KR101751501B1 - Repebody-Protein toxin Conjugate, Preparation Methods and Use Thereof - Google Patents

Abstract

The present invention relates to a protein toxin complex using a repebody and a method for producing the same. More particularly, the present invention relates to a protein toxin complex comprising a VLR (leucine-rich repeat) family protein having an alpha helical capping motif Protein toxin conjugate conjugated with a substance having the same activity as a lipoprotein toxin that exhibits substrate specificity by linking an LRR structure derived from a variable lymphocyte receptor to a variable region, Methods and uses thereof. The lipid-protein-toxin complex according to the present invention is a combination of lipid-specific high substrate specificity, excellent tissue and tumor penetration ability, and a protein toxin having biological activity. Thus, the lipid-protein toxin complex has superior efficacy And has the advantage of having specific selectivity compared to the case of using as a protein toxin alone. By using this, it is possible to bind a lipoprotein that specifically binds to a protein overexpressed in a cancer cell, Lt; RTI ID = 0.0 > tumor < / RTI >

Description

Repubody-protein toxin conjugate, preparation method thereof, and use thereof " Repebody-Protein toxin conjugate, Preparation Methods and Use Thereof &

The present invention relates to a Repebody-Protein toxin conjugate, and more particularly, to a protein having an alpha helical capping motif and a VLR (leucine-rich repeat) family protein at the N-terminus of the LRR The present invention relates to a Lipid body-protein toxin complex in which a toxin protein is bound to a protein derivative having a Lipid body or a Lipid body as a basic skeleton, wherein the LRR structure originating from a variable lymphocyte receptor is linked to a variable region and exhibits substrate specificity, .

Protein toxins derived from bacteria and plants are composed of protein only and have a strong cell death effect. Therefore, although there has been an attempt to use protein toxins for cancer treatment and the like, there has been a strong in vivo side effect due to the absence of the function of targeting cancer cells alone. Thus, attempts have been made to combine binding proteins and toxins to develop cancer therapeutic proteins. Binding proteins can target cancer cells and adhere to the surface of cancer cells, and protein toxins can kill the cells to reduce side effects. The possibility of treating cancer of a target therapeutic agent made by such a method has already been proven. (Drug Discovery Today. 2011 April; 16 (11/12): 495-503)

Antibodies, which are typical binding proteins, have a high specific binding capacity to target proteins, have a long half-life and low toxicity, but they are expensive to produce in mammalian cell lines and generally have a large protein with a molecular weight of 150 kDa (2 light chains, 2 heavy chains) are connected to a disulfide bond, which is a disadvantage in that the thermodynamic stability is lower than that of other simple proteins.

In order to overcome these disadvantages, a new protein skeleton which can be specifically bound to a specific target protein and can be produced easily and inexpensively in microorganisms such as Escherichia coli, and which is very stable thermodynamically has been actively conducted Bar, dozens of novel protein structures have emerged (Urlich et al., Cancer Genomics Proteomics, 2013). As a representative example, Adnectin, which is the tenth domain of Fibronectin type 3, was developed by Adnexus and contracted with BMS to transfer technology to BMS in 2007. Affibody , Lipocalin skeleton Anticalin, and plasma protein Tetranectin-based Atrimers were developed. Avimers using various membrane receptors, the A-domain, were developed by Avidia and transferred to Amgen in 2006 for a technology transfer of $ 3.8 million . In addition, DARPins (developed by Molecular Partner and transferred to Roche on a scale of US $ 1 billion in 2013) based on Centrin and Ankyrin repeat miotif using FN3 domain, Fynomers using SH3 domain of Fyn protein, Nanobody (developed by Ablynx, Inc., and a Nanobody-drug polymer with Spirogen) is being actively studied.

Under these circumstances, the inventors have successfully developed a non-antibody protein scaffold, a repibody that can replace an existing antibody. Lipid bodies are one-fifth the size of antibodies and are produced in large quantities in E. coli, and animal tests confirm that immunogenicity is negligible. In addition, it has been proved that heat and pH stability are very excellent, the binding force to the target substance can be easily increased up to the pico-mole level, and the specificity to the target substance is very excellent.

The inventors of the present invention have conducted studies to develop a more effective binding protein-protein toxin conjugate according to the above-mentioned research flow. In the case of the lipofibrate-protein toxin complex using the lipoprotein as the binding protein, That is, E. coli is expressed in a soluble form so that it can be easily mass-produced at a low cost and that the T _m of the lipid body is thermodynamically stable at 85 ° C. or higher. The lipid body has a molecular weight of 25 ~ 30 kDa, it is confirmed that the therapeutic effect can be sufficiently exhibited as a protein toxin conjugate that specifically kills the target cell by binding to the target protein while retaining the excellent tissue penetration ability when developed as a diagnostic and therapeutic protein , Thereby completing the present invention.

Korean Patent Laid-Open Publication No. 10-2011-0099600 A1 (2011.09.08.) International Patent Publication No. WO2013-129852 A1 (Sep.

 Drug Discovery Today. 2011 April; 16 (11/12): 495-503.  Proc Natl Acad Sci U S 2012 Feb 28; 109 (9): 3299-304.  Cancer Genomics Proteomics. 2013 Jul-Aug; 10 (4): 155-68.

It is an object of the present invention to provide a recombinant microorganism which comprises a recombinant microorganism and a recombinant microorganism into which the vector is introduced and a recombinant microorganism into which the vector is introduced, And a method for producing the same.

Another object of the present invention is to provide a method for preventing or treating cancer, comprising administering a composition for preventing or treating cancer containing the Lippi-body-toxin protein complex and a composition for preventing or treating cancer comprising the Lippi-body- And to provide a method of treatment.

