KR101477123B1 - Antibody Binding Peptide-Ferritin Fusion Protein and Use Thereof - Google Patents

Abstract

The present invention is a nanoparticle comprising 24 antibody-binding peptide-ferritin fusion protein monomers, wherein 6 partial structures with 4-fold symmetry point are formed by ferritin, and 4-fold symmetry point Of the antibody binding peptide is fused in the range of +/- 10 aa of the antibody, and four antibody binding peptides are positioned in the vicinity of the 4-fold symmetry point. And its use.
The present invention relates to a method for producing a fusion protein by introducing a sequence of an antibody binding peptide into ferritin, and using the antibody to bind the ferritin to the ferritin without damaging the structure of the antibody, the biochip, drug delivery system, Can be utilized.

Description

Antibody Binding Peptide-Ferritin Fusion Protein and Use Thereof "

The present invention relates to a fusion protein of an antibody-binding peptide and ferritin (FcBP-ferritin) and uses thereof.

The present invention also relates to nanoparticles comprising 24 antibody-binding peptide-ferritin fusion protein monomers, wherein six partial structures with a 4-fold symmetry point are formed by ferritin, and 4- Nanoparticles characterized in that antibody-binding peptides are fused in the ± 10 aa (amino acid) range of the fold symmetry point and four antibody-binding peptides are located near the 4-fold symmetry point; And its use.

Ferritin is a protein that stores iron and is widely found in prokaryotes and eukaryotes. The molecular weight of ferritin is about 500,000 Da. It consists of heavy chain and light chain. It has self-assembly ability and shows unique characteristics to form spherical particles. Ferritin is a protein in which 24 monomers (a single monomer or a monomer composed of one of the heavy chain or light chain) are assembled to form a huge spherical tertiary structure. In the case of human ferritin, the outer diameter is about 12 nm and the inner diameter is about 8 nm. Ferritin may be dispersed into monomers depending on pH conditions and may form nanoparticles with 24 monomers combined, which can capture a variety of substances in ferritin (Wang, Z .; Li, C .; Ellenburg, M ; Soistman, E .; Ruble, J .; Wright, B .; Ho, JX; and Carter, DC, (2006) Acta Crystallographica Section D: Biological Crystallography , 62: 800-806). In general, ferritin in the form of nanoparticles has iron oxide inside, but also manganese oxide (Mn (O) OH and Mn ₃ O ₄ ), cobalt oxide (Co (O) OH and Co ₃ O ₄ ), Cr (OH) ₃ , Ni (OH) ₃ , In ₂ O ₃ , FeS, CdS, CdSe, (Meldrum, FC, and Mann, S., (1993) J ) have reported cases of the manufacture of ferrites containing various inorganic metals such as selenoquinone (ZnSe) (Mackle, P .; Charnock, JM; Garner, .. Am Chem Soc, 115: 8471; Meldrum, FC; Douglas, T .; Levi, S .; Arosio, P .; and Mann, S., (1995) J. Inorg Biochem, 58:.... 59 Iwahori, K., Yamashita, I., (2005) Bull . Chem . Soc . Jpn . , 78: 2075, Okuda, M .; Iwahori, K . ; Tsukamoto, R . ; I, and Yoshimura, H., (2003) Biotechnol. Bioeng . , 84: 187; Ueno, T .; Suzuki, M .; Goto, T .; Matsumoto, T .; Nagayama, K .; and Watanabe, Y (2004) Angew . Chem . Int . Ed . , 43: 2527 ). In addition, there has also been reported a case of synthesizing ferritin by using a peptide selectively binding to a metal, for example, using a peptide binding to silver (Kramer, RM; Li, C .; Carter, DC ; Stone, MO; and Naik, RR, (2004) J. Am . Chem. Soc . , 126 (13): 282). It has also been reported that ferritin, which captures the MRI contrast agent Gd-HPDOTA (gadolinium- [10- (2-hydroxypropyl) -1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid] (2002) Angew. Chem . Int . Ed . , 41: 1017). Thus, ferritin can introduce various functional groups through molecular biology, The ferritin structure incorporating various functional groups has the advantage that it can be modified by an appropriate functional group by an optional method and can be provided with various physicochemical properties as needed. For example, When the peptide sequence is introduced at the proper position of the ferritin, the corresponding metal ion can be selectively bound and transported along with the protein. In this process, even when a single formula is introduced into the ferritin protein monomer, phrase The protein nanoparticles is 24 have a uniform formula, and consequently to a transporter functions to transport the 24 metal ions with a Ferritin is a very sophisticated form of nanoparticles.

A target-oriented drug delivery system is a technique designed to selectively deliver a drug to a treatment site so as not to expose the healthy tissue to the drug, but to exhibit an excellent therapeutic effect with only a small amount of the drug. Using a targeted drug delivery system can maximize the effectiveness of drug treatment by concentrating the drug on a specific area of a diseased human body, and it can minimize side effects caused by toxic drugs such as anticancer drugs. In addition, in recent years, attempts have been made to utilize a target-oriented drug delivery system for disease diagnosis. More recently, the development of Theragnosis (Therapy + Therapy) technology, which can simultaneously perform treatment and diagnosis using a target-oriented drug delivery system, is actively under way. That is, by attaching a molecular imaging probe to a target-oriented drug delivery system and imaging the tracer using non-invasive in vivo imaging, The technology that can be done is TerraGinosis technology.

Among the nanostructures used in pharmaceuticals, liposomes are mainly composed of phospholipid, which is a constituent of a biological cell membrane, and can collect water-soluble or insoluble (hydrophobic) drugs therein. Liposomes have the advantage of being able to control various constituents and surface components, but they are highly toxic to cells and have problems such as phagocytosis of phagocytes when they are injected into the circulation system. As a technique for solving these problems, a stealth liposome has been developed in which a liposome is bound to PEG at the terminal of a phospholipid or a liposome surface is coated with PEG or a polysaccharide. Micelles are carriers composed of chain-like molecules having both hydrophilic and hydrophobic sites. In the center of the aqueous phase, the hydrophobic moieties are gathered together to form a spherical shape. Usually, the micelles are collected at the central portion to increase solubility and bioavailability do. In the case of anticancer drugs, nanoparticle drug delivery systems selectively accumulate in cancer tissues through the enhanced permeability and retention (EPR) effect, which is a passive targeting method. In general, cancer tissue is supplied with nutrients and oxygen through the new blood vessels. Since blood vessels are formed within a short period of time, unlike normal cells, they have a loose structure. Through the loose blood vessels formed in cancer tissues, nanoparticles of several tens to several hundreds of nanometers can accumulate around cancer tissues and have a slow release rate and stay in cancer tissues for a long time. This phenomenon is called enhanced permeability and retention (EPR) do. However, this effect is a passive method using the environment around the cancer, so it is difficult to expect a certain effect at all times. Therefore, in order to achieve more effective targeting, a method using a drug delivery system having an antibody capable of recognizing an antigen or a receptor expressed at a target site has been developed. In addition, combining a selective antibody with a variety of contrast media for chemotherapy and radiotherapy for chemotherapy can reduce systemic side effects, increase the effectiveness of the treatment, and improve the accuracy of the image. Currently, the chemical covalent bond forming method used as an immobilization method for an antibody for targeting is poor in reproducibility and may impair the inherent function of the antibody. In the case of biologically derived medicines as well as general medicines, structural changes in the manufacturing process must be accompanied by a clear identification of structural changes, drug efficacy and toxicity testing in order to obtain approval from the KFDA. Therefore, it is almost impossible to apply a target-oriented drug delivery system using antibodies to human body without ensuring reproducibility of antibody immobilization.

On the other hand, biochips are also called biological arrays in which biological substances such as nucleic acids and proteins are fixed on a template surface. Among these, a protein chip means a biochip that integrates tens or thousands of different peptides or proteins capable of reacting with a specific biomaterial on a solid surface and binds to various biomolecules to analyze them effectively. In recent years, it has been applied to diagnosis of disease, construction of a high-speed screening system for new drug candidates, study of protein expression pattern, and discovery of new protein markers. A nucleic acid or a protein capable of binding together with a protein, an antigen, an antibody, an affinity ligand, a cell receptor, an enzyme, a nucleic acid, or a low molecular substance immobilized on a substrate can be used for the biochip. Biochip technology has become an important tool that enables large scale and high throughput in biological research as a result of the development and integration of information analysis technology.

An important factor in protein chip technology is activation of the protein, exposure of the active site, high integration, and specific binding to the target material. Proteins can be attached to the surface of various types of protein chip solid substrates by diffusion, adsorption / absorption, covalent bonding, and affinity. However, since proteins are generally randomly integrated on the surface of a substrate and the structure of the protein is easily modified, the activity of the protein is inhibited. As a result, binding with surface molecules becomes impossible and specific binding is not efficient. Since the binding specificity between the protein probe and the target substance applied to the protein chip is determined very sensitively according to the structure of the protein, it is necessary to immobilize the protein having the active structure in a position and direction suitable for the high sensitivity recognition of the target substance. However, in the immobilization step, an ineffective immobilization process, such as masking of the active site or instability due to non-specific protein clustering or reduction in the degree of integration, has been recognized as an impediment to high sensitivity detection.

Currently, among the biochip technologies using nanoparticles, it is a technology that binds proteins, peptides, antigens, and antibodies to the surface of semiconductor quantum dots (QDs). The quantum dot has a narrow range of light emitting regions and has an advantage that the light emitting region can be easily controlled. However, there is a disadvantage that the manufacturing process is complicated, the production cost is high, and the quantum dot manufacturing environment is different from the stabilization condition of the biomaterial.

A first object of the present invention is to provide nanoparticles comprising 24 antibody-binding peptide-ferritin fusion protein monomers; And a production method thereof.

A second object of the present invention is to provide an antibody-binding peptide-ferritin fusion protein; A nucleic acid encoding the same; A recombinant vector comprising the nucleic acid; And a transformant transformed with the nucleic acid or the recombinant vector.

A third object of the present invention is to provide a drug delivery system using nanoparticles according to the present invention; Protein chips; Diagnostic kit; And an antigen detection method.

The first aspect of the present invention is a nanoparticle comprising 24 antibody-binding peptide-ferritin fusion protein monomers, wherein six partial structures with a 4-fold symmetry point are formed by ferritin, Binding peptide is fused in the range of +/- 10 aa (amino acid) of the 4-fold symmetric point, and four antibody-binding peptides are located in the vicinity of the 4-fold symmetry point.

At this time, the antibody is preferably bound to the antibody-binding peptide.

A second aspect of the present invention is to provide a method for the preparation of nanoparticles by self-assembly of ferritin, wherein the ferritin has a 4-fold symmetry point of ± 10 aa. An antibody-binding peptide-ferritin fusion protein in which the antibody-binding peptide is fused; A nucleic acid encoding the same; A recombinant vector comprising the nucleic acid; And a transformant transformed with the nucleic acid or the recombinant vector.

