WO2011071279A2 - Bpb-기반 카르고 운반 시스템 - Google Patents

Bpb-기반 카르고 운반 시스템 Download PDF

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WO2011071279A2
WO2011071279A2 PCT/KR2010/008645 KR2010008645W WO2011071279A2 WO 2011071279 A2 WO2011071279 A2 WO 2011071279A2 KR 2010008645 W KR2010008645 W KR 2010008645W WO 2011071279 A2 WO2011071279 A2 WO 2011071279A2
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type
bpb
delivery system
trp
peptide
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PCT/KR2010/008645
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English (en)
French (fr)
Korean (ko)
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WO2011071279A3 (ko
Inventor
전상용
김성현
박세호
김동규
박진호
어 쏘우페이
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광주과학기술원
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Priority to JP2012543020A priority Critical patent/JP5677453B2/ja
Priority to US13/515,163 priority patent/US20120321697A1/en
Publication of WO2011071279A2 publication Critical patent/WO2011071279A2/ko
Publication of WO2011071279A3 publication Critical patent/WO2011071279A3/ko

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • the present invention relates to a BPB-based cargo delivery system.
  • Antibodies are immunoglobulin proteins, a type of plasma protein produced by B cells, that specifically inactivate and inactivate antigens by specifically recognizing and binding to specific sites of antigens.
  • the FDA has approved 21 monoclonal antibodies, and antibodies such as Rituximab and Herceptin have been effective in more than 50% of patients who have not seen any response in other treatments. Has demonstrated successful clinical treatment of lymphoma, colon cancer or breast cancer using monoclonal antibodies.
  • the total market for therapeutic antibodies is estimated to grow at an annual average rate of 203 ⁇ 4) from $ 10 billion in 2004 to $ 30 billion in 2010, and the market is expected to grow exponentially.
  • the development of new drugs using antibodies is active because the drug development period is short, investment costs are low, and side effects can be easily predicted.
  • the antibody is a herbal medicine, the human body is hardly affected, and the half-life in the body is overwhelmingly long compared to low molecular weight drugs, so the patient is friendly. Despite this usefulness, monoclonal antibodies in humans are recognized as foreign antigens and can cause severe allergic or hypersensitivity reactions.
  • the production cost is high, and thus the price of the therapeutic agent is rapidly increased, and there are a wide range of methods for culturing and purifying the antibody. Since technology in the field is protected by various intellectual property rights, expensive licensing fees must be paid.
  • Antibody-replacement protein is a recombinant protein made to have constant and variable regions like an antibody.
  • a small and stable protein is replaced with a random sequence of amino acids to make a library, which is then screened for the target material, thereby providing high affinity and good Substances with specificity can be found.
  • avimer and affibody in antibody replacement proteins have been reported to have a picomol affinity for a target substance. It is reported that these antibody replacement proteins are small and stable and can penetrate deep into cancer cells and generally produce less immune responses.
  • antibody replacement proteins There are currently 40 antibody replacement proteins that have been developed. Among them, the antibody replacement proteins that are being commercialized by venture companies and multinational pharmaceutical companies are fibronectin type m domain, lipocalin, LDLR-A domain, crystallin, protein, and ankyrin repeat ( Ankyrin repeat), BPTI is used in the protein and has a high affinity of several nanomolar to picomolar to the target. Adnectin, Avimer, and Kunitz domains are currently undergoing FDA clinical trials.
  • the present invention focused on peptide-based antibody replacement proteins that are different from antibody replacement proteins using proteins up to now.
  • Peptides have been widely used in place of antibody therapeutics due to their proper pharmacokinetics, mass productivity, low toxicity, antigenic inhibition and low production cost compared to antibodies.
  • the advantages of peptides as therapeutic drugs are low production costs, high safety and responsiveness, relatively low patent royalties, and less exposure to unwanted immune systems, which can inhibit the production of antibodies to the peptides themselves. Deformation through is easy and accurate. However, most peptides are not used in many applications because they show lower affinity and specificity for specific protein targets than antibodies. There is a disadvantage.
  • the inventors have sought to develop a system capable of delivering various substances intracellularly or to the cell surface based on target binding and specificity.
  • peptides are randomly bound to both ends of a structural stabilization site having a relatively rigid peptide backbone, and when the two peptides are jointly bound to a target molecule, significantly increased binding capacity and specificity
  • a bipodal peptide binder (BPB) can be obtained, and the binding of the cargo to the BPB can only be carried on the cell surface or within the cell based on the target binding and specificity of the BPB.
  • the present invention provides a BPB-based Cargo Delivery System comprising:
  • BBP Bipodal Peptide Binder
  • a structure stabilizing region comprising parallel, antiparallel or parallel lei and antiparallel amino acid strands having interstrand noncovalent bonds formed thereon;
  • the present invention provides a method of delivering a cargo comprising the step of treating a BPB-based Cargo Delivery System to an individual, tissue, or cell.
  • BPB Bipodal Peptide Binder
  • the inventors have sought to develop a system capable of delivering various substances intracellularly or to the cell surface based on target binding and specificity.
  • the peptides are randomly bound to both ends of the structural stabilization site having a relatively rigid peptide backbone, and when the two peptides are jointly bound to the target molecule, the binding ability and specificity are greatly increased.
  • Bipodal peptide binder having Only when binding the cargo of the transport target, it was confirmed that various substances can be transported into the cell surface or cells based on the target binding and specificity of the BPB.
  • the basic strategy of the present invention is to connect a peptide that is bound to a target at both ends of a rigid peptide backbone.
  • the rigid peptide backbone acts to stabilize the overall structure of the bipodal peptide provider and enhances the binding of the target binding site I and the target binding site ⁇ to the target molecule.
  • Structural stabilization sites available in the present invention include parallel, antiparallel or parallel and antiparallel amino acid strands, interstrand hydrogen bonds, electrostatic interactions, hydrophobic interactions, van der Waals interactions, pi-pi Protein structure motifs in which non-covalent bonds are formed by interaction, cation-pi interaction, or a combination thereof.
  • Non-covalent bonds formed by hydrogen bonds, electrostatic interactions, hydrophobic interactions, van der Waals interactions, pi-pi interactions, cation-pi interactions, or a combination of these strands are the rigidity of the structural stabilization site. Contribute to.
  • interstrand non-covalent bonds at the structure stabilization site include hydrogen bonds, hydrophobic interactions, van der Waals interactions, pi-pi interactions or combinations thereof.
  • covalent bond there may be a covalent bond to the structured stabilization site.
  • disulfide bonds may be formed at the structured stabilization site to further increase the robustness of the structure stabilized site.
  • the increase in firmness by such covalent bonds is given in consideration of the specificity and affinity of the bipodal peptide binder for the target.
  • the amino acid strands of the structure stabilization site are linked by a linker.
  • linker refers to the material that connects the strands.
  • the turn sequence in ⁇ -hairpin serves as a linker
  • a substance connecting two C-terminus of leucine zipper eg, , Peptide linkers
  • the linker connects the parallel, antiparallel or parallel and antiparallel amino acid strands.
  • At least two strands (preferably two strands) arranged in parallel fashion, at least two strands (preferably two strands) arranged in antiparallel fashion, and at least three strands arranged in parallel and antiparallel fashion.
  • the linker (preferably three strands) is connected by the linker.
  • the linker is a turn sequence or peptide linker.
  • the turn sequence is ⁇ -turn, ⁇ -turn, ⁇ -turn, -3 ⁇ 4 or ⁇ -loop ° 1 (Venkatachalam CM (1968), Biopolymers, 6, 1425-1436; Nemethy G and Printz MP. (1972), Macro / no 1 ecu les, 5, 755-758; Lewis PN et al., (1973), Biochim. Biophys.Acta, 303, 211-229; Toniolo C. (1980) CRC Crit. Rev. Biochem., 9, 1-44; Richardson JS. (1981), Adv. Protein Chem., 34, 167-339; Rose GD et al., (1985), Adv.
  • the turn sequence used in the present invention is ⁇ -turn.
  • ⁇ -turn When ⁇ -turn is used as the turn sequence, it is preferably a type I, type ⁇ , type ⁇ , type ⁇ ', type m or type ⁇ turn sequence, more preferably type I, type ⁇ , type ⁇ , type ⁇ 'turn sequence, even more preferably a type ⁇ or type ⁇ ' turn sequence, most preferably a type ⁇ turn sequence (BL Sibanda et al., J. Mo I. Biol., 1989, 206, 4 , 759-777; BL Sibanda et al., Methods Enzymol., 1991, 202, 59-82).
  • the turn sequence in the present invention is H. Jane Dyson et al. , Eur. J. Biochem. 255: 462-471 (1998), which is incorporated herein by reference.
  • Available for the turn sequence include the following amino acid sequences: X-Pro-Gly-Glu- Val; Ala-X-Gly-Glu-Val (X is selected from 20 amino acids).
  • X is selected from 20 amino acids.
  • the peptide linker connects two strands arranged in parallel or two strands arranged in an antiparallel manner. desirable.
  • Peptide linkers can be used in any known in the art.
