FUSION POLYPEPTIDES FOR INHIBITING ANGIOGENESIS, FUSION PROTEIN NANOCAGES HAVING MULTIVALENT PEPTIDES FOR INHIBITING ANGIOGENESIS, AND THERANOSTIC USE THEREOF
20220331453 · 2022-10-20
Inventors
Cpc classification
A61K49/0054
HUMAN NECESSITIES
C07K14/78
CHEMISTRY; METALLURGY
C07K16/28
CHEMISTRY; METALLURGY
C07K2319/75
CHEMISTRY; METALLURGY
G01N33/543
PHYSICS
C07K2319/735
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention provides fusion polypeptides for inhibiting angiogenesis, fusion protein nanocages having peptides for inhibiting angiogenesis, and diagnostic and therapeutic (theranostic) uses thereof.
Claims
1. A fusion polypeptide for inhibiting angiogenesis, i) consisting of an anti-angiogenic peptide; a helix-based polypeptide represented by SEQ ID NO 1, which is linked to the peptide; a hydrophilic elastin-based polypeptide (hydrophilic EBP) linked to the helix-based peptide; and a helix-based polypeptide represented by SEQ ID NO 2, which is linked to the hydrophilic EBP, ii) consisting of a helix-based polypeptide represented by SEQ ID NO 1; a first hydrophilic EBP linked to the peptide; an anti-angiogenic peptide linked to the first hydrophilic EBP; a second hydrophilic EBP linked to the anti-angiogenic peptide; and a helix-based polypeptide represented by SEQ ID NO 2, which is linked to the second hydrophilic EBP, or iii) consisting of an anti-angiogenic peptide; a helix-based polypeptide represented by SEQ ID NO 1, which is linked to the peptide; a hydrophilic EBP linked to the helix-based polypeptide; an anti-angiogenic peptide linked to the hydrophilic EBP; a hydrophilic EBP linked to the anti-angiogenic peptide; and a helix-based polypeptide represented by SEQ ID NO 2, which is linked to the hydrophilic EBP.
2. The fusion polypeptide for inhibiting angiogenesis of claim 1, wherein the anti-angiogenic peptide is an anti-Flt1 peptide [SEQ ID NO 3] or a PEDF (pigment epithelial-derived factor) 34-mer [SEQ ID NO 4].
3. The fusion polypeptide for inhibiting angiogenesis of claim 1, wherein the hydrophilic EBP is represented by one of SEQ ID NOS 5 to 14.
4. The fusion polypeptide for inhibiting angiogenesis of claim 1, wherein the i) consists of [anti-Flt1 peptide of SEQ ID NO 3]-[helix-based polypeptide represented by SEQ ID NO 1]-[hydrophilic EBP]-[helix-based polypeptide represented by SEQ ID NO 2].
5. The fusion polypeptide for inhibiting angiogenesis of claim 1, wherein the ii) consists of [helix-based polypeptide represented by SEQ ID NO 1]-[hydrophilic EBP]-[EDF 34-mer of SEQ ID NO 4]-[hydrophilic EBP]-[helix-based polypeptide represented by SEQ ID NO 2].
6. The fusion polypeptide for inhibiting angiogenesis of claim 1, wherein the iii) consists of [anti-Flt1 peptide of SEQ ID NO 3]-[helix-based polypeptide represented by SEQ ID NO 1]-[hydrophilic EBP]-[EDF 34-mer of SEQ ID NO 4]-[hydrophilic EBP]-[helix-based polypeptide represented by SEQ ID NO 2].
7. The fusion polypeptide for inhibiting angiogenesis of claim 1, wherein the i) is represented by SEQ ID NO 16, the ii) is represented by SEQ ID NO 17, and the iii) is represented by SEQ ID NO 18.
8. A composition for treating a disease caused by angiogenesis, comprising the fusion polypeptide for inhibiting angiogenesis according to claim 1.
9. The composition for treating a disease caused by angiogenesis of claim 8, wherein the disease caused by angiogenesis is one or more selected from a group consisting of diabetic retinopathy, retinopathy of prematurity, macular degeneration, choroidal neovascularization, neovascular glaucoma, ocular disease caused by corneal neovascularization, corneal transplantation rejection, corneal edema, corneal opacity, cancer, hemangioma, angiofibroma, rheumatoid arthritis and psoriasis.
10. A fusion protein nanocage having a peptide for inhibiting angiogenesis, prepared as the helix-based polypeptide represented by SEQ ID NO 1 and the helix-based polypeptide represented by SEQ ID NO 2 in the fusion polypeptide for inhibiting angiogenesis according to claim 1 self-assemble.
11. The fusion protein nanocage having a peptide for inhibiting angiogenesis of claim 10, wherein the nanocage has a multivalent fusion polypeptide for inhibiting angiogenesis.
12. A theranostic nanoprobe for a disease caused by angiogenesis, comprising: a fluorescent dye; and the fusion protein nanocage having a peptide for inhibiting angiogenesis according to claim 10, wherein the fluorescent dye is held in the nanocage.
13. A theranostic nanoprobe for a disease caused by angiogenesis, comprising: a Raman dye-bound metal nanoparticle; and the fusion protein nanocage having a peptide for inhibiting angiogenesis according to claim 10, wherein the Raman dye-bound metal nanoparticle is held in the nanocage.
