NON-IMMUNOGENIC PROTEIN NANOPARTICLES WITH CANCER TARGETING ACTIVITY USING ABMUMIN-BINDING PEPTIDE

Abstract

The present invention relates to recombinant self-assembling protein nanoparticles presenting an albumin-binding peptide at the surface. For the recombinant self-assembling protein nanoparticles according to the present invention, an albumin-binding peptide can reduce the immunogenicity of the recombinant self-assembling protein nanoparticles because the albumin-binding peptide is presented at the surface, and thus binds to an albumin protein present in vivo, and the albumin-binding peptide can also provide the cancer delivery function of the recombinant self-assembling protein nanoparticles because the albumin-binding peptide binds to albumin around cancer. Simultaneously, the binding of the albumin-binding peptide to albumin can significantly increase the in vivo residence time of the recombinant self-assembling protein nanoparticles, thus increasing the potential for use in various medical applications.

Claims

1. A recombinant self-assembling protein nanoparticle presenting an albumin-binding peptide at the surface thereof.

2. The recombinant self-assembling protein nanoparticle of claim 1, wherein the recombinant self-assembling protein further comprises a linker peptide.

3. The recombinant self-assembled protein nanoparticle of claim 1, wherein the recombinant self-assembling protein further comprises a gold ion adsorbable peptide and a superparamagnetism-inducing peptide.

4. The recombinant self-assembling protein nanoparticle of claim 1, wherein the recombinant self-assembling protein nanoparticle further comprises a target-oriented peptide.

5. The recombinant self-assembled protein nanoparticle of claim 1, wherein the self-assembling protein is a hepatitis B virus core protein, a tobacco mosaic virus protein, a Thermoplasm acidophilum-derived proteasome (tPTS), or an Escherichia coli-derived DNA binding protein (eDPS).

6. The recombinant self-assembling protein nanoparticle of claim 1, wherein the recombinant self-assembling protein nanoparticle is a hepatitis B virus core protein.

7. The recombinant self-assembled protein nanoparticle of claim 1, wherein the recombinant self-assembling protein is a hepatitis B virus core protein, and the albumin-binding peptide is located at a spike site of the hepatitis B virus core protein.

8. The recombinant self-assembling protein nanoparticle of claim 1, wherein the recombinant self-assembling protein is a hepatitis B virus core protein, and the albumin-binding peptide is fused to some monomers of the hepatitis B virus core protein.

9. A non-immunogenic pharmaceutical composition comprising: the recombinant self-assembling protein nanoparticle according to claim 1; and a pharmaceutically acceptable carrier.

10. A contrast agent composition comprising: the recombinant self-assembling protein nanoparticle according to claim 1; a labeling material for imaging; and a pharmaceutically acceptable carrier.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0056] The above and other objects, features and advantages of the present invention will become more apparent to those of ordinary skill in the art by describing in detail exemplary embodiments thereof with reference to the accompanying drawings, in which:

[0057] FIG. 1 is a schematic view illustrating the structure of a hepatitis B virus capsid protein nanoparticle including an albumin-binding peptide according to the present invention;

[0058] FIG. 2 is a set of transmission electron microscope (TEM) photographs of hepatitis B virus capsid protein nanoparticles (HBVC) and hepatitis B virus capsid protein nanoparticles (HBVC-ABP) including an albumin-binding peptide and a set of graphs illustrating the sizes of nanoparticles according to the dynamic light scattering (DLS) analysis;

[0059] FIG. 3 illustrates ELISA results confirming whether hepatitis B virus capsid protein nanoparticles (HBVC-ABP) including an albumin-binding peptide bind to albumin;

[0060] FIG. 4A illustrates results of measuring the interaction of proteins in sera through size comparison using DLS after B hepatitis virus capsid protein nanoparticles (HBVC)(FIG. 4A) are introduced into actual human sera; and FIG. 4B illustrates results of measuring the interaction of proteins in sera through size comparison using DLS after hepatitis B virus capsid protein nanoparticles (HBVC-ABP) including an albumin-binding peptide are introduced into actual human sera;