  • the sequence of a suitable peptide linker may be selected in consideration of the following factors: ( a ) the ability to be applied to flexible extended conformation; (b) the ability to not produce secondary structures that interact with biological target molecules; And (c) the absence of hydrophobic residues or residues with charges that interact with the biological target molecule.
  • Preferred peptide linkers include Gly, Asn and Ser residues. Other neutral amino acids such as Thr and Ala can also be included in the linker sequence. Suitable amino acid sequences for linkers are Maratea et al., Gene 40: 39-46 (1985); Murphy et al. , Proc. Natl. Acad Sci. USA 83: 8258-8562 (1986); US Patent Nos. 4, 935, 233, 4,751,180 and 5,990,275.
  • the peptide linker sequence may consist of 1-50 amino acid residues.
  • the structural stabilization site is a hairpin, ⁇ -hairpin, beta-turn, linker-linked ⁇ -sheet or linker-linked leucine zipper, more preferably the structure-stabilization site is ⁇ -hairpin or ⁇ -sheets linked by linkers, most preferably ⁇ -hairpins.
  • ⁇ -hairpin refers to the simplest protein motif comprising two ⁇ strands, the two ⁇ strands representing an antiparallel alignment with each other. In this ⁇ -hairpin the two ⁇ strands are generally linked by turn sequences.
  • the turn sequence applied to the ⁇ -hairpin is a type I, type 1 ', type ⁇ , type ⁇ ', type m or type ⁇ turn sequence, more preferably type I, type 1 ', type ⁇ , A type ⁇ 'turn sequence, even more preferably a type ⁇ or type ⁇ ' turn sequence, and most preferably a type ⁇ turn sequence.
  • X-Pro-Gly-Glu-Val Or a turn sequence represented by Ala_X—Gly-Glu-Val (X is selected from 20 amino acids) can also be used for ⁇ -hairpins.
  • the type I turn sequence is Asp-Asp-Ala- Thr-Lys-Thr
  • the type ⁇ turn sequence is Glu-Asn-Gly-Lys
  • the type ⁇ turn sequence is X-Pro -Gly-Glu-Val
  • Ala-X-Gly-Glu-Val X is selected from 20 amino acids
  • the type ⁇ 'turn sequence is Glu-Gly— Asn-Lys or Glu-D-Pro-Asn -Lys.
  • Peptides with ⁇ -hairpin formulations are well known in the art. See, eg, US Pat. No. 6,914,123 and Andrea G. Cochran et al. , PNAS, tryptophan zipper disclosed in 98 (10): 5578-5583, template-fixed ⁇ -hairpin mimetic disclosed in W02005 / 047503, ⁇ disclosed in US Pat. No. 5,807,979. Hairpin variants are well known. In addition, peptides having ⁇ -hairpin conformation are described by Smith & Regan (1995) Science 270: 980-982; Chou & Fassman (1978) Annu. Rev. Biochem.
  • tryptophan zipper is used.
  • the tryptophan zipper used in the present invention is represented by the following general formula (I):
  • Xi-Trp (X 2 ) X3-X4-X5 (X'2) X6-X7 3 ⁇ 4 is Ser or Gly-Glu, X 2 and X ' 2 are independently of each other Thr, His, Val, lie, Phe or Tyr, X 3 is Trp or Tyr, is Type I, Type ⁇ , Type ⁇ , Type ⁇ ′ or type m or type ⁇ turn sequence, 3 ⁇ 4 is Trp or Phe, 3 ⁇ 4 is Trp or Val, and X 7 is Lys or Thr-Glu.
  • 3 ⁇ 4 in the general formula! is Ser or Gly-Glu
  • 3 ⁇ 4 and ⁇ ' 2 are independently of each other Thr, His or Val
  • 3 ⁇ 4 is Trp or Tyr
  • 3 ⁇ 4 is Type I, Type 1' , Type ⁇ or type ⁇ ′ turn sequence
  • 3 ⁇ 4 is Trp or Phe
  • 3 ⁇ 4 is Trp or Val
  • X 7 is Lys or Thr-Glu.
  • 3 ⁇ 4 is Ser or Gly-Glu
  • 3 ⁇ 4 and X'2 are independently of each other Thr, His or Val
  • 3 ⁇ 4 is Trp
  • X4 is type I, type I ', type ⁇ or type ⁇ ′ turn sequence
  • 3 ⁇ 4 is Trp
  • 3 ⁇ 4 is Trp
  • X 7 is Lys or Thr-Glu.
  • Thr is 3 ⁇ 4 is Trp, is type ⁇ or type ⁇ 'turn sequence, 3 ⁇ 4 is Trp, 3 ⁇ 4 is Trp and ⁇ 7 is Lys.
  • Xi is Ser, 3 ⁇ 4 and X'2 are Thr, 3 ⁇ 4 is Trp, is a type ⁇ turn sequence (ENGK) or type ⁇ 'turn sequence (EGNK), and 3 ⁇ 4 is Trp 3 ⁇ 4 is Trp and X 7 is Lys.
  • ENGK type ⁇ turn sequence
  • EGNK type ⁇ 'turn sequence
  • amino acid sequences of tryptophan zippers suitable for the present invention are described in SEQ ID NOs: 1 to 3 and 5 to 10.
  • ⁇ -hairpin peptides usable as structural stabilization sites in the present invention are peptides derived from B1 domainin of protein G, ie GB1 peptides.
  • the structural stabilization site is preferably represented by the following general formula ⁇ :
  • 3 ⁇ 4 is Arg, Gly-Glu or Lys—Lys
  • 3 ⁇ 4 is Gin or Thr
  • 3 ⁇ 4 is type I, type ⁇ , type ⁇ , type ⁇ 'or type m or type ⁇ turn sequence
  • 3 ⁇ 4 is Gin, Thr- Glu or Gln-Glu. More preferably, the structure stabilization site of general formula ⁇ is represented by the following general formula ⁇ :
  • 3 ⁇ 4 is Gly-Glu or Lys-Lys
  • 3 ⁇ 4 is type I, type 1 ', type ⁇ , type ⁇ ' or type ⁇ or type ⁇ turn sequence
  • 3 ⁇ 4 is Thr-Glu or Gln-Ghi.
  • Exemplary amino acid sequences of GB1 ⁇ -hairpins suitable for the present invention are described in SEQ ID NOs: 4 and 14 to 15.
  • ⁇ -hairpin peptides usable as structural stabilization sites in the present invention are HP peptides.
  • the structural stabilization site is preferably represented by the following general formula m:
  • Xi is Lys or Lys-Lys
  • X 2 is Trp or Tyr
  • 3 ⁇ 4 is Val or Thr
  • is type I type ⁇ , type ⁇ , type ⁇ 'or type m or type ⁇ turn sequence
  • 3 ⁇ 4 is Trp Or Ala
  • 3 ⁇ 4 is Trp or Val
  • X 7 is Ghi or Gln-Glu.
  • ⁇ -hairpin peptide that can be used as a structural stabilization site in the present invention is represented by the following general formula IV:
  • Xi is Lys-Thr or Gly
  • 3 ⁇ 4 is Trp or Tyr
  • 3 ⁇ 4 is type I, type ⁇ ', type ⁇ , type ⁇ ' or type m or type m 'turn sequence
  • X4 is
  • Exemplary amino acid sequences of ⁇ -hairpins of Formulas III and IV are set forth in SEQ ID NOs: 11-12, 15, and 16-19.
  • a hairpin alpha-hairpin, beta-hairpin, gamma-hairpin, P-hairpin, etc.
  • beta-turns may be used as structural stabilization sites.
  • a ⁇ -sheet connected by a linker may be used as the structure stabilization site.
  • two or more amino acid strands, which are parallel or antiparallel, preferably antiparallel, are in an extended form, and hydrogen bonds are formed between the amino acid strands.
  • ⁇ -sheet structure two adjacent ends of two amino acid strands are connected by a linker.
  • linker various turn-sequences or peptide linkers described above may be used. If the turn-sequence is used as a linker, the ⁇ _turn sequence is most preferred.
  • leucine zippers or leucine zippers linked by linkers may be used as structural stabilization sites.
  • Leucine zippers are conservative peptide domains that cause parallel dimerization of two ⁇ -chains, and are generally dimerization domains found in proteins involved in gene expression (“Leucine scissors”. Glossary of Biochemistry and Molecular Biology ( (1997) Ed. David M. Glick.London: Portland Press; Landschulz WH, et al. (1988) Science 240: 1759-1764).
  • Leucine zippers generally comprise a heptad repeat sequence, with the leucine residues located at the fourth or fifth.
  • Specific examples of leucine zippers used in the present invention are described in SEQ ID NO: 39 Sequence.
  • Each half of the leucine zipper consists of short ⁇ -chains with direct leucine contact between the ⁇ -chains.
  • the leucine zipper in the transcription factor generally consists of a hydrophobic leucine zipper site and a basic site (site that interacts with the main groove of the DNA molecule). When the leucine zipper is used in the present invention, the basic site is not necessarily required.
  • two adjacent ends of two amino acid strands may be linked by a linker.
  • linker various turn-sequences or peptide linkers described above may be used, and preferably, a peptide linker that does not affect the structure of the leucine zipper is used.
