this situation, in body, target proves to have come by induced tissue damage, preferably by causing ischemic and then building the method for blood supply.There are many methods can accomplish this point in different tissues.Such as, the blood flow flowing to mouse hind leg can with the of short duration blocking-up of simple tourniquet.Or, interim clamp can be used on the artery leading to kidney.Ischemical reperfusion injury is induced, as shown in mouse, rat, dog and pig by temporary interruption coronary artery in heart.In induced animal model, the exemplary process of tissue injury is summarized in table 1.
the exemplary process that table 1. damages for inducing ischemia-reperfusion
Animal model for ischemical reperfusion injury is described in further detail in the following references::
Greenberg etc., the 7th chapter .Mousemodelsofischemicangiogenesisandischemia-reperfusio ninjury (mouse model of ischemic blood vessels generation and ischemical reperfusion injury) .MethodsEnzymol.444:159-74 (2008).
Chimenti etc., Myocardialinfarction:animalmodels (myocardial infarction: animal model) .MethodsMol.Med.98:217-26 (2004).
BlackSC, Invivomodelsofmyocardialischemiaandreperfusioninjury:app licationtodrugdiscoveryandevaluation (In vivo model of myocardial infarction and reperfusion injury: be applied to drug development and evaluation) .J.Pharmacol.Toxicol.Methods43 (2): 153-67 (2000).
Establish targeting specific by such as Chen etc., FASEBJ.4 (12): 3033-39 (1990) described compares clamping and does not clamp bispecific fusion protein in kidney (or target polypeptide structural domain) and deposit, or as Zbinden etc., Am.J.Physiol.HeartCirc.Physiol.292:H1891-H1897 (2007) is described to be compared process and deposits with bispecific fusion protein (or target polypeptide structural domain) in untreated hind leg, uses radiolabeled bispecific fusion protein (or target polypeptide structural domain).Or, bispecific fusion protein (or target polypeptide structural domain) can detect in uniform formation with ELISA, maybe can with bispecific fusion protein (or the target polypeptide structural domain) real time imagery being marked with suitable imaging metal (as Tc99, Y or Gd).The specific deposition in heart damage region can as Dumont etc., Circulation102 (13): 1564-8 (2000) described mensuration.Prove that the exemplary process of targeting proteins damaged tissue is shown in table 2.
table 2. proves target damaged tissue
As mentioned above, some target polypeptide structural domain comprises the antibody of binding target molecule (as DNA, myosin, cardiac myosin, c-Met, palatelet-selectin, ICAM-1).In some embodiments, described target structural domain is antimyosin antibody (as the R11D-10 for Autopsy Cases sphaeroprotein, the 2G4-sD7 for cardiac myosin heavy chain, 1B2 and 5C2, the 2F4 for Autopsy Cases sphaeroprotein for Autopsy Cases immunoglobulin heavy chain, the monoclonal antibody for myosin, B7 antibody, B7scFv or other antibody known in the art).In some embodiments, some target polypeptide structural domain comprises the scFv antibody of binding target molecule.Such as, described target structural domain can be anti-DNASl-1scFv (aDNASl1, SEQIDNO:1 or 73) anti-DNASI-22scFv (aDNASl22, SEQIDNO:2).Representational described antibody and scFv antibody comprise or have and is provided as SEQIDNO:1,2,30,73 and the sequence of 76-80 herein.In some embodiments, representational described antibody and scFv antibody nucleic acids sequence comprise or have the sequence being provided as SEQIDNO220-224 herein.
Obvious function associated antibodies or can also substitute and is used as target polypeptide structural domain.Antibody interacts mainly through the amino-acid residue and target antigen being positioned at six weights and light chain complementarity determining area (CDR).For this reason, the diversity of the aminoacid sequence in CDR between independent antibody is higher than the sequence outside CDR.Be responsible for most of antibody-antigene due to CDR sequence to interact, produce the modified antibody of simulating initial antibodies character by the CDR sequence of combination from an antibody with the Frame sequence from different antibodies.This Frame sequence can available from the open DNA database comprising germline antibody gene sequence.
Therefore, herein institute provides one or more CDR of target polypeptide domain sequence can for generation of reservation initial target to the function associated antibodies of polypeptide domain in conjunction with feature.In one embodiment, one or more be selected from SEQIDNO:1,2,30,73 and the CDR district of 76-80 and known person framework region and CDR to recombinate the target polypeptide structural domain merging and produce other recombined engineerings and transform.Described heavy and light chain variable framework region can derived from identical or different antibody sequence.CDR district easily by with database as known array comparison in Vbase and IMGT is identified.The target polypeptide structural domain obtained and SEQIDNO:1,2,30,73 and the target polypeptide structural domain of 76-80 have one or more CDR.In some embodiments, described target polypeptide structural domain comprise SEQIDNO:1,2,30,73 and 76-80 described at least one CDR of sequence.
Well known antibody weighs and light chain CDR3 structural domain plays particularly important in the binding specificity/affinity of antibody to antigen.Therefore, in some embodiments, the antibody comprising specific antibodies described herein weight and/or light chain CDR3 is generated.Described antibody also can comprise weight and/or light chain CDR1 and/or CDR2 of antibody disclosed herein.
The CDR1 of above-mentioned engineered antibody, 2 and/or 3 districts can comprise those exact amino acid sequence disclosed herein.But, those of ordinary skill in the art should understand may have some deviations with accurate CDR sequence, particularly CDR1 and CDR2 sequence, it can tolerate and more changes than CDR3 sequence and do not change epitope specificity (described deviation is that such as conservative amino acid replaces).Therefore, in other embodiments, described engineered antibody can be made up of one or more CDR1 and CDR2, and described CDR1 and CDR2 has such as 80%, 90%, 95%, 98%, 99% or 99.5% homogeny with specifying the corresponding CDR of antibody herein.
In another embodiment, one or more CDR residue can be changed combine to modify thus obtain more favourable combination rate or more favourable in conjunction with dissociation yield.Use this strategy, superelevation binding affinity can have been obtained (as K d=10 -10or less) antibody.Affinity mature technology well known in the art can be used for changing CDR district, and then just the binding molecule obtained is screened in required combination change.Therefore, along with CDR changes, binding affinity and immunogenic change can be monitored and mark, thus obtaining and to combine with regard to best of breed and the antibody of reduced immunogenicity optimization.
One or more framework or joining region (i.e. the non-CDR residue) that can also resist body weight and/or variable region of light chain are modified, as long as the antigen binding affinity after these modifications does not significantly reduce.
Activation structure territory
Any polypeptide regulating cellular network activity or cell is raised to another location from a position is detected in described activation structure territory.In some embodiments, described activation structure territory is by carrying out activation signal Signal Transduction Pathways in conjunction with cell surface receptor.In some embodiments, some activation structure territory is any agonist of growth factor polypeptide, cytokine antagonist polypeptide (as chemokine polypeptides) or acceptor or integrin binding partner.Obvious this adjustment can be increase or the minimizing of cellular network activity, and described activity is as induced cell proliferation, induced cell growth, promotion cell survival and/or inhibited apoptosis.In some embodiments, other factors or cell (as stem cell) can be raised in described activation structure territory.
Growth factor polypeptide can detect the activation of growth regulation factor acceptor (as HGF or IGF acceptor).Some this peptide species is wild type hepatocytes somatomedin (HGF) or HGF α chain (as GENBANK accession number P14210), or it retains the derivative of at least 10% wild-type biology activity, as by measure in suitable target cell corresponding growth factor receptors activation measure.Can activation be assessed, such as, by measuring receptor kinase or downstream albumen as the phosphorylation of AKT, basic as described in Nishi etc., Proc.Natl.Acad.Sci.USA95:7018 – 7023 (1998).MTT and CTG known in the art also can be used to test.
In some embodiments, described activation structure territory is somatomedin.In some embodiments, described activation structure territory comprises aforementioned or protein variant.Representative activation structure territory includes but not limited to fibroblast growth factor (FGF), desmocyte growth factor-21 (FGF1), FGF2 (FGF2, also referred to as Prostatropin (bFGF)), the FGF2 (FGF2-146aa) of 146aa, the FGF2 (FGF2-157aa) of 157aa, fibroblast growth factor 4 (FGF4), fibroblast growth factor 7 (FGF7), Urogastron (EGF), rhIGF-1 (IGF), type-1 insulin like growth factor (IGF1), IMA-IGF2BP3-001 (IGF2), pHGF (HGF), pHGF-NK1 structural domain (HGF-NK1), pHGF-K1 structural domain (HGF-K1), pHGF-NK2 structural domain (HGF-NK2), pHGF-K2 structural domain (HGF-K2), neuregulin (NRG, also referred to as tune albumen (HRG)), neuregulin-β ectodomain (NRG1 β-ECD), neuregulin-1 β EGF spline structure territory (NRG1 β-EGF), thymosin, extrasin beta 4 (Tbeta4), granulocyte colony-stimulating factor (G-CSF), STEM CELL FACTOR (SCF, also referred to as mast cell growth factor (MGF)), periostin, vascular endothelial growth factor (VEGF, also referred to as VEGF-A (VEGF-A)), VEGF-A-121 (VEGF-A-121), VEGF-A-165 (VEGF-A-165), vascular endothelial growth factor-B (VEGF-B), vascular endothelial growth factor-B-167 (VEGF-B-167), VEGF-C (VEGF-C), stroma cell derivative factor (SDF), CXCL12 (SDF-1), Stromal cell-derived factor-1α (SDF-1 α), Thr6 PDGF BB (PDGF), PDGF-AA (PDGF-AA), PDGF-AB (PDGF-AB), PDGF-BB (PDGF-BB), teratoma derivative growth factor (TDGF), teratoma derivative growth factor 1 (TDGF1), nerve growth factor (NGF), β-nerve growth factor (β-NGF), Brain Derived Neurotrophic Factor (BDNF), neurotrophic factor-3 (NT-3), thrombopoietin (TPO), transforming growth factor-beta 1 (TGF-β 1), transforminggrowthfactor-β2 (TGF-β 2), Delicious peptide (BMP), bone morphogenesis protein-2 (BMP2), strand BMP-2 (scBMP2), Bone morphogenetic protein 3 (BMP3), bone morphogenetic protein 4 (BMP4), activin A, second born of the same parents element, beta-catenin, dickkopf homologue 1 (DKK1), erythropoietin (EPO), tethelin (GH), heparin binding epidermal growth factor like growth factor (HBEGF), Regular Insulin, interleukin (IL), interleukin 6 (IL-6), IL-10 (IL-10), interleukin-13 3 (IL-33), leukaemia inhibitory factor (LIF), MCP 1 (MCP1, also referred to as CCL2), PTN (PTN), tumor necrosis factor-alpha (TNF-α), Wnt, Wnt1, Wnt2, Wnt3a, Wnt7a, Wnt8a, Wnt11, or have specific antibody to described activator receptor, its variant, its isotype, its fragment, with its combination.In some embodiments, described activation structure territory is designed to comprise somatomedin strand or somatomedin structural domain.Such as, described activation structure territory is designed to comprise the somatomedin structural domain (as BMP-2) of 2 of linking together through joint (as GGGGSGGGGSGGGGS (SEQIDNO:103)) or more copy.
Representative growth factor polypeptide has sequence described in SEQIDNO:3-9,32-40 or 50-64 herein.Representative somatomedin can by nucleic acid sequence encoding described in this paper SEQIDNO:187-211.
As above with regard to some target polypeptide structural domains CDR discuss, also consider with the activation structure territory of SEQIDNO:3-9,32-40 or 50-64 or change the activation structure territory having one or more structural domain, module or aminoacid sequence.This structural domain, module or aminoacid sequence can be identified and this activation structure territory can with knowing technique construction.Therefore, in some embodiments, described activation structure territory comprises at least one structural domain of sequence described in SEQIDNO:3-9,32-40 or 50-64, module or aminoacid sequence or change.Similarly, cytokine antagonist polypeptide regulates the activation of corresponding cytokine receptor, measures by same way.
In some embodiments, described activation structure territory is the growth factor polypeptide in conjunction with growth factor receptors on cell surface.Representational described growth factor receptors is following acceptor: Urogastron (EGF), neuregulin/tune albumen (NRG/HRG), fibroblast growth factor (FGF), rhIGF-1 (as IGF-I), Thr6 PDGF BB (PDGF), vascular endothelial growth factor (VEGF) and its isotype (as VEGF-A or VEGF-C), teratoma derivative growth factor 1 (TDGF1), transforming growth factor-alpha (TGF-α), transforming growth factor-beta (TGF-β) and its isotype are as TGF-β 1 or TGF-β 2, thrombopoietin (THPO) or periostin.Acceptor described in other comprises hypertrophy/stem cell factor acceptor (SCFR), hepatocyte growth factor receptor (HGF acceptor, i.e. c-Met), ErbB-2, ErbB-3, ErbB-4, high-affinity trk C, BDNF/NT-3 growth factor receptors, NT-3 growth factor receptors or Vascular endothelial growth factor receptor-1 (VEGFR-I).
Representative cytokine receptor comprises such as FL cytokine receptor, the public chain acceptor of cytokine receptor γ, interleukin-10 receptor alpha chain, interleukin-10 receptor β chain, IL-12 receptor β-1 chain, IL-12 receptor β-2 chain, interleukin-13 receptor alpha-1 chain, interleukin-13 receptor alpha-2 chain, interleukin-17 acceptor, interleukin-17 B acceptor, IL-21 receptor precursor, interleukin-1 receptor I type, interleukin-1 receptor II type, Interleukin 2 Receptor α chain, Interleukin 2 Receptor β chain, interleukin-3 receptor alpha chain, interleukin-4 receptor α chain, IL-5 receptor alpha chain, interleukin-6 receptor alpha chain, interleukin-6 receptor β chain, IL-7 receptor alpha chain, high-affinity interleukin-8 acceptor A, high-affinity interleukin-8 acceptor B, IL-9 acceptor, IL-18 acceptor 1, interleukin-1 receptor sample 1 precursor, interleukin-1 receptor sample 2, toll sample acceptor 1, toll sample acceptor 2, toll sample acceptor 5, CX3C Chemokine Receptors 1, C-X-C CC chemokine receptor 3 type, C-X-C Chemokine receptor4 type, C-X-C chemokine receptor 5 type, C-X-C Chemokine Receptors 6 type, C-C Chemokine Receptors 1 type, C-C Chemokine Receptors 2 type, CCR3 type, C-C Chemokine receptor4 type, C-C Chemokine Receptors 6 type, C-C Chemokine Receptors 7 type precursor, C-C Chemokine Receptors 8 type, C-C Chemokine Receptors 9 type, C-C Chemokine Receptors 10 type, C-C Chemokine Receptors 11 type, Chemokine Receptors sample 2, with chemokine XC acceptor.Other activation structure territories are following acceptors: Solute Carrier organic anion transporter family member 1A2 (SLCO1A2), Sphingosine kinase 1 (SPHK1), minopontin (SPP1), also referred to as osteopontin (OPN), oncoprotein 53 (TP53), TnT 1 type (TNNT1), TSPY sample albumen 2 (TSPYL2), Nampt, WAP4 disulfide linkage Core domain 1 (WFDC1), extrasin beta 4, without wing MMTV integration site family member 11 (WNT11).Representative activation structure territory to comprise in such as phylaxin, the cell-derived proliferation-associated genes 1 (SIPA1) Yin sub-– 1 (SDF-1), signal induction of matrix and other parts listed above any one and substantially retains above-mentioned part in conjunction with homoreceptor ability and derivative.
Integrin is the acceptor regulating other cells of a certain cell attachment or its surrounding tissue.Integrin in conjunction with cell surface and extracellular matrix component as fibronectin, vitronectin, collagen and ln.Representative integrin comprises such as α 1β 1, α 2β 1, α 4β 1, α 5β 1, α 6β 1α lβ 2, α mβ 2, α iIbβ 3, α vβ 3, α vβ 5, α vβ 6, α 6β 4.
As initial testing, bispecific fusion protein (or its activation structure territory) and suitable acceptor in conjunction with available technological assessment known in the art.In a representative test, by being proved to combine by suitable solid support with the restructuring extracellular domain bag of suitable acceptor.The receptor extracellular domain of described bispecific fusion protein activation structure territory nonrecognition is used as Specificity control.Not there is the support substrate of any sessile receptor with comparing yet.Similar with the above-mentioned method in conjunction with ischemic associated molecule, prove dose-dependently bind receptor by the standard operation scheme corresponding with solid support used and combination technology.In addition, also carry out changing and combine and specificity monitor by the research as competitor of the scale of construction or the soluble target molecule that comprises increase level.Or described bispecific fusion protein is fixed on upholder and in conjunction with the solvable extracellular domain of corresponding acceptor for proving the specific combination of dose-dependently.
The binding affinity of described bispecific fusion protein and described receptors bind and kinetics is combined and dissociation rate is also measured by standard technique and (has the uncorrelated fusion rotein contrasting activation structure territory with other negative control molecule, lack the fusion rotein in activation structure territory) and positive control molecule (wild-type receptor part of recombinating, as somatomedin or cytokine) make comparisons.The balance of described bispecific fusion protein and kinetics incorporating parametric also with do not merge wild-type part survey same parameters and make comparisons to measure described part and whether affect this part normally in conjunction with its corresponding acceptor with the fusion of other molecules.Described Information Availability is in the effective dose measuring described bispecific fusion protein.
It is viewed that bispecific fusion protein is significantly higher than (as at least 100 times) negative control in conjunction with the affinity of fixed growth factor acceptor or cytokine receptor.In addition, its interactional antibody competition can be blocked with excessive soluble polypeptide, soluble receptors or Binding peptide or acceptor with the combination of sessile receptor.Preferably, described bispecific fusion protein with native ligand in conjunction with the affinity binding growth factor in its acceptor 10 0 times or cytokine receptor.
