KR20180008349A - A novel ferritin nanocage whose half life is extended and use thereof - Google Patents

Abstract

The present invention is to provide a fusion protein in which 2 to 12 peptides consisting of an amino acid sequence represented by sequence number 1 on C-terminal of self-assembly proteins are continuously connected or peptides consisting of amino acid sequences represented by sequence number 3 or 4 are connected to extend the half-life and improve tumor targeting ability of recombinant nanocage, and uses thereof.

Description

FIELD OF THE INVENTION [0001] The present invention relates to ferritin nanocage having increased half-life and a ferritin nanocage having half-

The present invention relates to a novel nanocage and its use, and more particularly to a novel ferritin nanocage having an increased half-life and a use thereof.

Protein-based nanocages are being developed in the field of nanomedicine because of their excellent structural and stability properties, high biocompatibility, low toxicity profile, and versatility for combining functional moieties through precise deformation Because. To date, modifications through genetic and chemical strategies have led to drug encapsulation, delivery to target tissues, and protein-based nanocage as well as the role of the carrier, as well as factors that may contribute to the pharmacologically active agent itself, such as release kinetics ). ≪ / RTI > This type of substance is representative of ferritin nanocage, and the naturally-derived material is the only platform for drug and vaccine delivery, diagnosis, biomineralization scaffolding and more. In addition, in addition to the features of protein-based nanocages, ferritin nanocages have the following desirable characteristics: (i) high production levels as soluble recombinant proteins in Escherichia coli ; (ii) uniform size and shape distribution at 10 nm scale; (iii) reversible degradation / assembly in a pH-dependent manner to encapsulate the drug in a nanocage core; (iv) eight hydrophilic channels that mediate the transfer of metal cations into and out of the cage.

To date, a number of studies have been carried out to genetically chemically bind dyes, peptides, proteins and antibody fragments and to bind a number of metals for Magnetic Resonance Imaging (MRI), Positron Emission Tomography (PET), optical imaging, drug delivery systems and pharmacologically active agents These features were used to encapsulate the drug. However, despite many promising features and applications in nanomaterials, ferritin nanocage has the problem of rapid degradation of all protein-based therapeutics and a short half-life. In general, ferritin is known to be rapidly removed from plasma after intravenous infusion in an animal model study. This reduces the residence time of the ferritin nanocage in the circulation as well as the high accumulation in the liver and the rapid elimination by the kidney as well as ferritin receptors present in many cell types. In particular, important challenges and limitations in clinical development of protein-based nanocages are in vivo stability, absorption, distribution, metabolism and excretion (ADME). Thus, recent studies have explored ways to overcome the short half-life of ferritin nanocage, and for example, PEGylation, a well-known method for extending half-life, has been applied to ferritin for tumor targeting. However, despite several beneficial properties, PEGylation has several disadvantages: (i) an additional chemical reaction is required for conjugation to the protein surface; (ii) it is difficult to produce a homogeneous product, resulting in a low yield and a high manufacturing cost. (iii) the biological function of the protein can be reduced during random PEGylation. For this reason, recently, the use of PAS (proline, alanine, serine) polypeptides to report the formation of PASylated ferritin nanocages has been reported and encapsulation of doxorubicin has shown a longer retention time of doxorubicin in the circulatory system, but an accurate analysis of the pharmacokinetics of nanocage (E. Falvo et al., Biomacromolecules 17, 514-522, 2016, G. Fracasso et al., J Control Release 239, 10-18, 2016).

Disclosure of Invention Technical Problem [8] The present invention has been made to solve the above-mentioned problems, and it is an object of the present invention to provide a novel nanocage having improved stability in blood, half-life and tumor targeting ability.

It is another object of the present invention to provide a novel cancer therapeutic agent using the novel nanocage. However, these problems are exemplary and do not limit the scope of the present invention.

According to one aspect of the present invention, there is provided a peptide comprising 2 to 12 peptides each consisting of the amino acid sequence of SEQ ID NO: 1 consecutively connected to the C-terminus of the self-assembled protein or comprising the amino acid sequence of SEQ ID NO: 2 to 4 A fusion protein is provided.

According to another aspect of the present invention, there is provided a pharmaceutical composition for treating cancer, wherein the fusion protein comprises a nanocage produced by self-assembly, as an active ingredient.

According to another aspect of the present invention, there is provided a nanocage in which the fusion protein is self-assembled.

According to another aspect of the present invention, there is provided a drug carrier comprising the nanocage and a drug to be delivered.

According to one embodiment of the present invention as described above, the effect of producing a novel nanocage and a drug delivery system containing the nanocage can be realized, as compared with the conventional nanocage, in which stability in blood, half-life, and tumor targeting ability are improved. Of course, the scope of the present invention is not limited by these effects.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram showing improved half-life and tumor targeting capacity in blood of recombinant nanocages (LCFNs) prepared in the present invention. FIG.
2 is a schematic diagram illustrating a 3D model of a series of LCFNs by computer simulation using a Modellar (v 9.2) program. XTEN (red) -fused human ferritin subunit (cyan) by flexible linker peptide was prepared based on the wtFN cage structure. (A) LCFN36; (B) LCFN72; (C) LCFN144; (D) LCFN288.
3 is a schematic diagram showing a 3D model of wtFNs and LCFNs presented with affibody by computer simulation using a Modellar (v 9.2) program.
Fig. 4 shows the structures of pT7-wtFN, pT7-LCFN36, pT7-LCFN72, pT7-LCFN144 and pT7-LCFN288, showing the structure of the fused expression vector for the production of recombinant wtFN and LCFNs.
5 is a photograph of a gel showing the recombinant nanocage purified through SDS-PAGE analysis. (A) wtFN, (B) LCFN36; (C) LCFN72; (D) LCFN144; (E) LCFN288.
6 is a transmission electron micrograph showing a uniform spherical cage structure of the purified nanocage. (A) wtFN, (B) LCFN36; (C) LCFN72; (D) LCFN144; (E) LCFN288.
Figure 7 is a graph showing the oligomeric state of recombinant wtFN and LCFNs analyzed by size exclusion chromatography (SEC).
8 is a graph showing the nano-scale size of the purified nanocage measured by dynamic light scattering analysis (DLS). (A) wtFN, (B) LCFN36; (C) LCFN72; (D) LCFN144; (E) LCFN288.
FIG. 9 is a pharmacokinetic analysis of the recombinant wtFN and LCFNs of the present invention, wherein the recombinant nanocage was administered intravenously to a mouse model, and sera was separated to perform Western blot analysis.
FIG. 10 is a graph showing blood plasma concentrations obtained by separating serum after administration of the recombinant wtFN and LCFNs of the present invention to a mouse model.
FIG. 11 is a graph showing fluorescence images (A) obtained by culturing Cy5.5-labeled recombinant nanocage and macrophages (BMDMs) and the degree of binding (B).
FIG. 12 is a graph showing quantitative analysis of fluorescence image (A) obtained by culturing Cy5.5-labeled recombinant nanocage and endothelial cells (HUVEC) and binding degree (B).
FIG. 13 is a photograph of the fluorescence intensities of in vivo near-infrared (NIR) fluorescence in time after injection of wtFN, Affibody-FN and Affibody-LCFN into a mouse model. In the photograph, the circle indicates the tumor site.
FIG. 14 is a graph showing the analysis of the near-infrared fluorescence intensity of the tumor region over time. FIG.
Figure 15 is a near-infrared fluorescence image of an incised major organs containing liver, lung, spleen, kidney, heart, and intestine following wt FN, Affibody-FN and Affibody-LCFN infusion in a mouse model.
FIG. 16 is a near-infrared fluorescence image of an incision tumor after injection of wt FN, Affibody-FN and Affibody-LCFN into a mouse model.
Fig. 17 is a graph showing quantitative analysis of near-infrared fluorescence intensity of a tumor excised from a mouse model. Fig.

Definition of Terms

As used herein, the term "self-assembled protein" refers to a protein capable of forming nanoparticles by forming multimers by regular arrangement at the same time as expression without the aid of a particular inducer it means. Self-assembling proteins include sHsp (small heat shock protein), ferritin, vault, P6HRC1-SAPN, M2e-SAPN, MPER-SAPN, and various virus or bacteriophage capsid proteins. The self-assembled proteins are well described in Hosseinkhani et al. (Chem. Rev., 113 (7): 4837-4861, 2013). This document is incorporated herein by reference in its entirety.

As used herein, the term " XTEN peptide "is an artificial peptide designed to improve the performance of proteins and peptides while avoiding many of the disadvantages of PEG. XTEN is a highly hydrophilic and unstructured protein consisting of only alanine, glutamine, glycine, proline, serine and threonine residues, and the recombinant fusion protein to which XTEN is added is known to enhance the in vivo half-life of non-fusion natural proteins (Schellenberger et al ., Nat. Biotechnol . 27: 1186-1990, 2009; Alters et al ., PloS One 7: e50630, 2012; Cleland et al ., J. Pharm. Sci . 101: 2744-2754, 2012; et al, PloS One 5:. e10175, 2010) has been made using the same protein drug development attempts. However, it is not known whether it is applicable to nanocage, a more complex protein complex, except in the case of a single protein.

As used herein, the term "ferritin" is a storage protein of iron in an animal, which is distributed in the liver, spleen, bone marrow and the like and has a molecular weight of 504 kDa. And ferritin is involved in the absorption and storage of iron in vivo.

