WO2020085766A1 - Novel peptide binding specifically to human serum albumin, and uses thereof - Google Patents

Abstract

The present invention relates to a novel peptide binding specifically to human serum albumin, more specifically to a peptide binding specifically to human serum albumin, polynucleotides coding for the polypeptide, an expression vector comprising the polynucleotides, a transgenic organism into which the expression vector has been introduced, and a method for producing the polypeptide by means of the transgenic organism. The novel peptide according to the present invention binds specifically to human serum albumin and increases the in-blood circulation time and half-life of low molecular weight protein or peptide to increase the bioavailability thereof, and thus can be broadly applied as a new system that increases the effectiveness of drug treatment.

Description

Novel polypeptides specifically binding to human plasma albumin and uses thereof

The present invention relates to novel polypeptides that specifically bind to human plasma albumin and uses thereof, and more particularly, to polypeptides that bind to human plasma albumin, methods for producing the polypeptides, and uses thereof.

Chemical synthetic drugs and low molecular weight proteins and peptides are being developed worldwide to treat various diseases. However, since these therapeutic agents have a real low therapeutic effect due to rapid in vitro discharge, in vivo instability, and low bioavailability such as disease targeting ability, they are ultimately solved to improve drug efficacy and lower toxicity. Development is required.

A drug delivery system that can increase drug efficacy and lower toxicity, such as a drug delivery system loaded with anti-cancer drugs such as doxorubicin or paclitaxel in biocompatible liposomes or albumin, is raising expectations as an alternative to existing drug treatment methods. Development and clinical evaluation of delivery systems are being conducted. However, only a handful of drug delivery platforms are being promoted for clinical application and industrialization because their effectiveness has been proven more than existing drug treatment methods. In particular, in the case of using biopolymers, nanoparticles, and antibodies that are being developed to increase drug delivery efficiency, methods of chemically binding drugs to the Fc region of antibodies, biopolymers, and albumin have been researched and put into practical use, but mostly overseas and multinational pharmaceutical companies This is a patent problem occupied by A and cannot be used for new drug development and drug delivery system development. In addition, due to the chemical synthesis of most drug delivery systems and chemical bonding methods according to the properties of the drug, the homologous / large scale of the drug delivery systems that are industrially applicable is not easy, and the toxicity of drug delivery systems injected outside the body is toxic. And an immune response.

When developing a general-purpose drug delivery system, a new concept drug delivery platform capable of mass production without being associated with patents from overseas and multinational pharmaceutical companies is essential. In particular, it should be possible to more easily and uniformly manufacture and mass-produce not only low-molecular chemical drugs, but also small-molecular-weight recombinant protein or peptide therapeutics that have recently received attention. In order to commercialize the drug delivery system for improving drug performance, these problems are solved as well as dramatically increasing the circulation time in blood of low molecular weight chemical drugs and protein / peptide therapeutics and delivering drugs as disease targets. The development of a universal drug delivery platform that maximizes treatment efficacy is essential.

Human plasma albumin (HSA), one of the high-molecular substances in vivo, is not only excellent in biocompatibility, but also a highly stable protein with a half-life in the blood of 19 days. It has been actively researched as a drug delivery platform to improve the in vivo stability of various drugs such as obesity, autoimmune diseases and infectious diseases.

The albumin-based drug delivery system is divided into three categories as follows. i) albumin is synthesized directly in vitro to bind to a drug, ii) albumin nanoparticle type, iii) antibody or peptide that binds albumin to a drug. However, among these albumin-based drug delivery systems, the method of synthesizing albumin in vitro and delivering the drug by combining with other drugs may cause an autoimmune disease to albumin in vivo due to an immune response when injected into the body. The albumin nanoparticle type is difficult to mass-produce using E. coli and is not suitable for additional manufacturing processes. The albumin binding domain is also difficult to develop as a new drug delivery platform because patents are preempted by overseas pharmaceutical companies and research institutes (KR 10-2015-0058454).

Against this background, the present inventors have developed a new polypeptide that specifically binds human plasma albumin using a novel protein skeleton called a repebody. The Lipid Body is composed of LRR (Leucin-rich repeat) module, is 1/5 of the size of the antibody, is capable of mass production in E. coli, and has very excellent heat and pH stability. In addition, the Lipid body that specifically binds to the target substance can be easily selected using a phage display, and the binding force to the target can be very easily increased up to the pico-mole level.

Accordingly, the present inventors, in order to develop a new polypeptide that specifically binds to human plasma albumin, as a result of earnest efforts, produced a mutant library using a specific Lipid body, and are specific to human plasma albumin through module-based binding capacity increase New polypeptides with binding power were selected. It was confirmed that the developed polypeptide has a very high specificity and binding ability to human plasma albumin, and does not inhibit the binding of albumin with a receptor that is involved in the circulation mechanism of albumin when binding with albumin. In addition, when injected into mice in a form combined with human plasma albumin, it was confirmed that the circulation time of the protein in the blood increased from less than 30 minutes to 24 hours, thereby completing the present invention.

In addition, when the low molecular weight protein drug growth hormone and Glucagon-like peptide-1 were genetically engineered and expressed in the lipobody, it was confirmed that the circulation time of the growth hormone in the mouse blood was significantly increased, and it was previously expressed in E. coli. As a result of expressing genetically linked human plasma albumin-binding lipobody to mouse interleukin-7, which was difficult, it was confirmed that expression was possible in E. coli, and mouse interleukin-7 of the expressed conjugate maintained activity by maintaining binding to the receptor. Thus, the present invention was completed.

The above information described in this background section is only for improving the understanding of the background of the present invention, and thus does not include information that forms prior art already known to those of ordinary skill in the art. It may not.

Summary of the invention

An object of the present invention is to use a lipobody that specifically binds to human plasma albumin, a polynucleotide encoding the lipobody, a vector containing the polynucleotide, a recombinant microorganism in which the vector is introduced, and the recombinant microorganism. It provides a method for producing a Lipibody.

Another object of the present invention is to provide a lipobody-protein complex in which the lipobody and the bioactive protein are linked through a linker.

In order to achieve the above object, the present invention provides a repebody that is represented by the amino acid sequence of any one of SEQ ID NOs: 2 to 12 and specifically binds to human plasma albumin.

The present invention also, the polynucleotide encoding the lipobody, a recombinant vector containing the polynucleotide, a recombinant microorganism in which the recombinant vector is introduced, and (i) culturing the recombinant microorganism to express the lipobody; And (ii) recovering the expressed lipid body.

The present invention also provides a lipobody-protein conjugate in which the lipobody and the bioactive protein are linked through a linker.

Figure 1 is a schematic diagram showing the overall structure of the amino acid residues constructing a random library for the development of a lipid body that binds to human plasma albumin in the present invention.

Figure 2 shows the results of measuring the binding force of the finally selected Lipid body clones after performing phage display bio-panning on human plasma albumin using the phage library constructed in the present invention by surface plasmon resonance (SPR) It is a figure showing.

Figure 3 is a diagram showing the results of measuring the binding force of a lipobody that specifically binds to human plasma albumin developed in the present invention by an enzyme-linked immunoprecipitation assay (Enzyme-Linked Immunosorbent Assay, ELISA).

4 is a view showing the results of measuring the binding force of the Lipid body specifically binding to human plasma albumin developed in the present invention by a pull-down method.