[0061] FIG. 5A is a result illustrating the establishment of experimental groups for an in vivo experiment of B hepatitis virus capsid protein nanoparticles (HBVC) and hepatitis B virus capsid protein nanoparticles (HBVC-ABP) including an albumin-binding peptide; and FIG. 5B is the blood concentration (FIG. 5B) of interleukin-1 beta of each experimental group in FIG. 5A;

[0062] FIG. 6A is a result illustrating the establishment of experimental groups to investigate in vivo antibody induction according to the injection of protein nanoparticles; and FIG. 6B is the in vivo antibody concentration of each experimental group in FIG. 6A; and

[0063] FIG. 7A illustrates NIR fluorescence images of mice intravenously injected with recombinant HBVC particles labeled with Cy 5.5. FIG. 7B illustrates the NIR fluorescence intensity over time from tumors in the mice of FIG. 7A. FIG. 7C illustrates an ex vivo near-infrared fluorescence image of 5 major organs and tumors extracted from mice after the intravenous injection.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

[Example 1] Construction of Expression Vector for Biosynthesis of HBV Capsid-Derived Nanoparticles

[0064] FIG. 1 is a schematic view illustrating the structure of a hepatitis B virus capsid protein nanoparticle (HBVC-ABP) including an albumin-binding peptide according to the present invention.

[0065] According to the vector schematic view described in the following Table 1, hepatitis B virus capsid protein nanoparticles (HBVC) and hepatitis B virus capsid protein nanoparticles (HBVC-ABP) including the albumin-binding peptide according to the present invention were prepared through PCR. All prepared plasmid expression vectors were purified on an agarose gel, and then the sequence was confirmed through complete DNA sequencing.

[0066] An expression vector capable of expressing the respective protein nanoparticles was constructed by sequentially inserting the PCR products thus prepared into a pT7-7 expression vector.

[0067] The expression vectors of the respective protein nanoparticles proceeded as pT7-HBVC and pT7-HBVC-ABP.

[0068] A clone encoding the synthesis of N-NdeI-H6(hexahistidine)-HBVcAg(1-78)-linker(G4SG4T)-ABP(DDEWLCGWRPLCIDEILR)-HBVcAg(81-149)-linker(G4SG4T)-ClaI-C was obtained by inserting an albumin-binding peptide (DDEWLCGWRPLCIDEILR) instead of Asp79-Arg80 which is the loop 79-80th amino acid sequence between HBV core protein (HBVC) genes.

[0069] Further, in order to verify the actual cancer targeting capability of actual ABP, the present inventors used HBVC-aff+ particles that had already been verified for cancer targeting capability in the Examples. For the HBVC-affi+ particles configured by the present inventors in the past, a vector was constructed so as to facilitate surface presentation by tandem insertion of an affibody binding to an epidermal growth factor receptor (EGFR) known to be overexpressed in cancer into the loop 79-80th amino acid sequence between HBV core protein (HBVC) genes at a spike and inserting a linker at both ends of the affibody. The aforementioned vector is as follows. NH2-H6-linker 1 (ASSLRQILDSQKMEWRSNAGGS)-linker 2 (G3S G3TG3SG3)-Y6-HBVcAg (1-78)-linker 3 (G4SG4T)(affibody peptide (VDNKFNKEMWAAWEEIRNLPNLNGWQMTAFIASLVDDPSQSANLAEAKKL NDAQAPK)2-linker 4-HBVcAg (81-149)-COOH