As used herein, the term "ferritin heavy chain protein " (hereinafter abbreviated as FTH) refers to a protein that constitutes the heavy chain subunit of ferritin, a major intracellular iron storage protein in prokaryotes and eukaryotes , And the ferritin protein consists of 24 subunits of the ferritin heavy chain and the light chain, respectively. The main function of ferritin proteins is to store iron in a water-soluble, non-toxic state. The ferritin heavy chain protein has been known to self-assemble into 24 subunits without light chain protein to form hollow internal nanoparticles (Cho et al ., Biochem. Biophys. Res. Commun . 327 (2): 604-608, 2005) . The ferritin heavy chain protein can act as a nanocage by loading other drugs into the empty internal space of self-assembled nanoparticles, and due to these properties, is being studied for the purpose of drug delivery.

As used herein, the term "nanocage " refers to hollow nanoparticles, including inorganic nanocages and organic nanocages, where inorganic nanocages are formed from silver nanoparticles in boiling water Hollow gold nanoparticles produced by reaction with chloroauric acid (HAuCl ₄ ), and organic nanoparticles include protein nanocages such as ferritin self-assembled nanocages.

As used herein, the term "diabetes mellitus" is a type of metabolic disorder such as insufficient secretion of insulin or a failure to function normally. It is characterized by hyperglycemia in which blood glucose concentration is elevated. It causes symptoms and signs and excretes glucose from the urine.

The term " intrinsically disordered proteins " (IDPs), as used herein, refers to proteins that lack a fixed or trimeric three-dimensional structure, large multi-domain proteins linked by flexible linkers, random coils, and a partially structured state in a partially unstructured state. In the present invention, four XTEN peptides fused to the C-terminal of ferritin having a 4-3-2 axisymmetric structure form a cluster peptide bundle and are named 'IDP clouds', and 24 monomer subunits form a ferritin nanocage Thus, a total of six IDP clouds are present on the ferritin surface.

As used herein, the term " antibody mimetics ", as used herein, refers to an " antibody mimetic "in which two heavy and two light chains form a quaternary structure of a hetero- (Abbreviated as Fab, F (ab ') 2, Fab' or an artificial fragment in which a variable region of a heavy chain and a light chain is linked by a linker), which contains a minimum unit for maintaining antigen binding ability Antibody fragments (V _H H, V _NAR, etc.) derived from camel or cartilaginous fish composed of only a heavy chain without a light chain, such as a single-chain variable fragment (scFv) or Affilin, Affibody, Affimin, Affibin, Alphabody, Anticlin, Avimer, DARpin, Fynomoer, Kunitz domain peptide, , Nanobodies, monobodies, and variable lymphocyte receptors (VLRs) Like antibody analogs prepared from non-antibody-derived protein scaffolds. Unlike antibodies, single chain-based antibody mimetics include XTEN-ferritin, a constituent protein of the XTEN-nanocage, according to one embodiment of the present invention. By being operably linked, it can be used in the manufacture of nanocages with enhanced binding capacity to certain antigens and improved half-life.

As used herein, the term "affibody" is a type of artificial antibody that uses 58 amino acid residues, the IgG binding region, called the Z domain, of Staphylococcus aureus protein A (SPA). The epibadi is composed of alpha helices and lacks disulfide bonds. It is known that clonazepam is capable of specific binding to various antigens by randomly mutating 13 amino acids located in two alpha-helices associated with the binding capacity of the parent protein domain.

As used herein, the term "long circulating ferritin nanocages" (LCFNs) refers to a long-circulating ferritin nanocage with increased in vivo half-life, based on the crystal structure of the human ferritin heavy chain (2FHA) - refers to fused ferritin protein.

DETAILED DESCRIPTION OF THE INVENTION

According to one aspect of the present invention, there is provided a peptide comprising 2 to 12 peptides each consisting of the amino acid sequence of SEQ ID NO: 1 consecutively connected to the C-terminus of the self-assembled protein or comprising the amino acid sequence of SEQ ID NO: 2 to 4 A fusion protein is provided.

The ferritin may be a small heat shock protein (sHsp), ferritin, vault, P6HRC1-SAPN, M2e-SAPN, MPER-SAPN or a viral or bacteriophage capsid protein. Or a ferritin light chain protein.

In the fusion protein, the ferritin heavy chain protein may be composed of the amino acid sequence of any one of SEQ ID NOS: 5 to 16, wherein the virus or bacteriophage capsid protein is selected from the group consisting of bacteriophage MS2 capsid protein, bacteriophage P22 capsid protein, Qβ bacteriophage capsid protein, CCMV A capsid protein, a capsid protein, a CPMV capsid protein, an RCNMV capsid protein, an ASLV capsid protein, a HCRSV capsid protein, a HJCPV capsid protein, a BMV capsid protein, a SHIV capsid protein, an MPV capsid protein, an SV40 capsid protein, an HIV capsid protein, Protein, or a rotavirus VP6 protein.

In the fusion protein, a target protein to be delivered may be added to the N-terminus of the self-assembling protein or the C-terminus of the peptide, and the target protein is selected from the group consisting of Glucagon-like peptide-1 (GLP-1), EPO erythropoietin, human growth hormone, human colony stimulating factor (hCSF), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), interferon alpha, interferon beta, interferon gamma, interleukin 2, 11, factor I, factor II, factor III, factor IV, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, osteogenic protein 2, TGF-beta, or a single chain antibody analogue to a cancer antigen.

In the fusion protein, the single chain antibody analogue scFv, (scFv) _2, V _H H, V _NAR, Ke pilrin (Affilin), Ke Peabody (Affibody), Ke pimeo (Affimer), Ke pitin (Affitin), Alpha, alpha, alpha, alpha, alpha, beta, alpha, alpha, alpha, ), A variable lymphocyte receptor (VLR), a Minibody, a Diabody, a Tetrabody, a Triabody, or a Peptabody and the cancer antigen is AFP, CA-125, CEA, CA19-9, BCR- ABL fusion gene, BRCA1, hCG, HER2, or PSA.

According to another aspect of the present invention, there is provided a pharmaceutical composition for treating cancer, comprising a nanocage produced by self-assembly of a fusion protein as an active ingredient.

According to another aspect of the present invention, there is provided a nanocage in which the fusion protein is self-assembled.

According to another aspect of the present invention, there is provided a drug carrier comprising the nanocage and a drug to be delivered.

For example, anticancer drugs can be used for chemotherapy, and anti-inflammatory drugs such as steroids, COX2 inhibitors and TNF-alpha inhibitors can be used for the treatment of inflammatory diseases. A hypoglycemic agent such as DPP-4 inhibitor, insulin or GLP-1 may be used. In order to treat hypertension, a vasodilator may be used. For the treatment of cancer or infectious diseases, an immunosuppressive agent, Immunosuppressants may be used for the treatment of grafts, L-DOPA or dopamine agonists, MAO-B inhibitors may be used for the treatment of Parkinson's disease, and cholinesterase inhibitors may be used to improve cognitive performance in Alzheimer's patients , Blood coagulation factors may be used for the treatment of hemophilia patients, and for the treatment of various neurological diseases Channel blockers such as T-type calcium channel blockers or N-type calcium channel blockers may be used.

The fusion protein may include a linker peptide between the ferritin heavy chain protein and the Xten protein, wherein the linker peptide is (G4S) n, (GSSGGS) n, KESGSVSSEQLAQFRSLD (SEQ ID NO: 26), EGKSSGSGSESKST (EAAAK) n, CRRRRRREAEAC (SEQ ID NO: 29), A (EAAAK) ₄ ALEA (EAAAK) ₄ A (SEQ ID NO: 30), GGGGGGGG (SEQ ID NO: 31), GGGGGG (SEQ ID NO: 37), TRHRQPRGWE (SEQ ID NO: 38), AGNRVRRSVG (SEQ ID NO: 33), AEAAAKEAAAAKA (SEQ ID NO: 34), PAPAP (SEQ ID NO: 35), (Ala- Pro) n, VSQTSKLTRAETVFPDV (SEQ ID NO: 39), RRRRRRRR (SEQ ID NO: 40), GFLG (SEQ ID NO: 41), or GSSGGSGSSGGSGGGDEADGSRGSQKAGVDE (SEQ ID NO: 42).

In addition, for efficient purification of the fusion protein, a tag peptide for purification may be further added at the N-terminal or C-terminal. The tag peptide comprises a HisX6 peptide, a GST peptide, a FLAG peptide (DYKDDDK, SEQ ID NO: 44), a streptavidin binding peptide, a V5 epitope peptide (GKPIPNPLLGLDST, SEQ ID NO: 45), a Myc peptide (EQKLISEE, ), Or HA peptide (YPYDVPDYA, SEQ ID NO: 47).

The drug delivery or pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. The composition comprising a pharmaceutically acceptable carrier may be various oral or parenteral formulations, but is preferably a parenteral formulation. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, . In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The drug delivery or pharmaceutical composition may be administered orally or parenterally from the group consisting of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, And can have any one of the formulations selected.

The drug carrier or pharmaceutical composition of the present invention may be administered orally or parenterally. When administered parenterally, the drug carrier or pharmaceutical composition may be administered through various routes such as intravenous injection, intranasal inhalation, intramuscular injection, intraperitoneal administration, It is possible.

The drug delivery vehicle or pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount.

The term "pharmaceutically effective amount " as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will vary depending on the species and severity, age, sex, , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts. The pharmaceutical composition of the present invention may be administered at a dose of 0.1 mg / kg to 1 g / kg, more preferably at a dose of 1 mg / kg to 500 mg / kg. On the other hand, the dose can be appropriately adjusted according to the age, sex and condition of the patient.

The drug carrier or pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art.