Bispecific fusion protein (with its activation structure territory) also has the ability regulating and associate receptor activation.The cell model evaluation of the available such as ischemia-reperfusion of described activity, described model uses cultured myocyte as neonatal cardiac myocytes (NRVM) or clone.Simulated ischemia (SI) generally comes initial by the anoxic in metabolic poison (deoxyglucose and hyposulfite) and metabolite (high potassium, lactic acid salt, low pH) or anaerobic chamber.Reperfu-sion is by being resuspended in oxidation buffer liquid to simulate.Develop external Human adult cardiomyocytes group ischemia model, described model provide 2 kinds of main ischemic component-anoxics and metabolite accumulation-without any exogenous metabolism inhibitor or metabolite.Table 3 shows exemplary process for proving that bispecific fusion protein prevents myocardial cell injury, promotes the ability of cardiac stem cells growth, motion or differentiation and/or promotion repair of damaged tissues.
table 3. activity rating method
Spontaneous growth Summing Factor cytokine can be used as activation structure territory.But, obviously the polypeptide portion that this native sequences and sequence change can also be used, prerequisite is that described polypeptide retains the ability (as used one of following test) activating homoreceptor, this kind of polypeptide can detect activated receptor, preferably activates described acceptor to observe with regard to native ligand at least 1% (preferably at least 10%) degree.Some activation structure territory of binding growth factor receptor is provided as SEQIDNO:3-9,32-40 and 50-64 in this article.Fusion rotein activity containing described sequence is well known (as Hashino etc., J.Biochem.119 (4): 604-609 (1996); Nishi etc., Proc.Natl.Acad.Sci.USA95:7018 – 23 (1998)).
Activation structure territory can be selected with regard to application-specific according to required treatment result.Such as, containing FGF2, VEGF α or its substantially retain in conjunction with the part of homoreceptor ability or the activation structure territory of derivative generally can be used for increase vasculogenesis.For increasing survival and for cytodifferentiation (regeneration) object, the activation structure territory containing IGF, HGF or NRG1 (or its part or derivative) can being used.
In certain situation, the activity simultaneously evaluating activation structure territory and target polypeptide may be needed.ELISA can be advantageously used in this object.
The substrate adsorbs of target polypeptide (as DNA), in elisa plate, is then closed with the suitable buffer containing BSA.Then add described bispecific fusion protein, then add the restructuring substrate (such as, if activator is somatomedin, then substrate is restructuring homoreceptor or receptor fragments (extracellular domain)) in described activation structure territory.This substrate carries out fluorescent mark for detecting or detecting with the traget antibody of the acceptor regions of not remarkably influenced ligand binding.
The activity in vivo of described bispecific fusion protein is generally evaluated by signal transmission change in the molecule of this bispecific fusion protein activation structure territory adjustment by detecting.This generally includes the change of cell surface receptor phosphorylation state or downstream mediator as Phosphorylated-AKT or phosphorylation-ERK, as the flow cytometry of treated cell, immunofluorescence, ELISA, phosphorylation mark or Western analyze detect.Other functional evaluation comprise tests (reduced number of TUNEL positive cell) or DNA ladder by dyeing and the number of viable cells test of identification of morphology, the Level of Apoptosis test being combined (through immunofluorescence) or flow cytometry by annexin V, the test of detection Caspase Activity, TUNEL-.In each situation, if bispecific fusion protein induces remarkable (as at least 2 times) level of the modulated molecule of described testing inspection, functionally active or phosphorylation to change, then it works in vivo.The available any clinical relevant criterion evaluation of the reparation of damaged tissue in patient.Such as, detect the reparation of blocking tissue by quantitates cell number as myocyte, inoblast number or scar amount, or by the output of heart function or the function test of configuration aspects, comprise LVEDP, LVDP ,+dp/dT, LV weight, room volume and diastole left ventricle aneurysm.Know these methods evaluated and describe in detail in the literature.Generally, if produce remarkable (as at least 2 times) change in any described clinical evaluation, think that bispecific fusion protein repairs damaged tissue.
Transformation period instrumentality
It will be understood by those skilled in the art that the dual specificity protein being used for the treatment of application may not show optimum serum half-life due to its low relative molecular amount.Therefore, in some treatment use, the transformation period regulating described dual specificity protein may be needed.In some embodiments, for realizing described dual specificity protein in ill damage or the accumulation damaging organic region, this dual specificity protein coupling has transformation period instrumentality.This transformation period instrumentality can increase the Half-life in vivo of described fusion rotein.Such as, the transformation period of the dual specificity protein containing transformation period instrumentality is about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours or larger.In some embodiments, the transformation period of the dual specificity protein containing transformation period instrumentality is about 24 hours or larger.In some embodiments, the transformation period of the dual specificity protein containing transformation period instrumentality is about 1 week or larger.
Described target polypeptide structural domain directly can be connected through peptide bond with activation structure territory.In some embodiments, they can connect through transformation period instrumentality.In a preferred embodiment, described transformation period instrumentality is polypeptide.Therefore, described transformation period instrumentality can have 2 ends, i.e. a N-terminal and a C-terminal.In some embodiments, described transformation period instrumentality connects described target polypeptide structural domain at an end through peptide bond and connects described activation structure territory at another end through peptide bond.In some embodiments, described joint to be connected with described target polypeptide domain C end at N-terminal and to be connected with described activation structure territory N-terminal at C-terminal.In other embodiments, described joint to be connected with described target polypeptide structural domain at C-terminal and to be connected with described activation structure territory at N-terminal.In other embodiments, described transformation period instrumentality connects at one of described dual specificity protein end.Such as, in some embodiments, described transformation period instrumentality is connected with described activation structure territory N-terminal at C-terminal.In other embodiments, described transformation period instrumentality connects at described target domain C end.In other embodiments, described transformation period instrumentality can be connected with described activation structure territory C-terminal at N-terminal.In other embodiments, described transformation period instrumentality can be connected with described target domain C end at N-terminal.
In some embodiments, described transformation period instrumentality is designed to promote described bispecific fusion protein size and exceedes about 70kDa or equivalent radius minimizes to make kidney remove.In some embodiments, described transformation period instrumentality be designed by the receptor-mediated recirculation of FcRn or in conjunction with serum component as human serum albumin (HSA) extend as described in the bispecific fusion protein transformation period.
Preferably, described transformation period instrumentality is non-immunogenic in people.Described transformation period instrumentality can be human serum protein or its retain the derivative of at least 50% sequence thereto on the region that is made up of at least 100 continuous amino acids." sequence thereto " used herein refers in the background of many Nucleotide or peptide sequence and reference sequence, when described polynucleotide or peptide sequence carry out optimum comparison, these polynucleotide or peptide sequence identical or there is specified percentage reference sequence in the identical Nucleotide in corresponding position or residue.
In some embodiments, described transformation period instrumentality is modified by the glycosylation of one or more glycosylation site existing in this transformation period instrumentality.Such as, following amino acid: l-asparagine, Serine, Threonine can add or remove to change the glycosylation of described transformation period instrumentality.In some embodiments, the glycosylation of transformation period instrumentality described in described dual specificity protein can regulate the transformation period of this dual specificity protein.In some embodiments, described transformation period instrumentality sequence is modified to reduce glycosylation.This modification comprises Asn (N) and is replaced by Gln (Q) or Ala (A), and/or Ser (S) or Thr (T) is replaced by Ala (A).
Human serum albumin (HSA) has natural long serum half-life, and part is because it is in conjunction with FcRN and recirculation.HSA is albumen the abundantest in blood and is presented at safety in people.In some embodiments, the l-asparagine of HSA position 503 can remove amide group and reduce the transformation period, replaces remove by N503Q.In some embodiments, the halfcystine C34 of HSA can be replaced to Serine or L-Ala (S or A) to remove free cysteine and to make selectivity disulfide formation minimum.In some embodiments, described transformation period instrumentality is the modified forms of the modification HSA Domain III (mHSA_dIII) having N503Q replacement and extra terminal glycine.Described modified forms retains HSA in conjunction with the characteristic of FcRn increases serum half-life.In some embodiments, described transformation period instrumentality comprise with human serum albumin aminoacid sequence (SEQIDNO:12) at least 70%, 80%, 85%, 90% or 95% identical at least 100 continuous amino acids.In some embodiments, described transformation period instrumentality comprise SEQIDNO:10,12, sequence described in 24-28,65 or 67.In some embodiments, described transformation period instrumentality nucleotide sequence comprises sequence described in SEQIDNO:212-215.
In some embodiments, described transformation period instrumentality comprise with human a-fetoprotein (AFP) aminoacid sequence (SEQIDNO:29,68) at least 70%, 80%, 85%, 90% or 95% identical at least 100 continuous amino acids.In some embodiments, the N-connection glycosylation site of AFP is removed by N251Q replacement.In some embodiments, described transformation period instrumentality comprises sequence described in SEQIDNOs:29,68 or 69.In some embodiments, described transformation period instrumentality nucleotide sequence comprises sequence described in SEQIDNO:216.
In some embodiments, described transformation period instrumentality comprise with vitamin D binding protein (VDBP) aminoacid sequence at least 70%, 80%, 85%, 90% or 95% identical at least 100 continuous amino acids.In some embodiments, the N-connection glycosylation site of VDBP removes by N288Q or N288T replacement.In some embodiments, described transformation period instrumentality comprises sequence described in SEQIDNO:66.In some embodiments, described transformation period instrumentality nucleotide sequence comprises sequence described in SEQIDNO:219.
In some embodiments, described transformation period instrumentality comprise with people's transthyretin (TTR) aminoacid sequence at least 70%, 80%, 85%, 90% or 95% identical at least 100 continuous amino acids.In some embodiments, transthyretin is modified to remove N118N-glycosylation site.In some embodiments, described transformation period instrumentality is the monomeric form of TTR.
In some embodiments, described transformation period instrumentality comprise with people Fc aminoacid sequence at least 70%, 80%, 85%, 90% or 95% identical at least 100 continuous amino acids.The Fc structural domain of antibody has the natural ability in conjunction with FcRn, and the transformation period is extended.In some embodiments, the engineered one-tenth of Fc structural domain of antibody is not in conjunction with Fc (γ) R.In an illustrative embodiments, the engineered one-tenth Q of Fc structural domain replaces N397 (N297Q variant).In some embodiments, described transformation period instrumentality is called the mono-poly-variant form of the Fc of scFc.Such as, native dimericization forms the IgG heavy chain subgroup of Fc is hinge-CH2-CH3.In some embodiments, Fc structural domain is engineered is shaped as strand, and this is by connecting hinge-CH2-CH3 with flexible joint if GGGGSGGGGSGGGGSGGGGS (SEQIDNO:104) is to produce hinge-CH2-CH3-joint-hinge-CH2-CH3 chain.In an illustrative embodiments, the engineered one-tenth Q of strand Fc (scFc) replaces N397 and replaces C220 (N297Q, C220S) with S.In some embodiments, scFc structural domain comprises sequence described in SEQIDNO:71.In some embodiments, described transformation period instrumentality nucleotide sequence comprises sequence described in SEQIDNO:218.
In some embodiments, described transformation period instrumentality comprise with PASization aminoacid sequence at least 70%, 80%, 85%, 90% or 95% identical at least 100 continuous amino acids.PASization be simulation PEGization proline(Pro)-, L-Ala-and/or Serine enriched sequence (see WO/2008,155134).The polypeptide segment of proline(Pro), L-Ala and/or Serine is formed with the semi-structured three-dimensional structure territory of large hydraulic radius, thus reduces fusion rotein removing.In some embodiments, PASization aminoacid sequence is about 200,300,400,500 or 600 amino acid lengths.Such as, PASization is 20 times of repetitions of aminoacid sequence ASPAAPAPASPAAPAPSAPA (SEQIDNO:105).
In some embodiments, described transformation period instrumentality comprise with albumin bound people from territory antibody (albudAb) aminoacid sequence (SEQIDNO:70) at least 70%, 80%, 85%, 90% or 95% identical at least 100 continuous amino acids.Albumin bound domain antibodies increases the fusion rotein transformation period (see WO2008/096158) by Non-covalent binding serum albumin.In some embodiments, described albumin bound people from territory antibody engineering becomes remove C-terminal l-asparagine thus remove Lys-ArgKex2 protease site.In some embodiments, described transformation period instrumentality nucleotide sequence comprises sequence described in SEQIDNO:217.
Representational described transformation period instrumentality comprise SEQIDNO:10,12,14-29,45-49,65-71 or 105 arbitrary described those.
In some embodiments, described transformation period instrumentality can be modified into and cysteine residues is replaced to Serine or alanine residue to reduce the ability forming disulfide linkage.
Described transformation period instrumentality can be included in or be coupled in bispecific fusion protein separately or with short (as 2-20 amino-acid residue) linker polypeptide.In some embodiments, at one or two end, described linker polypeptide occurs at the N-terminal of described transformation period instrumentality, C-terminal or N-terminal and C-terminal.Suitable short linker polypeptide for described joint N-terminal comprises such as dipeptides such as – Gly-Ala-, and (GA) is with – Ala-Ser-(AS).Suitable short linker polypeptide for described joint C-terminal comprises such as dipeptides such as – Leu-Gln-, and (LQ) is with – Thr-Gly-(TG).In some embodiments, described linker is longer than 2 amino acid.Such as, described linker is 5,10,5,20,30,40,50,60,70,80,90,100 amino acid lengths.Preferably, described linker is (as glycine rich) or structurizing flexibly (as the enrichment of α spiral).In some embodiments, described linker or peptide linker have sequence described in SEQIDNO:41-42,87-91 or 244.In some embodiments, described linker based on people's albumen as transthyretin.
SEQIDNO:46-49 is described in the SEQIDNO:45 transformation period instrumentality of N and the representative linker dipeptides of C-terminal.But linker (if there is) can be positioned at either one or two end of described transformation period instrumentality described in obvious described short linker polypeptide and SEQIDNO:95-104 or 182-184.
Compare the fusion rotein without transformation period instrumentality, the transformation period that some preferred transformation period instrumentality provides described bispecific fusion protein to extend.Transformation period instrumentality can with the test evaluation measuring physiological condition stability inferior to the effect of transformation period.Such as, bispecific fusion protein can in serum (as human serum) 37 DEG C hatch 120 hours, when hatching beginning and shift out sample in every 24 hours afterwards.Then, above-mentioned binding tests is carried out to detect the functional bispecific fusion rotein level of each time point.Then compare this level to contrast to provide the transformation period from the bispecific fusion protein level built without (or with different transformation period instrumentalities) in transformation period instrumentality situation.
Selectable unit and representative bispecific fusion protein
Obvious element selective is in addition to the foregoing included in institute herein and provides in bispecific fusion protein.Described element can exist for multiple object, comprises and promotes expression, preparation or purifying bispecific fusion rotein or run target function.Such as, the leading polypeptide of N-terminal can be there is.Representative leading polypeptide comprises or has SEQIDNO:41-42,87-91 or 244 arbitrary described sequences.Bispecific fusion protein or alternatively can also comprise polyhistidine (as six Histidines) label to promote purifying.This label comprises at least six Histidine continuous amino acid residues, and can be positioned at C or N-terminal.In some embodiments, the C-terminal of described bispecific fusion protein comprises hexahistidine tag.Also can there are other amino-acid residues in polyhistidine with residue bispecific fusion protein junction.Some bispecific fusion protein provided herein comprise SEQIDNO:43,44 or 92-94 described in containing the polypeptide of C-terminal polyhistidine.
Some bispecific fusion protein has and meets one of following universal architecture (from N-terminal to C-terminal, left-to-right display):
Or
Or
Or
Or
Or
Representative bispecific fusion protein comprises (from N-terminal to C-terminal):
(a) leading polypeptide (such as comprise or there is sequence described in SEQIDNO:41-42,87-91 or 244);
(b) target polypeptide structural domain (such as comprise or have SEQIDNO:1,2,30,31,72,73, sequence described in 76-83 or 85-86);
(c) optional short linker polypeptide;
(d) transformation period instrumentality (such as comprise or have SEQIDNO:10,12,14-29,45-49,65-71 or 105 arbitrary described sequences);
(e) optional short linker polypeptide;
(f) activation structure territory (such as comprise or there is the arbitrary described sequence of SEQIDNO:3-9,32-40 and 50-64); With
(g) polypeptide containing polyhistidine (such as containing the polypeptide of six Histidines, as comprised or have the polypeptide of sequence as described in SEQIDNO:43-44 or 92-94).
Such as, described in some, bispecific fusion protein comprises (from N-terminal to C-terminal):
(a) leading polypeptide (such as comprise or there is sequence described in SEQIDNO:41-42,87-91 or 244);
(b) target polypeptide structural domain (such as comprise or have SEQIDNO:1,2,30,31,72,73, sequence described in 76-83 or 85-86);
(c) transformation period instrumentality (such as comprise or have SEQIDNO:10,12,14-29,45-49,65-71 or 105 arbitrary described sequences);
(d) optional short linker polypeptide;
(e) activation structure territory (such as comprise or there is the arbitrary described sequence of SEQIDNO:3-9,32-40 and 50-64); With
(f) polypeptide containing polyhistidine (such as containing the polypeptide of six Histidines, as comprised or have the polypeptide of sequence as described in SEQIDNO:43-44 or 92-94).
