JP2008162981A - Biotinylated or homing peptide presentation-type bionanocapsule - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a bionanocapsule easily making interactions with various biomolecules. <P>SOLUTION: The bionanocapsule includes biotin/biotin-binding protein/homing peptide-modified hepatitis B virus surface antigen protein or a modified product thereof. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a homing peptide-presenting bio-nanocapsule. The present invention also relates to biotinylated bio-nanocapsules. Furthermore, the present invention relates to a bio-nanocapsule that presents a biorecognition molecule via biotin. The bio-nanocapsule of the present invention is suitably used for a drug delivery system (DDS) and a gene delivery system (GDS).

  Bionanocapsules are nano-sized proteoliposomes that contain drugs, genes, proteins, etc. and can be delivered to any cell or tissue in the body. Unlike the liposome, this bio-nanocapsule is based on a virus infection system, so that the substance introduction efficiency into the cell is remarkably high. Since the original type of bio-nanocapsule is specific to the human liver, a pre-S peptide derived from the HBsAg protein (functioning as a human liver-specific recognition molecule) can be delivered to retargeted cells and tissues other than the liver. It was necessary to exchange with another biorecognition molecule (antibody, cytokine, receptor, etc.) (Patent Documents 1 to 3).

On the other hand, it has been shown that peptides generally called homing peptides, particularly peptides having 3 amino acid residues or more and less than 15 amino acid residues can recognize specific organs and cells in vivo and can be used for in vivo pinpoint targeting. (Non-patent documents 1 to 3). However, these homing peptides are isolated from random peptide libraries using phages, and are currently used only for applications in which the peptides remain in the state of the phages or drugs or fluorescent substances are directly bound to the peptides. No homing peptide portion was isolated from a phage library and bound to a macromolecule (liposome, gene, RNA, protein, etc.) and applied to in vivo delivery.
WO01 / 64930 WO03 / 082330 WO03 / 082344 Joyce JA, Laakkonen P., Bernasconi M., Bergers G., Ruoslahti E., and Hanahan D. (2003) CANCER CELL 4, 393-403. Kolonin M, Pasqualini R, Arap W. Current Opinion in Chemical Biology 2001, 5: 308-313 Zhang L, Hoffman JA, and Ruoslahti E Circulation 2005; 112: 1601-1611

  An object of the present invention is to provide a bio-nanocapsule that can easily interact with various biomolecules.

  Another object of the present invention is to provide a bio-nanocapsule excellent in biorecognition ability.

  As a result of repeated studies in view of the above problems, the present inventor has made biotinylated or biotin-binding protein a HBsAg protein constituting a bio-nanocapsule or a biotin-binding protein, and avidin, streptavidin, neutravidin, etc. By connecting biotinylated biorecognition molecules via biotin-binding proteins of biotin or biotinylated biorecognition molecules linked to bionanocapsules, the substance in the capsule It has been found that any cell / tissue can be targeted without substantially compromising the introduction of the protein into the body. It was also found that cells / tissues can be targeted by presenting the homing peptide on the bio-nanocapsule.

The present invention provides the following biotinylated bionanocapsule, bionanocapsule linked with biotin-binding protein, and biorecognition molecule-presenting bionanocapsule.
1. A bio-nanocapsule comprising a hepatitis B virus surface antigen (HBsAg) protein or a variant thereof linked to a homing peptide.
2. Item 2. The bio-nanocapsule according to Item 1, wherein the homing peptide is linked to the HBsAg protein or a variant thereof via a biotin-biotin-binding protein-biotin bond.
3. Item 2. The bio-nanocapsule according to Item 1, wherein the homing peptide is contained in a fusion protein linked directly or via an appropriate amino acid sequence to the HBsAg protein or a variant thereof.
4). Item 2. The bio-nanocapsule according to Item 1, wherein the HBsAg protein or a variant thereof includes a binding domain with an antibody Fc region, and a fusion protein including a homing peptide and an antibody Fc region is linked to the surface antigen protein.
5. Item 5. The biotinylated bio-nanocapsule according to Item 4, wherein the domain is a ZZ tag.
6). A biotinylated bio-nanocapsule comprising a biotin-modified HBsAg protein or a variant thereof.
7). A biotin-binding protein-biotinylated bio-nanocapsule comprising a biotin-modified HBsAg protein or a variant thereof and a biotin-binding protein.
8). Item 2. The biotinylated bio-nanocapsule according to item 1, wherein the HBsAg protein or a variant thereof includes a domain that is binding to an Fc region of an antibody.
9. Item 9. The biotinylated bio-nanocapsule according to Item 8, wherein the domain is a ZZ tag.
Ten. A bio-nanocapsule containing an HBsAg protein or a variant thereof chemically linked to a biotin-binding protein.
11． Item 11. The bio-nanocapsule according to Item 10, wherein the biotin-binding protein is bound to the HBsAg protein or a variant thereof via a crosslinking agent.

  According to one preferred embodiment of the present invention, biotinylated bionanocapsules and bionanocapsules linked with biotin-binding proteins can be obtained. This biotinylated bio-nanocapsule can link any biotinylated biorecognition molecule via a biotin-binding protein such as avidin / streptavidin / neutravidin. Moreover, the bio-nanocapsule linked with a biotin-binding protein can link any biotinylated biorecognition molecule. By connecting biotin via an appropriate spacer, a large number of biological recognition molecules can be connected without impairing the biological recognition ability. The bio-nanocapsule in which the biotinylated bio-nanocapsule of the present invention is linked to a biotin-binding protein is preferably used for introducing a substance encapsulated therein into a cell, but in addition to this, a nucleic acid (DNA, RNA), protein (reception) The body or its ligands, enzymes) and any suitable biorecognition molecule for interacting with any substance such as a biologically active substance. Conventional bio-nanocapsules have biorecognition molecules linked in advance to the HBsAg protein or a variant thereof, and even when a ZZ tag is linked, proteins other than antibodies cannot be linked. Each time, it was necessary to recreate the expression vector for recombinant organisms as a fusion protein with the HBsAg protein. On the other hand, the biotinylated bio-nanocapsule of the present invention is desirable from the viewpoint of efficiency because it can cope with the connection of any biological recognition molecule via a (biotin)-(biotin-binding protein)-(biotin) bond. Similarly, according to the bio-nanocapsule to which the biotin-binding protein of the present invention is linked, it is possible to cope with linking of any biological recognition molecule via a (biotin-binding protein)-(biotin) bond.

  According to another preferred embodiment of the present invention, a homing peptide-presenting bio-nanocapsule is provided. The homing peptide-presenting bio-nanocapsule can present a large number of homing peptides (either one type or two or more types) via biotin, so that a substance encapsulated in the capsule can be simultaneously introduced into a plurality of cells. Moreover, by introducing the homing peptide at an appropriate density, the introduction efficiency can be further improved, and it has high utility as an introduction agent for encapsulated substances such as nucleic acids and proteins. Furthermore, if a plurality of antigenic substances are presented on the cell surface, they are useful as vaccines. Since homing peptides are small molecules, unlike antibodies, there is an advantage that antigenicity is less likely to be a problem. The homing peptide can be surface-displayed on the bio-nanocapsule as a fusion protein of ZZ tag and Fc region, biotin and biotin-binding protein, or HBsAg protein. Furthermore, the homing peptide can be chemically bound to the bio-nanocapsule via a bivalent or multivalent cross-linking agent.

  In this specification, the bio-nanocapsule may be abbreviated as “BNC”. “BNC” is sometimes used to mean a bio-nanocapsule biotinylated.

  BNC means an HBsAg protein or a variant thereof and nano-sized particles having a phospholipid membrane as a constituent element, and has a structure in which the HBsAg protein or a variant thereof is stuck into the phospholipid membrane. Substances introduced into cells such as nucleic acids, proteins, and drugs may be encapsulated inside the BNC. As used herein, “unencapsulated BNC” refers to BNC in which the introduced substance is not encapsulated.

  Examples of the HBsAg protein or a variant thereof, which is a component of BNC, include an HBsAg protein or a variant thereof. The HBsAg protein may be combined with the hepatitis B virus core antigen (HBcAg) protein to form unencapsulated BNC. The HBsAg protein or a variant thereof may have a sugar chain. As a method of encapsulating the introduction substance such as nucleic acid, protein, and drug in the unencapsulated BNC, electroporation may be performed. The liposome containing the introduction substance and the unencapsulated BNC that is biotinylated or not are fused. The introduced substance may be enclosed inside.

