Polypeptide-drug conjugate that binds to il4ra and use thereof

Abstract

The present invention relates to a polypeptide-drug conjugate that binds to IL4Ra and a use thereof, and provides: a polypeptide comprising a ligand that specifically binds to IL4Ra and a temperature-responsive disordered domain; and a polypeptide-drug conjugate in which a cytotoxic drug is bound to the polypeptide. More specifically, the polypeptide, in which an IL4RA-binding ligand and a temperature-responsive disordered domain are repetitively linked, exhibits high selectivity in binding to IL4Ra, and the polypeptide-drug conjugate, in which a cytotoxic drug is bound to the polypeptide, demonstrates significantly enhanced cancer cell-killing efficacy and anti-tumor activity compared to unbound drugs or conventional anticancer agents, and has been confirmed to suppress drug resistance in cancer cells that are resistant to the anticancer agent temozolomide. Accordingly, the polypeptide-drug conjugate can be provided as an effective anticancer therapeutic agent for the treatment of tumors overexpressing IL4Ra and as a pharmaceutical composition for inhibiting drug resistance in cancer cells exhibiting resistance to anticancer drugs.

Description

Polypeptide drug conjugates binding to IL4Ra and uses thereof

[0001] The present invention relates to a polypeptide drug conjugate that binds to interleukin 4 receptor alpha (IL4Ra) and uses thereof, and relates to a polypeptide-drug conjugate in which an IL4Ra binding ligand and a polypeptide comprising an atypical domain having temperature-sensitive properties and an apoptotic drug are covalently bonded, and to medical uses thereof.

[0002] Pancreatic cancer is a highly malignant disease with an average survival time of 9-15 months for patients and a 5-year survival rate of less than 16%. The first-line treatment for pancreatic cancer is FOLFIRINOX, a triple combination therapy combining three anticancer drugs: 5-fluorouracil, irinotecan, and oxaliplatin. However, it has no selectivity for cancer cells, and the rate of penetration into the tumor is very low due to the development of desmoplasia. In addition, it induces drug resistance, so the effectiveness of the anticancer drug is very limited. To overcome the fibrous connective tissue of pancreatic cancer and inject high concentrations into the tumor, a local treatment method using endoscopic ultrasound-guided fine-needle injection is being developed.

[0003] Glioblastoma is a rare disease that occurs in the brain of adults. It is a highly malignant disease with a median survival time of less than 15 months and a 5-year survival rate of less than 7%. Combination chemoradiation therapy, which uses radiation and temozolomide simultaneously, is the standard treatment for brain tumors such as glioblastoma or diffuse intrinsic pontine glioma. However, this standard treatment is not only nonspecific and cannot distinguish between cancer cells and normal cells, but also induces resistance to temozolomide due to a drug release mechanism mediated by P-glycoprotein.

[0004] Topotecan or panobinostat, which are used in chemotherapy for brain tumors, and irinotecan, which is used in the treatment of pancreatic cancer, can also inhibit the proliferation of cancer cells or induce apoptosis, but they have side effects of killing not only cancer cells but also normal cells because they lack cell selectivity. Various therapeutic drugs, including antibodies, antibody-drug conjugates (e.g., ABT-414 or AMG-595), and small molecule synthetic drugs (e.g., irinotecan, topotecan, or panobinostat), are being tried to treat brain tumor patients. However, due to the existence of the blood-brain barrier, a special cell membrane structure with very low drug permeability, anticancer drugs cannot penetrate into the tumor tissue, resulting in very low anticancer cure rates.

[0005] Convection-enhanced delivery has been developed to deliver high concentrations of drugs to brain tumor tissues by bypassing the blood-brain barrier. By applying convection-enhanced delivery, anticancer drugs such as irinotecan, topotecan, or panobinostat, which are small molecule synthetic drugs, can be directly injected into brain tumors. However, since the injected anticancer drugs diffuse or leak out through the P-glycoprotein-mediated drug release mechanism, the half-life of the drugs inside the brain tumor is very short, less than 3 hours, and therefore the anticancer efficacy of the injected drugs is very limited.

[0006] IL4Ra is specifically overexpressed in the cell walls of tumor cells, including brain tumors, breast cancer, and pancreatic cancer, including glioblastoma and diffuse intrinsic glioma, and the treatment and prognosis of patients with IL4Ra overexpression are poor.

[0007] A peptide that binds to a receptor present on the surface of a cancer cell is called a ligand, and the ligand binds to the receptor through a non-covalent bond. If a polypeptide has one ligand, it is called a monovalent polypeptide, and the binding strength between the monovalent polypeptide and the receptor is called affinity. If a polypeptide has multiple ligands, it is called a multivalent polypeptide, and the binding strength between the multivalent polypeptide and the receptor is called avidity, and affinity and binding strength are expressed as the equilibrium dissociation constant (Kd). Compared to monovalent polypeptides, multivalent polypeptides can have increased selectivity and binding avidity for receptors.

[0008] Against this backdrop, to solve the above-mentioned problems, an innovative anticancer drug development strategy is needed that provides a thermosensitive multivalent polypeptide-drug conjugate with increased selectivity and binding affinity for the IL4Ra receptor, which directly administers and accumulates drugs inside the tumors of pancreatic cancer and brain tumors in which IL4Ra is specifically expressed, and then selectively internalizes the drugs into cancer cells, overcomes side effects of systemic toxicity and anticancer drug resistance, and allows the anticancer drugs to remain inside the tumor for a long period of time and maintain a sustained anticancer effect.

[0009] The purpose of the present invention is to provide a ligand that specifically binds to IL4Ra, comprising an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, and a polypeptide that specifically binds to IL4Ra, wherein the ligand and a temperature-sensitive atypical domain comprising an amino acid sequence represented by SEQ ID NO: 3 are repeatedly linked.

[0010] In addition, another object of the present invention is to provide a ligand-biotin conjugate or a polypeptide-biotin conjugate in which biotin is bound to the ligand or the polypeptide, or a ligand-fluorophore conjugate or a polypeptide-fluorophore conjugate in which a fluorescent substance is bound to the ligand or the polypeptide.

[0011] In addition, another object of the present invention is to provide a composition for detecting IL4Ra, a composition for diagnosing cancer in which IL4Ra is overexpressed, or a composition for imaging cancer cells in which IL4Ra is overexpressed, comprising the ligand, the polypeptide, the ligand-biotin conjugate, the polypeptide-biotin conjugate, the ligand-fluorophore conjugate, or the polypeptide-fluorophore conjugate as an active ingredient.

[0012] In addition, another object of the present invention is to provide a ligand-drug conjugate or a polypeptide-drug conjugate in which a drug is bound to the ligand or the polypeptide.

[0013] In addition, another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer in which IL4Ra is overexpressed, comprising the ligand-drug conjugate or the polypeptide-drug conjugate as an active ingredient.

[0014] In addition, another object of the present invention is to provide a pharmaceutical composition for suppressing resistance to temozolomide in a brain tumor patient, comprising the ligand-drug conjugate or the polypeptide-drug conjugate as an active ingredient.

[0015] To achieve the above purpose, the present invention provides a ligand that specifically binds to IL4Ra, comprising an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.

[0016] In addition, the present invention provides a polypeptide that specifically binds to IL4Ra, wherein the ligand and a temperature-sensitive atypical domain comprising an amino acid sequence represented by SEQ ID NO: 3 are repeatedly linked.

[0017] In addition, the present invention provides a polynucleotide encoding the ligand or the polypeptide, a recombinant vector comprising the polynucleotide, and a transformant isolated by transformation with the recombinant vector.

[0018] Additionally, the present invention provides a ligand-biotin conjugate in which biotin is bound to the ligand.

[0019] In addition, the present invention provides a polypeptide-biotin conjugate in which biotin is bound to the polypeptide.

[0020] In addition, the present invention provides a ligand-fluorescent substance conjugate in which a fluorescent substance is bound to the ligand.

[0021] In addition, the present invention provides a polypeptide-fluorescent substance conjugate in which a fluorescent substance is bound to the polypeptide.

[0022] In addition, the present invention provides a composition for detecting IL4Ra comprising the ligand, polypeptide, ligand-biotin conjugate, polypeptide-biotin conjugate, ligand-fluorophore conjugate or polypeptide-fluorophore conjugate as an active ingredient.

[0023] In addition, the present invention provides a composition for diagnosing cancer in which IL4Ra is overexpressed, comprising the ligand, polypeptide, ligand-biotin conjugate, polypeptide-biotin conjugate, ligand-fluorophore conjugate or polypeptide-fluorophore conjugate as an active ingredient.

[0024] In addition, the present invention provides a composition for imaging IL4Ra-overexpressing cancer cells, comprising the ligand, polypeptide, ligand-biotin conjugate, polypeptide-biotin conjugate, ligand-fluorophore conjugate or polypeptide-fluorophore conjugate as an active ingredient.

[0025] In addition, the present invention provides a ligand-drug conjugate in which a drug is bound to the ligand.

[0026] In addition, the present invention provides a polypeptide-drug conjugate in which a drug is bound to the polypeptide.

[0027] In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer in which IL4Ra is overexpressed, comprising the ligand-drug conjugate or the polypeptide-drug conjugate as an active ingredient.

[0028] In addition, the present invention provides a pharmaceutical composition for suppressing resistance to temozolomide in a brain tumor patient, comprising the ligand-drug conjugate or the polypeptide-drug conjugate as an active ingredient.