Ferritin nanocages are not only easy to manipulate by genetic or chemical strategies, but also have properties such as uniform particle size distribution, ideal physical properties, high biocompatibility and low toxicity profiles, is a new platform for biomineralization scaffolds. However, short half-life remains a challenge for clinically applying ferritin-based nanomedicine. Accordingly, the present inventors have recognized the above problems and tried to propose a solution to prolong the half-life of the ferritin nanocage by preparing rationally designed long circulating ferritin nanocages (LCFNs) having Intrinsically Disordered Protein (IDP) ) Were developed. The half-life extended ferritin nanocage of the present invention was designed and constructed by 3D modeling prediction, and the pharmacokinetic parameters including half life, removal rate, and average residence time were evaluated through qualitative and quantitative analysis. LCFNs have a 10-fold increase in half-life and overall improved pharmacokinetic parameters compared to wild-type ferritin nanocages (wtFN), which is associated with lower binding capacity to bone marrow-derived macrophages (BMDMs) and endothelial cells . In addition, the tumor targeting moiety, epidermal growth factor receptor (EGFR) -targeted pebibody peptides, was fused to LCFNs for potential evaluation as a teraginous plasmid, and tumor targets -LCFNs were used for effective targeting through the active and passive traits, It has been successfully accumulated in tumor tissue by the shielding effect of my IDP. Thus, the strategy can be applied to other protein-based nanocapsules in the field of nanomedicine (FIG. 1).

Hereinafter, the present invention will be described in more detail by way of examples. It should be understood, however, that the invention is not limited to the disclosed embodiments, but may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, Is provided to fully inform the user.

Example 1: Modeling of LCFN fusion proteins

We used the MODELLER v9.12 program as the crystal structure of the human ferritin heavy chain (PDB 2FHA, SEQ ID NOS: 5 and 17) as the template and the structure of the subunits of long circulating ferritin nanocages (LCFNs) And the XTEN peptide structure was calculated using a PEPFOLD server (J. Maupetit et al., Nucleic Acids Res. 37 W498-W503. 2009). In addition, since the PEPFOLD server permits submission within 30 amino acids for de novo peptide structure calculations, the repeating unit of XTEN-36 (30 amino acids) was used, and the structure calculated using the MODELLER program was a long XTEN Peptides (SEQ ID NOS: 1-4). A total of 1,000 monomeric structures were then generated and assembled into LCFNs based on the wFt cage structure (PDB 3A68) using PyMOL, and the affibody-fusion wtFN and LCFNs structures were assembled with anti-EGFR affibody ) Structure (PDB 1H0T, SEQ ID NOS: 48 and 49), and the model with the lowest energy and lowest energy was selected.

Example 2: Preparation of LCFNs

For the production of wtFN and LCFNs based on the modeling data of the LCFN fusion protein, the following six gene clones were prepared by PCR using appropriate primers (Figure 2):

i) N- Nde I-6x Histidine tag- (hFH) - Hind III -C;

Ⅱ) N- Nde I-6x Histidine tag- (hFH) - Xho I-linker- Bam HI- (XTEN36) - Hind Ⅲ-C;

Ⅲ) N- Nde I-6x Histidine tag- (hFH) - Xho I-linker- Bam HI- (XTEN72) - Hind Ⅲ-C;

Ⅳ) N- Nde I-6x Histidine tag- (hFH) - Xho I-linker- Bam HI- (XTEN144) - Hind Ⅲ-C;

v) N- Nde I-6x Histidine tag- (hFH) - Xho I-linker- Bam HI- (XTEN288) - Hind Ⅲ-C.

In addition, two additional gene clones were prepared to produce the nanocage containing the peptibody peptide (Figure 3):

vi) N- Nde I-6x Histidine tag- (hFH) - Bam HI-Affibody-Xho I-C;

vⅱ) N- Nde I-6x Histidine tag- (hFH) - Xho I-linker- Bam HI- (XTEN144) - Hind Ⅲ- Affibody- Cla I-C.

The hFH gene was cloned using a cDNA clone, and the affibody gene specifically binding to the XTEN gene and EGFR was provided through the gene synthesis service of Cosmogenetech (Seoul, Korea), and a glycine-rich linker (SEQ ID NO: 18 ) Were cloned using an overlap and overlap PCR method. Then, the gene clone was ligated with pT7-7 plasmid (pT7-wtFN, pT7-LCFN36, pT7-LCFN72, pT7-LCFN144, pT7-LCFN288, pT7-FN- PT7-LCFN144-Affibody encoding the synthesis of recombinant LCFNs). Subsequently, after completion of the sequencing, the expression vector was transformed into E. coli (BL21 (DE3) [F-ompThsdSB (rB-mB-)]) with an ampicillin resistant selection marker (Fig. The information on the primers used in the above cloning is shown in Table 1 below.

Classification primer The nucleic acid sequence (5 '-> 3') order
number hFH




(NdeI) H6 hFH (HindIII) F CATATGCATCACCATCACCATCACGACCGCGTCCACCTC 50 (NdeI) H6 hFH (HindIII) R AAGCTTTTAGCTTTCATTATCACTGTC 51 (NdeI) H6 hFH (XhoI) F CATATGCATCACCATCACCATCACGACCGCGTCCACCTC 52 (NdeI) H6 hFH (XhoI) R CTCGAGGCTTTCATTATCACTGTC 53 (NdeI) H6 hFH (BamHI) F CATATGCATCACCATCACCATCACGACCGCGTCCACCTC 54 (NdeI) H6 hFH (BamHI) R GGATCCGCTTTCATTATCACTGTC 55 XTEN






XTEN36 F GGATCCGAGGGTGGTAGCGAAGGCTCTGAAGGTGAGGGTAGC 56 XTEN36 R AAGCTTTTAACCCTCACCAGAACCCTCAGAGCCCTCACCTTCGGAACC 57 XTEN72 F GGATCCGAGGGTGGTAGCGAAGGCTCTGAAGGTGAG 58 XTEN72 R AAGCTTTTAGCCTTCGCCAGAGCCTTCGGAACCTTCACC 59 XTEN144 F GGATCCGGGGGCACCTCCACCCCT 60 XTEN144 R AAGCTTTTAGCCAGGGGCTGTGGAACT 61 XTEN288 F GGATCCGGCGGCACTTCT 62 XTEN288 R AAGCTTTTAACCACCGCC 63 Affibody




(BamHI) Affibody (XhoI) F GGATCCGTGGATAACAAATTTAAC 64 (BamHI) Affibody (XhoI) R CTCGAGTTAGTGGATAACAAATTT 65  (HindIII) Affibody (ClaI) F AAGCTTGTGGATAACAAAA 66  (HindIII) Affibody (ClaI) R ATCGATTTATTTCGGCGC 67  (BamHI) XTEN144 (HindIII) F GGATCCGGGGGCACCTCCACCCCT 68  (BamHI) XTEN144 (HindIII) R AAGCTTGCCAGGGGCTGTGGAACT 69

Example 3: Recombinant protein expression and purification

The present inventors synthesized recombinant nanocages (wtFN and LCFNs) in Escherichia coli and then analyzed the yield and purity using SDS-PAGE and confirmed protein multimer formation by fast protein liquid chromatography (FPLC). Then, the recombinant protein-transformed cells were cultured at 37 ° C in a 2xYT culture medium containing ampicillin until an OD600 of 0.6 was reached, and protein expression was induced for 12 hours after addition of 1 mM IPTG. After induction, cells were obtained by centrifugation and the pellet was resuspended in lysis buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 10 mM Imidazole, pH 8.0) and homogenized with an ultrasonic processor. The recombinant nanocage was then purified through a Ni-NTA chromatography step and the buffer was exchanged with PBS and further analyzed using SDS-PAGE.

As a result, the expression of purified recombinant nanocages (wtFN and LCFNs) was observed (Fig. 5) and their molecular weight was found to be greater than the calculated value based on the amino acid sequence (see Table 2).

wtFN LCFN36 LCFN72 LCFN144 LCFN288 Theoretical M.W. (kDa) 21.5 28.6 31.7 38.2 51.6 Experimental M.W. (kDa) ~ 21.5 ~ 33 ~ 37 ~ 58 ~ 90

Example 4: Transmission electron microscopy (TEM) analysis

Transmission electron microscopy (TEM) analysis of the recombinant nanocage LCFNs prepared in the present invention was carried out using a Tecnai transmission electron microscope (Tecnai TEM). LCFNs samples were analyzed on copper grids with a carbon film (Electron microscopy science, US) As a single drop and negatively stained using a uranyl acetate solution. The TEM images were then acquired using a CCD camera and FEI imaging software installed on Philips Tecnai 20.

As a result, the structure of LCFNs showed the formation of a cage structure with a uniform size distribution (Fig. 6). This result implies that ferritin monomer C-terminal fusion of long XTEN peptide with large hydraulic volume successfully forms spherical nanocage of uniform size.

Example 5 Size Exclusion Chromatography (SEC) and Dynamic Light Scattering (DLS) Analysis

The oligomeric state of the recombinant nanocage (LCFNs) prepared in the present invention was analyzed by size exclusion chromatography (SEC, Atka purifier 100) with a Superdex 200 10/300 GL column and elution of LCFN The elution profile was monitored after measuring the absorbance at 280 nm as compared to the wild type nanocage (wtFN). In addition, the hydrodynamic size of LCFNs and wtFN was analyzed by dynamic light scattering (DLS) analysis and the zeta potential was measured using a Zetasizer Nano ZS (Malvern Instruments, Ltd., UK).