[0070] [Table 1] Construct of expression vector for each nanoparticle

TABLE-US-00001 Protein nanoparticle Expression vector HBVC NH.sub.2-NdeI-H6-HBVC-ClaI-COOH HBVC- , NH.sub.2-NdeI-H6(hexahistidine)-HBVcAg(1-78)-linker(G4SG4T)- ABP ABP(DDEWLCGWRPLCIDEILR)-HBVcAg(81-149)-linker(G4SG4T)-ClaI-COOH HBVC- NH2-H6- linker 1 (ASSLRQILDSQKMEWRSNAGGS)- linker 2 (G3S affi+ G3TG3SG3)-Y6-HBVcAg (1-78)- linker 3 (G4SG4T)-(affibody peptide (VDNKFNKEMWAAWEEIRNLPNLNGWQMTAFIASLVDDPSQSANLAEAKKLNDAQAPK)2- linker 4-HBVcAg (81-149)-COOH

[Example 2] Biosynthesis of Candidate Protein Nanoparticles

[0071] An E. coli strain BL21(DE3)[F-ompThsdSB(rB-mB-)] was each transformed with the prepared expression vector, and ampicillin-resistant transformants were selected. The transformed E. coli was cultured in flasks (250 mL Erlenmeyer flasks, 37 C., 150 rpm) containing 50 mL of a Luria-Bertani (LB) medium (containing 100 mgL-1 ampicillin). When medium turbidity (OD600) reached about 0.4 to 0.5, the expression of the recombinant gene was induced by adding isopropyl--D-thiogalactopyranosid (IPTG) (1.0 mM) and biotin (100 uM) for biotinylation at the N-terminus of the protein. After culturing at 20 C. for 16 to 18 hours, a bacterial cell precipitate was collected by centrifuging the cultured E. coli at 4,500 rpm for 10 minutes, and then suspended in 5 ml of a lysis solution (10 mM Tris-HCl buffer, pH 7.5, 10 mM EDTA) and disrupted using an ultrasonic cell disruptor (Branson Ultrasonics Corp., Danbury, Conn., USA). After disruption, centrifugation was performed at 13,000 rpm for 10 minutes, and then the supernatant and the insoluble aggregate were separated. Purification was performed according to Example 3 using the separated supernatant.

[Example 3] Purification of Protein Nanoparticles and Attachment of Fluorescent Material

[0072] The supernatant obtained in Example 2 was purified through a 3-step process. First, 1) Ni2+-NTA affinity chromatography using binding of histidine and nickel fused and expressed in the recombinant protein, and then 2) the recombinant protein was concentrated and a fluorescent material was attached through buffer exchange, and 3) finally, sucrose gradient ultracentrifugation was performed in order to separate only the self-assembled protein nanoparticles to which the fluorescent material was attached. Detailed description for each step is as follows.

[0073] 1) Ni2+-NTA Affinity Chromatography

[0074] In order to purify the recombinant protein, the cell pellet was re-suspended in 5 mL of a lysis buffer (pH 8.0, 50 mM sodium phosphate, 300 mM NaCl, 20 mM imidazole) by collecting cultured E. coli by the same method described above, and cells were disrupted using an ultrasonic cell disruptor. After only the supernatant was separated by centrifuging the disrupted cell solution at 13,000 rpm for 10 minutes, each recombinant protein was separated using Ni2+-NTA columns (Qiagen, Hilden, Germany)(wash buffer: pH 8.0, 50 mM sodium phosphate, 300 mM NaCl, 80 mM imidazole/elution buffer: pH 8.0, 50 mM sodium phosphate, 300 mM NaCl, 200 mM imidazole).

[0075] 2) Concentration and Buffer Exchange and Fluorescent Material Attachment Processes

[0076] 3 ml of the recombinant protein eluted through Ni2+-NTA affinity chromatography was put into a ultracentrifugal filter (Amicon Ultra 100K, Millipore, Billerica, Mass.), and centrifuged at 5,000 g until 1 ml of the solution remained on the column at 5,000 g. Thereafter, in order to attach cy5.5 which is an NIR fluorescent material, the protein particles were subjected to buffer exchange with a sodium bicarbonate (0.1 M, pH 8.5) buffer, and a fluorescent material was attached at room temperature for 12 hours.