[0029] The present invention relates to a polypeptide drug conjugate binding to IL4Ra and a use thereof. According to the present invention, a polypeptide-drug conjugate comprising an IL4Ra binding ligand and a thermosensitive atypical domain exhibits significantly improved cancer cell killing activity compared to unbound exatecan, unbound SN38, and the existing anticancer agent irinotecan, and not only exhibits potent anti-tumor efficacy in an orthotopic glioblastoma animal model, but also has an anticancer effect of inhibiting tumor growth and prolonging animal survival compared to the small molecule anticancer agent topotecan. Therefore, the polypeptide-drug conjugate can be provided as an effective anticancer therapeutic agent for cancer treatment.

[0030] In addition, since the polypeptide-drug conjugate was confirmed to have an effect of inhibiting drug resistance of glioblastoma cancer cells exhibiting temozolomide resistance, the polypeptide-drug conjugate can be provided as an effective anticancer therapeutic agent for cancer treatment and a pharmaceutical composition for inhibiting drug resistance of cancer cells exhibiting drug resistance to anticancer agents.

[0031] The polypeptide-biotin conjugate according to the present invention, in which a polypeptide and biotin are combined, bind highly selectively to IL4Ra and thus can be utilized to specifically detect cells expressing IL4Ra.

[0032] The polypeptide-AZDye647 conjugate or the polypeptide-carboxyfluorescein conjugate, in which the polypeptide according to the present invention is combined with the fluorescent compound AZDye647 or carboxyfluorescein, selectively and concentration-dependently binds to IL4Ra, and thus can be utilized as a cancer diagnostic agent for specifically detecting cancer cells expressing IL4Ra and diagnosing cancer.

[0033] Figure 1 is a graph of high pressure liquid chromatography analysis of a ligand represented by sequence number 1 (A) and sequence number 2 (B).

[0034] Figure 2 shows the structure and absorbance spectrum (C) of ligand (SEQ ID NO: 1)-biotin conjugate (A) and ligand (SEQ ID NO: 2)-biotin conjugate (B).

[0035] Figure 3 shows the structure (A) and absorbance spectrum (B) of a polypeptide (SEQ ID NO: 4)-biotin conjugate.

[0036] Figure 4 is a graph showing the determination of the equilibrium dissociation constant (Kd) between IL4Ra and ligand (SEQ ID NO: 1) (A) and ligand (SEQ ID NO: 2) (B).

[0037] Figure 5 is a graph showing the determination of the equilibrium dissociation constant (Kd) between IL4Ra and a polypeptide (SEQ ID NO: 4).

[0038] Figure 6 shows the structure (A) of the polypeptide (SEQ ID NO: 4)-AZDye647 conjugate, the absorbance spectrum (B), and the graph (C) of the results of treatment of glioblastoma U87-MG cells with 0.125 μM, 0.25 μM, and 0.5 μM of the polypeptide (SEQ ID NO: 4)-AZDye647 conjugate and analysis using a fluorescence flow cytometer 1 hour later.

[0039] Figure 7 shows the structure (A) of a polypeptide (SEQ ID NO: 4)-carboxyfluorescein conjugate, the absorbance spectrum (B) and the internalization of the polypeptide (SEQ ID NO: 4)-carboxyfluorescein conjugate 1 hour after treatment of glioblastoma U87-MG cells with 0.1 μM, 0.25 μM, 0.5 μM and 1.0 μM of the polypeptide (SEQ ID NO: 4)-carboxyfluorescein conjugate, taken by fluorescence microscopy (C).

[0040] Figure 8 is a synthetic process of linker-drug MPA-SN3 (A), MEC-SN38 (B), MPA-exatecan (C), and MEC-exatecan (D).

[0041] Figure 9 is a synthetic process of linker-drug MVCP-exatecan.

[0042] Figure 10 is a synthesis of linker-drug doxorubicin-hydrazone (A) and polypeptide (SEQ ID NO: 5)-hydrazone-doxorubicin conjugate (B).

[0043] Figure 11 is a synthesis of linker-drug pyridyldisulfide-succinic acid-SN38.

[0044] Figure 12 shows the structure (A) and UV-visible absorbance spectrum (B) of a polypeptide (SEQ ID NO: 4)-MPA-SN38 conjugate.

[0045] Figure 13 shows the structure (A) and UV-visible absorbance spectrum (B) of a polypeptide (SEQ ID NO: 4)-MVCP-exatecan conjugate.

[0046] Figure 14 shows the scatter plot of SF8628 cell death graph (A) and glioblastoma U87-MG cell death graph (B) in response to ligand (SEQ ID NO: 1)-MVCP-exatecan (a) and ligand (SEQ ID NO: 2)-MVCP-exatecan (b) and irinotecan (c).

[0047] Figure 15(A) is a graph of scattered endogenous SF8628 cell death of polypeptide (SEQ ID NO: 4)-MPA-SN38 (▲), SN38 (▼) and irinotecan (●), Figure 15(B) is a graph of scattered endogenous SF8628 cell death of polypeptide (SEQ ID NO: 4)-MPA-SN38 (▲), SN38 (▼) and irinotecan (●), Figure 15(C) is a graph of scattered endogenous SF8628 cell death of polypeptide (SEQ ID NO: 4)-MVCP-exatecan (▲), exatecan (▼) and irinotecan (●), and Figure 15(D) is a graph of scattered endogenous SF8628 cell death of polypeptide (SEQ ID NO: 4)-MVCP-exatecan (▲), exatecan (▼) and irinotecan (●) in glioblastoma. This is a graph of U87-MG-Luc2 cell death.

[0048] Figure 16 is a graph showing the IC50 (1.356 nM) of polypeptide (SEQ ID NO: 4)-MPA-SN38 (A) and IC50 (10.31 nM) of SN38 (B) against human glioblastoma T98G cells resistant to temozolomide, showing that polypeptide (SEQ ID NO: 4)-MPA-SN38 overcomes anticancer drug resistance.

[0049] Figure 17 is a graph showing the structure of a polypeptide (SEQ ID NO: 5)-TSA-SA-SN38 conjugate (A), and the IC50 (0.4329 μM) and IC50 (0.7606 μM) values ​​of the polypeptide (SEQ ID NO: 5)-TSA-SA-SN38 conjugate (a) and SN38 (b) against pancreatic cancer PANC-1 cells.

[0050] Figure 18 is a graph of the temperature-sensitive phase transition characteristics of a polypeptide (SEQ ID NO: 4)-MPA-SN38 conjugate.

[0051] Figure 19 is a test schedule for evaluating the anti-tumor efficacy of a polypeptide (SEQ ID NO: 4)-MPA-SN38 conjugate using an orthotopic glioblastoma animal model.

[0052] Figure 20 is a photograph showing the monitoring of bioluminescence changes over time after administration of a negative control group (A), topotecan (B), and polypeptide (SEQ ID NO: 4)-MPA-SN38 conjugate (C) to an orthotopic transplantation glioblastoma animal model.

[0053] Figure 21 is a graph showing the monitoring of tumor proliferation inhibition (A), body weight (B), and survival rate (C) according to administration of a negative control group (●), topotecan (■), and polypeptide (SEQ ID NO: 4)-MPA-SN38 (▲) in an anti-tumor test using an orthotopic glioblastoma animal model.

[0054] The present invention provides a ligand that specifically binds to IL4Ra, comprising an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.

[0055] Specifically, the ligand specifically binding to IL4Ra has an amino acid sequence represented by the general formula (1) X1-X2-X3-X4-X5-X6-X7-X8-X9-X10 (general formula 1), wherein 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids are substituted, deleted and / or added without abolishing the IL4Ra binding ability of the ligand, X1 is R, K, H, N, Q or can be deleted, X2 is F, Y or can be deleted, X3 is R, K, H, N or Q, X4 is R, K, H, N or Q, X5 is R, K, H, N or Q, X6 is L, I, V or N, X7 is D or E, X8 is R, K, H, N or Q, and X9 is N or Q , and X10 can be C or deleted.

[0056] More specifically, X1 is R, K or can be deleted, X2 is F, Y or can be deleted, X3 is R, K or Q, X4 is R, K or Q, X5 is R, K or Q, X6 is L, V or N, X7 is D or E, X8 is R, K or Q, X9 is N or Q, and X10 is C or can be deleted.

[0057]

[0058] In addition, the present invention provides a polypeptide that specifically binds to IL4Ra, wherein the ligand and a temperature-sensitive atypical domain comprising an amino acid sequence represented by SEQ ID NO: 3 are repeatedly linked.

[0059] Specifically, the polypeptide may have an amino acid sequence represented by the following general formula (2) M[(IL4Ra binding ligand)(VGVPG)uCGVPG(VGVPG)v(IL4Ra binding ligand)(VGVPG)w]x(IL4Ra binding ligand)(VGVPG)yCGVPG(VGVPG)zW (general formula 2).

[0060] More specifically, in the general formula (2), the temperature-sensitive atypical domain is composed of (VGVPG), which is an amino acid sequence represented by SEQ ID NO: 3, wherein u, v, w, x, y and z may be integers of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, which are the number of repetitions of SEQ ID NO: 2. Even more specifically, the temperature-sensitive atypical domain is composed of (VGVPG), which is an amino acid sequence represented by SEQ ID NO: 3, wherein u may be 4, 5 or 6, v may be 4, 5 or 6, w may be 9, 10 or 11, x may be 1, 3 or 5, y may be 4, 5 or 6, and z may be 4, 5 or 6. More specifically, the temperature-sensitive atypical domain is composed of an amino acid sequence (VGVPG) represented by SEQ ID NO: 3, wherein u may be 5, v may be 4, w may be 10, x may be 3, y may be 5, and z may be 4.