In order to accomplish the above object, the present invention provides a modified repeating module of VLR (Variable Lymphocyte Receptor) protein and an N-terminal of LRR (Leucine rich repeat) family protein having an alpha helical capping motif and a C - Provides a Repebody-protein toxin conjugate with a protein-toxin conjugate with a terminally fused repebody.

The present invention also provides a method for producing the Lipid body-protein toxin complex using the polynucleotide encoding the Lipid body-protein toxin complex, a vector comprising the polynucleotide, a recombinant microorganism into which the vector is introduced, and the recombinant microorganism .

The present invention also provides a composition for preventing or treating cancer comprising the Lippi-body-protein toxin complex as an active ingredient.

The Lipid-protein-toxin complex according to the present invention is a combination of a bacterial exotoxin having biological activity and a high substrate specificity and superior tissue and tumor penetration ability provided by Lipid body, And has the advantage of minimizing toxicity by having a specific selectivity for target cells as compared with the case of using only a protein toxin, and is also expressed in a soluble form in E. coli, It is useful for mass-production, thermodynamically stable, and excellent tissue penetration, and is useful for killing target cells by specifically binding to a target protein.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a structural schematic diagram of a Lipid body-protein toxin complex
Figure 2 shows the results of in vitro cytotoxicity of the anti-EGFR Lipid-Pseudomonas exotoxin A complex to the HCC827 cell line. PE40 is part of Pseudomonas exotoxin A. The anti-EGFR lipid bodies are designated as A11, AC1, EgA, and EgH9, and have increasingly strong binding affinity for EGFR in the order of A11, AC1, EgA, and EgH9. D3 is a control group with no binding to EGFR, and the vertical axis is the cell survival rate (%).
FIG. 3 shows the results of in vitro cytotoxicity of the anti-EGFR Lipid-Pseudomonas exotoxin A complex on the HT-29 cell line.
4 shows the results of in vitro cytotoxicity of 'EgH9-PE40' in A431, HepG2 and SW620 cell lines of anti-EGFR Lipibody-Pseudomonas exotoxin A complex. SW620 is a cell line that does not express EGFR on the cell surface.
FIG. 5 shows the results of inhibition of tumor growth of an in vivo xenografted mouse efficacy test on HT-29, wherein the drug was administered at a total of 5 times at 2-day intervals.
FIG. 6 shows the results of mouse weight measurement in the experiment of FIG.

The present invention provides a novel Lipid body-protein toxin complex in which a protein toxin is directly bound through gene binding to a 'protein comprising a lipid body or a lipid body as a basic framework'.

The term " lipibody " of the present invention means a water-soluble fusion polypeptide fused with an N-terminal and an LRR (Leucine rich repeat) family protein of an internulin protein and a polypeptide partially modified by the basic structure thereof, Which was first developed by Professor Kim Hak-Sung of KAIST and named as "Repebody" (Korean Patent Publication No. 10-2011-0099600 A1, Korean Patent Publication No. 1356075, International Patent Publication No. WO2013-129852 A1 and Lipibody Proc Natl Acad Sci US A. 2012 Feb 28; 109 (9): 3299-304). More specifically, the Lipid body is a microbial-derived, N-terminal of a Leucine rich repeat (LRR) family protein having an alpha helical capping motif, a modified repetitive module of a vaciable lymphocyte receptor protein and a VLR protein Terminus of the target protein. The lipid body has a characteristic of specifically binding to a specific target protein such as, for example, a receptor specifically expressed or overexpressed in cancer cells, and a repetitive module All of the proteins belonging to the LRR family having the amino acid sequence of SEQ ID NO: 1, and all fusion LRR family proteins that have improved their biological properties. Also, the N-terminal of the interleukin protein, which is a component of the lipid body, can be selected at the N-terminal having a high degree of structural similarity depending on the kind of the LRR family protein that can be fused, By selecting an amino acid, it is possible to change the amino acid of the corresponding module.

In one aspect, the present invention provides a modified repeat module of the VLR (Variable Lymphocyte Receptor) protein and an N-terminal of a Leucine rich repeat (LRR) family protein having an alpha helical capping motif and a C- Protein toxin conjugate in which the fusion toxin is bound to a fusion protein of the fusion protein.

The lipid body constituting the lipid-protein toxin complex in the present invention may be any of the known lipid body monomers described above or may be a lipid body multimer composed of two or more lipid body monomers (hereinafter referred to as "lipid body multimer") And an organic / inorganic compound such as polyethylene glycol (PEG) or a protein such as an Fc protein is introduced into the lipid body monomer or the lipid body multimer. In addition to the aforementioned lipid body in which the Fc protein or PEG is introduced into the lipid body monomer or oligomer, or each of them, various kinds of proteins, specific amino acid sequences and organic / inorganic compounds, etc. can be obtained by methods well known in the field of the present invention , And the resultant lipid bodies can be used without limitation as long as they do not deviate from the object of the present invention.

The term "protein toxin " of the present invention refers to toxic substances produced in living cells or organisms. Toxins include bacterial toxins, plant toxins, and animal toxins. But are not limited to, Pseudomonas exotoxin A, diphtheria antitoxin, botulism toxin, tetanus antitoxin, heterotoxin, cholera toxin, cytotoxin, lysine, and the like.