  • Random amino acid sequences are joined to both ends of the structure stabilization site described above.
  • the random amino acid sequence forms target binding site I and target binding site ⁇ .
  • One of the greatest features of the present invention is to prepare a peptide binder in a bipodal manner by connecting the target binding site I and the target binding site ⁇ to both ends of the structure stabilization site.
  • the target binding site I and the target binding site ⁇ cooperatively bind to the target, thereby greatly increasing the affinity for the target.
  • the amino acid number n of the target binding site I is not particularly limited, preferably an integer of 2-100, more preferably an integer of 2-50, even more preferably an integer of 2-20, most preferably Is an integer from 3 to 10.
  • the amino acid number m of the target binding site ⁇ is not particularly limited, preferably an integer of 2-100, more preferably an integer of 2-50, even more preferably an integer of 2-20, most preferably Is an integer from 3 to 10.
  • the target binding site I and the target binding site ⁇ may each contain different or the same number of amino acid residues.
  • the target binding site I and the target binding site ⁇ may contain different or identical amino acid sequences, and preferably include different amino acid sequences.
  • the amino acid sequence included in the target binding site I and / or target binding site ⁇ is a linear amino acid sequence or a cyclic amino acid sequence.
  • at least one amino acid residue among the amino acid sequences included in the target binding site I and / or the target binding site ⁇ is an acetyl group, a fluorenyl methoxy carbonyl group, or a formyl group. It may be modified into palmitoyl group, myristyl group, stearyl group or polyethylene glycol (PEG).
  • the bipodal peptide binder of the present invention bound to a biological target molecule can be used for the regulation of physiological reactions in vivo, detection of in vivo substances, in vivo molecular imaging, in vitro cell imaging and drug delivery targeting. It can also be used as an escort molecule.
  • the structure stabilization site, the target binding site I or the target binding site ⁇ (more preferably, the structure stabilization site, even more
  • the cargo is bonded to the linker of the structural stabilization site.
  • the cargo is a compound, a chemical, a biopharmaceutical, an inorganic particle, a nanoparticle, a protein, a peptide, a nucleic acid molecule, a lipid, a carbohydrate, a liposome and a label molecule which generates a detectable signal. It includes, but is not limited to.
  • Labels that generate the detectable signal include T1 contrast material (eg Gd chelate compound), T2 contrast material (eg superparamagnetic material (eg magnetite, Fe 3 0 4 , Y-Fe 2 0 3) manganese ferrite, cobalt ferrite And nickel ferrite)), radioactive isotopes (e.g., U C, 15 0, 13 N, P 32 , S 35 , 44 Sc, 45 Ti, 118 I, 136 La, 198 T1, 200 T1, 205 Bi, and 20 3 ⁇ 4O , Including but not limited to fluorescent materials (fluorescein, phycoerythrin, rhodamine, lissamine, and Cy3 and Cy5), chemiluminescence groups, magnetic particles, mass labels or electron-dense particles It doesn't happen.
  • T1 contrast material eg Gd chelate compound
  • T2 contrast material eg superparamagnetic material (eg magnetite, Fe 3 0 4 ,
  • the chemicals include, for example, anti-inflammatory drugs, analgesics, anti-arthritis agents, antispasmodics, antidepressants, antipsychotics, neurostabilizers, anti-anxiety drugs, antagonists, antiparkin's disease drugs, cholinergic agonists, anticancer agents, antiangiogenic agents, Immunosuppressants, antiviral drugs, antibiotics, appetite suppressants, analgesics, anticholinergic agents, antihistamines, antimigraine, hormones, coronary, cerebrovascular or peripheral vasodilators, contraceptives, antithrombotics, diuretics, antihypertensives, cardiovascular diseases , Cosmetic ingredients (eg, anti-wrinkle agents, anti-aging agents and skin whitening agents) and the like, but are not limited thereto.
  • Cosmetic ingredients eg, anti-wrinkle agents, anti-aging agents and skin whitening agents
  • the biopharmaceuticals include insulin, insulin-like growth factor 1 (IGF-1), growth hormone, erythropoietin, granulocyte-colony stimulating factors (G-CSFs), and GM-CSFs (gr anu 1 ocy te / mac r ophage-co 1 ony stimulating factors, interferon alpha, interferon beta, interferon gamma, interleukin-1 alpha and beta, interleukin-3, interleukin-4, interleukin-6, interleukin-2, epidermal growth factors (EGFs), calcitonin calcitonin, adrenocorticotropic hormone (ACTH), tumor necrosis factor (TNF), atobisban, buserelin, cetrorelix, deslorelin, desmopressin (desmopressin), dynorphin A (1-13), elcatonin, eleidosin, epifitabide ⁇ ! ⁇ acid ⁇ , GHR
  • Target binding site I and / or target binding site ⁇ may comprise amino acid sequences that bind to various targets.
  • Targetable by the bipodal peptide binders of the present invention include biochemicals, peptides, polypeptides, nucleic acids, carbohydrates, lipids, cells and tissues such as biological targets, compounds, metal or nonmetallic materials, preferably It is a biological target.
  • the biological target to which the target binding site binds is preferably a biochemical, peptide, polypeptide, glycoprotein, nucleic acid, carbohydrate, proteoglycan, lipid or glycolipid.
  • biochemicals to which the target binding site binds include various in vivo metabolites (eg, ATP, NADH, NADPH, carbohydrate metabolites, lipid metabolites and amino acid metabolites).
  • in vivo metabolites eg, ATP, NADH, NADPH, carbohydrate metabolites, lipid metabolites and amino acid metabolites.
  • Exemplary peptides or polypeptides to which a target binding site binds include enzymes, ligands, receptors, biomarkers, hormones, transcription factors, growth factors, immunoglobulins, signaling proteins, binding proteins, ion channels, antigens, adhesion proteins, structural proteins. , Including, but not limited to, regulatory proteins, toxin proteins, cytokines and blood coagulation factors.
  • the target of the bipodal peptide binder is Fibronectin extra domain B (ED-B), VEGFCvascular endothelial growth factor), Vascular endothel ial growth factor receptor (VEGFR), vascular cell adhesion molecule-1 (VCAM1), nicot inic acetylcholine receptor (nAchR), human serum albumin (HSA), MyD88, Epi dermal Growth Factor Receptor (EGFR), HER2 / neu, CD20, CD33, CD52, Epipithelial Cell Adhesion Molecule (EPCAM), TNF-a (Tumor Necrosis Factor ⁇ a), IgE (Immunoglobulin E), CDllA (a -chain of lymphocyte function-associated antigen 1), CD3, CD25, Glycoprotein Ilb / IIIa, Integrin, Alpha ⁇ fetoprotein (AFP), ⁇ 2 ⁇ (Bet ⁇ microglobul in), BTA (B1 adder Tumor Antigens
  • Exemplary nucleic acid molecules to which the target binding site binds include, but are not limited to, gDNA, mRNA, cDNA, rRNACribosomal RNA (rDNA), ribosomal DNA (rDNA), and tRNA.
  • Exemplary carbohydrates bound by the target binding site are in vivo carbohydrates, including, but not limited to, monosaccharides, disaccharides, trisaccharides, and polysaccharides.
  • Exemplary lipids to which the target binding site binds include, but are not limited to, fatty acids, triacylglyceres, sphingolipids, gangliosides, and cholesterol.
  • the bipodal peptide binder of the present invention may bind to a biomolecule (eg, a protein) exposed to a cell surface, but may also bind to a biomolecule (eg, a protein) in a cell and regulate activity of the biomolecule.
  • a biomolecule eg, a protein
  • a biomolecule eg, a protein
  • the bipodal peptide binder targets intracellular proteins, preferably the bipodal peptide binder further comprises a cell transmembrane peptide (CPP).
  • CPP cell transmembrane peptide
  • the CPP includes various CPPs known in the art, for example, HIV-1 Tat protein, oligoarginine, ANTP peptide, HSV VP22 transcriptional regulator protein, MTS peptide derived from vFGF, Penetratin, Transportan, Pep-1 Peptide, Pep-7 peptide, Buforin II, MAP (model amphiphatic peptide), k-FGF, Ku 70, pVEC, SynBl or ⁇ ⁇ 1, but is not limited thereto.
  • CPP includes various CPPs known in the art, for example, HIV-1 Tat protein, oligoarginine, ANTP peptide, HSV VP22 transcriptional regulator protein, MTS peptide derived from vFGF, Penetratin, Transportan, Pep-1 Peptide, Pep-7 peptide, Buforin II, MAP (model amphiphatic peptide), k-FGF, Ku 70, pVEC, SynBl or ⁇ ⁇ 1, but is not limited thereto
  • bipodal peptide binders bound to CPP enter the cell to bind to and regulate (eg, inhibit) the activity.
  • the bipodal peptide binder of the present invention is typically a construct of "one strand of the N-target binding site I-structure stabilization site-linker-the other strand of the structure stabilization site-target binding site ⁇ -C".
  • the bipodal peptide binder of the present invention in the bipodal peptide binder of the present invention, between the target binding site I and one strand of the structure stabilization site and / or between the other strand-target binding site ⁇ of the structure stabilization site, It includes a structure influence inhibiting region that blocks interstructural effects between structural stabilization sites.