5 is a view showing the size exclusion chromatography (Size Exclusion Chromatography) results confirming that the binding of the aA1C4 lipid body selected in the present invention is very stable with human plasma albumin.

6 is a view showing the results of confirming by the pull-down assay (Pull-down assay) that the aA1C4 lipid body and human plasma albumin conjugate selected in the present invention stably maintains the binding for 96 hours or more under the condition of 37 degrees provided with plasma. .

7 is a view showing the results of the size exclusion chromatography (Size Exclusion Chromatography) confirmed that the aA1C4 lipid body and human plasma albumin conjugate selected in the present invention stably maintains the binding for at least 96 hours under the condition of 37 degrees provided with plasma. to be.

8 is a view showing the results confirmed by immunoprecipitation assay (ELISA) that the aA1C4 Lipid Body selected in the present invention binds to human plasma albumin most specifically among plasma albumin of each animal and can also bind to monkey plasma albumin. .

FIG. 9 shows that when the aA1C4 lipid body selected in the present invention is bound to human plasma albumin, binding to the neonatal Fc receptor (FcRn) is not inhibited at pH 6.0, but binding is inhibited at pH 7.4. It is a figure showing the results confirmed by immunoprecipitation assay (ELISA).

10 is labeled with the radioactive isotope (Technetium-99m, ^99m Tc) after the aA1C4 lipobody selected in the present invention is combined with human plasma albumin and circulated in the blood for more than 18 hours when injected into the tail vein of the mouse It is a figure showing the result of confirming the existence.

11 is a diagram showing the distribution of major organs in the body per hour when injected into the tail vein of a mouse after labeling an albumin that does not bind to albumin with a radioisotope (Technetium-99m, ^99m Tc).

Figure 12 shows the main organ distribution in the body per hour when injected into the tail vein of a mouse by labeling the aA1C4 lipobody selected in the present invention with a radioisotope (Technetium-99m, ^99m Tc) and binding to human plasma albumin. It is a drawing shown.

13 is a view evaluating the behavior of the body when injected into the tail vein of a mouse by binding to human plasma albumin after labeling the aA1C4 lipobody selected in the present invention with a radioisotope (Technetium-99m, ^99m Tc).

Figure 14 is aa1C4 selected from the present invention, after expressing the low molecular weight protein drug, growth hormone (Human Growth Hormone) genetically engineered to form a conjugate with human plasma albumin, and then injected into the mouse body by intravenous injection This diagram shows the result of comparing the circulation time in blood with growth hormone.

Figure 15 is a low molecular weight protein drug Glucagon-like peptide-1 is genetically linked to the aA1C4 lipobody selected in the present invention, and then expressed to form a conjugate with human plasma albumin, and injected into the mouse body by intravenous injection. It is a diagram showing the results of comparing my circulation time with GLP-1 linked to a Lipibody that does not bind to human plasma albumin.

Figure 16 shows the result of comparing the binding force to the receptor with the mouse interleukin-7 by immunoprecipitation assay by expressing in E. coli after genetically engineering the mouse interleukin-7, a low-molecular protein drug, to the selected aA1C4 lipobody in the present invention. It is a drawing.

Detailed description of the invention and preferred embodiments

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known in the art and commonly used.

In the present invention, a polypeptide that specifically binds to human plasma albumin is selected, and an increase in circulation time in blood using the polypeptide is selected.

In the present invention, screening of polypeptides specifically binding the polypeptides included in the library for development of the LipidBody to human plasma albumin through biopanning using phagemid was performed, and the work of improving their binding power was performed. . As a result, a polypeptide specific for human plasma albumin and having a high binding force was prepared to confirm the effect.

That is, in an embodiment of the present invention, in order to develop a novel polypeptide capable of specifically binding to human plasma albumin, the N-terminal, variable lymphocyte receptor (VLR) of the internalin B protein ), A Lipibody library was constructed that randomly contains a repeat module of a polypeptide in which the Leucine-rich repeat (LRR) protein portion is fused.

In addition, the library may be in the form of a phagemid containing a polynucleotide encoding the polypeptide. The term "phagemid" of the present invention means a circular polynucleotide molecule derived from phage, a virus that hosts E. coli, as a host, and includes sequences of proteins and surface proteins necessary for propagation and propagation. Recombinant phagemids can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation can be performed using enzymes and the like generally known in the art. The phagemid may include a signal sequence or a leader sequence for secretion in addition to expression control elements such as a promoter, an operator, an initiation codon, a termination codon, and an enhancer, and mainly label the phage surface by fusing the desired protein with the surface protein of the phage Can be used in a method. The promoter of phagemid is mainly inducible, and may also include a selectable marker for selecting host cells. For the purpose of the present invention, the phagemid contains MalEss, DsbAss or PelBss, a signal sequence or leader sequence for expressing and secreting a polynucleotide encoding the polypeptide constituting the library, and expressing a recombinant protein on the surface of the phage. The sequence described in the inventor's prior patent (No. 10-2012-0019927) comprising a polynucleotide encoding a gp3 domain which is a type of surface protein of M13 phage for expression of histidine-tag and phage surface for identification It may be a polynucleotide of No. 2, but is not particularly limited thereto.

The present inventors used the phage display method using a library prepared based on the nucleic acid sequence corresponding to SEQ ID NO: 1 containing the phagemid to novel polypeptides in the form of a lipid body having excellent binding ability to human plasma albumin (SEQ ID NO: 2, 3, 4, 5) were selected. Among them, SEQ ID NO: 2, which has the most excellent physical properties, was selected as a candidate for increasing binding power.

Subsequently, as a result of performing site directed mutagenesis to increase the binding capacity to human plasma albumin, a novel polypeptide having increased binding capacity (SEQ ID NO: 6) was selected, and it was confirmed that the binding power was very high (FIG. 2).

Accordingly, the present invention, in one aspect, is represented by the amino acid sequence of any one of SEQ ID NOs: 2 to 12, and relates to a repebody that selectively binds to human plasma albumin.

The term “lipibody” of the present invention refers to a water-soluble fusion polypeptide in which the N-terminal and leucine rich repeat (LRR) family proteins of an internal protein are fused, and a partially modified polypeptide based on the same. First developed by KAIST's Professor Kim Hak-Sung's team and named as 'Repebody' (Republic of Korea Patent Publication No. 10-2011-0099600 A1, Republic of Korea Registered Patent Publication 1356075, International Publication Patent Publication WO2013-129852 A1 and Lippie Body) Proc Natl Acad Sci US A. 2012 Feb 28; 109 (9): 3299-304). More specifically, the Lipid body is a microorganism-derived, N-terminal of the Leucine rich repeat (LRR) family protein having an alpha helical capping motif, a modified repeat module and a VLR protein of a variable lymphocyte receptor (VLR) protein. The C-terminal of the fused, this Lipid body has the property of specifically binding to a specific target protein, such as a receptor specifically expressed or over-expressed only in cancer cells, and repeat modules. It may include all proteins belonging to the LRR family and all fusion LRR family proteins that have improved their biophysical properties. In addition, the N-terminus of the internal protein, which is a component of the lipobody, can be selected from the N-terminus with high structural similarity according to the type of LRR family protein that can be fused, and is most stable through calculation of binding energy, etc. It is possible to change the amino acid of the corresponding module by selecting the amino acid.