Other bispecific fusion proteins comprise (from N-terminal to C-terminal):
(a) leading polypeptide (such as comprise or there is sequence described in SEQIDNO:41-42,87-91 or 244);
(b) activation structure territory (such as comprise or there is the arbitrary described sequence of SEQIDNO:3-9,32-40 and 50-64);
(c) optional short linker polypeptide;
(d) transformation period instrumentality (such as comprise or have SEQIDNO:10,12,14-29,45-49,65-71 or 105 arbitrary described sequences);
(e) optional short linker polypeptide;
(f) target polypeptide structural domain (such as comprise or have SEQIDNO:1,2,30,31,72,73, sequence described in 76-83 or 85-86);
(g) polypeptide containing polyhistidine (such as containing the polypeptide of six Histidines, as comprised or have the polypeptide of sequence as described in SEQIDNO:43-44 or 92-94).
Other bispecific fusion proteins comprise (from N-terminal to C-terminal):
(a) leading polypeptide (such as comprise or there is sequence described in SEQIDNO:41-42,87-91 or 244);
(b) transformation period instrumentality (such as comprise or have SEQIDNO:10,12,14-29,45-49,65-71 or 105 arbitrary described sequences);
(c) optional short linker polypeptide;
(d) activation structure territory (such as comprise or there is the arbitrary described sequence of SEQIDNO:3-9,32-40 and 50-64);
(e) optional short linker polypeptide;
(f) target polypeptide structural domain (such as comprise or have SEQIDNO:1,2,30,31,72,73, sequence described in 76-83 or 85-86);
(g) polypeptide containing polyhistidine (such as containing the polypeptide of six Histidines, as comprised or have the polypeptide of sequence as described in SEQIDNO:43-44 or 92-94).
Other bispecific fusion proteins comprise (from N-terminal to C-terminal):
(a) leading polypeptide (such as comprise or there is sequence described in SEQIDNO:41-42,87-91 or 244);
(b) transformation period instrumentality (such as comprise or have SEQIDNO:10,12,14-29,45-49,65-71 or 105 arbitrary described sequences);
(c) optional short linker polypeptide;
(d) target polypeptide structural domain (such as comprise or have SEQIDNO:1,2,30,31,72,73, sequence described in 76-83 or 85-86);
(e) optional short linker polypeptide;
(f) activation structure territory (such as comprise or there is the arbitrary described sequence of SEQIDNO:3-9,32-40 and 50-64);
(g) polypeptide containing polyhistidine (such as containing the polypeptide of six Histidines, as comprised or have the polypeptide of sequence as described in SEQIDNO:43-44 or 92-94).
Other bispecific fusion proteins comprise (from N-terminal to C-terminal):
(a) leading polypeptide (such as comprise or there is sequence described in SEQIDNO:41-42,87-91 or 244);
(b) target polypeptide structural domain (such as comprise or have SEQIDNO:1,2,30,31,72,73, sequence described in 76-83 or 85-86);
(c) optional short linker polypeptide;
(d) activation structure territory (such as comprise or there is the arbitrary described sequence of SEQIDNO:3-9,32-40 and 50-64);
(e) optional short linker polypeptide;
(f) transformation period instrumentality (such as comprise or have SEQIDNO:10,12,14-29,45-49,65-71 or 105 arbitrary described sequences);
(g) polypeptide containing polyhistidine (such as containing the polypeptide of six Histidines, as comprised or have the polypeptide of sequence as described in SEQIDNO:43-44 or 92-94).
Other bispecific fusion proteins comprise (from N-terminal to C-terminal):
(a) leading polypeptide (such as comprise or there is sequence described in SEQIDNO:41-42,87-91 or 244);
(b) activation structure territory (such as comprise or there is the arbitrary described sequence of SEQIDNO:3-9,32-40 and 50-64);
(c) optional short linker polypeptide;
(d) target polypeptide structural domain (such as comprise or have SEQIDNO:1,2,30,31,72,73, sequence described in 76-83 or 85-86);
(e) optional short linker polypeptide;
(f) transformation period instrumentality (such as comprise or have SEQIDNO:10,12,14-29,45-49,65-71 or 105 arbitrary described sequences);
(g) polypeptide containing polyhistidine (such as containing the polypeptide of six Histidines, as comprised or have the polypeptide of sequence as described in SEQIDNO:43-44 or 92-94).
In some embodiments, described short linker polypeptide comprises sequence described in SEQIDNO:95-104 or 182-184.
In some embodiments, described optional short linker polypeptide is dipeptides (Gly-Ala; Ala-Ser; Leu-Gln; Or there is the polypeptide of aminoacid sequence listed by SEQIDNO:95-104 and 182-184 Thr-Gly).
Representative bispecific fusion protein includes but not limited to: aDNASI1_mHSA_IGF1, aPS4A7_mHSA_IGF1, aDNASI1_mHSA_HGF (NK1), aPS4A7_mHSA_HGF (NK1), AnxV_mHSA_FGF2, AnxV_mHSA_NRG1b (EGF), aDNASI1_mHSA_FGF2, aDNASI1_mHSA_NRG1b (EGF), AnxV_mHSA_VEGFB (111), AnxV_mHSA_VEGFB (167), AnxV_mHSA_HGF (NK1), AnxV_mHSA_IGF1, IGF1_mHSA_AnxV, HGF (NK1) _ mHSA_AnxV, NRG1b (EGF) _ mHSA_AnxV, FGF2_mHSA_AnxV, VEGFB (167) _ mHSA_AnxV, VEGFB (111) _ mHSA_AnxV, IGF1_mHSA_B7scFv, IGF1_mHSA_Syt1, IGF1_mHSA_aDNASI1, NRG1b (EGF) _ mHSA_B7scFv, NRG1b (EGF) _ mHSA_Syt1, NRG1b (EGF) _ mHSA_aDNASI1, FGF2_mHSA_B7scFv, FGF2_mHSA_Syt1, FGF2_mHSA_aDNASI1, B7scFv_mHSA_IGF1, Syt1_mHSA_IGF1, aDNASI1_mHSA_IGF1, B7scFv_mHSA_NRG1b (EGF), Syt1_mHSA_NRG1b (EGF), B7scFv_mHSA_FGF2, Syt1_mHSA_FGF2.Representative bispecific fusion protein can have SEQIDNO; 106,108,110,112,118,120,124,126,128,130,132,134,136,140,142,144,146,148,150,152,154,156,158,160,162,164,166,168,170,172,174,176,178, sequence described in 180, or can by tool SEQIDNO:107,109,111,113,119,121,125,127,129,131,133,135,137,141,143,145,147,149,151,153,155,157,159,161,163,165, the nucleic acid encoding of sequence described in 167,169,171,173,175,179 or 181.
Representative bispecific fusion protein containing non-binding target polypeptide includes but not limited to: DAscFv_mHSA_IGF1, DAscFv_mHSA_HGF (NK1), AnxVm1234_mHSA_VEGFB (111), AnxVm1234_mHSA_VEGFB (167), AnxVm1234_mHSA_HGF (NK1), AnxVm1234_mHSA_IGF1, AnxVm1234_mHSA_NRG1b (EGF), AnxVm1234_mHSA_FGF2, HGF (NK1) _ mHSA_AnxVm1234, NRG1b (EGF) _ mHSA_AnxVm1234, FGF2_mHSA_AnxVm1234, VEGFB (167) _ mHSA_AnxVm1234, VEGFB (111) _ mHSA_AnxVm1234, IGF1_mHSA_DAscFv, NRG1b (EGF) _ mHSA_DAscFv, FGF2_mHSA_DAscFv, DAscFv_mHSA_NRG1b (EGF) and DAscFv_mHSA_FGF2.Representative bispecific fusion protein can have SEQIDNO114,116,122,138,185,246,248,254,258,260,262,264,272,274, sequence described in 276 or can by tool SEQIDNO:115,116,123,139,186,247,249,255,259,261, the nucleic acid encoding of sequence described in 263,265,273,275 or 277.
Prepare bispecific fusion protein
Bispecific fusion protein using standard techniques synthesizes, and comprises liquid phase and Solid phase peptide synthesis and recombinant DNA technology.For solid phase synthesis, the C-terminal amino acid of described sequence connects insoluble upholder, and remaining amino acid adds successively.For being longer than about 50 amino acid whose polypeptide, shorter region can be synthesized in this way, then the longer polypeptide of concentrated formation.The method forming peptide bond by activating C-terminal (as used coupling agent N, N'-dicyclohexylcarbodiimide) is well known.
For recombinant DNA technology, the DNA of encoding bispecific fusion rotein is prepared chemically or is prepared by the DNA be separated be connected each fusion rotein part of encoding.The DNA of each section of encoding bispecific fusion rotein can be separated and de novo synthesis from known.The method of direct chemical synthesising DNA is well known, and described synthesis automatic DNA synthesizer DNA routine is carried out.Chemosynthesis produces single stranded polynucleotide, and it is by being transformed into double-stranded DNA with complementary sequence hybridization or with archaeal dna polymerase.Although chemical synthesising DNA is generally limited to the sequence shorter than bispecific fusion protein, obviously bispecific fusion protein obtains by connecting comparatively short data records in frame completely.Or the DNA sequence dna of encoding bispecific fusion rotein is by clone's preparation.Clone technology is well known, and be described in detail in such as Standard reference works, as Sambrook etc., MolecularCloning:ALaboratoryManual (" molecular cloning: laboratory manual ") (the 3rd edition), CSH Press (ColdSpringHarborLaboratoryPress) (2001).DNA part can link together in frame generation complete encoding sequence.
Once obtain the DNA of encoding bispecific fusion rotein, described DNA can be cloned into the carrier of expressing for protokaryon or eukaryotic host cell.Technology DNA being included in described carrier is known for those of ordinary skill in the art.In this expression vector, the DNA operability of encoding bispecific fusion rotein connects the nucleotide sequence (as suitable promoter and termination signal (as needs)) needed for expressing.Promotor is the nucleotide sequence (being usually located at the 5' of encoding sequence) instructing the encoding sequence of adjacent connection to transcribe.Termination signal can be the terminator codon and/or the transcription termination signal that stop translation.Other regulatory elements (as enhancer element) also can be present in expression vector.This carrier is preferably plasmid or virus vector.Preferably, expression vector also comprises the selective marker of giving and selecting resistance.This makes in carrier to its karyomit(e) described in cell energy stable integration and growth forms stove point, itself so that can clone and increase in clone.Multiple choices known in the art mark, comprises the gene such as provided penbritin, methotrexate, mycophenolic acid, aminoglycoside G-418, Totomycin and puromycin-resistant.Those of ordinary skill in the art understand very much protein expression can numerous expression systems, comprise intestinal bacteria (E.coli), other host bacteriums, yeast and multiple higher eucaryotic cells as COS, CHO, HeLa and myeloma cell line.
Host cell standard method conversion or transfection contain the carrier of bispecific fusion protein coding DNA.Expressing in host cell makes DNA be transcribed into corresponding mRNA, then translates mRNA and produces bispecific fusion protein.
Once express, described bispecific fusion protein according to standard method purifying, can comprise such as ammonium sulfate precipitation or affinity column chromatography.With regard to pharmaceutical application, preferably at least about the substantially pure composition of 90-95% homogeneity, most preferably 98-99% or higher homogeneity.Once partial purification or reach homogeneous as required, if be used for the treatment of application, described polypeptide should substantially containing intracellular toxin.
Pharmaceutical composition
The present invention also provides the pharmaceutical composition that pharmacologically can accept vehicle containing at least one bispecific fusion protein described herein and at least one.Described composition can be used for treating suffer from tissue injury or damage risk in a organized way patient to prevent tissue injury or reparation or regenerating damaged tissue.Described patient comprises the patient such as experiencing myocardial infarction, renal impairment and/or ischemic stroke.As needs, in described pharmaceutical composition, also can comprise other activeconstituentss, as stem cell or other promote the reagent of repair of damaged tissues.
Term used herein " physiology can accept " to refer to be ratified by Federal Regulatory Agencies or state government or generally acknowledge for animal at " American Pharmacopeia " (U.S.Pharmacopeia) or other and particularly for the pharmacopeia of people in list.The thinner, adjuvant, vehicle or the supporting agent that give described bispecific fusion protein showed in term " vehicle ".It can be sterile liquid that physiology can accept vehicle, Ru Shui and oil, comprises those (as peanut oil, soybean oil, mineral oil or sesame oil) that oil, animal, plant or synthesis are originated.When pharmaceutical composition intravenously gives, water is preferred vehicle.Salt brine solution and dextrose and aqueous glycerin solution can also be used as liquid carrier, specific for injection solution.Suitable drug excipient comprises such as starch, glucose, lactose, sucrose, gelatin, Fructus Hordei Germinatus, rice, flour, whiting, silica gel, sodium stearate, glyceryl monostearate, talcum, sodium-chlor, skim-milk, glycerine, propylene, ethylene glycol, water and ethanol.As needs, described composition also can comprise a small amount of wetting agent or emulsifying agent or pH buffer reagent.
Can be used for anyly suitablely giving mode by compounding pharmaceutical composition, comprise such as parenteral, the inside and outside use of nose, oral or local and give, as by transdermal means, for the process of preventing and/or treating property.What these compositions can adopt multiple applicable mode of administration knows any one in form, as solution, suspension, emulsion, tablet, pill, capsule, powder agent, aerosol and sustained release preparation.Described composition can be mixed with suppository, with traditional binders and vehicle as triglyceride.Oral preparations can comprise standard vehicles as phannaceutical grades of mannitol, lactose, starch, Magnesium Stearate, soluble saccharin, Mierocrystalline cellulose, magnesiumcarbonate etc.Suitable medicine gives pattern and vehicle example is described in " Remington:TheScienceandPracticeofPharmacy (Lei Mingdun: pharmaceutical science and put into practice) " A.R.Gennaro and compiles, Donald Lippincott Williams Louis Wilkins publishing company (LippincottWilliams & Wilkins) (the 21st edition, 2005) of philadelphia, pa.
Usually, pharmaceutical composition parenteral provided herein gives (as by intravenously, intramuscular or subcutaneous injection) or oral administration or topical application.Give for parenteral, described bispecific fusion protein can suspend or be dissolved in described vehicle.General preferred sterile aqueous carrier, as water, buffered water, salt solution or phosphate buffered saline (PBS).In addition, aseptic, fixing oil can be used as solvent or suspension medium.For this purpose, the fixing oil of any gentleness can be used, comprise monoglyceride or the triglyceride of synthesis.In addition, lipid acid such as oleic acid can be used for preparing injectable composition.Pharmaceutically acceptable auxiliary substance also can include roughly physiological condition in, as pH regulator and buffer reagent, tension regulator, dispersion agent, suspension agent, wetting agent, stain remover, sanitas, local anesthetic and buffer reagent.
In one preferred embodiment, described pharmaceutical composition preparation gives patient (as people) for intravenously.Usually, the medicine that intravenously gives is sterile isotonic water-based buffered soln.When needing, described composition also can comprise solubilizing agent and local anesthetic if lignocaine is to alleviate the pain of injection site.Generally, described composition separately provides or mixes in unit dosage, such as, indicate dry lyophilized powder in the sealing of active agent content (as sealing gland) container such as ampoule or medicine bag or without aqueous concentrate.When described composition infusion gives, the available infusion bottle containing sterile pharmaceutical grade water or salt solution distributes.When the injection of described composition gives, the Injectable sterile water of an ampoule or salt solution can be provided thus described composition can mix before administration.
Be intended to can be provided as such as tablet, lozenge, lozenge, water-based or oleaginous suspension, dispersible powder or granule, emulsion, hard or soft capsule or syrup or elixir for the composition of oral application.Described composition also can comprise one or more components as sweeting agent, odorant, tinting material and sanitas.Tablet comprises activeconstituents, is mixed with the physiology being suitable for producing tablet and can accepts vehicle.Described vehicle comprises such as inert diluent, granulating and disintegrating agent, tackiness agent and lubricant.The preparation of oral application can be provided as hard gelatin capsule, wherein said activeconstituents mixed inert solid diluent, or is provided as soft gelatin capsule, wherein said activeconstituents mixing water or oily medium.Aqueous suspension comprises active material, is mixed with the vehicle that one or more are suitable for producing aqueous suspension.This vehicle comprises suspension agent and dispersion or wetting agent.Be suitable for, by adding water and prepare the dispersible powder of aqueous suspension and granule providing activeconstituents, being mixed with dispersion or wetting agent, suspension agent and one or more sanitass.
Oleaginous suspension is by being suspended from vegetables oil (as peanut oil, sweet oil, sesame oil or Oleum Cocois) or mineral oil such as whiteruss is prepared by activeconstituents.Pharmaceutical composition also can adopt O/w emulsion form.Oil phase can be vegetables oil or mineral oil or its mixture.Suitable emulsifying agent comprises the natural gum of such as natural generation, the phosphatide of natural generation and acid anhydride.
Pharmaceutical composition comes degerming by conventional sterilization techniques, or can sterile filtration.Aseptic aqueous solution can pack use or freeze-drying, and freeze-dried products combines with sterile aqueous carrier before administration.The pH of aqueous pharmaceutical compositions is generally 3-11, more preferably 5-9 or 6-8, most preferably 7-8, as 7-7.5.
Bispecific fusion protein provided herein is generally present in pharmaceutical composition with a certain concentration, thus give patient's single dose can delivery treatments significant quantity.Treatment significant quantity is the amount producing obvious patient benefit, as can be detected reparation or regenerating damaged tissue or reducing tissue injury symptom.Treatment significant quantity can close to the amount being enough to realize detecting tissue repair or regeneration in one or more animal model exemplified by table 3.But obvious many factors can affect treatment significant quantity, comprise the activity of bispecific fusion protein used; Patient age, body weight, general health, sex and diet; Administration time and approach; Excretion rate; Anyly to treat, as drug regimen simultaneously; With type and the seriousness of tissue injury in patients receiving treatment.Optimal dosage can be determined by conventionally test and well known method.Dosage range is generally every dosage and is about 0.5mg – and is about 400mg bispecific fusion protein (as every dosage 0.5mg, 1mg, 2mg, 5mg, 10mg, 50mg, 100mg, 200mg, 300mg or 400mg).Generally, dosage level scope is preferably provided to be the composition that every kg body weight is about that 0.1mg-is about 100mg.In some embodiments, dosage unit form comprises about 10mg-and is about 100mg bispecific fusion protein.