[0077] 3) Sucrose Gradient Ultra-High Speed Centrifugation

[0078] After a solution including 60%, 50%, 40%, 30%, and 20% sucrose was each prepared by adding sucrose at each concentration to a PBS (2.7 mM KCl, 137 mM NaCl, 2 mM KH.sub.2PO.sub.4, 10 mM Na.sub.2HPO.sub.4, pH7.4) buffer, 2 ml of the sucrose solution at each concentration (60 to 20%) was each put into tubes for ultra-high speed centrifugation (Ultra-Clear 13.2 ml tube, Beckman) starting from the solution at high concentration, and then after the tubes were filled with 1 ml of the recombinant protein solution present in the prepared buffer for self-assembly, ultra-speed centrifugation was performed at 4 C. and 35,000 rpm for 16 hours (Ultracentrifuge L-90k, Beckman). After centrifugation, a pipette was carefully used to replace the buffer of the recombinant in the upper layer (40 to 50% sucrose solution part) using an ultracentrifugal filter and a PBS buffer as specified in 2).

[Example 4] Analysis of Structure of Prepared Protein Nanoparticles

[0079] In order to analyze the structure of the recombinant protein nanoparticles purified after being subjected to the aforementioned process, the recombinant protein was photographed by a transmission electron microscope (TEM). First, a purified protein sample which had not been stained was placed on carbon-coated copper electron microscope grids, and then naturally dried. In order to obtain stained images of the protein nanoparticles, electron microscope grids including the naturally dried sample were incubated with a 2% (w/v) aqueous uranyl acetate solution at room temperature for 10 minutes, and washed three to four times with distilled water. Protein nanoparticle images were observed using Philips Technai 120 kV electron microscope. In addition, the sizes of the nanoparticles were measured through a dynamic light scattering (DLS) analysis.

[0080] FIG. 2 is a set of transmission electron microscope (TEM) photographs of hepatitis B virus capsid protein nanoparticles (HBVC) and hepatitis B virus capsid protein nanoparticles (HBVC-ABP) including an albumin-binding peptide and a set of graphs illustrating the sizes of nanoparticles according to the dynamic light scattering (DLS) analysis.

[0081] As can be seen in the TEM photographs, it was confirmed that spherical nanoparticles were formed, and through the dynamic light scattering (DLS) analysis, it was confirmed that the HBVC and the HBVC-ABP were formed as spherical nanoparticles having a size of 34.34.2 nm and 38.42.5 nm, respectively.

[Example 5] Proof of Presence or Absence of Binding of Prepared Protein Nanoparticle HBVC-ABP with Albumin Through ELISA

[0082] First, 2 ug/ml human serum albumin (HSA) was bound onto a 96-well high binding plate. And then, the plates to which HSA was bound were treated with hepatitis B virus capsid protein nanoparticles (HBVC) at various concentrations and hepatitis B virus capsid protein nanoparticles (HBVC-ABP)(0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, and 1.5 mg/ml) including an albumin-binding peptide, and allowed to react for 1 hour. Subsequently, anti-His tag primary antibodies were bound for 1 hour, and then secondary antibodies to which HRP had been bound were bound for 1 hour, and then a TMB solution was added to each well. The change in color after the treatment with the TMB solution was observed, and it was confirmed whether the ABP bound to actual albumin by measuring absorbance at 450 nm.

[0083] FIG. 3 illustrates ELISA results confirming whether hepatitis B virus capsid protein nanoparticles (HBVC-ABP) including an albumin-binding peptide bind to albumin. It can be seen that the hepatitis B virus capsid protein nanoparticles (HBVC) do not bind to albumin, and thus there is no change in absorbance, whereas the absorbance is increased by the binding of the hepatitis B virus capsid protein nanoparticles (HBVC-ABP) including the albumin-binding peptide to albumin as the treatment concentration thereof is increased.