In the present invention, the protein toxin can be connected to the C-terminus of the lipid body through a spacer for the purpose of preserving the original structure of the protein and maximizing the activity of the toxin. Although the spacers are generally composed of amino acids, the type and length thereof are not particularly limited, and a (G3S) n spacer having a flexable property as in one embodiment of the present invention, i.e., three glycines and one serine (EAAAK) n (where E is glutamate, A is alanine, K is lysine, and n is generally one or more integers, or G4S Glycine and serine can be varied in number and arrangement.) In order to introduce more hydrophilic properties in consideration of physical properties, various spacer (s) containing charged amino acids such as DRDD (where D is aspartate and R is arginine) May also be used.

In the present invention, an endoplasmic reticulum residue sequence may be used to increase the penetration ability of the protein toxin into the cytoplasm. The ER retention sequence is generally composed of amino acids, but the type and length thereof are not particularly limited. As in one embodiment of the present invention, KDEL (K is Lysine, D is aspartate, E is glutamate, L is leucine ) May be used. The endoplasmic reticulum sequence may be linked to the C-terminus of the protein toxin to increase intracellular penetration of the protein toxin.

The Lipid body of the Lipid body-protein toxin complex according to the present invention may be selected from the group consisting of CD19, CD20, CD21, CD22, CD37, CD70, CD72, CD79a / b, CD180, CD30, CD33, CD43, CD56, CD74, CD138, endothelin B receptor, EGFR , CRPTO, FAP, mesothelin, G2D, 5T4, alpha v beta6, CD174, CD227 (MUC-1), CD326 (Epcam), ED-B, CD227 (MUC-1), nectin- , GPNMB, LIV1A, MUC16 (CA125), TIM-1 (CDX-014), GD2, GPNMB, PMEL17, SMA, STEAP-1, TENB2 and CAIX .

For example, when the Lippi-body protein-toxin complex of the present invention is developed as an anticancer therapeutic agent, the target protein is a specific or over-expressed protein in cancer cells, including but not limited to a non-Hodgkin lymphoma (CD30), CD20, CD37, CD70, CD72, CD79a / b, and CD180 in the case of non-Hodgkin's lymphoma and CD19, CD20, (CD45), CD74, CD138, and CD134 are the major indications for CD34, CD43, and acute-lymphoid leukemia, respectively, when the primary indications are CD43 and multiple myeloma as the primary indications. EGFR, CD56, CD326, CRIPTO, FAP, mesothelin, G2D, and EGFR were the major indications for lung cancer, endothelin B receptor, head and neck cancer, (EGFR), CD74, CD174, CD227 (MUC-1), CD326 (Epcam), and CRIPTO are the major indications for EGFR, EGFRvIII, and colorectal / rectal cancer when primary indications are 5T4 and alpha v beta6 and glioblastoma , FAP, and ED-B, and CD74, CD227 (MUC-1), nectin-4 (ASG-22ME) and alpha v beta6 when the primary indication for pancreatic cancer is HER2, CD174, GPNMB (CA125), TIM-1 (CDX-014), and mesothelin and melanoma are the main indications for ovarian carcinoma, ovarian cancer, and CRITTO, nectin-4 (ASG-22ME) 1, and TENB2 for primary indications of prostate cancer; CAIX and TIM-1 (CDX) for predominant indi- rect kidney cancer; and GX2, GPNMB, ED-B, PMEL17 and endothelin B receptor -014), mesothelioma (mesothelioma) and mesothelin can be the main indications.

The above indications and target proteins do not necessarily have to be identical, and in particular, EGFR and HER2 do not need to be limited to the indications that are overexpressed in various cancers.

In one embodiment of the present invention, an anti-EGFR Lipid body derivative-protein toxin conjugate using Lipid Binding to EGFR (Endothelial Growth Factor Receptor) was prepared and its efficacy was confirmed.

In the present invention, the Lippi-body protein-toxin complex may be characterized by being represented by an amino acid sequence of any one of SEQ ID NOS: 1 to 4.

The present invention also relates to polynucleotides encoding the Lippi-body protein toxin complex.

The present invention also relates to a vector comprising said polynucleotide.

As used herein, the term "vector" means a DNA product containing a nucleotide sequence of a polynucleotide encoding the desired protein operably linked to a suitable regulatory sequence so as to be capable of expressing the protein of interest in a suitable host cell. The regulatory sequence may include a promoter capable of initiating transcription, any operator sequence for modulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating termination of transcription and translation, And can be variously manufactured. The promoter of the vector may be constitutive or inducible. The vector may be transcribed into a suitable host, then replicated or functional, independent of the host genome, and integrated into the genome itself.

The vector used in the present invention is not particularly limited as long as it is replicable in the host cell, and any vector known in the art can be used. Examples of commonly used vectors include plasmids in the natural or recombinant state, phagemids, cosmids, viruses and bacteriophages. For example, pWE15, M13, λMBL3, λMBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, and Charon21A can be used as the phage vector or cosmid vector, and pBR, pUC and pBluescriptII , pGEM-based, pTZ-based, pCL-based, pET-based, and the like. 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 vector and the like can be used. Most preferably, pACYC177, pCL, pCC1BAC vectors can be used.

The present invention also relates to a recombinant microorganism into which a vector containing the polynucleotide or the polynucleotide is introduced.

The term " recombinant microorganism " in the present invention means that a vector having a polynucleotide encoding at least one target protein is introduced into a host cell, or a polynucleotide encoding at least one target protein is introduced into a microorganism so that the polynucleotide is integrated into a chromosome Means a cell infected with a trait to express a target protein, and may be any cell such as a eukaryotic cell, a prokaryotic cell, etc., but is not limited to, a bacterial cell such as Escherichia coli, Streptomyces or Salmonella typhimurium; Yeast cells; Fungal cells such as Pichia pastoris; Insect cells such as Drosophila and Spodoptera Sf9 cells; CHO, COS, NSO, 293, and Bowmanlanoma cells.