The term "Variable Lymphocyte Receptor (VLR)" of the present invention refers to a type of LRR family protein expressed in eel and chilled fish, and is an immune function protein expressed in eel and chilled fish and binds to various antigenic substances. It can be usefully used as a skeleton. Polypeptide in which the N-terminal and VLR protein of the internaline B protein are fused increases in water solubility and expression level than the VLR protein in which the internaline B protein is not fused, and thus can be used to manufacture new protein therapeutics based thereon.

The term "LRR (Leucine Rich Repeat) protein" in the present invention means a protein composed of a combination of modules in which leucine is repeated at a constant position, (i) has one or more LRR repeat modules, and (ii) the LRR. The repeat module consists of 20 to 30 amino acids, and (iii) the LRR repeat module has a "LxxLxxLxLxxN" in a conservative pattern, where L is alanine, glycine, phenylalanine, tyrosine, leucine, isoleucine, valine and tryptophan. Hydrophobic amino acids, such as N, asparagine, glutamine, serine, cysteine or threonine, x refers to any amino acid, (iv) LRR family protein refers to a protein having a three-dimensional structure such as horseshoe. The LRR family protein of the present invention has a sequence whose sequence is unknown or has a sequence unknown to the natural world through a design such as consensus design, in addition to finding using a newly derived mRNA or cDNA in vivo. It may include all of the variants having a skeleton structure.

In the present invention, the term "internaline B protein" is a type of LRR family protein expressed in Listeria strains. The N-terminal structure differs from other LRR family proteins in which different hydrophobic cores are uniformly distributed over all molecules. It is known to have a stable expression even in microorganisms. Stable expression of LRR family proteins in microorganisms because the N-terminus of the internal protein is not only the microorganism-derived N-terminal region, which is most important for folding of the repeat module, but also has a stable shape including alpha helix. Can be effectively used for

In the present invention, the term "N-terminus of an interlining protein" refers to the N-terminus of an interlining protein required for water-soluble expression and folding of a protein, and includes an alpha helical capping motif and a repeating module of interlining proteins it means. The N-terminus of an interlining protein includes, without limitation, the N-terminus of an interlining protein required for soluble expression and folding of the protein, for example, an alpha helical capping motif "ETITVSTPIKQIFPDDAFAETIKANLKKKSVTDAVTQNE (SEQ ID NO: 13)" and a repeat module can do. In addition, the repeat module pattern may include "LxxLxxLxLxxN (SEQ ID NO: 14)". L of the repeating module pattern is alanine, glycine, phenylalanine, tyrosine, leucine, isoleucine, valine or tryptophan; N is asparagine, glutamine, serine, cysteine or threonine, x means any hydrophilic amino acid. The N-terminal of the internal protein can be selected from the N-terminal having a high structural similarity according to the type of LRR family protein that can be fused. Variations are possible.

The term "human plasma albumin" in the present invention is a protein that occupies the majority of the substances constituting the remaining plasma except for blood cells such as red blood cells and white blood cells in the blood, and serves to regulate the osmotic pressure of blood and ions in the blood It also serves to transport fatty acids. Albumin, along with immunoglobulins, is known to have a very long half-life of about 30 days in the blood, involving the neonatal Fc receptor (FcRn).

In another aspect, the present invention is a polynucleotide encoding the Lipid body; It relates to a recombinant vector comprising the polynucleotide and a recombinant microorganism in which the polynucleotide or the recombinant vector is introduced.

In the present invention, the polynucleotide is not particularly limited thereto, but may be a polynucleotide encoding any one of the amino acid sequences of SEQ ID NOs: 2 to 12, 70% or more with the polynucleotide, more preferably 80 % Or more, and more preferably, a polynucleotide having a nucleotide sequence having 90% or more homology, but is not particularly limited thereto.

The term "vector" of the present invention means a DNA preparation containing a base sequence of a polynucleotide encoding the target protein operably linked to a suitable regulatory sequence so that the target protein can be expressed in a suitable host. The regulatory sequence includes a promoter capable of initiating transcription, any operator sequence to regulate such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation. After transformation into a suitable host, the vector can replicate or function independently of the host genome and can be integrated into the genome itself.

The vector used in the present invention is not particularly limited as long as it can be cloned among hosts, and any vector known in the art can be used. Examples of commonly used vectors include natural or recombinant plasmids, phagemids, cosmids, viruses and bacteriophage. For example, pWE15, M13, λMBL3, λMBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, and Charon21A can be used as a phage vector or cosmid vector, and pBR-based, pUC-based, and pBluescriptII-based plasmid vectors. , pGEM system, pTZ system, pCL system and pET system. The vector usable in the present invention is not particularly limited, and known expression vectors can be used. Preferably, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, pET-21a, pET-32a, pcDNA3.1 vector or the like can be used. Most preferably, pET-21a, pET-32a, and pcDNA3.1 vectors can be used.

The term "recombinant microorganism" of the present invention means a cell in which a vector having a gene encoding one or more target proteins is introduced into a host cell and the trait is infected to express the target protein, and all cells such as eukaryotic cells, prokaryotic cells, etc. It may be, but is not particularly limited to, E. coli, Streptomyces, Salmonella typhimurium and other bacterial cells; Yeast cells; Fungal cells such as Pichia pastoris; Insect cells such as Drozophila and Spodoptera Sf9 cells; Animal cells such as CHO, COS, NSO, 293, Bow melanoma cells; Or it can be a plant cell. The host cell usable in the present invention is not particularly limited, but E. coli can be preferably used as the host cell. Most preferably, E. coli BL21 (DE3) and OrigamiB (DE3) can be used as host cells.

The term "transformation" in the present invention means that a vector containing a polynucleotide encoding a target protein is introduced into a host cell so that the protein encoded by the polynucleotide in the host cell can be expressed. The transformed polynucleotide includes all of them, whether they can be inserted into the host cell chromosome or located outside the chromosome, as long as it can be expressed in the host cell. In addition, the polynucleotide includes DNA and RNA encoding a target protein. The polynucleotide may be introduced into a host cell and expressed in any form as long as it can be expressed. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette (Expression cassette), which is a gene construct containing all elements necessary for self-expression. The expression cassette usually includes a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of an expression vector capable of self-replicating. In addition, the polynucleotide may be introduced into a host cell in its own form, and may be operably linked to a sequence required for expression in the host cell.

In another aspect, the present invention, (i) culturing the recombinant microorganism to express the Lipid body; And (ii) recovering the expressed lipid body.

In the above method, the step of culturing the recombinant microorganism is not particularly limited, but is preferably performed by a known batch culture method, a continuous culture method, a fed-batch culture method, and the culture conditions are not particularly limited thereto. Proper pH (pH 5 to 9, preferably pH 6 to 8, most preferably pH 6.8) using a basic compound (eg sodium hydroxide, potassium hydroxide or ammonia) or an acidic compound (eg phosphoric acid or sulfuric acid) It can be controlled, oxygen or oxygen-containing gas mixture can be introduced into the culture to maintain aerobic conditions, and the culture temperature can be maintained at 20 to 45 ° C, preferably 25 to 40 ° C, for about 10 to 160 hours Cultivation is preferred. The polypeptide produced by the culture may be secreted into the medium or remain in the cell.