14. The theranostic nanoprobe of claim 12, wherein the disease caused by angiogenesis is one or more selected from a group consisting of diabetic retinopathy, retinopathy of prematurity, macular degeneration, choroidal neovascularization, neovascular glaucoma, ocular disease caused by corneal neovascularization, corneal transplantation rejection, corneal edema, corneal opacity, cancer, hemangioma, angiofibroma, rheumatoid arthritis and psoriasis.
15. The theranostic nanoprobe of claim 13, wherein the disease caused by angiogenesis is one or more selected from a group consisting of diabetic retinopathy, retinopathy of prematurity, macular degeneration, choroidal neovascularization, neovascular glaucoma, ocular disease caused by corneal neovascularization, corneal transplantation rejection, corneal edema, corneal opacity, cancer, hemangioma, angiofibroma, rheumatoid arthritis and psoriasis.
Description
BRIEF DESCRIPTION OF DRAWINGS
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MODE FOR CARRYING OUT THE INVENTION
Example 1: Materials
[0094] A pET-21a (+) vector and BL21 (DE3)E. coli cells were obtained from Novagen Inc. (Madison, Wis., U.S.). Top10 competent cells and calcein-AM were purchased from Invitrogen (Carlsbad, Calif., U.S.), and HUVECs were purchased from Lonza (Basel, Switzerland). All customized oligonucleotides were synthesized by Cosmo Gene Tech (Seoul, South Korea) and human recombinant VEGF-165 (rhVEGF.sub.165) was obtained from R&D Systems (Minneapolis, U.S.). CIP (calf intestinal alkaline phosphatase), BamHI and XbaI were purchased from Thermo Fisher Scientific (Waltham, Mass., U.S.). AcuI and BseRI were purchased from New England Biolabs (Ipswich, Mass., U.S.). T4 DNA ligase was purchased from Elpis Bio-tech (Taejeon, South Korea). DNA miniprep, gel extraction and PCR purification kits were purchased from Geneall Biotechnology (Seoul, South Korea). Dyne Agarose High was purchased from DYNE BIO, Inc. (Seongnam, South Korea). The Top10 cells were grown in TB DRY media obtained from MO BIO Laboratories, Inc. (Carlsbad. Calif., U.S.). The BL21 (DE3) cells were grown in Circle Grow media purchased from MP Biomedicals (Solon, Ohio, U.S.). The HUVECs cells were grown with EGM-2 Bullet Kit and EBM-2 purchased from Lonza (Basel, Switzerland). Ready Gels (Tris-HCl 2-20% precast gels) were purchased from Bio-Rad (Hercules, Calif., U.S.). PBS (phosphate-buffered saline, pH 7.4) and ampicillin were purchased from Sigma-Aldrich (St Louis, Mo., U.S.). Matrigel was purchased from BD Biosciences (San Diego, Calif., U.S.). Human recombinant VEGF.sub.165 protein and human recombinant VEGF R1/Flt-1 F.sub.c were purchased from R&D System (Minneapolis, Minn., U.S.). Gold(III) chloride trihydrate, sodium borohydride, ascorbic acid and malachite green isothiocyanate were purchased from Sigma-Aldrich (St Louis, Mo., U.S.).
Example 2: Notation for Different EBP Blocks and Block Polypeptides Thereof
[0095] Different EBPs having a pentapeptide repeat unit Val-Pro-(Gly or Ala)-X.sub.aa-Gly [VP(G or A)XG] (SEQ ID NO 26) are named as follows. The X.sub.aa may be any amino acid other than Pro. First, a pentapeptide repeat of Val-Pro-Ala-X.sub.aa-Gly (VPAXG) (SEQ ID NO 26, where the G or A is A) having plasticity is defined as an elastin-based polypeptide with plasticity (EBPP). On the other hand, a pentapeptide repeat of Val-Pro-Gly-X.sub.aa-Gly (VPGXG) (SEQ ID NO 26, where the G or A is G) is called an elastin-based polypeptide with elasticity (EBPE). Second, in [X.sub.iY.sub.jZ.sub.k].sub.n, the capital letters in the parenthesis represent the single-letter amino acid codes of guest residues, i.e., the amino acids at the fourth position (X.sub.aa or X) of the EBP pentapeptide, and subscripts corresponding to the capital letters indicate the ratio of the guest residues in an EBP monomer gene as repeat units. The subscript number n in [X.sub.iY.sub.jZ.sub.k].sub.n (i+j+k=6) represents the total number of repeats of EBP of the present disclosure, i.e., [VPGXG VPGXG VPGXG VPGXG VPGXG VPGXG] (SEQ ID NO 27) or [VPAXG VPAXG VPAXG VPAXG VPAXG VPAXG] (SEQ ID NO 28). For example, EBPP[A.sub.1G.sub.4I.sub.1].sub.6 is an EBPP block consisting of 6 repeats of [VPAXG VPAXG VPAXG VPAXG VPAXG VPAXG] (SEQ ID NO 28), in which the ratio of Ala, Gly and Ile at the fourth guest residue position (X.sub.aa) is 1:4:1. Finally, a diblock polypeptide of EBPP with another peptide is named according to the composition of each block in brackets with a hyphen between the blocks, e.g., as EBPP[A.sub.1G.sub.4I.sub.1].sub.6-HPC5.