  In this specification, the size of BNC, introduced substance (nucleic acid, protein, drug, etc.) may be measured by an electron microscope or an atomic force microscope (AFM), such as Zetasizer Nano-ZS (Malvern Instruments) May be measured optically.

  When the HBsAg protein is expressed in eukaryotic cells, the protein is expressed and accumulated as a membrane protein on the endoplasmic reticulum membrane, and is preferably released as lumens or extracellularly as hollow nanoparticles. As eukaryotic cells, mammalian cells such as mammals and insects, yeasts and the like can be applied. Since such particles do not contain any HBV genome, they are extremely safe for the human body.

  In one preferred embodiment, the unencapsulated BNC prior to biotinylation is 70-90 parts by weight hepatitis B virus protein or variant thereof, 5-15 parts by weight lipid, 5-15 parts by weight sugar chain. Consists of A sugar chain is added when it is produced in a eukaryotic cell, and varies depending on the eukaryotic cell used. BNC phospholipid membranes include phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), etc. For example, in BNC produced in yeast, phosphatidylcholine (PC) ) Is the main component. The composition of the phospholipid membrane varies depending on the type of eukaryotic cells used for production, the presence or absence of fusion with liposomes, and the like. In one embodiment of the present invention, eukaryotic cells used to produce unencapsulated BNC include mammalian cells (CHO cells, HEK293 cells, COS cells, etc.), insect cells (Sf9, Sf21, HighFive strains, etc.). Yeast (Saccharomyces, Pichia, Hansenulla, Schizosaccharomyces, Candida), etc., and the endoplasmic reticulum membrane of these eukaryotic cells is considered to be a phospholipid membrane of hollow nanoparticles.

  S protein (226 amino acids), which is included in HBsAg protein and is a component of S particle, has particle forming ability. Pre-S2 consisting of 55 amino acids added to S particles is M protein (M particle constituent protein), and Pre-S1 consisting of 108 amino acids (subtype y) or 119 amino acids (subtype d) in M protein L protein (L particle constituent protein) is added.

  In the present specification, unless otherwise specified, the numbering of amino acid positions in the Pre-S1 region is performed based on subtype y of 108 amino acids. One skilled in the art readily recognizes the corresponding position for 119 amino acids (subtype d).

  L protein and M protein have the ability to form particles, as does S protein. Therefore, the two regions of PreS1 and PreS2 may be arbitrarily substituted, added, deleted, or inserted. For example, by using a modified protein that lacks the hepatocyte recognition site of human and chimpanzee contained in position 3-77 (subtype y) of the Pre-S1 region, it is possible to obtain BNC that loses hepatocyte recognition ability. it can. In addition, since the PreS2 region includes a site that recognizes hepatocytes via albumin, this albumin recognition site can also be deleted. In the present invention, when the homing peptide is surface-presented and linked to BNC, it is preferable to delete the hepatocyte recognition site and albumin recognition site or to lose these functions. When biotinylated BNC is used for uses other than substance introduction into cells (for example, vaccine use), the recognition site may or may not remain in these.

  Since the S protein (226 amino acids) is responsible for particle forming ability, it is necessary to modify the S protein so as not to impair the particle forming ability. For example, even if S protein 107-148 is deleted, it retains particle-forming ability (J. Virol. 2002 76 (19), 10060-10063), so substitution, addition, deletion, insertion, etc. may be performed. Similarly, the hydrophobic 154-226 residues at the C-terminal part can retain the particle forming ability even when substitution, addition, deletion, insertion, and the like are performed. On the other hand, 8-26 residue part (transmembrane region TM1) and 80-98 residue part (TM2) of S protein are transmembrane helix (transmembrane sequence), and this region is not mutated or deleted, Addition, substitution, etc. are preferably performed leaving a hydrophobic residue so as to maintain transmembrane properties.

  In one preferred embodiment, various variants of HBsAg protein are widely encompassed as long as they have the ability to form hollow nanoparticles, and taking HBsAg protein as an example, any number of PreS1 and PreS2 regions Substitution, deletion, addition, insertion, and the like. Regarding S protein, one or several or plural, for example, 1 to 120, preferably 1 to 50, more preferably 1 to 20, more preferably 1 to 10, especially 1 to 5 amino acids may be substituted, added, deleted or inserted. As a method for introducing mutations such as substitution, addition, deletion and insertion, DNA encoding the protein can be used, for example, in cytospecific mutagenesis (Methods in Enzymology, 154, 350, 367-382 (1987); , 468 (1983); Nucleic Acids Res., 12, 9441 (1984)), chemical synthesis means such as the phosphate triester method and phosphate amidite method (for example, using a DNA synthesizer) ( J. Am. Chem. Soc., 89, 4801 (1967); 91, 3350 (1969); Science, 150, 178 (1968); Tetrahedron Lett., 22, 1859 (1981)). For example, as a modified HBsAg protein, an HBsAg (Q129R, G145R) protein or a further modified version thereof is preferred because of its low antigenicity. Codon selection can be appropriately determined in consideration of the codon usage of the host.

As a variant of the HBsAg protein, a protein capable of binding an antibody Fc domain (for example, a ZZ tag derived from Staphylococcus aureus Protein A) can also be used. The ZZ tag is defined as an amino acid sequence having the ability to bind to the Fc region of immunoglobulin G, and consists of the following two repeated sequences (sequence of ZZ tag:
VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK).

  The ZZ tag is rich in Lys (K), and can be biotinylated at the side chain when biotinylating later, so that a large number of biotin can be bound (see FIGS. 7 and 8). In addition to the ZZ tag, a modified HBsAg protein into which a peptide rich in Lys (K) residues is introduced is preferable because the efficiency of biotinylation is increased. Biotinylated amino acids (positions) can be detected by digesting the HBsAg protein or its variant with a protease such as trypsin or lysyl endopeptidase C, if necessary, and analyzing with LC-MS, MS / MS, etc. Can be determined.

  The biotinylated BNC of the present invention can be obtained by biotinylating a non-biotinylated BNC that can be obtained by expressing an HBsAg protein or a variant thereof in a eukaryotic cell with an appropriate biotinylating agent.

The biotinating agent is not particularly limited as long as it binds biotin directly or via an appropriate spacer. A preferred biotinylating agent is bound via a spacer having an aliphatic amide structure such as —NH— (CH ₂ ) _n —CO— (n = 1 to 6) or a polyalkylene glycol structure. Polyalkylene glycols include polyethylene glycol (PEG), polypropylene glycol (PPG), polybutylene glycol (PBG), (PEG)-(PPG)-(PEG) block copolymer, (PPG)-(PEG)-( (PPG) block copolymer, (PEG)-(PBG)-(PEG) block copolymer, (PBG)-(PEG)-(PBG) block copolymer, and the like, preferably PEG, PPG, (PEG)-(PPG)-(PEG) block copolymer, (PPG)-(PEG)-(PPG) block copolymer, more preferably PEG. A preferred PEG structure is represented by the following formula:
-(CH ₂ CH ₂ O) _n-
(In the formula, n represents an integer of 2 to 500, preferably 2 to 100, more preferably 2 to 50, still more preferably 4 to 10.)
In one preferred embodiment, the preferred spacer of the biotinating agent of the present invention has a polyalkylene glycol structure. The polyalkylene glycol structure is preferably bonded to biotin and HBsAg protein or a variant thereof via an ester, amide or thioether bond, preferably via an amide bond.

In another preferred embodiment, biotinating agents can be used, for example, having the following structure:
X- (CH ₂ ) _m1 -NH- {CO (CH ₂ ) _n NH} _m2- (biotinyl)
(In the formula, X reacts with an amino group such as sulfosuccinimide oxycarbonyl group, succinimide oxycarbonyl group, tetrafluorophenoxycarbonyl, cyanomethyloxycarbonyl, p-nitrophenyloxycarbonyl, I, Br, Cl, etc. An active ester residue capable of forming an amide (NHCO) or aminoalkyl group, and a halogen atom, m1 represents an integer of 2 to 6, and m2 is 0 to 50, preferably 1 to 10, more preferably 0 to 5 More preferably, it represents an integer of 0 to 3.)
Specific biotinating agents include, for example, Pierce's EZ-Link Sulfo-NHS-Biotin, EZ-Link Sulfo-NHS-LC-Biotin, EZ-Link Sulfo-NHS-LC-LC-Biotin, EZ-Link- Various biotinating agents such as NHS-PEO ₄ -Solid Phase Biotinylation Kit are exemplified.

