KR20160080832A - Repebody Derivative-Drug Conjugates, Preparation Methods and Use Thereof - Google Patents

Abstract

The present invention relates to a drug conjugate using a repebody derivative and a method for preparing the same. Particularly, the present invention relates to a repebody derivative-drug conjugate (RDC) comprising a repebody having substrate specificity through the connection of a leucine-rich repeat (LRR) structure derived from a variable lymphocyte receptor (VLR) as a variable domain to an N-terminal of an LRR family protein having an alpha spiral capping motif, and an active substance, such as a drug, toxin, ligand and detection label, conjugated with the repebody. The present invention also relates to a method for preparing the repebody derivative-drug conjugate. The RDC according to the present invention shows high substrate specificity and excellent tissue and tumor permeability provided by the repebody in combination with a biologically active compound, and thus has significantly higher effect as compared to a repebody alone and specific selectivity as compared to the compound alone. Thus, it is expected that a conjugate of the repebody that is bound specifically to a protein over-expressed in cancer cells with a toxin killing cells can destroy only cancer cells selectively and can be used for treatment for tumors.

Description

[0001] The present invention relates to a lipobody derivative-drug conjugate, a preparation method thereof, and a use thereof.

The present invention relates to a Repebody Derivative-Drug Conjugates, and more particularly to a method for producing a leucine-rich repeat (LRR) family protein having an alpha helical capping motif and a VLR lymphocyte receptor) is linked to a variable region and a protein derivative having a Lipid body or a Lipid body showing substrate specificity is attached to a lipid body conjugated with a substance having activity such as drug, toxin, ligand and detection label (RDC), a preparation method thereof, and a use thereof.

Since the development of monoclonal antibodies from hybridoma cells by Kohler and Milstein in 1975, efforts have been made to develop them as therapeutic antibodies.

However, antibody-drug conjugates (ADCs) that bind toxins to antibodies have been studied since the antibody itself does not kill cancer cells. MMAE (monomethyl auristatin E) was added to antibodies targeting CD30, (Brentuximab vedotin) was developed by Seattle Genetics of the United States and approved by the FDA in 2011 for indications for relapsed AML (Acute Myeloid Lymphoma). In 2013, HER2 (Human EGFR Receptor 2 (Ado-trastuzumab emtansine) conjugated with DM1, a toxin of the maytansinoid family, has been developed by Roche, Switzerland, in Herceptin, a therapeutic antibody targeting the HER2-positive metastatic breast cancer patient who is resistant to herceptin And received approval from the FDA.

On the other hand, antibodies used in antibody therapeutics and ADCs have a high specific binding capacity to target proteins, have a long half-life and low toxicity, but are expensive to produce in mammalian cell lines and generally have a molecular weight of 150 kDa (2 light chains, 2 heavy chains) are linked to disulfide bonds, 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 Inc. and contracted with BMS to transfer technology to BMS in 2007. Affibody , Anticalin with Lipocalin skeleton and Atrimers with plasma protein Tetranectin were developed. Avimers using A-domains of various membrane receptors were developed by Avidia and transferred to Amgen in 2006 for a technology transfer of $ 3.8 million .

DARPins based on Centrin and Ankyrin repeat miotif using the FN3 domain (developed by Molecular Partner and transferred to Roche in the amount of US $ 1 billion in 2013), Fynomers using the SH3 domain of Fyn protein, Nanobody (developed by Ablynx, Inc., and Nanobody-drug polymer with Spirogen) is being actively studied.

Korean Patent Laid-Open Publication No. 10-2011-0099600 A1 (2011.09.08.) International Patent Publication No. WO2013-129852 A1 (Sep. U.S. Published Patent Application No. US2012-0308584 A1 (2012.12.06.)

 Proc Natl Acad Sci U S 2012 Feb 28; 109 (9): 3299-304.  Cancer Genomics Proteomics. 2013 Jul-Aug; 10 (4): 155-68.

The inventors of the present invention have been conducting studies to develop a more effective protein-drug complex in accordance with the above-mentioned research flow. In the conventional antibody-drug complexes, the complexes of lipid-drug complexes when creating a, that the advantages of the repeater body, that is, points can be expressed in soluble form in E. coli (soluble form) easily mass-produced at low cost and, in the repeater body T _m is thermodynamically very stable with less than 85 ℃ , Lipid Bodies are 25 ~ 30 kDa in molecular weight and have excellent tissue penetration ability when developed as a diagnostic and therapeutic protein, but they are drug complexes that specifically kill target cells by binding to target proteins. A new form of the drug complex, the Repibody derivative-Drug Conjugate e, RDC), and completed the present invention.

The present invention relates to a method of binding a drug to a protein comprising a lipid body or a lipid body as a basic skeleton (hereinafter referred to as a "lipid body derivative") using an enzyme or the like, Thereby providing a body derivative-drug complex.

Lipibodies are water-soluble fusion polypeptides fused with N-terminal and LRR (Leucine rich repeat) family proteins of interleukin proteins, and partially modified polypeptides based on them. (Korean Patent Publication No. 10-2011-0099600 A1, Korean Patent Publication No. 1356075, International Patent Publication No. WO2013-129852 A1 and Lipid body-related article 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.

The Lipid body derivative constituting the Lipid body derivative-drug complex in the present invention may be a known Lipid body monomer or a Lipid body multimer composed of two or more Lipid body monomers (hereinafter referred to as "Lipid body multimer") And also an organic / inorganic compound such as a protein such as Fc protein or polyethylene glycol (PEG) is introduced into these lipid body monomers or lipid body dimers, each of which can contain an amino acid You can have additional motifs.

That is, even if the lipid body is used as a monomer in the form of a lipid body derivative within the object of the present invention, it is not a problem. However, in the case of the lipid-body monomer, the molecular weight is 25-30 kDa, the rate of renal clearence can be fast, and the blood half-life can also be short. Thus, when it is necessary to slow the rate of excretion in the kidney or to make the blood half-life longer, it may be desirable to make the lipid body in a multimeric form to have a molecular weight of ~ 60 kDa, a threshold of renal excretion, By linking Fc protein (~ 50 kDa) to the Lipid body multimer, the molecular weight is increased to slow the renal excretion rate, and the FcRn (Fc neonatal receptor) binding site in the Fc protein binds to FcRn of endothelial cells, In addition, it is possible to increase the half-life of the blood through the mechanism of release into the blood, and in particular, a multimer composed of the same type of Lipid body monomer can improve the binding ability to the target protein. In the case of a multimer composed of a heterologous Lipid body monomer It is possible to simultaneously bind two or more target proteins and to develop them as a more effective therapeutic agent . In the case of PEGylation, attempts have been made to increase the half-life by increasing the molecular weight by binding to a protein having a short half-life of blood, which is small in size from the past. Many successful cases have been reported (representative examples include C- (Pegasys), the same method can be used to increase the half-life of blood by PEGylation by introducing PEG into lipid-body monomers or multimers.

In addition to the lipid body monomers or oligomers, or the Lipid body derivatives described above in which Fc protein or PEG is introduced into each of them, various kinds of proteins or specific amino acid sequences and organic / inorganic compounds can be prepared by methods well known in the field of the present invention Etc., and the resulting lipid body derivatives can be used without limitation within the scope of the present invention.

One of the preferred forms of the Lipid body derivative-drug complex according to the present invention is a form in which a drug is bound to a Lipid body derivative using an enzyme. The Lipid body derivative-drug complex used in the preparation of a Lipid body derivative- Should have an amino acid motif that the enzyme recognizes.

The Lipid body derivative-drug complex according to the present invention may be obtained by directly binding the Lipid body derivative and the drug without a specific enzyme, or may be obtained by binding using a specific enzyme.

In the present invention, when a drug is directly bound to a lipid body derivative without using an enzyme, the drug may be bound through the cysteine, lysine, or glycan present in the lipid body derivative, or artificially introduced into the cysteine, selenocysteine, unnatural amino acid may be substituted or added to another amino acid in the corresponding lipid body derivative to bind the drug.

Among the direct drug binding methods described above, when a drug is directly bound to a cysteine present in a lipid body derivative, the lipid body generally has four cysteines at the C-terminal region and usually forms two intramolecular disulfide bonds , A reducing agent such as DTT or TCEP to generate a free thiol group, and then reacting with a linker-drug having a maleimide group to form an RDC through thiol-maleimide linkage.