In addition, the culture medium used is sugar and carbohydrates (e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose), fats and fats (e.g. soybean oil, sunflower seeds) as carbon sources. Oil, peanut oil and coconut oil), fatty acids (e.g. palmitic acid, stearic acid and linoleic acid), alcohols (e.g. glycerol and ethanol) and organic acids (e.g. acetic acid) can be used individually or in combination. ; Nitrogen sources include nitrogen-containing organic compounds such as peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean meal and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and Ammonium nitrate) or the like may be used individually or in combination; As the phosphorus source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, the corresponding sodium-containing salt, and the like can be used individually or in combination; Other metal salts (eg magnesium sulfate or iron sulfate), amino acids and essential growth-promoting substances such as vitamins may be included.

The method for recovering the polypeptide produced in the culturing step of the present invention recovers the desired lipobody from the culture medium using a suitable method known in the art according to a culture method, for example, a batch, continuous or fed-batch culture method. can do.

On the other hand, when using the Lipid body of the present invention, it is expected that the circulation time or the half-life of a chemical drug or a peptide / protein-based drug with a short half-life when injected into the blood can be significantly increased to increase the therapeutic efficacy of the drug. . In addition, it was expected that expression in E. coli would be improved when peptide / protein-based drugs, which are difficult to express in E. coli, were expressed by linking them to the Lipibody of the present invention.

That is, in another embodiment of the present invention, after the human blood growth cycle (hGH) and Glucagon-like peptide-1 (GLP-1) having a short circulation time in the blood are combined with the lipid body, the complex is combined with human plasma albumin, Increased circulation time in the blood was confirmed by measuring the amount of the drug remaining in the blood by periodically collecting blood after injecting it into the mouse or rat body by tail vein injection. At this time, the drug was labeled with a radioisotope prior to injection into the animal to measure the amount of radioisotope remaining at a specific time, or the amount of the drug remaining was relatively calculated using an immunoprecipitation assay (ELISA). Additionally, mouse interleukin-7 was linked to the lipobody to confirm binding to the receptor after expression.

As a result, it was confirmed that the half-life of the complex bound to the lipid body increased dramatically compared to the human growth hormone not bound to the lipid body (FIG. 14). In addition, in the case of Glucagon-like peptide-1, it was confirmed that binding to the lipid body significantly increased half-life compared to that of a lipid body that does not bind to albumin (FIG. 15). In the case of mouse interleukin-7, it is very difficult to express in E. coli, but it was expressed in E. coli when expressed in association with the lipobody, and the immunoprecipitation method (ELISA) shows that the expressed mouse interleukin-7 maintains binding to the receptor. It was confirmed using (Fig. 16).

Therefore, in another aspect, the present invention relates to a repeatbody-bioactive molecule conjugate in which the lipobody and the bioactive substance are bound through a linker.

In the present invention, the linker is generally composed of amino acids, but the type and length are not particularly limited, and (G3S) n spacer having flexible properties as in one embodiment of the present invention, that is, glycine 3 and 1 serine can be arranged repeatedly, or a GSAGSAAGSG linker or an alpha-helix linker with rigid properties (EAAAK) n (where E is glutamate, A is alanine, K is lysine, G is glycine, S Is a serine, and n is generally an integer of 1 or more, but in the case of G3S, a variety of glycine and serine variants and arrangements are possible), and DRDD (here D) to introduce more hydrophilic properties in consideration of physical properties. Aspartate, R is arginine) and various linkers containing charged amino acids can also be used.

In the present invention, "bioactive substance" is any substance that exhibits physiological activity in vivo, and any substance capable of interacting with various substances in the body to modulate their function or activity can be used, but as a preferred example , Nucleic acids, nucleotides, proteins, polypeptides, peptides, amino acids, sugars, lipids, vitamins, toxins, and drugs, but can also include smaller molecules that make up these substances.

In the present invention, the term “bioactive polypeptide”, “bioactive protein”, “active protein”, “protein” or “protein drug” refers to a polypeptide or protein that exhibits an antagonistic effect on a physiological phenomenon in vivo. Can be used interchangeably.

Specifically, bioactive proteins include human growth hormone, growth hormone releasing hormone, growth hormone releasing peptide, interferons and interferon receptors (eg, interferon-α, -β and -γ, water-soluble type I interferon receptor, etc.), colony stimulation Factor, interleukins (e.g., interleukin-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -13, -14, -15, -16, -17, -18, -19, -20, -21, -22, -23, -24, -25, -26, -27, -28, -29, -30, etc.) Interleukin receptors (eg IL-1 receptor, IL-4 receptor, etc.), enzymes (eg glucocerebrosidase, iduronate-2-sulfatase), α -Galactosidase-A (α-alactosidase-A), α-L-iduronidase, butyrylcholinesterase, chitinase, glutamate decarboxylase , Imiglucerase, lipase, uricase, platelet-activator acetyl Platelet-activating factor acetylhydrolase, neutral endopeptidase, myeloperoxidase, etc., interleukin and cytokine binding proteins (e.g. IL-18bp, TNF-binding protein, etc.) , Macrophage activator, macrophage peptide, B cell factor, T cell factor, protein A, allergen suppressor, cell necrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressor, metastatic growth factor, alpha-1 anti Trypsin, albumin, α-lactalbumin, apolipoprotein-E, erythropoietic factor, hyperglycosylated erythropoietic factor, angiopoeitins, hemoglobin, thrombin, thrombin receptor activating peptide, Thrombomodulin, blood factors Ⅶ, Ⅶa, VIII, Ⅸ, and XIII, plasminogen activators, fibrin-binding peptides, urokinase, streptokinase, hirudin (hirud in), protein C, C-reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, platelet-derived growth factor, epithelial cell growth factor, epidermal cell growth factor, angiostatin, angiotensin ), Bone growth factor, bone growth promoting protein, calcitonin, insulin, atriopeptin, cartilage inducer, elcatonin, connective tissue activator, tissue factor pathway inhibitor, follicle stimulating hormone , Luteinizing hormone, luteinizing hormone releasing hormone, nerve growth factors (e.g., nerve growth factor, ciliary neurotrophic factor, axogenesis factor-1, Glucagon- like-pepetide (GLP-1), brain-natriuretic peptide, glial derived neurotrophic factor, netrin, neurotrophic inhibitor (neurophil inhibitor factor), neural factors, neurturin, etc., parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growth factor, adrenal cortical hormone, glucagon, cholecystokinin, pancreatic polypeptide, gastrin release peptide , Corticotropin release factor, thyroid-stimulating hormone, autotaxin, lactoferrin, myostatin, receptors (e.g. TNFR (P75), TNFR (P55), IL-1 receptor, VEGF receptor , B cell activator receptor, etc., receptor antagonists (e.g., IL1-Ra, etc.), protein toxins (Pseudomonas aeruginosa exotoxin A, diphtheria toxin, botulinum toxin, tetanus antitoxin, heterogeneous toxin, cholera toxin, siguatoxin, gel Glonin or lysine), cell surface antigens (e.g. CD 2, 3, 4, 5, 7, 11a, 11b, 18, 19, 20, 23, 25, 33, 38, 40, 45, 69, etc.) , Monoclonal antibodies, polyclonal antibodies, antibody fragments (eg scFv, Fab, Fab ', F (ab') 2 and Fd), virus-derived It includes various kinds such as new antigen.

Particularly preferred physiologically active polypeptides are human growth hormone, interferon (interferon-α, -β, -γ, etc.), granulocyte colony stimulator, erythrocyte, which are frequently administered when administered to the human body for the purpose of treatment or prevention of disease. Production factors and antibody fragments, and most preferably interferon-α. In addition, any derivative or derivative is included in the scope of the bioactive polypeptide of the present invention, as long as it has substantially the same or increased function, structure, activity or stability as the natural form of the bioactive polypeptide.