  The biotinating agent may be reacted with the biotinating agent as described above and BNC at 1 to 37 ° C., preferably at room temperature. The HBsAg protein or a variant thereof constituting the biotinylated BNC of the present invention binds 0.1 to 20, preferably 2 to 10, more preferably 3 to 8 biotin residues per HBsAg protein or variant thereof. Yes. The present inventor has found that even when a biotin residue is introduced into almost all Lys residues of the HBsAg protein or a variant thereof, the activity of introducing a substance into a cell or tissue is not impaired. Many Lys residues are present on the outer surface of BNC and are thought to be involved in the recognition of negatively charged cell surfaces due to their positively charged groups (epsilon-amino groups), but this finding is surprising. Met.

  The biotin-binding protein used in the present invention is not particularly limited as long as it can link a biotinylated biorecognition molecule such as a biotinylated HBsAg protein or a variant thereof and a biotinylated homing peptide, preferably avidin, streptavidin, or neutravidin. In addition, other biotin bonds such as streptavidin, neutravidin (NeutrAvidin), and fusion proteins with multiple avidins attached (may be chemically combined with a linker). Sex proteins are widely encompassed. The biotin-binding protein should be neutral or weakly acidic with a buffer pH of neutral or weakly acidic to prevent nonspecific adsorption with cells or tissues.

  BNC in which a homing peptide is linked via a biotin-biotin-binding protein-biotin bond has an activity of introducing a substance into the cell similar to the fusion protein in which the homing peptide is directly linked to the HBsAg protein or a variant thereof. Therefore, biotinylated BNC is useful as an intermediate for determining which homing peptide (which may be a wide range of cell recognition molecules) is suitable for cell recognition.

  As the linker or crosslinking agent used for linking BNC and homing peptide, or linking BNC and biotin-binding protein, a divalent or polyvalent linker or crosslinking agent is preferably used. Such linker / crosslinking agent is not particularly limited as long as it can be used for linking proteins or peptides, and known linker / crosslinking agents can be widely used. Specifically, N- (4-maleimidobutyryloxy) succinimide (GMBS), N- (6-maleimidocaproyloxy) succinimide (EMCS), N- (8-maleimidocapryloxy) succinimide (HMCS), N -(11-maleimidoundecanoyloxy) succinimide (KMUS), N-((4- (2-maleimidoethoxy) succinyl) oxy) succinimide (MESS), N- (4-maleimidobutyryloxy) sulfosuccinimide sodium salt Proteins / peptides such as (sulfo-GMBS), N- (6-maleimidocaproyloxy) sulfosuccinimide sodium salt (sulfo-EMCS), N- (8-maleimidocapryloxy) sulfosuccinimide sodium salt (sulfo-HMCS) upper Active esters such as N-hydroxysuccinimide esters, sulfo-N-hydroxysuccinimide esters, phenol esters, which react with mino groups; alkyl or aryl isocyanates or isothiocyanates which react with amino groups on proteins / peptides, or on proteins / peptides Illustrative are bivalent or polyvalent linker / crosslinkers having two or more reactive groups in the linker / crosslinker molecule such as maleimide or α-haloamide that react with sulfhydryl groups.

  When the homing peptide is directly used as a fusion protein with BNC, the DNA encoding the HBsAg protein or a variant thereof and the DNA encoding the homing peptide are linked in-frame via the DNA encoding the spacer peptide as necessary. By incorporating this into a vector or the like and expressing it in a eukaryotic cell, a BNC that recognizes any target cell can be obtained.

  HBsAg protein variants include antigenicity (variants in which the sites involved in antigenicity such as epitopes have been deleted / replaced), immunogenicity, particle structure stability, tissue / cell selectivity, and liposomes. A variant with improved fusion ability may be used.

  In a preferred embodiment of the present invention, the particle size of the unencapsulated BNC is about 20 to 130 nm, and the particle size of the BNC encapsulating the introduction material is usually about 40 to 500 nm, depending on the size of the introduction material. For example, it is about 40 to 400 nm, more preferably about 40 to 200 nm, particularly about 40 to 150 nm.

  The BNC of the present invention to which a biorecognition molecule is bound is useful as a substance that specifically introduces an introduced substance into a specific cell. For example, if BNC encapsulating the introduced substance is administered into the body by intravenous injection or the like, the particles circulate in the body, and are targeted to target cells by molecules selective / specific for hepatocytes or other cells presented on the particle surface. Guided material is introduced into the target cell.

  In addition, the BNC of the present invention can be preferably used as a cell introduction reagent by mixing with target cells in vitro.

  The substance to be introduced into the cell is not particularly limited. For example, various drugs that are introduced into the cell and cause physiological action, for example, physiologically active proteins such as hormones, lymphokines, cytokines, enzymes, etc .; antigenic proteins that act as vaccines; Genes expressed in cells, polynucleotides such as plasmids, or polynucleotides and oligonucleotides (including RNAi) involved in specific gene expression that induces or induces expression; various genes introduced for gene therapy and Examples include antisense DNA / RNA. The “gene” to be introduced includes not only DNA but also RNA (including siRNA). The substance to be introduced is preferably a high molecular weight physiologically active substance such as a protein or gene, but can be applied to various low molecular weight drugs (especially difficult-to-absorbable drugs and drugs that require selective toxicity). Also preferable results can be obtained. The introduced substance may be mixed with a stabilizer and enclosed in BNC, or BNC and stabilizer may be mixed. In addition, genes and proteins may be natural or synthesized, and may be modified genes and proteins.

  Examples of biological recognition molecules include antibodies, antigens, physiologically active peptides, lymphokines, cytokines, sugar chains, receptors, homing peptides, virus-derived coat proteins, microorganism-derived membrane proteins, and the like. The biotinylated BNC of the present invention can bind many biotins per HBsAg protein or a variant thereof. Therefore, the degree of BNC cell recognition can be altered by controlling the number of biotin per protein or variant thereof. In addition, by introducing two or more types of biorecognition molecules of different types, the introduced substance can be introduced into two or more types of cells / organs / tissues.

  Biotinylated BNC can bind biotin-binding protein first and then link biotinylated biorecognition molecule, but preferably biotin-binding protein and biotinylated biorecognition molecule are bound in advance. Can be reacted with biotinylated BNC to produce a BNC on which a biorecognition molecule is surface-presented.

  In particular, when a homing peptide is used as a biorecognition molecule, a BNC with low antigenicity and few side effects can be obtained. The number of amino acids in the homing peptide is 3 to 30, preferably 3 to 25, more preferably 3 to 15, and still more preferably 4 to 10.

  BNC is nano-sized but very large particles (molecular weight of about 6 million) compared to proteins (molecular weight is almost 100,000 or less). There are known examples of homing peptides linked to low molecular weight drugs, but when BNC is linked, the BNC is very large, so the homing peptide's biological recognition ability is expected to be impaired. The inventor has found that the encapsulated substance in the BNC can be introduced into the cell with high efficiency by presenting the homing peptide on the BNC surface.

  Further, if the homing peptide and the Fc region of IgG are fused, the BNC that finally presented the homing peptide can be obtained through binding to the ZZ tag of BNC.

  The homing peptide is not particularly limited as long as it recognizes any cell / organ / tissue in the living body, and examples thereof include the following.

  Human homing peptides are for example (Steps toward mapping the human vasculature by phage display.Arap W, Kolonin MG, Trepel M, Lahdenranta J, Cardo-Vila M, Giordano RJ, Mintz PJ, Ardelt PU, Yao VJ, Vidal CI, Chen L, Flamm A, Valtanen H, Weavind LM, Hicks ME, Pollock RE, Botz GH, Bucana CD, Koivunen E, Cahill D, Troncoso P, Baggerly KA, Pentz RD, Do KA, Logothetis CJ, Pasqualini R. Nat Med 2002 Feb; 8 (2): 121-7.), And a methodology for identifying homing peptides for various human cells (each tissue, organ) by methods such as phage display has been established. Thus, by linking the human homing peptide thus identified to BNC, a specific tissue / organ can be targeted.