Also, among the direct drug-binding methods described above, when a drug is directly bound to a lysine present in a lipid body derivative, the lipid body usually has 18-19 Lysines, and half of them are solvent-accessible (For antibodies, there are usually 80-90 Lysines, of which about half are known to be solvent-accessible.) Approximately 10 Lysines can bind drugs via amide bond formation, (Drug-Repebody Ratio) is usually higher than 6, the half-life of the RDC may be shortened or the toxicity may be caused. Therefore, the DRR should be 3 to 4 on average And to adjust the maximum DRR to be less than six.

Another example of the direct drug binding method mentioned above is to introduce a cysteine or selenocysteine that is not originally present by substituting a specific amino acid of the lipid body with a cysteine or selenocysteine or by inserting it into a specific position and then reducing it to a suitable condition to expose the thiol group And the drug is bound thereto by using maleimide or the like.

Another direct drug binding method is to substitute a specific amino acid of Lipid body with unnatural amino acid having a special functional group in addition to 20 or more amino acids present in nature, And a method of binding a drug to a specific functional group. One way to do this is to produce proteins in vitro without using cells. In other words, when translating a specific codon into a protein, it is possible to introduce an unnatural amino acid having a desired functional group at a desired position by inserting a tRNA capable of introducing an unnatural amino acid. On the other hand, site-specific labeling can be performed by developing cell lines that can use and translate unnatural amino acids. In this technique, the cell line recognizes a tRNA capable of recognizing an amber codon translated into a stop codon and an unnatural amino acid, and is manipulated so as to express an aminoacyl-tRNA synthase linked to the amber codon tRNA, The lipid body has an unnatural amino acid at a specific position. This unnatural amino acid can have a specific functional group, which can bind the drug. For example, when the unnatural amino acid is para-acetyl phenylalanine, the drug can be bound through oxime bond formation. Drug binding via click chemistry is also possible depending on the functional group.

Meanwhile, when a drug is bound using an enzyme, an amino acid motif that can be recognized by the enzyme is present in the lipibody derivative. The enzymes include, but are not limited to, soratease, FGE (Formylglycine Generating Enzyme) , A transglutaminase or an isoprenoid transferase.

Among these enzymes, sortase is an enzyme derived from Gram-positive bacteria and fixes the protein to peptidoglycan. Using this specificity, the amino acid called LPXTG, which can recognize sortase at the C-terminal of the light chain and / After introducing the motif, tags with 3-5 glycines can be attached to the sortase.

Among the above enzymes, FGE recognizes the amino acid motif CXPXR and converts the cysteine in the corresponding sequence into a formyl-glycine having an aldehyde group. Therefore, a CXPXR sequence is introduced into a desired region of the lipid body, After constructing the cell line, the RDC can be prepared by attaching a tag with an amine group to the lipid body having the aldehyde functional group obtained in the cultivation process, and then adding the drug to the lipid body.

Of these enzymes, transglutaminase (TG) catalyzes the coupling of free primary amines to glutamine sidechain acyl groups. Based on the fact that the native glutamine present in the lipid body is not recognized by TG in the sequence of the posterior and posterior amino acid sequences, LLQG, an amino acid motif that is newly recognized by TG in various parts of the lipid body, have.

Hereinafter, the Lipid body derivative-drug complex obtained by using the isoprenoid transferase disclosed in US Patent Publication No. US2012-0308584 A1 as an enzyme will be exemplified and exemplified in detail, but the Lipid body derivative-drug complex Are not limited to those described above.

When the isoprenoid transferase among the above enzymes is used, the amino acid motif that the enzyme recognizes is not limited, but is CAAX, XXCC, XCXC or CCXX located at the C-terminal of the lipid body derivative. Here, C is an amino acid that determines cysteine, A is an aliphatic amino acid independently, and X is an enzyme substrate specificity. When FTase is used as an isoprenoid transferase as in the specific examples of the present invention, , And -CVIM introduced at the C-terminal may be used as one form that satisfies the condition of the CAAX motif.

The amino acid motifs recognized by the isoprenoid transferase may 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 reactivity of the enzyme. Here, the spacer is generally composed of amino acids, but the type and length thereof are not particularly limited. Seven glycines may be used as in one embodiment of the present invention. A (G4S) n spacer having a flexable property, (EAAAK) n (where E is glutamate, A is alanine, K is lysine, and n is the number of amino acid residues in general, In the case of G4S, glycine and serine can be varied in number and arrangement.) In order to introduce more hydrophilic properties in consideration of physical properties, DRDD (where D is aspartate and R is arginine) A variety of spacers, including charged amino acids, may also be used.

The amino acid motifs in the present invention include, but are not limited to, CAAX, XXCC, XCXC or CCXX, C is cysteine, A is an aliphatic amino acid independently, X is an amino acid that determines that the motif is a substrate of the enzyme, Lt; / RTI > to the C-terminus of the lipid body. The type and length of the spacer are not particularly limited, and may preferably be composed of one or more amino acids and compounds, and most preferably, seven glycines can be used as in one embodiment of the present invention .

In the present invention, the amino acid motif may be bound to the drug via one or more linkers, although not limited thereto. Preferably, the linker may be combined with FPP-carbonyl analog which is an analogue of farnesyl pyrophosphate (FPP).

In the present invention, the linker is an alkylene having 1 to 50 carbon atoms, and may satisfy at least one of the following (i) to (iv).

(i) said alkylene comprises at least one unsaturated bond,

(ii) said alkylene comprises at least one heteroarylene,

(iii) the carbon atom of the alkylene is substituted with at least one heteroatom selected from nitrogen (N), oxygen (O) and sulfur (S)

(iv) the alkylene is further substituted with alkyl having 1 to 20 carbon atoms.

In the present invention, the linker (L) may comprise at least one isoprenyl derivative unit represented by the following formula (A) which can be recognized by an isoprenoid transferase.

(A)

In the present invention, the linker (L) may be a 1,3-dipolar cycloaddition reaction, a hetero-diels reaction, a nucleophilic substitution reaction, Aldol-type carbonyl reactions, additions to carbon-carbon multiple bonds, oxidation reactions or click rections. Unit. ≪ / RTI > In the present invention, the coupling unit may be formed by a reaction between acetylene and an azide, or a reaction between an aldehyde or a ketone group and a hydrazine or a hydroxylamine. In the present invention, the bonding unit may be represented by the following formula (B), (C) or (D).

[Chemical Formula B]

≪ RTI ID = 0.0 &

[Chemical Formula D]

L ₁ is a single bond or alkylene having 1 to 30 carbon atoms;

R < ₁₁ > is hydrogen or alkyl having 1 to 10 carbon atoms;

L ₂ is alkylene having 1 to 30 carbon atoms.

In the present invention, the linker (L) may further comprise a linking unit represented by the formula (E) or (F).

(E)

- (CH ₂ ) _r (W (CH ₂ ) _q ) _p -

[Chemical Formula F]

- (CH ₂ CH ₂ V) _x -

W is -O-, -S-, -NR ₂₁ -, -C (O) NR ₂₂ -, -NR ₂₃ C (O) -, -NR ₂₄ SO ₂ - or -SO ₂ NR ₂₅ -;

V is -O-, C ₁ -C ₈ alkylene or -NR ₂₁ -;

R ₂₁ to R ₂₅ are each independently hydrogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl C ₆ -C ₂₀ aryl or C ₁ -C ₆ alkyl C ₃ -C ₂₀ heteroaryl;

r is an integer from 1 to 10;

q is an integer from 0 to 10;

p is an integer from 1 to 10; And

x is an integer of 1 to 10;

In the present invention, the drug may be but is not limited to a drug, a toxin, an affinity ligand, a detection probe, or a combination thereof.