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

The present invention also provides a method for producing a recombinant microorganism, comprising the steps of (i) culturing the recombinant microorganism to express the Lippi-body protein toxin complex; And (ii) recovering the expressed Lipid body-protein toxin complex.

In the above method, the step of culturing the transformant 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 like, and the culture conditions are not particularly limited thereto (PH 5 to 9, preferably pH 6 to 8, most preferably pH 6.8) using a basic compound such as sodium hydroxide, potassium hydroxide or ammonia or an acidic compound such as phosphoric acid or sulfuric acid, And the oxygen or oxygen-containing gas mixture can be introduced into the culture to maintain aerobic conditions, and the incubation temperature can be maintained at 20 to 45 캜, preferably 25 to 40 캜, for about 10 to 160 hours Lt; / RTI > The polypeptide produced by the culture may be secreted into the medium or left in the cells.

In addition, the culture medium used may be a carbon source such as sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats such as soybean oil, sunflower seeds Alcohols such as glycerol and ethanol, and organic acids such as acetic acid, etc. may be used individually or in combination with one or more of the following: ; Examples of nitrogen sources include nitrogen-containing organic compounds such as peptone, yeast extract, juice, malt extract, corn steep liquor, soybean meal and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, Ammonium nitrate) may be used individually or in combination; As the phosphorus source, potassium dihydrogenphosphate, dipotassium hydrogenphosphate and the corresponding sodium-containing salt may be used individually or in combination; Other metal salts such as magnesium sulfate or iron sulfate, amino acids and vitamins.

The method for recovering the Lippi-body toxin protein complex produced in the present invention can be carried out by a suitable method known in the art according to a culture method, such as a batch, continuous or fed-batch culture method, Can be collected.

The present invention, in another aspect, relates to a composition for preventing or treating disease comprising a lipid-protein-toxin complex as an active ingredient.

In the present invention, the disease may include, but is not limited to, all diseases known in the art, including gastrointestinal diseases, respiratory diseases, cardiovascular diseases, kidney diseases, endocrine diseases, immune diseases, blood diseases, Metabolic disorders, sexually transmitted diseases, cancer, psychiatric disorders, diseases associated with addiction or surgical diseases. More specifically, the present invention relates to a pharmaceutical composition for preventing or treating diseases such as cold, pneumonia, tuberculosis, AIDS, plague, osteoarthritis, and the like, including cancers such as brain cancer, leukemia, stomach cancer, colon cancer, rectal cancer, liver cancer, lung cancer, esophageal cancer, prostate cancer, breast cancer, Other diseases such as infectious diseases such as prion diseases, hepatitis B, atherosclerosis, hernias, smallpox, anemia, poliomyelitis, allergy, asthma, diabetes, arteriosclerosis, nephropathy, stroke, Alzheimer's disease and obesity.

In the present invention, the disease is preferably cancer, more preferably a non-Hodgkin lymphoma, a non-Hodgkin lymphoma, an acute-myeloid leukemia, A cancer selected from the group consisting of acute-lymphoid leukemia, multiple myeloma, head and neck cancer, lung cancer, glioblastoma, large intestine / rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, melanoma, , Renal cancer, and mesothelioma.

The term "treatment" as used herein means to inhibit or alleviate the disease or one or more symptoms caused therefrom by the administration of the composition, as well as to prevent the progress of the disease or treatment of reversing the symptoms of the disease. The term "prevention" in the present invention means any action that inhibits disease or delay the onset of the disease by administration of the composition.

In the present invention, the lipid-toxin protein complex, which has been developed in the present invention, is combined with a substrate of a lipid body. For example, when the lipid body is EGFR, the lipid body and the EGFR are combined , And the toxin protein acts on the cell overexpressing EGFR to prevent or treat the disease.

The composition for preventing or treating disease comprising the Lippi-body protein-toxin complex of the present invention as an active ingredient may further comprise a pharmaceutically acceptable carrier and may be formulated together with the carrier.

In the present invention, the amount of the lipid-protein-toxin complex in the composition is not limited, but may be added in an amount of 0.01 to 95% by weight based on the total weight of the composition.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not irritate the organism and does not interfere with the biological activity and properties of the administered compound. Examples of the pharmaceutical carrier which is acceptable for the composition to be formulated into a liquid solution include sterilized and sterile water suitable for the living body such as saline, sterilized water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

The composition for preventing or treating cancer comprising the polypeptide of the present invention and a pharmaceutically acceptable carrier can be applied to any formulation containing it as an active ingredient and can be manufactured into oral or parenteral formulations. The pharmaceutical formulations of the present invention may be administered orally, rectally, nasal, topical (including under the ball and tongue), subcutaneous, vaginal or parenteral (intramuscular, subcutaneous And intravenous), or forms suitable for administration by inhalation or insufflation.

Examples of formulations for oral administration comprising the composition of the present invention as an active ingredient include tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs can do. A binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin, an excipient such as dicalcium phosphate, a disintegrating agent such as corn starch or sweet potato starch, Calcium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax. In the case of a capsule formulation, in addition to the above-mentioned materials, a liquid carrier such as a fatty oil may be further contained.

Examples of the formulations for parenteral administration containing the composition of the present invention as an active ingredient include injection forms such as subcutaneous injection, intravenous injection or intramuscular injection, suppository injection method, or aerosol agent for inhalation through a respirator . ≪ / RTI > For formulation into injectable formulations, the compositions of the present invention may be formulated as solutions or suspensions in water with stabilizers or buffers in water, and formulated for unitary administration of ampoules or vials. For injection into suppositories, it may be formulated into rectal compositions such as suppositories or enema preparations, including conventional suppository bases such as cocoa butter or other glycerides. When formulated for spraying, such as an aerosol formulation, a propellant or the like may be formulated with the additive such that the water-dispersed concentrate or wet powder is dispersed.