  • At the site of rotation are amino acids that are relatively free of rotation of ⁇ and ⁇ in the peptide molecule.
  • the amino acids with relatively free rotation of ⁇ and ⁇ are glycine, alanine and serine.
  • the bipodal peptide binder will have a random sequence, indicating that there is no sequence preference or no designated (or fixed) amino acid residue at any position of target binding site I and / or target binding site ⁇ . it means.
  • a library of bipodal peptide binders can be used in a split-synthesis method (Lam et al. (1991) Nature 354: 82; WO 92/00091) carried out on a solid support (eg, polystyrene or polyacrylamide resin). Can be built accordingly.
  • the library of bipodal peptide binders is constructed in a cell surface display manner (eg phage display, bacterial display or yeast display).
  • the library of bipodal peptide binders may be prepared through a display method based on plasmids, bacteriophages, phagemids, yeasts, bacteria, mRNA or ribosomes.
  • Phage display is a technique for displaying various polypeptides in the form of proteins fused to coat proteins on the surface of the phage (Scott, JK and Smith, GP (1990) Science 249: 386; Sambrook, J. et al., Molecular Cloning. A Laboratory Manual, 3rd ed.Cold Spring Harbor Press (2001); Clackson and Lowman, Phage Display, Oxford University Press (2004)). Random peptides are displayed by fusing the gene to be expressed in gene m or gene VI of filamentous phage (eg, M13).
  • Phageimide may be used for the fiji display.
  • Phageimide is a plasmid vector with one copy of the bacterial origin of replication (eg, ColEl) and the intergenic site of the bacteriophage. DNA fragments cloned in this phagemid are propagated like plasmids.
  • a preferred embodiment of the present invention comprises the following steps: (i) Gene m or gene ⁇ of a phage coat protein (eg, filamentous phage such as M13) A fusion gene in which a gene encoding a coat protein) and a gene encoding a bipodal peptide binder are fused; And constructing a library of expression vectors comprising transcriptional regulatory sequences (eg, lac promoters) operably linked to the fusion gene; (ii) introducing said expression vector library into a suitable host cell; (iii) culturing the host cell to form recombinant phage or phagemid virus particles so that the fusion protein is displayed on the surface; (iv) contacting a biological target molecule with the viral particle to bind the particle to the target molecule; And (V) separating particles not bound to the target molecule.
  • a phage coat protein eg, filamentous phage such as M13
  • the method of producing an expression vector comprising a bipodal peptide binder gene may be carried out according to methods known in the art.
  • known phagemids or phage vectors e.g. pIGT2, fUSE5, fAFFl, fd-CATl, m663, fdtetDOG, pHENl, pComb3, pComb8, pCANTAB 5E (Pharmacia), LamdaSurfZap, pIF4 PM48, PM52, PM54, fdH) And p8V5
  • phagemids or phage vectors e.g. pIGT2, fUSE5, fAFFl, fd-CATl, m663, fdtetDOG, pHENl, pComb3, pComb8, pCANTAB 5E (Pharmacia), LamdaSurfZap, pIF4 PM48, PM52, PM54, f
  • phage display methods are performed using filamentous phage, but lambda phage display (W095 / 34683; US Pat. No. 5,627,024), T4 phage display (Ren et al. (1998) Gene 215: 439; Zhu (1997) CAN 33: 534, and T7 phage display (US Pat. No. 5,766,905) can also be used to build a library of bipodal peptide binders.
  • the method of introducing the vector library into a suitable host cell can be carried out according to a variety of transformation methods, most preferably according to the electroporation method (see, for example, US Pat. Nos. 5,186,800, 5,422,272, 5,750,373).
  • Suitable hosts for the present invention are gram negative bacterial cells such as E. coli, and suitable E. coli hosts include JM101, E. coli, ⁇ 2 strain 294, E. coli strain W3110 and E. coli XL-lBlue (Stratagene). Including, but not limited to. Host cells are preferably prepared as competent cells prior to transformation (Sambrook, J. et al., Molecular Cloning. A Laboratory Manual, 3rd ed.
  • Selection of transformed cells is generally And cultured in a medium containing antibiotics (eg, tetracycline and ampicillin)
  • the selected transformed cells are further cultured in the presence of helper phage to generate recombinant phage or phagemid virus particles.
  • helper phage include, but are not limited to, Ex helper phage, M13-K07, M13-VCS, and R408.
  • bipodal 3 ⁇ 4-tide binders of the present invention are described in SEQ ID NO: 20-38 and 40-41.
  • the present invention provides a nucleic acid molecule encoding the aforementioned bipodal peptide binder.
  • the present invention provides a vector for expression of a bipodal peptide binder comprising a nucleic acid molecule encoding a bipodal peptide binder.
  • the present invention provides a transformant comprising a vector for expression of a bipodal peptide binder.
  • nucleic acid molecule is meant to encompass DNA (gDNA and cDNA) and RNA molecules inclusively, and the nucleotides, which are the basic structural units in nucleic acid molecules, are modified from sugar or base sites, as well as natural nucleotides. Analogs (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 9.0: 543-584 (1990)).
  • the vector of the present invention is a powerful promoter capable of transferring transcription to the nucleic acid molecule in addition to the nucleic acid molecule encoding the bipodal peptide binder (e.g., ac promoter, promoter, / adJV5 promoter, I PP promoter, ⁇ ⁇ promoter, ⁇ / promoter, rac5 promoter, ⁇ promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.), ribosome binding site and transcription / detox termination sequence for initiation of translation .
  • ac promoter e.g., promoter, / adJV5 promoter, I PP promoter, ⁇ ⁇ promoter, ⁇ / promoter, rac5 promoter, ⁇ promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.
  • the vector of the invention may further comprise a signal sequence (eg pelB) on the 5'-direction of the nucleic acid molecule encoding the bipodal peptide binder.
  • a signal sequence eg pelB
  • the vector of the present invention further includes a tagging sequence (eg, myc tag) for confirming that the bipodal peptide binder is well expressed on the surface of the phage.
  • the vector of the present invention comprises a phage coat protein, preferably a gene of a filamentous phage such as M13 or a gene encoding a gene coat protein.
  • the vector of the present invention comprises an origin of replication of bacteria (eg ColEl) and / or a bacteriophage.
  • the vector of the present invention may include antibiotic resistance genes commonly used in the art as a selection marker, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neo Resistance genes for mycin and tetracycline.
  • the transformants of the present invention are preferably Gram-negative bacterial cells such as E. coli, and suitable E. coli hosts are JM101, E. coli 12 strain 294, E. coli strain W3110 and E. coli XL-lBlue (Stratagene ), But is not limited thereto.
  • the method of carrying the vector of the present invention into a host cell may be performed by the CaCl 2 method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114 (1973)), one method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114 (1973); and Hanahan, D., J. Mol. Biol., 166: 557-580 (1983)) and electroporation methods (US Pat. Nos. 5,186,800, 5,422,272, 5,750,373) and the like.
  • bipodal peptide binders of the present invention exhibit very low levels (eg, nM levels) of K D values (dissociation constants) to provide peptides that exhibit very high affinity to biological target molecules.
  • K D values dissociation constants
  • bipodal peptide binders exhibit about 10 2 -10 5 times (preferably about 10 3 -10 4 times) high affinity as compared to binders made in a monopodal manner.
  • the bipodal peptide binder of the present invention not only has use as a medicament, but also detects substances in vivo, in vivo molecular imaging, in vitro cells It can be used to target imaging and drug delivery, and can also be used as an escort molecule.
  • the present invention provides a BPB-based cargo delivery system.
  • the bipodal peptide binders of the present invention thus exhibit very low levels (eg, nM levels) of K D values (dissociation constants), resulting in very high affinity to the target.
  • the BPB-based cargo delivery system of the present invention can deliver various substances into the cell surface or cells based on the target binding and specificity of BPB.
  • La shows a schematic diagram of a bipodal-peptide binder comprising ⁇ -hairpin as a structural stabilization site.
  • FIG. Lb shows a schematic diagram of a bipodal-peptide binder comprising ⁇ -sheets linked by linkers as structural stabilization sites.
  • FIG. Lc shows a schematic diagram of a bipodal peptide binder comprising leucine zippers linked by linkers as structural stabilization sites.
  • Figure ID shows a schematic of a bipodal-peptide binder comprising a leucine-rich motif linked by a linker as a structural stabilization site.
  • FIG. 2 shows a strategy for cloning bipodal peptide binder libraries.
  • the pelB signal sequence myc tag, is a tagging sequence to confirm that the target gene is well expressed on the surface of the phage.
  • the lac promoter was used as a promoter.
  • FIG. 3 shows the biopanning results of ED-B straptavidin and BSA for input phage during pyronectin ED-B biopanning.
  • FIG. 4 shows ELISA results of ED-B and BSA of 60 recombinant phages recovered in the third step of biopanning of bipodal peptide binder library during pyronectin ED-B biopanning.
  • Figure 5a is a result of measuring the affinity of a specific bipodal peptide binder that binds to the pyronectin ED-B protein.