Exemplary drugs include, but are not limited to, TARCEVA (Genentech / OSI Pharm.), VELCADE (Millenium Pharm.), FASLODEX (AstraZeneca), SUINTl Pfizer, FEMARA, Novartis, GLEEVEC Novartis, PTK787 / ZK 222584 (Novartis), Eloxatin (Sanofi), 5-fluorouracil (5-FU, leucovorin) , Sirolimus (RAPAMUNE; Wyeth), LASPARNIV (TYKERB, GSK572016; GlaxoSmithKline), Lonapanib (SCH 66336), Sorafenib (BAY43-9006; Bayer Labs.), ), AG1478, AG1571 (SU 5271; Sugen), alkylating agents such as thiotepa and CYTOXAN cyclophosphamide; Alkyl sulphonates, such as, for example, clay plates, impro sulphates and poly sulphates; Aziridines, such as benzodopa, carbobucone, metouredopa, and uredopa; Ethyleneimine and methylamelamine including althretamine, triethylene melamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolromelamine; Acetogenins (especially borax thaxin and boraxacinone); Camptothecin (inducing synthetic analogue topotecan); Bryostatin; Calistatin; CC-1065 (including adozelesin, chaselesin and non-gelsin synthetic analogs thereof); Cryptophycin (especially cryptophycin 1 and cryptophycin 8); Dolastatin; Two okamycins (including synthetic analogs, KW-2189 and CB1-TM1); Eleuterobin; Pancreatistin; Sacodictin; Spongistatin; Nitrogen mustards such as chlorambucil, chlorpavine, chlorophosphamide, estramustine, ifposamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novivicin, fenestrel , Fred Nimustine, Troposphamide, Uracil Mustard; Nitrite ureas such as camustine, chlorothoxin, potemustine, lomustine, nimustine and lemnystine; Antibiotics such as enediyne antibiotics such as calicheamicin, especially calicheamicin gamma 1 and calicheamicin omega I1 (see, for example, Agnew, Chem Intl ed Engl., 33: 183- 186 (1994)) and dynaminin, which induces Dynaminicin A. Bisphosphonates such as Claudronate, Esferramycin, Nioxanostatin chromophore and related chromoprotein enanedin antibiotic chromophore, Accliminomycin, Ev But are not limited to, thymomycin, anthramycin, azaserine, bleomycin, cactinomycin, carabicin, caninomycin, caspofilin, chromomycin, dactinomycin, -L-norleucine, ADRLIMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin and deoxy doxorubicin), epirubicin, Rome, Mitomycin, such as mitomycin C, mycophenolic acid, nogalamycin, olibomycin, perfromycin, papyrimycin, puromycin, couelramycin, rodurubicin, streptomycin, streptozocin, Metabolites such as 5-fluorouracil (5-FU), folic acid analogues such as denonfterin, methotrexate, proteopterin, ciprofloxacin, Pyrimidine analogues such as asitabine, azacytidine, 6-azauridine, thiamine, thymidine, thymine, But are not limited to, carotene, camphor, cytarabine, dideoxyuridine, doxifluridine, enocitabine and fluoxurdine; androgens such as carrousosterone, dromoglomerolone propionate, epithiostanol, And testolactone, an anti-adrenal, e. G., Ami Glutamic acid, glutamic acid, glutamic acid, glutamic acid, glutamic acid, glutamic acid, glutamic acid, glutetiimide, mitotan and triostane; Deferoxamine; democheline; diaziquone; elforinitine; Elliptinium acetate; Epothilone; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Ronidainine; Mytansinoids such as mytansine and ansamitoxin; Mitoguazone; Mitoxantrone; Fur monotherapy; Nitraerine; Pentostatin; Phenamate; Pyra rubicin; Truncate tron; 2-ethylhydrazide; Procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); Lauric acid; Liqin; Xanthopyran; Spirogermanium; Tenuazonic acid; Triazicone; 2,2 ', 2 "-trichlorotriethylamine, tricothexene (especially T-2 toxin, veracoline A, loridine A and angiidine), urethane, vindesine, Taxotene, such as TAXOL (R) paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ) (American Pharmaceutical Partners, Schaumber, I11.) And TAXOTERE® dock laundry cells (Rhone-Poulenc Rorer, Antony, France), cloranvac; gemcitabine, 6-thio The present invention relates to a method for the treatment and / or prophylaxis of cancer, which comprises administering to a mammal a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of guanine, mercaptopurine, platinum analogs such as cisplatin, carboplatin, vinblastine, platinum, etoposide, Seed; Deatrexate; Daunomycin; Aminopterin; Zeloda; Ibandronate CPT-11, topoisomerase inhibitor RFS 2000, difluoromethylorhornin (DFMO), retinoids such as retinoic acid, capecitabine, and pharmaceutically acceptable salts, solvates, acids Or derivatives thereof.

Additional medicaments include, but are not limited to, (i) for example, tamoxifen (including NOLVADEX [0117] ® tamoxifen), raloxifene, droloxifene, 4-hydroxy tamoxifen, trioxifen, Anti-hormone agents that act to regulate or inhibit hormone action on tumors, such as anti-estrogens and selective estrogen receptor modulators (SERMs), including LY117018, onapristone and FAREATON? (ii) aromatase inhibitors that inhibit aromatase enzymes, such as 4 (5) -imidazoles, aminoglutethimides, MEGASE (R) megestrol acetate, AROMASIN (R) exemestane , FEMARA choletrozole and ARIMIDEX® anastrozole; (iii) anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; As well as troxacytavin (1,3-dioxolane nucleoside cytosine analog); (iv) an aromatase inhibitor; (v) protein kinase inhibitors; (vi) lipid kinase inhibitors; (vii) inhibiting signaling pathway gene expression associated with antisense oligonucleotides, particularly adherent cells, such as PKC-alpha, Raf, H-Ras; (viii) ribozymes, such as VEGF inhibitors, such as ANGIOZYME ribozyme and HER2 expression inhibitors; (ix) a vaccine, for example, a gene therapy vaccine; ALLOVECTIN® vaccines, LEUVECTIN vaccines and VAXID vaccines; PROLEUKIN (R) L-2; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH; (x) anti-angiogenic agents such as, for example, AVASTIN, Genentech; And (xi) a pharmaceutically acceptable salt, solvate, acid or derivative thereof,

.

In some embodiments, a cytokine may be used as the agent. Cytokines are small cell-signaling protein molecules secreted by a number of cells and are a category of signaling molecules that are widely used for intracellular information exchange. These include monocaine, lymphocaine, traditional polypeptide hormones, and the like. Examples of cytokines include, but are not limited to, growth hormones such as human growth hormone, N-methionyl human growth hormone and bovine growth hormone; Parathyroid hormone; Thyroxine; insulin; Proinsulin; Relaxin; Prolaxin; Glycoprotein hormones such as vesicle stimulating hormone (FSH), thyroid stimulating hormone (TSH) and luteinizing hormone (LH); Liver growth factor fibroblast growth factor; Prolactin; Placental lactogen; Tumor necrosis factor-a and -beta; Mueller-inhibiting material; Mouse gonadotropin binding peptides; Inhivin; Actibin; Vascular endothelial growth factor; Integrin; Thrombopoietin (TPO); Nerve growth factors such as NGF-beta; Platelet-growth factor; Transforming growth factor (TGF), such as TGF-a and TGF-beta; Insulin-like growth factor-I and -II; Erythropoietin (EPO); Bone inducer; Interferons, such as interferon-a, -beta and-gamma; Colony stimulating factor (CSF), such as macrophage-CSF (M-CSF); Granulocyte-macrophage-CSF (GM-CSF); And granulocyte-CSF (G-CSF); IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, -10, IL-11, IL-12; Tumor necrosis factors such as TNF-a and TNF-beta; And other polypeptide factors including LIF and a kit ligand (KL). As used herein, the term cytokine also includes recombinant cell cultures and biologically active equivalents of native sequence cytokines or from natural sources.

The term "toxin" refers to toxic substances produced in living cells or organisms. The toxin may be a small molecule, peptide or protein capable of causing a disease upon contact with, or absorption by, a biological macromolecule, e. G., An enzyme or a body tissue that interacts with the cell receptor. Toxins include plant toxins and animal toxins. Examples of animal toxins include, but are not limited to, diphtheria antitoxin, botulism toxin, tetanus toxin, heterotoxin, cholera toxin, tetrodotoxin, blevetoxin, and cytochrome. 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), mitacinoids (EP 1391213 , ACR 2008, 41, 98-107), calicheamicin (US 2009105461, Cancer Res. 1993, 53, 3336-3342), daunomycin, doxorubicin, methotrexate, vindesine, SG2285 (Cancer Res. 17, 6849-6858), dolastatin, the dolastatin analogue auristatin (US563548603), cryptophysin, camptothecin, rijsin derivatives, CC-1065 analogs or derivatives, doxamycin, endian antibiotics, Esperamicin, epothilone, and toxoid. The toxin can exhibit cytotoxicity and cell growth inhibitory activity by tubulin binding, DNA binding, and topoisomerase inhibition.