[Example 6] Verification of Presence or Absence of Binding of Prepared Protein Nanoparticle HBVC-ABP with Actual Albumin in Serum

[0084] After B hepatitis virus capsid protein nanoparticles (HBVC) and hepatitis B virus capsid protein nanoparticles (HBVC-ABP) including an albumin-binding peptide were introduced into human sera, the interaction of proteins in sera was measured through size comparison using the DLS.

[0085] FIG. 4 illustrates results of measuring the interaction of proteins in sera through size comparison using the DLS after B hepatitis virus capsid protein nanoparticles (HBVC)(FIG. 4A) and hepatitis B virus capsid protein nanoparticles (HBVC-ABP) including an albumin-binding peptide (FIG. 4B) are introduced into actual human sera.

[0086] It can be confirmed that in the serum mixed with the HBVC, the HBVC peak is maintained even after 24 hours, whereas in the serum mixed with the HBVC-ABP, the HBVC-ABP particle peak disappears within 1 hour after the reaction. It can be seen that the HBVC-ABP particles bind to albumin in serum to increase particle size, and thus the peak thereof appears while being mixed with the protein peak present at the rear part. (FIGS. 4 (A) and (B))

[Example 7] Verification of Immunogenicity of Prepared Protein HBVC-ABP

[0087] After it was proved through the Examples that HBVC-ABP actually bound to albumin, it was proved through an in vivo experiment whether the ABP expressed on the surface of the HBVC, which has various advantages when injected in vivo, but is limited in use due to high immunogenicity, could reduce the immunogenicity of the HBVC by binding to albumin in vivo.

Example 7-11

[0088] Interleukin-1 beta is a cytokine induced when a foreign material is injected in vivo to activate an in vivo macrophage by an immune response. Accordingly, it was intended to observe an increase or decrease in immunogenicity due to the injection of the foreign material by measuring the concentration of interleukin-1 beta.

[0089] FIG. 5 is a result illustrating the establishment (FIG. 5A) of experimental groups for an in vivo experiment of B hepatitis virus capsid protein nanoparticles (HBVC) and hepatitis B virus capsid protein nanoparticles (HBVC-ABP) including an albumin-binding peptide and the blood concentration (FIG. 5B) of interleukin-1 beta of each experimental group.

[0090] A control PBS, HBVC, and an experimental group HBVC-ABP material were injected intravenously into three animals per group at a concentration unit of 0.5 mg/ml for units of 3 days, 1 day, 12 hours, 9 hours, 6 hours, 3 hours, and 1 hour, and then finally, blood of all the experimental groups was collected. Thereafter, the change in concentration of an in vivo cytokine induced by HBVC and HBVC-ABP was measured as time passed after the material was injected using an ELISA kit (Mouse IL-1 ELISA Ready-SET-Go!, Cat. No. 88-7013-22, eBioscience) capable of detecting interleukin-1 beta.

Example 7-2

[0091] Antibody titer measurement is the most commonly used method for measuring the immunogenicity of the current material. When a foreign material is injected in vivo, an antibody against the corresponding foreign material is induced, so that the immunogenicity of the foreign material may be inferred by the amount of induced antibody.

[0092] FIG. 6 is a result illustrating the establishment (FIG. 6A) of experimental groups to investigate the in vivo antibody induction according to the injection of protein nanoparticles and the in vivo antibody concentration (FIG. 6B) of each experimental group.

[0093] A control PBS, HBVC, and an experimental group HBVC-ABP material were injected intraperitoneally into three animals per group at a concentration unit of 0.5 mg/ml twice at an interval of 2 weeks, and then finally, blood of all the experimental groups was collected. Thereafter, in order to detect the antibodies induced by HBVC and HBVC-ABP, 2 ug/ml HBVC was bound to a 96-well plate (Nunc MaxisorpELISAplate), and then anti-HBVC and anti-HBVC-ABP antibodies present in blood were induced to bind by reacting the extracted blood therewith.

[0094] As a result of induction, it was confirmed that HBVC-ABP particles could actually induce the effect of reducing immunogenicity by verifying that antibodies induced by HBVC particles were present in a larger amount in blood than antibodies induced by HBVC-ABP.