Example 3: Preparation of Modified pET-21a Vector for Seamless Gene Cloning
[0096] A pET-21a vector was treated with XbaI, BamHI and CIP at 37° C. for 20 minutes in FastDigest buffer and then dephosphorylated. The restriction enzyme-treated plasmid DNA was purified using a PCR purification kit and then eluted with deionized water. Two oligonucleotides with XbaI- and BamHI-compatible sticky ends were designed as follows.
TABLE-US-00001 (SEQ ID NO 19) 5′-ctagaaataattttgtttaactttaagaaggaggagtacatatggg ctactgataatgatcttcag-3′. (SEQ ID NO 20) 5′-gatcctgaagatcattatcagtagcccatatgtactcctccttctt aaagttaaacaaaattattt-3′.
[0097] The two oligonucleotides were annealed in T4 DNA ligase buffer by heating at 95° C. for 2 minutes and then cooled slowly to room temperature over 3 hours. The annealed double-stranded DNA (dsDNA), i.e., a DNA insert, was ligated into the multiple cloning site (MCS) of the linearized pET-21a vector by treating with T4 DNA ligase in T4 DNA ligase buffer and incubating at 37° C. for 30 minutes. For seamless cloning and expression, the gene recombinant pET-21a (mpET-21a) vector was transformed into Top10 competent cells, which were then plated on an SOC (super optimal broth with catabolite repression) plate treated with 50 μg/mL ampicillin. The DNA base sequence of the mpET-21a vector was verified by fluorescent dye terminator DNA sequencing (Applied Biosystems Automatic DNA Sequencer ABI 3730).
Example 4: Synthesis of EBP Monomers and Oligomerization Thereof
[0098] The base sequences of EBPPs including a ‘pentapeptide repeat unit’ Val-Pro-Ala-X.sub.aa-Gly (SEQ ID NO 26, where the A or G is A), in which the ratio of Ala, Gly and Ile at the fourth residue is 1:4:1, were designed to optimize T.sub.t below a physiological temperature. A pair of oligonucleotides for encoding EBPP[A.sub.1G.sub.4I.sub.1].sub.1 were annealed in T4 DNA ligase buffer by heating at 95° C. for 3 minutes and then cooled slowly to room temperature over 3 hours. The mpET-21a cloning vector was treated with BseRI and CIP at 37° C. for 30 minutes and then dephosphorylated. The restriction enzyme-treated plasmid DNA was purified using a PCR purification kit and then eluted with deionized water. The annealed dsDNA and the linearized mpET-21a cloning vector were treated with T4 DNA ligase in T4 DNA ligase buffer and then ligated by incubating at 16° C. for 30 minutes. The ligated plasmid was transformed into Top10 chemically competent cells and then plated onto an SOC plate treated with 50 μg/mL ampicillin. DNA base sequence was then confirmed by DNA sequencing. A gene was prepared until the repeat number was 6, i.e., EBPP[A.sub.1G.sub.4I.sub.1].sub.6.
[0099] EBP sequences having a pentapeptide repeat unit Val-Pro-(Gly or Ala)-X.sub.aa-Gly (SEQ ID NO 26) in which the fourth residue were varied with different molar ratios were designed at DNA level to optimize T.sub.t below a physiological temperature. The amino acid sequences of EBPs having various pentapeptide repeat units are shown in Table 1.
TABLE-US-00002 TABLE 1 Amino acid sequences of EBP EBP Amino acid sequence SEQ ID NO EBPE[A.sub.1G.sub.4I.sub.1] VPGGG VPGAG VPGGG VPGGG VPGIG VPGGG 5 EBPP[A.sub.1G.sub.4I.sub.1] VPAGG VPAAG VPAGG VPAGG VPAIG VPAGG 6 EBPE[K.sub.1G.sub.4I.sub.1] VPGGG VPGKG VPGGG VPGGG VPGIG VPGGG 7 EBPP[K.sub.1G.sub.4I.sub.1] VPAGG VPAKG VPAGG VPAGG VPAIG VPAGG 8 EBPE[D.sub.1G.sub.4I.sub.1] VPGGG VPGDG VPGGG VPGGG VPGIG VPGGG 9 EBPP[D.sub.1G.sub.4I.sub.1] VPAGG VPADG VPAGG VPAGG VPAIG VPAGG 10 EBPE[E.sub.1G.sub.4I.sub.1] VPGGG VPGEG VPGGG VPGGG VPGIG VPGGG 11 EBPP[E.sub.1G.sub.4I.sub.1] VPAGG VPAEG VPAGG VPAGG VPAIG VPAGG 12
[0100] PEDF cDNA fragment 34-mer (130-23) was amplified from human PEDF cDNA fragments by PCR by using the following primers.