  The BNC of the present invention encapsulating the introduced substance brings BNC close to target cells and tissues in vivo and in vitro, and has various fusion substances (genes, siRNAs) inside BNC due to its strong fusion activity with the cell membrane of BNC itself. (Including RNA, compounds or drugs, proteins) are introduced into cells with high efficiency.

  BNC of the present invention surface-displayed with a homing peptide can pinpoint large molecules such as genes and proteins, and unlike the liposomes, it can be distributed evenly throughout the body without being accumulated in the liver and finally accumulated at the target site It is. Furthermore, it is possible to significantly suppress the antigenicity of BNC.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, it cannot be overemphasized that this invention is not limited to these Examples.

Example 1: Delivery of 100-nm polystyrene beads to cells with homing peptide-fused Fc protein and ZZ-BNC (1) Plasmid construction and purification of Fc-fused LyP-1 homing peptide (LyP-1 / Fc protein) Expression protein A LyP-1 / Fc fusion protein vector in E. coli was constructed using a vector pET22b (+) having a signal sequence (pelB signal) that transfers E. coli to the E. coli periplasm fraction. CDNA encoding the Fc domain of human IgG was inserted into pET22b (+), and a sequence encoding LyP-1 peptide (CGNKRTRGC) was inserted at the N-terminus.

The LyP-1 / Fc fusion protein expression vector was transformed into Escherichia coli BL21 (DE3) strain and cultured overnight at 37 ° C. in 10 mL LB medium (containing 100 μg / ml ampicillin). The next day, it was diluted to 1 L with LB medium (containing 100 μg / ml ampicillin), and when A ₆₀₀ reached 0.7, induction was performed with 400 mM IPTG. After induction, the cells were cultured at 17 ° C. for 5 hours, and osmotic shock was performed with 30 mM Tris-HCl, 40% sucrose, 2 mM EDTA, pH 7.2, 100 ml. Centrifugation was performed at 12000 rpm for 20 minutes, and only the cells were collected. To the collected cells, 100 ml of 0.5 mM MgSO ₄ was added, and proteins present in the periplasm fraction were released on ice. After centrifugation at 12000 rpm for 20 minutes, the supernatant was recovered, and the recovered supernatant was dialyzed overnight against 20 mM Tris-HCl pH8.0. Protein G Sepharose 4 Fast Flow (Amersham Biosciences) was packed in a column, the dialyzed sample was passed through, and eluted with 0.1 M glycine buffer pH 2.6 to obtain purified LyP-1 / Fc protein.

  The obtained purified LyP-1 / Fc protein was analyzed by SDS-12.5% PAGE, and Western blotting was performed. When detected with an HRP-labeled anti-human IgG Fc mouse-derived monoclonal antibody (1: 5000) (MP Biomedicals, Inc.), a band was detected around 26 kDa (FIG. 1: left lane, purified LyP-1 / Fc protein; Right lane, purified Fc protein). Protein was quantified using BICINCHONINIC ACID PROTEIN ASSAY KIT (Sigma), concentrated by ultrafiltration using Amicon Ultra 10000 MWCO (MILLIPORE) to a final concentration of about 300 μg / ml, and stored at 4 ° C.

(2) Accumulation of LyP-1 / Fc protein in human breast cancer cells
MDA-MB-435 human breast cancer cells and HEK293 human fetal kidney cells (each 1 × 10 ⁵ cells) were seeded in 500 μl DMEM (containing 10% FBS) and cultured overnight in a 37 ° C. CO ₂ incubator. The next day, LyP-1 / Fc protein and Fc protein were added to a final concentration of 4 μM (at DW), and cultured in a 37 ° C., 5% CO ₂ incubator for 7 hours. After washing with PBS three times and fixing with 4% PFA (paraformaldehyde), PBS containing 0.25% (v / v) Triton-X100 was added and allowed to stand at room temperature for 15 minutes. 100 μl of FITC-labeled anti-human IgG Fc antibody (MP Biomedicals, Inc.) diluted 1/300 times with a blocking solution was added and allowed to stand at room temperature for 1 hour. After washing with 0.03% Triton-X100-containing PBS three times, FITC-derived fluorescence was observed using a confocal laser scanning microscope.

  As shown in FIG. 2A, it was confirmed that LyP-1 / Fc protein was accumulated only in MDA-MB-435 cells. The percentage of LyP-1 / Fc protein and Fc protein accumulated in each cell was measured by (the number of cells with fluorescence / total number of cells). LyP-1 / Fc protein into MDA-MB-435 cells 80.9%, Fc protein accumulation was 9.1%, LyP-1 / Fc protein accumulation in HEK293 cells was 9.7%, and Fc protein accumulation was 10.5%. Each fluorescence value was measured with the multi-fluorescence plate lidar VARIOSKAN (Thermo electron corporation). The value of MDA-MB-435 cells with LyP-1 / Fc protein added was the same as that when Fc protein was added to the same cells. The value was 2.3 to 4.5 times the value in other cells (FIG. 2B). From the above results, it was shown that LyP-1 / Fc protein accumulates specifically in MDA-MD-435 cells by the action of LyP-1 peptide.

(3) In vitro interaction between ZZ protein and LyP-1 / Fc protein
In PBS, 1 mol of BNC (ZZ-BNC) presenting ZZ protein was mixed at a ratio of LyP-1 / Fc protein of 1, 2, 5, 10, 20 mol. Next, it is sufficient to immunoprecipitate anti-HBs mouse-derived monoclonal antibody-binding beads (1 mol ZZ-BNC) included in the IMX HBsAg Dynapack (Abbott Japan) kit that measures the amount of HBsAg protein in patient blood. The mixture was mixed and allowed to stand at 4 ° C. for 60 min. Thereafter, the mixture was centrifuged at 15000 rpm for 20 minutes, and the supernatant and the precipitate were collected. The precipitate was washed 3 times with PBS. The supernatant and precipitate were analyzed by SDS-12.5% PAGE, and Western blotting was performed using an HRP-labeled anti-human IgG Fc mouse-derived monoclonal antibody (1: 5000) (MP Biomedicals, Inc.) (FIG. 3). As a result, free LyP-1 / Fc protein was not observed when 1 mol of LyP-1 / Fc protein was mixed with 1 mol of ZZ-BNC. However, when LyP-1 / Fc protein 2, 5, 10, 20 mol was mixed with 1 mol of ZZ-BNC, free LyP-1 / Fc protein was detected. From the above results, it was suggested that LyP-1 / Fc protein binds to ZZ-BNC 1 mol in vitro at a ratio of 1-2 mol.

(4) Retargeting ZZ-BNC to human breast cancer cells using LyP-1 / Fc protein
MDA-MB-435 human breast cancer cells (1 × 10 ⁵ cells) were seeded in 500 μl DMEM (containing 10% FBS) and cultured overnight in a 37 ° C., 5% CO ₂ incubator. Mix 1 mol of LyP-1 / Fc protein with 1 mol of ZZ-BNC, and let stand at 4 ° C for 1 hour. Add ZZ-BNC 5 μg of ZZ-BNC / well mixture to ZZ-BNC / LyP-1 / Fc mixture. Was added to the cell culture supernatant. After 7 hours, the cells were washed 3 times with PBS, fixed with 4% PFA, and then allowed to stand at room temperature for 15 minutes using PBS containing 0.25% (v / v) Triton-X100. 100 μl each of primary antibody (anti-ZZ-BNC rabbit-derived polyclonal antibody) diluted 1 / 100-fold with a blocking solution was added and allowed to stand at room temperature for 1 hour. After washing 3 times with 0.03% Triton-X100-containing PBS, 100 μl each of secondary antibody (Alexa546-labeled anti-rabbit IgG antibody) diluted 1/2000 times with blocking solution was added and allowed to stand at room temperature for 1 hour. . After washing three times with 0.03% Triton-X100-containing PBS, fluorescence was observed using a confocal laser scanning microscope. As a negative control, the same experiment was performed using Fc protein instead of LyP-1 / Fc protein. As a result, it was confirmed that ZZ-BNC was incorporated into MDA-MB-435 cells to which LyP-1 / Fc protein-presenting ZZ-BNC was added (FIG. 4). On the other hand, ZZ-BNC could not be confirmed in MDA-MB-435 cells supplemented with Fc protein-presenting ZZ-BNC. (The number of cells confirmed to be ZZ-BNC / total number of cells), LyP-1 / Fc protein-presenting ZZ-BNC was accumulated in human breast cancer cells at 80.3%, Fc protein-presenting ZZ-BNC The accumulation was 12.3%. From the above results, it was found that LyP-1 / Fc protein-presenting ZZ-BNC was incorporated into MDA-MD-435 cells with high efficiency.