A third aspect of the present invention is a method for producing a recombinant expression vector comprising the steps of: i) preparing a recombinant expression vector comprising the nucleic acid according to the present invention; (Ii) preparing a transformant transformed with the recombinant expression vector; Iii) expressing the antibody-binding peptide-ferritin fusion protein from the transformant of step ii); And iv) purifying the expressed antibody-binding peptide-ferritin fusion protein by self assembly to form six partial structures with a 4-fold symmetry point, wherein the fusion protein nanoparticle And a method for producing the same.

A fourth aspect of the present invention provides a target-oriented drug delivery system characterized in that the drug is delivered to the interior or surface of the nanoparticles according to the present invention.

A fifth aspect of the present invention provides a protein chip comprising nanoparticles according to the present invention.

In a sixth aspect of the present invention, there is provided a method for preparing a protein chip, comprising: preparing a protein chip according to the present invention; Immobilizing an antibody against an antigen to be detected on the protein chip; Reacting a sample with a protein chip comprising the immobilized antibody; And measuring an antigen-antibody reaction.

A seventh aspect of the present invention provides a diagnostic kit comprising a protein chip according to the present invention.

Hereinafter, the present invention will be described in detail.

Ferritin, a biologically-derived nanostructure, has six sub-structures structurally having a 4-fold symmetry point when 24 ferritin monomers form nanoparticles, and the same portion of each protein monomer (Amino acid sequence) (see Fig. 2). Therefore, if you are modifying a protein part that forms a symmetry point, four equations will be encountered in the same place.

The antibody binding peptide (FcBP) may be a peptide which recognizes a specific site of the Fc domain of the antibody and can specifically bind to or bind to the site. That is, it may be a peptide which can bind in vitro, preferably in vivo, with sufficient affinity and specificity to the Fc domain. The bonding may be by non-covalent bonding such as hydrogen bonding, ionic bonding, hydrophobic bonding, Van der Waals bonding, have. Linear or internally disulfide bond, and in the case of a circular peptide which forms a disulfide bond therein, it may contain two or more cysteine residues separated by one or more amino acids. Any of those capable of specifically recognizing the Fc region of the antibody to be adjacent or binding can be used without limitation, but a non-limiting example is described in EP 1757701A1. On the other hand, the contents described in EP 1757701A1 are incorporated herein. The Fc position-selective peptide may be naturally expressed, but is not limited thereto, and may be artificially synthesized.

In addition, the antibody-binding peptides of the present invention can be used for peptide derivatives, such as peptide mimetics and analogues (for example, peptide derivatives), as well as peptides that can specifically bind to or bind to specific sites of the antibody Fc domain peptide analogues).

The antibody-binding peptide may be composed of about 40 amino acids or less, preferably 30 amino acids or less, and more preferably 10 to 15 amino acids. As already mentioned, the amino acid may be an amino acid derivative such as an amino acid mimetic or the like in addition to the known 20 amino acids.

The mimetics or analogs of the peptides or amino acids include non-amino acid compound structures having a structure similar to a native amino acid or an antibody binding peptide according to the present invention. The mimic or analog is defined as a material exhibiting similar physical properties, such as size, charge or hydrophobicity, represented by the corresponding amino acid or peptide in the appropriate spatial orientation. As a specific example, a peptide mimetic compound may be a compound in which the amide bond present between one or more amino acids is replaced by a carbon-carbon bond or other bond known in the art.

Thus, the term "amino acid " of the present invention encompasses L-amino acids or their residues that occur naturally in the broad concept, as well as D-amino acids and chemically modified amino acids. For example, the above-mentioned amino acid mimetics and the like are included, and the mimetics and the like in the present invention may include functional equivalents.

The antibody-binding peptide of the present invention is composed of, for example, 11 amino acids, and peptides having a ring-like structure formed by 1 and 11 amino acid disulfide bonds in the molecule, and immunoglobulin (G) G, IgG) in the Fc region. This is shown in Table 1.

The antibody-binding peptides can be prepared by phage-display. The term "phage-display" in the present invention is an experimental technique for studying protein-protein, protein-peptide and protein-DNA interactions using bacteriophages to link proteins and their coding genetic information. The phage-display can be used to produce constrained and unconstrained peptide libraries. The library can be used to identify and select peptides that bind to a particular target molecule. That is, peptides that bind specifically to the Fc region of the antibody can be identified and screened using the above-described Pigment-Display technique, which can be referred to as antibody-binding peptides.

Using the antibody-binding peptide previously developed by molecular biology and the structural characteristics of the above-mentioned ferritin, the present inventors have succeeded in introducing an antibody-binding peptide near the symmetry point of the ferritin protein so that four antibody-binding peptides are present on the surface of the ferritin nanoparticles And the ferritin structure with new functions was developed.

As a result of measuring the binding force between the antibody and the nanoparticles formed by the self assembly of the antibody-bound peptide-ferritin fusion protein thus prepared, a very high binding force which was not observed in the conventional single antibody binding peptide was observed. In Example 3, the antibody bound to the antibody-bound peptide-ferritin nanoparticles was confirmed to be not destroyed by binding the two proteins even if the buffer solution was allowed to flow for 2 hours or more. In addition, it was confirmed that the antibody remains in the nanoparticles even when the antibody-bound peptide-ferritin nanoparticles conjugated with human antibodies are exposed to an excess of rabbit serum.

In addition, using the antibody-binding peptide-ferritin nanoparticles according to the present invention, it was possible to induce the formation of a strong nanoparticle-antibody complex through self-assembly without structural damage of the antibody by simple mixing with the antibody.

Generally, when the antibody-binding peptide-ferritin fusion protein is expressed, the nanoparticles can be purified while being formed, so that the mixing of the antibody can be performed after the nanoparticles are formed by self-assembly. Even before nanoparticle formation, nanoparticles can form even when antibody is mixed with an antibody-binding peptide-ferritin fusion protein. When an antibody is mixed with an antibody-binding peptide-ferritin fusion protein, formation of nanoparticles may be delayed due to steric hindrance caused by antibodies, but if a portion of the antibody attached to the antibody-binding peptide- ferritin fusion protein is dissociated after a long time, Particle-antibody complexes. ≪ / RTI > Accordingly, it is also within the scope of the present invention to form the nanoparticles after mixing the antibody-binding peptide-ferritin fusion protein with the antibody.

As described above, the protein nanoparticles having the antibody-binding peptide introduced into the ferritin according to the present invention induce the formation of a strong nanoparticle-antibody complex through self-assembly by simple mixing with the antibody, Chip as well as form a target-oriented drug delivery system. In addition, a new type of target-directed teraginous agent can be formed by simple mixing with various antibodies (see Figure 1).

Antibody binding Peptides - Ferritin The fusion protein  Containing nanoparticles

The antibody-binding peptide-ferritin fusion protein according to the present invention can form spherical nanoparticles by self-assembly of 24 pieces. At this time, the diameter of the nanoparticles may be 1 to 20 nm.

At this time, among the fusion protein monomers forming nanoparticles according to the present invention, ferritins may be homologous or heterologous. In addition, all of the ferrites may be derived from the same source, and ferritins derived from different sources may be mixed. Ferritin may be microorganism-derived ferritin, such as bacteria, or eukaryotic cell-derived ferritin. Preferably human-derived ferritin.

Among the fusion protein monomers according to the present invention, the ferritin may be a mutein in which one or more amino acid residues in the native amino acid sequence are substituted with cysteine or cysteine is additionally inserted. Cysteine itself has a high affinity for heavy metals, and further includes a thiol group having high reactivity, so that it can be easily coupled with other reactors, thereby facilitating the introduction of a drug directly or through a linker. Preferably, the cysteine is selected from the group consisting of a second Ser (serine), a 19th Ser (serine), a 61st Glu (glutamic acid), a 68th Lys (lysine), a 102nd Ala or an amino acid residue containing at least one of cysteine or cysteine at the 1st and 2nd, 161st, 162nd or 174th positions of the amino acid sequence of Pyrococcus furiosus , More than one sequence may be inserted. The inserted sequence may be a single cysteine residue or a GGC sequence, but may be used without restriction if it contains one or more cysteines and does not alter the structure of the native protein. Substitution or insertion into the cysteine may be performed using any method known to those skilled in the art without limitation, but preferably by site-directed mutagenesis.

For example, the second serine, 19 th serine, 102 th alanine, or 113 th Asp (aspartic acid) of the human-derived ferritin amino acid sequence is substituted with one or more cysteines, or Pyrococcus one or more amino acids between the first and second amino acids of the amino acid sequence of furiosus to form nanoparticles comprising cysteine capable of binding to a drug exposed to the external surface. Further, human-derived ferritin 61st amino acid sequence of Glu (glutamic acid), 68 beonjjae Lys (lysine) or 137th Glu (glutamic acid) with one or more cysteine or a substitution, Pyrococcus it is possible to prepare nanoparticles comprising cysteine capable of binding to the drug exposed on the inner surface by further inserting at least one amino acid sequence including cysteine or cysteine at the 161nd , 162th or 174th positions of the amino acid sequence of furiosus .

The antibody-binding peptide-ferritin fusion protein of the present invention has a 4-fold symmetry point in the ferritin of ± 10 aa. Lt; RTI ID = 0.0 > antibody-binding < / RTI > At this time, four antibody binding peptides are located in the vicinity of one 4-fold symmetry point. In addition, the nanoparticles according to the present invention may have one or more antibodies bound to four antibody-binding peptides located near one 4-fold symmetry point.

At this time, both ends of the antibody-binding peptide may be independently fused to each other in the range of +/- 10 aa of the 4-fold symmetry point of the ferritin, either through a linker or without a linker. By adjusting the length and / or amino acid composition of the linker, the spacing and orientation between antibody-binding peptides near the 4-fold symmetry point can be controlled. Non-limiting examples of linkers include (Gly) ₅ or (GlySer) ₃ . The length of the linker is adjustable by adjusting the number of amino acids introduced.

Antibody binding Peptides - Ferritin Fusion protein

One aspect of the present invention relates to a method of preparing a ferritin nanoparticle having a 4-fold symmetry point of ± 10 aa. Conjugated peptide-ferritin fusion protein in which the antibody-binding peptide is fused to the antibody-binding peptide-ferritin fusion protein.

In addition, one aspect of the present invention provides a nucleic acid encoding an antibody-binding peptide-ferritin fusion protein according to the present invention, a recombinant vector comprising the nucleic acid, and a transformant transformed with the nucleic acid or the recombinant vector.

Further, one aspect of the present invention is a method for producing a recombinant expression vector comprising: i) preparing a recombinant expression vector comprising a nucleic acid according to the present invention; (Ii) preparing a transformant transformed with the recombinant expression vector; Iii) expressing the antibody-binding peptide-ferritin fusion protein from the transformant of step ii); And iv) purifying the expressed antibody-binding peptide-ferritin fusion protein by self assembly to form six partial structures with a 4-fold symmetry point, wherein the fusion protein nanoparticle And a method for producing the same.