TABLE-US-00003 (forward) (SEQ ID NO 21) 5′-AAAGGATCCCCCTACTGGTAATGCTCTTCAGTCTAGAGAT-3′ (reverse) (SEQ ID NO 22) 5′-CACGACCAACGGCTACTGATAGTGATCTTCAGCTAGCGAT-3′
[0101] The forward primer had an XbaI restriction enzyme site at 3′-end, and the reverse primer had a NheI restriction enzyme site at 5′-end. The two primers had AcuI restriction enzyme and recognition sites for seamless cloning of a gene including EBPP[A.sub.1G.sub.4I.sub.1].sub.n. An insert gene was constructed by treating the amplified gene fragments of PEDF 34-mer with XbaI and NheI in CutSmart buffer at 37° C. for 2 hours. After the treatment, the product was electrophoresed on agarose gel and the insert gene was purified using a gel extraction kit. The pET-21a cloning vector was treated with XbaI and CIP at 37° C. for 1 hour and then dephosphorylated. The restriction enzyme-treated DNA was purified using a PCR purification kit and eluted with deionized water. The restriction enzyme-treated PEDF 34-mer gene fragment and the linearized pET-21a cloning vector were ligated by treating with T4 DNA ligase in T4 DNA ligase buffer and incubating at 16° C. for 30 minutes. The ligation product was transformed into Top10 chemically competent cells, which were then plated on an SOC plate treated with 50 μg/mL ampicillin. The transformant was initially screened by diagnostic restriction enzyme treatment on agarose gel and further confirmed by DNA sequencing as described above.
Example 6: Construction of HPC (Helix-Based Protein Cage)-Encoding Gene in Cloning Vector
[0102] For fusion with genes of other peptides between helical bundles (ferritin A, B, C and D) and a short fifth helix (ferritin E), genes encoding the four helical bundles and a gene encoding the fifth helix were cloned into mPET-21a. The genes encoding the four helical bundles, having XbaI and BamHI restriction enzyme sites at both ends, were delivered to the pUCIDT vector. An insert gene was constructed by treating the plasmid including the four helical bundles with 10 U of XbaI and 10 U of BamHI in CutSmart buffer at 37° C. for 2 hours. After the treatment, the product was electrophoresed on agarose gel and the insert gene was purified using a gel extraction kit. A total of 4 μg of the mpET-21a cloning vector was treated with restriction enzymes and then dephosphorylated with 15 U of XbaI, 10 U of BamHI and 10 U of FastAP thermosensitive alkaline phosphatase at 37° C. for 1 hour. The plasmid DNA was purified using a PCR purification kit and eluted with 40 μL of distilled, deionized water. After treating 90 pmol of the gene fragments of the four helical bundles and 30 pmol of the linearized mpET-21a cloning vector with 1 U of T4 DNA ligase in T4 DNA buffer, ligation was performed by incubating at 16° C. for 30 minutes. The ligated product was transformed into Top10 competent cells and then plated onto an SOC plate treated with 50 μg/mL ampicillin. The transformant was initially screened by diagnostic restriction enzyme treatment on agarose gel and further confirmed by DNA sequencing as described above. A 57-bp gene encoding the fifth helix, having sticky ends of GG-5′ and 3′-CC was constructed through hybridization. A pair of oligonucleotides (50 μL, 2 μM) encoding the fifth helix were heated at 95° C. for 3 minutes in T4 DNA ligase buffer and then slowly cooled to room temperature over 3 hours. A total of 4 μg of the mpET-21a cloning vector was treated with restriction enzymes and then dephosphorylated by treating with 15 U of BseRI and 10 U of FastAP as a thermosensitive alkaline phosphatase at 37° C. for 30 minutes. The plasmid DNA was purified using a PCR purification kit and eluted with 40 μL of distilled, deionized water. After treating 90 pmol of annealed double-stranded DNA (dsDNA) and 30 pmol of the linearized mpET-21a cloning vector with 1 U of T4 DNA ligase in T4 DNA buffer, ligation was performed by incubating at 16° C. for 30 minutes. The ligated product was transformed into Top10 chemically competent cells and then plated onto an SOC plate treated with 50 μg/mL ampicillin. DNA sequence was confirmed by DNA sequencing.
Example 7: Construction of Gene of Anti-Flt1-HPC4-EBP-PEDF 34-Mer-HPC5 Fusion Protein Cage
[0103] A pair of oligonucleotides encoding an anti-Flt1 peptide acting as a VEGFR1 antagonist were synthesized chemically by Cosmo Genetech (Seoul, Korea) and linked to an oligonucleotide cassette with sites recognized by AcuI and BseRI. For seamless cloning, the oligonucleotide cassette encoding the anti-Flt1 peptide was designed rationally to have no BseRI, XbaI, AcuI and BamHI restriction sites. Each plasmid containing EBPP[A.sub.1G.sub.4I.sub.1].sub.6 having BseRI, XbaI, AcuI and BamHI restriction sites, PEDF 34-mer, the fragments of four helical bundles of HPC and the fragment of the fifth helix of HPC and the oligonucleotide cassette were used to create genes for a fusion polypeptide library of anti-Flt1-four helical bundles (HPC4)-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-PEDF 34-mer-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-fifth helix (HPC5). For encoding of the fusion polypeptide library, a plasmid vector encoding the fifth helix was treated with 15 U of BseRI in CutSmart buffer at 37° C. for 1 hour. The restriction enzyme-treated plasmid DNA was purified using a PCR purification kit and then dephosphorylated by treating with 10 U of FastAP as a thermosensitive alkaline phosphatase in CutSmart buffer at 37° C. for 1 hour. The restriction enzyme-treated and dephosphorylated plasmid DNA was purified using a PCR purification kit and then eluted with 40 μL of distilled, deionized water. For construction of an insert gene, 4 mg of the EBPP[A.sub.1G.sub.4I.sub.1].sub.6 gene was treated with 10 U of BseRI and 15 U of AcuI in CutSmart buffer at 37° C. for 1 hour. After the treatment, the product was electrophoresed on agarose gel and the insert gene was purified using a gel extraction kit. 90 pmol of the purified insert gene and 30 pmol of the linearized vector were ligated by treating with 1 U of T4 DNA ligase in T4 DNA ligase buffer and incubating at 16° C. for 30 minutes. The ligated product was transformed into Top10 chemically competent cells, which were then plated onto an SOC plate treated with 50 μg/mL ampicillin. The transformant was initially screened by diagnostic restriction enzyme treatment on agarose gel and further confirmed by DNA sequencing. A plasmid vector including the insert DNA was constructed by the method described above. For construction of the insert gene, PEDF 34-mer was prepared by PCR and restriction enzyme treatment and the oligonucleotide cassette encoding the anti-Flt1 peptide was used as the insert gene. The insert gene of the four helical bundles was prepared by the same method as the EBPP[A.sub.1G.sub.4I.sub.1].sub.6 insert gene. Ligation was performed as described above.