  • Figure 5b is the result of measuring the affinity of a specific bipodal peptide binder that binds to VEGF.
  • Figure 5c is the result of measuring the affinity of a specific bipodal peptide binder that binds to VCAM1.
  • Figure 5d is a result of measuring the affinity of a specific bipodal tempide binder that binds to the nicotinic acetylcholine receptor (nAchR).
  • Figure 5e is the result of measuring the affinity of a specific bipodal peptide binder that binds to HSA Human Serum Albumin.
  • Figure 6a is a result of measuring the absorbance of the recombinant phage having a bipodal peptide binder ELISA for a number of proteins for specificity test for pyronectin ED-B. Results from the left bar are for straptavidin, ED-B, acetylcholine al, BSA, VCAM, TNF- ⁇ , thrombin, myoglobulin, lysozyme and bispartin.
  • Figure 6b is a result of measuring the absorbance of the recombinant phage having a bipodal peptide binder ELISA for a variety of proteins to test for specificity for VEGF.
  • Figure 6c is a result of measuring the absorbance of the recombinant phage having a bipodal peptide binder ELISA for a variety of proteins for specificity test for VCAM1.
  • Figure 6d is a result of measuring the absorbance of the recombinant phage having a bipodal peptide binder ELISA for a number of proteins to test the specificity for the nAchR fragment peptide.
  • Figure 6e is a result of measuring the absorbance of the recombinant phage having a bipodal peptide binder for the specificity test for HSA by performing an ELISA for a number of proteins.
  • Figure 6f is a result of measuring the absorbance of the recombinant phage having a bipodal peptide binder ELISA for a variety of proteins to test for specificity for MyD88.
  • bipodal peptide binders specific for cancer biomarker fibronectin ED-B. Over time, it appears that bipodal peptide binders accumulate in cancer. When the individual organs are separated and measured for fluorescence, they also accumulate in the cancer.
  • 11A shows purification of GST-BPB fusion protein.
  • Lib is affinity measurement results for Fibronectin EDB of GST-BPB. 12A shows purification of TNFa-BPB.
  • FIG. 13A shows the results of analyzing the encapsulation efficiency of 9R / siRNA in BPB CSS- LS.
  • the green box shows the liposomal fraction containing the 9R / siRNA complex and the red box shows the free unencapsulated siRNA fraction.
  • Figure 13b is the result of analyzing the uptake of BPB CSS- LS in the EDB over-expressing cell line. Negative represents untreated cells, LS represents cells treated with liposomes only, CSS-LS represents cells treated with BPB CSS -LS.
  • Figure 13c is the result of analyzing the uptake of BPB CSS- LS in the EDB non-expressing cell line. Negative represents untreated cells, LS represents cells treated with liposomes only, CSS-LS represents cells treated with BPB CSS -LS.
  • Figure 13d is the result of analyzing the VEGF-C siRNA knockdown efficiency in MCF-7 cells.
  • 14a shows the results of conjugation of DSPE—PEG 2000 -Mal and BPB (SSS) peptide using MALDI.
  • Figure 14b is a result of ELS analysis observed the change in hydrodynamic size of SPI0N-BPB for a week.
  • Figure 14c is a TEM image of (a) DSPE-PEG2000 coated SPION and (b) BPB conjugated DSPE- PEG2000 coated SPION.
  • Figure 14d is the result of T2-weighted MR phantom study of DSPE-PEG 20 oo coated SPION (DSPE-SPION) and BPB conjugated DSPE-PEG2000 coated SPION (SSS-SPION).
  • FIG. We show the results of (a) MRI image and (b) T2 signal of cells treated with DSPE-PEG 2000 coated SPION and BPB conjugated DSPE-PEG2000 coated SPION.
  • 14F is a confocal microscope image of cells treated with DSPE-PEG 2000 coated SPION and BPB conjugated DSPE-PEG2000 coated SPION.
  • Figure 14g is the MTT assay results of DSPE-PEG 2000 coated SPION and BPB conjugated DSPE-PEG2000 coated SPION.
  • '14h is an MRI image of a brain cancer animal model of DSPE-PEG 2000 coated SPION and BPB conjugated DSPE-PEG2000 coated SPION.
  • 15A is a TEM image of Gold Nanoparticle—BPB.
  • Figure 15b is the result of measuring the difference in the amount of Au in the cells using ICP-MS after the same concentration of BPB conjugated gold nanoparticles using U87MG cells.
  • Figure 15c is a micrograph of silver enhancement after treatment of (a) GNP or (b) BPB-GNP to U87MG cells.
  • Beta-Fl (5'-TOTATGCGGCCCAGCTGGCC (NNK) 6 GGATOTGGACATGGGAAAACGGAAAA-3 ') and Beta-Bl (5'-
  • PIGT2 phagemid vector was reacted with Sfil and Notl for 4 hours each, followed by addition of CIAP (Calf Intestinal Alkaline Phosphatase) (NEB, Ipswich) for 1 hour, followed by PCR. Purification using a purification kit. These were quantified by UV-Vis spectroscopy (Ultrospec 2100pro, Amersham Bioscience) to 2.9 insert genes using T4 DNA ligase (Bioneer, Dae j eon, Korea), pIGT2 phagemid vector 12 / g and 15 at 18 ° C. After connecting for a period of time, precipitated with ethanol to dissolve DNA with TE buffer 100 ⁇ .
  • E. col / XL1-BLUE cells (American Type Culture Collection, Manassas, USA) were plated on LB agar-plates. The colonies grown in agar plate medium were inoculated in LB medium of 5 and then incubated for one day with mixing at 37 rpm at a speed of 200 rpm. Cultured 10 cells were inoculated in 2 LB sages and cultured in the same manner until the absorbance was 0.3-0.4 at a wavelength of 600 nm. The incubated flask was left on ice for 30 minutes, then centrifuged at 4,000 ⁇ g for 20 minutes at 4 ° C. to remove all supernatants except the sunk cells and suspended in 1 sterile sterile distilled water.
  • Electroporation was performed by dispensing 12 jug of phagemid vector and 25 100 ⁇ by connecting the insert DNA 2.9 i to the bipodal peptide binder. Competent cells were dissolved on ice, mixed with 200 ⁇ of competent cells and mixed with solution 4 ⁇ , then placed in a prepared 0.2 cm cuvette and placed on ice for 1 minute.
  • the electroporator (BioRad, Hercules, CA) was programmed at 25 ⁇ at 25 yF and 2.5 kV and the prepared cuvette was drained and placed in an electroporator and pilsed (time constant is 4.5—5 msec). Thereafter immediately placed in 1 LB medium containing 20 mM glucose prepared at 37 ° C.
  • a total of 25 i cells obtained were transferred to 100 test tubes. After an incubation at 200 rpm at 37 ° C for one hour, incubate 10 to determine the number of libraries. Ampicillin agar medium was plated. The remaining cells were put in 1 LB of 20 mM glucose and 50 / zg / ampicillin and incubated at 30 ° C for one day. Centrifugation at 4 ° C. at 4,000 ⁇ g for 20 minutes to remove all supernatants except the precipitated cells, resuspended in LB of 40 1 and stored glycerol at -80 ° C with a final concentration of 203 ⁇ 4 or more.
  • Recombinant phage was produced in a bipodal peptide binder library stored at -80 ° C. Ampicillin (50 / g / m £) and 20 mM glucose were added to a 500-LB liquid medium in 500 flasks, and then the library 1 ⁇ stored at -80 ° C was added at 150 rpm at 37 ° C for one hour. Cultures were mixed at speed. Ex helper phage (Ig therapy, Chuncheon, Korea) of 1X10 11 pfu was added thereto and incubated under the same conditions for one hour.
  • the supernatant was removed by centrifugation for 10 minutes at 1,000 ⁇ g, and ampicillin (50 zg / m ⁇ ) and kanamycin (25 ⁇ LB liquid medium containing 25) were incubated for one day to produce recombinant phage.
  • ampicillin 50 zg / m ⁇
  • kanamycin 25 ⁇ LB liquid medium containing 25
  • 25 m £ of PEG / NaCl was mixed with 100 ⁇ and left on ice for 1 hour, followed by centrifugation at 10,000Xg for 20 minutes at 4 ° C. Carefully remove and resuspend the pellet with 2 PBSCpH 7.4).
  • Fibronectin ED-B VEGFCvascular endothelial growth factor (VEGKvascular cell adhesion molecule-1), nicotinic acetylcholine receptor (nAchR), human serum albumin (HSA) and MyD88 to be used in the examples were prepared as follows.
  • VEGFCvascular endothelial growth factor VEGFCvascular endothelial growth factor
  • nAchR nicotinic acetylcholine receptor
  • HSA human serum albumin
  • MyD88 MyD88
  • the common hapaek PCR reaction a by (5 minutes at 94 ° C, 30 cycles at 55 ° C 30 sec, 1 min and 30 sec at 94 ° C at 72 ° C) and then made to EDB insert using the PCR purification kit Purification by The EDB insert gene and the pET28b vector were subjected to restriction enzymes to link the EDB insert gene to the pET28b vector (Novagen). The insert DNA of about 2 was reacted with BamHKWR, Ipswich) and 71 ⁇ 2fe / (NEB, Ipswich) for 4 hours and then purified using a PCR purification kit.