As a result, the recombinant nanocages (LCFNs) of the present invention exhibited an elution profile of size exclusion chromatography (SEC) similar to wt FN that did not form multimers and residual oligomers (FIG. 7). As a result of the dynamic light scattering analysis (DLS), the size of the recombinant nanocage (LCFNs) prepared was slightly larger than that of the wild-type ferritin nanocage of about 15 nm to 11 nm, (Fig. 8). In addition, as shown in Table 3 below, the surface charge of LCFNs modified by a hydrophilic layer was measured neutral.

wtFN LCFN36 LCFN72 LCFN144 LCFN288 Zeta potential (mV) 9.1 ± 0.6 9.5 ± 0.1 8.3 ± 0.4 9.8 ± 0.3 8.3 ± 0.5

Example 6: Western blot analysis

All experiments on mouse models used in the present invention were carried out in compliance with the relevant laws and institutional guidelines of KIST (KIST), and the laboratory committee approved the experiments. In addition, for the pharmacokinetics analysis, the recombinant nanocage (3 mg / ml) of the present invention added to dPBS (100 μl) was added to the athymic nude of 7-week old (20 g, Orient, Seoul, Korea) The mice were anesthetized at a certain point of time after the injection, blood was collected using citric acid solution (3.8%, pH 7.2) as an anticoagulant, centrifuged for 10 minutes at 4 ° C and 13,000 rpm, It was left on ice until separation. Subsequently, the separated serum was diluted with dPBS and SDS-PAGE loading buffer for Western blot analysis, and the denatured sample was analyzed by electrophoresis and the resulting gel was transferred to a semi-wet nitrocellulose membrane And blocked with 5% skim milk of buffer (50 mM Tris-Cl, 150 mM NaCl, 0.1% Tween 20, pH 7.6). The membrane was then treated with anti-ferritin heavy chain primary antibody (Abcam, ab65080) and secondary anti-rabbit IgG (Sigma Aldrich A0545) and incubated with Clarity Western ECL substrate and images were obtained using Biorad Chemidoc .

As a result, wtFN was no longer detected after 6 hours, but LCFNs were detected up to 48 hours and the residence time of the recombinant nanocage in vivo was increased (FIG. 9).

Example 6: Pharmacokinetics Characterization

In order to quantify the pharmacokinetic properties of the recombinant nanocage prepared in the present invention, the N-hydroxysuccinimide (NHS) ester group of Cy5 dye (Lumiprobe) is reacted with the amine group of the lysine residues on the outer surface of the nanocage (amine groups). The nano-cage was mixed with Cy5-NHS at a molar ratio of 1:24 in 0.1 M sodium bicarbonate (pH 8.4) and incubated at 25 ° C for 1 hour. The separation of free Cy5 was carried out by chromatography (PD-10, GE Healthcare). Subsequently, the pharmacokinetic experiment was diluted in bicarbonate buffer (100 μl), and the recombinant nanocage (10 mg / ml) was intravenously injected through the tail vein of the mouse. Blood was collected at 0-48 hours after the injection and the serum was separated according to the above-mentioned method. The separated sera were analyzed in a volume of 50 mu l in a black 96-well microplate and luorescence detection was performed using a fluorescence microplate reader (Perkin Elmer EnVision 2103) at excitation and emission wavelengths of 620 and 665 nm, And the quantified serum concentration (mgml-1) of the nanocage calculated from standard curves was analyzed with WinNonlin software (version 5.0) using a compartment-independent model.

As a result, serum concentration of intravenously administered recombinant nanocage (LCFNs) in the mouse model was higher than wtFN over time and increased with the size of IDP clouds (FIG. 10). Further, as indicated for analysis of the pharmacokinetic profile of the wtFN and LCFNs Table 4, it showed that the half-life (t _1/2) of LCFNs is increased according to the magnitude of the IDP clouds especially inde a half-life of wtFN 1 sigan On the other hand, LCFN144 and LCFN288 exhibited a long half-life of about 10 times at 11.7 and 10.0 hours, respectively, and the clearance rate was also slow.

wtFN LCFN36 LCFN72 LCFN144 LCFN288 C _max ^a (ug.ml ^-1 ) 293.0 217.8 869.0 835.5 816.0 t _1/2 ^b (hr) 1.0 3.2 5.4 11.8 10.1 AUC ^c (mg.hr.ml ^-1 ) 0.2 3.2 4.7 7.2 6.5 AUC _inf ^d (mg.hr.ml ^-1 ) 0.2 0.7 4.7 7.5 6.7 MRT ^e (hr) 0.8 0.7 6.3 10.9 10.8 V _d ^f (ml.kg ^-1 ) 370.2 5.2 82.5 113.4 108.0 Cl ^g (ml.hr ^-1. Kg ^-1 ) 324.0 334.4 10.6 6.7 7.4

Example 7: Endothelial cell and macrophage binding assay

Bone marrow (BM) cells were isolated from Balb / c mice for endothelial cell and macrophage binding analysis of the recombinant nanocage of the present invention, and single cell suspensions were treated with macrophage colony stimulating factor (M-CSF ), 10% fetal bovine serum, L-glutamine penicillin, and streptomycin. BMDM was then cultured in fresh medium supplemented with M-CSF (10 ng / ml) and adherent cells were harvested on day 8. Human umbilical vein endothelial cells (HUVEC) used in the present invention were purchased from ATCC and cultured in endothelial basal culture medium. Meanwhile, the nanocage was labeled with Cy5.5 NHS dye (GE Healthcare) and the fluorescence intensity was measured using a fluorescence microplate reader. Then, to evaluate the cell binding of wtFN and LCFN144, macrophages or endothelial cells were labeled with 5M CellTracker Green and plated on 35 mm glass bottom plates. Subsequently, the macrophages (3.3 × 10 ⁶ ) or endothelial cells were incubated with 400 nM wtFN and LCFN 144, cultured at 4 ° C. for a predetermined period of time (15 minutes for endothelial cells and 1 hour for macrophages) (Nikon, UK) and the obtained images were analyzed using ImageJ software.

As a result, a remarkable decrease in recombinant nano-cage LCFN144 was observed in macrophages compared to wtFN, and image quantitative analysis showed that the binding to macrophages was reduced by 618 fold (Fig. 11). In addition, endothelial cells similarly showed a reduced binding of 332-fold compared to wtFN (FIG. 12). These results suggest that IDP clouds can reduce the elimination of recombinant nanocage by mainly responsible for the ferritin surface masking of cellular ferritin receptors.

Example 8: Analysis of tumor accumulation effect of LCFNs

To analyze the tumor accumulation characteristics of the epidermal growth factor receptor (EGFR) -targeted peptibody peptide fused to the C-terminal of the recombinant nanocage (LCFNs) reconstituted in Example 2, the eXplore Optix System In vivo biodistribution assays were performed using Cy5.5-labeled wtFN and LCFN144 with Advanced Research Technologies Inc., CA (n = 3) and the fluorescence intensities of the nanocage samples ) Were adjusted to the same values based on data acquired by a fluorimetric microplate reader. Subsequently, wt5N and LCFN144 labeled with Cy5.5 were intravenously injected intravenously into each 4T1 tumor-bearing nude mouse via tail vein and the in vivo system imaging of mice was performed using the eXplore Optix System (Advanced) Research Technologies Inc., CA) at various time points (0-24 hours). For quantitative analysis of tumor fluorescence intensity, the tumor's NIRF intensity (total photons per square centimeter per solid angle (p / s / cm ² / sr)] was calculated using the Analysis Workstation software (ART Advanced Research Technologies Inc) ROI) analysis. The mice were sacrificed at 24 hours after the injection, the tumors were excised and the main organs (liver, lung, spleen, kidney, heart, and intestine) were imaged using a KODAK Image Station (4000 MM, Kodak, New Haven, CT) And the quantitative fluorescence intensity was analyzed in the same manner as described above.

As a result, the NIR fluorescence intensity of the anti-EGFR epitope-LCFN was higher in the tumor-bearing mouse model than in the anti-EGFR afibody-FN and wtFN nanocage, over time 13). Quantitative analysis showed that Apibody-FN was 1.58 times higher than that of wtFN and 2.44 times higher than that of affibody-LCFN (Fig. 14). In addition, in vivo biodistribution imaging analysis and quantitative fluorescence intensity analysis of the tumor model and major organs of the mouse model were the same as above (Figs. 15 and 17). Thus, the above results suggest that recombinant nanocage (anti-EGFR apiabody-LCFN) is effectively accumulated in tumor tissue by effective shielding effect of IDP cloud mediator in effective targeting and in vivo delivery through active and passive methods .

In conclusion, nanocage prepared by fusing ferritin nanoparticles with XTEN peptide and afibody has improved half-life in blood and superior tumor targeting ability in vivo as compared with conventional nanocage, and thus can be used as an effective cancer therapeutic agent .