As used herein, the term "vector" refers to a nucleic acid construct comprising an essential control element operatively linked to the expression of a nucleic acid insert, as an expression vector capable of expressing a desired protein in a suitable host cell. The present invention can produce a recombinant vector comprising a nucleic acid encoding an antibody-binding peptide-ferritin fusion protein.

Meanwhile, the nucleic acid encoding the antibody-binding peptide-ferritin fusion protein or the recombinant vector comprising the nucleic acid according to the present invention may be transformed or transfected into a host cell.

The recombinant vector of the present invention can be obtained by linking (inserting) the nucleic acid of the present invention into an appropriate vector. The vector into which the nucleic acid of the present invention is to be inserted is not particularly limited as long as it can be replicated in the host. For example, plasmid DNA, phage DNA, etc. may be used. Specific examples of plasmid DNA include commercial plasmids such as pCDNA3.1 + (Invitrogen). Other examples of plasmids that can be used in the present invention include E. coli-derived plasmids (pYG601BR322, pBR325, pUC118 and pUC119), Bacillus subtilis subtilis) - a plasmid origin (YEp13, YEp24 and YCp50) - derived plasmid (pUB110 and pTP5), and yeast. Specific examples of phage DNA include lambda-phages (Charon4A, Charon21A, EMBL3, EMBL4, lambda gt10, lambda gt11 and lambda ZAP). In addition, animal viruses such as retrovirus, adenovirus or vaccinia virus, insect viruses such as baculovirus may also be used.

As a vector of the present invention, a fusion plasmid (for example, pJG4-5) to which a nucleic acid expression-activating protein (such as B42) is linked can be used. Such fusion plasmids include GST, GFP, His- tag, Myc-tag, etc. However, the fused plasmid of the present invention is not limited by the above examples.

In order to insert the nucleic acid of the present invention as a vector, a method of inserting the purified DNA into a restriction site or a cloning site of a suitable vector DNA by cleaving the DNA with an appropriate restriction enzyme can be used.

The nucleic acid of the present invention is preferably operably linked to a vector. The vector of the present invention may further comprise a promoter and a nucleic acid of the present invention in addition to a cis element such as an enhancer, a splicing signal, a poly A addition signal, a selection marker ), A ribosome binding sequence (SD sequence), and the like. Examples of selectable markers include, but are not limited to, chloramphenicol resistant nucleic acids, ampicillin resistant nucleic acids, dihydrofolate reductase, neomycin resistant nucleic acids, etc., but limiting the additional components operatively linked by the above examples no.

As used herein, the term "transformation" refers to the introduction of DNA as a host and DNA replication as a factor of a chromosome or by chromosomal integration, introducing an external DNA into a cell, It means a phenomenon that causes change.

Any transformation method of the present invention can be used, and can be easily carried out according to a conventional method in the art. In general, transfection methods include CaCl ₂ Precipitation method, CaCl ₂ Hanahan method, which increases the efficiency by using a reducing material called DMSO (dimethyl sulfoxide), electroporation, calcium phosphate precipitation method, protoplast fusion method, agitation method using silicon carbide fiber, agrobacterium mediated transformation method , Transformation with PEG, dextran sulfate, lipofectamine and drying / inhibition mediated transformation methods.

The method for transforming the nucleic acid encoding the antibody-binding peptide-ferritin fusion protein of the present invention or a vector containing the same is not limited to the above examples, and the transformation or transfection methods commonly used in the art may be used without limitation .

A transformant of the present invention can be obtained by introducing a nucleic acid encoding an antibody-binding peptide-ferritin fusion protein as a target nucleic acid or a recombinant vector containing the nucleic acid as a host.

The host is not particularly limited as long as it is capable of expressing the nucleic acid of the present invention. Specific examples of the host which can be used in the present invention include bacteria belonging to the genus Escherichia such as E. coli; Bacillus subtilis Bacillus genus such as subtilis ; Pseudomonas Pseudomonas such as putida ; Lactobacillus such as Lactobacillus and Enterococcus; Saccharomyces S. cerevisiae ), skiing investigation, Schizosaccharomyces yeast such as pombe ; Animal cells and insect cells.

When a bacterium such as Escherichia coli is used as a host, the recombinant vector of the present invention can autonomously replicate in a host, and is composed of a promoter, a ribosome binding sequence, a nucleic acid of the present invention, and a transcription termination sequence have.

The promoter of the present invention may be any promoter as long as the nucleic acid of the present invention is expressed in a host such as Escherichia coli. For example, Escherichia coli or phage-derived promoters such as trp promoter, lac promoter, PL promoter or PR promoter; Escherichia coli-infected phage-derived promoters such as the T7 promoter may be used. Artificially modified promoters such as the tac promoter may also be used.

The nucleotide sequence encoding the antibody-binding peptide-ferritin fusion protein according to the present invention can be optionally linked to appropriate regulatory nucleotide sequence (s) using methods known in the art to control the expression of the coding sequence For example, Sambrook et al . , 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, Cold Spring Harbor Laboratory Press, NY).

As used herein, the term "operably linked" indicates that the nucleotide sequence elements are linked in a functional relationship. For example, a "operably linked" promoter is one in which the regulatory element is in the proper position and orientation relative to the nucleotide coding sequence to control RNA polymerase initiation and expression of the nucleic acid (e.g., Effect. The promoter region may be a promoter present in any prokaryotic cell.

In some embodiments, the antibody-binding peptide-ferritin fusion protein coding sequence may be operably linked to a nucleotide sequence encoding a signal peptide (SP) such that the gene product is secreted outside the bacteria and introduced into the culture medium.

In order to facilitate the purification of the target protein recovered in the present invention, the plasmid vector may further include other sequences as necessary. The sequence that may be further included may be a tag sequence for protein purification and may be a tag sequence such as glutathione S-transferase (Pharmacia, USA), MBP (Maltose binding protein, USA), FLAG (IBI, USA) and hexa histidine hexahistidine; Quiagen, USA), and most preferably MBP. However, the types of sequences necessary for purifying the target protein are not limited by the above examples.

Further, in the case of a fusion protein expressed by a vector containing the fusion sequence, it can be purified by affinity chromatography. For example, when glutathione-S-transferase is fused, glutathione, which is a substrate of the enzyme, can be used. When MBP is used, desired protein can be easily recovered using an amylose column.

The host cells transformed by the above method for expressing the antibody-binding peptide-ferritin fusion protein of the present invention can be cultured by a conventional method used in the art. For example, the transformant expressing the antibody-binding peptide-ferritin fusion protein can be cultured in various media, fed-batch culture, continuous culture, and the like. The method of culturing the transformant of the present invention is not limited. The carbon source that may be contained in the medium for the growth of the host cell may be appropriately selected according to the judgment of a person skilled in the art depending on the type of the transformant prepared and a suitable culture condition is adopted to control the culture time and amount .

When a suitable host cell is selected and the culture conditions are established, the transformant in which the target protein has been successfully transformed will produce the antibody-binding peptide-ferritin fusion protein, and the antibody-binding peptide- Peptide-ferritin fusion proteins can be secreted into the cytoplasm, periplasmic space, or extracellular space of host cells. The target protein may also be expressed in soluble or insoluble form.

Proteins expressed in or outside the host cell can be purified in a conventional manner. Examples of purification methods include salting out (eg, ammonium sulfate precipitation, sodium phosphate precipitation, etc.), solvent precipitation (eg, protein fraction precipitation using acetone or ethanol), dialysis, gel filtration, ion exchange, reverse phase column chromatography Chromatography, ultrafiltration and the like can be used alone or in combination to purify the protein of the present invention.

Antibody binding Peptides - Ferritin The fusion protein  The term binding to the contained nanoparticles sieve

The type of antibody bound to the antibody binding peptide in the nanoparticles of the present invention is not limited as long as it can bind to the antibody binding peptide and includes Fc capable of binding to the antibody binding peptide or a fragment or derivative / Antibodies, fragments, derivatives or analogs of antibodies. The antibody is preferably IgG.

Antibody derivatives include, but are not limited to, antibodies modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, induction by known protecting / blocking groups, proteolysis, intracellular ligands or other proteins. Any of a variety of chemical modifications may be performed by known techniques including, but not limited to, specific chemical degradation, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. Additionally, the derivative may contain one or more non-classical amino acids.

Antibodies include both polyclonal and monoclonal antibodies as well as specific recombinant antibodies such as chimeric, humanized or human antibodies.

In the present invention, the antibody may be a receptor-specific antibody or a ligand-specific antibody. In the present invention, the antibody may also be a receptor-specific antibody that does not prevent ligand binding but prevents receptor activation.

The antibody binding to the nanoparticle according to the present invention may be a therapeutic antibody, an antibody capable of binding with a separate therapeutic agent or diagnostic agent, an antibody for targeting without therapeutic effect, It may be an antibody capable of antibody reaction.

The therapeutic or diagnostic agent may bind to the antibody, but may also be carried by the nanoparticles according to the present invention.

Meanwhile, about 30 therapeutic antibodies have been approved by the FDA and their safety is very high because they closely resemble those of in vivo IgG. Therapeutic antibodies are used in a wide range of disease treatments (eg, transplant rejection, cancer, autoimmune diseases and inflammation, heart disease, infectious infection, etc.) and these antibodies are specific for receptor proteins or antigenic proteins And thus the specificity is very high. Therefore, combining a molecular imaging probe or a drug delivery vehicle with a therapeutic antibody can turn the therapeutic agent into a teraginous agent capable of monitoring the therapeutic process as well as the effect of the drug combination. In addition, by combining a molecular imaging probe or a drug delivery system with a simple targeting antibody, it is possible to develop a teraginosis preparation for diagnosis, treatment, or simultaneous diagnosis and treatment. The present invention can provide a technology for converting target ferritin into an FcBP fusion protein, introducing a molecular imaging probe, a therapeutic drug, etc., and then developing a target-oriented teraginousism material together with the antibody.

The drug carried by the nanoparticles of the present invention

Drugs delivered by the nanoparticles of the invention include therapeutic agents, diagnostic agents / detection agents.

Non-limiting examples of therapeutic agents include antibodies, antibody fragments, drugs, toxins, nucleic acid hydrolases, hormones, immunomodulators, chelators, boron compounds, photoactive agents or dyes, and radioisotopes do.

Non-limiting examples of diagnostic / detection agents include radioactive isotopes, dyes (e.g., biotin-streptavidin complexes), contrast agents, fluorescent compounds or fluorescent proteins, and magnetic resonance imaging Enhancer (paramagnetic ion). Preferably, the diagnostic agent comprises a radioactive isotope, a magnetic resonance imaging (MRI) enhancement agent, and a fluorescent compound. In order to load the antibody component with radioactive metal or paramagnetic ions, it may be necessary to react with a reactant having a long tail attached to many of the chelating groups to couple the ions. The tail may be a polymer such as polylysine or polacacaride or a polymer such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrin, polyamine, crown ether, bis-thiosemic A chain having a pendant group capable of bonding with a chelating group such as a thiosemicarbazone, polyoximes, and a group known to be useful for the above purpose. The chelate is bound to the antibody using standard chemistry. The chelate can normally be conjugated to the antibody by a minimal loss of immunoreactivity and by a group capable of forming a bond to the molecule with minimal assembly and / or internal cross-linking.