The term "ligand" refers to a molecule capable of forming a complex with a target biomolecule. An example of a ligand is a molecule that binds to a predetermined position of a target protein and transmits a signal. It can be a substrate, an inhibitor, a stimulant, a neurotransmitter or a radioisotope.

"Detectable moiety" or "marker" refers to a composition that is detectable by spectroscopic, photochemical, biochemical, immunochemical, radioactive or chemical means. For example, useful labels include ³² P, ³⁵ S, fluorescent dyes, electron-dense reagents, enzymes (such as are commonly used in ELISAs), biotin-streptavidin, dioxygenine, Or a nucleic acid molecule having a sequence complementary to the target. The detectable moiety often generates a measurable signal, e.g., a radioactive, chromogenic or fluorescent signal, which can be used to quantify the amount of bound detectable moiety in the sample. Quantitation of the signal is accomplished by, for example, scintillation counting, density counting, flow cell analysis, ELISA, or direct analysis (one or more peptides can be assayed) by mass spectrometry of circular or subsequently digested peptides. Those skilled in the art are familiar with the techniques and detection means for the label compounds of interest. These techniques and methods are conventional and well known in the art.

As used herein, the term "probe" is intended to encompass all types of electromagnetic radiation, such as (i) providing a detectable signal, or (ii) interacting with a first probe or a second probe to generate a first or second (Iii) stabilize the interaction with the antigen or ligand, or increase the binding affinity; or (iv) electrophoretic mobility, such as by physical parameters such as charge, hydrophobicity, or the like, Refers to a substance capable of affecting cell-invasion action, or (v) capable of regulating ligand affinity, antigen-antibody binding or ion complex formation.

In the present invention, when the Lipid body derivative-drug complex is developed as an anti-cancer therapeutic agent, the target protein is a specific or overexpressed protein in cancer cells. When the non-Hodgkin lymphoma is the main indication, (CD30), acute-myeloid leukemia (leukemia), acute myelogenous leukemia (CD), and the like. In the case of non-Hodgkin's lymphoma, CD30, CD21, CD22, CD37, CD70, CD72, CD79a / CD43 and CD43, and acute-lymphoid leukemia are the main indications for CD43 and multiple myeloma for CD56, CD74, CD138, and endothelin B receptor, EGFR, EGFR, CD56, CD326, CRIPTO, FAP, mesothelin, G2D, 5T4, and EGFR are the main indications for head and neck cancer (EGFR), CD74, CD174, CD227 (MUC-1), CD326 (Epcam), CRIPTO, FAP and EGFR, EGFRvIII, and colorectal / rectal cancers are the major indications for alpha v beta6, glioblastoma, CD74, CD227 (MUC-1), nectin-4 (ASG-22ME), and alpha v beta6 were the major indications for ED-B and pancreatic cancer, and HER2, CD174, GPNMB, CRIPTO, (CA125), TIM-1 (CDX-014), mesothelin and melanoma were the main indications for ovarian cancer, 1, and TENB2 for primary prostate cancer, CAIX and TIM-1 (CDX-014) for predominantly renal cancer, , Mesothelioma (mesothelioma), and mesothelin.

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, the anti-EGFR Lipibody derivative-drug (anti-EGFR RDC, ERDC) using Lipid Binding to EGFR (Endothelial Growth Factor Receptor) was prepared and its efficacy was confirmed.

The present invention also provides a method for producing a protein comprising the steps of: (a) preparing a protein to which an amino acid motif capable of recognizing an enzyme is bound to one or more C-termini of a lipid body derivative; (b) preparing a functionalized protein by reacting the protein prepared in step (a) with one or more substrates containing a first functional group using an enzyme; And (c) reacting the functionally-shrunk protein of step (b) with a conjugate having a second functional group bound to the drug to produce a lipid-derivative-drug complex; To a process for preparing a protein-drug conjugate.

In the present invention, the second functional group is not limited but can be bound to the drug by one or more linkers.

In the present invention, the reaction between the functionalized protein and the drug can be formed by the reaction of acetylene with azide, or the reaction of an aldehyde or ketone group with hydrazine or hydroxylamine.

The sequence variant of each of the Lipid body monomers and the multimer composed thereof, the Fc protein and the PEG-linked forms thereof, can be obtained by removing a part of the carboxy terminal thereof, adding a new amino acid sequence to the carboxy terminal, An amino acid sequence recognizable by isoprenoid transferase may be combined after the new amino acid sequence is added after the part is removed.

On the other hand, in the C-terminal CAAX sequence (C is cysteine, A is an aliphatic amino acid independently, and X is an amino acid which determines that the motif is a substrate of the enzyme), which is a sequence recognizable by the isoprenoid transferase , And may further include a step of cutting off the -AAX unit after the step (b).

The present invention also provides a method for producing a protein comprising the steps of: (a) preparing a protein to which an amino acid motif capable of recognizing an enzyme is bound to one or more C-termini of a lipid body derivative; (b) preparing a conjugate by binding one or more drugs to a substrate of the enzyme; (c) reacting the protein produced in step (a) with the conjugate obtained in step (b) to prepare a lipid-derivative-drug complex; To a process for preparing a protein-drug conjugate.

Through the above-described production methods, a lipid-derivative-drug conjugate having substrate specificity can be homogeneously produced.

The present invention also relates to a pharmaceutical composition for diagnosis, prevention and treatment of diseases using the Lipid body derivative-drug complex. 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, And 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 dosage form of the pharmaceutical composition may be an oral formulation selected from the group consisting of powders, granules, tablets, capsules, suspensions, emulsions, and syrup, though not limited thereto.

In the present invention, the amount of the lipid body derivative-drug 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. The composition may be formulated in the form of an oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like, an external preparation, a suppository and a sterile injection solution. The composition may further include a carrier, an excipient and a diluent. The lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, But are not limited to, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil in the form of tablets, capsules, powders, granules, Can be used.

When the composition is formulated, it is formulated using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, interfacial drugs and the like which are commonly used. Solid formulations for oral administration may include tablets, lozenges, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, , Sucrose, lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like. Since the composition has little toxicity and side effects, it can be safely used for prolonged use for preventive or therapeutic purposes.

The composition is not limited to other components other than those containing the composition as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as an additional ingredient. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. As other flavoring agents, natural flavoring agents (tau martin), stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) have. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 mg of the lipid body derivative-drug complex of the present invention. In addition to the above-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. The proportion of such additives is not limited, but is preferably selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

In the present invention, the composition using the Lipid body derivative-drug complex may be used to provide a conventionally known method for diagnosing disease diagnosis, though not limited thereto. The composition may be directly administered to a subject for obtaining disease diagnosis information or may be attached to a substrate of a biochip prepared for detecting genes, proteins, cells and the like known as disease-specific targets, and the gene, It is possible to utilize the method for providing information of disease diagnosis having high accuracy and reliability through a method of detecting the same at the same time.

The present invention also relates to a polynucleotide encoding said Lipid body derivative.

The present invention also relates to a recombinant vector comprising said polynucleotide. In the present invention, the type of the vector is not limited within a commonly used range and can be selected and used.

The present invention also relates to a microorganism transformed with said recombinant vector. In the present invention, the microorganism for transformation is not particularly limited, but microorganisms capable of overexpressing the recombinant vector can be selected and used. Escherichia coli , have.

The lipid-derivative-drug complex (RDC) according to the present invention is a combination of a drug having biological activity and a high substrate specificity, excellent tissue and tumor penetration ability provided by the lipid body, It has an advantage in that it has the advantage of minimizing the toxicity by having a specific selectivity for the target cell as compared with the case of using the compound alone and it has an advantage in that it is soluble form ), Which can be easily mass-produced at low cost, is extremely stable thermodynamically, and has excellent tissue penetration ability, and has an advantage of being able to specifically bind to a target protein, The advantage of the complex is that it provides a new form of drug complex.