[0061] Preferably, the polypeptide may be composed of any one amino acid sequence selected from the group consisting of SEQ ID NO: 4 to SEQ ID NO: 6, but is not limited thereto.

[0062] The ligand or polypeptide of the present invention can be readily prepared by chemical synthesis methods known in the art. Representative methods include, but are not limited to, liquid or solid phase synthesis, fragment condensation, and F-MOC or T-BOC chemistry.

[0063] In addition, the ligand or polypeptide of the present invention can be produced by genetic engineering methods. First, a DNA sequence encoding the ligand or polypeptide is synthesized according to a conventional method. The DNA sequence can be synthesized by PCR amplification using appropriate primers. Alternatively, the DNA sequence can be synthesized by a standard method known in the art, for example, using an automatic DNA synthesizer (e.g., sold by Biosearch or Applied Biosystems). The produced DNA sequence is inserted into a vector containing one or more expression control sequences (e.g., promoter, enhancer, etc.) that are operatively linked to the DNA sequence and control the expression of the DNA sequence, and a host cell is transformed with the recombinant expression vector formed thereby. The resulting transformant is cultured in an appropriate medium and under conditions to allow the DNA sequence to be expressed, and a substantially pure peptide encoded by the DNA sequence is recovered from the culture. The recovery can be performed using a method known in the art (e.g., chromatography). The term “substantially pure peptide” as used above means that the peptide according to the present invention does not substantially contain any other protein derived from the host.

[0064] In the present invention, the ligand or polypeptide is a concept including functional variants thereof. The term "functional variant" refers to any similar sequence in which some amino acid substitutions occur at amino acid positions that do not affect the properties of the ligand or polypeptide of the present invention that specifically bind to IL4Ra.

[0065]

[0066] Additionally, the present invention provides a polynucleotide encoding the polypeptide.

[0067] The above “polynucleotide” is a polymer of deoxyribonucleotides or ribonucleotides existing in single-stranded or double-stranded form. It encompasses RNA genome sequences, DNA (gDNA and cDNA), and RNA sequences transcribed therefrom, and includes analogs of natural polynucleotides unless otherwise specified.

[0068] The polynucleotide comprises not only a nucleotide sequence encoding the polypeptide, but also a complementary sequence to the sequence. The complementary sequence includes not only a perfectly complementary sequence but also a substantially complementary sequence.

[0069] Additionally, the polynucleotide may be modified. Such modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides. A polynucleotide encoding the amino acid sequence is also interpreted to include a nucleotide sequence that exhibits substantial identity to the nucleotide sequence. Such substantial identity may be a sequence that exhibits at least 80% homology, at least 90% homology, or at least 95% homology when the nucleotide sequence and any other sequence are aligned to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art.

[0070]

[0071] In addition, the present invention provides a recombinant vector comprising the polynucleotide.

[0072] In addition, the present invention provides a transformant isolated by transformation with the recombinant vector.

[0073] In the present invention, “vector” means a self-replicating DNA molecule used to carry a clone gene (or other piece of clone DNA).

[0074] In the present invention, “recombinant vector” refers to a plasmid, viral vector or other vector known in the art capable of expressing an inserted nucleic acid in a host cell, and may be a polynucleotide encoding a peptide of the present invention operably linked to a conventional expression vector known in the art. The recombinant vector may generally include a replication origin capable of proliferating in a host cell, one or more expression control sequences (e.g., promoter, enhancer, etc.) that regulate expression, a selective marker, and a polynucleotide encoding a peptide of the present invention operably linked to the expression control sequence. The transformant may be one transformed by the recombinant vector.

[0075] Preferably, the transformant can be obtained by introducing a recombinant vector containing a polynucleotide encoding the peptide of the present invention into a host cell by a method known in the art, for example, but not limited to, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene gun, and other known methods for introducing nucleic acids into cells.

[0076]

[0077] Additionally, the present invention provides a ligand-biotin conjugate in which biotin is bound to the ligand.

[0078] In addition, the present invention provides a polypeptide-biotin conjugate in which biotin is bound to the polypeptide.

[0079] In addition, the present invention provides a ligand-fluorescent substance conjugate in which a fluorescent substance is bound to the ligand.

[0080] In addition, the present invention provides a polypeptide-fluorescent substance conjugate in which a fluorescent substance is bound to the polypeptide.

[0081] Preferably, the fluorescent material may be, but is not limited to, AZDye647 or carboxyfluorescein.

[0082]

[0083] In addition, the present invention provides a composition for detecting IL4Ra comprising the ligand, polypeptide, ligand-biotin conjugate, polypeptide-biotin conjugate, ligand-fluorophore conjugate or polypeptide-fluorophore conjugate as an active ingredient.

[0084]

[0085] In addition, the present invention provides a composition for diagnosing cancer in which IL4Ra is overexpressed, comprising the ligand, polypeptide, ligand-biotin conjugate, polypeptide-biotin conjugate, ligand-fluorophore conjugate or polypeptide-fluorophore conjugate as an active ingredient.

[0086] Preferably, the cancer in which IL4Ra is overexpressed may be, but is not limited to, glioblastoma, diffuse intrinsic pontine tumor, brain tumor, breast cancer, pancreatic cancer, liver cancer, bone cancer, ovarian cancer, biliary tract cancer, colon cancer, head and neck cancer, bladder cancer, stomach cancer, kidney cancer, uterine cancer, prostate cancer, spinal cord cancer, lung cancer, or skin cancer.

[0087] As used herein, "diagnosis" means confirming the presence or characteristics of a pathological condition. For the purposes of the present invention, diagnosis means confirming the presence or characteristics of cancer.

[0088] Diagnosis of cancer using the present invention can be made by reacting the ligand, polypeptide, ligand-biotin conjugate, polypeptide-biotin conjugate, ligand-fluorophore conjugate or polypeptide-fluorophore conjugate of the present invention with the relevant tissue or cell obtained directly by blood, urine or biopsy and detecting the binding thereof.

[0089]

[0090] In addition, the present invention provides a composition for imaging IL4Ra-overexpressing cancer cells, comprising the ligand, polypeptide, ligand-biotin conjugate, polypeptide-biotin conjugate, ligand-fluorophore conjugate or polypeptide-fluorophore conjugate as an active ingredient.

[0091] Preferably, the ligand or polypeptide may be labeled with, but is not limited to, a chromogenic enzyme, a radioisotope, a chromophore, a luminescent material, a fluorescent material, a magnetic resonance imaging material, a superparamagnetic particle, or an ultrasuper paramagnetic particle.

[0092] More preferably, the fluorescent material may be, but is not limited to, AZDye647 or carboxyfluorescein.

[0093] Cancer cell imaging and cancer diagnosis can be used for purposes other than initial diagnosis of cancer, including, but not limited to, monitoring progression, treatment progress, and response to therapeutic agents. The ligand or polypeptide may be provided in a labeled state to facilitate confirmation, detection, and quantification of binding, as described above.

[0094]

[0095] In addition, the present invention provides a ligand-drug conjugate in which a drug is bound to the ligand.

[0096] In addition, the present invention provides a polypeptide-drug conjugate in which a drug is bound to the polypeptide.

[0097] Preferably, the drug may be an anticancer agent, and more preferably, at least one selected from the group consisting of Exatecan, DXD, deruxtecan, SN38, Topotecan, Doxorubicin, Monomethyl Auristatin E (MMAE), Monomethyl Auristatin F (MMAF), and Mal-PEG4-VA-PBD, but is not limited thereto.

[0098] Preferably, the ligand or polypeptide can be linked to the drug via a linker, wherein the linker has a C2, C3, C4, C5, C6, C7, C8 or C9 carbon to the nitrogen atom of the maleimide functional group and can be a compound linked to the drug via an ester bond, an amide bond, a carbamate bond or a hydrazone bond, and more preferably, the linker is maleimidyl propionic acid (3-Maleimidopropionic Acid; MPA), 1-(2-aminoethyl)maleimide, N-(2-hydroxyethyl)maleimide, 6-maleimidohexanoic acid, succinic acid or maleidocaproyl-valine-citrulline-paranitroaminobenzoic acid. (Mc-Val-Cit-Pab; MVCP), but is not limited thereto.

[0099]

[0100] In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer in which IL4Ra is overexpressed, comprising the ligand-drug conjugate or the polypeptide-drug conjugate as an active ingredient.

[0101] Preferably, the cancer in which IL4Ra is overexpressed may be, but is not limited to, glioblastoma, diffuse intrinsic pontine tumor, brain tumor, breast cancer, pancreatic cancer, liver cancer, bone cancer, ovarian cancer, biliary tract cancer, colon cancer, head and neck cancer, bladder cancer, stomach cancer, kidney cancer, uterine cancer, prostate cancer, spinal cord cancer, lung cancer, or skin cancer.

[0102]

[0103] In addition, the present invention provides a pharmaceutical composition for suppressing resistance to temozolomide in a brain tumor patient, comprising the ligand-drug conjugate or the polypeptide-drug conjugate as an active ingredient.

[0104] The pharmaceutical composition of the present invention can be manufactured using pharmaceutically suitable and physiologically acceptable auxiliary agents in addition to the active ingredient, and the auxiliary agents can be solubilizers such as excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, glidants, or flavoring agents. The pharmaceutical composition of the present invention can be preferably formulated as a pharmaceutical composition by additionally including one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration. In the composition formulated as a liquid solution, acceptable pharmaceutical carriers are sterile and biocompatible, and can be used as a mixture of saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders and lubricants can be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions and emulsions, pills, capsules, granules or tablets.