(5) Co-localization of ZZ-BNC and lysosomal marker MDA-MB-435 human breast cancer cells infected with the above LyP-1 / Fc protein-presenting ZZ-BNC were treated with blocking solution in the same manner. Primary antibody (anti-Lamp2 mouse-derived monoclonal antibody (clone H4B4; DSHB, Iowa City, IA) and anti-ZZ-BNC rabbit-derived polyclonal antibody) diluted with 100-fold and secondary antibody (Alexa546 diluted with 1 / 2000-fold with blocking solution) -Immunostaining was performed with labeled anti-rabbit IgG antibody and Cy3-labeled anti-mouse IgG antibody (Amersham Biosciences)). As a result, fluorescence derived from Alexa546 was observed inside the region exhibiting fluorescence derived from Cy3 (FIG. 5). From the above, it was shown that ZZ-BNC was introduced into cells with high efficiency by LyP-1 / Fc protein and existed in late endosomes and lysosomes in the cells.

(6) Delivery of rhodamine-labeled polystyrene beads (FluoSpheres; diameter 100 nm) to MDA-MB-435 cells using ZZ-BNC and LyP-1 / Fc protein
2 mg of 2 mg / ml FluoSpheres (Molecular Probes) was mixed in one vial of COATSOME EL-01-A (Nippon Yushi Co., Ltd.) and allowed to stand at room temperature for 30 minutes. 1.6 μl of liposome-fluorescent bead solution was diluted to 100 μl with distilled water, added to lyophilized ZZ-BNC (100 mg) and thawed, and both ZZ-BNC and liposome lipid content were prepared to a final concentration of 1 μg / μl. . 1 mol of purified LyP-1 / Fc protein is mixed with 1 mol of ZZ-BNC and left to stand at 4 ° C for 1 hour, and then MDA-MB-435 cells prepared as described above have 5 μg of ZZ-BNC per well. Was added as follows. After culturing at 37 ° C. for 7 hours in a 5% CO ₂ incubator, the plate was washed three times with PBS, and fluorescence was observed using a confocal laser scanning microscope. As a result, fluorescence of LyP-1 / Fc protein-presenting ZZ-BNC was observed in 67% of all cells (FIG. 6). On the other hand, ZZ-BNC alone was 25%, and liposome alone was 8%. From the above, it was shown that LyP-1 / Fc-presenting ZZ-BNC can introduce a large substance such as FluoSpheres (diameter 100 nm) specifically and efficiently into human breast cancer cells.

Example 2: In vivo pinpoint delivery of fluorescent substance with biotinylated homing peptide, biotinylated ZZ-BNC and streptavidin (1) Biotin labeling of ZZ-BNC
The ZZ-BNC solution was concentrated to a concentration of 600 μg / ml or higher using an ultrafiltration filter (Millipore). 100 μl of ZZ-BNC was biotinylated at room temperature for 30 minutes using NHS-Sulfo-Biotination Kit (Pierce) according to the protocol attached to the manufacturer, and the amino group and N-terminal amino group of the lysine residue were labeled with biotin. Free unreacted biotin was removed by centrifuging at 1,000 × g for 2 minutes using Zeba ^™ Desalt Spin Columns (Pierce, which can be replaced with a column packed with Sephadex G25 or G50) (FIG. 7).

(2) HABA assay to measure biotin uptake level
180 μL of HABA / avidin solution (Pierce) was pipetted into microplate wells, absorbance was measured at 500 nm, and recorded as A500HABA / avidin. Next, 20 μL of biotinylated sample was added to the well containing the HABA / avidin solution, and after stirring well, absorbance was measured at 500 nm. When the measurement was approximately constant for at least 15 seconds, it was recorded as an A500HABA / avidin / biotin sample. By applying the absorbance change from A500HABA / avidin to the A500HABA / avidin / biotin sample to the formula provided by Pierce, the amount of biotin contained in the sample can be calculated.

(3) Preparation of LyP-1 peptide (CNKRTRGGC) -presenting ZZ-BNC Powdered biotinylated LyP-1 peptide (Biotin-GGCNKRTRGGC / Ruoslahti et al., PNAS, 101, 9381-9386 (2004) (Peptides at Invitrogen Synthesis (purity 95% or more)) diluted with distilled water to 140 ng / μl (15 μl) and 1 mg / ml streptavidin (Seikagaku Corporation) prepared with distilled water are mixed and reacted at room temperature for 30 minutes. Then, a streptavidin-biotinylated LyP-1 peptide complex was formed, and then biotinylated ZZ-BNC (100 μg as the amount of protein) was added to the above streptavidin-biotinylated LyP-1 peptide complex, followed by 30 at room temperature. The reaction was allowed to proceed for minutes to prepare LyP-1 peptide-presenting ZZ-BNC (FIG. 8).

(4) Preparation of RhoPamine B-containing LyP-1 peptide-presenting ZZ-BNC 2 ml of Rhodamine B (ALDRICH) diluted to 0.5 mg / ml with distilled water is mixed in one vial of COATSOME EL-01-A (Nippon Yushi Co., Ltd.) And allowed to react for 30 minutes at room temperature. Thereafter, gel filtration was performed using a spin column filled with Sephacryl HR-S500, and free rhodamine B was removed to prepare rhodamine B-containing liposomes. 10 μl of this reaction solution is mixed with the above LyP-1 peptide-presenting ZZ-BNC, and rhodamine B-containing liposomes and LyP-1 peptide are presented so that the amount of phospholipid and ZZ-BNC protein contained in the liposomes are equal. Type ZZ-BNC was mixed to prepare Rhodamine B-containing LyP-1 peptide-presenting type ZZ-BNC.

(5) Accumulation of biotinylated LyP-1 peptide in MDA-MB-435 cells
MDA-MB-435 cells (human-derived breast cancer cells) and WiDr cells (human-derived colorectal cancer cells) are spread on 1 x 10 ⁵ cells each on a 24-well collagen-coated plate (IWAKI) and placed in 500 μl DMEM (containing 10% FBS) And cultured at 37 ° C. overnight in a 5% CO ₂ incubator. 140 ng of biotinylated LyP-1 peptide and 7 μg of FITC-labeled streptavidin (Molecular Probes) were reacted in distilled water at room temperature for 30 minutes to form a FITC-labeled streptavidin-biotinylated LyP-1 peptide complex and added to each well . After 7 hours, the plate was washed 3 times with PBS, and fluorescence was observed using a confocal laser scanning microscope. As a result, it was observed that the FITC-labeled streptavidin-biotinylated LyP-1 peptide complex was specifically accumulated only in MDA-MB-435 cells (FIG. 9). On the other hand, the biotinylated GGCRPPR peptide (Peptide synthesis request was Invitrogen / Ruoslahti et al., Circulation, 112, 1601-1611. (2005)), which was a negative control, did not accumulate at all in both cells.

(6) Production of mice carrying human-derived breast cancer cells
MDA-MB-435 in cells (human breast cancer cells) 10cm collagen-coated plates (IWAKI) were plated on DMEM (10% FBS-containing), and cultured for several days at 37 ° C. CO ₂ incubator, the 1 × 10 ⁷ cell Obtained. Xenograft mixed with 500 μl of PBS (-) and 500 μl of Matrigel, injected subcutaneously into the back of Nude BALB / can nu / nu mice, grown for about 2 weeks, and has tissue derived from MDA-MB-435 cells (diameter 7-10 mm) A model mouse was prepared.