In the present invention, the drug is not limited to these, but alotinib (TARCEVA; Genentech / OSI Pharm.), Borezomib (VELCADE; MilleniumPharm.), Fulvestrant (FASLODEX; AstraZeneca), sutente (SU11248) ; Pfizer), letrozole (FEMARA; Novartis), imatinib mesylate (GLEEVEC; Novartis), PTK787 / ZK 222584 (Novartis), oxaliplatin (Eloxatin; Sanofi), 5-fluorouracil (5-FU, leucovorin) , Rapamycin (Sirolimus, RAPAMUNE; Wyeth), Lapatinib (TYKERB, GSK572016; GlaxoSmithKline), Lonapanib (SCH 66336), Sorafenib (BAY43-9006; Bayer Labs.), Gefitinib (IRESSA; Astrazeneca ), AG1478, AG1571 (SU 5271; Sugen), alkylating agents such as thiotepa and CYTOXAN® cyclophosphamide; Alkyl sulfonates such as busulfan, improsulfan and piposulfan; Aziridine, for example benzodopa, carbocuone, meturedopa and uredopa; Ethyleneimine and methylammelamine, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; Acetogenins (especially bullataxin and bullataxinone); Camptothecin (inducing a synthetic analogue topotecan); Bryostatin; Callistatin; CC-1065 (including its adozelesin, chazelesin and biselesin synthetic analogs); Cryptophycins (particularly cryptophycin 1 and cryptophycin 8); Dolastatin; Duokamycin (including synthetic analogues, KW-2189 and CB1-TM1); Eleuterobin; Pancreatitis; Sacodictin; Spongistatin; Nitrogen mustards, e.g. chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembikin, phenesterine , Prednistine, trophosphamide, uracil mustard; Urea nitrites, such as carmustine, chlorozotoxin, potemustine, lomustine, nimustine and ranimustine; Antibiotic agents, eg, enedine antibiotics (eg, calicheamicin, especially calicheamicin gamma 1I and calicheamicin omega I1 (see eg Agnew, Chem Intl ed Engl., 33: 183- 186 (1994)) and multimycin, which induces multimycin A; bisphosphonates, such as clodronate; esperamicin, niokazinostatin chromophores and related chromoprotein enenidine antibiotic chromophores, aclacinomycins, vices Tinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carabicin, kaninomycin, casino filin, chromomycin, dactinomycin, daunorubicin, detorvucin, 6-diazo-5-oxo -L-norleucine, ADRLIMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin and deoxydoxorubicin), epirubicin, esolerubicin, and micelle Roman, Mitomycin, for example, mitomycin C, mycophenolic acid, nogalamycin, olibomycin, peflomycin, portpyromycin, puromycin, cuelamycin, rodurubicin, streptomycin, streptozosin, tuber Cydine, ubenimex, ginostatin and zorubicin; anti-metabolites such as 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, Trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine and thiguanine; pyrimidine analogs such as acitabine, azacitidine, 6-azauridine, Camopur, cytarabine, dideoxyuridine, doxyfluridine, enositabine and phloxuridine; androgens such as callosterone, dromostanolone propionate, epithiostanol, mepithiostan And testolactones; anti-adrenal such as amino Glutethimide, mitotan and trirostan; folic acid supplements such as folinic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; enyluracil; amsaclean; vestlabusil; bisantrene; Edtraxate; depotamine; demecolsin; diazicone; elfornitine; Elliptinium acetate; Epothilone; Etogluside; Gallium nitrate; Hydroxyurea; Lentinan; Rhonidinin; Maytansinoids such as maytansin and ansamitoxin; Mitoguazone; Mitoxantrone; Fur group; Nitraerin; Pentostatin; Phenamet; Pyrarubicin; Rosoksatron; 2-ethylhydrazide; Procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); Lajoksan; Lysine; Sizopyran; Spirogermanium; Tenoazonic acid; Triazion; 2,2 ', 2 "-trichlorotriethylamine; trichothecene (especially T-2 toxin, veracurin A, loridine A and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; Mitoractol; fibrobroman; thornycin; arabinoside ('Ara-C'); cyclophosphamide; thiotepa; taxoid, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ) No ABRAXANE ™ cremophor, albumin processed nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumber, I11.) And TAXOTERE® docetaxel (Rhone-Poulenc Rorer, Antony, France); Chloranbucil; gemcitabine; 6-thio Guanine; mercaptopurine; platinum analogs such as cisplatin, carboplatin; vinblastine; platinum; etoposide, ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine; novatron; tenipo Seed; deatrexate; daunomycin; aminopterin; geloda; ibandronay T; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DFMO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, solvates, acids thereof Or derivatives.

Additional drugs are not limited to these, but (i), for example, tamoxifen (including NOLVADEX® tamoxifen), laroxifene, drooxifene, 4-hydroxytamoxifene, trioxyphene, keoxyphene, LY117018, Anti-hormonal agents that act to modulate or inhibit hormonal action on tumors, such as anti-estrogen and selective estrogen receptor modulators (SERMs), including onaripristone and FAREATON 'toremifene; (ii) Aromatase inhibitors that inhibit the aromatase enzyme, which modulates estrogen production in the adrenal glands, for example 4 (5) -imidazole, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® Exemestane , FEMARA ㄾ letrozole and ARIMIDEX® anastrozole; (iii) anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; As well as troxacitabine (1,3-dioxolane nucleoside cytosine analog); (iv) aromatase inhibitors; (v) protein kinase inhibitors; (vi) lipid kinase inhibitors; (vii) antisense oligonucleotides, particularly those that inhibit gene expression in signaling pathways associated with adherent cells, eg PKC-alpha, Raf, H-Ras; (viii) ribozymes such as VEGF inhibitors such as ANGIOZYME ribozyme and HER2 expression inhibitors; (ix) vaccines, such as gene therapy vaccines; ALLOVECTIN® vaccine, LEUVECTIN vaccine and VAXID vaccine; PROLEUKIN®rlL-2; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH; (x) anti-angiogenic agents such as Bevacizumab (AVASTIN, Genentech); And (xi) pharmaceutically acceptable salts, solvates, acids or derivatives thereof.

The term "toxin" refers to a toxic substance produced in living cells or organisms. Toxins can be biological macromolecules, for example small molecules, peptides or proteins that can cause disease upon contact or absorption by body tissues that interact with enzymes or cell receptors. Toxins include plant toxins and animal toxins. Examples of animal toxins include, but are not limited to, diphtheria antitoxins, botulinum toxins, tetanus antitoxins, heterotoxins, cholera toxins, tetrodotoxins, brebetoxins, siguatoxins. Examples of plant toxins include, but are not limited to, lysine and AM-toxin.

Examples of small molecule toxins include, but are not limited to, auristatin, geldanamycin (Kerr et al., 1997, Bioconjugate Chem. 8 (6): 781-784), mitashinoids (EP 1391213, ACR 2008) , 41, 98-107), calicheamicin (US2009105461, Cancer Res. 1993, 53, 3336-3342), daunomycin, doxorubicin, methotrexate, vindesine, SG2285 (Cancer Res. 2010, 70 (17), 6849 -6858), dolastatin, auristatin of the dolastatin analogue (US563548603), cryptophycin, camptothecin, lysine derivative, CC-1065 analog or derivative, duokamycin, endian antibiotic, esperamicin, Epothilone and toxoid. The toxin may exhibit cytotoxic and cell growth inhibitory activity by tubulin binding, DNA binding, topoisomerase inhibition, and the like.