FIG. 1 shows the result of immunofluorescence detection of cell-specific binding using MCF7 (EGFR low expression breast cancer cell line), A431 (EGFR overexpressing cancer cell line) and HCC827 (EGFR overexpressing lung cancer cell line).
2 is a conceptual diagram of a lipid body derivative-drug complex structure
Figure 3 is a HIC-HPLC peak confirmation result of each of the steps of preparing the Lipid body derivative-drug complex (C) through the intermediate step (B) activated by the Farnesyl analog from the Lipid body derivative (A).
FIG. 4 shows the results of in vitro cytotoxicity of A431 and MCF-7 of the anti-EGFR-lipid body-drug complex ERDC-V1 prepared using Erep-V1, an anti-EGFR Lipid body derivative.
FIG. 5 is a graph showing the effect of the anti-EGFR lipid body derivative Erep-V1 and the lipid body derivative-drug complexes ERDC-V1 and ERDC prepared using Erep-V3 and Erep-V3, -V2, a result obtained by measuring the in vitro cytotoxicity against the A431 ERDC-V3 respectively.
Figure 6 shows the results of in vitro cytotoxicity of ERDC-V1, ERDC-V2 and ERDC-V3 against HCC827.
Figure 7 is a graph showing the ability of ERDC-V3 to inhibit tumor growth in an in vivo xenotransplantation mouse efficacy test against HCC827.
8 shows the result of measuring the mouse weight in the experiment of FIG.

Hereinafter, the present invention will be described in detail with reference to examples. However, these are for the purpose of illustrating the present invention in more detail, and the scope of the present invention is not limited by the following examples.

Example 1 Identification of Specific Binding Ability of EGFR-Specific Lipid Bodies

Lipibody (KR2014-0062391, hereinafter referred to as RP-E1V1) having the polypeptide sequence of SEQ ID NO: 1 was overexpressed with 0.5 mM IPTG as a host cell by OrigamiB (DE3) (Novagen, USA), and affinity and gel permeation chromatography The protein was purified purely through the gel. To confirm whether RP-E1V1 specifically binds to cancer cells expressing EGFR, cell-specific binding was detected using MCF7 (EGFR low expression breast cancer cell line), A431 (EGFR overexpressing epidermal cancer cell line), and HCC827 (EGFR overexpressing lung cancer cell line) Respectively. RP-E1V1 was labeled with fluorescein, and each cancer cell was treated at a concentration of 1 μg / mL for about 3 hours. Then, confocal microscopy (confocal microscopy) Were observed.

As shown in Fig. 1, fluorescence was hardly observed in MCF7, whereas A431 and HCC827 showed strong fluorescence signals. This result implies that Lipidase RP-E1V1 binding to the extracellular domain of EGFR can bind specifically to the cell.

[Example 2] Preparation of lipibody derivative

(7) glycine at the C-terminus of RP-E1V1 and cysteine-valine-isoleucine-methionine as the CAAX motif in order to obtain a derivative of the Lipid body of SEQ ID NO: 2 Hereinafter referred to as Erep-V1).

The gene encoding Erep-V1 was cloned into the 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 Erep-V2 (SEQ ID NO: 4), Erep-V3 (SEQ ID NO: 6), and the negative control Erep that did not bind to EGFR, which improved the binding ability to EGFR using Erep- -NV (SEQ ID NO: 8).

[Example 3] Production of isoprenoid substrate Compound A

The target compound A was prepared in the same manner as described in U.S. Patent Publication No. US2012-0308584 A1.

[Example 4] Prednylation method

The prenylation of the lipid body was performed using the Farnesyl analog (Compound A) and FTase (# 344145, Calbiochem, USA). The prenylation reaction was carried out at 30 ° C for 12 hours using 50 mM Tris-HCl (pH 7.4) buffer containing 5 mM MgCl 2, 10 μM ZnCl 2 and 5 mM DTT. After the reaction was completed, it was desalted with a PD-10 desalting column (GE Lifescience), concentrated using Vivaspin 2, and the buffer was replaced with PBS. Subsequently, proteins were quantified by Nanodrop and BCA method.

1. Prenylation of Lipid body derivatives

The lipid body was prenylated using Compound A and FTase according to the method described above. The material thus prepared was named " prenylated lipibody derivative ". The prenylated Lipid body derivatives obtained by predilining Erep-V1, Erep-V2, Erep-V3 and Erep-NV are Erep-V1-Compound A, Erep-V2-Compound A, Erep- NV-Compound A < / RTI > Among them, the structure of 'Erep-V1-Compound A' which is a substance produced by the reaction of Erep-V1 and Compound A is as follows.

Erep-V1-Compound A

[Example 5] Preparation of Linker-toxin Compound B

Preparation of compound 1b

Compound 1a (10 g, 59.3 mmol) was dissolved in DMF (90 mL) at room temperature under a nitrogen atmosphere, sodium azide (5.78 g, 88.9 mmol) was added and the mixture was stirred at 100 ° C for 13 hours. After completion of the reaction, chloroform (200 mL) and distilled water (300 mL) were added and extracted. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was subjected to column chromatography to obtain Compound 1b (10.3 g, 99%).

(M, 2H), 3.40 (t, J = 5.4 MHz, 2H), 2.20 (m, 2H), 3.70-3.68 , J = 6.0MHz, 1H)

Preparation of compound 1c

CBr4 (21.4 g, 64.6 mmol) was dissolved in MC (100 mL) at 0 DEG C under a nitrogen atmosphere and then Triphenylphosphine (16.9 g, 64.6 mmol) dissolved in MC (100 mL) and compound Ib (10.3 g, 58.7 mmol) And the mixture was stirred at room temperature for 13 hours. After completion of the reaction, MC (300 mL) and distilled water (300 mL) were added and extracted, and the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was subjected to column chromatography to give compound 1c (12 g, 85%).

J = 6.4 MHz, 2H), 3.72-3.67 (m, 6H), 3.48 (t, J = 6.0 MHz, 2H), 3.40 (t, J = 4.8 MHz, CDCl3) 2H)

Preparation of compound 1d

Compound 1c (1 g, 4.20 mmol) was dissolved in acetonitrile at room temperature under a nitrogen atmosphere, N-Boc-Hydroxyamine (643 mg, 4.82 mmol) and DBU (0.659 mL, 4.41 mmol) were added and the mixture was stirred at 60 ° C for 13 Lt; / RTI > After completion of the reaction, MC (300 mL) and distilled water (300 mL) were added and extracted, and the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was subjected to column chromatography to give compound 1d (748 mg, 70%).

(M, 2H), 3.74-3.69 (m, 6H), 3.42 (t, J = 4.8 < RTI ID = 0.0 & , ≪ / RTI > 2H), 1.49 (s, 9H). EI-MS m / z: 291 (M < + >).

Preparation of Compound 1e

Compound 1d (200 mg, 0.688 mmol) was dissolved in methanol (5 mL), Pd / C (10%) (70 mg) was added, and the mixture was stirred under a hydrogen atmosphere for 3 hours. After the reaction was completed, the mixture was Celite filtered and concentrated under reduced pressure to give compound 1e (180 mg, 98%).

J = 5.2MHz, 1H), 2.88 (t, J = 5.2MHz, 1H) 2.81 (m, 2H), 3.74-3.62 (t, J = 5.2 MHz, IH), 1.64 (s, 2H), 1.48 (s, 9H). EI-MS m / z: 265 (M < + >).

Preparation of Compound 2a

5-formyl salicylic acid (2 g, 12.038 mmol) was dissolved in DMF at 0 ° C under nitrogen atmosphere and then 2- (trimethylsilyl) ethanol (1.72 mL, 12.038 mmol), DMAP (147 mg, 1.204 mmol) 12.038 mmol) was added. The mixture was stirred at room temperature for 12 hours. After the reaction was completed, ethyl acetate (100 mL) and distilled water (100 mL) were added. The organic layer thus obtained was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was subjected to column chromatography to obtain Compound 2a (1.6 g, 50%).

_{^{1 H NMR (400MHz, CDCl 3}} ) δ 11.38 (s, 1H), 9.77 (s, 1H), 7.48 (d,, J = 8.4MHz, 1H), 6.61 (dd, J = 8.4, 2.0MHz, 1H) , 6.53 (d, J = 2.0 MHz, 1H), 5.36-5.25 (m, 4H), 4.23 (m,

Preparation of compound 2b

Compound 2a (60 mg, 0.225 mmol) was dissolved in acetonitrile (2 mL) at room temperature under a nitrogen atmosphere, and then Compound 3 (prepared in the same manner as described in 90 mg, 0.225 mmol, US 2012/030858) (209 mg, 0.900 mmol) and molecular sieves (90 mg). The mixture was stirred at ambient temperature for 2 hours. After completion of the reaction, ethyl acetate (50 mL) and distilled water (30 mL) were added. The organic layer thus obtained was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was subjected to column chromatography to give compound 2b (103 mg, 79%).