[0105] The pharmaceutical formulation form of the pharmaceutical composition of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions, and sustained-release formulations of the active compound, etc. The pharmaceutical composition of the present invention may be administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. The effective amount of the active ingredient of the pharmaceutical composition of the present invention refers to the amount required for the prevention or treatment of a disease. Therefore, it can be adjusted according to various factors including the type of disease, the severity of the disease, the types and contents of the active ingredient and other ingredients contained in the composition, the type of formulation, and the patient's age, body weight, general health condition, sex and diet, administration time, administration route and secretion rate of the composition, treatment period, and concurrently used drugs.

[0106] Below, the present invention is described in detail using examples that do not limit the scope of the invention. The following examples are intended to concretize the invention and do not limit or restrict the scope of the invention. Therefore, anything that a specialist in the technical field can easily infer from the detailed description and examples of the invention is interpreted as falling within the scope of the invention.

[0107]

[0108] <Example 1> Synthesis and high-pressure liquid chromatography (HPLC) analysis of IL4Ra ligand

[0109] The amino acid sequences of the ligand (SEQ ID NO: 1) and ligand (SEQ ID NO: 2) that specifically bind to IL4Ra are shown in Table 1.

[0110] Sequence number Amino acid sequence 1 RYRKRLDRNC 2 (Head-to-tail cyclic) RYRKRLDRNC

[0111] RYRKRLDRNC ligands represented by SEQ ID NO: 1 and (head-to-tail cyclic)RYRKRLDRNC ligands represented by SEQ ID NO: 2 were synthesized by solid-phase peptide synthesis.

[0112] Purity was analyzed using high-pressure liquid chromatography, and molecular weight was measured using the MALDI-TOF method and compared with the theoretically predicted molecular weight.

[0113] As a result, the purities of the ligand (SEQ ID NO: 1) and ligand (SEQ ID NO: 2) were 93.0% and 94.9%, respectively, as shown in Fig. 1.

[0114] As a result, the actual molecular weights were 1380.4 kDa and 1362.4 kDa, as shown in Table 2.

[0115] Sequence numberTheoretical molecular weight (Da)Actual molecular weight (Da)11379.61380.421361.61362.4

[0116]

[0117] <Example 2> Preparation of IL4Ra binding polypeptide

[0118] 1. Cloning of the IL4Ra binding polypeptide gene

[0119] The amino acid sequences of the polypeptides represented by SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 and the genes of the polypeptides represented by SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 were compiled into a sequence list.

[0120] The polypeptide gene was synthesized by continuous solid-phase synthesis and ligated to linear pET26b(+) to generate novel expression vectors pET26b(+)-1, pET26b(+)-2, and pET26b(+)-3, which were transformed into E. coli expression strains by heat shock and stored at -80°C.

[0121]

[0122] 2. IL4Ra binding polypeptide expression

[0123] Transformed E. coli cells stored at -80°C were inoculated into starter culture (250 mL flasks containing 50 mL of medium supplemented with 100 μg / mL ampicillin) and cultured overnight at 37°C with shaking. The starter culture was centrifuged at 3,000 g for 15 minutes at 4°C and resuspended in 10 mL of fresh medium. 5 mL of the starter culture suspended in expression culture (4 L flasks containing 1 L of medium with 100 μg / mL ampicillin) was inoculated and cultured with shaking at 37°C. When the optical density (OD) at 600 nm reached approximately 0.8, IPTG (final concentration: 1 mM) was added to induce expression. After 3 hours of expression induction, cells were collected by centrifugation at 3,000 g for 20 minutes at 4°C.

[0124]

[0125] 3. IL4Ra binding polypeptide purification

[0126] The collected E. coli cells were resuspended in 35 mL of cold PBS buffer (pH 7.4) and disrupted using sonication at 4°C. The cell lysate was centrifuged at 15,000 g for 15 minutes at 4°C, and undissolved cell debris was removed. After adding NaCl (2.5 M) to the cell lysate, the IL4Ra binding polypeptide was aggregated at room temperature. The aggregated protein was separated from the solution by centrifugation at 10,000 g for 15 minutes at 40°C. The supernatant was removed, and the pellet was dissolved in cold PBS buffer, and additional reverse transition cycling was performed to obtain a high-purity IL4Ra binding polypeptide.

[0127]

[0128] 4. Confirmation of IL4Ra binding polypeptide molecular weight

[0129] The mass of the purified polypeptide (SEQ ID NO: 4) was analyzed using a mass spectrometry Q Exactive Plus instrument, and the measured molecular weight (35587.79 kDa) was confirmed to be almost identical to the theoretical molecular weight (35589.06 kDa).

[0130]

[0131] <Example 3> Synthesis of IL4Ra binding ligand-biotin conjugate

[0132] RYRKRLDRNC ligand (2.6 mg, 1.88 μmol) represented by SEQ ID NO: 1 was placed in a round-bottom flask (10 mL), dissolved in DMF (2 mL) and sodium phosphate solution (0.7 mL), and then maleimide-biotin solution (25 mg / mL, 45.0 μL) was added, and the reaction mixture was stirred at room temperature for 1 hour. The ligand (SEQ ID NO: 1)-biotin conjugate was purified by recrystallization.

[0133] A ligand (SEQ ID NO: 2)-biotin conjugate was synthesized using the same method as the above ligand (SEQ ID NO: 1)-biotin conjugate synthesis.

[0134] As a result, the structures of the IL4Ra binding ligand (SEQ ID NO: 1)-biotin conjugate and the ligand (SEQ ID NO: 2)-biotin conjugate synthesized as in Fig. 2 are as in Fig. 2(A) and Fig. 2(B), respectively, and the maximum absorbance of the ligand-biotin conjugate was confirmed at a wavelength of 275 nm as in Fig. 2(C).

[0135]

[0136] <Example 4> Synthesis of a polypeptide (SEQ ID NO: 4)-biotin conjugate

[0137] Polypeptide (SEQ ID NO: 4) (99.8 μM, 0.25 mL) and DMF (3.47 mL) were mixed in a round-bottom flask (20 mL), and a maleimide-biotin solution (25 mg / mL, 180.3 μL) was added. The reaction mixture was stirred at room temperature for 4 h. The polypeptide (SEQ ID NO: 5)-biotin conjugate was purified by recrystallization.

[0138] As a result, the structure of the polypeptide (SEQ ID NO: 4)-biotin conjugate is as shown in Fig. 3(A), and the maximum absorbance of the polypeptide-biotin conjugate was confirmed at a wavelength of 275 nm as shown in Fig. 3(B).

[0139]

[0140] <Example 5> Determination of the equilibrium dissociation constant (Kd) between IL4Ra binding ligand and IL4Ra

[0141] IL4Ra solution (1.0 ug / ml) was prepared using phosphate-buffered saline and plated in a 96-well plate (Nunc MaxiSorp ™ 100 μL was added to each flat-bottom plate. After 2 hours, the IL4Ra solution was removed and washed twice with washing solution (380 μL, Phosphate buffer saline containing 0.05% (v / v)) and then blocking solution (200 μL, Block ™Casein) was added. After 1 hour, the blocking solution was removed, washed twice with washing solution (380 μL), and then the ligand (SEQ ID NO: 1)-biotin conjugate solution (100 μL) was added to the wells at concentrations of 60, 30, 15, 7.5, 3.75, 1.875, 0.938, 0.469, 0.234, 0.117, 0.059, 0.029, 0.015, 0.007, and 0.000 μM. After 1 hour, the ligand (SEQ ID NO: 1)-biotin conjugate solution was removed, washed three times with washing solution (380 μL), and streptavidin-HRP solution (100 μL, 1:1000 dilution) was added. After 1 hour, the streptavidin-HRP solution was removed, washed three times with washing solution (380 μL), TMB Substrate solution (100 μL) was added, and the absorbance at 650 nm was measured to be approximately 0.7 while monitoring the development of a blue color. Then, sulfuric acid solution (0.5 M, 100 μL) was added and the absorbance was measured at 450 nm. The absorbance according to the concentration of the ligand (SEQ ID NO: 1)-biotin conjugate was analyzed to determine the equilibrium dissociation constant (Kd).

[0142] The equilibrium dissociation constant (Kd) of the ligand (SEQ ID NO: 2)-biotin conjugate was determined in the same manner as the equilibrium dissociation constant (Kd) of the ligand (SEQ ID NO: 1)-biotin conjugate was determined.

[0143] As a result, as shown in Fig. 4, the equilibrium dissociation constant (Kd) of the ligand (SEQ ID NO: 1)-biotin conjugate (A) for IL4Ra was 98.11 nM, and the equilibrium dissociation constant (Kd) of the ligand (SEQ ID NO: 2)-biotin conjugate (B) was 444.0 nM.