In the present invention, the lipobody-complex may be used to control various physiological activities, and for example, it may be used as a composition for treating cancer containing the lipobody-complex as an active ingredient.

In the present invention, the cancer is non-Hodgkin lymphoma, non-Hodgkin lymphoma, acute-myeloid leukemia, acute-lymphoid leukemia, multiple myeloma ( Group consisting of multiple myeloma, head and neck cancer, lung cancer, glioblastoma, colorectal / rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, melanoma, prostate cancer, kidney cancer and mesothelioma It can be characterized by being selected from.

The term "treatment" in the present invention means that the administration of the composition not only suppresses or alleviates one or more symptoms caused by cancer or its treatment, but also prevents the treatment of cancer or the progression of cancer that reverses the symptoms of cancer. In the present invention, the term "prevention" refers to all actions that suppress the disease or delay the onset of the administration of the composition.

The prevention or treatment of cancer in the present invention is achieved by improving the in vivo duration of the complex by combining the lipobody-complex developed in the present invention with albumin, for example, when the complex is an antibody against a mutant protein, the lipid body And albumin to prevent and treat cancer by inhibiting the activity of the mutant protein.

The composition for preventing or treating cancer containing the lipobody-complex of the present invention as an active ingredient may further include a pharmaceutically acceptable carrier, and may be formulated together with the carrier.

In the present invention, the amount of the lipid body-complex in the composition is not limited, but may be added at 0.01 to 95% by weight of the total composition weight.

The term "pharmaceutically acceptable carrier" in the present invention refers to a carrier or diluent that does not stimulate the organism and does not inhibit the biological activity and properties of the administered compound. As a pharmaceutical carrier that is acceptable in a composition formulated as a liquid solution, as a sterile and biocompatible material, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants can be added to make formulations for injection, pills, capsules, granules or tablets, such as aqueous solutions, suspensions and emulsions.

The composition for preventing or treating cancer comprising the polypeptide of the present invention and a pharmaceutically acceptable carrier is applicable to any formulation containing it as an active ingredient, and can be prepared as an oral or parenteral formulation. The pharmaceutical formulations of the present invention are oral, rectal, nasal, topical (including cheek and sublingual), subcutaneous, vaginal or parenteral; intramuscular and subcutaneous. And forms suitable for administration by inhalation (including intravenous) or administration by inhalation or insufflation.

Formulations for oral administration comprising the composition of the present invention as an active ingredient include, for example, tablets, troches, lozenges, water-soluble or oily suspensions, preparation powders or granules, emulsions, hard or soft capsules, syrups or elixirs. can do. For formulation into tablets and capsules, formulations such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, and stearic acid masne It may contain a lubricant such as calcium, calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax, and in the case of capsule formulations, it may further contain a liquid carrier such as fatty oil in addition to the above-mentioned substances.

Formulations for parenteral administration comprising the composition of the present invention as an active ingredient include subcutaneous injections, intravenous injections, intramuscular injections, injectable forms, suppository injection methods, or sprays, such as aerosols that enable inhalation through a respiratory system. It can be formulated as. In order to formulate an injectable formulation, the composition of the present invention can be prepared as a solution or suspension by mixing in water with a stabilizer or buffer, and formulated for unit administration of ampoules or vials. To inject into a suppository, it can be formulated into a composition for rectal administration such as a suppository or enema containing a conventional suppository base such as cocoa butter or other glycerides. When formulated for spraying, such as aerosols, propellants and the like may be combined with additives to disperse the concentrated dispersion or wet powder.

The composition for preventing or treating cancer comprising the lipobody-complex of the present invention as an active ingredient is a method for preventing or treating cancer, including administering the same, and those skilled in the art who understand the contents of the present invention are within the scope of the present invention. You can see that it belongs. The present invention also relates to a method for preventing or treating cancer, comprising administering the lipobody-complex. The present invention also relates to the cancer prophylactic or therapeutic use of the lipobody-complex. The present invention also relates to a method of using the above Lipibody-complex for preparing a medicament for the prevention or treatment of cancer.

The term "administration" in the present invention means introducing the pharmaceutical composition of the present invention to a patient in any suitable way. The route of administration of the composition of the present invention can be administered through various routes, oral or parenteral, as long as it can reach the target tissue, specifically, oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, It can be administered in a conventional manner via transdermal, intranasal, inhalation, intraocular or intradermal routes.

The treatment method of the present invention includes administering a composition for preventing or treating cancer of the present invention in a pharmaceutically effective amount. It is obvious to a person skilled in the art that the appropriate total daily dosage can be determined by the treating physician within the proper medical judgment. The specific therapeutically effective amount for a particular patient includes the specific composition, patient's age, weight, general health status, gender and diet, time of administration, including the type and extent of response to be achieved and, in some cases, whether other agents are used. It is preferable to apply differently according to various factors including the route of administration and the secretion rate of the composition, the treatment period, the drug used with or concurrently with the specific composition, and similar factors well known in the pharmaceutical field. Therefore, it is preferable to determine the effective amount of the composition for preventing or treating cancer suitable for the purpose of the present invention in consideration of the above.

It is apparent to those skilled in the art that the lipobody-complex of the present invention can be used for various purposes depending on the type of bioactive protein to be bound as well as cancer prevention or treatment.

For example, when the body and insulin of the present invention are combined, it will be apparent that the lipid body-insulin complex can be combined with albumin to dramatically improve the duration in the body, thereby reducing the number of insulin doses of diabetics.

For example, in the case of the Lipid Body-GLP-1 complex in which Lipid Body and GLP-1 of the present invention are combined, the number of times GLP-1 is administered to a diabetic patient by significantly improving the duration of GLP-1 in combination with albumin It will be obvious that it can be reduced.

For example, in the case of the lipobody-interleukin-7 complex in which the lipobody and interleukin-7 of the present invention are combined, it will be apparent that it is possible to modulate the inflammatory response by significantly improving the duration of interleukin in the body by combining with albumin. .

For example, in the case of the Lipid Body-Human Growth Hormone complex in which the Lipid Body and Human Growth Hormone of the present invention are combined, it can be combined with albumin to dramatically improve the body's duration of the Human Growth Hormone to control the function of Human Growth Hormone. Will be self-evident.

As the specific parts of the present invention have been described in detail above, it will be apparent to those of ordinary skill in the art that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Example

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example 1: Construction of a library for the screening of a specific binding lipid body to human plasma albumin using phage display

Phage display was performed using a library phage already constructed in the prior patent (No. 10-2012-0019927) for the selection of a lipobody that specifically binds to human plasma albumin (FIG. 1). 1 is a schematic diagram showing the overall structure of amino acid residues intended to construct a random library.

Mutagenic primers for library construction were synthesized to replace the selected amino acids with NNK degenerate codons.

Subsequently, by using the primers, an overlap polymerase chain reaction (overlap PCR) was performed for the two modules to obtain library DNA, and of SEQ ID NO: 2 described in the prior patent (No. 10-2012-0019927) of the present inventor The final library phagemid was obtained by inserting it into phagemid pBEL118N.