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

<110> Korea Institute of Science and Technology <120> A novel ferritin nanocage whose half life is extended and use          the <130> PD17-5509 <150> KR 10-2016-0090234 <151> 2016-07-15 <160> 69 <170> Kopatentin 2.0 <210> 1 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> xten 36 <400> 1 Glu Gly Gly Ser Glu Gly Ser Glu Gly Glu Gly Gly Ser Glu Gly Ser Gly   1 5 10 15 Glu Gly Gly Gly Ser Glu Gly Ser Glu Gly Glu Gly Ser Glu Gly              20 25 30 Ser Gly Glu Gly          35 <210> 2 <211> 72 <212> PRT <213> Artificial Sequence <220> <223> xten 72 <400> 2 Glu Gly Gly Ser Glu Gly Ser Glu Gly Glu Gly Gly Ser Glu Gly Ser Gly   1 5 10 15 Glu Gly Gly Gly Ser Glu Gly Ser Glu Gly Glu Gly Ser Glu Gly              20 25 30 Ser Gly Glu Gly Glu Gly Gly Ser Glu Gly Ser Glu Gly Glu Gly Gly          35 40 45 Ser Glu Gly Ser Glu Gly Glu Gly Gly Glu Gly Ser Gly Glu Gly Gly Glu      50 55 60 Gly Ser Glu Gly Ser Gly Glu Gly  65 70 <210> 3 <211> 144 <212> PRT <213> Artificial Sequence <220> <223> xten 144 <400> 3 Gly Thr Ser Thr Pro Gly Ser Ser Ser Ser Ser Ser Gly Thr Ser Ser   1 5 10 15 Ser Gly Glu Ser Ser Thr Ala Pro Gly Thr Ser Pro Ser Gly Glu Ser              20 25 30 Ser Thr Ala Pro Gly Ser Thr Ser Ser Thr Ala Glu Ser Pro Gly Pro          35 40 45 Gly Ser Thr Ser Glu Ser Ser Ser Ser Ser Ser Ser Ser Thr Ser Ser Ser Thr Ser      50 55 60 Ser Thr Ala Glu Ser Pro Gly Pro Gly Thr Ser Pro Ser Gly Glu Ser  65 70 75 80 Ser Thr Ala Pro Gly Thr Ser Thr Pro Glu Ser Gly Ser Ala Ser Pro                  85 90 95 Gly Ser Thr Ser Ser Thr Ala Glu Ser Pro Gly Pro Gly Thr Ser Pro             100 105 110 Ser Gly Glu Ser Ser Thr Ala Pro Gly Thr Ser Pro Ser Gly Glu Ser         115 120 125 Ser Thr Ala Pro Gly Thr Ser Pro Ser Gly Glu Ser Ser Thr Ala Pro     130 135 140 <210> 4 <211> 288 <212> PRT <213> Artificial Sequence <220> <223> xten 288 <400> 4 Gly Thr Ser Thr Pro Gly Ser Ser Ser Ser Ser Ser Gly Thr Ser Ser   1 5 10 15 Ser Gly Glu Ser Ser Thr Ala Pro Gly Thr Ser Pro Ser Gly Glu Ser              20 25 30 Ser Thr Ala Pro Gly Ser Thr Ser Ser Thr Ala Glu Ser Pro Gly Pro          35 40 45 Gly Ser Thr Ser Glu Ser Ser Ser Ser Ser Ser Ser Ser Thr Ser Ser Ser Thr Ser      50 55 60 Ser Thr Ala Glu Ser Pro Gly Pro Gly Thr Ser Pro Ser Gly Glu Ser  65 70 75 80 Ser Thr Ala Pro Gly Thr Ser Thr Pro Glu Ser Gly Ser Ala Ser Pro                  85 90 95 Gly Ser Thr Ser Ser Thr Ala Glu Ser Pro Gly Pro Gly Thr Ser Pro             100 105 110 Ser Gly Glu Ser Ser Thr Ala Pro Gly Thr Ser Pro Ser Gly Glu Ser         115 120 125 Ser Thr Ala Pro Gly Thr Ser Pro Ser Gly Glu Ser Ser Thr Ala Pro     130 135 140 Gly Thr Ser Thr Pro Gly Ser Ser Ser Ser Ser Ser Gly Thr Ser Ser 145 150 155 160 Ser Gly Glu Ser Ser Thr Ala Pro Gly Thr Ser Pro Ser Gly Glu Ser                 165 170 175 Ser Thr Ala Pro Gly Ser Thr Ser Ser Thr Ala Glu Ser Pro Gly Pro             180 185 190 Gly Ser Thr Ser Glu Ser Ser Ser Ser Ser Ser Ser Ser Thr Ser Ser Ser Thr Ser         195 200 205 Ser Thr Ala Glu Ser Pro Gly Pro Gly Thr Ser Pro Ser Gly Glu Ser     210 215 220 Ser Thr Ala Pro Gly Thr Ser Thr Pro Glu Ser Gly Ser Ala Ser Pro 225 230 235 240 Gly Ser Thr Ser Ser Thr Ala Glu Ser Pro Gly Pro Gly Thr Ser Pro                 245 250 255 Ser Gly Glu Ser Ser Thr Ala Pro Gly Thr Ser Pro Ser Gly Glu Ser             260 265 270 Ser Thr Ala Pro Gly Thr Ser Pro Ser Gly Glu Ser Ser Thr Ala Pro         275 280 285 <210> 5 <211> 182 <212> PRT <213> Homo sapiens <400> 5 Thr Thr Ser Ser Thr Ser Gln Val Arg Gln Asn Tyr His Gln Asp Ser   1 5 10 15 Glu Ala Ala Ile Asn Glu Ile Asn Leu Glu Leu Tyr Ala Ser Tyr              20 25 30 Val Tyr Leu Ser Met Ser Tyr Tyr Phe Asp Arg Asp Asp Val Ala Leu          35 40 45 Lys Asn Phe Ala Lys Tyr Phe Leu His Gln Ser His Glu Glu Arg Glu      50 55 60 His Ala Glu Lys Leu Met Lys Leu Gln Asn Gln Arg Gly Gly Arg Ile  65 70 75 80 Phe Leu Gln Asp Ile Lys Lys Pro Asp Cys Asp Asp Trp Glu Ser Gly                  85 90 95 Leu Asn Ala Met Glu Cys Ala Leu His Leu Glu Lys Asn Val Asn Gln             100 105 110 Ser Leu Leu Glu Leu His Lys Leu Ala Thr Asp Lys Asn Asp Pro His         115 120 125 Leu Cys Asp Phe Ile Glu Thr His Tyr Leu Asn Glu Gln Val Lys Ala     130 135 140 Ile Lys Glu Leu Gly Asp His Val Thr Asn Leu Arg Lys Met Gly Ala 145 150 155 160 Pro Glu Ser Gly Leu Ala Glu Tyr Leu Phe Asp Lys His Thr Leu Gly                 165 170 175 Asp Ser Asp Asn Glu Ser             180 <210> 6 <211> 181 <212> PRT <213> Mus musculus <400> 6 Thr Thr Ala Ser Pro Gl Gln Val Arg Gln Asn Tyr His Gln Asp Ala   1 5 10 15 Glu Ala Ala Ile Asn Glu Ile Asn Leu Glu Leu Tyr Ala Ser Tyr              20 25 30 Val Tyr Leu Ser Met Ser Cys Tyr Phe Asp Arg Asp Asp Val Ala Leu          35 40 45 Lys Asn Phe Ala Lys Tyr Phe Leu His Gln Ser His Glu Glu Arg Glu      50 55 60 His Ala Glu Lys Leu Met Lys Leu Gln Asn Gln Arg Gly Gly Arg Ile  65 70 75 80 Phe Leu Gln Asp Ile Lys Lys Pro Asp Arg Asp Asp Trp Glu Ser Gly                  85 90 95 Leu Asn Ala Met Glu Cys Ala Leu His Leu Glu Lys Ser Val Asn Gln             100 105 110 Ser Leu Leu Glu Leu His Lys Leu Ala Thr Asp Lys Asn Asp Pro His         115 120 125 Leu Cys Asp Phe Ile Glu Thr Tyr Tyr Leu Ser Glu Gln Val Lys Ser     130 135 140 Ile Lys Glu Leu Gly Asp His Val Thr Asn Leu Arg Lys Met Gly Ala 145 150 155 160 Pro Glu Ala Gly Met Ala Glu Tyr Leu Phe Asp Lys His Thr Leu Gly                 165 170 175 His Gly Asp Glu Ser             180 <210> 7 <211> 181 <212> PRT <213> Rattus norvegicus <400> 7 Thr Thr Ala Ser Pro Gl Gln Val Arg Gln Asn Tyr His Gln Asp Ala   1 5 10 15 Glu Ala Ala Ile Asn Glu Ile Asn Leu Glu Leu Tyr Ala Ser Tyr              20 25 30 Val Tyr Leu Ser Met Ser Cys Tyr Phe Asp Arg Asp Asp Val Ala Leu          35 40 45 Lys Asn Phe Ala Lys Tyr Phe Leu His Gln Ser His Glu Glu Arg Glu      50 55 60 His Ala Glu Lys Leu Met Lys Leu Gln Asn Gln Arg Gly Gly Arg Ile  65 70 75 80 Phe Leu Gln Asp Ile Lys Lys Pro Asp Arg Asp Asp Trp Glu Ser Gly                  85 90 95 Leu Asn Ala Met Glu Cys Ala Leu His Leu Glu Lys Ser Val Asn Gln             100 105 110 Ser Leu Leu Glu Leu His Lys Leu Ala Thr Asp Lys Asn Asp Pro His         115 120 125 Leu Cys Asp Phe Ile Glu Thr Tyr Tyr Leu Ser Glu Gln Val Lys Ser     130 135 140 Ile Lys Glu Leu Gly Asp His Val Thr Asn Leu Arg Lys Met Gly Ala 145 150 155 160 Pro Glu Ala Gly Met Ala Glu Tyr Leu Phe Asp Lys His Thr Leu Gly                 165 170 175 His Gly Asp Glu Ser             180 <210> 8 <211> 170 <212> PRT <213> Ovis aries <400> 8 Thr Thr Ala Ser Pro Gl Gln Val Arg Gln Asn Tyr His Gln Asp Ser   1 5 10 15 Glu Ala Ala Ile Asn Glu Ile Asn Leu Glu Leu Tyr Ala Ser Tyr              20 25 30 Val Tyr Leu Ser Met Ser Tyr Tyr Phe Asp Arg Asp Asp Val Ala Leu          35 40 45 Lys Asn Phe Ala Lys Tyr Phe Leu His Gln Ser His Glu Glu Arg Glu      50 55 60 His Ala Glu Arg Leu Met Lys Leu Gln Asn Gln Arg Gly Ala Arg Ile  65 70 75 80 Phe Leu Gln Asp Ile Lys Lys Pro Asp Arg Asp Asp Trp Glu Asn Gly                  85 90 95 Leu Asn Ala Met Glu Cys Ala Leu Cys Leu Glu Arg Ser Val Asn Gln             100 105 110 Ser Leu Leu Glu Leu His Lys Leu Ala Thr Glu Lys Asn Asp Pro His         115 120 125 Leu Cys Asp Phe Ile Glu Thr His Tyr Leu Asn Glu Gln Val Glu Ala     130 135 140 Ile Lys Glu Leu Gly Asp His Ile Thr Asn Leu Arg Lys Met Gly Ala 145 150 155 160 Leu Trp Ile Gly His Gly Arg Val Val Leu                 165 170 <210> 9 <211> 180 <212> PRT <213> Sus scrofa <400> 9 Thr Thr Ser Cys Ser Ser Gln Val Arg Gln Asn Tyr His Gln Asp Ser   1 5 10 15 Glu Ala Ala Ile Asn Glu Ile Asn Leu Glu Leu Tyr Ala Ser Tyr              20 25 30 Val Tyr Leu Ser Met Ser Tyr Tyr Phe Asp Arg Asp Asp Val Ala Leu          35 40 45 Lys Asn Phe Ala Lys Tyr Phe Leu His Gln Ser His Gly Gly Arg Gly      50 55 60 His Ala Glu Lys Leu Met Lys Leu Gln Thr Gln Arg Gly Ala Arg Ile  65 70 75 80 Phe Leu Gln Asp Ile Met Lys Pro Glu Arg Asp Asp Trp Glu Asn Gly                  85 90 95 Leu Thr Ala Met Glu Phe Ala Leu His Val Val Lys Asn Val Tyr Gln             100 105 110 Ser Leu Leu Glu Leu His Lys Leu Ala Thr Asp Lys Asn Asp Pro His         115 120 125 Leu Cys Asp Phe Ile Glu Thr His Tyr Leu His Glu Gln Val Lys Ala     130 135 140 Ile Lys Glu Leu Gly Asp His Ile Thr Asn Leu His Arg Met Gly Ala 145 150 155 160 Pro Glu Tyr Gly Met Ala Glu Tyr Leu Phe Asp Lys His Thr Leu Gly                 165 170 175 Ser Ser Glu Ser             180 <210> 10 <211> 185 <212> PRT <213> Cricetulus griseus <400> 10 Thr Thr Ala Leu Thr Thr Ala Ser Ser Ser Gln Val Arg Gln Asn   1 5 10 15 Tyr His Gln Asp Ser Glu Ala Ala Ile Asn Arg Gln Ile Asn Leu Glu              20 25 30 Leu Tyr Ala Ser Tyr Val Tyr Leu Ser Ser Ser Cys Tyr Phe Asp Arg          35 40 45 Asp Asp Val Ala Leu Lys Asn Phe Ala Lys Tyr Phe Leu His Gln Ser      50 55 60 