Examples of the fluorescent substance for diagnosis and detection include fluorescent compounds such as rhodamine, Alexa derivatives, cyanine derivatives, FAM, TAMRA, FITC, PE, PerCP, APC, coumarin or derivatives thereof, or GFP, eGFP, CFP, eCFP, YFP , RFP, and the like. And preferably a cyanine derivative such as Cy3, Cy3.5, Cy5, Cy5.5 and Cy7. The fluorescent substance can be bound to the nanoparticles of the present invention directly or through a linker.

In particular, useful metal-chelate combinations include diagnostic isotopes and 2-benzyl-DTPA and monomethyl and cyclohexyl analogs thereof used in the general energy range of 60 to 4,000 keV, and include, for example, imaging agents and / is a radioactive isotope is used as ^{125 I, 131 I, 123 I} , 124 I, 62 Cu, 64 Cu, 67 Cu, 186 Re, 188 Re, 82 Rb, 177 Lu, 18 F, 153 Sm, 213 Bi, ¹¹¹ In, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁹ Sr, ¹⁶⁹ Er, ¹⁹² Ir, ¹¹¹ In ,, ⁹⁰ Y, ⁹⁹ mTc, ⁹⁴ mTc, ¹¹ C, ¹³ N, ¹⁵ O, ⁷⁶ Br. Representative transition metal ions such as manganese (Mn), iron (Fe), and gadolinium (Gd) in non-radioactive metals are useful as MRIs as paramagnetic materials. However, since the ions and the radioactive isotopes are highly toxic, they can be used in combination with a chelating agent or the like. The chelating agent may be complexed with a macrocyclic chelating agent such as DTPA, NOTA, DOTA, MS325, HPDO3A, EDTA, NTA and TETA depending on the kind of metal, And the like. Preferably a radionuclide of gallium, yttrium and copper, respectively, and the metal-chelate complex can be prepared very steadily by aligning the ring size with the metal of the object. Ring-shaped chelates such as macrocyclic polyethers useful for stable binding with nuclides such as ²²³ Ra used in RAIT (radiation and imaging technology) may also be included in the scope of the present invention.

Preferably, the use of the nanoparticles comprising muteins substituted or inserted with cysteine in the natural type ferritin of the present invention can facilitate the introduction of a drug including the metal element as a diagnostic and / or therapeutic agent. As described above, cysteine itself has a high affinity for heavy metals, and further includes a thiol group having high reactivity, so that it can be easily coupled with other reactors, thereby facilitating introduction of a drug directly or via a linker . Therefore, if a chelating agent that binds to the metal ions listed above is modified to include maleimide or acetamide capable of binding with the thiol group of cysteine, it can form a covalent bond with the cysteine exposed to the inside / outside of the nanoparticle . Meanwhile, for the same reason as above, nanoparticles prepared using the cysteine-introduced mutein have not only metal elements but also other kinds of drugs such as antibodies, antibody fragments, drugs, toxins, nucleic acid hydrolases, (For example, biotin-streptavidin complex), a contrast agent, a fluorescent compound, or a fluorescent protein or the like, such as a therapeutic agent and a therapeutic agent such as a therapeutic agent, a modulator, a chelator, a boron compound, a photoactive agent or a dye, The delivery of the formulation / detection agent can also be facilitated.

Immunoconjugates are conjugates of therapeutic or diagnostic agents and antibody components. The diagnostic agent may comprise a radioactive or non-radioactive label, a contrast agent (magnetic resonance imaging, computed tomography, or a contrast agent suitable for ultrasound), and the radioactive label may be gamma-, beta-, alpha-, It may be a positron emission isotope.

An immunomodulator is a therapeutic agent as defined herein and is typically an immune response cascade, such as macrophage, B-cell, and / or T-cell, that is proliferating or activating in the immune response cascade Stimulate the cells. An example of such an immunomodulator is cytokine. Those skilled in the art will understand that interleukins and interferons are a type of cytokine that stimulates T-cell or other immune cell activity.

Protein chip and antigen detection method

One aspect of the present invention provides a protein chip comprising nanoparticles for immobilizing antibodies according to the present invention. At this time, the antibody may be bound to the antibody-binding peptide in the nanoparticles.

According to another aspect of the present invention, there is provided a method for preparing a protein chip, Immobilizing an antibody against an antigen to be detected on the protein chip; Reacting a sample with a protein chip comprising the immobilized antibody; And measuring an antigen-antibody reaction.

In the protein chip of the present invention, the nanoparticles for immobilizing antibodies of the present invention may be immobilized on a solid substrate. The solid substrate is not limited as long as it is used in a biochip. Preferably, however, commonly used polymers or gels such as gold, glass, modified silicone, tetrafluoroethylene, polystyrene, and polypropylene can be used. The surface of the substrate may also be surface treated with polymers, plastics, resins, carbohydrates, silicas, silica inducers, carbon, metals, inorganic glass and membranes. The substrate not only serves as a support but also provides a place where a binding reaction between the immobilized antibody and the antigen takes place. The size of the substrate and the position, size, and shape fixed on the substrate can be changed according to the purpose of the analysis, a spotting machine, a scanner, and the like.

In the present invention, a method known in the art can be applied to the method for detecting an antigen, in addition to the use of the protein chip of the present invention. More specifically, the immobilized antibody on the protein chip of the present invention is collected from a sample, The antigen can be detected by reacting with a blood or a body fluid to confirm whether or not the antigen-antibody reaction has occurred. In order to confirm whether the antigen-antibody reaction has occurred, different coloring materials may be used depending on the type of antibody, and at the same time, various kinds of antigens may be searched using fluorescence of various colors.

The antigen to be detected may be any bioactive material that can be detected by an immunoassay, for example, an autoantibody, a ligand, a natural extract, a peptide, a protein, a metal ion, , Natural drugs, metabolites, dielectrics, viruses and viruses, and bacteria and viruses, but not always limited thereto. The sample to be tested for the presence or absence of such an antigen or its concentration may be used without any treatment or diluted with a suitable buffer solution. After incubating for a suitable period of time, the target substance which has not been bound to the capture antibody and other substances in the sample which are not capable of binding can be further washed and washed before the next step.

The present invention relates to a method for producing a fusion protein by introducing a sequence of an antibody binding peptide into ferritin, and using the antibody to bind the ferritin to the ferritin without damaging the structure of the antibody, the biochip, drug delivery system, Can be utilized.

1 is a diagram showing a conceptual diagram for explaining the present invention.
2 is a diagram showing the structure of microorganism-derived ferritin rotated by 90 °.
FIG. 3 is a schematic diagram showing a ferritin-antibody-binding peptide fusion protein and a process for producing nanoparticles formed therefrom.
FIG. 4 is a chart of the purification of ferritin using size-exclusion chromatography. FIG.
FIG. 5 is a transmission electron microscope (TEM) analysis result for confirming the morphology of the spherical ferritin-FcBP complex.
FIG. 6 is a result of measurement of binding of ferritin-antibody-binding peptide complex and antibody using SPR (surface plasmon resonance). FIG.
FIG. 7 is a measurement result of the binding of a ferritin-antibody-binding peptide complex and an antibody using QCM (quartz crystal microbalance).
FIG. 8 is a fluorescence microscope photograph showing the results of treatment of Herceptin-CY3 and human IgG-CY3 against SKBR3 (HER2 / neu +) and MCF10A according to Example 4-1.
9 is a fluorescence image of a ferritin-FcBP-Herceptin complex applied to SKBR3 (HER2 / neu +) according to Example 4-2.
10 is a fluorescence image of a ferritin-FcBP-Herceptin complex applied to MCF10A (HER2 / neu-) according to Example 4-3.
FIG. 11 shows the result of a targeting experiment in which conditions for mixing ferritin-FcBP and Herceptin were changed in order to establish the stabilization condition of the ferritin-FcBP-Herceptin complex. The newly applied ferritin-FcBP-Herceptin complex in SKBR3 (HER2 / neu +) showed a decrease in non-selective protein precipitation compared to the previous experiment.
12 is a structural diagram of human-derived ferritin.
Fig. 13 shows the results of a 15% SDS-PAGE gel confirming overexpression and purification of human-derived ferritin into which an antibody-binding peptide has been introduced, according to Example 6. (lane 5: ferritin derived from the final purified human)
FIG. 14 shows the result of SPR analysis of the binding force between human-derived ferritin and human-derived antibody (herceptin, IgG1) into which an antibody-binding peptide has been introduced.
FIG. 15 is a diagram showing the result of purification and substitution of a cysteine residue in human-derived ferritin-FcBP into which an antibody-binding peptide has been introduced to introduce a specific ligand (drug or imaging contrast agent): (a) E61C b) E137C, (c) A102C and (d) S19C.
FIG. 16 is a diagram showing the result of overexpression of a cysteine residue added to microorganism-derived ferritin-FcBP into which an antibody-binding peptide has been introduced to introduce a specific ligand (drug or imaging contrast agent): (a) and Cysteine was introduced after the 161st and 174th amino acids in 164C and 177C, respectively, resulting in parityin containing cysteine residues at 164th and 177th amino acid sequences, and (c) This is the result for paritin into which cysteine is introduced at the second position of the amino acid sequence.
17 is a view showing an example of a chelate capable of binding with a metal ion including a terminal end of a reactive group capable of binding to cysteine introduced into ferritin-FcBP according to the present invention.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1: Antibody binding peptide-ferritin recombinant DNA productions methods and amino acid sequence analysis for fusion protein production

The recombinant DNA preparation method for producing antibody-bound peptide-ferritin fusion protein is as follows (FIG. 3).

Ferritin derived from Pyrococcus furiosus (SEQ ID NO: 1) was used in the modeling step. The ferritin gene derived from the microorganism was amplified with primers P1 (5'-aaaacatatgttgagcgaaagaatgctcaaggc-3 ') and P2 (SEQ ID NO: 3; 5'-aaaaggatccttactctcctccctgc-3') and then ligated with vector pET 30 (b). The cloned ferritin gene was identified by sequencing.

An antibody binding peptide sequence (SEQ ID NO: 4) was introduced with linker amino acids between positions 144 and 145 of the ferritin protein.

SEQ ID NO: 4: CGGGGGG DCAWHLGELVWCT GGGGGAS

The underlined portion is an antibody binding peptide (FcBP), and the sequence at the end of FcBP is a linker.