Example 8: Expression and Purification of EBP and Fusion Protein Cage Genes
[0104] E. coli strain BL21 (DE3) cells were transformed with each vector containing EBP and fusion protein cages, and then inoculated in 50 mL of CircleGrow medium supplemented with 50 μg/mL ampicillin. Preculture was performed in a shaking incubator at 200 rpm overnight at 37° C. Then, after inoculating 50 mL of TB DRY medium with 500 mL of CircleGrow medium supplemented with 50 μg/mL ampicillin, the cells were cultured in a shaking incubator at 200 rpm for 16 hours at 37° C. When absorbance (optical density) at 600 nm (OD.sub.600) reached 1.0, overexpression of the polypeptide was induced by adding IPTG to a final concentration of 1 mM. The cells were centrifuged at 4500 rpm for 10 minutes at 4° C. The expressed EBPs and block polypeptides thereof were purified by ITC as described above. The cell pellet including the EBPs was resuspended in 30 mL of PBS buffer and the EBPs were purified with PBS. The cell pellet including the fusion protein cage was resuspended in 30 mL of PBS containing 3 M urea and then purified with PBS containing 3 M urea to denature the helical structure of NPC. The cells were lysed by sonication (VC-505, Sonic and materials Inc., Danbury, Conn.) on ice bath for 10 seconds with 20-second intervals. The cell lysate was centrifuged in a 50-mL centrifuge tube at 13000 rpm for 15 minutes at 4° C. to precipitate insoluble debris. The supernatant containing soluble EBPs was transferred to a new 50-mL centrifuge tube and centrifuged with 0.5% w/v PEI at 13000 rpm for 15 minutes at 4° C. to precipitate nucleic acid contaminants. The inverse phase transition of the EBPs was triggered by adding NaCl at a final concentration of 3 M, and the aggregated EBPs were separated from the lysate solution by centrifuging at 13000 rpm for 15 minutes at 4° C. The aggregated EBPs were resuspended in cold buffer and centrifuged at 13000 rpm for 15 minutes at 4° C. to remove aggregated protein contaminants. These aggregation and resuspension processes were repeated 5-10 times until the EBP purity reached about 95% when determined by SDS-PAGE.
[0105]
TABLE-US-00004 TABLE 2 SEQ ID NO 1 HPC4 SSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGF YFDRDDVALEGVSHFFRELAEEKREGYERLLKMQ NQRGGRALFQDIKKPAEDEWGKTPDAMKAAMAL EKKLNQALLDLHALGSARTDPHLCDFLETHFLDEE VKLIKKMGDHLTNLHRLGG SEQ ID NO 2 HPC 5 PEAGLGEYLFERLTLKHD SEQ ID NO 3 Anti-FM peptide GNQWFI SEQ ID NO 4 PEDF 34-mer DPFFKVPVNKLAAAVSNFGYDLYRVRSSTSPTTN SEQ ID NO 6 EBPP[A.sub.1G.sub.4I.sub.1].sub.1 VPAGG VPAAG VPAGG VPAGG VPAIG VPAGG
[0106]
Example 9: Characterization of EBPs and Anti-Flt1-HPC4-EBP-PEDF 34-Mer-HPC5 Fusion Protein Cage
[0107] The purity of EBPs and the fusion protein cage was determined by SDS-PAGE. The effect of temperature on the inverse phase transition of the EBPs and fusion protein cage at 25 μM in PBS was investigated by measuring OD.sub.350 using the Cary 100 Bio UV/Vis spectrophotometer equipped with a multi-cell thermoelectric temperature controller (Varian Instruments, Walnut Creek, Calif.) from 10 to 85° C. at a heating rate of 1° C./min. The self-assembly and thermal sensitivity of the fusion protein cage were identified using a temperature-controlled Nano ZS90 (ZEN3690) dynamic light scattering (DLS) instrument (Malvern instruments, Worcestershire, UK). Their hydrodynamic radius (R.sub.H) at 25 μM in PBS was measured in 11 successive runs at each temperature in a temperature range from 20 to 60° C. at a heating rate of 1° C./min. Their T.sub.t was defined as the onset temperature of phase transition and was calculated from each DLS plot.