His Glu Glu Arg Glu His Ala Glu Lys Leu Met Lys Leu Gln Asn Gln  65 70 75 80 Arg Gly Gly Arg Ile Phe Leu Gln Asp Ile Lys Lys Pro Asp Arg Asp                  85 90 95 Asp Trp Glu Ser Gly Leu Asn Ala Met Glu Cys Ala Leu His Leu Glu             100 105 110 Lys Ser Val Asn Gln Ser Leu Leu Glu Leu His Lys Leu Ala Thr Asp         115 120 125 Lys Asn Asp Pro His Leu Cys Asp Phe Ile Glu Thr His Tyr Leu Asn     130 135 140 Glu Gln Val Lys Ser Ile Lys Glu Leu Gly Asp His Val Thr Asn Leu 145 150 155 160 Arg Lys Met Gly Ala Pro Glu Ala Gly Met Ala Glu Tyr Leu Phe Asp                 165 170 175 Lys His Thr Leu Gly His Ser Glu Ser             180 185 <210> 11 <211> 182 <212> PRT <213> Macaca mulatta <400> 11 Thr Thr Ser Ser Thr Ser Gln Val Arg Gln Asn Tyr His Gln Asp Ser   1 5 10 15 Glu Ala Ala Ile Asn Glu Ile Asn Leu Glu Leu Tyr Ala Ser Tyr              20 25 30 Val Tyr Leu Ser Met Ser Tyr Tyr Phe Asp Arg Asp Asp Val Ala Leu          35 40 45 Lys Asn Phe Ala Lys Tyr Phe Leu His Gln Ser His Glu Glu Arg Glu      50 55 60 His Ala Glu Lys Leu Met Lys Leu Gln Asn Gln Arg Gly Gly Arg Ile  65 70 75 80 Phe Leu Gln Asp Ile Lys Lys Pro Asp Tyr Asp Asp Trp Glu Ser Gly                  85 90 95 Leu Asn Ala Met Glu Cys Ala Leu His Leu Glu Lys Asn Val Asn Gln             100 105 110 Ser Leu Leu Glu Leu His Lys Leu Ala Thr Asp Lys Asn Asp Pro His         115 120 125 Leu Cys Asp Phe Ile Glu Thr His Tyr Leu Asn Glu Gln Val Lys Ala     130 135 140 Ile Lys Glu Leu Gly Asp His Val Thr Asn Leu Arg Lys Met Gly Ala 145 150 155 160 Pro Glu Ser Gly Leu Ala Glu Tyr Leu Phe Asp Lys His Thr Leu Gly                 165 170 175 Asp Ser Asp Asn Glu Ser             180 <210> 12 <211> 185 <212> PRT <213> Bos taurus <400> 12 Glu Pro Glu Pro Pro Leu Asn Pro Pro Lys Pro Ile Arg Gln Asn Tyr   1 5 10 15 Cys Pro Lys Cys Glu Ala Thr Val Asn Ser His Ala Ala Leu Glu Phe              20 25 30 His Ala Ser Phe Gln Cys Leu Ala Met Ala Phe Tyr Leu Asp Cys Asp          35 40 45 Asp Met Ala Leu Lys His Phe Ser Arg Phe Phe Leu Leu Cys Ser His      50 55 60 Glu His Ser Glu Arg Ala Glu Asn Leu Leu Phe Leu Gln Asn Gln Arg  65 70 75 80 Gly Gly Arg Thr Cys Phe Leu Asp Ile Arg Lys Pro Glu Thr Gln Gln                  85 90 95 Arg Glu Ser Gly Leu Gln Ala Met Gln Asp Ile Leu His Leu Glu Lys             100 105 110 Cys Val Asn Gln Ser Leu Leu Asn Leu Tyr Gln Leu Ala Thr Asp Ser         115 120 125 Ser Asp Ala His Leu Cys His Phe Leu Glu Thr His His Leu Asp Gln     130 135 140 Gln Val Lys Phe Ile Lys Glu Leu Gly Tyr Val Ser Asn Leu Ser Asn 145 150 155 160 Val Glu Ser Leu Glu Gly Ser Leu Ala Glu Tyr Val Phe Asp Lys Leu                 165 170 175 Thr Leu Gly Asp Gly Asp Lys Asn Asp             180 185 <210> 13 <211> 182 <212> PRT <213> Gorilla gorilla <400> 13 Thr Thr Ser Ser Thr Ser Gln Val Arg Gln Asn Tyr His Gln Asp Ser   1 5 10 15 Glu Ala Ala Ile Asn Ser Glen Ile Asn Leu Glu Leu Tyr Ala Ser Tyr              20 25 30 Val Tyr Leu Ser Met Ser Tyr Tyr Phe Asp Arg Asp Asp Val Ala Leu          35 40 45 Lys Asn Phe Ala Lys Tyr Phe Leu His Gln Ser His Glu Glu Arg Glu      50 55 60 His Ala Glu Lys Leu Met Lys Leu Gln Asn Gln Gln Gly Gly Arg Ile  65 70 75 80 Phe Leu Gln Asp Ile Lys Lys Pro Asp Cys Asp Asp Trp Glu Ser Gly                  85 90 95 Leu Asn Ala Met Glu Cys Ala Leu His Leu Glu Lys Asn Val Asn Gln             100 105 110 Ser Leu Leu Glu Leu His Lys Leu Ala Thr Asp Lys Asn Asp Pro His         115 120 125 Leu Cys Asp Phe Ile Glu Thr His Tyr Leu Asn Glu Gln Val Lys Ala     130 135 140 Ile Lys Glu Leu Gly Asp His Val Thr Asn Leu Arg Lys Met Gly Ala 145 150 155 160 Pro Glu Ser Gly Leu Ala Glu Tyr Leu Phe Asp Lys His Thr Leu Gly                 165 170 175 Asp Ser Asp Asn Glu Ser             180 <210> 14 <211> 182 <212> PRT <213> Pan paniscus <400> 14 Thr Thr Ser Ser Thr Ser Gln Val Arg Gln Asn Tyr His Gln Asp Ser   1 5 10 15 Glu Ala Ala Ile Asn Glu Ile Asn Leu Glu Leu Tyr Ala Ser Tyr              20 25 30 Val Tyr Leu Ser Met Ser Tyr Tyr Phe Asp Arg Asp Asp Val Ala Leu          35 40 45 Lys Asn Phe Ala Lys Tyr Phe Leu His Gln Ser His Glu Glu Arg Glu      50 55 60 His Ala Glu Lys Leu Met Glu Leu Gln Asn Gln Leu Gly Gly Arg Ile  65 70 75 80 Phe Leu Gln Asp Ile Lys Lys Pro Asp Cys Asp Asp Trp Glu Ser Arg                  85 90 95 Leu Asn Ala Met Glu Cys Ala Leu His Leu Glu Lys Asn Val Asn Gln             100 105 110 Ser Leu Leu Glu Leu His Lys Leu Ala Thr Asp Lys Asn Asp Pro His         115 120 125 Leu Cys Asp Phe Ile Glu Thr His Tyr Leu Asn Glu Gln Val Lys Ala     130 135 140 Ile Lys Glu Leu Gly Asp Gln Val Thr Asn Leu Arg Lys Met Gly Ala 145 150 155 160 Pro Glu Ser Gly Leu Ala Glu Tyr Leu Phe Asp Lys His Thr Leu Gly                 165 170 175 Asp Ser Asp Asn Glu Ser             180 <210> 15 <211> 182 <212> PRT <213> Pan troglodytes <400> 15 Thr Thr Ser Ser Thr Ser Gln Val Arg Gln Asn Tyr His Gln Asp Ser   1 5 10 15 Glu Ala Ala Ile Asn Glu Ile Asn Leu Glu Leu Tyr Ala Ser Tyr              20 25 30 Val Tyr Leu Ser Met Ser Tyr Tyr Phe Asp Arg Asp Asp Val Ala Leu          35 40 45 Lys Asn Phe Ala Lys Tyr Phe Leu His Gln Ser His Glu Glu Arg Glu      50 55 60 His Ala Glu Lys Leu Met Lys Leu Gln Asn Gln Arg Gly Gly Arg Ile  65 70 75 80 Phe Leu Gln Asp Ile Lys Lys Pro Asp Cys Asp Asp Trp Glu Ser Gly                  85 90 95 Leu Asn Ala Met Glu Cys Ala Leu His Leu Glu Lys Asn Val Asn Gln             100 105 110 Ser Leu Leu Glu Leu His Lys Leu Ala Thr Asp Lys Asn Asp Pro His         115 120 125 Leu Cys Asp Phe Ile Glu Thr His Tyr Leu Asn Glu Gln Val Lys Ala     130 135 140 Ile Lys Glu Leu Gly Asp His Val Thr Asn Leu Arg Lys Met Gly Ala 145 150 155 160 Pro Glu Ser Gly Leu Ala Glu Tyr Leu Phe Asp Lys His Thr Leu Gly                 165 170 175 Asp Ser Asp Asn Glu Ser             180 <210> 16 <211> 182 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > <400> 16 Thr Thr Ser Ser Thr Ser Gln Val Arg Gln Asn Tyr His Gln Asp Ser   1 5 10 15 Glu Ala Ala Ile Asn Glu Ile Asn Leu Glu Leu Tyr Ala Ser Tyr              20 25 30 Val Tyr Leu Ser Met Ser Tyr Tyr Phe Asp Arg Asp Asp Val Ala Leu          35 40 45 Lys Asn Phe Ala Lys Tyr Phe Leu His Gln Ser His Glu Glu Arg Glu      50 55 60 His Ala Glu Lys Leu Met Lys Leu Gln Asn Gln Arg Gly Gly Arg Ile  65 70 75 80 Phe Leu Gln Asp Ile Lys Lys Pro Asp Cys Asp Asp Trp Glu Ser Gly                  85 90 95 Leu Asn Ala Met Glu Cys Ala Leu His Leu Glu Lys Asn Val Asn Gln             100 105 110 Ser Leu Leu Glu Leu His Lys Leu Ala Thr Asp Lys Asn Asp Pro His         115 120 125 Leu Cys Asp Phe Ile Glu Thr His Tyr Leu Asn Glu Gln Val Lys Ala     130 135 140 Ile Lys Glu Leu Gly Asp His Val Thr Asn Leu Arg Lys Met Gly Ala 145 150 155 160 Pro Glu Ser Gly Leu Ala Glu Tyr Leu Phe Asp Lys His Thr Leu Gly                 165 170 175 Asp Ser Asp Asn Glu Ser             180 <210> 17 <211> 567 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > <400> 17 catcaccatc accatcacac gaccgcgtcc acctcgcagg tgcgccagaa ctaccaccag 60 gactcagagg ccgccatcaa ccgccagatc aacctggagc tctacgcctc ctacgtttac 120 ctgtccatgt cttactactt tgaccgcgat gatgtggctt tgaagaactt tgccaaatac 180 tttcttcacc aatctcatga ggagagggaa catgctgaga aactgatgaa gctgcagaac 240 caacgaggtg gccgaatctt ccttcaggat atcaagaaac cagactgtga tgactgggag 300 agcgggctga atgcaatgga gtgtgcatta catttggaaa aaaatgtgaa tcagtcacta 360 ctggaactgc acaaactggc cactgacaaa aatgaccccc atttgtgtga cttcattgag 420 acacattacc tgaatgagca ggtgaaagcc atcaaagaat tgggtgacca cgtgaccaac 480 ttgcgcaaga tgggagcgcc cgaatctggc ttggcggaat atctctttga caagcacacc 540 ctgggagaca gtgataatga aagctaa 567 <210> 18 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> Lingker peptide <400> 18 Gly Ser Ser Gly Gly Gly Gly Ser   1 5 10 15 Glu Ala Asp Gly Ser Arg Gly Ser Gln Lys Ala Gly Val Asp Glu              20 25 30 <210> 19 <211> 93 <212> DNA <213> Artificial Sequence <220> <223> Lingker peptide <400> 19 ggcagctctg gtggcagcgg tagctctggc ggtagcggcg gtggcgatga agcggacggt 60 agccgcggct ctcagaaagc gggtgtggat gaa 93 <210> 20 <211> 111 <212> DNA <213> Artificial Sequence <220> <223> XTEN 36 <400> 20 gagggtggta gcgaaggctc tgaaggtgag ggtagcgaag gttccggtga aggcgaaggt 60 ggcagcgagg gttccgaagg tgagggctct gagggttctg gtgagggtta a 111 <210> 21 <211> 219 <212> DNA <213> Artificial Sequence <220> <223> XTEN 72 <400> 21 gagggtggta gcgaaggctc tgaaggtgag ggtagcgaag gttccggtga aggcgaaggt 60 ggcagcgagg gttccgaagg tgagggctct gagggttctg gtgagggtga aggtggctct 120 gaaggctccg aaggtgaagg cggttctgaa ggcagcgaag gcgaaggcgg cgaaggctcc 180 ggtgaaggtg aaggttccga aggctctggc gaaggctaa 219 <210> 22 <211> 441 <212> DNA <213> Artificial Sequence <220> <223> XTEN 144 <400> 22 gggggcacct ccacccctga gagcggctcc gcatccccag gaacctcccc ttccggagag 60 agcagcaccg ctcctggaac cagccccagc ggagagagct ccaccgctcc aggaagcacc 120 agctccaccg ccgagagccc tggaccaggc tccacctccg agagcccttc cgggaccgca 180 