(5'-aaactagtctggtggcccggtggtggtggtggtgctagcgacagtcctcaaatattgttc-3 '; 5'-aaactagttctcccaggtgccatgcacagtcaccaccaccaccaccacctggcaaactttaacttgtcc-3') of SEQ ID NO: 13 and P4 of SEQ ID NO: 14 to insert the DNA sequence encoding the FcBP sequence into the gene of ferritin derived from microorganism After amplification, restriction enzyme SpeI was treated and circular plasmid DNA was obtained through the splicing junction. The insertion of the DNA sequence encoding the FcBP sequence was confirmed by DNA sequencing.

E. coli (Rosetta DE3, Novagen) was transformed with plasmid DNA corresponding to ferritin-FcBP. IPTG (Isopropyl-β-D-thio-galactoside) was added to 1 mM and incubated at 18 ° C. for 16 hours Expression of the protein was induced.

Example  2: Ferritin- FcBP Purification and characterization of

Ferritin-FcBP-expressing E. coli was suspended in cell lysate (50 mM Tris pH 7.5, 250 mM NaCl, 5% glycerol, 0.5% 2-mercaptoethanol) and disrupted with an ultrasonic generator. This was centrifuged and only the supernatant was heated at 100 ° C for 30 minutes and centrifuged to separate the supernatant again. The separated supernatant was passed through a Superose-6 (GE Healthcare) column using 50 mM Na ₂ HPO _4, pH 6.5 and 100 mM NaCl to purify the final ferritin-FcBP protein. The purified protein was analyzed by SDS-PAGE analysis for the size of the protein monomer and the purity of the purified protein.

Purified ferritin-FcBP had a structure of aggregation of 24 monomers. The size of the complex was confirmed by confirming the elution time of the protein in Superose-6 (FIG. 4) The shape of the ferritin-FcBP complex formed was confirmed (Fig. 5).

Example  3: ferritin-antibody binding Peptides  Measurement of antibody binding capacity of complex

Example 3-1: SPR using H IgG and PyFn - FcBP ( Surface Plasmon Resonance

BIAcore 3000 analyzer (BIACORE, Uppsala, Sweden) was used to measure the degree of cross-linking between H IgG and PyFn-FcBP by the SPR method. Biosensor analysis was performed using a CM5 sensor chip (BIACORE, Uppsala, Sweden) activated with EDC / NHS (N '- (3-dimethylaminopropyl) carbodiimide hydrochloride / N-hydroxysuccinimide) at a flow rate of 10 μl / / Ml in 10 mM sodium acdetate, pH 4.5) for 7 minutes. The remaining active area on the surface of the sensor chip was deactivated by the addition of 1.0 M ethanolamine (pH 8.0).

PyFn-FcBP was diluted in buffer solution (1 × PBS, pH 7.4) at each concentration and flowed at a flow rate of 30 μl / min to confirm the binding sensor and the dissociation sensorgram (FIG. 6). In order to correct the sensorgram caused by nonspecific binding with the sensor chip, a completely deactivated cell was used only with BSA (bovine serum albumin) and the non-specific binding was corrected by subtracting the sensor gram of BSA from the sensorgram of PyFn-FcBP . The surface of the sensor chip was regenerated by flowing regeneration buffer (20 mM NaOH).

Example  3-2: R IgG   And PyFn - FcBP Using SPR  Measurement method

BIAcore 3000 analyzer (BIACORE, Uppsala, Sweden) was used as in Example 3-1 to measure the degree of mutual bonding between R IgG and PyFn-FcBP by the SPR method.

(20 μg / ㎖ in 10 (10 μg / ml) in a CM5 sensor chip (BIACORE, Uppsala, Sweden) activated with EDC / NHS mM sodium acdetate, pH 4.5) for 7 minutes. The remaining active area on the surface of the sensor chip was deactivated by the addition of 1.0 M ethanolamine (pH 8.0).

PyFn-FcBP was diluted in buffer solution (1 × PBS, pH 7.4) at each concentration, and flowed at a flow rate of 30 μl / min, and a binding sensorgram and a dissociation sensorgram were confirmed (FIG. In order to correct the sensorgram caused by nonspecific coupling with the sensor chip, the cell completely deactivated by BSA was used and the non-specific binding was corrected by subtracting the sensor gram of BSA from the sensorgram of PyFn-FcBP. The surface of the sensor chip was regenerated by flowing a regeneration buffer solution (20 mM NaOH).

Example 3-3: Antibody (IgG H) by SPR measurement and protein PyFn - FcBP Interprotein Interaction Measurement

To confirm whether the SPR method of Example 3-1 selectively measured the binding of the FcBP portion of the PyFn-FcBP protein to the antibody Fc domain, the antibody protein immobilized on the CM5 sensor chip was added with PyFn-FcBP protein Were measured at 500, 250, 100, 50, 25, 10, and 5 nM concentrations. As a result, as shown in FIG. 6, the PyFn-FcBP protein shows a sensorgram specifically binding to the antibody Fc domain, and the increase of the RU value according to the amount of PyFn-FcBP bound is increased in proportion to the concentration of PyFn-FcBP . These results show that the increase curve of the binding sensorgram of the above SPR measurement is due to the interaction between the H IgG Fc domain and the PyFn-FcBP protein.

Example 3-4: Antibody by SPR measurements (R IgG) protein and PyFn - FcBP Interprotein Interaction Measurement

In order to confirm whether the SPR method of Example 3-2 selectively measured the binding of the FcBP portion of the PyFn-FcBP protein to the antibody Fc domain, the antibody protein immobilized on the CM5 sensor chip was added with PyFn-FcBP protein Were flowed at a concentration of 500, 250, 100, 50, 25, and 10 nM, and the binding sensorgrams were investigated. As a result, as shown in FIG. 7, the PyFn-FcBP protein showed a sensorgram specifically binding to the antibody Fc domain, and the increase of the RU value according to the amount of PyFn-FcBP bound was increased in proportion to the concentration of PyFn-FcBP . These results show that the increase curve of the binding sensorgram of the above SPR measurement is due to the interaction between the R IgG Fc domain and the PyFn-FcBP protein.

Example  4: ferritin-antibody binding Peptides  Identification of target orientation using complex

Example  4-1: Cell selection, antibody selection, initial experimental conditions

To confirm whether ferretin-FcBP selectively transfers Herceptin, an antibody treatment agent, to specific cells, SKBR3 (HER2 / neu +), a breast cancer cell overexpressing HER2 / neu receptor, was selected and HER2 / neu receptor MCF10A < / RTI > Herceptin is a commercially available breast cancer treatment, which is a monoclonal antibody that inhibits downstream signaling by binding to the HER2 / neu receptor and thus has anti-cancer effects. SKBR3 was cultured in Dulbecco's Modified Eagle's Medium (DMEM, Gibco) containing 10% FBS (fetal bovine serum) and 1% penicillin / streptomycin. MCF10A was cultured in DMEM / F12 (Gibco) medium containing 5% horse serum, 20 ng / ml EGF, 0.5 mg / ml hydrocortisone, 100 ng / ml cholera toxin, 10 μg / ml insulin and 1% penicillin / streptomycin Lt; / RTI >

Ferritin-FcBP, fluorescein-5-maleimide (Invitrogen), antibody Herceptin, and human IgG, CY3 (GE Healthcare) were used for ferritin-FcBP and ferritin-FcBP. The method of introducing the fluorescent substance is as follows. A fluorescent substance corresponding to about 10 times or more the number of moles of the protein was reacted at room temperature for 1 to 2 hours in the presence of phosphate buffer (pH 7.5 to 8.0). Excess fluorescent material not participating in the reaction was removed using PD10 (GE Healthcare), a desalting column, and only the protein with fluorescent substance was recovered. The recovered protein was quantitated by Bradford assay.

Prior to this experiment, Herceptin-CY3 and human IgG-CY3 were treated with SKBR3 (HER2 / neu +), and Herceptin-CY3 was bound to MCF10A. (Fig. 8). A fluorescence microscope, DeltaVisionRT (Applied Precision), was used to confirm binding.

Example  4-2: SKBR3 ( HER2 / neu +)

SKBR3 was cultured in DMEM (Gibco) medium containing 10% FBS and 1% penicillin / streptomycin. Herceptin-CY3, ferritin-FcBP-fluorescein-5-maleimide, human IgG-CY3 and ferritin-FcBP-fluorescein-5-maleimide were reacted for 15 minutes in the presence of DMEM to bind each antibody to ferritin. Two types of antibody-ferritin were treated with SKBR3, reacted for 30 minutes, washed with PBS, and fluorescent images were observed (FIG. 9). Fluorescence images showed that the fluorescence of CY3 and fluorescein was observed simultaneously in the samples treated with Herceptin-CY3 and ferritin-FcBP-fluorescein-5-maleimide. SKBR3 cells were targeted by Herceptin and the ferritin structure and Herceptin Respectively.

Example  4-3: MCF10A ( HER2 / neu -)

MCF10A was cultured in DMEM / F12 (Gibco) medium containing 5% horse serum, 20 ng / ml EGF, 0.5 mg / ml hydrocortisone, 100 ng / ml cholera toxin, 10 μg / ml insulin and 1% penicillin / streptomycin Lt; / RTI > Herceptin-CY3 and ferritin-FcBP were reacted with fluorescein-5-maleimide, human IgG-CY3 and ferritin-FcBP with fluorescein-5-maleimide in the presence of DMEM for 15 min to bind each antibody to ferritin. MCF10A was treated with two types of antibody-ferritin, reacted for 30 minutes, washed with PBS, and observed for fluorescence image (FIG. 10). At this time, no fluorescence was observed in the MCF10A cell line.

FIG. 11 is a fluorescence image showing the result of treatment with SKBR3 cell line after changing the binding conditions of ferritin-FcBP and Herceptin to solve the problem of protein precipitation as a result of the experiment in FIG. Ferritin-FcBP-fluorescein-5-maleimide was reacted for 15 minutes in the presence of DMEM containing 0.05% TWEEN 20 to allow each antibody to bind to ferritin. The antibody-ferritin was treated with SKBR3 and reacted for 30 minutes, followed by washing with PBS, and fluorescence images were observed. As a result, it was observed that excessive protein precipitation was reduced.

Example  5: Human-derived ferritin-antibody binding Peptides  Composite manufacturing

The human-derived ferritin genes (SEQ ID NOS: 5 and 6, Fig. 12) were distributed from the 21st Century Frontier Human Gene Bank (clone number: hMU001607, hMU011497). The human-derived ferritin gene was amplified using primers P1 (5'-gggaattc catatg agctcccagattcgtcagaat-3 ') and P2 (SEQ ID NO: 8; 5'-ccgctcgagttagtcgtgcttgagagtgagcct-3') and then digested with restriction enzymes NdeI, XhoI And cloned into vector pET 28 (a). The cloned ferritin gene was identified by sequencing.