Pharmaceutical composition can be packed and be used for the treatment of or prevent tissue injury (as treatment myocardial infarction or renal impairment).The pharmaceutical preparation of packaging comprises the container of at least one pharmaceutical composition described herein of Preservation therapy significant quantity and indicates contained composition to be used for the treatment of the specification sheets (as label) of patient tissue damage (as treatment myocardial infarction or renal impairment).Pharmaceutical composition can be packaged in multiple single dosage unit, respectively containing the fixed amount bispecific fusion protein in packing.Or described container can preserve the pharmaceutical composition of multiple doses.
Methods for the treatment of
Described pharmaceutical composition can give patient (preferred mammal, as ox, pig, horse, chicken, cat, dog, or more preferably people) to treat the pathological tissue damage of patient.In the context of the invention, term " treatment " comprises prevention and therapy administration.In prophylactic application, pharmaceutical composition described herein given susceptible or have the patient of development pathological tissue damage risk with prevention, delay in addition or reduce tissue injury seriousness.In therapeutic application, carry out treating reducing pathological tissue damage seriousness or regenerating tissues after injury.In some embodiments, described pharmaceutical composition can give with other treatment combinatorial association.
Representative pathological tissue damage comprises the tissue injury after damage to cardiac tissue (as myocardial infarction associated injury), Renal tissues damage and ischemic stroke (as cerebral ischemia, also referred to as cerebral infarction, critical limb ischemia or other ischemics).In some embodiments, described pharmaceutical composition to be used for after tissue or organ damage protective tissue from infringement and/or regenerating tissues and/or blood supply.
In some embodiments, described pharmaceutical composition can be given such as, with prevention, delay, minimizing or treatment autoimmune disease, systemic lupus erythematosus (SLE), also referred to as lupus.SLE is autoimmune disease, wherein many tissues or system under attack and inflammation, such as joint, skin, liver, kidney, hemocyte, heart, lung, neural system, blood vessel.Immunity system generates autoantibody, specific for nucleoprotein and DNA.In some embodiments, described pharmaceutical composition can give the object that needs with protective tissue after injury from infringement and regenerating tissues.In some embodiments, described pharmaceutical composition can be combined with existing immunosuppression or other treatment and given.
In some embodiments, described pharmaceutical composition can give the object of needs with prevention, delay, minimizing or treatment type i diabetes.In type i diabetes, in the immune system destruction pancreas of body self, generate the β cell of Regular Insulin.In some embodiments, described pharmaceutical composition can give the object of needs to regenerate β cell.In some embodiments, described pharmaceutical composition can be treated to combine with type i diabetes known in the art and be given.
In some embodiments, described pharmaceutical composition can give the object of needs with prevention, delay, minimizing or treated tissue or organ collpase.Such as, described pharmaceutical composition can be used for the treatment of brain, spinal cord or nerve degeneration as alzheimer's disease, Parkinson's disease, multiple sclerosis or amyotrophic lateral sclerosis (ALS), also referred to as Lu Geli creutzfeldt jakob disease.In some embodiments, described pharmaceutical composition can be combined with existing treatment known in the art and given.
In some embodiments, described pharmaceutical composition can give the object of needs with prevention, delay, minimizing or treatment bone and/or cartilage related disorder.In some embodiments, described pharmaceutical composition can be used for Regenerated Bone and/or cartilaginous tissue.Described pharmaceutical composition can be combined with existing treatment known in the art and given.
Any one in multiple known delivery system can be used for giving bispecific fusion protein, comprises and is such as encapsulated in liposome, particulate, microcapsule, the reconstitution cell expressing described bispecific fusion protein, receptor-mediated or retrovirus or other nucleic acid carriers.Described bispecific fusion protein gives by any approach that facilitates, such as by infusion or bolus injection, by adsorbing through epithelium or mucocutaneous lining (oral mucosa, rectum and intestinal mucosa etc.), and can give together with other biological promoting agent.Give can be whole body or local.In addition, may need, by any suitable pathways, described bispecific fusion protein is introduced central nervous system, comprise Intraventricular and intrathecal injection; Intracerebral ventricle injection can be promoted by intraventricular catheter, such as in conjunction with storage as Ao Maye (Ommaya) storage.Lung also can be used to give, such as, by using sucker or atomizer, and have the preparation of propellant.
In a particular implementation, region bispecific fusion protein local of the present invention being given needs treatment may be needed; This realizes by local infusion, topical application (as combined with wound dressing after surgery), injection, conduit mode, suppository routes or graft mode in such as operation, described graft has porous, non-porous or colloidal material, comprises film as silicon rubber (sialastic) film or fiber.In another embodiment, vesica as lipid physical efficiency be used for sending as described in bispecific fusion protein.In another embodiment, described bispecific fusion protein is sent in controlled release system; Such as, this controlled release system can be placed in therapeutic targets (as experienced or the biological organs of damage risk in a organized way) or its near.The ordinary skilled artisan that bases on practicality of described delivery system.
In some embodiments, bispecific fusion protein provided herein effectively treats pathological tissue damage, and this is by the ability of stem cell recruitment to damaged tissue at least partly because of it.In some situation, sufficient stem cell can reside in patient (as resident cardiac stem cells).But in some embodiments, giving stem cell (as bone marrow derived autologous stem cells) altogether may be useful.This stem cell can give before or after described bispecific fusion protein, or can give (in same medicine composition or the composition that separates) simultaneously.
In some embodiments, bispecific fusion protein provided herein effectively improves tissue survival.In some embodiments, described bispecific fusion protein can be given and target particular organization or organ (as heart).Then, described dual specificity protein is accumulated in particular organization or organ (heart contrary with another organ) by target structural domain binding target molecule related tissue.Once in conjunction with described target molecule, described bispecific fusion protein can be separated with this target molecule, leaves and associates (as damaged cell or " risky " cell) with the target molecule of similar paracrine mode and different tissues cell, growth factor receptors or cytokine receptor again.
As mentioned above, optimal dosage depends on some factor known in the art, but general range is every dosage is about 0.5mg-and is about 400mg bispecific fusion protein (as every dosage 10mg, 50mg, 100mg, 200mg, 300mg or 400mg).The bispecific fusion protein of certain dose (in aforementioned pharmaceutical compositions) treatability gives patient, per hour, every day, weekly, monthly or once a year or repeatedly (as per hour, every day, weekly, monthly or annual 2,4,5,6,7,8,9,10,11 or 12 times).More generally give every day or single dose weekly, comprising scope is the bispecific fusion protein amount that every kg body weight is about that 0.1mg – is about 100mg.
In other embodiments, pharmaceutical composition containing bispecific fusion protein can give patient, dosage range is weekly about 0.1mg-about 2500mg weekly, about 0.1mg-about 10mg weekly weekly, about 1mg-about 100mg weekly weekly, weekly about 10mg-about 500mg weekly, weekly about 100mg-about 2500mg weekly, about 10mg-about 100mg weekly weekly, or about 100mg-about 1000mg weekly weekly.Or, can give the pharmaceutical composition containing bispecific fusion protein, dosage range is every other day about 0.1mg-every other day about 500mg, every other day about 1mg-every other day about 75mg, every other day about 10mg-every other day about 50mg, or every other day about 20mg-every other day about 40mg.Or, can give the pharmaceutical composition containing bispecific fusion protein, dosage range is weekly 3 about 0.1mg-3 about 100mg weekly, weekly 3 about 1mg-3 about 75mg weekly, 3 about 10mg-3 about 50mg weekly weekly, or 3 about 20mg-3 about 40mg weekly weekly.
In other embodiments, the pharmaceutical composition containing bispecific fusion protein is given Mammals (as people), continuous 1,2,3 or 4 hour; Every day 1,2,3 or 4 times; Every other day or every three, four, five or six days; 1,2,3,4,5,6,7,8,9 or 10 time weekly; Twice weekly; Monthly 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 or 30 time; Monthly twice; Every six months 1,2,3,4,5,6,7,8,9 or 10 time; Annual 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19 or 20 time; Or annual twice.Obviously the pharmaceutical composition containing bispecific fusion protein can but must not give with different frequency during treatment plan.
The following example provides by way of illustration and is not construed as limiting.Except as otherwise noted, all reagent and solvent are normal business level and do not need to be further purified to use.Use conventional improvement, the method that the following example provides can be changed to prepare and use other bispecific fusion proteins in the scope of the invention and pharmaceutical composition by those of ordinary skill in the art.
Embodiment
The preparation of the representative bispecific fusion protein of embodiment 1.
Prepare bispecific fusion protein, wherein target polypeptide structural domain is in conjunction with DNA and activation structure territory is NRG1.Described 2 structural domains are connected by modification human serum albumin (HSA) joint.NRG1 and the HSA joint N-terminal including short linker polypeptide in recombinates and to be merged and anti-DNAscFv and the modification HSA joint C-terminal of including extra short linker polypeptide in are recombinated and merged.Described modification HSA joint comprises 2 aminoacid replacement.The cysteine residues of natural HSA position 34 is mutated into Serine and is oxidized to reduce this site the potential albumen heterogeneity caused.May be reduced the pharmacology transformation period to going the natural HSA position 503 place l-asparagine of amide group sensitivity, it is mutated into glutamine.The circulating half-life that described bispecific fusion protein extends given by described modification HSA joint.
The external activity of embodiment 2. bispecific fusion protein
2 kinds of composition activities (wherein said target polypeptide structural domain is in conjunction with DNA and described activation structure territory is NRG1) ELISA test of representative bispecific fusion protein prepared by embodiment 1,2 arms that described ELISA is designed to only when this bispecific fusion protein are simultaneously active in conjunction with producing during its substrate.ELISA is basic as Stokes etc., J.Clin.Pathol.35 (5): 566 – 573 (1982) and Gripenberg etc., Scand.J.Immunol.1:151-157 carries out described in (1978).More specifically, the PBS solution of 1-50ng/ml bispecific fusion protein is added plate well (anti-DS-DNA antibody ELISA test kit (the international Alpha diagnostic companies (AlphaDiagnosticInternational) of preadsorption DNA, AutogenBioclear company of Britain (DistbyAutogenBioclear, UK))) and hatch according to manufacturer's guide and wash until add the step detecting antibody.In this stage, PBS/1%BSA/0.05% tween (Tween) solution of 100 μ l1-50ng/ml biotinylation goat-anti people NRG1-β 1 (peace enlightening biology (R & DSystems) BAF377) (antibody of ' activating sub-arm ') is added the porose and incubated at room of institute 1 hour, wash in the PBS having 0.05% tween 20.The 100 μ l Streptavidin-HRP that dilute in PBS (the liquid storage 2ug/ml of 1:200 dilution, (peace enlightening biology 890803)) add each hole and incubated at room 30 minutes.After washing eventually in the PBS having 0.05% tween 20, add 100 μ lSuperSignalELISAPico chemical luminous substrates (according to manufacturer's specification sheets, Pierre Si (Pierce), cat#34077) and (Hewlett-Packard (Packard) or analogous instrument are measured luminous (representing positive signal) at Fusion microplate reader.
Be significantly higher than (at least high 100 times) by measured signal amount in the hole of bispecific fusion protein and there is no DNA or the negative control containing dead arm (namely not comprising activation structure territory or target polypeptide structural domain).In addition, find that signal changes along with the bispecific fusion protein amount adding hand-hole.
The activity in vivo of embodiment 3. bispecific fusion protein
The activity in vivo of representative bispecific fusion protein prepared by embodiment 1 is measured by the signal intensity in detection fusion protein activation structural domain institute adjustment module.For the activation structure territory of this fusion rotein NRG1, by detecting that comparing untreated or simulation process heart increase with phosphorylation ErbB-3 in described Bispecific fusion thing process heart cell evaluates activity.Trachea cannula, ventilation and thoracotomy are postoperative produces myocardial infarction by ligation arteria coroaria sinistra (LCA) in C57BL/6 mouse.Coronary occlusion is confirmed by the variable color of acute inspection left ventricular wall, the closed front Electrocardiographic ST assessment of chest.Sham-operation mouse accepts identical surgical procedure and does not carry out LCA ligation.
Be shifted out the mouse after normal mouse or induced myocardial infarction, through contrast and the heart of bispecific fusion protein process mouse, 4% paraformaldehyde is fixed, embedding, section and installation, as Dhein, Mohr and Delmar, PracticalMethodsinCardiovascularResearch (" cardiovascular research hands-on approach "), described in 2005,473rd page (Springer Verlag (Springer) in New York).Phosphorylation-ErbB3 antibody (CST (CellSignalingTechnology); Massachusetts Bei Fuli) for passing through Immunofluorescence test phosphorylation-ErbB3.Observe phosphorylation-ErbB3 level that treated heart compares untreated heart and increase by 2 times or more and deixis activates son.Cell number (every visual field quantity, or total percentage), every cell signal intensity of display increase or both increase.
The embodiment 4. mouse tissue injury repairing of bispecific fusion protein
Embodiment 1 give the mouse after myocardial infarction containing the composition of representative bispecific fusion protein, myocardial infarction is induced as mentioned above.By intravenous injection (as tail vein).After giving, heart function is evaluated as follows.By Chloral Hydrate (400mg/kg body weight, i.p.) anesthetized mice, with micro-tip pressure sensor (model SPR-671, meter La Er (Millar)) intubate right carotid with the LV+ measuring left ventricle (LV) pressure and close in chest preparation and-dP/dt.Make comparisons to confirm affect heart function with bispecific fusion protein process with the measurement of untreated control mouse gained.Observe the remarkable improvement of heart function, by these measurement for Evaluation of at least one.
The expression and purification of embodiment 5. fusion rotein
Design, expression and purification are containing the fusion rotein in target structural domain, transformation period instrumentality and activation structure territory.Target structural domain and the activation structure territory of multiple combination are assembled with mHSA (SEQID10) transformation period instrumentality, different short connecting peptides sequence, not the homopolypeptide leader sequence of different directions.With regard to each aminoacid sequence design and synthesis DNA sequence dna, consider that the codon of expection expression biological (as CHO or pichia pastoris phaff (Pichiapastoris)) uses, comprises or avoid the demand of specific Restriction Enzyme recognition site, other codon optimized factors known in the art.DNA sequence dna builds and/or is assembled in expression plasmid, described Plastid transformation to expressing in biology, fusion rotein process LAN.Then, each fusion rotein differently combines purifying, comprises Reactive blue Sepharose Chromatography, Ni affinity chromatography, anion-exchange chromatography and size exclusion chromatography.
Coding intact fusion protein or include in fusion rotein part DNA (as individual target structural domain, transformation period Regulatory domain or activation structure territory) buy from commercial source (BioBasic, DNA2.0).Clearly define aminoacid sequence.By constraint condition as codon use and restriction site (require or forbid) are pass on to supplier.The whole DNA sequence dna of interest encodes albumen is selected to avoid low expression (as avoiding the high secondary structure of mRNA level in-site) and supplier's preference according to these constraint conditions, general strategy by supplier from the theoretical library of isocoding sequence.In certain situation, codon is used and is applicable to independent CHO or pichia pastoris phaff.In other situations, the sub-use table of the combination pin of rare codon in arbitrary bio distribution is avoided in application.In certain situation, supplier provides the fusion rotein of the total length in expression vector.In other situations, need to be subcloned into interested expression vector.Subclone operation traditional method completes, and uses II type Restriction Enzyme and DNA ligase (New England's biology laboratory (NewEnglandBiolabs)).Other molecular clonings are carried out to generate the fusion rotein having the target in alternative combinations and direction, activation and transformation period Regulatory domain with these technology and polymerase chain reaction (PCR).Fusion rotein with on DNA level between functional domain the one or more II type restriction sites design of junction to replace or to reset any functional domain flexibly.When needing, restriction site or connector area add in sequence by including PCR the primer in.
In certain situation, albumen PichiaPink expression system (hero company (Invitrogen) A11151 test kit) is expressed in pichia pastoris phaff.Gene yeast saccharomyces cerevisiae (Saccharomycescerevisiae) α-conjugative element secretion signal of interest encodes albumen is by cloning with secreting, expressing recombinant protein in pPink α-HC plasmid frame.In other situations, albumen Selexis/CHO cloning system is purified.The gene clone of interest encodes albumen is in Selexis carrier and be transfected into polyclone CHO-K1 cell to express recombinant protein.PPink α-HC plasmid comprises bacterial origin of replication (pUC) and resistance marker (penbritin) for propagation with select in intestinal bacteria circular plasmids.It also comprises TRP2 gene and ADE2 gene, and described TRP2 gene is used for the integration of target linearized vector during being transformed into pichia spp, and described ADE2 gene comprises for the adenine auxotrophic in complementary pichia spp.High level after AOX1 promotor guarantees methanol induction is transcribed and is guaranteed effective Transcription Termination with CYC1 sequence.Described plasmid integration can carry out the selection of viability driving to ADE2-defective type pichia spp and screen according to colony colour on the substratum lacking VITAMIN B4.High copy intasome presents white, and low copy intasome presents pink colour or redness, this is because accumulate purine precursors in pichia spp vacuole.Generate with regard to albumen and select white colony, and in some cases, screen several bacterium colony at the front just small-scale of scale operation (liter) (milliliter) albumen formation efficiency.PPink α-HC plasmid figure and details can available from hero companies.