[Example 8] Verification of Cancer Targeting Capability Through NIR Image Analysis of Prepared Protein

[0095] In order to verify the cancer targeting capability of the albumin-binding peptide, the cancer target capabilities of HBVC protein nanoparticles (also represented by HBVC (aff, ABP)) and HBVC-ABP protein nanoparticles prepared in the Examples, and protein nanoparticles (hereinafter referred to as HBVC (aff+)) prepared by replacing the spike site with a tandem sequence of an affibody peptide having strong and specific affinity for human epidermal growth factor receptor I (EGFR) instead of the albumin-binding peptide were compared with one another. EGFR is overexpressed on the surface of a wide range of tumor cells including U87MG. On the surfaces of HBVC, HBVC-ABP protein nanoparticles, and HBVC (affi+) protein nanoparticles, a fluorescent material Cy 5.5 was attached. After the degrees of fluorescence of the prepared materials were uniformly adjusted, the material was administered intravenously to U87MG(glioblastoma)-bearing 5-week-old nude mice, and then the mice were monitored at predetermined time points for 24 hours using an IVIS spectrum imaging system (Caliper Life Sciences, Hopkinton, Mass.).

[0096] FIG. 7 illustrates the tumor targeting and in vivo distribution of HBVC-ABP in mice. FIG. 7A illustrates NIR fluorescence images of mice intravenously injected with recombinant HBVC particles (HBVC (aff, ABP), HBVC (aff+), and ABP-HBVC) labeled with Cy 5.5. FIG. 7B illustrates the NIR fluorescence intensity over time from tumors in the mice of FIG. 7A. FIG. 7C illustrates an ex vivo near-infrared fluorescence image of 5 major organs and tumors extracted from mice at the time point of 48 hours after the intravenous injection.

[0097] As can be seen in FIGS. 7A and 7B, remarkably large amounts of Cy5.5-ABP-HBVC and HBVC (aff+) were delivered to tumors as compared to Cy5.5-HBVC (aff, ABP)(no affibody and albumin-binding peptide), and in particular, ABP-HBVC was retained longer in tumors. This suggests that the role of serum albumin as a major energy and nutrient source for tumor growth will prolong the retention of ABP-HBVC in tumors and further enhance the tumor targeting performance of ABP-HBVC. In addition, since the albumin-binding peptide binds to albumin, the albumin-binding peptide has a longer half-life in vivo than other peptides.

[0098] The ex vivo near-infrared fluorescence image of 5 major organs and tumors of FIG. 7C shows considerable accumulation of ABP-HBVC and HBVC (aff+) in the liver. EGFR is also expressed at high levels on the surface of hepatocytes, and the liver is a major albumin-producing organ. It can be seen that the amount of ABP-HBVC accumulated in tumors is much larger than the amount of the albumin-binding peptide-free HBVC (aff, ABP). Furthermore, a remarkable amount of HBVC (aff+) was detected in the kidneys, indicating that HBVC (aff+) is removed from the body by kidney excretion more rapidly than ABV-HBVC.

[0099] For the recombinant self-assembling protein nanoparticles according to the present invention, an albumin-binding peptide can reduce the immunogenicity of the recombinant self-assembling protein nanoparticles because the albumin-binding peptide is presented at the surface, and thus binds to an albumin protein present in vivo, and the albumin-binding peptide can also provide the cancer delivery function of the recombinant self-assembling protein nanoparticles because the albumin-binding peptide binds to albumin around cancer. Simultaneously, the binding of the albumin-binding peptide to albumin can significantly increase the in vivo residence time of the recombinant self-assembling protein nanoparticles, thus increasing the potential for use in various medical applications.

[0100] It will be apparent to those skilled in the art that various modifications can be made to the above-described exemplary embodiments of the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention covers all such modifications provided they come within the scope of the appended claims and their equivalents.