An antibody binding peptide sequence was introduced between positions 157 and 158 of the ferritin protein. Directed mutagenesis (Invitrogen) using primer Q1 (SEQ ID NO: 9; 5'-ctccacaggctgggtggcgactgtgcatggcacctgggagaggctgggctggcgagtat-3 ') and Q2 (SEQ ID NO: 10; 5'-atactcgcccagcccagcctctcccaggtgccatgcacagtcgccacccagcctgtaggag-3' Respectively. Binding peptide is introduced by performing second site designation mutagenesis using Q3 (SEQ ID NO: 11; 5'-gactgtgcatggcacctgggagacggtctggtgcaccgaggctgggctggcgagtat-3 ') and Q4 (SEQ ID NO: 12; 5'-atactcgcccagcccagcctcggtgcaccagaccagttctcccaggtgccatgcacagtc-3' Ferritin gene was constructed. Plasmid DNA corresponding to human-derived ferritin-FcBP was transformed with Escherichia coli (Rosetta DE3, Novagen), IPTG (Isopropyl-β-D-thio-galactoside) was added to 1 mM and incubated at 18 ° C. for 16 hours To induce protein expression.

Example  6: Human derived ferritin- FcBP Purification and characterization of

Escherichia coli expressing human ferritin-FcBP was suspended in cell lysate (50 mM Tris pH 7.5, 250 mM NaCl, 5% glycerol, 0.5% β-mercaptoethanol) and disrupted with an ultrasonic generator. The supernatant was centrifuged and the supernatant was purified by a batch method using Talon resin (Clontech). Protein purification using the batch method will be described in detail below. After rinsing the membrane with 1 ml of a Talon resin (1 ml) with 10 ml of column buffer (50 mM Tris pH 7.5, 250 mM NaCl, 5% glycerol, 0.05% β-mercaptoethanol), the supernatant Slowly shake at 4 ° C for 1 hour to adsorb the protein to the talon resin. After removing the aqueous solution by filtration, the Talon resin was washed twice with column buffer (10 ml), 10 ml of elution buffer (100 mM imidazole) was added, and the mixture was slowly rocked for 1 hour Filtration was repeated one more time using 5 ml of elution buffer to increase the yield. Protein purified by the above method was quantitated by Bradford assay, and protein over-expression was estimated through a 15% SDS-PAGE gel to confirm the overexpression and purification of human-derived ferritin into which the antibody-binding peptide was introduced (Fig. 13).

Example  7: Person origin  Ferritin-antibody binding Peptides  Measurement of antibody binding capacity of complex

BIAcore 3000 analyzer (BIACORE, Uppsala, Sweden) was used to measure the degree of cross-linking between H IgG and human derived ferritin-FcBP by the SPR method. Biosensor analysis was performed using a CM5 sensor chip (BIACORE, Uppsala, Sweden) activated with EDC / NHS (N '- (3-dimethylaminopropyl) carbodiimide hydrochloride / N-hydroxysuccinimide) at a flow rate of 10 μl / / Ml in 10 mM sodium acdetate, pH 4.5) for 7 minutes. The remaining active area on the surface of the sensor chip was deactivated by the addition of 1.0 M ethanolamine (pH 8.0).

Human-derived ferritin-FcBP was diluted in buffer solution (1 × PBS, pH 7.4) at various concentrations, and flowed at a flow rate of 30 μL / min, and binding sensor and dissociation sensorgrams were confirmed (FIG. 14). In order to correct for sensorgrams caused by nonspecific binding with the sensor chip, cells completely deactivated with BSA were used and non-specific binding was corrected by subtracting the sensorgram of BSA from the sensorgram of human-derived ferritin-FcBP. The surface of the sensor chip was regenerated by flowing a regeneration buffer solution (20 mM NaOH).

Example  8: Person origin  Ferritin-antibody binding Peptides  To the complex was added cysteine ( cysteine ) Introduction of moiety

To introduce a cysteine residue at a specific position of human-derived ferritin (SEQ ID NO: 35) -FcBP, spot-directed mutagenesis was carried out using the following primers. SEQ ID NO: 36 is the amino acid sequence of the human-derived ferritin-antibody binding peptide complex, and includes the linker sequences (CGGGGGGD and TGGGGGAS) at both ends of the antibody binding peptide (CAWHLGELVWC). To introduce cysteine into the nanostructure, 61 E (Glu), 68 K (Lys), and 137 E (Glu) were replaced with cysteine. To introduce cysteine into the nanostructure, Ser), 19 (Ser), 102 (Ala), and 113 (D) were replaced with cysteine. The substituted sites are based on native type ferritin sequences that are not complexes with antibody binding peptides. The base sequences of the primers used are as follows.

E61C forward: 5'-gccgaggagaagcgctgcggctacgagcgtctcctg-3 ', E61C reverse (SEQ ID NO: 16): 5'-caggagacgctcgtagccgcagcgcttctcctcggc-3', K68C forward (SEQ ID NO: 17): 5'-ggctacgagcgtctcctgtgcatgcaaaaccaccgt-3 ', K68C reverse (SEQ ID NO: 18): 5'-acgctggttttgcatgcacaggagacgctcgtagcc-3 ', E137C forward (SEQ ID NO: 19): 5'-actcacttcctagattgcgaagtgaagcttatcaag-3', E137C reverse (SEQ ID NO: 20): 5'-cttgataagcttcacttcgcaatctaggaagtgagt- (SEQ ID NO: 23): 5'-gaggcagccgtcaactgcctggtcaatttgtacctg-3 ', S19C reverse (SEQ ID NO: 22): 5'-ggcagccatatgagctgccagattcgtcagaat- ): 5'-caggtacaaattgaccaggcagttgacggctgcctc-3 ', A102C forward (SEQ ID NO: 25): 5'-atgaaagctgccatgtgcctggagaaaaagctgaac-3', A102C reverse (SEQ ID NO: 26): 5'-gttcagctttttctccaggcacatggcagctttcat-3 ', D113C forward 5'-aaccaggcccttttgtgccttcatgccctgggttct-3 ', D113C reverse No. 28): 5'-agaacccagggcatgaaggcacaaaagggcctggtt-3 '.

PCR for the spot-directed mutagenesis was performed by denaturation at 95 ° C for 30 seconds, priming at 45 ° C for 1 minute, and elongation at 68 ° C for 12 minutes after initial denaturation at 95 ° C for 5 minutes The DNA was amplified by repeating 17-19 times, and DpnI, which is a restriction enzyme, was treated to remove the template DNA, followed by transformation into E. coli and confirmation of the DNA base sequence. Using this method, a human-derived ferritin-FcBP gene having cysteine inserted at various positions was prepared. Plasmid DNA corresponding to cysteine-inserted ferritin-FcBP was transformed with Escherichia coli (Rosetta DE3, Novagen), added with IPTG (Isopropyl-β-D-thio-galactoside) Or 37 < 0 > C for 3 hours to induce protein expression. In the case of E61C, E137C, S19C, A102C, and D113C, K68C and S2C were expressed in the form of insoluble protein when cultured at 18 ° C for 16 hours. (Table 2).

Example  9: Cysteine introduced Person origin  The ferritin- FcBP Purification of

Escherichia coli expressing human-derived ferritin-FcBP with cysteine prepared in Example 8 was suspended in a cell lysate (50 mM Tris pH 7.5, 250 mM NaCl, 5% glycerol, 0.5% beta -mercaptoethanol) , And disrupted with an ultrasonic generator. The supernatant was centrifuged and the supernatant was purified by a batch method using Talon resin (Clontech). Protein purification using the batch method will be described in detail below. After rinsing the activated talon resin (1 ml) twice with 10 ml of a column buffer (50 mM Tris pH 7.5, 250 mM NaCl, 5% glycerol, 0.05% beta-mercaptoethanol), the separated supernatant And the mixture was slowly shaken at 4 캜 for 1 hour to adsorb the protein to the talon resin. After removing the aqueous solution by filtration, the Talon resin was washed twice with column buffer (10 ml), 10 ml of elution buffer (100 mM imidazole) was added, and the mixture was slowly rocked for 1 hour. And 5 mL of elution buffer to repeat the procedure one more time to increase the yield. Protein purified by the above method was quantitated by Bradford assay, and protein over-expression was estimated through a 15% SDS-PAGE gel to confirm the overexpression and purification of human-derived ferritin into which the antibody-binding peptide was introduced (Fig. 15).

Example  10: Pyrococcus furiosus  Derived ferritin-antibody binding Peptides  Cysteine in the complex Residue  Introduction (microbial ferritin- FcBP )

Ferritin (SEQ ID NO: 37) -For the introduction of a cysteine residue at a specific position in FcBP, spot-directed mutagenesis was carried out using the following primers. SEQ ID NO: 38 is the amino acid sequence of the human-derived ferritin-antibody binding peptide complex, and includes the linker sequences (CGGGGGGD and TGGGGGAS) at both ends of the antibody binding peptide (CAWHLGELVWC). In order to introduce cysteine into the interior of the nanostructure, an amino acid sequence (GGC) containing at least one cysteine residue was inserted at positions 161 and 174, and in order to introduce cysteine to the outside of the nanostructure, Cysteine was inserted between amino acids. The inserted site is based on a native type ferritin sequence that is not a complex with an antibody binding peptide. The base sequences of the primers used are as follows.

164C (In) forward (SEQ ID NO: 29): 5'-gataaggagttgagtgcgggcggttgcagagctccaaagctccca-3 ', 164C (In) reverse (SEQ ID NO: 30): 5'-tgggagctttggagctctgcaaccgcccgcactcaactccttatc-3' 5'-taagaaggagatatacatatgtgcttgagcgaaagaatgctcaag-3 ', 2C (Ex) reverse 5'-gagctcgaattcggatccttagcaaccgccctctcctccctgcattaagag-3', 2C (Ex) forward (SEQ ID NO: 33): 5'-taagaaggagatatacatgggttgctgagatggggttgctaggatcgattcgagctc- (SEQ ID NO: 34): 5'-cttgagcattctttcgctcaagcacatatgtatatctccttctta-3 '.

Cysteine-inserted ferritin-FcBP gene was constructed by performing spot-directed mutagenesis. Plasmid DNA corresponding to cysteine-inserted ferritin-FcBP was transformed with E. coli (BL21 DE3, Novagen), IPTG (Isopropyl-β-D-thio-galactoside) For induction of protein expression. In the case of ferritin-FcBP, three kinds of mutants inserted at different positions of cysteine residues were prepared. These mutants were confirmed to be expressed as a water soluble protein when cultured at 18 ° C for 16 hours (FIG. 16) .