_{^{1 H NMR (400MHz, CDCl 3}} ) δ 9.93 (s, 1H), 8.22 (d, J = 2.0MHz, 1H), 7.97 (dd, J = 6.4, 2.0MHz, 1H), 7.26 (d, J = 9.2 2H), 4.24 (d, J = 9.2 MHz, 1H), 3.70 (s, 3H), 2.06-2.04 (m, 9H) , 1.14-1.08 (m, 2H), 0.07 (s, 9H)

Preparation of compound 2c

Compound 2b (100 mg, 0.171 mmol) was dissolved in 2-propanol (0.3 mL) and chloroform (1.5 mL) at 0 ° C under a nitrogen atmosphere and silica gel (720 mg) and sodium borohydride (16 mg, 0.427 mmol) . The mixture was stirred for 3 hours. After the reaction was completed, ethyl acetate (50 mL) and distilled water (20 mL) were added. The organic layer thus obtained was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was subjected to column chromatography to give compound 2c (94 mg, 94%).

_{^{1 H NMR (400MHz, CDCl 3}} ) δ 7.71 (d, J = 2.0MHz, 1H), 7.45 (dd, J = 6.4, 2.0MHz, 1H), 7.17 (d, J = 8.4MHz, 1H), 5.40- 2H), 4.40-4.29 (m, 2H), 4.18 (d, J = 8.8 MHz, 1H), 3.74 (s, 3H) , 2.08-2.04 (m, 9H), 1.14-1.09 (m, 2H), 0.08 (s, 9H)

Preparation of compound 2d

Compound 2c (90 mg, 0.154 mmol) was dissolved in DMF (1.0 mL) at 0 ° C under a nitrogen atmosphere and bis (4-nitrophenyl) carbonate (94 mg, 0.308 mmol) and DIPEA (40 uL, 0.231 mmol) . The mixture was stirred at room temperature for 3 hours. After the reaction was completed, ethyl acetate (50 mL) and distilled water (20 mL) were added. The organic layer thus obtained was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was subjected to column chromatography to give compound 2d (104 mg, 90%).

_{^{1 H NMR (400MHz, CDCl 3}} ) δ 8.28 (m, 2H), 7.80 (d, J = 2.4MHz, 1H), 7.53 (dd, J = 6.4, 2.0MHz, 1H), 7.37 (m, 2H), 2H), 4.20 (d, J = 8.8 Hz, 1H), 5.41-5.33 (m, 3H), 5.25 (M, 2H), 0.08 (s, 9H), < RTI ID = 0.0 &

Preparation of Compound 2e

Compound 2d (1.5 g, 2.00 mmol) was dissolved in DMF (8 mL) at room temperature under a nitrogen atmosphere, followed by the addition of MMAF-OMe (1.34 g, 1.80 mmol) followed by HOBT (54 mg, 0.4 mmol), pyridine mL) and DIPEA (0.383 mL, 2.2 mmol). The mixture was stirred at room temperature for 12 hours. After completion of the reaction, ethyl acetate (200 mL) and distilled water (300 mL) were added. The organic layer thus obtained was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was subjected to column chromatography to give compound 2e (1.7 g, 70%).

EI-MS m / z: 1357 (M ^< ^{+ &} gt ^; ).

Preparation of compound 2f

Compound 2e (1.7 g, 1.253 mmol) was dissolved in THF (15 mL) at 0 ° C under a nitrogen atmosphere, tetrabutylammonium fluoride (1M in THF) (2.5 mL, 2.506 mmol) Lt; / RTI > After completion of the reaction, ethyl acetate (200 mL) and distilled water (300 mL) were added. The organic layer thus obtained was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was subjected to column chromatography to obtain Compound 2f (1.34 g, 85%).

EI-MS m / z: 1257 (M ^< ^{+ &} gt ^; ).

Preparation of 2g compound

Compound 2f (1.34 g, 1.066 mmol) and compound 2n (384 mg, 1.28 mmol) were dissolved in DMF (10 mL) at 0 ° C under a nitrogen atmosphere and then DIPEA (464 L, 2.665 mmol) and PyBOP (832 mg, 1.599 mmol) And the mixture was stirred at room temperature for 4 hours. After completion of the reaction, ethyl acetate (200 mL) and distilled water (300 mL) were added. The organic layer thus obtained was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was subjected to column chromatography to obtain 2 g of a compound (1.2 g, 75%).

EI-MS m / z: 1503 (M ^< ^{+ &} gt ^; ).

Preparation of compound 2h

2 g (530 mg, 0.352 mmol) of the compound was dissolved in methanol (10 mL) at -10 ° C under a nitrogen atmosphere, and then LiOH (147 mg, 7.98 mmol) dissolved in water (8 mL) was slowly added and stirred for 1 hour. After the reaction was complete, chloroform (200 mL) and distilled water (30 mL, pH 2) were added. The organic layer thus obtained was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was subjected to column chromatography to give compound 2h (260 mg, 45%).

EI-MS m / z: 1349 (M ^< ^{+ &} gt ^; ).

Preparation of Compound 2i (Compound B)

Compound 2h (260 mg, 0.192 mmol) was dissolved in methylene chloride (4 mL) and water (2 mL) at 0 ° C under nitrogen atmosphere and 4M-HCl (in dioxane, 4 mL) was added and stirred at 0 ° C for 1 hour . After completion of the reaction, concentration under reduced pressure gave compound 2i (210 mg, 85%).

EI-MS m / z: 1249 (M ^< ^{+ &} gt ^; ).

[Example 6] Drug conjugation using compound B

1. Drug conjugation of prenylated lipibody derivatives

Compound B was dissolved in DMSO to prepare a 10 mM solution. The mixture was mixed at a ratio of 1:15 for the oxime coupling of the prenylated lipibody and toxin, and the mixture was reacted at 30 ° C. for 24 hours. After completion of the reaction, the solution was desalted on a PD-10 column, concentrated using Vivaspin 2, and quantified by Nanodrop and BCA method.

The structure of the ERDC-V1 prepared by using the representative Erep-V1-compound A and the compound B is as follows.

ERDC-V1

[Test Example 1] In vitro cytotoxicity analysis of ERDC (anti-EGFR RDC)

1. Cell line

(EGFR-overexpressing squamous cell carcinoma cell line), MCF-7 cell line (EGFR low expression breast cancer cell line), and HCC-827 cell line (EGFR overexpressing non-small cell lung cancer cell line). Cell lines were cultured according to the recommended specifications provided in commercially available cell lines.

2. Test sample

Anti-EGFR lipidate-drug conjugate (ERDC), anti-EGFR lipid body, and MMAF used for ERDC preparation.

3. Test method

The in vitro activity of ERDC was measured using the above-described cell line (anti-EGFR lipid body as a control substance and MMAF, a toxin used in the production of ERDC) was used. A431 and MCF7 cells were plated 1x10 ⁴ per well, HCC827 cells play in a 96-well culture plates ^three 5x10. After 24 hours of incubation, the lipid body, drug and conjugate were added at various concentrations. After 72 hours, the number of viable cells was counted using SRB dye. Absorbance was measured at 540 nm using SpectraMax 190 (Molecular Devices, USA).

4. Test results

ERDC-V1, an anti-EGFR lipidate derivative-drug complex (FIG. 2) prepared according to the present invention, was analyzed by HIC-HPLC for confirmation of production (FIG. 3) Was superior to the control anti-EGFR Lipibody Erep-V1 on the A431 squamous cell carcinoma cell lines overexpressing the cell line, whereas it did not show cytotoxicity on the MCF-7 breast cancer cell line with low EGFR expression 4). Erep-V1 and Erep-V3 were used as the parental Erep-V2 and Erep-V3 lipid body derivatives, respectively, which had the ability to bind to EGFR constantly and were then used to express the anti-EGFR lipibody derivative- drug complexes ERDC-V1, ERDC- -V3, and their efficacy was compared through in vitro cytotoxicity experiments. As a result, it was confirmed that ERDC-V3 exhibited the best cell death effect in A431 cells (FIG. 5).

The excellent cytotoxicity of ERDC-V3 was also verified in an experiment with HCC827 (non-small cell lung cancer cell line), another cell line that overexpresses EGFR (FIG. 6).

[Test Example 2] In vivo tumor suppression test of ERDC-V3

1. Test sample

In vivo xenotransplantation mouse efficacy studies were performed using PBS buffer, Cetuximab (anti-EGFR antibody), Erep-V3 (anti-EGFR lipibody derivative, version 3), ERDC-V3 (anti-EGFR lipibody derivative- drug complex) Respectively.