(7) Specific accumulation of Rhodamine B-containing LyP-1 peptide-presenting ZZ-BNC into MDA-MB-435 cell-derived tissue Rhodamine B-containing LyP-1 peptide-presenting ZZ-BNC (50 mg as the amount of ZZ-BNC protein) Was administered from the tail vein of a Xenograft model mouse having MDA-MB-435 cell-derived tissue. As negative controls, Rhodamine B-containing ZZ-BNC not presenting LyP-1 (ZZ-BNC protein amount 50 mg), Rhodamine B-containing liposomes 10 μl, Rhodamine B 10 μl prepared to 0.5 mg / ml each in distilled water 125 μl What was made into was used. After administration, excitation was performed at a wavelength of 545 nm using an In Vivo imaging apparatus OV100 (OLYMPUS), and fluorescence was observed at a wavelength of 570-625 nm. In the mice administered with Rhodamine B-containing LyP-1 peptide-presenting ZZ-BNC, accumulation in the transplanted tumor was confirmed from about 3 hours after administration (FIGS. 10 and 11). This accumulation was confirmed up to about 12 hours after administration. On the other hand, accumulation in the transplanted tumor could not be confirmed in the mice administered with rhodamine B-containing ZZ-BNC, rhodamine B-containing liposome, and rhodamine B not presenting LyP-1 (FIG. 8). From the above, it was found that BNC presenting a homing peptide can deliver a pinpoint substance in vivo.

(8) Specific accumulation of rhodamine B-containing cardiovascular endothelial cell-specific homing peptide-presenting ZZ-BNC in the mouse heart Homing peptide that specifically recognizes mouse heart-derived vascular endothelial cells (CRPPR: Zhang L, Hoffman JA, and Ruoslahti E Circulation 2005; 112: 1601-1611) containing 15 μl of a biotinylated GGCRPPR peptide (Peptide synthesis request is Invitrogen) to 140 ng / μl with distilled water and 1 μg / ml streptavidin (Seikagaku Corporation) The mixture was reacted at room temperature for 30 minutes to form a streptavidin-biotin-labeled LyP-1 peptide complex. Next, biotinylated ZZ-BNC (100 μg as the amount of protein) was mixed with the whole amount of the above streptavidin-biotin-labeled LyP-1 peptide complex and reacted at room temperature for 30 minutes to prepare GGCRPPR peptide-presenting ZZ-BNC. Next, rhodamine B-containing GGCRPPR peptide-presenting ZZ-BNC was prepared by the same method as “(4) Preparation of rhodamine B-containing LyP-1 peptide-presenting ZZ-BNC”. 50 mg of rhodamine B-containing GGCRPPR peptide-presenting ZZ-BNC as a protein amount was administered from the tail vein of BALB / c mice (♀). As negative controls, rhodamine B-containing ZZ-BNC not showing GGCRPPR (ZZ-BNC protein amount 50 mg), rhodamine B-containing liposomes 10 μl, rhodamine B 10 μl prepared to 0.5 mg / ml each in distilled water to 125 μl What was done was used. Eight hours after administration, the heart was removed from each mouse, excited with an In Vivo imaging apparatus OV100 (OLYMPUS) at a wavelength of 545 nm, and fluorescence was observed at a wavelength of 570-625 nm. As a result, strong fluorescence was observed in the mouse heart administered with rhodamine B-containing GGCRPPR peptide-presenting ZZ-BNC (FIG. 12). In addition, when the same heart was resin-embedded and fixed (using Technovit), the sections were observed for fluorescence. As a result, accumulation of fluorescence was confirmed around the blood vessels of the mouse heart administered with rhodamine B-containing GGCRPPR peptide-presenting ZZ-BNC ( FIG. 13). From the above, it has been found that homing peptide-presenting BNC can deliver substances pinpoint not only in cancer tissues but also in normal tissues in vivo.

Example 3: Difference in biokinetics between bio-nanocapsules and liposomes Rhodamine B-containing liposomes (Lp-Rho) and rhodamine B-containing BNCs (BNC-Lp-Rho) are the LyP-1 described in "Example 2 (7)". Is equivalent to rhodamine B-containing ZZ-BNC (50 mg as the amount of ZZ-BNC protein) and rhodamine B-containing liposome 10 μl. These were administered from the tail vein of BALB / c mice (♀), excited with a wavelength of 545 nm by an In Vivo imaging apparatus OV100 (OLYMPUS), and fluorescence was observed at a wavelength of 570-625 nm. As a result, within 60 minutes of administration, most of the administered rhodamine B-containing liposomes were trapped in the liver (most of the existing DDS carriers behave the same), but the localization of rhodamine B-containing BNC in the liver was Not observed (Figure 14). Thereafter, it was strongly suggested that both were secreted from bile into the intestinal tract by 300 minutes and behaved as feces. Moreover, although the mouse | mouth 60 minutes after administration was observed with the In Vivo imaging device (Xenogen), the same result was shown. Based on the above, it was considered that BNC was not trapped in the liver immediately after administration, but circulated throughout the body in the bloodstream, unlike liposomes. This property of BNC is very effective when retargeting to various sites in the body using a homing peptide and is not found in other DDS carriers including liposomes.

Example 4: Retargeting of BNC using RIP1-Tag2 tumor homing sequence CRGRRST (1) Construction of plasmid pGEX-6P-1-GFP-RT1 for GFP-RT1 expression
4 μg of pGEX-6P-1-GFP plasmid DNA was cleaved with HindIII 24U and EcoRI 32U, and a fragment of about 5.8 kb was separated by 1% agarose (1XTAE buffer) electrophoresis and purified with Nucleospin column (MACHEREY-NAGEL). The linker has an EcoRI sticky end on the 5 'side and a HindIII sticky end on the 3' side, and RIP1-Tag2 tumor homing sequence CRGRRST (referred to as RT1: Stage-specific vascular markers revealed by phage display in a mouse model of pancreatic Islet tumorigenesis. The following synthetic DNA designed to encode Joyce JA, Laakkonen P., Bernasconi M., Bergers G., Ruoslahti E., and Hanahan D. (2003) CANCER CELL 4 , 393-403.) The synthesis was commissioned (HPLC purification) to Bio-One Co., Ltd.

Array:
RIPTag-CRGRRST-U (28Nts) 5'-AATTCG TG C CGC GG T CGC CGC AGT ACT A-3 '
RIPTag-CRGRRST-L (28Nts) 3'- GC AC G GCG CC A GCG GCG TCA TGA TTCGA-5 '
CRGRRST
A restriction enzyme SacII recognition sequence was introduced in the underlined portion so that the linker-inserted plasmid could be easily searched. Mix 10 μl each of 100 μM RIPTag-CRGRRST-U and RIPTag-CRGRRST-L synthetic DNA solution into a 500 μl tube, heat at 95 ° C. for 5 minutes, then heat block of BLOCK INCUBATOR (KOKEN RIKA BP-4100) containing the tube The double-stranded DNA was annealed by allowing it to stand at room temperature and slowly cooling it for about 20 minutes. Both this mixed solution and plasmid DNA purified by Nucleospin column were combined using ligation high (TOYOBO), and transformed into DH5α competent cells prepared according to Reference 2. The colony lysis PCR using pGEX primer was used to confirm the presence or absence of the inserted colony. The plasmid DNA purified by the QIAGEN plasmid mini kit (QEAGEN) was introduced with the base sequence designed by SacII digestion and sequencing. Confirmed that.