Example  11: Cysteine ferritin-antibody binding Peptides  In the complex Metal ligand  Introduction

Bacterial or human-derived protein nanoparticles were dissolved in oxygen-free 50 mM phosphate buffer (pH 7.5), treated with 10 mM TCEP for 30 minutes, and the excess organic reagent was removed with a PD10 column to recover the protein. To the recovered protein nanoparticle solution was added (S) -2- (4- (2-bromoacetamido) benzyl) -DOTA ((S) -2- ) -DOTA, diethylene triamine pentaacetic acid-maleimidoethylamide (DTPA-MEA), or diethylene triamine pentaacetic acid-bromoacetamide- bromoacetamidoethylamide (DTPA-BAEA) and the like were mixed and allowed to stand at room temperature for 10 hours or at 4 占 폚 for 24 hours. Excess of unreacted reagent was removed with a PD10 column to prepare protein nanoparticles having metal ion coordination ligands attached thereto. Ligand binding was confirmed by mass spectrometry.

Example  12: ferritin-antibody binding Peptides - Metal ligand  Introduction of metal ions into the complex

A metal ion corresponding to 1.1 equivalents of the ligand was added to the ferritin-antibody-bound peptide-metal ligand complex and used for imaging or therapy, or after purification with PD10 desalting column.

<110> Korea Research Institute of Bioscience and Biotechnology          UNIST Academy-Industry Research Corporation <120> Antibody binding peptide - Ferritin Fusion Protein and use          the <130> PA120875 / KR <150> KR 10-2011-0104339 <151> 2011-10-12 <160> 38 <170> Kopatentin 2.0 <210> 1 <211> 525 <212> DNA <213> Pyrococcus furiosus <220> <221> gene &Lt; 222 > (1) .. (525) <223> Ferritin <400> 1 atgttgagcg aaagaatgct caaggcttta aatgaccagc taaacaggga gctttattct 60 gcatatctat actttgccat ggctgcctac tttgaagatc ttggccttga aggtttcgcc 120 aactggatga aggctcaggc tgaagaagag attgggcatg cactgaggtt ctacaactac 180 atctacgatc gcaatggtag ggttgagctt gatgaaattc caaagcctcc aaaggagtgg 240 gagagcccat taaaagcttt tgaagctgct tacgagcatg agaaattcat atgcaagtcc 300 atatatgaat tggcagcttt agcagaggag gaaaaagatt actcgacgag ggcattctta 360 gagtggttta tcaacgagca ggttgaggaa gaggccagcg taaagaaaat actggacaag 420 ttaaagtttg ccaaggacag tcctcaaata ttgttcatgc ttgataagga gttgagtgcg 480 agagctccaa agctcccagg gctcttaatg cagggaggag agtaa 525 <210> 2 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer P1 for amplification of Pyrococcus furiosus-derived ferritin <400> 2 aaaacatatg ttgagcgaaa gaatgctcaa ggc 33 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer P2 for amplification of Pyrococcus furiosus-derived ferritin <400> 3 aaaaaggatc cttactctcc tccctgc 27 <210> 4 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> linker-FcBP-linker <400> 4 Cys Gly Gly Gly Gly Gly Gly Asp Cys Ala Trp His Leu Gly Glu Leu   1 5 10 15 Val Trp Cys Thr Gly Gly Gly Gly Gly Ala Ser              20 25 <210> 5 <211> 552 <212> DNA <213> human <220> <221> gene &Lt; 222 > (1) .. (552) <223> Ferritin 1 <400> 5 atgacgaccg cgtccacctc gcaggtgcgc cagaactacc accaggactc agaggccgcc 60 atcaaccgcc agatcaacct ggagctctac gcctcctacg tttacctgtc catgtcttac 120 tactttgacc gcgatgatgt ggctttgaag aactttgcca aatactttct tcaccaatct 180 catgaggaga gggaacatgc tgagaaactg atgaagctgc agaaccaacg aggtggccga 240 atcttccttc aggatatcaa gaaaccagac tgtgatgact gggagagcgg gctgaatgca 300 atggagtgtg cattacattt ggaaaaaaat gtgaatcagt cactactgga actgcacaaa 360 ctggccactg acaaaaatga cccccatttg tgtgacttca ttgagacaca ttacctgaat 420 gagcaggtga aagccatcaa agaattgggt gaccacgtga ccaacttgcg caagatggga 480 gcgcccgaat ctggcttggc ggaatatctc tttgacaagc acaccctggg agacagtgat 540 aatgaaagct aa 552 <210> 6 <211> 528 <212> DNA <213> human <220> <221> gene &Lt; 222 > (1) .. (528) <223> Ferritin 2 <400> 6 atgagctccc agattcgtca gaattattcc accgacgtgg aggcagccgt caacagcctg 60 gtcaatttgt acctgcaggc ctcctacacc tacctctctc tgggcttcta tttcgaccgc 120 gatgatgtgg ctctggaagg cgtgagccac ttcttccgcg aactggccga ggagaagcgc 180 gagggctacg agcgtctcct gaagatgcaa aaccagcgtg gcggccgcgc tctcttccag 240 gacatcaaga agccagctga agatgagtgg ggtaaaaccc cagacgccat gaaagctgcc 300 atggccctgg agaaaaagct gaaccaggcc cttttggatc ttcatgccct gggttctgcc 360 cgcacggacc cccatctctg tgacttcctg gagactcact tcctagatga ggaagtgaag 420 cttatcaaga agatgggtga ccacctgacc aacctccaca ggctgggtgg cccggaggct 480 gggctgggcg agtatctctt cgaaaggctc actctcaagc acgactaa 528 <210> 7 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> primer P1 for amplification of human ferritin <400> 7 gggaattcca tatgagctcc cagattcgtc agaat 35 <210> 8 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer P2 for amplification of human ferritin <400> 8 ccgctcgagt tagtcgtgct tgagagtgag cct 33 <210> 9 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> primer Q1 for 1st site-directed mutagenesis <400> 9 ctccacaggc tgggtggcga ctgtgcatgg cacctgggag aggctgggct gggcgagtat 60                                                                           60 <210> 10 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> primer Q2 for 1st site-directed mutagenesis <400> 10 atactcgccc agcccagcct ctcccaggtg ccatgcacag tcgccaccca gcctgtggag 60                                                                           60 <210> 11 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> primer Q3 for 2nd site-directed mutagenesis <400> 11 gactgtgcat ggcacctggg agaactggtc tggtgcaccg aggctgggct gggcgagtat 60                                                                           60 <210> 12 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> primer Q4 for 2nd site-directed mutagenesis <400> 12 atactcgccc agcccagcct cggtgcacca gaccagttct cccaggtgcc atgcacagtc 60                                                                           60 <210> 13 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> primer P3 for FcBP insertion <400> 13 aaactagtct ggtgcaccgg tggtggtggt ggtgctagcg acagtcctca aatattgttc 60                                                                           60 <210> 14 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> primer P4 for FcBP insertion <400> 14 aaactagttc tcccaggtgc catgcacagt caccaccacc accaccaccc ttggcaaact 60 ttaacttgtc c 71 <210> 15 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> E61C forward <400> 15 gccgaggaga agcgctgcgg ctacgagcgt ctcctg 36 <210> 16 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> E61C reverse <400> 16 caggagacgc tcgtagccgc agcgcttctc ctcggc 36 <210> 17 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> K68C forward <400> 17 ggctacgagc gtctcctgtg catgcaaaac cagcgt 36 <210> 18 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> K68C reverse <400> 18 acgctggttt tgcatgcaca ggagacgctc gtagcc 36 <210> 19 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> E137C forward <400> 19 actcacttcc tagattgcga agtgaagctt atcaag 36 <210> 20 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> E137C reverse <400> 20 cttgataagc ttcacttcgc aatctaggaa gtgagt 36 <210> 21 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> S2C forward <400> 21 ggcagccata tgagctgcca gattcgtcag aat 33 <210> 22 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> S2C reverse <400> 22 attctgacga atctggcagc tcatatggct gcc 33 <210> 23 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> S19C forward <400> 23 gaggcagccg tcaactgcct ggtcaatttg tacctg 36 <210> 24 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> S19C reverse <400> 24 caggtacaaa ttgaccaggc agttgacggc tgcctc 36 <210> 25 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> A102C forward <400> 25 atgaaagctg ccatgtgcct ggagaaaaag ctgaac 36 <210> 26 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> A102C reverse <400> 26 gttcagcttt ttctccaggc acatggcagc tttcat 36 <210> 27 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> D113C forward <400> 27 aaccaggccc ttttgtgcct tcatgccctg ggttct 36 <210> 28 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> D113C reverse <400> 28 agaacccagg gcatgaaggc acaaaagggc ctggtt 36 <210> 29 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> 164C (In) forward <400> 29 gataaggagt tgagtgcggg cggttgcaga gctccaaagc tccca 45 <210> 30 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> 164C (In) reverse <400> 30 tgggagcttt ggagctctgc aaccgcccgc actcaactcc ttatc 45 <210> 31 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> 177C (In) forward <400> 31 ctcttaatgc agggaggaga gggcggttgc taaggatccg aattcgagct c 51 <210> 32 <211> 51 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > 177C (In) reverse <400> 32 gagctcgaat tcggatcctt agcaaccgcc ctctcctccc tgcattaaga g 51 <210> 33 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> 2C (Ex) forward <400> 33 taagaaggag atatacatat gtgcttgagc gaaagaatgc tcaag 45 <210> 34 <211> 45 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > 2C (Ex) reverse <400> 34 cttgagcatt ctttcgctca agcacatatg tatatctcct tctta 45 <210> 35 <211> 175 <212> PRT <213> human <220> <221> PEPTIDE <222> (1) (175) <223> Ferritin <400> 35 Met Ser Ser Gln Ile Arg Gln Asn Tyr Ser Thr Asp Val Glu Ala Ala   1 5 10 15 Val Asn Ser Leu Val Asn Leu Tyr Leu Gln Ala Ser Tyr Thr Tyr Leu              20 25 30 Ser Leu Gly Phe Tyr Phe Asp Arg Asp Asp Val Ala Leu Glu Gly Val          35 40 45 Ser His Phe Phe Arg Glu Leu Ala Glu Glu Lys Arg Glu Gly Tyr Glu      50 55 60 Arg Leu Leu Lys Met Gln Asn Gln Arg Gly Gly Arg Ala Leu Phe Gln  65 70 75 80 Asp Ile Lys Lys Pro Ala Glu Asp Glu Trp Gly Lys Thr Pro Asp Ala                  85 90 95 Met Lys Ala Met Ala Leu Glu Lys Lys Leu Asn Gln Ala Leu Leu             100 105 110 Asp Leu His Ala Leu Gly Ser Ala Arg Thr Asp Pro His Leu Cys Asp         115 120 125 Phe Leu Glu Thr His Phe Leu Asp Glu Glu Val Lys Leu Ile Lys Lys     130 135 140 Met Gly Asp His Leu Thr Asn Leu His Arg Leu Gly Gly Pro Glu Ala 145 150 155 160 Gly Leu Gly Glu Tyr Leu Phe Glu Arg Leu Thr Leu Lys His Asp                 165 170 175 <210> 36 <211> 197 <212> PRT <213> Artificial Sequence <220> <223> human ferritin-FcBP fusion protein <400> 36 Met Ser Ser Gln Ile Arg Gln Asn Tyr Ser Thr Asp Val Glu Ala Ala   1 5 10 15 Val Asn Ser Leu Val Asn Leu Tyr Leu Gln Ala Ser Tyr Thr Tyr Leu              20 25 30 Ser Leu Gly Phe Tyr Phe Asp Arg Asp Asp Val Ala Leu Glu Gly Val          35 40 45 Ser His Phe Phe Arg Glu Leu Ala Glu Glu Lys Arg Glu Gly Tyr Glu      50 55 60 Arg Leu Leu Lys Met Gln Asn Gln Arg Gly Gly Arg Ala Leu Phe Gln  65 70 75 80 Asp Ile Lys Lys Pro Ala Glu Asp Glu Trp Gly Lys Thr Pro Asp Ala                  85 90 95 Met Lys Ala Met Ala Leu Glu Lys Lys Leu Asn Gln Ala Leu Leu             100 105 110 Asp Leu His Ala Leu Gly Ser Ala Arg Thr Asp Pro His Leu Cys Asp         115 120 125 Phe Leu Glu Thr His Phe Leu Asp Glu Glu Val Lys Leu Ile Lys Lys     130 135 140 Met Gly Asp His Leu Thr Asn Leu His Arg Leu Gly Gly Gly Gly Gly Gly 145 150 155 160 Gly Gly Asp Cys Ala Trp His Leu Gly Glu Leu Val Trp Cys Thr Gly                 165 170 175 Gly Gly Gly Gly Glu Ala Gly Leu Gly Glu Tyr Leu Phe Glu Arg Leu             180 185 190 Thr Leu Lys His Asp         195 <210> 37 <211> 174 <212> PRT <213> Pyrococcus furiosus <220> <221> PEPTIDE <222> (1). (174) <223> Ferritin <400> 37 Met Leu Ser Glu Arg Met Leu Lys Ala Leu Asn Asp Gln Leu Asn Arg   1 5 10 15 Glu Leu Tyr Ser Ala Tyr Leu Tyr Phe Ala Met Ala Ala Tyr Phe Glu              20 25 30 Asp Leu Gly Leu Glu Gly Phe Ala Asn Trp Met Lys Ala Gln Ala Glu          35 40 45 Glu Glu Ile Gly His Ala Leu Arg Phe Tyr Asn Tyr Ile Tyr Asp Arg      50 55 60 Asn Gly Arg Val Glu Leu Asp Glu Ile Pro Lys Pro Pro Lys Glu Trp  65 70 75 80 Glu Ser Pro Leu Lys Ala Phe Glu Ala Ala Tyr Glu His Glu Lys Phe                  85 90 95 Ile Cys Lys Ser Ile Tyr Glu Leu Ala Ala Leu Ala Glu Glu Glu Glu Lys             100 105 110 Asp Tyr Ser Thr Arg Ala Phe Leu Glu Trp Phe Ile Asn Glu Gln Val         115 120 125 Glu Glu Glu Ala Ser Val Lys Lys Ile Leu Asp Lys Leu Lys Phe Ala     130 135 140 Lys Asp Ser Pro Gln Ile Leu Phe Met Leu Asp Lys Glu Leu Ser Ala 145 150 155 160 Arg Ala Pro Lys Leu Pro Gly Leu Leu Met Gln Gly Gly Glu                 165 170 <210> 38 <211> 201 <212> PRT <213> Artificial Sequence <220> <223> Pyrococcus furiosus ferritin-FcBP fusion protein <400> 38 Met Leu Ser Glu Arg Met Leu Lys Ala Leu Asn Asp Gln Leu Asn Arg   1 5 10 15 Glu Leu Tyr Ser Ala Tyr Leu Tyr Phe Ala Met Ala Ala Tyr Phe Glu              20 25 30 Asp Leu Gly Leu Glu Gly Phe Ala Asn Trp Met Lys Ala Gln Ala Glu          35 40 45 Glu Glu Ile Gly His Ala Leu Arg Phe Tyr Asn Tyr Ile Tyr Asp Arg      50 55 60 Asn Gly Arg Val Glu Leu Asp Glu Ile Pro Lys Pro Pro Lys Glu Trp  65 70 75 80 Glu Ser Pro Leu Lys Ala Phe Glu Ala Ala Tyr Glu His Glu Lys Phe                  85 90 95 Ile Cys Lys Ser Ile Tyr Glu Leu Ala Ala Leu Ala Glu Glu Glu Glu Lys             100 105 110 Asp Tyr Ser Thr Arg Ala Phe Leu Glu Trp Phe Ile Asn Glu Gln Val         115 120 125 Glu Glu Glu Ala Ser Val Lys Lys Ile Leu Asp Lys Leu Lys Phe Ala     130 135 140 Lys Cys Gly Gly Gly Gly Gly Gly Asp Cys Ala Trp His Leu Gly Glu 145 150 155 160 Leu Val Trp Cys Thr Gly Gly Gly Gly Gly Ala Ser Asp Ser Pro Gln                 165 170 175 Ile Leu Phe Met Leu Asp Lys Glu Leu Ser Ala Arg Ala Pro Lys Leu             180 185 190 Pro Gly Leu Leu Met Gln Gly Gly Glu         195 200