  • CIAP was added and reacted for 1 hour, and then purified using a PCR purification kit. Add them with a molar ratio of about 1: 3 to the vector and insert, connect them for 10 hours at 18 ° C using T4 DNA ligase (Bioneer, Dae j eon, Korea), and attach to XL-1 competent cells. After transformation, the cells were plated in agar medium containing kanamycin.
  • VEGF_FK5'-ATAGAATTCGCACCCATGGCAGAA-3 ') and VEGF_B1 (5'- ⁇ 00 ⁇ 0 ⁇ 00000 ⁇ 0 ( ⁇ ⁇ ⁇ 0 ⁇ 0 ⁇ 0 ⁇ 0 ⁇ 0 ⁇ 0 ⁇ 00-3')) were synthesized, VEGF-F1 20 pmol, VEGF-B1 20 pmol, 2.5 mM dNT 4 ⁇ , ExTaq DNA polymerase 1 (10 U) and 10 x PCR buffer 5 ⁇ were mixed to make a mixture with distilled water added to a total of 50 ⁇ .
  • the mixture was subjected to PCR reaction (5 min at 94 ° C, 30 cycles: 30 seconds at 55 ° C., 1 minute at 72 ° C. and 30 seconds at 94 ° C.) to make a VEGF insert and then purified using a PCR purification kit.
  • the VEGF insert gene was transferred to the pET32a vector (Novagen).
  • the VEGF insert gene and the pET32a vector were treated with restriction enzymes for ligation.
  • the PCR purification kit was reacted with ⁇ oA7 (NEB, Ipswich) and Hindi IK '' Ipswich for about 2 // g of insert DNA for 4 hours.
  • T4 DNA ligase (Bioneer, Dae j eon, Korea) was used for ligation at 18 ° C. for 10 hours, transformed into XL-1 competent cells, and plated in agar medium containing ampicillin. The colonies grown on agar plate medium were inoculated in 5 LB medium and incubated at 37 ° C. at 200 rpm for one day, followed by plasmid purification kit (GeneAU, Seoul, Korea) to purify the plasmid, It was sequenced to see if cloning was successful.
  • the Korea Biotechnology Research Institute received the human VCAM1 gene. Restriction enzymes were treated with the VCAM1 insert gene and the pET32a vector to link the VCAM1 insert gene to the pET32a vector. These allergens and inserts were added in a molar ratio of about 1: 3, ligation was performed at 18 ° C for 10 hours using T4 DNA ligase (Bioneer, Dae j eon, Korea), XL-1 competent cells After transformation, the cells were plated in agar medium containing ampicillin.
  • a pET28b vector cloned with fibronectin ED-B was transformed into BL21 cells and plated in agar medium containing kanamycin. After while heunhap then inoculated with the colonies grown on the agar plate medium on LB medium kanamycin (25 zg / m £) 5 contains a ⁇ at 37 ° C at a rate of 200 rpm to incubate for one day, kanamycin (25 ⁇ g / m £) was transferred to 50 LB medium containing and incubated for 3 hours. Cultured E.
  • Lysis buffer 50 mM sodium phosphate, pH 8.0
  • coli ⁇ by using a sonicator, and centrifuge for 1 hour at 15,000 Xg for supernatant to Ni-NTA. It binds to affinity resins (Elpisbio, Dae j eon, Korea). The resin was washed with Lysis buffer and then obtained by eluting the N-terminal His-tag ED-B protein using Illution buffer (50 mM sodium phosphate (pH 8.0), 300 mM NaCl and 300 mM imidazole). The protein thus obtained was purified by gel filtration using a Superdex75 column (GE Healthcare, United Kingdom) and PBS (pH 7.4) buffer to obtain high-purity ED-B protein.
  • Illution buffer 50 mM sodium phosphate (pH 8.0), 300 mM NaCl and 300 mM imidazole.
  • Illution buffer 50 mM sodium phosphate (pH 8.0), 300 mM NaCl and 300 mM imidazole.
  • Biotin was linked to ED-B protein for biopanning. 6 mg of sulfo-NHS-SS-biotin (PIERCE, Illinois, USA) and 1.5 rag of ED-B protein were reacted at room temperature under 0.1 M sodium borate (pH 9.0) for 2 hours, and the reaction was not followed. Biotin-EDB protein was purified by gel filtration using a Superdex75 column and PBS (pH 7.4) buffer to remove -SS-biotin.
  • PET32a vectors cloned from VEGF121 and VCAM1 were transformed into AD494 cells and plated in agar medium containing ampicillin.
  • Ampicillin the colonies grown in an agar plate medium (after 5 and after inoculation in LB medium in a 25 comprises from 37 ° C heunhap at a rate of 200 rpm for one day culture, containing ampicillin (25 a
  • VEGF121 was obtained by cutting between VEGF and Trx with thrombin to obtain pure VEGF.
  • HSA was purchased from Genetex Company (Irvine).
  • Biotin-SGEWVIKEARGWKHWVFYSCCPTTPYLDITYH 32 mer
  • nAchR Nicot inic acetylcholine receptor
  • Anigen Korea, Kwangju
  • Human MyD88 was implemented in Santa Cruz Biotechnology (sc-4540 WB) (California).
  • Ampicillin 50 ⁇ / ⁇ and 20 mM after the combined wave the glucose, was added to Ex helper phages of 2X10 10 pfu by heunhap at a rate of 200 rpm for one hour at 37 ° C, and were cultured. Culture for l, 10 min 000Xg After centrifugation, the supernatant is removed and precipitated. Heunhap cells ampicillin (50 ⁇ / i) in and kanamycin (30 ° C and resuspended in 25 40 M LB liquid medium containing a rate of 200 rpm and incubated for one day. 4,000Xg the culture medium for 20 min and 4 Centrifugation was carried out under conditions of ° C.
  • VEGF, VCAMl-trx, HSA and MyD 88 (5 t / g /) were placed in 10 wells of 96 well ELISA plates (Corning) at 50 ⁇ , then allowed to stand overnight at 4 ° C. After blocking for 2 hours at room temperature, the solution was discarded and washed three times with 0. PBST.
  • 800 ⁇ and 103 ⁇ 4> BSA 200 ⁇ containing bipodal peptide binder recombinant phage were mixed and transferred to 10 wells in which VEGF, VCAMl-Trx, and HSA were combined and left at room temperature for 1 hour.
  • Ex helper phage was added to the 2X10 10 pfu by heunhap at a rate of 200 rpm for one hour at 37 ° C, and were cultured. After centrifugation of the culture broth at l, 000Xg for 10 minutes, the supernatant was removed and the precipitated cells were ampicillin (50 And resuspended in kanamycin (40 LB liquid medium containing 25) and incubated for one day at 30 ° C. at 200 rpm. Cultures were centrifuged at 4,000 ⁇ g, 20 minutes and 4 ° C.
  • ELISA of each input phage of the bipodal peptide binder library was performed on straptavidin, BSA and ED-B.
  • 10 ig / strapavidin was put in 18 wells at 50 ⁇ for each well and 10 g / r BSA was put in 9 wells at 50 for each well and left at 4 ° C. for one day.
  • the next day only 9 wells of 18 wells with straptavidin were washed three times with 0.1% PBST (tween-20), and biotin ED-B (10; «g /) was added and left at room temperature for 1 hour.
  • TMBXBD Science tetramethylbenzidine
  • Plaques were inoculated into 4 LB-ampicillin (50 glv) cultures using sterilized tips, followed by shaking culture at 37 ° C for 1 day to purify plasmids using plasmid fram kits for commissioning (Genotech, Dae j eon, Korea).
  • the sequencer primer used the vector sequence 5'-GATTACGCCAAGCTTTGGAGC-3 '.
  • Example 7 Cancer Targeting of a Bipodal Peptide Binder Specific for Pirbonectin ED-B, a Cancer Biomarker
  • MyD88 is an intracellular protein
  • 9 arginine (Anigen, Korea), which is an eel 1 penetrating peptide, is used in the EDC / lysine residue of the loop of the bipodal peptide binder.
  • NHS Sigma
  • Cartilage cells were treated with IL-lbeta (10 ng / ml) (R & D systems, Minneapolis), which activates the activity of MyD88.
  • MyD88-specific bipodal peptide binder (peptide 1 of Table 3f) was treated with chondrocytes 10 ⁇ , mRNA was isolated after 12 hours, and then RT-PCR was performed for MMP-13 and GAPDH. In addition, the cartilage cells are destroyed to obtain intracellular proteins, followed by anti-MMP 13 antibody (Abeam, ab3208, Cambridge) and a semi-dry transfer machine (Amersham Bioscience, Piscataway) were subjected to Western blotting to determine the amount of MMP-13.
  • the structure stabilization site of the bipodal peptide binder As the structure stabilization site of the bipodal peptide binder, a stable beta-hairpin motif was used.
  • tryptophan zippers (Andrea et al., Proc. Natl. Acad. Sci. 98: 5578—5583 (2001)), which stabilize the beta-hairpin motif structure by the interaction of tryptophan-tryptophan amino acids, were used.