ccaggcagca cttccagcac cgctgagtcc ccaggacctg gcacatcccc ttccggggag 240 agctccactg ctcctgggac ctccacccca gagtccgggt cagctagccc agggtccacc 300 tcctccacag ccgaatcccc tgggcctggg acatccccat ccggggagtc ctctaccgcc 360 ccagggacat cccctagcgg cgagtcatca acagccccag ggacctcccc cagcggcgag 420 agttccacag cccctggcta a 441 <210> 23 <211> 879 <212> DNA <213> Artificial Sequence <220> <223> XTEN 288 <400> 23 ggcggcactt ctaccccgga atccggcagc gcaagccctg gtacctcccc ttccggtgag 60 agctctaccg ctccgggcac ctctccgagc ggtgagtctt ccaccgctcc gggttctacc 120 agctccaccg cagagagccc aggtccgggt tccacttccg aatccccgtc cggtaccgca 180 ccaggttcca cctccagcac cgctgaatcc ccaggccctg gcactagccc ttccggcgag 240 agctctacgg ctccgggtac gtctacccca gaatccggtt ccgctagccc gggctctacc 300 tcctccactg ctgaatcccc gggtcctggc acctccccat ccggtgaatc ctctaccgcg 360 ccgggtactt ctccgagcgg cgagtccagc acggcgccgg gtacctcccc gagcggtgaa 420 tctagcactg caccgggtac tagcactccg gaaagcggtt ccgcatcccc gggtaccagc 480 ccgtctggcg aatctagcac cgcgccgggc acttcccctt ctggtgaatc cagcaccgca 540 ccgggtagca cttccagcac cgcggaaagc ccgggtccag gctctactag cgaatctccg 600 tctggcaccg ccccaggttc tacctctagc accgccgaat ctccgggtcc gggcacgagc 660 ccgtctggtg agtcttctac tgcgccgggc acgtccaccc cggaatctgg ctctgcgtct 720 ccgggcagca cttcttctac cgcggaatct ccgggcccgg gcaccagccc gagcggcgaa 780 tcttctacgg ccccgggcac ttctccgtct ggtgaatctt ccaccgcccc gggcacgtct 840 ccgtccggtg aatcttctac tgctccgggc ggtggttaa 879 <210> 24 <211> 30 <212> PRT <213> Artificial Sequence <220> GLP-1 <400> 24 Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln   1 5 10 15 Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly              20 25 30 <210> 25 <211> 90 <212> DNA <213> Artificial Sequence <220> GLP-1 <400> 25 gctgaaggga cctttaccag tgatgtaagt tcttatttgg aaggccaagc tgccaaggaa 60 ttcattgctt ggctggtgaa aggccgagga 90 <210> 26 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Lingker peptide <400> 26 Lys Glu Ser Gly Ser Ser Ser Ser Glu Gln Leu Ala Gln Phe Arg Ser   1 5 10 15 Leu Asp         <210> 27 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Lingker peptide <400> 27 Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr   1 5 10 <210> 28 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Lingker peptide <400> 28 Gly Ser Ala Gly Ser Ala Ala Gly Ser Gly Glu Phe   1 5 10 <210> 29 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Lingker peptide <400> 29 Cys Arg Arg Arg Arg Arg Arg Glu Ala Glu Ala Cys   1 5 10 <210> 30 <211> 46 <212> PRT <213> Artificial Sequence <220> <223> Lingker peptide <400> 30 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Lys   1 5 10 15 Glu Ala Ala Ala Lys Ala Leu Glu Ala Glu Ala Ala Ala Lys Glu Ala              20 25 30 Ala Ala Ala Ala Ala Ala Ala Lys Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala          35 40 45 <210> 31 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Lingker peptide <400> 31 Gly Gly Gly Gly Gly   1 5 <210> 32 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Lingker peptide <400> 32 Gly Gly Gly   1 5 <210> 33 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Lingker peptide <400> 33 Gly Gly Gly Gly Ser   1 5 <210> 34 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Lingker peptide <400> 34 Ala Gla Ala Ala Ala Ala Ala Ala Ala Lys Ala   1 5 10 <210> 35 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Lingker peptide <400> 35 Pro Ala Pro Ala Pro   1 5 <210> 36 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Lingker peptide <400> 36 Val Ser Gln Thr Ser Lys Leu Thr Arg Ala Glu Thr Val Phe Pro Asp   1 5 10 15 Val     <210> 37 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Lingker peptide <400> 37 Pro Leu Gly Leu Trp Ala   1 5 <210> 38 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Lingker peptide <400> 38 Thr Arg His Arg Gln Pro Arg Gly Trp Glu   1 5 10 <210> 39 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Lingker peptide <400> 39 Ala Gly Asn Arg Val Arg Arg Ser Val Gly   1 5 10 <210> 40 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Lingker peptide <400> 40 Arg Arg Arg Arg Arg Arg Arg Arg   1 5 <210> 41 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Lingker peptide <400> 41 Gly Phe Leu Gly   One <210> 42 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> Lingker peptide <400> 42 Gly Ser Ser Gly Gly Gly Gly Ser   1 5 10 15 Glu Ala Asp Gly Ser Arg Gly Ser Gln Lys Ala Gly Val Asp Glu              20 25 30 <210> 43 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> HisX6 peptide <400> 43 His His His His His   1 5 <210> 44 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> FLAG peptide <400> 44 Asp Tyr Lys Asp Asp Asp Lys   1 5 <210> 45 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> V5 epitope peptide <400> 45 Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr   1 5 10 <210> 46 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Myc peptide <400> 46 Glu Gln Lys Leu Ile Ser Glu Glu   1 5 <210> 47 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> HA peptide <400> 47 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala   1 5 <210> 48 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Affibody <400> 48 Val Asp Asn Lys Phe Asn Lys Glu Met Trp Ala Ala Trp Glu Glu Ile   1 5 10 15 Arg Asn Leu Pro Asn Leu Asn Gly Trp Gln Met Thr Ala Phe Ile Ala              20 25 30 Ser Leu Val Asp Asp Ser Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala          35 40 45 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Glu Phe Val Asp Asn Lys      50 55 60 Phe Asn Lys Glu Met Trp Ala Ala Trp Glu Glu Ile Arg Asn Leu Pro  65 70 75 80 Asn Leu Asn Gly Trp Gln Met Thr Ala Phe Ile Ala Ser Leu Val Asp                  85 90 95 Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn             100 105 110 Asp Ala Gln Ala Pro Lys         115 <210> 49 <211> 354 <212> DNA <213> Artificial Sequence <220> <223> Affibody <400> 49 gtggataaca aatttaacaa agaaatgtgg gcggcgtggg aagaaattcg taacctgccg 60 aacctgaacg gctggcagat gaccgcgttt attgcgagcc tggtggatga tccgagccag 120 agcgcgaacc tgctggcgga agcgaaaaaa ctgaacgatg cgcaggcgcc gaaagaattc 180 gtggataaca aatttaacaa agaaatgtgg gcggcgtggg aagaaattcg taacctgccg 240 aacctgaacg gctggcagat gaccgcgttt attgcgagcc tggtggatga tccgagccag 300 agcgcgaacc tgctggcgga agcgaaaaaa ctgaacgatg cgcaggcgcc gaaa 354 <210> 50 <211> 39 <212> DNA <213> Artificial Sequence <220> (NdeI) H6 hFH (HindIII) F <400> 50 catatgcatc accatcacca tcacgaccgc gtccacctc 39 <210> 51 <211> 27 <212> DNA <213> Artificial Sequence <220> (NdeI) H6 hFH (HindIII) R <400> 51 aagcttttag ctttcattat cactgtc 27 <210> 52 <211> 39 <212> DNA <213> Artificial Sequence <220> (NdeI) H6 hFH (XhoI) F <400> 52 catatgcatc accatcacca tcacgaccgc gtccacctc 39 <210> 53 <211> 24 <212> DNA <213> Artificial Sequence <220> (NdeI) H6 hFH (XhoI) R <400> 53 ctcgaggctt tcattatcac tgtc 24 <210> 54 <211> 39 <212> DNA <213> Artificial Sequence <220> (NdeI) H6 hFH (BamHI) F <400> 54 catatgcatc accatcacca tcacgaccgc gtccacctc 39 <210> 55 <211> 24 <212> DNA <213> Artificial Sequence <220> (NdeI) H6 hFH (BamHI) R <400> 55 ggatccgctt tcattatcac tgtc 24 <210> 56 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> XTEN36 F <400> 56 ggatccgagg gtggtagcga aggctctgaa ggtgagggta gc 42 <210> 57 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> XTEN36 R <400> 57 aagcttttaa ccctcaccag aaccctcaga gccctcacct tcggaacc 48 <210> 58 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> XTEN72 F <400> 58 ggatccgagg gtggtagcga aggctctgaa ggtgag 36 <210> 59 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> XTEN72 R <400> 59 aagcttttag ccttcgccag agccttcgga accttcacc 39 <210> 60 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> XTEN144 F <400> 60 ggatccgggg gcacctccac ccct 24 <210> 61 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> XTEN144 R <400> 61 aagcttttag ccaggggctg tggaact 27 <210> 62 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> XTEN288 F <400> 62 ggatccggcg gcacttct 18 <210> 63 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> XTEN288 R <400> 63 aagcttttaa ccaccgcc 18 <210> 64 <211> 24 <212> DNA <213> Artificial Sequence <220> (BamHI) Affibody (XhoI) F <400> 64 ggatccgtgg ataacaaatt taac 24 <210> 65 <211> 24 <212> DNA <213> Artificial Sequence <220> (BamHI) Affibody (XhoI) R <400> 65 ctcgagttag tggataacaa attt 24 <210> 66 <211> 18 <212> DNA <213> Artificial Sequence <220> (HindIII) Affibody (ClaI) F <400> 66 aagcttgtgg ataacaaa 18 <210> 67 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> (HindIII) Affibody (ClaI) R <400> 67 atcgatttat ttcggcgc 18 <210> 68 <211> 24 <212> DNA <213> Artificial Sequence <220> (BamHI) XTEN144 (HindIII) F <400> 68 ggatccgggg gcacctccac ccct 24 <210> 69 <211> 24 <212> DNA <213> Artificial Sequence <220> (BamHI) XTEN144 (HindIII) R <400> 69 aagcttgcca ggggctgtgg aact 24