(2) Expression and purification of GFP-RT1 protein
pGEX-6P-1-GFP-RT1 was transformed into BL21-CodonPlus (registered trademark) (STRATAGENE) competent cell (High efficiency transformation of Escherichia coli with plasmids. Inoue H., Nojima H., and Okayama H. (1990) Gene 96, 23-28.) And the resulting single clone was expressed as follows. 2xYT medium 37 ° C Overnight 10ml of the culture solution is inoculated into 1L of 2xYT medium, cultured at 37 ° C to OD ₆₀₀ = 0.9, final expression is induced with 0.2 mM IPTG, cultured at 20 ° C for 6 hours, then collected did.
Purification from bacterial cells basically follows the literature (Solubilization and Purification of Enzymatically Active Glutathione S-Transferase (pGEX) Fusion Proteins. Frangioni JV and Neel BG (1993) Analytical Biochemistry 210 , 179-187.) Went to. The cells were solubilized in 42.5 ml of STE buffer (11.8 mM Tris-Cl pH 8.0, 176.5 mM NaCl, 1.18 mM EDTA), and lysozyme was added to a final concentration of 100 μg / ml. Lysis for minutes. Add DTT to 5 mM and Sodium Sarkosyl to 1.5% (both final concentrations), mix with vortex mixer, and then use Ultrasonic disruptor UD-201 (use trace amount TP-040, OUTPUT 4, DUTY 60)) ( TOMY) was sonicated for 1-2 minutes. After removing the insoluble material by centrifugation at 15,460 xg 4 ° C for 30 minutes, Triton X-100 was added to a final concentration of 1.6% and added to Glutathione sepharose 4B (800 μl with 75% slurry) (GE Healthcare Life Sciences). Adsorption was carried out while rotating at 2 ° C. for 2 hours. Econo-Pac Column (BIO-RAD) was filled with a solubilized solution containing Glutathione sepharose 4B, the non-adsorbed fraction was added again, washed with 60 ml of PBS buffer, and PreScission Protease Cleavage buffer (50 mM Tris-Cl pH7 .0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT) 12 ml. PreScission Protease 15 μl (30 U) was added to the same buffer 750 μl, and cut at 4 ° C. for 16 hours. GFP-RT1 cleaved from Glutathione S-transferase was eluted by pretreatment with 750 μl of PreScission Protease Cleavage buffer at room temperature for 10 minutes three times, and concentrated to about 600 μl with Centricon YM-10, 10 kDa NMWL (MILLIPORE). The final purified product was passed through a 0.22 μm filter, and the protein concentration was determined by BCA assay. About 1.6 mg of purified protein was obtained from 1 L of E. coli culture solution.

(3) Measurement of in vivo homing activity of GFP-RT1
Creation of RIP1-Tag2 mice was according to the literature (Heritable formation of pancreatic beta-cell tumours in transgenic mice expressing recombinant insulin / simian virus 40 oncogenes. Hanahan D. (1985) Nature 315, 115-122.). A 0.18% (W / V) NEMBUTAL injection was administered intraperitoneally to 13.5-week-old RIP1-Tag2 mice, and 100-200 μl (about 300 μg) of purified protein was administered from the tail vein under anesthesia. Seven minutes later, the chest was opened, and 20 ml of PBS followed by 20 ml of 2% paraformaldehide (PFA) / PBS solution was perfused from the left ventricle to remove the pancreas. 4% PFA / PBS 4 ° C for 30 minutes, and then immersed in PBS, 12% Sucrose / PBS, 15% Sucrose / PBS, 18% Sucrose / PBS for 30 minutes each at room temperature, and then placed in OCT Compound (Tissue-Tek 4583). After embedding and freezing, 10 μm sections were prepared with Cryostat microtome (Leica). The sections were air-dried and fixed at 10 ° C. with aceton: methanol = 1: 1 stored at 4 ° C. After washing with PBS, blocking with 5% goat serum / PBS for 30 minutes at room temperature, then reacting with Anti-GFP (abcam ab6556) solution (1/300 dilution in 2.5% goat serum / PBS) at 4 ° C for 16 hours, washing with PBS Then, react with Alexa Fluor 594 Goat anti-rabbit IgG (Molecular Probes A-11012) solution (1/1000 dilution in 2.5% goat serum / PBS) for 45 minutes at room temperature, wash with PBS, and then VECTASHIELD Mounting Medium with DAPI (VECTOR LABORATORIES , INC. H-1200) and observed with a fluorescence microscope. (Figs. 16 and 17)
As a result, the homing peptide RT1 was found in the state of the phage library when isolated or by literature (Stage-specific vascular markers revealed by phage display in a mouse model of pancreatic islet tumorigenesis. Joyce JA, Laakkonen P., Bernasconi M. , Bergers G., Ruoslahti E., and Hanahan D. (2003) CANCER CELL 4 , 393-403.) Are known to localize in the cytoplasm of naturally occurring insulinoma in the pancreas of RIP1-Tag2 mice. However, the homing peptide RT1 fused with GFP was localized in Blood Lake inside the cancer tissue without being localized in the cytoplasm of Insulinoma. In other words, the results show that the homing peptide functions correctly in vivo in the form of a phage library, alone or in a form bound to a small molecule, but does not function correctly when bound to a polymer again.

(4) Construction of RT1-presenting BNC expression plasmid and transformation into yeast Next, a yeast vector for expressing BNC fused with the homing peptide (RT1) on the same polypeptide was prepared. First, pGLD-LAG-d50N-ZZ was synthesized from the original type BNC expression vector pGLDLIIP39-RcT (Kuroda S, Otaka S, Miyazaki T, Nakao M, Fujisawa Y. Hepatitis B virus envelope L protein particles. Synthesis and assembly in Saccharomyces cerevisiae, J Biol Chem. 1992 267 1953-61) and subcloning the BamHI-SalI fragment (2.5 kb) of pGLD-LAG-d50N-ZZ into the pUC119 BamHI, SalI site. Subsequently, the following linker that was 5 ′ phosphorylated was introduced into the NotI site. Specifically, 4 μg of plasmid pUC119-LAG-d50N was cleaved with NotI 32U, treated with Alkaline phosphatase (TaKaRa) 60U, extracted with phenol-chloroform, and purified by 0.8% agarose electrophoresis. The linker was 5′-phosphorylated with T4 polynucletide kinase for each synthetic DNA purified on a reverse phase column, extracted with phenol-chloroform, and then combined with ligation high (Toyobo). The following three fragments: XhoI-XbaI fragment (0.6 kb) of plasmid pUC119-LAG-d50N-RT1, XbaI-ScaI fragment (4 kb) of pGLD-LAG-WT, ScaI-XhoI fragment of pGLD-LAG-WT ( 6 kb) was digested with 3 μg of each plasmid DNA using 24 U of restriction enzyme, purified by 0.8% agarose electrophoresis, and ligated using ligation high. The obtained plasmid pGLD-LAG-d50N-RT1 was purified by Cscl ultracentrifugation, and the spheroplast method (Transformation of Yeast. Hinnen, A., Saccharomyces cerevisiae BY8238 (MATa leu2-3, 112 his4-519 can1 [ρ-] Hicks, JB, and Fink, GR (1978) Proc Natl Acad Sci US A. 75, 1929-1933).
NotI- RIPTag-CRGRRST-U
5'- GGCCGCTGCCGCGGCCGCCGCAGTACTAGC-3 '
3'- CGACGGCGCCGGCGGCGTCATGATCGGGCC-5 '
NotI- RIPTag-CRGRRST-L

(5) After selection and culture transformation of RT1-BNC high expression strain, 6 strains grown on SD-leu plate are suspended in High-Pi medium and cultured in 30 ml of 8S5N-P400 medium at 30 ° C for 3 days did. Add 2.5 ml of buffer A (containing 0.1% Tween 80, 100 μg / ml PMSF) and about 2.5 ml of glass beads to the obtained bacterial cells (0.8-2 g), and use a vortex mixer for 10 minutes. The cells were crushed by vigorous stirring. The disrupted supernatant obtained by centrifugation at 20000 xg was diluted 2000 times, and IMx (ABBOTT JAPAN CO., LTD) was measured to select the highest one. Western blotting with anti-ZZ-BNC antibody confirmed that RT1-BNC had a molecular weight of about 34 kDa and that it did not undergo significant protease degradation in yeast cells.

For large-scale culture, start culture at High-Pi medium 50 ml, OD ₆₀₀ = 0,65, culture at 30 ° C for 48 hrs, and then occupy 1/10 of the volume to new High-Pi medium 400 ml at OD ₆₀₀ = 6.60. After planting and culturing at 30 ° C for 24 hrs, one-tenth of the OD ₆₀₀ = 4.96 was transplanted to a new 8S5N-P400 medium 4L and cultured at 30 ° C for 72 hrs. Finally, OD ₆₀₀ = 16.28 and 26.5 g of cells per liter of the culture solution could be obtained.