2. Test method

Balb / C nude mice maridang HCC-827 cell line the administration of 5 x 10 ^6/200 μL and divided the six rats Group mouse that the tumor size 130 mm ^3. Each group was treated with PBS, Cetuximab (10 mg / kg), Erep-V3 (10 mg / kg), ERDC-V3 (1 mg / kg) and ERDC-V3 of the tumor for 20 days every 3 days size ((length x width) ^2/2) and the body weight was observed in the population.

3. Test results

Through the above examples, it was confirmed that ERDC-V3, a lipid body derivative-drug complex prepared by the method of the present invention, inhibits tumor growth most strongly compared to a conventional lipoprotein Erep-V3 or an antibody control 7), antibody control and lipofibrate show equivalent effects on tumor growth upon administration at the same dose. When ERDC-V3 was administered at 10 mg / kg, mice showed a slight decrease in body weight until day 14, but recovered thereafter (FIG. 8).

From this, it was confirmed that ERDC-V3 (anti-EGFR Repebody Drug Conjugate-V3), which is a lipid body derivative-drug complex, shows superior efficacy to inhibit cytotoxicity in vitro and cancer cell growth in vivo, .

<110> LegoChem Biosciences Inc. <120> Repebody-drug-conjugate and preparation method thereof <130> P14031590356 <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 249 <212> PRT <213> Artificial Sequence <220> <223> Repebody Version 1 <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 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 His His His His His                 245 <210> 2 <211> 260 <212> PRT <213> Artificial Sequence <220> <223> Repebody Version 1 Modified <400> 2 Met His His His His His Glu Thr Ile Thr Val Ser Thr Pro Ile   1 5 10 15 Lys Gln Ile Phe Pro Asp Asp Ala Phe Ala Glu Thr Ile Lys Ala Asn              20 25 30 Leu Lys Lys Lys Ser Val Thr Asp Ala Val Thr Gln Asn Glu Leu Asn          35 40 45 Ser Ile Asp Gln Ile Ile Ala Asn Asn Ser Asp Ile Lys Ser Val Gln      50 55 60 Gly Ile Gln Tyr Leu Pro Asn Val Arg Met Leu His Leu Pro Ser Asn  65 70 75 80 Lys Leu His Asp Ile Ser Ala Leu Lys Glu Leu Thr Asn Leu Thr Tyr                  85 90 95 Leu Met Leu His Tyr Asn Gln Leu Gln Ile Leu Pro Asn Gly Val Phe             100 105 110 Asp Lys Leu Thr Asn Leu Lys Glu Leu Tyr Leu Ser Glu Asn Gln Leu         115 120 125 Gln Ser Leu Pro Asp Gly Val Phe Asp Lys Leu Thr Asn Leu Thr Glu     130 135 140 Leu Asp Leu Ala Arg Asn Gln Leu Gln Ser Leu Pro Lys Gly Val Phe 145 150 155 160 Asp Lys Leu Thr Gln Leu Lys Asp Leu Arg Leu Tyr Glu Asn Gln Leu                 165 170 175 Lys Ser Val Pro Asp Gly Val Phe Asp Arg Leu Thr Ser Leu Gln Tyr             180 185 190 Ile Trp Leu His Asp Asn Pro Trp Asp Cys Thr Cys Pro Gly Ile Arg         195 200 205 Tyr Leu Ser Glu Trp Ile Asn Lys His Ser Gly Val Val Arg Asn Ser     210 215 220 Ala Gly Ser Val Ala Pro Asp Ser Ala Lys Cys Ser Gly Ser Gly Lys 225 230 235 240 Pro Val Arg Ser Ile Cys Pro Thr Gly Gly Gly Gly Gly Gly Gly                 245 250 255 Cys Val Ile Met             260 <210> 3 <211> 249 <212> PRT <213> Artificial Sequence <220> <223> Repebody Version 2 <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 His His His His His                 245 <210> 4 <211> 260 <212> PRT <213> Artificial Sequence <220> <223> Repebody Version 2 Modified <400> 4 Met His His His His His Glu Thr Ile Thr Val Ser Thr Pro Ile   1 5 10 15 Lys Gln Ile Phe Pro Asp Asp Ala Phe Ala Glu Thr Ile Lys Ala Asn              20 25 30 Leu Lys Lys Lys Ser Val Thr Asp Ala Val Thr Gln Asn Glu Leu Asn          35 40 45 Ser Ile Asp Gln Ile Ile Ala Asn Asn Ser Asp Ile Lys Ser Val Gln      50 55 60 Gly Ile Gln Tyr Leu Pro Asn Val Arg Tyr Leu Ala Leu Gly Gly Asn  65 70 75 80 Lys Leu His Asp Ile Ser Ala Leu Lys Glu Leu Thr Asn Leu Thr Tyr                  85 90 95 Leu Met Leu His Tyr Asn Gln Leu Gln Ile Leu Pro Asn Gly Val Phe             100 105 110 Asp Lys Leu Thr Asn Leu Lys Glu Leu Tyr Leu Ser Glu Asn Gln Leu         115 120 125 Gln Ser Leu Pro Asp Gly Val Phe Asp Lys Leu Thr Asn Leu Thr Glu     130 135 140 Leu Asp Leu Ala Arg Asn Gln Leu Gln Ser Leu Pro Lys Gly Val Phe 145 150 155 160 Asp Lys Leu Thr Gln Leu Lys Asp Leu Arg Leu Tyr Glu Asn Gln Leu                 165 170 175 Lys Ser Val Pro Asp Gly Val Phe Asp Arg Leu Thr Ser Leu Gln Tyr             180 185 190 Ile Trp Leu His Asp Asn Pro Trp Asp Cys Thr Cys Pro Gly Ile Arg         195 200 205 Tyr Leu Ser Glu Trp Ile Asn Lys His Ser Gly Val Val Arg Asn Ser     210 215 220 Ala Gly Ser Val Ala Pro Asp Ser Ala Lys Cys Ser Gly Ser Gly Lys 225 230 235 240 Pro Val Arg Ser Ile Cys Pro Thr Gly Gly Gly Gly Gly Gly Gly                 245 250 255 Cys Val Ile Met             260 <210> 5 <211> 249 <212> PRT <213> Artificial Sequence <220> <223> Repebody Version 3 <400> 5 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 His His His His His                 245 <210> 6 <211> 260 <212> PRT <213> Artificial Sequence <220> <223> Repebody Version 3 Modified <400> 6 Met His His His His His Glu Thr Ile Thr Val Ser Thr Pro Ile   1 5 10 15 Lys Gln Ile Phe Pro Asp Asp Ala Phe Ala Glu Thr Ile Lys Ala Asn              20 25 30 Leu Lys Lys Lys Ser Val Thr Asp Ala Val Thr Gln Asn Glu Leu Asn          35 40 45 Ser Ile Asp Gln Ile Ile Ala Asn Asn Ser Asp Ile Lys Ser Val Gln      50 55 60 Gly Ile Gln Tyr Leu Pro Asn Val Arg Met Leu His Leu Pro Ser Asn  65 70 75 80 Lys Leu His Asp Ile Ser Ala Leu Lys Glu Leu Thr Asn Leu Thr Tyr                  85 90 95 Leu Met Leu His Tyr Asn Gln Leu Gln Ile Leu Pro Asn Gly Val Phe             100 105 110 Asp Lys Leu Thr Asn Leu Lys Glu Leu Tyr Leu Ser Glu Asn Gln Leu         115 120 125 Gln Ser Leu Pro Asp Gly Val Phe Asp Lys Leu Thr Asn Leu Thr Glu     130 135 140 Leu Asp Leu Ala Arg Asn Gln Leu Gln Ser Leu Pro Lys Gly Val Phe 145 150 155 160 Asp Lys Leu Thr Gln Leu Lys Asp Leu Arg Leu Tyr Glu Asn Gln Leu                 165 170 175 Lys Ser Val Pro Asp Gly Val Phe Asp Arg Leu Thr Ser Leu Gln Tyr             180 185 190 Ile Trp Leu His Asp Asn Pro Trp Asp Cys Thr Cys Pro Gly Ile Arg         195 200 205 Tyr Leu Ser Glu Trp Ile Asn Lys His Ser Gly Val Val Arg Asn Ser     210 215 220 Ala Gly Ser Val Ala Pro Asp Ser Ala Lys Cys Ser Gly Ser Gly Lys 225 230 235 240 Pro Val Arg Ser Ile Cys Pro Thr Gly Gly Gly Gly Gly Gly Gly                 245 250 255 Cys Val Ile Met             260 <210> 7 <211> 273 <212> PRT <213> Artificial Sequence <220> <223> Repebody Comparison Sequence <400> 7 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 Ile Leu Thr Gly Asn                  85 90 95 Gln Leu Gln Ser Leu Pro Asn Gly Val Phe Asp Lys Leu Thr Asn Leu             100 105 110 Lys Glu Leu Val Leu Val Glu Asn Gln Leu Gln Ser Leu Pro Asp Gly         115 120 125 Val Phe Asp Lys Leu Thr Asn Leu Thr Tyr Leu Asn Leu Ala His Asn     130 135 140 Gln Leu Gln Ser Leu Pro Lys Gly Val Phe Asp Lys Leu Thr Asn Leu 145 150 155 160 Thr Glu Leu Asp Leu Ser Tyr Asn Gln Leu Gln Ser Leu Pro Glu Gly                 165 170 175 Val Phe Asp Lys Leu Thr Gln Leu Lys Asp Leu Arg Leu Tyr Gln Asn             180 185 190 Gln Leu Lys Ser Val Pro Asp Gly Val Phe Asp Arg Leu Thr Ser Leu         195 200 205 Gln Tyr Ile Trp Leu His Asp Asn Pro Trp Asp Cys Thr Cys Pro Gly     210 215 220 Ile Arg Tyr Leu Ser Glu Trp Ile Asn Lys His Ser Gly Val Val Arg 225 230 235 240 Asn Ser Ala Gly Ser Val Ala Pro Asp Ser Ala Lys Cys Ser Gly Ser                 245 250 255 Gly Lys Pro Val Arg Ser Ile Ile Cys Pro Thr His His His His             260 265 270 His     <210> 8 <211> 284 <212> PRT <213> Artificial Sequence <220> <223> Repebody Comparison Sequence Modified <400> 8 Met His His His His His Glu Thr Ile Thr Val Ser Thr Pro Ile   1 5 10 15 Lys Gln Ile Phe Pro Asp Asp Ala Phe Ala Glu Thr Ile Lys Ala Asn              20 25 30 Leu Lys Lys Lys Ser Val Thr Asp Ala Val Thr Gln Asn Glu Leu Asn          35 40 45 Ser Ile Asp Gln Ile Ile Ala Asn Asn Ser Asp Ile Lys Ser Val Gln      50 55 60 Gly Ile Gln Tyr Leu Pro Asn Val Arg Tyr Leu Ala Leu Gly Gly Asn  65 70 75 80 Lys Leu His Asp Ile Ser Ala Leu Lys Glu Leu Thr Asn Leu Thr Tyr                  85 90 95 Leu Ile Leu Thr Gly Asn Gln Leu Gln Ser Leu Pro Asn Gly Val Phe             100 105 110 Asp Lys Leu Thr Asn Leu Lys Glu Leu Val Leu Val Glu Asn Gln Leu         115 120 125 Gln Ser Leu Pro Asp Gly Val Phe Asp Lys Leu Thr Asn Leu Thr Tyr     130 135 140 Leu Asn Leu Ala His Asn Gln Leu Gln Ser Leu Pro Lys Gly Val Phe 145 150 155 160 Asp Lys Leu Thr Asn Leu Thr Glu Leu Asp Leu Ser Tyr Asn Gln Leu                 165 170 175 Gln Ser Leu Pro Glu Gly Val Phe Asp Lys Leu Thr Gln Leu Lys Asp             180 185 190 Leu Arg Leu Tyr Gln Asn Gln Leu Lys Ser Val Pro Asp Gly Val Phe         195 200 205 Asp Arg Leu Thr Ser Leu Gln Tyr Ile Trp Leu His Asp Asn Pro Trp     210 215 220 Asp Cys Thr Cys Pro Gly Ile Arg Tyr Leu Ser Glu Trp Ile Asn Lys 225 230 235 240 His Ser Gly Val Val Arg Asn Ser Ala Gly Ser Val Ala Pro Asp Ser                 245 250 255 Ala Lys Cys Ser Gly Ser Gly Lys Pro Val Arg Ser Ile Ile Cys Pro             260 265 270 Thr Gly Gly Gly Gly Gly Gly Gly Cys Val Ile Met         275 280