  • Variable regions were created in two portions by randomly arranging six amino acids in each of the N- and C-terminal portions of the backbone tryptophan zipper (FIG. La).
  • This is called a bipodal peptide binder and has variable regions on both sides so that it can be cooperatively attached to the antigen and thus have high affinity and specificity.
  • the structure stabilization site of the bipodal peptide binder may be configured in various ways as shown in FIGS.
  • Two random sequence oligonucleotides were synthesized into double chains by PCR and then cut into restriction enzymes Sfi ⁇ and Not ⁇ and cloned into a pIGT2 phagemid vector to construct a library of 8 ⁇ 10 8 or more (FIG. 2).
  • the bipodal peptide binder library was subjected to biopanning three to five times for fibronectin ED-B, VEGF, VCAM1, nAchR, and HSA proteins, and the ratio of output phage / input phage of phage peptides recovered at each panning step was determined. (Table la).
  • Each input phage in the library of bipodal peptide binders was subjected to ELISA for ED-B, strapavidin and BSA. Reactivity of the first input phage was similar in absorbance to ED-B, straptavidin and BSA, but from the second input phage, ED-B hop luminosity was 5.1 times higher than that of Straptavidin and 3.4 times higher than that of BSA. In the third input phage, ED-B absorbance was 22 times higher than that of straptavidin and 15 times higher than that of BSA, indicating that biopanning was successfully achieved for ED-B (FIG. 3 and Table 2).
  • Phages recovered at the highest output / input ratio during the panning stage of each library were obtained in the form of plaques. After amplifying 60 phages from each plaque, ELISA was performed on BSA (FIG. 4). Clones with higher absorbance than BSA were selected to request DNA sequencing. From this a peptide sequence specific to each overlapped protein was obtained (Table 3).
  • Example 13 Determination of affinity of fibronectin ED-B, VEGF, VCAM1, nAchR and HSA
  • peptides for fibronectin ED-B, VEGF, VCAM1, nAchR and HSA were synthesized and measured for affinity using the SPR Biacore system (Biacore AB, Uppsala, Sweden).
  • SPR Biacore AB Biacore AB, Uppsala, Sweden.
  • peptide 1 showed 620 nM
  • peptide 2 showed 75 nM
  • peptide 3 showed 2.5 ⁇
  • peptide 1 showed 60 nM
  • peptide 2 showed 326 nM (FIG. 5B).
  • the affinity of the fragment peptide of VCAM1 was measured and peptide 1 showed 318 nM (FIG. 5C).
  • Recombinant phage that specifically binds to each protein was tested for specificity using ELISA.
  • 5 g / m £ of each protein was added to the wells by 50 ⁇ , washed three times with 0.1% PBST (Tween-20) the next day and blocked for 2 hours at room temperature using 2% BSA. After that, the solution was discarded and washed three times with 0.1% PBST.
  • the recombinant phage having the peptide of the present invention was well mixed with 23 ⁇ 4 BSA, dispensed into wells containing 10 proteins by 100, and left at 27 ° C. for 2 hours.
  • Example 16 Binding Assays for Other ⁇ -Hairpins Peptides were synthesized to have N-terminal sequence (HCSSAV) and C-terminal sequence (IIRLEQ) of Peptide2 that specifically bind ED-B to other ⁇ -hairpin backbones GBlm3 and HP7 in addition to tryptophan zippers (Anigen, Korea). That is, the sequence of bipodal peptide binders including tryptophan zippers
  • the HCSSAVGSWTWENGKWTWKGI IRLEQ and the bipodal peptide binder containing GBlm3 are HCSSAVGKKFTYNPATGKFTVQEGI IRLEQ and the bipodal peptide binder containing HP7 is HCSSAVGKTWNPATGKWTEGI IRLEQ.
  • the affinity of each peptide was measured using BIAcore X (Biacore AB, Uppsala, Sweden). Biotin-EDB was flowed onto the straptavidin SA chip (Biacore AB, Uppsala, Sweden) by 2,000 RU and fixed.
  • PBS pH 7.4
  • the flow was measured for kinetics at various concentrations at 30 d / min and the affinity was calculated by BIAevaluation. It was confirmed that the affinity similar to tryptophan zipper (43 nM) was 84 nM (Fig. 8), which proves that all stable ⁇ -hairpin motifs are possible as structural stabilization sites.
  • Cy5,5 fluorescent dies were attached to a bipodal peptide binder targeting fibronectin ED-B, which is widely distributed in cancers, and then to human U87MG cancer mice. Intravenous administration was performed to check the fluorescence of the bipodal peptide binder targeting cancer using the IVIS machine (FIG. 10).
  • IVIS machine FIG. 10
  • Bipodal peptide binders specific for ED-B have been observed to accumulate in cancer tissues, indicating that the bipodal peptide binders of the present invention can be used for in vivo imaging.
  • the pGEX4T_l vector (GE Healthcare) containing the Glutathione S-transferase (GST) gene was cloned into the BPB gene sequence that recognizes Fibronectin EDB.
  • Phagemid vector obtained from phage attached to EDB protein using 2 ligonucleotides GST-FU5 '-ACCGGATCCCATTGTTCTAGT-3' and GST-Bl (5'-ATTCTCGAGTTAC ( ⁇ TCCTCCT (X-3 ')) PCR was carried out in Examples 1 and 3) to obtain a BPB peptide gene attached to Fibronectin EDB
  • the peptide sequence of this BPB peptide was HCSSAVGSWTWENG WTWKGI IRLEQ GST—F1 20 pmol, GST-B1 20 pmol, phagemid template lul, 2.5mM dNTP mixture 4 ⁇ 1, Ex Taq DNA polymerase 1 ⁇ 1 (10 U), 10 x PCR buffer 5 ⁇ 1 were mixed and a mixture of
  • BPB Insert gene and pGEX4T-1 vector were subjected to restriction enzyme treatment to link the PGEX4T-1 vector.
  • IPTG ImM isopropyl-i3-E hiogalactopyranoside
  • Supernatant was bound to GST affinity resin (Peptron) by storage at -80 o C for one day and then dissolved by E. coli using Sonicator and centrifuged at 15,000 g for 1 hour. The resin was washed with PBS buffer and collected by dropping the GST-BPB protein using Elution buffer (20 mM GSH in lOmM Tris-HCl, pH 8). The protein thus collected was subjected to gel filtration using Superdex75 column (Amersham) and PBS (pH7.4) buffer to collect high purity GST-BPB protein.
  • TNF a tumor necrosis factor a
  • Fibronectin EDB BPB gene sequences that recognize human TNF a (tumor necrosis factor a) and Fibronectin EDB were cloned into pET28b vector (Novagen).
  • Two oligonucleotides TNF-FKAATAAAACATATG TCTCGAACCCCGA) and TNF-B1 (ATGGATCCCAGGGCMTGATC) were used to amplify the TNFa gene in vector Gh75 (Cytokine Bank, Korea), which was cloned from human TNF ⁇ and cloned into pET28b.
  • BPB-F1 AAT GAATTC TCTTCCTCATCG (CTTCCTCATC ( ⁇ TTGTAGTTCTCCT BPB-B1) (AAT AAGCTT TCA
  • TTGCTCCAACCTAAT was used to PCR phagemid vectors (Examples 1 and 3) obtained from phage attached to EDB protein by template to obtain BPB peptide gene attached to Fibronectin EDB.
  • the peptide sequence of this BPB peptide is CSSP I QGSWTWENGKWTWKG I IRLEQ.
  • the BPB gene was cloned into pET28b vector containing TNFa.
  • the plasmid was using a plasmid preparation kit Ensure that you have purified, cloned the success of sequencing.
  • the pET28b vector cloned with TNFa-BPB was transformed into BL21 cells and plated in kanamycine agar medium.
  • Kanamycine 25ug / ml
  • 0D 0.6-0.8
  • IPTG ImM isopropyl- ⁇ -D-thiogalactopyranoside
  • TNFa-BPB protein was collected by dropping Elution buffer (50 mM sodium phosphate (pH 8.0), 300 mM NaCl and 300 mM imidazole). The collected proteins were collected using Superdex75 column and PBS (pH7.4) Gel filtration using a buffer yielded high purity TNFa-BPB protein.
  • Elution buffer 50 mM sodium phosphate (pH 8.0), 300 mM NaCl and 300 mM imidazole.
  • the collected proteins were collected using Superdex75 column and PBS (pH7.4) Gel filtration using a buffer yielded high purity TNFa-BPB protein.
  • TNFa-BPB binds to Fibronectin EDB was confirmed using an SPR machine. Affinity measurements were performed using BIAcore X. The biotin—EDB was flowed into the Streptavidin SA chip (Biacore) by 2000RU and fixed. PBS (pH 7.4) was used as the running buffer, and the kinetics were measured at various concentrations while the flow was held at 30 ul / min. The affinity was calculated by BIAevaluation.
  • the cytotoxicity of TNFa-BPB and TNF a was measured by MTT assay using L-M mouse fibroblasts. First, 5000 L-M mouse fibroblasts were placed on 96 well microplates, and after 18 hours, TNF ⁇ and TNFa-BPB were added at different concentrations, and MTT was performed 30 hours later to measure cytotoxicity.