[0144]

[0145] <Example 6> Determination of the equilibrium dissociation constant (Kd) between polypeptide (SEQ ID NO: 4) and IL4Ra

[0146] A polypeptide (SEQ ID NO: 4) solution (1.0 μg / ml) was prepared using sodium phosphate solution and added 100 μL to each 96-well plate. After 2 h, the polypeptide solution was removed, washed twice with washing solution (380 μL), and blocking solution (200 μL) was added. After 1 h, the blocking solution was removed, washed twice with washing solution (380 μL), and biotin-IL4Ra solution (100 μL) was added to the wells at concentrations of 150, 75, 37.5, 18.75, 9.38, 4.69, 2.34, 1.17, 0.59, 0.29, 0.15, 0.07, 0.04, and 0.00 nM. After 1 hour, the biotin-IL4Ra solution was removed, washed three times with washing solution (380 μL), and streptavidin-HRP solution (100 μL) was added. After 1 hour, the streptavidin-HRP solution was removed, washed three times with washing solution (380 μL), and TMB substrate solution (100 μL) was added. After a certain period of time, sulfuric acid solution (0.5 M, 100 μL) was added, and the absorbance was measured at a wavelength of 450 nm. The absorbance values ​​according to the concentration of biotin-IL4Ra were analyzed to determine the equilibrium dissociation constant (Kd).

[0147] As a result, as shown in Fig. 5, the equilibrium dissociation constant (Kd) of IL4Ra and the polypeptide (SEQ ID NO: 4) was 134.1 nM, and the Kd of the ligand RKRNDRN was 5.54 mM.

[0148] As a result, it was confirmed that the binding affinity of the polypeptide (SEQ ID NO: 4) to IL4Ra was 41.3 times stronger than the binding affinity of the ligand RKRNDRN to IL4Ra.

[0149]

[0150] <Example 7> Synthesis of Polypeptide (SEQ ID NO: 4)-AZDye647 Conjugate

[0151] Polypeptide (SEQ ID NO: 4) (99.8 μM, 0.5 mL) and DMF (1.0 mL) were mixed in a round-bottom flask (10 mL), and a maleimide-AZDye647 solution (1.0 mg / mL, 1.0 mL) was added. The reaction mixture was stirred at room temperature for 2 h. The polypeptide (SEQ ID NO: 4)-AZDye647 conjugate was purified by recrystallization.

[0152] As a result, the structure of the polypeptide (SEQ ID NO: 4)-AZDye647 conjugate is the same as 6(A), and the maximum absorbance of the polypeptide (SEQ ID NO: 4)-AZDye647 conjugate was confirmed at a wavelength of 650 nm, as in 6(B).

[0153] As a result of fluorescence flow cytometry analysis, as shown in Fig. 6(C), the polypeptide (SEQ ID NO: 4)-AZDye647 conjugate bound to glioblastoma U87-MG cells in a concentration-dependent manner.

[0154]

[0155] <Example 8> Synthesis of a polypeptide (SEQ ID NO: 4)-carboxyfluorescein conjugate

[0156] Polypeptide (SEQ ID NO: 4) (99.8 μM, 0.5 mL) and DMF (1.0 mL) were mixed in a round-bottom flask (10 mL), and 5(6)-carboxyfluorescein N-hydroxysuccinimide ester solution (50.0 mg / mL, 26.3 μL) was added. The reaction mixture was stirred at room temperature for 2 hours, and the polypeptide (SEQ ID NO: 4)-biotin conjugate was purified by recrystallization.

[0157] As a result, the structure of the polypeptide (SEQ ID NO: 4)-carboxyfluorescein conjugate is as shown in Fig. 7(A), and the maximum absorbance of the polypeptide (SEQ ID NO: 4)-carboxyfluorescein conjugate was confirmed at a wavelength of 500 nm as shown in Fig. 7(B).

[0158] As a result of fluorescence microscopy analysis, as shown in Fig. 7(C), the polypeptide (SEQ ID NO: 4)-carboxyfluorescein conjugate was internalized into glioblastoma U87-MG cells in a concentration-dependent manner.

[0159]

[0160] <Example 9> Synthesis of Maleimidylpropionyl-SN38 (MPA-SN38)

[0161] MPA-SN38 was synthesized by the synthetic route of Fig. 8(A).

[0162] SN38 (2.0 g, 5.1 mmol), 3-Maleimidopropionic Acid (1.0 g, 6.6 mmol), Dimethylaminopyridine (0.06 g, 0.5 mmol), and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride [N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, 2.0 g, 9.9 mmol] were treated in chloroform (100 mL), and the mixture was stirred at room temperature for 15 hours.

[0163] The organic layer was washed with water, dried using anhydrous MgSO4, and concentrated in vacuo to obtain the product.

[0164] The product was further purified by chromatography using a silica gel column.

[0165]

[0166] <Example 10> Synthesis of Maleimidoethylcarbamoyl-SN38 (MEC-SN38)

[0167] MEC-SN38 was synthesized using the synthetic route of Fig. 8(B).

[0168] 1. Synthesis of 4-nitrophenylSN38 carbonate

[0169] SN38 (0.39 g, 1.0 mmol), triethylamine (1.0 g, 10.0 mmol), and 4-nitrophenyl chloroformate (0.30 g, 1.5 mmol) were treated in chloroform (50 mL), and the mixture was stirred at room temperature for 12 hours. The organic layer was washed with water, dried over anhydrous MgSO4, and concentrated in vacuo to obtain the product.

[0170]

[0171] 2. Synthesis of Maleimidoethylcarbamoyl-SN38 (MEC-SN38)

[0172] 4-Nitrophenyl SN38 carbonate (0.28 g, 0.5 mmol), triethylamine (0.5 g, 5.0 mmol) and N-(2-aminoethyl)maleimide hydrochloride [N-(2-Aminoethyl)maleimide Hydrochloride, 0.13 g, 0.74 mmol] were treated in chloroform (25 mL), and the mixture was stirred at room temperature for 12 hours.

[0173] The organic layer was washed with water, dried using anhydrous MgSO4, and concentrated in vacuo to obtain the product.

[0174]

[0175] <Example 11> Synthesis of Maleimidylpropionyl-Exatecan (MPA-Exatecan)

[0176] MPA-exatecan was synthesized by the synthetic route of Fig. 8(C).

[0177] Exatecan mesylate (0.53 g, 1.0 mmol), N-Succinimidyl 3-propionate (0.62 g, 3.0 mmol), N,N-diisopropylethylamine (5.4 g, 43 mmol) and dimethylformamide (55 mL) were treated in chloroform (110 mL), and the mixture was stirred at 30°C for 1 hour.

[0178] The organic layer was washed with water, dried using anhydrous MgSO4, and concentrated in vacuo to obtain the product.

[0179] The product was further purified by chromatography using a silica column.

[0180]

[0181] <Example 12> Synthesis of Maleimidoethylcarbamoyl-exatecan (MEC-exatecan)

[0182] MEC-exatecan was synthesized by the synthetic route of Fig. 8(D).

[0183] 1. Synthesis of N-(2-(hydroxy)maleimidyethyl 4-nitrophenylcarbonate)

[0184] N-(2-(Hydroxy)maleimide (0.71 g, 5 mmol), 4-nitrophenyl chloroformate (1.11 g, 5.5 mmol), and triethylamine (1.01 g, 10 mmol) were treated with dichloromethane (100 mL) and stirred at room temperature for 24 h.

[0185] The organic layer was washed with water, dried using anhydrous MgSO4, and concentrated in vacuo to obtain the product.

[0186]

[0187] 2. Synthesis of Maleimidoethylcarbamoylexatecan (MEC-exatecan)

[0188] Exatecan mesylate (0.24 g, 0.45 mmol), N,N-diisopropylethylamine (5.4 g, 43 mmol), 1-hydroxy-7-azabenzotriazole hydrate (0.14 g, 0.89 mmol), and 2-(Maleimidylethyl) 4-nitrophenyl carbonate [0.14 g, 0.25 mmol] were treated in dimethylformamide (10 mL), and the mixture was stirred at room temperature for 4 hours. Water (100 mL) was slowly added to the reaction solution to form a solid, which was then filtered to recover the product.

[0189]

[0190] <Example 13> Synthesis of Maleimidocaproyl-L-valine-L-citrulline-p-aminobenzylcarbamoylexatecan (MVCP-exatecan)

[0191] MVCP-exatecan was synthesized by the synthetic route of Fig. 9.

[0192] Exatecan mesylate (0.27 g, 0.5 mmol), N,N-Diisopropylethylamine (0.13 g, 1.0 mmol), 1-Hydroxy-7-azabenzotriazole hydrate (0.23 g, 1.5 mmol), and maleidocaproyl-valine-citrulline-p-aminobenzylalcohol p-nitrophenyl carbonate (0.34 g, 0.5 mmol) were treated in dimethylformamide (20 mL), and the mixture was stirred at room temperature for 4 hours.

[0193] Water (200 mL) was slowly added to the reaction solution to form a solid, which was then filtered to recover the product.

[0194]

[0195] <Example 14> Synthesis of Pyridyldithiopropyhydrazon-Doxorubicin

[0196] Pyridyldithiophene hydrazone-doxorubicin was synthesized using the synthetic route of Fig. 10.

[0197] Doxorubicin hydrochloride (58.0 mg, 0.1 mmol) was dissolved in anhydrous methanol (10 mL) in a round-bottom flask. 3-(2-pyridinyldithio)propanoic acid hydrazide [27.5 mg, 0.12 mmole] dissolved in anhydrous methanol (2 mL) was added to the doxorubicin solution. Three drops of trifluoroacetic acid were added to the reaction mixture, and the reaction was stirred at room temperature overnight.

[0198] The sample was purified by removing excess MeOH using a rotary evaporator and precipitating the solvent with anhydrous acetonitrile.