Claims (14)

Repebody Derivative-Drug Conjugates.
The method according to claim 1,
Wherein the Lipid body derivative is a form in which the Lipid body monomer, the Lipid body multimer, or the Fc protein and / or the polyethylene glycol (PEG) is introduced into each of them.
3. The method of claim 2,
Wherein the Lipid body derivative is in the form of an additional amino acid motif which is recognizable by the enzyme.
The method of claim 3,
Wherein the amino acid motif is CAAX, XXCC, XCXC or CCXX and is linked directly or via a spacer to the C-terminus of the lipid body derivative wherein C is cysteine, A is independently an aliphatic amino acid, X is a substrate of the enzyme Wherein the amino acid is an amino acid that determines the specificity of the Lipid body derivative-drug complex.
5. The method according to any one of claims 1 to 4,
Wherein the Lipid body derivative is bound to the drug via one or more linkers.
6. The method of claim 5,
Wherein the linker has an alkylene structure of 1 to 50 carbon atoms satisfying at least one of the following (i) to (iv):
(i) said alkylene comprises at least one unsaturated bond,
(ii) said alkylene comprises at least one heteroarylene,
(iii) the carbon atom of the alkylene is substituted with at least one heteroatom selected from nitrogen (N), oxygen (O) and sulfur (S)
(iv) the alkylene is further substituted with alkyl having 1 to 20 carbon atoms.
The method according to claim 6,
Wherein said linker is a compound comprising at least one isoprenyl derivative unit of formula (A) which is recognizable by an isoprenoid transferase. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt;
(A)

8. The method according to claim 7, wherein the coupling unit can not be expressed by a structural formula (or a structural expression)
The linker may be a 1,3-dipolar cycloaddition reactions triazole, isoxazole, hetero-diels reactions, nucloephilic substitution reactions, A coupling unit formed by non-aldol type carbonyl reactions, additions to carbon-carbon multiple bonds, oxidation reactions or click rections, (E. G., The reaction of acetylene with azide); Wherein the compound is a compound further comprising a linking unit of aldehyde or ketone group and hydrazine or hydroxylamine.
9. The method of claim 8,
Wherein the binding unit is a compound represented by the following formula (B), (C) or (D).
[Chemical Formula B]

&Lt; RTI ID = 0.0 &

[Chemical Formula D]

In the formula,
L ₁ is a single bond or alkylene having 1 to 30 carbon atoms;
R &lt; ₁₁ &gt; is hydrogen or alkyl having 1 to 10 carbon atoms;
L ₂ is alkylene having 1 to 30 carbon atoms.
8. The method of claim 7,
Wherein the linker is a compound further comprising a linking unit represented by the formula (E) or (F).
(E)
- (CH ₂ ) _r (W (CH ₂ ) _q ) _p -
[Chemical Formula F]
- (CH ₂ CH ₂ V) _x -
W is -O-, -S-, -NR ₂₁ -, -C (O) NR ₂₂ -, -NR ₂₃ C (O) -, -NR ₂₄ SO ₂ - or -SO ₂ NR ₂₅ -;
V is -O-, C ₁ -C ₈ alkylene or -NR ₂₁ -;
R ₂₁ to R ₂₅ are each independently hydrogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkyl C ₆ -C ₂₀ aryl or C ₁ -C ₆ alkyl C ₃ -C ₂₀ heteroaryl;
r is an integer from 1 to 10;
q is an integer from 0 to 10;
p is an integer from 1 to 10; And
x is an integer of 1 to 10;
The method according to claim 6,
Wherein the linker is coupled to an FPP-carbonyl analog which is an analog of FPP (farnesyl pyrophosphate).
12. The method according to any one of claims 1 to 11,
Wherein the drug is a toxin, a ligand, a detection probe or a combination thereof.
The method of claim 3,
Wherein the enzyme is a Sortase, a Formylglycine Generating Enzyme (FGE), a Transglutaminase, or an isoprenoid transferase.
14. The method of claim 13,
Wherein the isoprenoid transferase is FTase (Farnesyltransferase) or GGTase (Geranylgeranyltransferase).

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