In another aspect, the present invention relates to a method for preventing or treating cancer, comprising administering a composition for preventing or treating cancer comprising the Lippi-body-toxin protein complex.

As used herein, the term "administering" means introducing the pharmaceutical composition of the present invention to a patient in any suitable manner. The administration route of the composition of the present invention may be administered through various routes of oral or parenteral administration as long as it can reach the target tissues. Specifically, oral administration, rectal administration, topical administration, intravenous injection, intraperitoneal injection, intramuscular injection, Transdermal, intranasal, inhalation, intra-ocular or intradermal routes.

The therapeutic method of the present invention includes administering a pharmaceutical effective amount of the composition for preventing or treating cancer of the present invention. It will be apparent to those skilled in the art that the appropriate total daily dose may be determined by the practitioner within the scope of sound medical judgment. The specific therapeutically effective amount for a particular patient will depend upon a variety of factors, including the type and extent of the response to be achieved, the specific composition, including whether or not other agents are used, the age, weight, general health status, sex and diet, The route of administration and the fraction of the composition, the duration of the treatment, the drugs used or co-used with the specific composition, and the like, well known in the medical arts. Therefore, the effective amount of the composition for preventing or treating cancer that is suitable for the purpose of the present invention is preferably determined in consideration of the above-mentioned matters.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example 1 Preparation of Lipid-Protein Toxin Complex

A gene encoding each complex was cloned into pET21a (Novagen, USA) expression vector and transformed into E. coli BL21 (DE3) (Novagen, USA). A single colony of Escherichia coli was inoculated into LB (Luria-Bertani) medium (Duchefa, Netherlands) to which 100 μg / mL of ampicillin antibiotic was administered and cultured at 37 ° C for 12 hours. After inoculating the culture medium in ampicillin -LB medium newly prepared and diluted to a concentration of 1/100 and similarly cultured at 37 ℃, when OD ₆₀₀ reaches 0.5 IPTG (isopropylβ-D-1 at a final concentration of 0.5 mM -thiogalactopyranoside) to induce protein expression. Then, the temperature was lowered to 18 ° C and cultured for 20 hours. The protein was purified purely by affinity and gel permeation chromatography.

Test Example 1. In vitro cytotoxicity analysis of Lipid body-protein toxin complex

1. Cell line

(EGFR-overexpressing colorectal cancer cell line), HepG2 cell (EGFR-expressing hepatocellular carcinoma cell line), SW620 cell (EGFR-overexpressing colorectal cancer cell line), and HCC827 cell (EGFR-overexpressing non-small cell lung cancer cell line) Expression colorectal cancer cell line) according to the recommended culture conditions.

2. Test sample

As one embodiment of the Lipid body-protein toxin complex, the anti-EGFR Lipid-Pseudomonas exotoxin A complex was used. Anti-EGFR lipid bodies have increasingly strong binding affinity for EGFR in the order of A11, AC1, EgA, and EgH9. D3 is a control group with no binding to EGFR. Pseudomonas exotoxin A was connected to the C-terminus of the lipid body, and the vesicle residue sequence (KDEL) was connected to the C-terminus of P. aeruginosa exotoxin A to vaporize the cytoplasmic penetration of the toxin.

3. Test method

The in vitro activity of ERDC was measured using the cell line described above (control group, Lipidase (D3) - Pseudomonas exotoxin A complex without binding to EGFR was used). Cells were plated in 96-well culture plates at 5 × 10 ³ cells. After 24 hours of incubation, the lipid-protein-toxin complex was added at various concentrations. After 72 hours, the number of viable cells was counted using MTT dye. Absorbance was measured at 560 nm using SpectraMax 190 (Molecular Devices, USA).

4. Test results

The selective cytotoxicity of the anti-EGFR Lipidase-Pseudomonas exotoxin A complex prepared according to the present invention was tested for HCC827 (FIG. 2), HT-29 (FIG. 3), A431, It was confirmed that the cytotoxicity was low in the HepG2 (Fig. 4) cell line, while the SW620 cell line (Fig. 4) in which EGFR was not expressed was found to have low cytotoxicity. The efficacy of the anti-EGFR Lipid-Pseudomonas exotoxin A complexes with various binding potencies was tested in in vitro cytotoxicity experiments and it was found that EgH9-PE40 with the highest binding capacity exhibited the best apoptotic effect (Figs. 2 and 3).

Test Example 2. In vivo tumor suppression test of the Lipid body-protein toxin complex

1. Test sample

PBS buffer (control), EgA-PE40, EgH9-PE40 (anti-EGFR lipid-protein toxin complex) were used for in vivo xenografted mouse efficacy studies.

2. Test method

Ten Balb / C nude mice were treated with 10 ⁷ HT-29 cell lines and 5 groups were divided into mice having tumor sizes of 50-100 mm ³ . Each group was administered 5 times, twice a day, once every two days with PBS, 3 g EgA-PE40, 6 g EgA-PE40, 2 g EgH9-PE40 and 4 g EgH9-PE40 and the body size and size of the tumor were observed for 28 days.

3. Test results

Through the above examples, it was confirmed that the Lipid body-protein toxin complex produced by the method of the present invention inhibited the growth of tumors more than the PBS control group (FIG. 5), and the weight of mice was significantly different between the groups (Fig. 6).