[0199]

[0200] <Example 15> Synthesis of pyridyldisulfide-succinic acid-SN38

[0201] Pyridyldisulfide-SA-SN38 was synthesized using the same synthetic route as in Fig. 11.

[0202]

[0203] 1. Synthesis of N-Hydroxysuccinic acid-SN38 (NHS-SA-SN38)

[0204] SN38 (1.64 g, 4.2 mmol), succinic anhydride (14.0 g, 0.14 mol), and N,N-diisopropylethylamine (31.5 mL, 181 mmol) were treated in dimethylformamide (130 mL), stirred at 40°C for 72 hours, and then NHS-SA-SN38 was purified by recrystallization.

[0205]

[0206] 2. Pyridyldisulfide-SA-SN38

[0207] Succinic acid-SN38 (0.968 g, 2.0 mmol), N-(3-dimethylaminopropyl)-N*?**?*-ethylcarbodiimide hydrochloride [N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, 1.74 g, 9.0 mmol], N-Hydroxysuccinimide (0.49 g, 4.26 mmol), 4-dimethylaminopyridine (0.24 g, 2.0 mmol), 2-(Pyridyldithio)ethylamine hydrochloride [2-(Pyridyldithio)ethylamine hydrochloride, 0.114 g, 0.61 mmol), Dimethylformamide (25 mL) were dissolved in dichloromethane. 35 mL) and stirred at room temperature for 12 hours.

[0208] The organic layer was washed with water, dried using anhydrous MgSO4, and concentrated in vacuo to obtain pyridyldisulfide-succinic acid-SN38.

[0209] Pyridyldisulfide-succinic acid-SN38 was further purified by chromatography using a silica gel column.

[0210]

[0211] <Example 16> Synthesis of IL4Ra-binding polypeptide (SEQ ID NO: 4)-MPA-SN38 conjugate

[0212] Polypeptide (SEQ ID NO: 4) (20.4 mM, 4 mL) and DMF (4.0 mL) were mixed in a round-bottom flask (20 mL), and MPA-SN38 solution (18.6 mM in DMSO, 4 mL) was added. The reaction mixture was stirred at room temperature for 12 h. The polypeptide (SEQ ID NO: 4)-MPA-SN38 conjugate was purified by recrystallization.

[0213] As a result, the structure of the polypeptide (SEQ ID NO: 4)-MPA-SN38 conjugate is as shown in Fig. 12(A), and the maximum absorbance of the polypeptide (SEQ ID NO: 4)-MPA-SN38 conjugate was confirmed at a wavelength of 360 nm as shown in Fig. 12(B).

[0214]

[0215] <Example 17> Synthesis of IL4Ra binding polypeptide (SEQ ID NO: 4)-MEC-SN38 conjugate

[0216] Polypeptide (SEQ ID NO: 4) (49.8 μM, 1.5 mL) and DMF (3.0 mL) were mixed in a round-bottom flask (10 mL), and MEC-SN38 solution (1.3 mM, 691.6 μL) was added. The reaction mixture was stirred at room temperature for 3 h. The polypeptide (SEQ ID NO: 4)-MEC-SN38 conjugate was purified by recrystallization.

[0217]

[0218] <Example 18> Synthesis of IL4Ra-binding polypeptide (SEQ ID NO: 4)-MPA-exatecan conjugate

[0219] Polypeptide (SEQ ID NO: 4) (49.8 μM, 1.5 mL) and DMF (3.0 mL) were mixed in a round-bottom flask (10 mL), and MPA-exatecan solution (5.1 mM, 176.3 μL) was added. The reaction mixture was stirred at room temperature for 12 h. The polypeptide (SEQ ID NO: 4)-MPA-exatecan conjugate was purified by recrystallization.

[0220]

[0221] <Example 19> Synthesis of IL4Ra-binding polypeptide (SEQ ID NO: 4)-MEC-exatecan conjugate

[0222] Polypeptide (SEQ ID NO: 4) (49.8 μM, 0.35 mL) and DMF (3.0 mL) were mixed in a round-bottom flask (10 mL), and MEC-exatecan solution (5.1 mM, 38.3 μL) was added. The reaction mixture was stirred at room temperature for 3 h. The polypeptide (SEQ ID NO: 4)-MEC-exatecan conjugate was purified by recrystallization.

[0223]

[0224] <Example 20> Synthesis of polypeptide (SEQ ID NO: 4)-MVCP-exatecan conjugate, polypeptide (SEQ ID NO: 5)-MVCP-exatecan conjugate, and peptide (SEQ ID NO: 6)-MVCP-exatecan conjugate

[0225] Polypeptide (SEQ ID NO: 4) (189.5 μM, 0.25 mL) and DMF (1.0 mL) were mixed in a round-bottom flask (10 mL), and MVCP-exatecan solution (15.9 mM, 238.4 μL) was added, and the reaction mixture was stirred at room temperature for 1 hour.

[0226] The polypeptide (SEQ ID NO: 4)-MVCP-exatecan conjugate was purified by recrystallization.

[0227] Polypeptide (SEQ ID NO: 5)-MVCP-exatecan conjugate and polypeptide (SEQ ID NO: 6)-MVCP-exatecan conjugate were synthesized in the same manner as the synthesis of the polypeptide (SEQ ID NO: 4)-MVCP-exatecan conjugate.

[0228] As a result, the structure of the polypeptide (SEQ ID NO: 4)-MVCP-exatecan conjugate is as shown in Fig. 13(A), and the maximum absorbance of the polypeptide (SEQ ID NO: 4)-MVCP-exatecan conjugate was confirmed at a wavelength of 365 nm as shown in Fig. 13(B).

[0229]

[0230] <Example 21> Synthesis of a polypeptide (SEQ ID NO: 5)-hydrazone-doxorubicin conjugate

[0231] Polypeptide (SEQ ID NO: 5) (282.0 μM, 2.4 mL) and DMF (4.0 mL) were mixed in a round-bottom flask (50 mL), and pyridyldithiopropyl hydrazone-doxorubicin (11.7 mg, 15.1 μmol) was added, and the reaction mixture was stirred at room temperature for 2 hours.

[0232] The polypeptide (SEQ ID NO: 5)-hydrazone-doxorubicin conjugate was purified by recrystallization.

[0233]

[0234] <Example 22> Synthesis of Polypeptide (SEQ ID NO: 5)-TEA-SA-Doxorubicin Conjugate

[0235] Polypeptide (SEQ ID NO: 5) (311.2 μM, 0.30 mL) and DMF (5.0 mL) were mixed in a round-bottom flask (20 mL), and pyridyldisulfide-succinic acid-SN38 (11.3 mg / mL, 0.65 mL) was added, and the reaction mixture was stirred at room temperature for 3 hours.

[0236] The polypeptide (SEQ ID NO: 5)-TEA-SA-doxorubicin conjugate was purified by the recrystallization method.

[0237]

[0238] <Example 23> Confirmation of cytotoxicity of ligand-MVCP-exatecan conjugate against human endothelium-derived pontine glioma cells

[0239] The cytotoxicity of the ligand (SEQ ID NO: 1)-MVCP-exatecan conjugate was confirmed using SF8628 cells with diffuse endothelial cell carcinoma.

[0240] To determine the IC50 value for cancer cells, SF8628 cells were cultured in DMEM medium containing 10% FBS, 2 mM L-glutamine, 50 μg / mL streptomycin, and 50 U / mL penicillin.

[0241] Cells were cultured in a culture chamber under conditions of 37°C, 95% relative humidity, and 5% CO2.

[0242] Cells were seeded at 1,000 cells per well in medium containing 10% FBS in a 96-well plate and cultured for 24 hours. After 24 hours of cell attachment, the culture medium was replaced with fresh DMEM medium (100 μL).

[0243] Ligand (SEQ ID NO: 1)-MVCP-exatecan was added to the wells at concentrations of 0, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 12.5, 5, 10, 25, 50, and 100 nM of exatecan, or irinotecan was added to the wells at concentrations of 10, 25, 50, 100, 250, 500, 1000, 2500, 5000, 10000, and 25000 nM of irinotecan.

[0244] After culturing for 144 h, the culture medium was removed, and the cells were washed once with 100 μL of cold DMEM medium. 10 μL of CCK-8 [2-(2-methoxy-4-nitrophenyl)-3-(4 nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] was added to each well and incubated for 1 h at 37°C, 95% relative humidity, and 5% CO2.

[0245] After incubation, the absorbance (OD) was measured at 450 nm using a microplate reader.

[0246] Cell viability curves were fitted to Sigmoidal, 4PL, X is log(Concentration) using GraphPad Prism 8.3.1.

[0247] The cytotoxicity of the ligand (SEQ ID NO: 2)-MVCP-exatecan conjugate was confirmed in the same manner as the cytotoxicity of the ligand (SEQ ID NO: 1)-MPA-exatecan conjugate.

[0248] As a result, the IC50 values ​​of the ligand-MVCP-exatecan conjugate (nM) and irinotecan (nM) for SF8628 cells were confirmed as shown in Table 3.

[0249] As a result, as shown in Table 3, it was confirmed that the cancer cell killing efficacy of the ligand (SEQ ID NO: 1)-MVCP-exatecan conjugate and the ligand (SEQ ID NO: 2)-MVCP-exatecan conjugate against the diffuse endogenous glioma SF8628 cells was stronger than that of irinotecan.