Claims (12)

A fusion protein in which 2 to 12 peptides composed of the amino acid sequence shown in SEQ ID NO: 1 are connected in series to the C-terminal of the self-assembled protein, or peptides composed of the amino acid sequence shown in SEQ ID NOs: 2 to 4 are connected. The method according to claim 1,
Wherein said self-assembling protein is a small heat shock protein (sHsp), ferritin, vault, P6HRC1-SAPN, M2e-SAPN, MPER-SAPN or a viral or bacteriophage capsid protein. 3. The method of claim 2,
Wherein the ferritin is a ferritin heavy chain protein or a ferritin light chain protein. The method of claim 3,
Wherein said ferritin heavy chain protein is comprised of an amino acid sequence of any one of SEQ ID NOS: 5 to 16. 3. The method of claim 2,
Wherein said virus or bacteriophage capsid protein is selected from the group consisting of bacteriophage MS2 capsid protein, bacteriophage P22 capsid protein, Qβ bacteriophage capsid protein, CCMV capsid protein, CPMV capsid protein, RCNMV capsid protein, ASLV capsid protein, HCRSV capsid protein, HJCPV capsid protein, A fusion protein that is a SHIV capsid protein, an MPV capsid protein, an SV40 capsid protein, an HIV capsid protein, an HBV capsid protein, an adenovirus capsid protein, or a rotavirus VP6 protein. The method according to claim 1,
Wherein the target protein to be delivered is added to the N-terminus of the self-assembling protein or to the C-terminus of the peptide. The method according to claim 6,
The target protein may be selected from the group consisting of Glucagon-like peptide-1 (GLP-1), EPO (erythropoetin), human growth hormone, human colony stimulating factor (hCSF), granulocyte colony stimulating factor (G- CSF), granulocyte macrophage colony stimulating factor Factor I, Factor II, Factor III, Factor IV, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI (interleukin 2) , Factor XII, Factor XIII, Osteogenic Protein 2, Osteogenic Protein 4, Insulin, TGF-beta, or a cancer antigen. 8. The method of claim 7,
The single chain antibody analogue _{scFv, (scFv) 2, V} H H, V NAR, Ke pilrin (Affilin), Ke Peabody (Affibody), Ke pimeo (Affimer), Ke pitin (Affitin), an alpha body (Alphabody), Antibodies such as Anticlin, Avimer, DARpin, Fynomoer, Kunitz domain peptide, nanobody, monobody, variable lymphocyte receptor VLR), Minibody, Diabody, Tetrabody, Triabody, or Peptabody. 8. The method of claim 7,
Wherein said cancer antigen is AFP, CA-125, CEA, CA19-9, BCR-ABL fusion gene, BRCA1, BRCA2, Beta-hCG, HER2 or PSA. A pharmaceutical composition for the treatment of cancer, comprising a nano-cage produced by self-assembly of the fusion protein of claim 9 as an active ingredient. 9. Nanocage in which the fusion protein of any one of claims 1 to 9 is self-assembled. 12. A drug delivery system comprising the nanocage of claim 11 and a delivery vehicle.

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