The present inventors completed the present invention by confirming that the Lipid body-protein toxin complex exhibits excellent cytotoxicity in vitro and inhibition of cancer cell growth in vivo .

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

                         SEQUENCE LISTING <110> Korea Advanced Institute of Science and Technology   <120> Repebody-Protein toxin Conjugate, Preparation Methods and Use        Thereof <130> P15-B315 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 624 <212> PRT <213> A11-PE40 <400> 1 Met Glu Thr Ile Thr Val Ser Thr Pro Ile Lys Gln Ile Phe Pro Asp 1 5 10 15 Asp Ala Phe Ala Glu Thr Ile Lys Ala Asn Leu Lys Lys Lys Ser Val             20 25 30 Thr Asp Ala Val Thr Gln Asn Glu Leu Asn Ser Ile Asp Gln Ile Ile         35 40 45 Ala Asn Asn Ser Asp Ile Lys Ser Val Gln Gly Ile Gln Tyr Leu Pro     50 55 60 Asn Val Arg Tyr Leu Ala Leu Gly Gly Asn Lys Leu His Asp Ile Ser 65 70 75 80 Ala Leu Lys Glu Leu Thr Asn Leu Thr Tyr Leu Met Leu His Tyr Asn                 85 90 95 Gln Leu Gln Ile Leu Pro Asn Gly Val Phe Asp Lys Leu Thr Asn Leu             100 105 110 Lys Glu Leu Tyr Leu Ser Glu Asn Gln Leu Gln Ser Leu Pro Asp Gly         115 120 125 Val Phe Asp Lys Leu Thr Asn Leu Thr Glu Leu Asp Leu Ser Tyr Asn     130 135 140 Gln Leu Gln Ser Leu Pro Glu Gly Val Phe Asp Lys Leu Thr Gln Leu 145 150 155 160 Lys Asp Leu Arg Leu Tyr Gln Asn Gln Leu Lys Ser Val Pro Asp Gly                 165 170 175 Val Phe Asp Arg Leu Thr Ser Leu Gln Tyr Ile Trp Leu His Asp Asn             180 185 190 Pro Trp Asp Cys Thr Cys Pro Gly Ile Arg Tyr Leu Ser Glu Trp Ile         195 200 205 Asn Lys His Ser Gly Val Val Arg Asn Ser Ala Gly Ser Val Ala Pro     210 215 220 Asp Ser Ala Lys Cys Ser Gly Ser Gly Lys Pro Val Arg Ser Ile Ile 225 230 235 240 Cys Pro Thr Glu Phe Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly                 245 250 255 Ser Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His             260 265 270 Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu         275 280 285 Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr     290 295 300 Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn 305 310 315 320 Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg                 325 330 335 Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu             340 345 350 Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala         355 360 365 Ala Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu     370 375 380 Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr 385 390 395 400 Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Asp Val Ser Phe Ser                 405 410 415 Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala His             420 425 430 Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr His Gly Thr         435 440 445 Phe Leu Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala Arg     450 455 460 Ser Gln Asp Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly Asp 465 470 475 480 Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala Arg                 485 490 495 Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro Arg Ser             500 505 510 Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro Glu         515 520 525 Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu Pro Leu Arg     530 535 540 Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg Leu Glu Thr 545 550 555 560 Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Val Val Ile Pro Ser Ala                 565 570 575 Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp Leu Asp Pro Ser Ser             580 585 590 Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala Ser         595 600 605 Gln Pro Gly Lys Pro Pro His His His His His Lys Asp Glu Leu     610 615 620 <210> 2 <211> 624 <212> PRT <213> AC1 to PE40 <400> 2 Met Glu Thr Ile Thr Val Ser Thr Pro Ile Lys Gln Ile Phe Pro Asp 1 5 10 15 Asp Ala Phe Ala Glu Thr Ile Lys Ala Asn Leu Lys Lys Lys Ser Val             20 25 30 Thr Asp Ala Val Thr Gln Asn Glu Leu Asn Ser Ile Asp Gln Ile Ile         35 40 45 Ala Asn Asn Ser Asp Ile Lys Ser Val Gln Gly Ile Gln Tyr Leu Pro     50 55 60 Asn Val Arg Tyr Leu Ala Leu Gly Gly Asn Lys Leu His Asp Ile Ser 65 70 75 80 Ala Leu Lys Glu Leu Thr Asn Leu Thr Tyr Leu Met Leu His Tyr Asn                 85 90 95 Gln Leu Gln Ile Leu Pro Asn Gly Val Phe Asp Lys Leu Thr Asn Leu             100 105 110 Lys Glu Leu Tyr Leu Ser Glu Asn Gln Leu Gln Ser Leu Pro Asp Gly         115 120 125 Val Phe Asp Lys Leu Thr Asn Leu Thr Glu Leu Asp Leu Ala Arg Asn     130 135 140 Gln Leu Gln Ser Leu Pro Lys Gly Val Phe Asp Lys Leu Thr Gln Leu 145 150 155 160 Lys Asp Leu Arg Leu Tyr Gln Asn Gln Leu Lys Ser Val Pro Asp Gly                 165 170 175 Val Phe Asp Arg Leu Thr Ser Leu Gln Tyr Ile Trp Leu His Asp Asn             180 185 190 Pro Trp Asp Cys Thr Cys Pro Gly Ile Arg Tyr Leu Ser Glu Trp Ile         195 200 205 Asn Lys His Ser Gly Val Val Arg Asn Ser Ala Gly Ser Val Ala Pro     210 215 220 Asp Ser Ala Lys Cys Ser Gly Ser Gly Lys Pro Val Arg Ser Ile Ile 225 230 235 240 Cys Pro Thr Glu Phe Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly                 245 250 255 Ser Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His             260 265 270 Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu         275 280 285 Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr     290 295 300 Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn 305 310 315 320 Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg                 325 330 335 Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu             340 345 350 Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala         355 360 365 Ala Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu     370 375 380 Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr 385 390 395 400 Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Asp Val Ser Phe Ser                 405 410 415 Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala His             420 425 430 Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr His Gly Thr         435 440 445 Phe Leu Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala Arg     450 455 460 Ser Gln Asp Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly Asp 465 470 475 480 Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala Arg                 485 490 495 Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro Arg Ser             500 505 510 Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro Glu         515 520 525 Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu Pro Leu Arg     530 535 540 Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg Leu Glu Thr 545 550 555 560 Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Val Val Ile Pro Ser Ala                 565 570 575 Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp Leu Asp Pro Ser Ser             580 585 590 Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala Ser         595 600 605 Gln Pro Gly Lys Pro Pro His His His His His Lys Asp Glu Leu     610 615 620 <210> 3 <211> 624 <212> PRT <213> EgA-PE40 <400> 3 Met Glu Thr Ile Thr Val Ser Thr Pro Ile Lys Gln Ile Phe Pro Asp 1 5 10 15 Asp Ala Phe Ala Glu Thr Ile Lys Ala Asn Leu Lys Lys Lys Ser Val             20 25 30 Thr Asp Ala Val Thr Gln Asn Glu Leu Asn Ser Ile Asp Gln Ile Ile         35 40 45 Ala Asn Asn Ser Asp Ile Lys Ser Val Gln Gly Ile Gln Tyr Leu Pro     50 55 60 Asn Val Arg Tyr Leu Ala Leu Gly Gly Asn Lys Leu His Asp Ile Ser 65 70 75 80 Ala Leu Lys Glu Leu Thr Asn Leu Thr Tyr Leu Met Leu His Tyr Asn                 85 90 95 Gln Leu Gln Ile Leu Pro Asn Gly Val Phe Asp Lys Leu Thr Asn Leu             100 105 110 Lys Glu Leu Tyr Leu Ser Glu Asn Gln Leu Gln Ser Leu Pro Asp Gly         115 120 125 Val Phe Asp Lys Leu Thr Asn Leu Thr Glu Leu Asp Leu Ala Arg Asn     130 135 140 Gln Leu Gln Ser Leu Pro Lys Gly Val Phe Asp Lys Leu Thr Gln Leu 145 150 155 160 Lys Asp Leu Arg Leu Tyr Glu Asn Gln Leu Lys Ser Val Pro Asp Gly                 165 170 175 Val Phe Asp Arg Leu Thr Ser Leu Gln Tyr Ile Trp Leu His Asp Asn             180 185 190 Pro Trp Asp Cys Thr Cys Pro Gly Ile Arg Tyr Leu Ser Glu Trp Ile         195 200 205 Asn Lys His Ser Gly Val Val Arg Asn Ser Ala Gly Ser Val Ala Pro     210 215 220 Asp Ser Ala Lys Cys Ser Gly Ser Gly Lys Pro Val Arg Ser Ile Ile 225 230 235 240 Cys Pro Thr Glu Phe Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly                 245 250 255 Ser Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His             260 265 270 Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu         275 280 285 Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr     290 295 300 Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn 305 310 315 320 Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg                 325 330 335 Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu             340 345 350 Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala         355 360 365 Ala Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu     370 375 380 Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr 385 390 395 400 Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Asp Val Ser Phe Ser                 405 410 415 Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala His             420 425 430 Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr His Gly Thr         435 440 445 Phe Leu Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala Arg     450 455 460 Ser Gln Asp Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly Asp 465 470 475 480 Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala Arg                 485 490 495 Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro Arg Ser             500 505 510 Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro Glu         515 520 525 Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu Pro Leu Arg     530 535 540 Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg Leu Glu Thr 545 550 555 560 Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Val Val Ile Pro Ser Ala                 565 570 575 Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp Leu Asp Pro Ser Ser             580 585 590 Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala Ser         595 600 605 Gln Pro Gly Lys Pro Pro His His His His His Lys Asp Glu Leu     610 615 620 <210> 4 <211> 624 <212> PRT <213> EgH9-PE40 <400> 4 Met Glu Thr Ile Thr Val Ser Thr Pro Ile Lys Gln Ile Phe Pro Asp 1 5 10 15 Asp Ala Phe Ala Glu Thr Ile Lys Ala Asn Leu Lys Lys Lys Ser Val             20 25 30 Thr Asp Ala Val Thr Gln Asn Glu Leu Asn Ser Ile Asp Gln Ile Ile         35 40 45 Ala Asn Asn Ser Asp Ile Lys Ser Val Gln Gly Ile Gln Tyr Leu Pro     50 55 60 Asn Val Arg Met Leu His Leu Pro Ser Asn Lys Leu His Asp Ile Ser 65 70 75 80 Ala Leu Lys Glu Leu Thr Asn Leu Thr Tyr Leu Met Leu His Tyr Asn                 85 90 95 Gln Leu Gln Ile Leu Pro Asn Gly Val Phe Asp Lys Leu Thr Asn Leu             100 105 110 Lys Glu Leu Tyr Leu Ser Glu Asn Gln Leu Gln Ser Leu Pro Asp Gly         115 120 125 Val Phe Asp Lys Leu Thr Asn Leu Thr Glu Leu Asp Leu Ala Arg Asn     130 135 140 Gln Leu Gln Ser Leu Pro Lys Gly Val Phe Asp Lys Leu Thr Gln Leu 145 150 155 160 Lys Asp Leu Arg Leu Tyr Glu Asn Gln Leu Lys Ser Val Pro Asp Gly                 165 170 175 Val Phe Asp Arg Leu Thr Ser Leu Gln Tyr Ile Trp Leu His Asp Asn             180 185 190 Pro Trp Asp Cys Thr Cys Pro Gly Ile Arg Tyr Leu Ser Glu Trp Ile         195 200 205 Asn Lys His Ser Gly Val Val Arg Asn Ser Ala Gly Ser Val Ala Pro     210 215 220 Asp Ser Ala Lys Cys Ser Gly Ser Gly Lys Pro Val Arg Ser Ile Ile 225 230 235 240 Cys Pro Thr Glu Phe Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly                 245 250 255 Ser Glu Gly Gly Ser Leu Ala Ala Leu Thr Ala His Gln Ala Cys His             260 265 270 Leu Pro Leu Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu         275 280 285 Gln Leu Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr     290 295 300 Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn 305 310 315 320 Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg                 325 330 335 Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu             340 345 350 Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala         355 360 365 Ala Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu     370 375 380 Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr 385 390 395 400 Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Asp Val Ser Phe Ser                 405 410 415 Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala His             420 425 430 Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr His Gly Thr         435 440 445 Phe Leu Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala Arg     450 455 460 Ser Gln Asp Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly Asp 465 470 475 480 Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala Arg                 485 490 495 Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro Arg Ser             500 505 510 Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro Glu         515 520 525 Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu Pro Leu Arg     530 535 540 Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg Leu Glu Thr 545 550 555 560 Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Val Val Ile Pro Ser Ala                 565 570 575 Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp Leu Asp Pro Ser Ser             580 585 590 Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala Ser         595 600 605 Gln Pro Gly Lys Pro Pro His His His His His Lys Asp Glu Leu     610 615 620