Example 10: In Vitro Tube Formation Assay of HUVECs Using Fusion Protein Cage
[0108] In vitro tube formation assay of HUVECs was performed using the fusion protein cages to evaluate their effects on the proliferation, migration and tube formation of the endothelial cells. For Matrigel coating, 200 μL of Matrigel was solidified on a 48-well plate by incubating at 37° C. for 30 minutes. For fluorescence labeling, HUVECs were incubated with 0.5 μM calcein-AM at 37° C. for 15 minutes. In order to investigate how the proliferation, migration and tube formation of the endothelial cells are affected, the calcein-labeled HUVECs were spread on the Matrigel-coated well plate at 4×10.sup.4 cells/well and were incubated with 50 ng/mL human recombinant rhVEGF.sub.165 and fusion protein cage at 37° C. for 4 hours. The tube formation of the HUVECs was imaged with a micromanipulator (Zeiss, Oberkochen, Germany) and quantified by measuring whole tube length in three random fields per well with an angiogenesis analyzer of the Image J lab software. The tube formation was repeated three times.
Example 11: HUVEC Imaging Using Fluorescent Dye-Conjugated Fusion Protein Cage
[0109] For in-vitro cell imaging, the fusion protein cage was labeled with Alexa Fluor 488 C5 maleimide. 50 μM of the fusion protein cage was incubated with 500 μM of Alexa Fluor C5 maleimide at room temperature for 2 hours in 0.01 M PBS containing 3 M urea and 10 mM DTT. The remaining Alexa Fluor 488 C5 maleimide was removed by dialyzing with PBS. The label was characterized with a fluorescence spectrum (Ex: 480; Em: 500-550, Ex slit 10 nm; Em slit 5 nm) in SDS-PAGE without staining with a fluorescent protein. HUVECs were spread onto a 48-well plate at 4×10.sup.4 cells/well and then cultured overnight. The HUVECs were incubated with 50 ng/mL human recombinant rhVEGF.sub.165 and dye-conjugated fusion protein cage at different concentrations for 15, 30 and 60 minutes at 37° C. The HUVECs were imaged in a bright-field mode with a micromanipulator (Zeiss, Oberkochen, Germany) using FITC.
Example 12: Synthesis of Gold Nanoparticles Inside Cage Structure of Fusion Protein Cage and Conjugation of Raman Dye to Gold Nanoparticles
[0110] After incubating 25 μM fusion protein cage with 0.2 mM HAuCl.sub.4 at room temperature for 3 hours in 0.01 M PBS, unbound HAuCl.sub.4 was removed from the surface by washing twice with PBS. Then, gold ions were removed by adding 1 mM NaBH.sub.4 to the gold seeds. The resulting solution was pale yellow. After water quenching for 3 hours, incubation was performed for 1 hour after further adding 0.2 mM HAuCl.sub.4. Then, 1 mM ascorbic acid was added so that the gold seeds grew into gold nanoparticles (AuNPs). The final solution was ruby red. The nanoparticles synthesized in the protein cage were characterized by measurement of absorbance at 350 to 900 nm and TEM imaging.
[0111] Due to conjugation of a Raman dye to the gold nanoparticles (AuNPs) in the protein cage structure, the pH of 0.01 M PBS buffer containing AuNP-protein was decreased to pH 6.0, lower than the pI of the fusion protein (6.029), as HCl was added. 10 μM MGITC was added to the 0.01M PBS buffer of pH 6.0. The pH of the buffer was increased to pH 7.4 at 1 hour after the addition of NaOH. Raman measurement was performed by Raman spectroscopy (Renishaw 2000, Renishaw, UK).
[0112] Results
[0113] A fusion protein cage consisting of anti-Flt1-peptide, PEDF 34-mer and EBP was constructed by cloning of a gene encoding the fusion protein cage and IPTG-induced overexpression of the fusion protein cage. The gene encoding the fusion protein cage was constructed by inserting genes encoding EBPP[A.sub.1G.sub.4I.sub.1].sub.6, PEDF 34-mer, four helices of HPC and anti-Flt1 peptide sequentially into a plasmid including the fifth helix of HPC. The insertion of each gene was confirmed by treatment with XbaI and AcuI restriction enzymes, agarose gel electrophoresis and DNA sequencing. The constructed four genes encoding the fusion protein cage, (a) HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.12-HPC5, (b) anti-Flt1-HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.12-HPC5, (c) HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-PEDF 34-mer-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-HPC5 and (d) anti-Flt1-HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-PEDF 34-mer-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-HPC5, are shown in
[0114] The fusion protein cage was expressed in E. coli and purified by inverse transition cycling (ITC). The purity and molecular weight of the purified fusion protein cage were identified by copper-stained SDS-PAGE in order to visualize protein bands (
[0115] The thermal sensitivity of the fusion protein cage was determined by measuring the absorbance of the polypeptide solution depending on increase in temperature in order to monitor phase transition. Turbidity profiles were obtained by measuring the absorbance of 25 μM fusion protein cage in 10 mM PBS at 350 nm while heating at a rate of 1° C./min (
TABLE-US-00005 TABLE 3 Transition temperature (° C.) Abs DLS HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.12-HPC5 (SEQ ID NO 15) 55.12 59.00 Anti-Flt1-HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.12-HPC5 52.27 55.18 (SEQ ID NO 16) HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-PEDF 34-mer-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-HPC5 44.27 43.18 (SEQ ID NO 17) Anti-Flt1-HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-PEDF 34-mer-EBPP[A.sub.1G.sub.4I.sub.1].sub.6- 44.37 49.00 HPC5 (SEQ ID NO 18)