The obtained library was introduced into E. coli XL1-Blue by electroporation to obtain transformants, thereby constructing a library having a diversity of 1.0x10 ⁸ levels.

Example 2: Selection of a polypeptide that binds to human plasma albumin through the panning process of the Lipibody library

Using the library constructed in Example 1, a polypeptide capable of binding to human plasma albumin was selected and purified. In order to select candidates capable of binding to human plasma albumin, human plasma albumin was added to the immunotube at a concentration of 100 μg / ml and coated at 4 ° C. for 12 hours. The coated immunotube was washed 3 times with PBS and blocked with PBS solution (TPBSA) containing 1% BSA and 0.05% Tween 20 at 4 ° C. for 2 hours. Then, the purified phage was added to the coated immunotube at a concentration of 10 ¹² cfu / ml and reacted at room temperature for 2 hours. After the reaction was completed, the mixture was washed 5 times with PBS solution (TPBS) containing 0.05% Tween 20 for 5 minutes in total. Finally, 1 mL of 0.2 M Glycine-HCl (pH 2.2) was added to the immunotube and reacted at room temperature for 15 minutes to elute phage expressing a Lipibody candidate capable of binding to human plasma albumin on the surface. The eluate was neutralized by adding 60 µl of 1.0 M Tris-HCl (pH 9.0), added to 15 mL (OD ₆₀₀ = 0.5) of the culture medium of Escherichia coli XL1-Blue, and then bio-panned on a 2xYT plate (Bio -panning) process was repeated 6 times in the same manner.

As a result, it was confirmed that phages that specifically bind to human plasma albumin were concentrated through each panning process. The above results confirmed that the library phage binding to human plasma albumin specifically increased.

Example 3: Confirmation and sequence analysis of specific binding to selected human plasma albumin

Enzyme-linked immunosorbent assay (ELISA) was performed using a 96-well plate coated with human plasma albumin and Bovine serum albumin (BSA) from the phage selected through the method of Example 2. As compared with BSA, 74 Lipid body candidates having the highest absorbance (OD450) of the wells coated with human plasma albumin were selected, and the amino acid sequence of each of them was checked. After removing the cysteine-inserted phage, 4 kinds of Lipid body phage Screening.

The sequence of each Lipibody is shown in Table 1 below.

Example 4: Performing a module-based affinity enhancement method for increasing the binding capacity of the Lipibody

In order to circulate blood for a long time by stably binding with human plasma albumin in the body, improvement to a lipid body having high binding power to human plasma albumin is required. Therefore, the module-based affinity enhancement technology constructed in the prior patent (No. 10-2012-0019927) was used to secure a repibody with increased binding power. First, based on the polynucleotide sequence encoding the polypeptide of the aA1 lipid body that binds to the human plasma albumin selected in Example 3, the module adjacent to the first library (LRRV1) is selected in the same manner as in Example 2 of the preceding patent. Four residues in the concave region were mutated.

A total of 7 Lipibody clones (SEQ ID NOs: 6 to 12, Table 2) having an increased binding capacity after six additional panning processes were obtained. As a result of measuring the dissociation constant through Surface plasmon resonance (SPR), 4 nM level It secured aA1C4 Lipid body having the highest binding force of (SEQ ID NO: 6, Figure 2). In addition, this high binding ability was confirmed once again through an immunoprecipitation enhancement method (ELISA) and a pull-down technique (FIGS. 3 and 4).

Example 5: Confirmation of the stability of the selected lipid body and human plasma albumin conjugate

An experiment was carried out to confirm that the selected aA1C4 lipobody and the conjugate of human plasma albumin remained stable. It was confirmed that the analysis was performed with a single protein as a result of performing size exclusion chromatography after mixing Libobody and human plasma albumin at a molar ratio of 1: 1 and reacting at 4 ° C for 2 hours. (Fig. 5). The conjugate of the resulting body and human plasma albumin was incubated at 37 ° C providing rat plasma at a concentration of 10% (v / v), and then 0, 1, 2, 3, 4, 5, 6, 12, 24 , After 48, 72, and 96 hours, it was confirmed that the conjugate was kept very stable as a result of performing a pull-down assay on histag (FIG. 6). In addition, size exclusion chromatography was performed for each time to confirm high stability (FIG. 7).

Example 6: Confirmation of the ability to distinguish between species of albumin of the selected Lipid body and the possibility of binding to monkey plasma albumin

Using the selected aA1C4 Lipibody, an experiment was performed to confirm whether it could bind to plasma albumin of other animals. Each plasma human albumin (Sigma Aldrich) is treated with 100 μL at 10 μg / mL in each well of a 96-well plate. Three wells were used for each species of albumin. After coating at 4 ° C. for 12 hours, the remaining region without protein was treated with PBS solution (TPBSA) containing 1% BSA and 0.05% Tween 20 to perform a blocking process at room temperature for 1 hour.

Thereafter, the selected aA1C4 re-body was treated with 10 μg / mL at 100 μL for 1 hour at room temperature, and then washed 5 times with TPBS. The biotin (Biotin) is connected to the antibody capable of capturing the Lipid body, and treated with 100 μL at 10 μg / mL for each well for 1 hour at room temperature and then washed 5 times with TPBS. Thereafter, streptavidin-horseradish peroxidase (HRP) was treated and reacted at room temperature for another hour. After washing 5 times with TPBS and washing 3 times with PBS, 3,3 ', 5,5'-Tetramethylbenzidine (TMB), a substrate of hrp, was added to proceed with the color development reaction, followed by treatment with 1N H ₂ SO ₄ The color reaction was stopped. Then, the light emission degree (OD ₄₅₀ ) was measured through a light emission measurement device.

As a result, it was confirmed that the developed lipobody binds most specifically to human plasma albumin and has binding power to primate monkey plasma albumin (Rhesus Monkey Albumin, Athens Research & Technology). In addition, it was confirmed that the rabbit plasma albumin (Rabbit Serum Albumin, Sigma Aldrich) also has a strong binding force (Fig. 8).

Example 7: Confirmation of whether the selected lipid body targeting human plasma albumin does not affect recycling through FcRn, a circulation mechanism in the albumin body when combined with albumin

When the lipobody targeting human plasma albumin obtained in Example 4 formed a conjugate with albumin, an experiment was carried out to confirm whether it inhibited the binding of albumin with the most important neonatal Fc receptor (FcRn) in albumin's circulatory mechanism. Was carried out.

Albumin has a long half-life in the body because it is not destroyed through binding with the FcRn (neonatal Fc receptor) and secreted back into the blood. Albumin and FcRn can maintain the binding force in the pH 6.0 environment, thereby avoiding the process of destruction by lysosomes, and in the pH 7.4 environment, the binding of FcRn is eliminated, and albumin can be secreted again into the blood. In order for the albumin-binding lipobody-based drug delivery system using a long half-life of albumin to be effective, it is necessary to combine albumin with albumin without disturbing the albumin lipid body in the circulation mechanism of the albumin. Through the ELISA reaction, it was confirmed that albumin-binding lipid bodies do not interfere with the binding of albumin and FcRn even when bound to human plasma albumin (FIG. 9).