(6) Purification of RT1-BNC Basically (Yamada T, Iwabuki H, Kanno T, Tanaka H, Kawai T, Fukuda H, Kondo A, Seno M, Tanizawa K, Kuroda S. Physicochemical and immunological characterization of hepatitis B virus envelope particles exclusively consisting of the entire L (pre-S1 + pre-S2 + S) protein. Vaccine. 2001 19: 3154-63.).
The cell disruption was repeated for 1 minute → 1 minute cooling 5 times. The disrupted supernatant was dialyzed with about 15 times the amount of external solution (PBS, 1 mM EDTA) for 2 hours, then with about 25 times the amount of external solution for 2 hours, and then with the same amount of external solution overnight. The heat treatment was performed at 70 ° C. for 20 minutes. The heat-treated supernatant was passed through a 0.45 μm filter, and then sulfated cellulose fine column equilibrated with 10 mM phosphate buffer pH 7.2 + 0.15 M NaCl (column diameter 1.6 × 20 cm, carrier CHISSO CORP., Flow rate 4 ml The non-adsorbed fraction was washed with the same equilibration buffer twice the column volume and then eluted with 10 mM phosphate buffer pH 7.2 + 1 M NaCl. The elution fraction was concentrated to 5 ml or less with Amicon Ultra; NMWL 100K (MILLIPORE) and equilibrated with PBS and 1 mM EDTA (column diameter 1.6 × 60 cm, carrier Sephacryl S-500 HR; GE Healthcare Life Sciences, flow rate 0.3 ml / min). The eluted fraction was concentrated with Amicon Ultra; NMWL 100K, (MILLIPORE), and further, endotoxin was removed using Acrodisc.Units with MustangTM Membrane PNM STG25E3 (PALL), and the protein concentration was determined by BCA assay (FIG. 19). And 20).

(7) Preparation of RT1-BNC encapsulated in pEmGFP plasmid
1 μmol of COATSOME EL (Japanese fat EL-01-D) (freeze-dried product) was dissolved in 1 ml of 250 μg / m plasmid pcDNA6.2 / C-EmGFP-GW / CAT (Invitrogen) solution. The mixture was mixed with the BNC solution as follows to form a complex at room temperature for 10 minutes.

(8) Measurement of RT1-BNC in vivo homing activity
After 150 μl of the complex was administered to RIP1-Tag2 mice and allowed to stand for 65 hours, in vivo homing activity was analyzed using anti-ZZ antibody and anti-GFP antibody (FIGS. 21 and 22).
As a result, peptide sequence RT1: CRGRRST that accumulates in RIP1-Tag2 tumor in a stage-specific manner was fused to GFP and BNC, and detected using two types of antibodies in three systems as shown in the table below. In both cases, the results suggested the accumulation of red blood cells in blood lake, which was formed specifically in tumor with advanced stage.

(9) Limitations of polymer-bound homing peptides
The RT1 sequence has been reported to be specifically accumulated in the RIP1-Tag2 tumor in the state of the phage library when isolated, alone or as a small molecule conjugate. There have been no reports of examples of accumulation in tumor by fusing again. Similarly, when an existing homing peptide is bound to a polymer (GFP), it is often experienced that it does not localize to the original target site. For example, NSSRDLG (Muller OJ, Kaul F, Weitzman MD, Pasqualini R, Arap W, Kleinschmidt JA, Trepel M. Random peptide libraries displayed on adeno-associated virus to select for targeted gene therapy vectors Nat Biotechnol. 2003; 21: 1040-6.) Does not go to the heart by fusing GST-GFP (Figure 23). SMSIARL (Proc Natl Acad Sci US A. 2002; 99: 1527-31. Targeting the prostate for destruction through a vascular address. Arap W, Haedicke W, Bernasconi M, Kain R, Similarly, Rajotte D, Krajewski S, Ellerby HM, Bredesen DE, Pasqualini R, Ruoslahti E.) does not go to the prostate by fusing GST-GFP (FIG. 24).
In other words, there are many homing peptides that function as isolated phage phages, alone or in a low-molecular-weight form, and some of them function even when a new polymer is bound. Therefore, the method as shown in Example 4 takes time, and it is difficult to obtain a large amount of modified BNC because it depends on the sequence of the presented homing peptide. A simple method for producing a homing peptide-presenting BNC as shown in Example 1 and Example 2 must be used.

  BNC presenting the biorecognition molecules (particularly homing peptides) of the present invention is capable of pinpoint delivery to various targets, delivering pharmaceuticals to target cells (DDS, GDS), and various target sites. The delivery of research reagents is possible.

Purification of LyP-1 / Fc protein; the left band is purified LyP-1 / Fc protein and the right band is purified Fc protein. Accumulation of LyP-1 / Fc protein on human breast cancer cells. In vitro interaction between ZZ-BNC and LyP-1 / Fc protein. Retargeting ZZ-BNC to human breast cancer cells using LyP-1 / Fc protein. Co-localization of ZZ-BNC and lysosomal markers in breast cancer cells. 100 nm diameter fluorescent bead delivery to MDA-MB 435 cells using ZZ-BNC and LyP-1-Fc protein. The schematic diagram which shows the method of biotinylating ZZ-BNC. The schematic diagram which shows the method to couple | bond a homing peptide to a biotinylated ZZ-BNC via streptavidin. Specific accumulation of biotinylated LyP-1 peptide on MDA-MB-435 cells. Specific accumulation of LyP-1-displaying BNC in human breast cancer cell-derived tumors (1) Specific accumulation of LyP-1-displaying BNC in human breast cancer cell-derived tumors (2) In vivo delivery of rhodamine B to the heart using homing peptide-presenting BNC. In Vivo Pinpoint Delivery of Rhodamine to Mouse Heart using Homing Peptide (5 aa) -displaying BNC (Systemic Injection) Bio-imaging Analysis of Systemically Injected Rhodamine-labeled BNC in Nude Mouse Construction of pGEX6P-1-GFP-RT1. GFP-RT1 accumulated in the blood lake of RIP1-Tag2 tumor by in vivo administration. GFP-RT1 did not accumulate in normal pancreatic tissues accumulated in the blood lake of RIP1-Tag2 tumor by in vivo administration and was not observed with GFP. Construction of pGLD-LAG-d50N-RT1. Purification of RT1-BNC. RT1-BNC purification process chart. RT1-BNC was not observed in ZZ-BNC accumulated in the blood lake of RIP1-Tag2 tumor by in vivo administration. Accumulation of RT1-BNC in the blood lake of RIP1-Tag2 tumor was detectable with GFP antibody, but not observed with ZZ-BNC. Cardiac GST-GFP-NSSRDLG: The heart was not targeted at all. prostate GFP-SMSIARL: The prostate signal was weak, and it was detected in tissues around the prostate and blood vessels in the liver.

Claims (11)

A bio-nanocapsule comprising a hepatitis B virus surface antigen (HBsAg) protein or a variant thereof linked to a homing peptide. The bio-nanocapsule according to claim 1, wherein the homing peptide is linked to the HBsAg protein or a variant thereof via a biotin-biotin-binding protein-biotin bond. The bio-nanocapsule according to claim 1, wherein the homing peptide is contained in a fusion protein linked to the HBsAg protein or a variant thereof directly or via an appropriate amino acid sequence. The bio-nanocapsule according to claim 1, wherein the HBsAg protein or a variant thereof comprises a binding domain with an antibody Fc region, and a fusion protein comprising a homing peptide and an antibody Fc region is linked to the surface antigen protein. . The biotinylated bio-nanocapsule according to claim 4, wherein the domain is a ZZ tag. A biotinylated bio-nanocapsule comprising a biotin-modified HBsAg protein or a variant thereof. A biotin-binding protein-biotinylated bio-nanocapsule comprising a biotin-modified HBsAg protein or a variant thereof and a biotin-binding protein. The biotinylated bio-nanocapsule according to claim 1, wherein the HBsAg protein or a variant thereof includes a domain that is binding to an Fc region of an antibody. The biotinylated bio-nanocapsule according to claim 8, wherein the domain is a ZZ tag. A bio-nanocapsule containing an HBsAg protein or a variant thereof chemically linked to a biotin-binding protein. 11. The bio-nanocapsule according to claim 10, wherein a biotin-binding protein and an HBsAg protein or a variant thereof are bound via a cross-linking agent.

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