[0116] The turbidity at T.sub.t is due to the self-assembled cage structure of the fusion protein cage. The absorbance was increased at temperatures above T.sub.t. The T.sub.t of HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.12-HPC5, anti-Flt1-HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.12-HPC5, HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-PEDF 34-mer-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-HPC5 and anti-Flt1-HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-PEDF 34-mer-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-HPC5 was 55.12, 52.24, 44.17 and 44.37° C., respectively. The T.sub.t of anti-Flt1-HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.12-HPC5 was decreased by 2.85° C. as compared to that of HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.12-HPC5. However, there was no significant difference in T.sub.t among HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-PEDF 34-mer-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-HPC5 and anti-Flt1-HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-PEDF 34-mer-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-HPC5. The fusion protein cage including PEDF 34-mer had a lower T.sub.t than the fusion protein cage not including PEDF 34-mer. The T.sub.t of HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-PEDF 34-mer-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-HPC5 and anti-Flt1-HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-PEDF 34-mer-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-HPC5 was lower than the T.sub.t of HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.12-HPC5 and anti-Flt1-HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.12-HPC5 by 10.85° C. and 7.90° C., respectively. HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-PEDF 34-mer-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-HPC5 and anti-Flt1-HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-PEDF 34-mer-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-HPC5 had the triblock EBPP[A.sub.1G.sub.4I.sub.1].sub.6-PEDF 34-mer-EBPP[A.sub.1G.sub.4I.sub.1].sub.6, and anti-Flt1-HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.12-HPC5 had the monoblock EBPP[A.sub.1G.sub.4I.sub.1].sub.12. Although the EBP length of the fusion protein cage was the same, the number of EBP blocks was 2 times for the triblock EBPP[A.sub.1G.sub.4I.sub.1].sub.6-PEDF 34-mer-EBPP[A.sub.1G.sub.4I.sub.1].sub.6 than that of the monoblock EBPP[A.sub.1G.sub.4I.sub.1].sub.12. The T.sub.t of the fusion polypeptide was decreased as the EBP block was split and the PEDF 34-mer was inserted.
[0117] The HPC based on the fusion protein cage self-assembled into a cage structure at temperatures lower than T.sub.t and was aggregated at temperatures higher than T.sub.t. Their self-assembly and aggregation were characterized by dynamic light scattering (DLS). The hydrodynamic radius (R.sub.H) of 25 μM fusion protein cage in 10 mM PBS in a temperature range from 25 to 60° C. was measured 11 successive runs at each temperature while heating at a rate of 1° C./min. Their T.sub.t was defined as the inflection point of each thermal plot in
[0118] The anti-angiogenic function of the fusion protein cage was investigated in vitro through migration and tube formation assays of HUVECs. For investigation of inhibitory effect on migration and tube formation of HUVECs, calcein-labeled HUVECs were treated for 4 hours with the fusion protein cage at different concentrations on Matrigel to which 50 ng/mL human recombinant VEGF-165 (rhVEGF165) was added. For evaluation of the relationship between the peptides for inhibiting angiogenesis (anti-Flt1 peptide and PEDF 34-mer) and concentrations, HUVECs were incubated with the fusion protein cage including 0.01 nM, 0.1 nM, 1 nM, 10 nM, 0.1 μM, 1 μM or 1 μM HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.12-HPC5. As a control, HUVECs were treated with HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.12-HPC5 with no peptide for inhibiting angiogenesis at different concentrations (0.01 nM, 1 nM, 0.1 μM). The inhibition of tube formation of the HUVECs under each condition is shown in
[0119] The tube length of the HUVECs treated with HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.12-HPC5 was similar to the tube length of the HUVECs incubated with rhVEGF165, regardless of the concentration of HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.12-HPC5. This result indicates that HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.12-HPC5 has no effect on the tube formation of HUVECs. Other three fusion protein cages except the HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.12-HPC5 showed inhibitory effect at various concentration ranges. The tube formation of the HUVECs incubated with anti-Flt1-HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.12-HPC5 was decreased as the concentration of anti-Flt1-HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.12-HPC5 was increased from 100 nM to 1 μM. HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-PEDF 34-mer-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-HPC5 inhibited the tube formation of HUVECs in the concentration range from 0.01 nM to 0.1 nM, where anti-Flt1-HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.12-HPC5 had no effect on the tube formation of HUVECs. The HUVECs treated with 1 nM anti-Flt1-HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-PEDF 34-mer-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-HPC5 showed the least tube formation, and their tube formation was decreased as the concentration of anti-Flt1-HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-PEDF 34-mer-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-HPC5 was increased from 0.01 nM to 1 nM. Anti-Flt1-HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-PEDF 34-mer-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-HPC5 effectively inhibited the tube formation of HUVECs at broad concentration ranges of the present disclosure. When the concentration of the fusion protein cage was higher than the effective concentration range, the HUVECs treated with the fusion protein cage including PEDF 34-mer peptide showed a similar degree of tube formation as the HUVECs incubated with rhVEGF165. According to a previous report, the PEDF 34-mer peptide induced the inhibition of angiogenesis by interfering with the expression of endogenous caspase inhibitor and c-FLIP (FLICE-like inhibitory protein) through inactivation of NFAT (nuclear factor of activated T cells) and activation of JNK (c-Jun N-terminal kinase) for inhibition of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). Meanwhile, JNK was reported as a positive regulator of angiogenesis in endothelial cells (ECs). The inhibition of INK attenuated the sprout growth of ECs in 3D capillary sprout culture, reduced the growth and migration of ECs, and decreased the protein level of transcription factor Egr-1, which is a gene regulator involved in cellular growth and migration. This report explains the reason why the fusion protein cage including PEDF 34-mer has no effect at higher concentrations in the tube formation of HUVECs assay.