Example 8: Confirmation of residence time and major organ distribution in blood of the selected Lipibody targeting human plasma albumin

It was confirmed how long the conjugates of the selected lipobody and albumin targeting human plasma albumin circulate in mouse blood. After labeling the radioisotope ( ^99m Tc) using his tag attached to the Lipibody, reacting with albumin to make a Lipibody-Albumin conjugate, and then injecting it through the tail vein of the mouse into the body, the existing Lipi Although the body was removed from the blood within about 30 minutes, it was confirmed that the selected combination of the Lipid Body and albumin continuously circulates blood in the mouse body for 18 hours or more (FIG. 10).

In addition, as a result of confirming the major organs in the mouse body, it was confirmed that the existing Lipibody was rapidly removed through the kidney, but it was confirmed that the amount of removed Lipid body and albumin was significantly reduced through the kidney (FIG. 11). , 12).

Example 9: Evaluation of the body behavior of a lipobody targeting human plasma albumin using radioisotopes

The behavior of the mouse body of the selected lipobody and albumin conjugates targeting human plasma albumin was confirmed using imaging equipment. After labeling the lipid body using an isotope ( ^99m Tc), it formed a conjugate with albumin and provided it with a tail vein injection method in the mouse body. As a result, most of the normal lipid bodies appeared to exist in the kidney, but with the developed lipid body It was confirmed that the albumin conjugate was distributed in the mouse body, not the kidney. In addition, it was confirmed that it was present in the mouse body up to 24 hours after administration (Fig. 13).

Example 10: Evaluation of increase in circulation time in the blood of a low-molecular drug using a lipobody targeting human plasma albumin

A human body growth hormone linked through a linker was connected to the lipid body (SEQ ID NO: 6) obtained in Example 4 to prepare a lipid body-protein complex (SEQ ID NO: 15).

SEQ ID NO: 15: Repebody-hGH conjugate

By grafting the selected Human Growth Hormone targeting human plasma albumin, the circulation time in mouse blood was confirmed using a sandwich immunoprecipitation assay (Sandwich ELISA). As a result of forming a conjugate with albumin and providing a tail vein injection method into the mouse body, the existing growth hormone was rapidly removed from the mouse's body within 30 minutes, but the growth hormone connected to the albumin-binding lipobody was designed for more than 24 hours. It was confirmed that the inside was purified (FIG. 14).

Example 11: Evaluation of increase in circulation time in the blood of a low-molecular drug using a lipobody targeting human plasma albumin 2

After modifying alanine 8 to glycine with Glucagon-like peptide-1 (GLP-1) linked via a linker to the lipobody (SEQ ID NO: 6) obtained in Example 4, and then connecting it, the lipobody-protein complex (SEQ ID NO: 16) Was produced.

SEQ ID NO: 16: Repebody-GLP-1 conjugate

Glucagon-like peptide-1 was conjugated to the selected lipobody targeting human plasma albumin, and the circulation time in mouse blood was confirmed using a sandwich immunoprecipitation assay (Sandwich ELISA). As a result of forming a conjugate with albumin and providing a method of intravenous tail injection into the mouse body, GLP-1 conjugated to a lipobody that does not bind albumin was rapidly removed from the mouse's body within 1 hour, but connected to an albumin-binding lipobody. It was confirmed that the GLP-1 was purified in the blood of the mouse for 24 hours or more (FIG. 15).

Example 12: Improvement of E. coli expression of low-molecular drugs using a lipobody targeting human plasma albumin

A lipid body-protein complex (SEQ ID NO: 17) was prepared by connecting to a mouse interleukin 7 (mIL-7) linked to a lipid body (SEQ ID NO: 6) obtained in Example 4 through a linker.

SEQ ID NO: 17: Repebody-mIL-7 conjugate

mIL-7 is known to be very difficult to express in E. coli. However, expression in E. coli conjugating mIL-7 to a selected lipobody targeting human plasma albumin became possible, and the Lipidbody-mIL-7 expressed using immunoprecipitation assay (ELISA) binds to the mIL-7 receptor It was confirmed that it was maintained (Fig. 16).

As the specific parts of the present invention have been described in detail above, it will be apparent to those of ordinary skill in the art that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

The novel lipobody according to the present invention specifically binds to human plasma albumin, and by using this, the bioavailability of the existing drug by dramatically increasing the half-life of the drug in which the circulation time in the existing blood was short It can be widely used as a new drug delivery system because it can improve bioavailability and improve the therapeutic effect of drugs for specific diseases.

Electronic file attached.

Claims (9)

1. Repebody that is represented by the amino acid sequence of any one of SEQ ID NOs: 2 to 12 and specifically binds to human plasma albumin.
2. A polynucleotide encoding the lipobody of claim 1.
3. Recombinant vector comprising the polynucleotide of claim 2.
4. A recombinant microorganism in which the polynucleotide of claim 2 or the recombinant vector of claim 3 is introduced.
5. (i) culturing the recombinant microorganism of claim 4 to express a lipobody; And

   (ii) A method of producing a lipid body of claim 1, comprising recovering the expressed lipid body.
6. A lipid body-bioactive material conjugate in which the lipid body of claim 1 and a bioactive substance are bound through a linker.
7. The method according to claim 6, wherein the linker is selected from the group consisting of (G3S) n, (EAAAK) n, DRDD and GSAGSAAGSG linkers, wherein the body is a repeatbody-bioactive material conjugate, wherein G Is glycine, S is serine, E is glutamic acid, A is alanine, K is lysine, D is aspartic acid, R is arginine, and n is an integer of 1 or more.
8. The method of claim 6, wherein the bioactive material is a nucleic acid, nucleotide, protein, polypeptide, peptide, amino acid, sugar, lipid, vitamin, toxin or drug, characterized in that the body-reactive body complex (repebody-bioactive material) complex conjugate).
9. The method of claim 8, wherein the protein is human growth hormone, growth hormone releasing hormone, growth hormone releasing peptide, interferon and interferon receptors, colony stimulating factor, interleukin, interleukin receptors, enzymes, macrophage activator, macrophage peptide , B cell factor, T cell factor, protein A, allergen suppressor, cell necrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressor, metastatic growth factor, alpha-1 antitrypsin, albumin, α-lactic Albumin, apolipoprotein-E, erythropoietic factor, high glycated erythropoietic factor, angiopoeitins, hemoglobin, thrombin, thrombin receptor activating peptide, thrombomodulin , Blood factors VII, VIIa, VIII, VII, and XIII, plasminogen activators, fibrin-binding peptides, eurokinase, streptokinase, hirudin (h irudin), protein C, C-reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, platelet-derived growth factor, epithelial cell growth factor, epidermal cell growth factor, angiostatin, angiotensin ), Bone growth factor, bone formation promoting protein, calcitonin, insulin, atriopeptin, cartilage inducer, elcatonin, connective tissue activator, tissue factor pathway inhibitor, follicle stimulating hormone , Luteinizing hormone, luteinizing hormone releasing hormone, nerve growth factors, Glucagon-like-pepetide (GLP-1), parathyroid hormone, relaxin, cyclin, somatomedin, insulin-like growth factor, adrenal cortical hormone, glucagon , Cholecystokinin, pancreatic polypeptide, gastrin-releasing peptide, corticotropin-releasing factor, thyroid-stimulating hormone, autotaxin, lactoferrin, Lipobody-physiology, characterized in that it is selected from the group consisting of myostatin, receptors, receptor antagonists, protein toxins, cell surface antigens, monoclonal antibodies, polyclonal antibodies, antibody fragments and virus derived vaccine antigens. Repebody-bioactive material conjugate.

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