[0250] Cancer cell SF8628 Anticancer Irinotecan ligand (SEQ ID NO: 1)-MVCP-exatecan ligand (SEQ ID NO: 2)-MVCP-exatecan IC50 (nM) 871.0 (a) 11.13 (b) 7.427 (c) Cancer cell killing efficiency (fold) 1.0 (a / a) 78.3 (a / b) 117.3 (a / c)

[0251]

[0252] <Example 24> Confirmation of cytotoxicity of ligand-MVCP-exatecan conjugate against human glioblastoma cells

[0253] The IC50 values ​​of ligand-MVCP-exatecan conjugate (nM) and irinotecan (nM) against glioblastoma U87-MG cells were determined using the same method as that used to determine the cytotoxicity of ligand-MVCP-exatecan conjugate against diffuse endothelial cell carcinoma SF8628 cells.

[0254] As a result, as shown in Table 4, it was confirmed that the cancer cell killing efficacy of the ligand (SEQ ID NO: 1)-MVCP-exatecan conjugate and the ligand (SEQ ID NO: 2)-MVCP-exatecan conjugate against glioblastoma U87-MG cells was stronger than that of irinotecan.

[0255] Cancer cell U87-MG anticancer Irinotecan ligand (SEQ ID NO: 1)-MVCP-exatecan ligand (SEQ ID NO: 2)-MVCP-exatecan IC50 (nM) 4,420 (a) 36.07 (b) 27.42 (c) Apoptosis efficiency (fold) 1.0 (a / a) 122.5 (a / b) 161.2 (a / c)

[0256]

[0257] <Example 25> Determination of IC50 value of irinotecan against human breast cancer cells, pancreatic cancer cells, diffuse intrinsic glioma cells, and glioblastoma cells

[0258] Breast cancer MDA-MB231 cells, pancreatic cancer PANC-1 cells, diffuse intrinsic glioma SF8628 cells, and glioblastoma U87-MG cells were cultured in DMEM medium containing 10% FBS, 2 mM L-glutamine, 50 μg / mL streptomycin, and 50 U / mL penicillin.

[0259] After culturing the cells in a culture chamber under conditions of 37°C, 95% relative humidity, and 5% CO2, 1,000 cells per well were seeded in a medium containing 10% FBS in a 96-well plate, cultured for 24 hours to allow the cells to attach, and then the culture medium was replaced with fresh DMEM medium (100 μL).

[0260] Irinotecan was added to the wells at concentrations of 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50, 100, 250, 500, 1000, 2500, 5000, 10000, and 25000 nM.

[0261] After culturing for 144 h, the culture medium was removed, and the cells were washed once with 100 μL of cold DMEM medium. 10 μL of CCK-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] solution was added to each well, and the cells were incubated for 1 h under conditions of 37°C, 95% relative humidity, and 5% CO2. The absorbance was measured at a wavelength of 450 nm using a microplate spectrophotometer.

[0262] Cell survival curves were analyzed using Sigmoidal, 4PL, and X is log(Concentration) nonlinear regression models using GraphPad Prism 8.3.1.

[0263] As a result, the IC50 values ​​of irinotecan for breast cancer MDA-MB231 cells, pancreatic cancer PANC-1 cells, diffuse intrinsic glioma SF8628 cells, and glioblastoma U87-MG cells were 2,036 nM, 1,032 nM, 871.0 nM, and 4,420 nM, respectively, as shown in Table 5 A.

[0264]

[0265] <Example 26> Determination of IC50 value of polypeptide (SEQ ID NO: 4)-MPA-SN38 conjugate against human breast cancer cells, pancreatic cancer cells, diffuse endothelial cell carcinoma cells, and glioblastoma cells

[0266] After culturing the cells in a culture chamber under conditions of 37°C, 95% relative humidity, and 5% CO2, 1,000 cells were seeded per well in a medium containing 10% FBS in a 96-well plate, cultured for 24 hours to allow the cells to attach, and then the culture medium was replaced with fresh DMEM medium (100 μL).

[0267] Polypeptide (SEQ ID NO: 4)-MPA-SN38 was added to the wells at concentrations of SN38 of 0, 0.01, 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50, and 100 nM.

[0268] After culturing for 144 hours, the culture medium was removed, the cells were washed once with 100 μL of cold DMEM medium, 10 μL of CCK-8 solution was added to each well, and the cells were cultured for 1 hour under conditions of 37°C, 95% relative humidity, and 5% CO2. The absorbance was measured using a microplate spectrophotometer at a wavelength of 450 nm.

[0269] Cell survival curves were analyzed using Sigmoidal, 4PL, and X is log(Concentration) nonlinear regression models using GraphPad Prism 8.3.1.

[0270] As a result, as shown in Table 5 B, it was confirmed that the cancer cell killing efficacy of the polypeptide (SEQ ID NO: 4)-MPA-SN38 conjugate against breast cancer MDA-MB231 cells, pancreatic cancer PANC-1 cells, diffuse intrinsic glioma SF8628 cells, and glioblastoma U87-MG cells was stronger than that of irinotecan.

[0271]

[0272] <Example 27> Determination of IC50 value of polypeptide (SEQ ID NO: 4)-MEC-SN38 conjugate against human endothelial cell lines

[0273] The IC50 of the polypeptide (SEQ ID NO: 4)-MEC-SN38 conjugate against the disseminated intrinsic pontine glioma SF8628 cells was determined in the same manner as that used to determine the cancer cell killing efficacy of the polypeptide (SEQ ID NO: 4)-MPA-SN38 conjugate against the disseminated intrinsic pontine glioma SF8628 cells.

[0274] As a result, it was confirmed that the cancer cell killing efficacy of the polypeptide (SEQ ID NO: 4)-MEC-SN38 conjugate against the diffuse endogenous glioma SF8628 cells was more potent than that of irinotecan, as shown in Table 5 C.

[0275]

[0276] <Example 28> Determination of IC50 value of polypeptide (SEQ ID NO: 4)-MPA-exatecan conjugate against human breast cancer cells, pancreatic cancer cells, diffuse in situ glioma cells, and glioblastoma cells

[0277] The IC50 values ​​of the polypeptide (SEQ ID NO: 4)-MPA-exatecan conjugate against breast cancer MDA-MB231 cells, pancreatic cancer PANC-1 cells, diffuse intrinsic glioma SF8628 cells, and glioblastoma U87-MG cells were determined in the same manner as the IC50 values ​​of the polypeptide (SEQ ID NO: 4)-MPA-exatecan conjugate against breast cancer MDA-MB231 cells, pancreatic cancer PANC-1 cells, diffuse intrinsic glioma SF8628 cells, and glioblastoma U87-MG cells.

[0278] As a result, as shown in Table 5 D, it was confirmed that the cancer cell killing efficacy of the polypeptide (SEQ ID NO: 4)-MPA-exatecan conjugate against breast cancer MDA-MB231 cells, pancreatic cancer PANC-1 cells, diffuse intrinsic glioma SF8628 cells, and glioblastoma U87-MG cells was stronger than that of irinotecan.

[0279]

[0280] <Example 29> Confirmation of the cancer cell killing efficacy of a polypeptide (SEQ ID NO: 4)-MEC-exatecan conjugate against human endothelium-derived pontine glioma cells.

[0281] The IC50 of the polypeptide (SEQ ID NO: 4)-MEC-exatecan conjugate against the disseminated intrinsic pontine glioma SF8628 cells was determined in the same manner as that used to determine the cancer cell killing efficacy of the polypeptide (SEQ ID NO: 4)-MPA-exatecan conjugate against the disseminated intrinsic pontine glioma SF8628 cells.

[0282] As a result, it was confirmed that the cancer cell killing efficacy of the polypeptide (SEQ ID NO: 4)-MEC-exatecan conjugate against the diffuse endogenous glioma SF8628 cells was more potent than that of irinotecan, as shown in Table 5 E.

[0283]

[0284] <Example 30> Determination of IC50 values ​​of polypeptide (SEQ ID NO: 4)-MVCP-exatecan conjugate, polypeptide (SEQ ID NO: 5)-MVCP-exatecan conjugate, and polypeptide (SEQ ID NO: 6)-MVCP-exatecan conjugate against human endothelium-forming glioma cells and glioblastoma cells

[0285] 1,000 cells per well of 96-well plates containing 10% FBS were seeded in a medium containing 10% FBS for diffuse intrinsic glioma SF8628 cells or glioblastoma U87-MG cells, and cultured for 24 hours to allow cell attachment. The culture medium was then replaced with fresh DMEM medium (100 μL).

[0286] Polypeptide (SEQ ID NO: 4)-MVCP-exatecan, polypeptide (SEQ ID NO: 5)-MVCP-exatecan or polypeptide (SEQ ID NO: 6)-MVCP-exatecan were added to the wells at concentrations of 0, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50 and 100 nM of exatecan.

[0287] After culturing for 144 h, the culture medium was removed, and the cells were washed once with 100 μL of cold DMEM medium. 10 μL of CCK-8 [2-(2-methoxy-4-nitrophenyl)-3-(4 nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] was added to each well, and the cells were incubated for 1 h under conditions of 37°C, 95% relative humidity, and 5% CO2. The absorbance was measured at a wavelength of 450 nm using a microplate spectrophotometer.

[0288] Cell survival curves were analyzed using Sigmoidal, 4PL, and X is log(Concentration) nonlinear regression models using GraphPad Prism 8.3.1.

[0289] As a result, it was confirmed that the cancer cell killing efficacy of the polypeptide (SEQ ID NO: 4)-MVCP-exatecan conjugate, the polypeptide (SEQ ID NO: 5)-MVCP-exatecan conjugate, and the polypeptide (SEQ ID NO: 6)-MVCP-exatecan conjugate against the diffuse endothelial glioma SF8628 cells and the glioblastoma U87-MG cells was stronger than that of irinotecan, as shown in Table 5 F, G, and H.