In other situations, albumen Selexis/CHO cloning system is purified.Exemplary expression vectors is pMP20K (SELEXIS) and exemplary cells system is CHO-kl-S (SELEXIS).PMP20K uses conventional genetic elements.Express by people GAPD promoters driven.Be called that the genetic elements of matrix association regions or MAR element controls chromatinic dynamic organization, near isolation, gene is from the impact of peripheral chromatin, thus increases dependence copy number, the genetic expression irrelevant with position.The display of MAR element improves the possibility of separating clone and increases generation stability, and described clone shows required expression level for generating recombinant protein.Except expression plasmid, antibiotic resistance plasmids (as pSV2-neo, SELEXIS) also can be used to select stable conversion.Linearizing (as with Pvul) expression plasmid, then QIAQUICK purifying (Kai Jie company (QIAGEN)).Liposome LTX (hero company) for being transfected into Chinese hamster ovary celI in OptiMemI (Ji Bu can (Gibco)).Transfectional cell recovers 2 days with containing the F12Hams substratum of 10%FBS and does not have selective pressure, then in addition selective pressure 4 days, becomes the serum free medium having selective pressure subsequently. (Sai Mo fly generation you (ThermoScientific)) merges BBA for SA-, has HT fill-in (Ji Bu can (GIBCO)).
After expression, by the combination of Reactive blue Sepharose Chromatography, Ni affinity chromatography, anion-exchange chromatography and size exclusion chromatography according to manufacturer's specification sheets (GE Medical Group) purifying protein.Monitor albumen by SDS-PAGE (SDS-PAGE) to generate.
Protein expression in pichia pastoris phaff and pass through chromatogram purification subsequently
The gene yeast saccharomyces cerevisiae α-conjugative element secretion signal (SEQID244,245) of interest encodes albumen is by cloning with secreting, expressing recombinant protein in pPink α-HC plasmid (being included in hero A11151 test kit) frame.In addition, the DNA of coding His6-label adds to described gene 3' end to select by Ni affinity chromatography purification of recombinant proteins.In brief, Plastid transformation becomes Competent PichiaPink bacterial strain 2 (hero company, article No. A11154), culture is at BMGY (buffering compound glycerin substratum=1% yeast extract, 2% peptone, 100mM potassiumphosphate, pH6.0, have 1.34% yeast nitrogen of ammonium sulfate, without amino acid, 0.0004% vitamin H, 1% glycerine) in shaking culture case 30 DEG C grow to OD600=2-6.Now, sedimentation cell, by (cushioning composite methanol substratum=1% yeast extract, 2% peptone, 100mM potassiumphosphate with BMMY, pH6.0, have 1.34% yeast nitrogen of ammonium sulfate, without amino acid, 0.0004% vitamin H, 0.5-1% methyl alcohol) replace substratum with 1/5 original culture volume and take inducible protein and express.Then, culture 20-30 DEG C of regrowth 24-48 hour in shaking culture case.Every 12-24 hour, adds culture by extra methyl alcohol (to final concentration 0.5-1% (v/v)).During results, cell by centrifugation, collects supernatant, and sterile filtration 4 DEG C are preserved until purifying (usually in results 3 days).
According to the following fusion rotein of following method purifying: IGF1_mHSA_AnxV (SEQID136,137); IGF1_mHSA_AnxVm1234 (SEQID138,139); NRG1b (EGF) _ mHSA_AnxV (SEQID142,143); NRG1b (EGF) _ mHSA_AnxVm1234 (SEQID254,255); FGF2_mHSA_AnxV (SEQID144,145).With Ni agarose 6 speed stream resin (GE medical treatment & health (Healthcare) 17-5318-04 by run by gravity; 1mL resin/50mL supernatant) according to manufacturer's specification sheets by Ni affinity chromatography purification of recombinant proteins.Then, the effluent from these purifying cushions exchange in 50mMNaCl, 20mMTris, pH7.0, with centrifugal concentrator, is loaded on the HiTrapBlueHP1mL cylinder (GE Medical Group, 17-0412-01) that balances in same buffer.Described albumen is according to manufacturer's specification sheets 20mMTris, and pH7.0,50mMNaCl, 30mM Sodium octoate is as elution buffer purifying.Merge the eluate from Ni affinity chromatography and blue-sepharose chromatogram and concentrated/buffering exchanges to PBS (100mM sodium phosphate, 150mMNaCl), in pH7.2, use centrifugal concentrator.Then, sample is loaded into HiPrep26/60SephacrylS-200 high resolution column (GEHealthcare17-1195-01), described albumen at PBS (100mM sodium phosphate, 150mMNaCl), with flow velocity 1.3mL/min wash-out in pH7.2.What collection was identified by (SDS-PAGE) contains proteins of interest part and concentrates with centrifugal concentrator.
Whole purity is evaluated by SDS-PAGE.Fig. 1 shows the SDS-PAGE of IGF1_mHSA_AnxV (136), IGF1_mHSA_AnxVm1234 (138), NRG1b (EGF) _ mHSA_AnxV (142) and NRG1b (EGF) _ mHSA_AnxVm1234.Swimming lane 1 corresponds to Protein Marker.Protein sample under the corresponding non reducing conditions of swimming lane 2,4,6.Protein sample under the corresponding reductive condition of swimming lane 3,5,7,9 (50mM dithiothreitol (DTT) (DTT)).As shown in Figure 1, described fusion rotein (SEQIDNO136) runs (expection MW=111kDa) with correct molecular weight (MW) on SDS-PAGE gel.Purity is >80%.Under not having DTT situation, occur some dimers (<10% total protein), described albumen runs into biobelt.Brachymemma may be the reason observing biobelt pattern.As shown in Figure 1, following protein I GF1_mHSA_AnxVm1234 (SEQIDNO138), NRG1b (EGF) _ mHSA_AnxV (SEQIDNO142), NRG1b (EGF) _ mHSA_AnxVm1234 (SEQIDNO254) run (expection MW=111kDa) with correct molecular weight (MW) on SDS-PAGE gel.The purity of these fusion roteins is higher than 80%.Under there is no DTT situation, there are some dimers (<10% total protein) and described dimer by adding DTT and eliminating.
After purifying, the purity of FGF2_mHSA_AnxV fusion rotein (SEQIDNO144) is about 50%.Described fusion rotein runs into biobelt, and one of them is at correct MW (120kDa), and another is at lower MW.This possibility of result shows that lower molecular weight band is brachymemma product.
With the Ni agarose 6 speed stream resin (GEHealthcare17-5318-04 by run by gravity; 1mL resin/50mL supernatant) according to manufacturer's specification sheets by Ni affinity chromatography purification of Recombinant fusion rotein AnxV_mHSA_FGF2 (SEQIDNO118).In conjunction with/lavation buffer solution by 20mM potassiumphosphate, pH7.4,500mMNaCl, 25mM imidazoles forms, elution buffer by 20mM potassiumphosphate, pH7.4,500mMNaCl, 450mM imidazoles composition.After purifying, evaluate purity by SDS-PAGE.Described fusion rotein runs (120kDa) with correct MW and shows purity higher than 80% on gel.
AnxV_mHSA_NRG1b (EGF) (SEQID120, 121), AnxVm1234_mHSA_NRG1b (EGF) (SEQID116, 117), AnxV_mHSA_IGF1 (SEQID134, 135), AnxVm1234_mHSA_IGF1 (SEQID114, 115), AnxVm1234_mHSA_FGF2 (SEQID264, 265), IGF1_mHSA_B7scFv (SEQID150, 151), aDNASI1_mHSA_FGF2 (SEQID124, 125), aDNASI1_mHSA_NRG1b (EGF) (SEQID126, 127), IGF1_mHSA_Syt1 (SEQID152, 153), Syt1_mHSA_IGF1 (SEQID170, 171), IGF1_mHSA_aDNASI1 (SEQID154, 155), NRG1b (EGF) _ mHSA_B7scFv (SEQID156, 157) according to following method purifying.Blue-sepharose 6 speed stream resin (GE medical treatment & health 17-0948-03) loads Econo-pac (Bole (Bio-Rad) 732-1010) post (1.5cm internal diameter with standard method; 4mL resin/post).Chromatographic grade 8 multi-channel peristaltic pump carries out.Described post is with containing 50mMNaCl, 0mMTris, and the damping fluid of pH7.0 (blue-sepharose lavation buffer solution) balances.The electroconductibility deionized water adjustment of protein expression supernatant is to mate blue-sepharose lavation buffer solution (measuring with conductometer).The supernatant of each protein expression culture is loaded on post with 4-5mL/min.Post 5-10 column volume blue-sepharose lavation buffer solution cleans.Then, albumen 5-10 column volume less salt (LS) elution buffer (20mMTris, pH7.1,50mMNaCl, 45mM Sodium octoate) wash-out.In certain situation (albumen has SEQINNo120,116,134,114,264), this elution step is divided into 5x1.5mL part (A1-5), is then 7x4mL part (B1-7).After low salt elution buffer wash-out, other albumen 5 column volume height salt (HS) elution buffer (20mMTris, pH7.1,1MNaCl, 45mM Sodium octoate) wash-outs.By centrifugal ultrafiltration (Sai Duolisi (Sartorius-Stedim), VS2022) concentrated SDS-PAGE analyzes the protein content of described part, with PD-10 post (GE17-0851-01) desalination in 0.1M potassiumphosphate, 0.15MNaCl, pH7.2.Collect the part containing proteins of interest.AnxV_mHSA_NRG1b (EGF) (SEQID120) fusion rotein part is analyzed by SDS-PAGE.Purified fusion rotein is about 50% pure.SDS-PAGE analyzes the biobelt on display gel.One band is positioned at the expection MW (112kDa) of total length fusion rotein, and a band is characterized by lower MW, and it can be pointed out to be brachymemma product.AnxVm1234_mHSA_NRG1b (EGF) (SEQID116) fusion rotein part is analyzed by SDS-PAGE.SDS-PAGE analyzes the biobelt on display about 50% purity and gel.One band is positioned at the correct MW (112kDa) of total length fusion rotein, and a band is characterized by lower MW, and it can be pointed out to be brachymemma product.AnxV_mHSA_IGF1 (SEQID134) fusion rotein part is analyzed by SDS-PAGE.SDS-PAGE analyzes the biobelt on display about 50% purity and gel.One band is positioned at the correct MW (111kDa) of total length fusion rotein, and a band is characterized by lower MW, and it can be pointed out to be brachymemma product.AnxVm1234_mHSA_IGF1 (SEQID114) fusion rotein part is analyzed by SDS-PAGE.SDS-PAGE analyzes the biobelt on display about 50% purity and gel.One band is positioned at the correct MW (111kDa) of total length fusion rotein, and a band is characterized by lower MW, and it can be pointed out to be brachymemma product.AnxVm1234_mHSA_FGF2 (SEQID264) fusion rotein part is analyzed by SDS-PAGE.SDS-PAGE analyzes the biobelt on display about 50% purity and gel.One band is positioned at the correct MW (120kDa) of total length fusion rotein, and a band is characterized by lower MW, and it can be pointed out to be brachymemma product.IGF1_mHSA_B7scFv (SEQID150) fusion rotein part is analyzed by SDS-PAGE.SDS-PAGE analyze display be greater than 50% purity and gel on biobelt.One band is positioned at the correct MW (102kDa) of total length fusion rotein, and a band is characterized by lower MW, and it can be pointed out to be brachymemma product.Fusion rotein aDNASI1_mHSA_FGF2 (SEQIDNO124) analyzes and shows the purity being less than 20% on SDS-PAGE, with the correct MW (110kDa) of corresponding full-length proteins.Occur that lower MW band shows that described albumen may be cut or brachymemma.Fusion rotein aDNASI1_mHSA_NRG1b (EGF) (SEQIDNO126) analyzes and shows the purity being less than 50% on SDS-PAGE, with the correct MW (110kDa) of corresponding full-length proteins.Occur that lower MW band shows that described albumen may be cut or brachymemma.Fusion rotein IGF1_mHSA_Syt1 (SEQIDNO152) analyzes and shows the purity of about 50% on SDS-PAGE, with the correct MW (91kDa) of corresponding full-length proteins.Occur that lower MW band shows that described albumen may be cut or brachymemma.Fusion protein S yt1_mHSA_IGF1 (SEQIDNO170) analyzes and shows the purity being less than 50% on SDS-PAGE, with the correct MW (91kDa) of corresponding full-length proteins.Occur that higher and lower MW band shows to there is dimerisation products and brachymemma product.Fusion rotein IGF1_mHSA_aDNASI1 (SEQIDNO154) analyzes and shows the purity of about 50% on SDS-PAGE, with the correct MW (102kDa) of corresponding full-length proteins.Fusion rotein NRG1b (EGF) _ mHSA_B7scFv (SEQIDNO156) analyzes and shows the purity being less than 50% on SDS-PAGE, the correct MW (102kDa) with corresponding full-length proteins and lower MW band may correspond to brachymemma product.
AnxV_mHSA (SEQID252,253) and AnxVm1234_mHSA (SEQID250,251) fusion rotein are according to following method purifying.Albumen is precipitated from Pichia anomala expression supernatant by ammonium phosphate (adding to final concentration 82%).Throw out is resuspended in PBS damping fluid and to PBS dialysed overnight.After dialysis, albumen is loaded at 50mMNaCl, 20mM potassiumphosphate, the HiPrep26/60 Sephacryl S-200 high resolution column (GE health medical treatment 17-1195-01) balanced in pH7.0.Albumen is wash-out in same buffer, analyzes the part from wash-out by SDS-PAGE, collects the part containing proteins of interest.Then, collected eluate loads (with 1mL/min flow velocity) to 1mLHiTrapQ agarose speed fluidization tower (GE health medical treatment 17-5053-01) balanced in 20mM potassiumphosphate, 50mMNaCl, pH7.0.Albumen with elution buffer (20mM potassiumphosphate, 50mMNaCl, pH7.0) with 1mL/min wash-out in 20 column volume gradients.Collect described part and analyzed by SDS-PAGE.Collect the part containing proteins of interest.Having and analyzing whole purity by SDS-PAGE under there is no reductive agent situation.Fusion rotein AnxV_mHSA (SEQIDNO252) analyzes and shows the purity being greater than 90% on SDS-PAGE, with the expection MW (104kDa) of corresponding full-length proteins.There are some dimers (<10% total protein) but eliminate when DTT exists.Fusion rotein AnxVm1234_mHSA (SEQIDNO252) analyzes and shows the purity being greater than 90% on SDS-PAGE, with the expection MW (104kDa) of corresponding full-length proteins.There are some dimers (<10% total protein) but eliminate when DTT exists.
Protein expression in Selexis/CHO expression system and pass through chromatogram purification subsequently
The stable SelexisCHO clone of expression proteins of interest is in 37 DEG C in serum free medium, and 5-8%CO2, cultivates in shaking culture case.Substratum for growing is: 1LEx-Cell tMcDCHO merges substratum (Sigma (Sigma), 14365C-1000ML), 40mL200mML-glutamine (hero company, 25030-081), 10mL100XHT fill-in (hero company, 11067-030).The inoculum density of cell is 0.3-0.5x106 cell/mL.Culture just dilutes once reach 2-4x106 cell/mL, until obtain required volume of culture (6L).Cell strengthens solution (1LddH2O, 35g Strengthening agent 5 (sea clone (HyClone), 30865.01), 20gD-glucose, regulate pH to 7.0 with NaOH) add 3-5 days afterwards at the final large culture (cell enhancing amount=7-12% culture) of inoculation.Culture vigor one drops to less than 90% (after dilution culture to its final volume ~ 1 week), and just collection contains the cell conditioned medium of secretory protein interested.Cell conditioned medium passes through harvested by centrifugation and sterile filtration.If purifying carried out in 1 week, supernatant is stored in 4 DEG C, otherwise described supernatant is stored in-80 DEG C.
Fusion rotein aDNASI1 (L23) _ mHSA_HGF (NK1) (SEQID110,111), DAscFv_mHSA_IGF1 (SEQID246,247), DAscFv_mHSA_HGF (NK1) (SEQID248,249) is according to following method purifying.The supernatant ddH2O1:0.5 expressed from Selexis/CHO dilutes, and passes through 5mL blue sepharose column 2 times.Albumen is wash-out in the damping fluid containing 45mM Sodium octoate, then dialyses to PBS.Then, albumen ddH2O1:1 dilutes and to be loaded on 1mLQ anion-exchange column and with slow gradient (gradient=10%B, wherein A=1:1PBS: water, B=is dissolved in the 1MNaCl of PBS, and PBS=standard Du Shi PBS, without Mg/Ca) wash-out.To collect containing the part of proteins of interest and freezing with aliquot sample.Whole purity is evaluated by SDS-PAGE.All purifying proteins show the purity higher than 90% and run into single tape on SDS-PAGE.Figure 14 shows the SDS_PAGE of aDNASI1 (L23) _ mHSA_HGF (NK1) (SEQID110) fusion rotein, has the single tape at 114kDa expection MW.Figure 15 shows the SDS_PAGE of DAscFv_mHSA_IGF1 (SEQID246) fusion rotein, has the single tape at 102kDa expection MW.Figure 15 shows the SDS_PAGE of DAscFv_mHSA_HGF (NK1) (SEQID248) fusion rotein, has the single tape at 115kDa expection MW.
The combination of embodiment 6. bispecific fusion protein and damaged cell
Generate the fusion rotein containing target structural domain, transformation period instrumentality and activation structure territory, verify that it is through the ability of target structural domain In-vitro specificity in conjunction with damaged cell.Target structural domain used is human annexin-V V (AnxV, SEQID31), and it is combined in apoptosis the phosphatidylserine becoming and be exposed to the outside cell surface.Specific binding is proved with regard to multiple fusion rotein, include the fusion rotein in different activation structure territory, the different fusion rotein (as N-terminal activation structure territory and C-terminal target structural domain, N-terminal target structural domain and C-terminal activation structure territory) merging direction.Damaged cell also with regard to different cell type combines proof specific binding, comprises myocardial cell and embryonic stem cell derivative (ESC derives) heart cell.In certain situation, cell damages with induced oxidation pressure with hydrogen peroxide (H2O2) thus simulates the cells in vivo compromise state after myocardial infarction.Containing the fusion rotein of the non-binding variant of annexin V (AnxVm1234, SEQID84) not in conjunction with damaged cell, prove that being subject to annexin V target structural domain with the combination of fusion rotein regulates and controls.In a word, these data presentation fusion roteins merged target structural domain by specific binding and specific delivery activation structure territory to damaged cell.