Claims (31)

A nanoparticle comprising 24 antibody-bound peptide-ferritin fusion protein monomers,
Six partial structures with a 4-fold symmetry point are formed by ferritin, and antibody-binding peptides are fused in the ± 10 aa (amino acid) range of the 4-fold symmetry point of ferritin, It is characterized by the presence of four antibody-binding peptides in the vicinity of the nanoparticle.
The method according to claim 1,
The ferrites that form the nanoparticles are homologous or heterogeneous.
The method according to claim 1,
Wherein the ferrites forming the nanoparticles are mutein wherein at least one amino acid residue in the native amino acid sequence is replaced by cysteine or further inserted with cysteine.
The method of claim 3,
Wherein the cysteine binds to the drug either directly or through a linker.
The method of claim 3,
The mutein is the second Ser (serine), the 19th Ser (serine), the 61st Glu (glutamic acid), the 68th Lys (lysine), the 102th Ala (alanine) of the native human-derived human ferritin amino acid sequence, Wherein at least one amino acid residue selected from the group consisting of Asp (aspartic acid) and 137th Glu (glutamic acid) is substituted with cysteine.
The method of claim 3,
The mutein is a natural type Pyrococcus furiosus Wherein the cysteine is inserted between the first and second positions of the ferritin amino acid sequence, or the GGC amino acid sequence is further inserted between positions 161 and 162 or after position 174 of the ferritin amino acid sequence.
The method of claim 3,
Wherein the mutein is at least one amino acid residue selected from the group consisting of a second Ser (serine), a 19th Ser (serine), a 102th Ala (alanine) and a 113th Asp (aspartic acid) of a native human-derived human ferritin amino acid sequence Cysteine, or a natural type Pyrococcus furiosus Cysteine is additionally inserted between the first and second amino acids of the ferritin amino acid sequence to contain a drug-binding cysteine exposed to the outer surface of the particle,
To 61st Glu (glutamic acid), 68 beonjjae Lys (lysine) and 137th Glu (glutamic acid) with one or more amino acid residue selected from the group consisting of ferritin amino acid sequence of natural-type human origin is substituted by cysteine, or native Pyrococcus wherein the GGC amino acid sequence is further inserted between positions 161 and 162 of the furiosus ferritin amino acid sequence, or after the 174th position, or both positions, and is exposed to the inner surface of the particle.
The method according to claim 1,
The nanoparticles characterized in that both ends of the antibody-binding peptide are fused independently or through a linker in the range of +/- 10 aa of the 4-fold symmetry point in the ferritin.
The method according to claim 1,
Wherein the antibody-binding peptide-ferritin fusion protein monomer is formed by self-assembly of 24 molecules.
The method according to claim 1,
Wherein the nanoparticles have a diameter of 1 to 20 nm.
11. The method according to any one of claims 1 to 10,
Wherein the antibody is bound to the antibody-binding peptide.
12. The method of claim 11,
The nanoparticle is characterized in that one or more antibodies are bound to four antibody binding peptides located near the four-fold symmetry point of one of the ferrites.
12. The method of claim 11,
Wherein the antibody is IgG.
An antibody-binding peptide-ferritin fusion protein in which ferritin is fused with antibody-binding peptides in the range of ± 10 aa of the 4-fold symmetry point of ferritin during the formation of nanoparticles by self-assembly of ferritin.
15. The method of claim 14,
Wherein the two ends of the antibody-binding peptide are fused independently or through a linker in the range of +/- 10 aa of the 4-fold symmetry point in the ferritin.
15. The method of claim 14,
A fusion protein characterized by being immobilized on an antibody.
15. A nucleic acid encoding the antibody-binding peptide-ferritin fusion protein according to claim 14 or 15.
17. A recombinant expression vector comprising the nucleic acid of claim 17.
A transformant transformed with the nucleic acid of claim 17 or a recombinant vector comprising said nucleic acid.
I) preparing a recombinant expression vector comprising the nucleic acid of claim 17;
(Ii) preparing a transformant transformed with the recombinant expression vector;
Iii) expressing the antibody-binding peptide-ferritin fusion protein from the transformant of step ii); And
iv) purifying the expressed antibody-binding peptide-ferritin fusion protein by self assembly to form six partial structures with a 4-fold symmetry point; Production method.
A drug delivery system characterized in that the drug is transported to the inside or the surface of the nanoparticle according to any one of claims 1 to 10.

22. The method of claim 21,
A drug delivery system capable of delivering a target-oriented drug by binding an antibody to an antibody-binding peptide in a nanoparticle.
22. The method of claim 21,
Wherein the drug is a therapeutic agent or a diagnostic / detection agent.
24. The method of claim 23,
Wherein said therapeutic agent is an antibody, antibody fragment, drug, toxin, nucleic acid hydrolase, hormone, immunomodulator, chelator, boron compound, photoactive agent, dye or radioactive isotope.
24. The method of claim 23,
Wherein the diagnostic / detection agent is a radioactive isotope, a dye, a contrast agent, a fluorescent protein, a fluorescent compound, or a magnetic resonance imaging (MRI) enhancer.
22. The method of claim 21,
Wherein the drug is enclosed within the nanoparticles, or is attached to the inner or outer surface of the nanoparticle, either directly or through a linker.
A protein chip comprising nanoparticles according to any one of claims 1 to 10.
28. The method of claim 27,
A protein chip in which an antibody is bound to an antibody-binding peptide in a nanoparticle.
27. A method for preparing a protein chip, comprising: preparing a protein chip of claim 27;
Immobilizing an antibody against an antigen to be detected on the protein chip;
Reacting a sample with a protein chip comprising the immobilized antibody; And
And determining whether the antigen-antibody reaction has occurred.
28. A diagnostic kit comprising the protein chip according to claim 27.
31. The method of claim 30,
A diagnostic kit in which an antibody is bound to an antibody-binding peptide in a nanoparticle.

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