Claims (18)

A repetitive module of a VLR (Variable Lymphocyte Receptor) protein and an N-terminal of an LRR (Leucine rich repeat) family protein having an alpha helical capping motif (capping mofit) and a repeating body ) And a protein toxin conjugate (Repebody-protein toxin conjugate).
The repebody-protein toxin conjugate according to claim 1, wherein the lipid body is composed of a monomer or a multimer.
[Claim 3] The Repebody-protein toxin conjugate according to claim 2, wherein the Lipid body monomer or the multimer is a form in which an Fc protein and / or a polyethylene glycol (PEG) is introduced into each of them.
The method of claim 1, wherein the Lipid body is selected from the group consisting of CD19, CD20, CD21, CD22, CD37, CD70, CD72, CD79a / b, CD180, CD30, CD33, CD43, CD56, CD74, CD138, endothelin B receptor, EGFR, (MUC-1), nectin-4 (ASG-22ME), HER2, GPNMB, MEK-1, FAP, mesothelin, G2D, 5T4, alpha v beta6, CD174, CD227 Characterized in that it specifically binds to a protein selected from the group consisting of LIV1A, MUC16 (CA125), TIM-1 (CDX-014), GD2, GPNMB, PMEL17, SMA, STEAP-1, TENB2 and CAIX Repebody-protein toxin conjugate.
The recombinant protein-toxin conjugate according to claim 1, wherein the protein toxin is selected from the group consisting of bacterial toxins, plant toxins and animal toxins.
6. The method of claim 5, wherein the protein toxin is selected from the group consisting of Pseudomonas exotoxin A, diphtheria antitoxin, botulism toxin, tetanus antitoxin, heterotoxin, cholera toxin, cytotoxin and lysine. Repebody-protein toxin conjugate.
2. The Lipid-protein toxin conjugate according to claim 1, wherein the Lipid body and the protein toxin are bound through a spacer.
The method of claim 7, wherein the spacer is selected from the group consisting of (G3S) n spacer, (EAAAK) n spacer, and DRDD spacer, 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. 2. A repebody-protein toxin conjugate according to claim 1,
4. The conjugate according to claim 1, wherein the repebody-protein toxin conjugate comprises an endoplasmic reticulum residue. 2. The repebody-protein toxin conjugate according to claim 1, wherein the repebody-protein toxin conjugate comprises an endoplasmic reticulum residue.
10. The repebody-protein toxin conjugate according to claim 9, wherein the ERC residual sequence is represented by an amino acid sequence of KDEL.
The method according to claim 1, The Repebody-protein toxin conjugate is represented by the amino acid sequence of any one of SEQ ID NOS: 1 to 4. The Repebody-protein toxin conjugate according to claim 1,
A polynucleotide encoding the lipid-protein toxin complex of claim 1.
12. An expression vector comprising the polynucleotide of claim 12.
12. A recombinant microorganism into which the polynucleotide of claim 12 has been introduced.
13. A recombinant microorganism into which the expression vector of claim 13 has been introduced.
(I) culturing the recombinant microorganism of claim 14 or 15 to produce the lipid-protein toxin complex of claim 1; And
(Ii) recovering the resulting Lipid body-protein toxin complex.
A composition for preventing or treating cancer, which comprises the lipid-protein toxin complex of claim 1 as an active ingredient.
18. The method of claim 17, wherein the cancer is selected from the group consisting of non-Hodgkin lymphoma, non-Hodgkin lymphoma, acute-myeloid leukemia, acute-lymphoid leukemia, (Eg, multiple myeloma, head and neck cancer, lung cancer, glioblastoma, large intestine / rectal cancer, pancreatic cancer, breast cancer, ovarian cancer, melanoma, prostate cancer, kidney cancer, and mesothelioma) &Lt; / RTI &gt; wherein said composition is selected from the group consisting of: &lt; RTI ID = 0.0 &gt;

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