The combination of fusion rotein and cell is observed with flow cytometry.By the apoptosis-induced necrocytosis of oxidative pressure processed with hydrogen peroxide (H2O2).By identifying apoptosis or dead cell with propidium iodide (PI) mark or with the commercially available cell apoptosis detection kit mark based on fluorescence annexin V.In certain situation, fusion rotein first with fluorescence dye covalent labeling for detecting after a while.In these situations, the specific binding of fusion rotein and apoptotic cell is that the two positive of PI and fusion-protein fluorescence proves by observation of cell, and is that PI is positive but be feminine gender with regard to fusion-protein fluorescence with the equivalent cells that the non-binding variant of fusion rotein is hatched.In other situations, described fusion rotein does not first carry out covalent labeling, uses on the contrary in protein fusion to resist in conjunction with the fluorescent mark two of transformation period instrumentality to detect this fusion rotein.In these situations, by display from the anti-fluorescent signal amount of fusion rotein two and from the commercially available detection kit based on annexin V fluorescent signal amount between High relevancy prove the specific binding of fusion rotein and apoptotic cell.
The specific binding of A.IGF1_mHSA_AnxV and apoptosis heart cell
Expression and purification fusion rotein IGF1_mHSA_AnxV (SEQID136) and IGF1_mHSA_AnxVm1234 (SEQID138) as described in Example 5.According to manufacturer's specification sheets AlexaFluor488 (AlexaFluor488 trace of albumin labelling kit, hero company, A30006) covalent labeling 2 kinds of albumen.HL-1 cell (WilliamC.Claycomb, Louisiana university health science center (LouisianaStateUniversityHealthSciencesCenter)) be the myocardial cell system with Human adult cardiomyocytes feature, described cell 1:3 is inoculated in the pre-coated 96 orifice plate (BD/Falcon of gelatin/fibronectin, 353072) perfect medium (Claycomb substratum (Sigma, 51800C), comprise 10%FBS (Sigma, 12103C), (hero/Ji Bu can for 2mML-glutamine, 25030), (hero/Ji Bu can for 100U/mL penicillin+100ug/mL Streptomycin sulphate, 15070), with 0.1mM norepinephrine (Sigma, A0937) to hatch in 37 DEG C and 5%CO2).After 2 days, the cell 0.1 milliliter/hole substratum being supplemented with 400uMH2O2 (Sigma, H1009) is raised again, hatches 15 minutes in 37 DEG C and 5%CO2.Then, from each hole, sucking-off supplements the substratum of H2O2 and replaces with perfect medium, and described cell hatches 20-24 hour at 37 DEG C and 5%CO2.Second day, media transfer to the 96 deep hole v base plate (American science company (USAScientific), 1896-1110) in each hole was to collect cast-off cells.Described cell PBS (Sigma, D8537) washing once, is then carried out trypsin treatment with 40 μ L0.025% trypsinase-EDTA and is placed in 37 DEG C of incubators.Cell detachment is monitored under the microscope and is added 100uL/ hole DMEM and 10%FBS with deactivation trypsinase.Cell is washed with cold PBS and is resuspended in 100 μ L binding buffer liquid (annexin V-FITC cell apoptosis detection kit component, BD bio-science (BDBiosciences), 556547).Then add AlexaFluor488 mark fusion rotein and on ice lucifuge hatch 1 hour.The positive control of apoptotic cell detects and obtains with annexin V-FITC cell apoptosis detection kit (BD bio-science (BDBiosciences), 556547).In 2 kinds of situations, add 3 μ L propidium iodides (PI) and hatched for last 15 minutes.Cell is analyzed on BDFACSCantoII flow cytometer, uses suitable being unstained with simple stain contrast with calibration.
Apoptotic cell is marked with annexin V-FITC from BD bio-science cell apoptosis detection kit and propidium iodide (PI) (Fig. 2 and 3) altogether.56% cell, in two positive quadrant, indicates late cell apoptosis or necrocytosis.The better separation positive and negative cohort.
IGF1_mHSA_AnxV (SEQID136) and IGF1_mHSA_AnxVm1234 (SEQID138) is respectively marked with AlexaFluor488, reaches the mark (DOL) of 7.1 and 8.1 dyes/mole albumen respectively.Apoptotic cell is also marked with 80ng (7.1nM) IGF1_mHSA_AnxV and PI (Figure 4 and 5) altogether, or 80ng (7.1nM) IGF1_mHSA_AnxVm1234 and PI (Fig. 6 and 7).The two positive peak (53% cell) that the display of IGF1_mHSA_AnxV labeled cell is better separated, very similar with cell apoptosis detection kit positive control, show described fusion rotein specific binding apoptosis or dead cell.On the other hand, IGF1_mHSA_AnxVm1234 labeled cell does not show two positive peak (0% cell), shows non-binding target arm by design not in conjunction with apoptosis or dead cell.Generally, these data presentation contain the fusion rotein energy specific binding apoptosis of annexin V (AnxV) as target structural domain or the heart cell of death, therefore can be used for sending the fusogen of biology (as treatment) effect or molecule as activation structure territory, to treat the heart tissue of damaged or infringement.
The specific binding of B.IGF1_mHSA_AnxV and apoptosis heart cell
For proving that the apoptosis heart cell observed of embodiment 6A combines the AnxV target structural domain that is specific in IGF1_mHSA_AnxV fusion rotein and is not the result of IGF1 structural domain in conjunction with cell surface IGF acceptor, repeat described experiment, include IGF1 pre-incubation step in saturated and any/all IGF1 cell surface receptor of blocking-up.Method therefor is identical with embodiment 6A, except H2O2 concentration becomes 200 μMs.In addition, mark before fusion rotein after cell is resuspended in binding buffer liquid but adding AlexaFluor488-, add 800nMIGF1 (Calbiochem, 407240) and also hatch 10 minutes to block any/all IGF1 cell surface receptor in advance with IGF1.
It is two positive that annexin V-FITC in Fig. 8 and 9 and PI positive control show about 40%, instruction late cell apoptosis or necrocytosis group.IGF1_mHSA_AnxV is in conjunction with apoptotic cell in Figure 10 and 11 display, and non-binding contrast fusions IGF1_mHSA_AnxVm1234 does not combine (Figure 12 and 13), as shown in embodiment 6A.Secondly, for proving IGF1_mHSA_AnxV without its IGF1 structural domain significantly in conjunction with cell, repeating described test, having IGF1 to block step.Figure 14 and 15 display is even under excessive IGF1 exists and after hatching with excessive IGF1, and IGF1_mHSA_AnxV is still in conjunction with apoptotic cell.These digital proofs AnxV target structural domain is responsible for the specific binding of protein fusions and apoptotic cell.
C.AnxV_mHSA and apoptosis ESC derives the specific binding of heart cell
AnxV_mHSA (SEQID252) and AnxVm1234_mHSA (SEQID250) is according to the direct coupling AlexFluor647 of manufacturer's specification sheets (AlexaFluor647 carboxylic acid, succinimide ester, hero company, A-20006).The basic acquisition as described in (Nature2008,453:524-8) such as Yang of embryonic stem cell derivative (ESC derives) heart cell (PeterZandstra, University of Toronto (UniversityofToronto)).Operation scheme is available from BauwensCL, on .TissueEngA part .2011 April 25, " GeometricControlofCardiomyogenicInductioninHumanPluripot entStemCells. (the cardiac-like muscle cell induction in Linear Control human pluripotent stem cells) ".Complete with the operation scheme of aforementioned serum-free directed differentiation cardioblast pedigree based on the hESC differentiation assembled.HESC aggregate size by forcing gathering definition cell concn to control in containing the AggreWellTM inset (Stemcell Technologies Inc. (CA) (STEMCELLTechnologies)) of micropore grain surface.In brief, hESC single cell suspension spin down in Aggrewells that feeder layer exhausts, density is 1000 cell/micropores.Cell can be assembled under anoxic conditions and spends the night in StemPro34, and described StemPro34 is supplemented with substratum based on Glutamax, xitix, Transferrins,iron complexes, penicillin/streptomycin, adds ROCK inhibitor and 0.5ng/mlBMP4.1st day, described substratum was replaced with there being the basic medium of 10ng/mlBMP4,3ng/ml activin A and 5ng/mlbFGF.4th day, cell shifted out from micropore, and washing with the DMEMF12 supplementing 5%KOSR and transfer in low bunch of plate (lowclusterplate) has in the basic medium of 10ng/mlVEGF and 150ng/mlDkk1.8th day, described substratum was replaced with there being the basic medium of 10ng/mlVEGF, 150ng/mlDkk1 and 5ng/mlbFGF.12nd day, described substratum again replaces (the same cell factor) and cell transferred to normal oxygen condition until the 16th day.
Even without H2O2 or Zorubicin process, heart cell shows measurable apoptosis group, based on PI mark and annexin V-FITC detection kit (Figure 16).Further interpolation Zorubicin does not increase apoptosis part.But apoptotic cell group is enough to the combination testing apoptosis targent fused protein.Heart cell group AnxV_mHSA or AnxVm1234_mHSA is hatched, and also hatches altogether by annexin V-FITC detection kit simultaneously.The fluorescent signal of AlexaFluor647 and the FITC signal strong correlation (Figure 17) of apoptosis detection kit on AnxV_mHSA fusions instead of AnxVm1234_mHSA fusions, prove that AnxV_mHSA specific binding apoptosis ESC derives heart cell.
D.AnxV_mHSA_NRG1b (EGF) and apoptosis ESC derive the specific binding of heart cell
The combination secondary detection scheme that fusion rotein AnxV_mHSA_NRG1b (SEQID120) and apoptosis ESC derive heart cell proves, instead of first changes fusion rotein with covalent attachment fluorophore.Described fusion rotein is with anti-HSA antibody (the sheep anti-human albumin antibodies of affinity purification, Bethyl laboratory (BethylLabs), A80-129A) detect, it is according to manufacturer's specification sheets AlexaFluor647 (AlexaFluor647 carboxylic acid, succinimide ester, hero company, A-20006) self covalent labeling.Heart cell AnxV_mHSA_NRG1b (EGF) is hatched, and hatches altogether as described in embodiment 6C by annexin V-FITC detection kit simultaneously.Described fusion rotein is by the anti-detection of anti-HSAAlexaFluor647 bis-.Fluorescent signal from AlexaFluor647 and the FITC signal strong correlation (Figure 18) from detection kit.This display AnxV_mHSA_NRG1b (EGF) derives heart cell in conjunction with apoptosis ESC.In addition, get rid of fusion rotein (" without the part " of Figure 18) but still not show between AlexaFluor647 signal with the FITC signal based on apoptosis any associates with the control experiment that secondary detection reagent is hatched, prove that original AlexaFluor647 fluorescent signal is the combination due to fusion rotein itself, instead of due to independent two anti-combinations.
The combination of embodiment 7. fusion rotein and its target
The process that treatment is positioned patient disease relevant range is limited to area-of-interest by target or molecular epitope abundant especially has wherein come.Such as, myocardial infarction can expose several target molecule (as DNA, cardiac myosin and phosphatidylserine) after tissue injury, can be used for this object.Generate the several fusion roteins contained the special target structural domain of these target molecules.Especially, annexin V and synaptotagmin can be used for target phosphatidylserine, and SI-1 single chain variable fragment (aDNASIscFv) is for target DNA.But, it will be understood by those skilled in the art that in fusion rotein that including binding domain in may cause each independent structural domain characteristic to be lost or change (as binding affinity change, biologic activity changes).For whether measurement function can maintain in fusion rotein disclosed herein, development and application is based on the external combination test of ELISA.In essence, although described test display target has strict washing to be still retained in microwell plate to suitable fusion rotein, this is because they interact with the homology target molecule arranged at hole surface.Immunochemistry carried out to the existence of fusion rotein quantitative.When there is no homology target molecule or non-homogeneous target molecule, do not expect reservation there is no unexpected target structural domain cross reactivity.With the reservation of homology target molecule with without the combination identity binding specificity removed and target function.
Microwell plate (Pierre Si (Pierce) 15041) epi-position bag quilt interested.Phosphatidylserine (PS, APL (AvantiPolarLipids) 840032) is evaporated to dry deposition by making 50 μ L/ hole 12.5 μ g/mL methanol solutions.DNA (SigmaD3664) is deposited by reagent (Pierre Si (Pierce) 17250) by the 10 μ g/mLDNA solution and DNA bag adding 50 μ L pre-mixing 1:1 in hole.Myosin deposits by hatching 10 μ g/mL solution in Du Shi PBS.All bags are reflected at the lower room temperature of 200rpm vibration and carry out 2 hours.After washing, add 250 μ L/ holes without protein blocking damping fluid (Pierce37572) and plate at incubated at room 3-4 hour.After further washing, Xiang Kongzhong adds 100 μ L chromatogram purification fusion roteins, and concentration range is 160ng/mL – 20 μ g/mL.In 10mMHepes, 140mMNaCl, 2.5mMCaCl2, pH7.4, room temperature was carried out in conjunction with 2 hours.After further washing, Xiang Kongzhong adds 100 μ L and detects antibody (the sheep anti-human albumin antibodies of coupling HRP, Bethyl laboratory (BethylLabs) A80-129P), 1:5 in PBST, 000 or 1:50,000 dilutes and hatches 30-60 minute.After further washing, apply 75 μ L peroxidase (PierceTMBUltra34028), after observing significantly colour developing, with 75 μ L stop bath (KPL50-85-05) cancellation reactions.The absorbancy in hole is reading 450nm reading in plate instrument (TecanM200Pro).All fusion roteins and Antibody Combination carry out in triplicate.All washing steps are made up of the dispersion of 4 circulations and suction 250 μ LPBST, have 5 second to soak and oscillation step, use automatic 96 orifice plates to wash trigger (Bai Teng company (Biotek), Elx405) between each circulation.The hole of the blank well (solvent, wrap by reagent or only damping fluid) and amixis albumen that comprise simulation package quilt is as negative control.
Fusion rotein as embodiment 5 generation is described in detail in detail.IGF1_mHSA_Syt1 (SEQID152) and IGF1_mHSA_AnxV (SEQID136) shows specific binding phosphatidylserine (Figure 19).ADNASI1_mHSA_FGF2 (SEQID124), aDNASI1_mHSA_NRG1b (EGF) (SEQID126) and IGF1_mHSA_aDNASI1 (SEQID154) show specific binding DNA (Figure 20).
Described fusion rotein display specific binding target molecule, after proving target domain fusion transformation period instrumentality and activation structure territory, reservation function combines and illustrates target structural domain (and target) width that can merge into fusion rotein.Particular target such as phosphatidylserine can by multiple binding domain for process, as annexin V and synaptotagmin.On the contrary, specific protein kind has larger member's diversity as antibody derives scFv (aDNASI1 is its member), and it combines corresponding various target molecule or epi-position.ScFvs successfully includes fusion rotein instruction in other fusion roteins, applies the possibility that antibody derives target.ADNASI1 structural domain is also presented at N or C-terminal merges direction and has function containing in the fusions in multiple activation structure territory.In a word, these result determination fusion rotein targets may be not limited to particular target epi-position, particular target to structural domain kind, certain translation direction or the specific molecule containing activation structure territory.
The adjustment of embodiment 8. cytoactive
The biological activity in the activation structure territory of purified fusion protein carrys out external display by measuring irriate cell middle and lower reaches signal transmission.Make comparisons in the effect of described fusion rotein and wild-type, non-fused activation structure territory.Generating multiplely has different activation structure territory, different target structural domain and different merge the fusion rotein in direction and be shown as biological activity.These digital proofs can generate the fusion rotein of biological activity and energy transfer cell path signal, and described path is as short survival or propagation path.
Test together with the commercially available acquisition in each fusion rotein and positive control, its activation structure territory, non-fused form.Use has the fusion rotein of active targeting structural domain (as AnxV) and non-binding control target structural domain (as AnxVm1234 or DAscFv), and the activity showing described activation structure territory is independent of the characteristic of described target structural domain and function.Cultivate cell to be stimulated, carry out serum starvation, then stimulate with fusion rotein.Wash away albumen subsequently, measured the cytoactive of phosphorylation-Akt (pAkt) or phosphorylation-Erk (pErk) by ELISA.