  • TNFa-BPB TNFa-BPB
  • TNFa-BPB was measured for affinity for Fibronectin EDB using the SPR biacore system. Affinity measurement showed that it had affinity of 80nM and could recognize Fibronectin EDB even in the presence of TNFa (FIG. 12B).
  • TNFn and TNFn-BPB The cytotoxicity of TNFn and TNFn-BPB in L-M mouse fibroblast cells was measured by MTT. TNFa-BPB is 5 times higher in cytotoxicity than TNFa (FIG. 12C).
  • 9R peptide and VEGF-C siRNA were distilled off in HEPES—buffered 5% glucose (pH 7.4) 250 ⁇ l and incubated at room temperature for 10 minutes. The solutions were combined and vortexed to obtain 500 ⁇ l 9 R / siRNA complexes at a +/ ⁇ charge ratio of 1: 6.
  • the 9R / siRNA-loaded BPB-1 ipos nes (BPB css ' S) and the control 9R / si RNA-1 oaded-1 i posomes were purified by size-exclusion chromatography (CL-4B column).
  • CL-4B column size-exclusion chromatography
  • the siRNA content of the Sepharose CL4B column eluent was analyzed by OHGreen (Invitrogen).
  • BPBc ⁇ LS loaded with 9R / siRNA was regenerated with Hepes Buffer Saline (HBS) to obtain an optimal scattering intensity.
  • Hydrodynamic diameter and zeta potential were measured by electrophoretic light scattering using an ELS 8000 device (Otsuka Electronics Korea, Seoul, South Korea).
  • MCF-7 cells were treated with 50 nM VEGF-C siRNA in lipofectamineCpositive control, 50 nM BPB CSS- LS and 100 nM BPB CSS- Transformed with LS. After 4 hours, the cells were further incubated for 24 hours, followed by RNA isolation, cDNA synthesis and real-time RT-PCR.
  • siRNA was embedded in BPB-liposome (CSS-LS) and separated using a size exculsion CL-4B column to remove free siRNA. Large siRNA / BPB-liposomes come out first, followed by free siRNAs. In this way, purified BPB-liposome containing siRNA can be obtained.
  • BPB-liposome and control liposome were treated in the same manner using PC3 and LnCap, which are EDB negative cells, they do not adhere to each cell in Figure 13c. This indicates that without the target protein EDB, no BPB-liposome is attached and that the BPB-1 iposome is very specific for EDB.
  • FIG. 13D it was confirmed in MCF-7 cells whether BPB-LS containing VEGF-C siRNA was able to inhibit VEGF-C mRNA in cells.
  • Treatment with BPB-LS containing 50nM and ⁇ VEGF-C siRNA showed that the cells could inhibit VEGF-C mRNA similarly to lipofectamine.
  • DSPE-PEG2000 polymer and BPB conjugated DSPE-PEG2000 and Rhodamine-DSPE (5: 94: 1) are added to DW, and Oleic SPI0N (superparamagnetic iron oxide nanoparticle, synthesized by pyrolysis method) is dissolved in Hexane.
  • Polymer solution 1 was added, sonication was performed using a probe type sonicator, and then purified using stirring, centrifuge and magnet. And the group without BPB was synthesized in the same manner as above.
  • B2B conjugated DSPE-PEG2000 coated oleic SPI0N and DSPE-PEG 200 () coated olei c SPI0N were packed into e-tubes by concentration, respectively, and the T2 Time was measured using a 3T MRI scanner (Magnetom Avanto, Siemens, Germany). Calculated.
  • Balb / c nude female were transplanted to 6 U87MG cells, 5 X 10 6 cells at the top of the right hind leg of mice weeks after grown about two weeks, when the size of the cancer being eoteul 3 about 100-150 ⁇ BPB conjugated DSPE-PEG2000 coated olei c SPI0N and After the tail vein administration of DSPE-PEG 2000 coated oleic SPION at 20 mg Fe / kg, the signal intensity of the cancerous region was monitored over time using 3T MRI.
  • the hydrodynamic size (measured in aqueous solution) is about 30 nm in size, and the size increases by about 20 nm when using DSPE conjugated with BPB (SSS peptide). It was.
  • Cytotoxicity test of DSPE-PEG2000 coated SPION and BPB conjugated DSPE- PEG 20 oo coated SPION through MTT experiment showed low cytotoxicity in both groups (FIG. 14g).
  • Gold nanoparticles having a size of about 5 nm were synthesized using gold ions as a reducing agent.
  • the 5 nm gold nanoparticles need to be coated with biocompatible polymer PEG to reduce the uptake to in vivo stability, ie RESCreticuloendothelial system, etc. and increase the efficiency of delivery to target cells. Therefore, in order to introduce thiol group into PEG, it was reacted with Hpoic acid containing di thiol group and carboxyl group.
  • lipoic-PEG was synthesized by first activating the carboxyl group of Hpoic acid with DCC (dicyclohexylcarbodi imide) and NHS (N—hydroxylsuccini imide) and then reacting with LOOug's N3 ⁇ 4-PEG-0CH 3 .
  • DCC dicyclohexylcarbodi imide
  • NHS N—hydroxylsuccini imide
  • Gold nanoparticles can overcome the disadvantages of conventional contrast agents (short imaging time, kidney toxicity), but gold nanoparticles also have a problem that they are not released into the body due to problems such as size.
  • gold nanoparticles coated with amphoteric ions which can act as PEG instead of PEG and do not significantly increase the hydrodynamic size of nanoparticles, reduce the hydrodynamic size of final gold nanoparticles to less than 8 nm in vitro. Nanoparticles were designed to escape.
  • the amphoteric ions used in this study can be combined with gold nanoparticles in the blood by introducing cyclic dithiool groups rather than mono-thiol groups. Increased stability between ions.
  • the structure of zwitterion ions is
  • the surface of the gold nanoparticles provides an optimal ratio between the zwitterion and the gold nanoparticles needed for the gold nanoparticles to stabilize under serum and 1M NaCl.
  • Surface Plasmon resonance Measured by observing the change in peak peak.
  • various ratios (1: 3000, 1: 5000, 1: 7000, 1: 10000, 1: 12000, 1: 15000) between gold nanoparticles and amphoteric ions were mixed and separated using a centrifuge after 12 hours of reaction. 24 hours after the addition of 10% serum and 1M NaCl, the maximum surface plasmon resonance peak was measured using a UV-Vis spectrum.
  • a cyclic dithiol group was also introduced in BPB.
  • Lipoic acid was reacted with DCC (N, N, -Dicyclohexylcarbodiimide) / NHS (N-hydroxl succinimides) and THF (tetrahydrofuran) for 72 hours to form lipoic-NHS and purified by toluene recrystallization.
  • Gold nanoparticles can overcome the shortcomings of iodide-based CT contrast agents, which are currently widely used, as well as kidney toxicity and short imaging time, and develop gold nanoparticles coated with biocompatible polymers capable of liver cancer imaging as next-generation CT contrast agents. Can be.
  • the surface of the gold nanoparticles was coated with zwitterion ions so that the gold nanoparticles could be stabilized in the blood when injected in vivo.
  • the ratio between the gold nanoparticles and the zwitterion is 1: 10000 or more, the surface plasmon coplanar peak of the gold nanoparticles is unchanged even after 24 hours with the addition of serum and 1 M NaCl, even in the environment of serum and 1 M NaCl. It was confirmed that the gold nanoparticles are stable.
  • Lipoic-BPB which introduces cyclic ditides into BPB, was isolated by HPLC and the compound was confirmed by MALDI-TOF.
  • DTX-NHS was dissolved in DMF immediately before the conjugation reaction, and dissolved by adding BPB. If not completely dissolved DMS0 was added little by little. Add base DIEA (di-isoethyl amine) to help free amine formation of BPB and at room temperature Reaction 12 hours. DIEA added more than 2 equivalents of BPB.
  • the BPB sequence is N-terminal acetylated BPB and the sequence is SSSPIQGSWTWENGKWTWRGIIRLEQ.
  • the DTX-NHS and BPB conjugation reaction solution was dried in a vacuum dryer, dissolved in 503 ⁇ 4> ACN and analyzed by HPLC. HPLC peak samples corresponding to the DTX-BPB conjugate were subjected to mass spectrometry (MALD T0F) to perform DTX-BPB. Conjugate formation was confirmed.

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US20140296479A1 (en) * 2011-04-08 2014-10-02 Gwangju Institute Of Science And Technology D-aptide and retro-inverso aptide with maintained target affinity and improved stability
WO2013061818A1 (ja) * 2011-10-25 2013-05-02 国立大学法人岡山大学 新規ペプチド複合体、そのハイブリッド複合体およびその用途
JPWO2013061818A1 (ja) * 2011-10-25 2015-04-02 国立大学法人 岡山大学 新規ペプチド複合体、そのハイブリッド複合体およびその用途
JP2014047156A (ja) * 2012-08-30 2014-03-17 Osaka Prefecture Univ Ｖｅｇｆ結合性融合ペプチド

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