[0120] The anti-Flt1-HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-PEDF 34-mer-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-HPC5 that showed the most effective anti-angiogenic function was conjugated with a fluorescent dye as an imaging probe. A maleimide-modified fluorescent dye was conjugated at the thiol of HPC, and the conjugation of the fluorescent dye to the polypeptide was qualitatively identified by SDS-PAGE. The result is shown in
[0121] The application of the fusion polypeptide as a cell imaging probe was studied through fluorescence imaging of HUVECs. Another application of the fusion polypeptide is to introduce a Raman dye for Raman sensing by synthesizing gold nanoparticles (AuNPs) inside the cage structure. The HPC of the fusion protein cage has a binding site for inorganic ions including gold ions. The anti-Flt1-HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-PEDF 34-mer-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-HPC5 which inhibited angiogenesis the most effectively and had a potential for a cell imaging probe was incubated with gold ions for coordination with HPC. The gold ions coordinated with HPC were reduced to gold seeds, and the gold seeds grew into gold nanoparticles by addition of a reducing agent.
[0122] The anti-Flt1-HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-PEDF 34-mer-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-HPC5, which is a fusion protein cage having a multivalent peptide for inhibiting angiogenesis, was controlled precisely at genetic level in order to expose anti-Flt1 peptide and PEDF 34-mer, which are anti-angiogenic peptides, without interfering with self-assembly to a cage structure. The two anti-angiogenic peptides, anti-Flt1 peptide and PEDF 34-mer, had different mechanisms for inhibition of angiogenesis, which are related with intracellular signaling. The anti-Flt1-peptide inhibited intracellular signaling for angiogenesis, and the PEDF 34-mer induced anti-angiogenic signals. These different mechanisms affected the administration dosage of the fusion protein cage in in-vitro tube formation assay. In in-vitro tube formation assay of HUVECs, the fusion protein cage decreased tube formation of HUVECs gradually as the concentration of the anti-angiogenic peptide was increased. Whereas the fusion protein cage having the anti-Flt1 peptide showed anti-angiogenic effect in μM ranges, the fusion protein cage having the PEDF 34-mer inhibited the tube formation of HUVECs in nM ranges. The anti-Flt1-HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-PEDF 34-mer-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-HPC5 fusion protein cage including the two anti-angiogenic peptides showed the highest inhibitory effect in a broad concentration range. The fluorescent dye-conjugated anti-Flt1-HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-PEDF 34-mer-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-HPC5 showed capability as a cell imaging probe for inhibition of cell extension investigated by HUVEC imaging. The AuNP-anti-Flt1-HPC4-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-PEDF 34-mer-EBPP[A.sub.1G.sub.4I.sub.1].sub.6-HPC5 exhibited Raman spectra after introduction of MGITC to AuNP surface. The fusion protein cage including two different anti-angiogenic peptide and HPC according to the present disclosure has a remarkable potential as a therapeutic peptide for against angiogenesis and for tracing the progress of angiogenesis-related diseases.
[0123] Fusion of self-assembled protein nanostructures with functional peptides has been studied for application in theranostics and nanomedicine. In the present disclosure, a fusion polypeptide consisting of a vascular endothelial growth factor receptor (VEGFR)-targeting peptide, a pigment epithelium-derived factor (PEDF) 34-mer peptide for inhibiting angiogenesis, a temperature-responsive elastin-based polypeptide (EBP) and a helix-based protein cage (HPC) was prepared by a genetic engineering technique, which was then overexpressed in E. coli and purified by a non-chromatographic inverse transition cycling (ITC) method. The VEGFR-target peptide and the anti-angiogenic PEDF 34-mer peptide were exposed in the self-assembled protein cage and the EBP was introduced as a non-chromatographic purification tag. The physical and chemical properties and DySA (dynamic self-assembly) of the fusion protein cage having the multivalent peptide for inhibiting angiogenesis were characterized. The fusion protein cage inhibited the migration and tube formation of human umbilical vein endothelial cells (HUVECs) on Matrigel, which showed possibility as a nanoscale biomedicine for inhibiting angiogenesis. The fusion protein cage having anti-angiogenic effect was prepared into a fluorescent nanoprobe or an inorganic-organic hybrid SERS nanoprobe by chemically conjugating a fluorescent dye to the protein cage or metal nanoparticles (MNPs). For example, gold, silver, copper or iron nanoparticles may be chemically conjugated with a Raman dye for labeling in applications to theranostics and nanomedicine. The fusion protein cage having a multivalent peptide for inhibiting angiogenesis according to present disclosure may be used as a therapeutic agent for uncontrolled retinal, corneal and choroidal neovascularization, tumor growth, cancer cell metastasis, diabetic retinopathy and asthma and for bioimaging and biosensing of the progress thereof in the field of theranostics and nanomedicine.