A. use the AKT of NRG1b (EGF) fusion rotein irritation cancer cell active
Fusion rotein NRG1b (EGF) _ mHSA_AnxV (SEQID142) and AnxV_mHSA_NRG1b (EGF) (SEQID120) generate as described in Example 5.Wild-type NRG1b (EGF) is available from peace enlightening biology (396-HB/CF).Human prostata cancer, epithelioid cell DU145 cell are with 25,000 cells/well is inoculated in 96 orifice plate (BD/Falcon, 353072) perfect medium (RPMI-1640 (hero/Ji Bu can), 11875) in, described substratum comprises 10%FBS (sea clone, SH30071), (hero/Ji Bu can for 2mML-glutamine, 25030) and 50U/mL penicillin+50ug/mL Streptomycin sulphate (hero/Ji Bu can, 15070), and 37 DEG C and 5%CO2 overnight incubation.Second day, sucking-off substratum, cell 0.1 milliliter/hole PBS (without calcium and magnesium, Sigma, D8537) washing, with 0.1 milliliter/hole RPMI-1640+0.5%FBS feeder cell again, described cell hatched 20-24 hour at 37 DEG C and 5%CO2.Next day, cell stimulates with through dilution fusion rotein or reference protein, adds 25 microlitres/hole to existing 0.1 milliliter/hole, continues 10 minutes at 37 DEG C and 5%CO2.Wash by sucking-off substratum from hole and with the cold PBS in 0.2 milliliter/hole and stop stimulating.Cell in 25 microlitres/hole complete M-PER lysis buffer (mammalian proteins extract reagent (Pierre Si/Sai Mo fly generation you, 78501), 150mMNaCl, protease inhibitor cocktail (Roche (Roche) completemini, 04693124001) and inhibitors of phosphatases (RochePhosSTOP, 04906837001) cracking), previously prepared.Sealing plate, cell 4 DEG C of cracking 30 minutes on orbital shaker, lysate quick-frozen in-78 DEG C of preservations on dry ice.384 hole white plate (MaxiSorp can agree (Nunc), 460372) anti-Akt capture antibody (clone SKB1, Mi Libo (Millipore), 05-591) wraps quilt, and sealing, room temperature preservation is spent the night.
Second day, the cell lysate that thaws is cleaning and closed elisa plate also.Collect the lysate that thaws, again clean elisa plate, by Akt standard substance or collected lysate and add elisa plate, described plate incubated at room 2 hours.Cleaning elisa plate, add anti-phosphorylation Akt and detect antibody (Biotinylated mouse mAb, cell signalling company (CellSignaling), 5102), plate was incubated at room 1.5 hours.Clean described plate, add Streptavidin-horseradish peroxidase (SA-HRP, peace enlightening is biological, 890803), plate was incubated at room 30 minutes.Clean plate again, add substrate (SuperSignalELISAPico chemical luminous substrate, Pierre Si/Sai Mo fly generation you, 37069), reading plate instrument reads luminous.PAkt standard curve fit line (bilogarithmic graph chi).
The activity of NRG1b (EGF) and NRG1b (EGF) _ mHSA_AnxV is shown in Figure 21.Commercially available wild-type NRG1b (EGF) and fusion rotein all show and have biological activity, stimulate pAkt path.Similarly, Figure 22 shows the activity of wild-type and reverse fusion rotein AnxV_mHSA_NRG1b (EGF).These results prove translation skill merges NRG1b (EGF) activation structure territory and mHSA and AnxV does not eliminate its biological activity, because NRG1b (EGF) fusion rotein of embodiment 5 expression and purification has biological activity.
B. use the AKT in IGF1 fusion rotein irritation cancer cell active
Fusion rotein IGF1_mHSA_AnxV (SEQID136), IGF1_mHSA_AnxVm1234 (SEQID138) and IGF1_mHSA_B7scFv (SEQID150) generate as described in Example 5.Wild-type IGF1 is available from Calbiochem (407240).Cultivate as described in embodiment 8A and stimulate DU145 cell.All 3 protein fusions based on IGF1 are presented in DU145 cancer cells biological activity, and pAkt stimulates similar wild-type IGF1 (see Figure 23-24).
C. use the AKT in IGF1 fusion rotein cardiac stimulus cell active
Fusion rotein IGF1_mHSA_AnxV (SEQID136) generates as described in Example 5.Wild-type IGF1 is available from Calbiochem (407240).HL-1 cell (WilliamC.Claycomb, Louisiana university health science center) be the myocardial cell system with Human adult cardiomyocytes feature, described cell is with 60, 000 cells/well is inoculated in the pre-coated 96 orifice plate (BD/Falcon of gelatin/fibronectin, 353072) perfect medium (Claycomb substratum (Sigma, 51800C), comprise 10%FBS (Sigma, 12103C), (hero/Ji Bu can for 2mML-glutamine, 25030), (hero/Ji Bu can for 100U/mL penicillin+100ug/mL Streptomycin sulphate, 15070), with 0.1mM norepinephrine (Sigma, A0937)) and in 37 DEG C and 5%CO2 overnight incubation.Washed cell also carries out ELISA operation scheme described in embodiment 8A.
IGF1_mHSA_AnxV fusion rotein is presented in heart cell biological activity, and its effect and wild-type IGF1 are quite (active see dose response; Figure 25).These digital proofs can generate the activation structure territory of merging transformation period instrumentality and target structural domain and described activation structure territory can retain the ability of its effective stimulus cell.
ERK in FGF2 fusion rotein cardiac stimulus cell is active
Available from embryonic stem cell (ESC, thered is provided by PeterZandstra laboratory, University of Toronto) myocardial cell carry out being separated and with 40, 000 cells/well is inoculated in the StemPro-34 substratum of pre-coated 96 orifice plates of gelatin, and (hero/Ji Bu can, 10639) in, described culture medium supplemented has 38.5XStemPro-34 nutritional supplement (providing together with StemPro-34 substratum), (hero/Ji Bu can for 2mML-glutamine, 25030), (hero/Ji Bu can for 50U/mL penicillin+50ug/mL Streptomycin sulphate, 15070), 0.4mM thioglycerin (Sigma, M6145), 50ug/mL xitix (Sigma, A4544), 150ug/mL Transferrins,iron complexes (SigmaT8158), (peace enlightening is biological for 10ng/mLVEGF, 293-VE), (peace enlightening is biological for 150ng/mLDKK-1, 5439-DK) with 5ng/mL basic FGF (FGF2, send general Tyke (PeproTech), 100-18b), and hatch at 37 DEG C and 5%CO2.Stimulate first 24 hours, described growth medium changes the StemPro-34 without nutritional supplement and somatomedin into.Cell stimulates and cracking as described in embodiment 8A.For ELISA, 96 holes are high wraps quilt, sealing in conjunction with ELISA blackboard p-ERK1/Erk2 capture antibody (peace enlightening is biological, DYC1018), and room temperature preservation is spent the night.Second day, lysate accepts ELISA operation scheme described in embodiment 8A, except adopting p-ERK1/Erk2 standard substance, (peace enlightening is biological, and p-ERK1 DYC1018)/Erk2 detects antibody, and (peace enlightening is biological, DYC1018) measurement activation Erk1/Erk2 level, instead of Akt standard substance and anti-phosphorylation Akt detect antibody.Just stimulate pERKESC to derive heart cell and compare fusion rotein AnxV_mHSA_FGF2 (SEQID118) with wild-type FGF2 and described fusion rotein shows biological activity (Figure 26).
The accumulation of embodiment 9. fusion rotein is accumulated along with apoptotic cell and cytoactive stimulation
Fusion rotein through its target structural domain specific binding cell and with after be proven in vitro through the ability of its activation structure field stimulation cell-signaling pathways.Target structural domain used is human annexin-V V (AnxV, SEQID31).AnnV can be combined in apoptosis the phosphatidylserine becoming and be exposed to external cell surface.Activation structure territory used is IGF1 (SEQID3), and it is in conjunction with the IGF1 acceptor that cell surface is expressed.Once combine, the initial intracellular signal transmission of described IGF1 acceptor.Fusion rotein is first in conjunction with apoptosis heart cell, and it simulates damaged cell state in the body after myocardial infarction.Then, in conjunction with the cell of fusion rotein for stimulating the IGF1 signal transmission in healthy heart cell, simulate the paracrine effect that described fusion rotein activates signal transmission in infarct or neighbouring adjacent impaired or healthy cell.Downstream targets-phosphorylation the Akt of IGF1 signal transmission is measured by ELISA.In conjunction with the Akt signal transmission in the fusion rotein energy cardiac stimulus cell of cell.Wild-type, non-fused IGF1 do not induce Akt signal transmission, show that the annexin V target structural domain of described fusion rotein is crucial to generation signal transmission.Equally, AnxV_mHSA fusion rotein does not stimulate Akt signal transmission, shows that target structural domain is not enough to for signal transmission itself.In a word, this fusion rotein of these data presentation is bi-functional, can selectively targeted damaged tissue and can through its activation structure territory with paracrine sample loading mode transfer cell path signal.Result prove fusion rotein in damaged tissue instead of health tissues specificity accumulation and with after through activation structure territory regulate survival or regeneration therapeutic action.
In the first step, described fusion rotein by experience apoptosis HL1 myocardial cell in annexin V-phosphatidylserine to combine and along with damaged cell accumulation.Apoptosis-induced necrocytosis is carried out by the oxidative pressure processed from hydrogen peroxide (H2O2).By completing the combination of fusion rotein and damaged cell with the cell incubation of the anoikis fusion rotein contained by H2O2 process cell growth medium.In second step, the biological activity in conjunction with the fusion rotein activation structure territory of cell carrys out in-vitro evaluation by stimulating with the fusion rotein in conjunction with cell through serum starvation myocardial cell.Clean with stops stimulate after, the downstream signal in irriate cell pass through ELISA measurement phosphorylation Akt (pAkt).The pAkt level of described fusion rotein induction and the commercially available acquisition in its activation structure territory, non-fused form and contain annexin V target structural domain but the fusion rotein lacking activation structure territory is made comparisons.
Fusion rotein IGF1_mHSA_AnxV (SEQID136) expression and purification as described in Example 5.HL-1 cell (WilliamC.Claycomb, Louisiana university health science center) be the myocardial cell system with Human adult cardiomyocytes feature, described cell is inoculated in the pre-coated 96 orifice plate (BD/Falcon of gelatin/fibronectin with 1:2, 353072) perfect medium (Claycomb substratum (Sigma, 51800C), comprise 10%FBS (Sigma, 12103C), (hero/Ji Bu can for 2mML-glutamine, 25030), (hero/Ji Bu can for 100U/mL penicillin+100ug/mL Streptomycin sulphate, 15070), with 0.1mM norepinephrine (Sigma, A0937) to hatch in 37 DEG C and 5%CO2).Next day, the cell 0.1 milliliter/hole substratum being supplemented with 400uMH2O2 (Sigma, H1009) is raised again, hatches 15 minutes in 37 DEG C and 5%CO2.Then, from each hole, sucking-off supplements the substratum of H2O2 and replaces with perfect medium, and described cell hatches 20-24 hour at 37 DEG C and 5%CO2.Second day, media transfer to the 96 deep hole v base plate (American science company, 1896-1110) in each hole was to collect cast-off cells.For each sample, the substratum from 3 holes is collected in 1 hole of 96 deep hole v base plates.Then, the cell fusion rotein of collection hatches 15 minutes in 37 DEG C and 5%CO2 under calcium (binding buffer liquid, annexin V-FITC apoptosis detection kit component, BD bio-science, 556547) exists.Cell in conjunction with fusion rotein is also washed once with PBS (Sigma, D8537) by centrifugation, and cell is resuspended in the DMEM in 100 μ L/ holes calcic (binding buffer liquid) afterwards.Be inoculated in pre-coated 96 orifice plates of gelatin/fibronectin and in advance the HL-1 cell of serum starvation subsequently with the resuspension cytositimulation 20 minute of 100 μ L/ holes in conjunction with fusion rotein.Then, wash irriate cell and carry out ELISA operation scheme described in embodiment 5.The healthy HL-1 cell not being exposed to H2O2 also carries out trypsin treatment to gather in the crops with 40uL/ hole 0.025% trypsinase-EDTA and is placed in 37 DEG C of incubators.Cell detachment is monitored under the microscope and is added 100uL/ hole DMEM and 10%FBS with deactivation trypsinase.For each sample, from the trypsin treatment cell harvesting in 3 holes in 1 hole of 96 deep hole v base plates.Cell is washed with cold PBS and is resuspended in 300 μ LDMEM.Then, cell fusion rotein is hatched under calcium exists, and processes as mentioned above, and for stimulating the HL-1 cell of inoculation and serum starvation in advance.Washing irriate cell also carries out ELISA operation scheme described in embodiment 8A.
Only in the cell stimulated by the apoptosis prey fusion protein containing target (AnxV) and activation (IGF1) structural domain, observe phosphorylation Akt level to increase, as shown in figure 27.Wild-type, non-fused IGF1 can not irritation cells, may be because thus IGF1 is not captured not in conjunction with apoptotic cell.It is quite active that fusion rotein and wild-type IGF1 have shown in embodiment 4C, and therefore the phosphorylation Akt level increase of prey fusion protein be can't help its effect difference and caused.Although the IGF1 acceptor that non-fused IGF1 can expose in conjunction with apoptotic cell in theory on the surface, it seems and do not retain enough somatomedins with inducement signal transmission, or somatomedin is the mode of signal transmission can not retain.Equally, AnxV_mHSA fusion rotein can not irritation cell.Although AnxV_mHSA fusion rotein as described in Example 6 can in conjunction with apoptotic cell, it can not send signal with paracrine sample loading mode, because it lacks activation structure territory.Not detecting that phosphorylation Akt level increases in the cell stimulated by any albumen of pre-mixing untreated cell, may be because of the cell surface exposure phosphatidylserine of healthy cell not used for prey fusion protein.Equally, although can in conjunction with the IGF1 acceptor on healthy cell surface, somatomedin be caught and is not enough to irritation cell.In a word, target and mobilizing function while fusion rotein described in data presentation.
Embodiment 10. is by target damaged cardiac tissue in fusion rotein body
The following hypothesis of test: on AnxV target structural domain specific binding necrosis and apoptosis cell the fusion rotein IGF1_mHSA_AnxV (SEQID:136) (embodiment 6) of phosphatidylserine compare in the damaged cardiac tissue of IGF1_mHSA_AnxVm1234 (SEQID:138) (not in conjunction with the variant of phosphatidylserine) after myocardial infarction accumulate more and longer.Induction experiments myocardial inyaretion (MI) in mouse, substances intravenous injection (IGF1_mHSA_AnxV, IGF1_mHSA_AnxVm1234 or only supporting agent contrast), 12, put to death animal after 24 or 72 hours, the protein accumulation in the infraction of heart, frontier district and far-end (not impaired) district is by ELISA and immunohistochemical observation.The IGF1_mHSA_AnxV that immunohistochemical methods display gives latter 24 hours is positioned in the frontier district of infarct border, and non-binding variant none observe in infraction, frontier district or far-end (health) district.ELISA data presentation targeting proteins IGF1_mHSA_AnxV compares that non-binding misfolded proteins IGF1_mHSA_AnxVm1234 accumulation degree in cardiac infarction and frontier district is higher and the time is longer.In the damaged cardiac tissue of these digital proof prototype target fusion roteins IGF1_mHSA_AnxV after myocardial infarction, specificity accumulates and continues, the activation structure territory that energy specific delivery merges.
By ligation arteria coroaria sinistra Induction experiments myocardial inyaretion (MI) in mouse, explain in detail as follows.After 60 minutes, remove ligation, allow cardiac perfusion.Medicine-feeding test material or supporting agent are completed by tail vein injection after impinging upon MI for 22 hours.15 mouse are often organized in following administration:
Group 1: only supporting agent contrast
Group 2:15 μ gIGF1_mHSA_AnxV
Group 3:15 μ gIGF1_mHSA_AnxVm1234
For each group, put to death 5 mouse in each following time: after administration 12,24 and 72 hours.For each group/time point, prepare 3 animals and be used for immunohistochemical methods and 2 for ELISA, target is qualification in cardiac infarction district or the special anti-HSA signal of IGF1_mHSA_AnxV or IGF1_mHSA_AnxVm1234 at edge.Detailed operation scheme is as follows.
Carry out in the lab space that animal work is leased in Waltham, Massachusetts ViviSource company by Biotrofix company.Operation scheme is assessed by ViviSourceIACUC and is ratified, and solves and records all animal welfare issues.90 (90) only male C57/B612 mouse in age in week within 7-10 days before research starts, order (comprising 15 for preliminary study, Charles River Laboratories (CharlesRiverLaboratories)).They can freely obtain food and water.Distribute to animal identification number, use the permanent marker on tail.Within one day, observe animal before the study, get rid of and it seems healthy poor those.Animal rearing, in indoor, provides filtrated air and 50% ± 20% relative humidity of 21 ± 2 DEG C.Described room autotimer, opens light/dark circulation and the cut out without evening twilight in 12 hours for 12 hours. 1/4 " high-grade maize rod is for padding grass and Bio-Huts tMfor mouse (BioServK3352) or mouse RunnelTM (BioServK3322, K3323) is placed in each cage.Animal Lab 5001 diet.Water provides arbitrarily.4-6 animal raised by each cage.
In operation day, mouse of weighing, anaesthetizes and induces with the 100%O2 containing isoflurane in resin glass room.Mouse is placed in the surgery surface on inherent regulation heating cushion.Mouse is suitably fixed in back (outside of belly upward), with suitable dimension inner catheter (22G) endotracheal intubation, maintains the 1.0-2.5% isoflurane anesthesia in 100%O2.The operation of anesthesia confirms the response that pin, heel and afterbody pinch horizontally through losing Eyelid reflex and lacking.
Scrape chest (bottom to just passing through on the right side of breastbone from back), vacuum removes fur, with Septisol process skin.From the left chest of breastbone to regio pectoris percutaneous incision skin mouth parallel with rib.Intercostal muscle between rib 5 and 6 cuts on left side of heart, adduction rib.Qualification heart (left ventricle and left atrium), opens pericardium.Left lung compresses to shift out from described region gently downwards.7-0 silk suture to be placed in around arteria coroaria sinistra and ligation on ~ 2mm aseptic polyethylene PE-10 run, to observe heart after ligation, whether lose color (turning white, the evidence as ischemic).Cut open suture line residue end, by removing ligation through PE conduit and silk suture cutting in Ischemia Time after 60 minutes.Wound moist is kept by covering opening with aseptic saline gauze soak of heating.Once remove suture, the suitable Reperfu-sion with regard to ischemic area observes heart.Left lung PEEP (positive end expiratory pressure) is re-inflated, and the rib on opposite closes with 6-0 nonabsorbable monofilament nylon suture.Muscle layer 6-0 absorbable suture closes, and then carries out skin closure in a continuous manner with 6-0 silk suture.
Injection buprenorphine (Bedford laboratory (BedfordLabs) TMLot:18655303) is to anaesthetize (0.05mg/kg, subcutaneous), cut off isoflurane, mouse is once generation autonomous respiration with regard to tube drawing, and the clean cage being placed in supplementary thermal is to restore.Post operation, animal remains on heating cushion until recover from anesthesia.Then it returns clean cage.Frequently them are observed and at least once a day afterwards in operation day (the 0th day).Animal before surgery the-1 day and the 0th day (operation day) weigh, and then weigh every day until put to death.
10 μ L etc. points of substances are with suitable concn (IGF1 group 1-3 defined above; Supporting agent, IGF1_mHSA_AnxV or IGF1_mHSA_AnxVm1234, without in endotoxic PBS)-80 DEG C preserve until use day.Preserve without endotoxic PBS and 4 DEG C.Each decile substances is thawed before facing injection.200 μ L add substances without endotoxic PBS (room temperature) and suction mixes for several times up and down, then use syringe 22 (+/-1) hour after MI without top clearance that 200 μ L are injected mouse through tail vein.
At the appointed time point (after administration 12,24 or 72 hours), mouse carries out euthanasia as follows: animal is placed in degree of depth ketamine/xylazine anesthesia.For every treatment group 3 animals, open thoracic cavity chest and apex puncture, about 0.1ml15%KCl injects left ventricle, and animal is poured into fixing by physiological saline and zinc formaldehyde subsequently.Collect heart, preserve 24-48 hour in zinc formaldehyde, be then transferred to 70% ethanol, 4 DEG C of preservations.Then sample is delivered to quality organization service company (MassHistologyServices) and carry out immunohistochemical methods detection.
For every treatment group 2 animals, animal saline infusions.Isolating cardiac and left ventricle salt solution clean.Heart is trimmed to just in time left and right ventricle, is divided into 4 shown in Fig. 6 .1 piece.Collect each piece, weigh, quick-frozen (in liquid nitrogen), then-80 DEG C are stored in through mark Eppendorf tube (often pipe 1 sample, therefore every heart 4 samples) and on dry ice, are transported to Yin Xi drugmaker (SilverCreekPharmaceuticals).
For the heart tissue of adaptive immune group and ELISA control experiment, other mouse several carry out euthanasia as mentioned above and do not impose operation, cut heart and as above rinse, in certain situation, the 15 μ l containing 2 μ g fusion rotein IGF1_mHSA_AnxV being injected directly in left ventricular wall.The heart being with or without injection albumen is as above fixed described in immunohistochemical methods preparation.
Immunohistochemical methods detection fusion albumen
Immunohistochemical methods carries out in the quality organization service company of Worcester, MA, and this is a histology experiment room meeting GLP and require.Use its processing and dyeing fixing organization with the standard operation scheme of detection specificity albumen.In brief, heart fixing in zinc formaldehyde is divided into ventricle also by alcohol and the dimethylbenzene conventional processing of standard series.Each heart is embedded in paraffin, cuts into slices prepare with each about 6 micron thickness and be placed on microslide on come card (Leica) slicing machine.For each heart, prepare 8 continuous square section and be placed on slide glass, skipping 100 μm, preparing another 8 sections, skip 100 μm, and this is repeated by heart length.
Dye to 2 slide glasss from each group 8, one shows morphology and another anti-HSA dyeing with HSA location with H & E, and DAPI redyes showed cell core.Conventional procedure is used for H & E and dyes.Specifically, be organized in dimethylbenzene and remove paraffin, clean up in alcohol, hydration in water, Harris's brazilwood extract dyeing.Clean described slide glass, 1% water-based eosin stains, dewaters in a series of alcohol, cleans up in a series of dimethylbenzene, and covered.Then, the slide glass observation by light microscope of decentraction addle flat slice is represented with the section of location containing infarct.
For the HSA location in heart tissue, clean in dimethylbenzene with those sections of adjoining of dyeing H & E, clean up in alcohol, hydration in water, by the goat-anti human albumin primary antibodie 4C overnight incubation of 1:200 dilution, rinse in PBS.The anti-sheep IgG of AlexaFluor594 donkey is the fluorescent-labeled antibody for goat-anti HSA antibody, and described antibody is used as two with 1:400 extent of dilution at 37C subsequently and anti-within 1 hour, locates to detect anti-HSA.Described slide glass rinses in PBS, and by ProLongGold anti-cancellation reagent covered, described reagent also comprises the DAPI dyeing for observing core.
For positive control, the slide glass from the heart of direct injection IGF1_mHSA_AnxV is also processed with anti-HSA operation scheme.In addition, quality organization service company dyeing human liver tissue sample, described tissue sample comprises natural HAS as the positive control detecting primary antibodie.Negative control comprises anti-HSA primary antibodie is omitted in processing direct injection mouse heart for HSA location, and processing is used for the originally heart (exposure of amixis albumen) of HSA location usually.
ELISA detection fusion albumen
Every heart 4 samples (Figure 28) are prepared as follows with regard to ELISA.Sample is transferred to the safe lock Eppendorf tube of Ai Bende (Eppendorf) and thaws on ice.In each pipe, add RIPA damping fluid, described damping fluid comprises 1:5 ratio (mg tissue sample): the stopping protease inhibitor cocktail (diluting 100 times in damping fluid) of (uL damping fluid).At least 100 μ L damping fluids are used for each sample.Also add ZROB05 and the ZROB10 pearl mixture of 50/50, adopt the pearl of 1:2 ratio: damping fluid.Pipe be placed in the BulletBlender tissue homogenizer of the speed of being set to 9 and homogenize 3 minutes, if homogenize is incomplete, then repeating this process.Centrifugal described sample is also got aliquot sample and is carried out BCA (bicinchoninic acid) Protein assay to measure the total protein of each sample.
Enzyme linked immunosorbent assay (ELISA) detection standard method completes.Specifically, at the 1st day, the Reacti-Bind plate anti-HSA coated antibody 4 DEG C bag of 1:50 dilution in 50 μ L/ hole Du Shi PBS is spent the night.2nd day, hole washed 4X with washing trigger (program 6) by PBS-T (PBS, 0.05% polysorbas20).Non-specific binding 200 μ L/ holes close 2 hours and hole washes 4X with washing trigger by PBS-T (PBS, 0.05% polysorbas20) without protein blocking damping fluid room temperature.Xiang Kongzhong adds typical curve sample or the test sample in 50 μ L/ holes (96 orifice plate).Test sample is diluted to the whole total protein concentration of 8.745mg/mL in RIPA damping fluid (+proteinase inhibitor).Sealing plate and 4 DEG C of overnight incubation.3rd day, hole washed 4X with washing trigger by PBS-T, and every hole adds 1:25 in the PBS-T of 100uL/ hole, and the goat-anti HSA-HRP of 000 dilution detects antibody and with 220rpm incubated at room 30 minutes on vibrator platform, lucifuge is protected.Hole washes 4X with washing trigger by PBS-T, and room temperature adds the 1 step UltraTMBELISA reagent of every hole 100uL, and plate hatches 25 minutes in room temperature lucifuge.Termination reaction is carried out by adding 100uLKLPTMB termination reagent.Color becomes yellow from blueness.After 5 minutes, reading plate instrument carries out absorbance reading with A450 wavelength.The absorbancy background value of the tissue that amixis albumen exposes is available from the originally cardiac samples generated by method same as described above.From all test specimens product absorbance subtracting background values to obtain difference.The typical curve of concentration-absorbancy relation is generated from the sample mixing certain limit known quantity fusion rotein.Then, the protein concentration of each test sample is by making comparisons measure with normal concentration-absorbancy relation curve.Measure 2 aliquot sample from each cardiac tests sample to evaluate measurement variability and these are bases (Figure 29) of standard deviation contained by ELISA data.
Fusion rotein used comprises IGF1_mHSA_AnxV (targeting proteins) and IGF1_mHSA_AnxVm1234 (non-binding variant), generates as described in Example 5.Supporting agent contrast is without endotoxic PBS (Sigma).Animal is male C57/B6 mouse, is 12 week age during order, and operation consent adapts to 7-10 days.The dyeing that immunohistochemical methods is used and antibody: primary antibodie is that goat-anti human albumin (HSA) intersects adsorb antibodies, affinity purification (BethylLabs080-229Alot#3); Fluorescent mark two resists for the anti-sheep IgG (H+L) (hero company, A11058) of AlexaFluor594 donkey; There is the anti-cancellation reagent of the ProLongGold of DAPI (hero company, P36931).ELISA agents useful for same comprises RIPA cracking and Extraction buffer (Pierre Si, 89901); Pierre Si stops protease inhibitor cocktail, without EDTA (Pierre Si, 78425); Pierre Si BCA test kit, 23227; Reacti-Bind plate (Pierre Si, 15041); Du Shi PBS (Sai Mo (Thermo), 28374); Without protein blocking damping fluid (Pierre Si, 37572); Anti-HSA coated antibody (Bethyllabs antibody A 80-229A); Goat-anti HSA-HRP detects antibody (Bethyl laboratory, A80-229P); 1 step UltraTMBELISA reagent (Sai Mo (Pierre Si), 34028); KLPTMB stops reagent (KLP, 50-85-05); Without protein blocking damping fluid (Pierre Si, 37572); Organize homogenize pearl (NA company (NextAdvance), ZROB05 and ZROB10).Animal surgery material therefor comprises buprenorphine (Bedford laboratory TMLot:18655303), isoflurane, ketamine, xylazine, zinc formaldehyde and 15%KCl.
Figure 29 is summarized in by ELISA detection target and non-binding variant fusion proteins.With regard to target (group 2) and non-binding variant (organizing 3) fusion rotein, by the albumen measured in the cardiac infarction+frontier district of 2 mouse and non-infarct albumen upon administration 3 times (12,24 and 72 hours) make comparisons.For each heart, add the albumen of measurement in sample A and B1-far-end (as Fig. 6-1 institute defines), represent the albumen of the non-infarct of heart.Equally, add the albumen measured in sample B1-infraction and B2, represent the albumen of cardiac infarction district and surrounding edge battery limit (BL).
As shown in figure 29, compare the level (group 2, lath) of the dirty distal area of same animal center, targent fused protein (group 2, secret note) IGF1_mHSA_AnxV significantly improves after injection for 12 and 24 hours in infarct.Infraction and non-infarct all cannot detect at 72 hours.By contrast, non-binding misfolded proteins IGF1_mHSA_AnxVm1234 (group 3, secret note) improved to some extent at 12 hours, reduced at 24 hours and cannot detect at 72 hours.Relatively 12 and 24 little target constantly and non-binding proteins, the target IGF1_mHSA_AnxV (secret note, group 2) in infraction+frontier district is higher than non-binding IGF1_mHSA_AnxVm1234 (secret note, group 3).The fusion rotein IGF1_mHSA_AnxV of these results card target damaged myocardium cell (combined by activity and die or phosphatidylserine that necrotic myocardium cell is relevant with adjusting) can enter with the concentration higher than non-binding variant test MI damaged heart region also retain wherein and the time longer.In addition, targent fused protein specific localization damaged heart region described in data presentation, proves the target effect through AnxV target structural domain.
Also prove that administration block after 24 hours and IGF1_mHSA_AnxV in frontier district accumulates higher than non-binding variant IGF1_mHSA_AnxVm1234 by immunohistochemical location containing the fusion rotein of HSA.Figure 30 shows infraction and the form of surrounding tissue, and to infarct border and the special positive staining of periinfarct frontier district place IGF1_mHSA_AnxV.Upper left side: H & E dyes and shows form.Infarct is positioned at central authorities, and edge is divided by black curve, is to live (200x amplification) in upper left and the lower right corner.Upper right side: with same magnification just containing a series of section in this region of HSA fusion rotein dyeing.Red instruction HSA location; The DAPI dyeing of blue indicator cells core.The more high-amplification-factor image of the same area is in lower-left (400x) and the display of bottom right (600x) angle.Positive signal is had in myocardial cell's (thin arrow) at blocking tissue's (intermediate gauge arrow) edge.Upper left side and top-right white edge are in roughly same position in 2 adjacent 6um slide glasss, and lower-left and lower right corner image are the amplifications of these frame areas adjacent.
By contrast, Figure 31 shows the identical information of non-binding mutant IGF1_mHSA_AnxVm1234, shows its minimum specific stain.Upper left side: H & E dyes and shows form.Infarct is positioned at the upper part of image, and edge divides (200x amplification) by black curve.Upper right side: with same magnification just containing this region contiguous slices of HSA fusion rotein dyeing.Red instruction HSA location; The DAPI dyeing of blue indicator cells core.The more high-amplification-factor image of the same area is in lower-left (400x) and the display of bottom right (600x) angle.Only to have powerful connections in myocardial cell's (thin arrow) at blocking tissue's (intermediate gauge arrow) edge and red blood corpuscle (thick arrow) signal.Upper left side and top-right white edge are in roughly same position in 2 adjacent 6um slide glasss, and lower-left and lower right corner image are the amplifications of these frame areas adjacent.
Figure 32 display is for confirming that anti-HSA antibody is to containing the specific contrast of HSA fusion rotein.Upper left side: the positive control in mouse heart, wherein IF1_mHSA_AnxV as described in direct injection.Scarlet instruction is containing the strong fix of HSA fusion rotein in its injection place.Upper right side: the negative control in mouse heart.Preparation identical with upper left side, comprises injection IGF1_mHSA_AnxV but need not anti-HSA primary antibodie dye.Do not find specific stain.Lower left: the second negative control in mouse heart.Albumen is not injected, as upper left side processing in heart.Only can find incarnadine background stainings.Lower right: the positive control in people's liver.People's liver generates the HSA of significant quantity.The specific stain of sample is spread all over anti-HSA antibody staining display.In all images: blue dyeing is the nuclear DAPI dyeing of instruction.
Embodiment described herein and embodiment should be understood only for illustrating object and its multiple modifications is included within the scope of the application's spirit and authority and appended claims.The all publication quoted herein, patent and patent application are included in for all objects by reference of text at this.

Claims (10)

1. a fusion rotein, it comprises:
A () has the target structural domain of binding specificity to tissue damaged's cell target molecule of being correlated with, wherein target structural domain is selected from: Synaptotagmin I, anti-phosphatidylserine antibody, PS4A7, lactadherin, antimyosin antibody, anti-DNA antibody, its variant, its fragment and its combination; With
B () has the activation structure territory of binding specificity to cell surface associated receptor in described tissue, wherein said activation structure territory is selected from lower group: fibroblast growth factor, Urogastron, neuregulin/tune albumen, rhIGF-1, pHGF, thymosin, granulocyte colony-stimulating factor, STEM CELL FACTOR, periostin, vascular endothelial growth factor, stroma cell derivative factor, Thr6 PDGF BB, teratoma derivative growth factor, hypertrophy/stem cell factor, high-affinity trk C, BDNF/N-3 growth factor receptors, NT-3 somatomedin, thrombopoietin, thymosin, Delicious peptide, interleukin, specific antibody is had to activator receptor, its variant, its isotype, its fragment and its combination,
Wherein, after described activation structure territory is exposed to described acceptor, this activation structure territory in conjunction with described acceptor to promote described tissue regeneration or survival.
2. fusion rotein as claimed in claim 1, it also comprises peptide transformation period instrumentality.
3. fusion rotein as claimed in claim 1 or 2, it is characterized in that, described target structural domain and described activation structure territory are in conjunction with the differing molecular on the differing molecular on same cell or different cell.
4. fusion rotein as claimed in claim 2, it is characterized in that, described transformation period instrumentality comprises the sequence from one of the following: human serum albumin, alpha-fetoprotein, vitamin D binding protein, transthyretin, single-chain antibody Fc structural domain, proline(Pro)-, L-Ala-and/or Serine enriched sequence, albumin bound domain antibodies, its variant, its fragment and its combination.
5. fusion rotein as claimed in claim 4, is characterized in that, the aminoacid sequence of described transformation period instrumentality as SEQIDNO:10,12, any one of 14-29,45-49,65-71 or 105 shown in.
6. fusion rotein as claimed in claim 1 or 2, is characterized in that, described target structural domain comprise as SEQIDNO:1,2,30, any one of 72-73,76-80,85 or 86 shown in sequence.
7. fusion rotein as claimed in claim 1 or 2, is characterized in that, the sequence shown in described activation structure territory comprises any one of SEQIDNO:3-9,32-40 or 50-64.
8. a pharmaceutical composition, it comprises the fusion rotein described in claim 1 or 2 of medicine suitable carrier and treatment significant quantity.
9. pharmaceutical composition according to claim 8 is preparing the purposes by promoting in the medicine of tissue injury in tissue regeneration therapies object in need.
10. the nucleic acid molecule of the fusion rotein described in claim 1 or 2 of encoding.
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CN114478802B (en) * 2022-01-28 2023-05-26 郑州大学 Chimeric antigen receptor and application thereof
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CN116693682A (en) * 2023-06-02 2023-09-05 涌源合生科技(深圳)有限公司 anti-Tau protein antibody, preparation method and application thereof
CN116693682B (en) * 2023-06-02 2024-04-19 涌源合生科技(深圳)有限公司 Anti-Tau protein antibody, preparation method and application thereof
CN117417454A (en) * 2023-12-19 2024-01-19 北京索莱宝科技有限公司 Anti-chicken IgY antibody and application thereof
CN117417454B (en) * 2023-12-19 2024-03-05 北京索莱宝科技有限公司 Anti-chicken IgY antibody and application thereof

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