[0290] Drug IC50 (nM) MDA-MD231 PANC-1 SF8628 U87-MGA Irinotecan 2,0361,03287 1.04,420 B Polypeptide (SEQ ID NO: 4)-MPA-SN38 1.255 1.739 0.46 102.138 C Polypeptide (SEQ ID NO: 4)-MEC-SN38--0.34 11-D Polypeptide (SEQ ID NO: 4)-MPA-exatecan 10.36 13.33 10.29 14.10 E Polypeptide (SEQ ID NO: 4)-MEC-exatecan--0.4389-F Polypeptide (SEQ ID NO: 4)-MVCP-exatecan--0.6164 1.309 G Polypeptide (SEQ ID NO: 5)-MVCP-exatecan--0.73791.401H Polypeptide (SEQ ID NO: 6)-MVCP-exatecan--0.66851.367

[0291]

[0292] <Example 31> Confirmation of overcoming drug resistance in human cancer cells with temozolomide drug resistance

[0293] To confirm the overcoming of anticancer drug resistance in cancer cells resistant to temozolomide, human glioblastoma T98G cells and polypeptide (SEQ ID NO: 4)-MPA-SN38 conjugate, polypeptide (SEQ ID NO: 4)-MVCP-exatecan conjugate, polypeptide (SEQ ID NO: 5)-MVCP-exatecan conjugate and polypeptide (SEQ ID NO: 6)-MVCP-exatecan conjugate were used.

[0294] The IC50 values ​​of the polypeptide (SEQ ID NO: 4)-MPA-SN38 conjugate, the polypeptide (SEQ ID NO: 4)-MVCP-exatecan conjugate, the polypeptide (SEQ ID NO: 5)-MVCP-exatecan conjugate and the polypeptide (SEQ ID NO: 6)-MVCP-exatecan conjugate against glioblastoma T98G cells were determined in the same manner as the cancer cell killing efficacy of the polypeptide (SEQ ID NO: 4)-MPA-SN38 conjugate against human cancer cells.

[0295] As a result, as shown in Fig. 16, the IC50 value of polypeptide (SEQ ID NO: 4)-MPA-SN38 (A) for glioblastoma T98G cells resistant to temozolomide was 1.356 nM, and the IC50 value of SN38 (B) was 10.31 nM, confirming that polypeptide (SEQ ID NO: 4)-MPA-SN38 overcomes anticancer drug resistance.

[0296] As a result, it was confirmed that the polypeptide (SEQ ID NO: 4)-MPA-SN38 conjugate, the polypeptide (SEQ ID NO: 4)-MVCP-exatecan conjugate, the polypeptide (SEQ ID NO: 5)-MVCP-exatecan conjugate, and the polypeptide (SEQ ID NO: 6)-MVCP-exatecan conjugate overcame the anticancer drug resistance of human glioblastoma T98G cancer cells, as shown in Table 6.

[0297] Drug IC50 (nM) Cancer cell killing efficacy (fold) SN38 10.31 (a) 1.0 (a / a) Exatecan 11.78 (b) 1.0 (b / b) Polypeptide (SEQ ID NO: 4) - MPA - SN38 1.36 (c) 7.58 (a / c) Polypeptide (SEQ ID NO: 4) - MVCP - Exatecan 1.55 (d) 7.6 (b / d) Polypeptide (SEQ ID NO: 5) - MVCP - Exatecan 1.07 (e) 11.0 (b / e) Polypeptide (SEQ ID NO: 6) - MVCP - Exatecan 0.97 (g) 12.1 (b / g)

[0298]

[0299] <Example 32> Confirmation of the cancer cell killing efficacy of a polypeptide (SEQ ID NO: 5)-hydrozone-doxorubicin conjugate on human pancreatic cancer cells.

[0300] To determine the IC50 values ​​for cancer cells, pancreatic cancer AsPc-1, Capan-1, Capan-2, PANC-1, SNU-213, SNU-324 or SNU-410 cells were cultured in DMEM medium containing 10% FBS, 2 mM L-glutamine, 50 μg / mL streptomycin and 50 U / mL penicillin.

[0301] Cells were seeded at 20,000 cells per well in a 96-well plate in medium containing 10% FBS and cultured for 24 h to allow cell attachment, after which the culture medium was replaced with fresh DMEM medium (100 μL). Polypeptide (SEQ ID NO: 5)-hydrozonated-doxorubicin was added to the wells at concentrations of 0, 0.025, 0.05, 0.1, 0.25, 0.5, 0.75, 1, 1.5, 2.5, 5, 10, and 20 μM of doxorubicin.

[0302] After culturing for 72 h, the culture medium was removed, and the cells were washed once with 100 μL of cold DMEM medium. 10 μL of CCK-8 [2-(2-methoxy-4-nitrophenyl)-3-(4 nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] was added to each well, and the cells were incubated for 1 h under conditions of 37°C, 95% relative humidity, and 5% CO2. The absorbance was measured at a wavelength of 450 nm using a microplate spectrophotometer.

[0303] Cell survival curves were analyzed using Sigmoidal, 4PL, and X is log(Concentration) nonlinear regression models using GraphPad Prism 8.3.1.

[0304] As a result, as shown in Table 7, it was confirmed that the cancer cell killing efficacy of the polypeptide (SEQ ID NO: 5)-hydrozone-doxorubicin conjugate against pancreatic cancer cells was stronger than that of doxorubicin.

[0305] Pancreatic cancer cells IC50 (μM) Doxorubicin polypeptide (SEQ ID NO: 5)-hydrozone-doxorubicin conjugate Cancer cell killing efficacy (fold) AsPc-10.18 0.00 46 39.1 Capan-1 > 1.00 26 > 3.8 Capan-2 > 1.00 67 > 1.5 PANC-1 > 1.00 19 > 5.2 SNU-2 134.60 50.47 9.8 SNU-3 240.48 690.05 468.9 SNU-4 100.85 0.17 5.0

[0306]

[0307] <Example 33> Confirmation of the cancer cell killing efficacy of the polypeptide (SEQ ID NO: 5)-TEA-SA-SN38 conjugate against human pancreatic cancer PANC-1 cells.

[0308] To determine the IC50 value for cancer cells, pancreatic cancer PANC-1 cells were seeded at 20,000 cells per well in a 96-well plate in medium containing 10% FBS and cultured for 24 h to allow cells to attach, after which the culture medium was replaced with fresh DMEM medium (100 μL). Polypeptide (SEQ ID NO: 5)-TEA-SA-SN38 was added to the wells at concentrations of SN38 of 0.01, 0.05, 0.1, 0.25, 0.5, 0.75, 1, 1.5, 2.5, 5, 7.5, 10, 25, 50, and 100 μM.

[0309] After culturing for 72 h, the culture medium was removed, and the cells were washed once with 100 μL of cold DMEM medium. 10 μL of CCK-8 [2-(2-methoxy-4-nitrophenyl)-3-(4 nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] was added to each well, and the cells were incubated for 1 h under conditions of 37°C, 95% relative humidity, and 5% CO2. The absorbance was measured at a wavelength of 450 nm using a microplate spectrophotometer.

[0310] Cell survival curves were analyzed using Sigmoidal, 4PL, and X is log(Concentration) nonlinear regression models using GraphPad Prism 8.3.1.

[0311] As a result, the structure of the polypeptide (SEQ ID NO: 5)-TSA-SA-SN38 conjugate as shown in Fig. 17 is Fig. 17(A), and the IC50 values ​​of the polypeptide (SEQ ID NO: 5)-TSA-SA-SN38 conjugate (a) and SN38 (b) for pancreatic cancer PANC-1 cells as shown in Fig. 17(B) were 0.4329 μM and 0.7606 μM, respectively.

[0312]

[0313] <Example 34> Confirmation of the temperature-sensitive properties of the polypeptide (SEQ ID NO: 4)-MPA-SN38 conjugate

[0314] The thermal transition properties of the polypeptide (SEQ ID NO: 4)-MPA-SN38 conjugate were determined by observing the absorbance of the conjugate solution at a wavelength of 700 nm while increasing or decreasing the temperature at a rate of 1 °C / min using an UV-Vis spectrophotometer. The transition temperature (Tt) was defined as the point where the maximum absorbance was 50%, and the transition temperature of the polypeptide (SEQ ID NO: 4)-MPA-SN38 conjugate was fitted to the following equation.

[0315] Tt = a · Ln([polypeptide (SEQ ID NO: 4)-MPA-SN38 conjugate]) + b

[0316] In the above formula, [polypeptide (SEQ ID NO: 4)-MPA-SN38 conjugate] is the concentration of the polypeptide (SEQ ID NO: 4)-MPA-SN38 conjugate, a is the slope, and b is the Tt of 1 μM of the polypeptide (SEQ ID NO: 4)-MPA-SN38 conjugate.

[0317] As a result, the reverse temperature transition of the polypeptide (SEQ ID NO: 4)-MPA-SN38 conjugate was confirmed at 34.5 degrees Celsius, as shown in FIG. 18.

[0318]

[0319] <Example 35> Evaluation of anticancer efficacy of polypeptide (SEQ ID NO: 4)-MPA-SN38 conjugate using a mouse model bearing human glioblastoma

[0320] 1. To test the anti-tumor efficacy of polypeptide (SEQ ID NO: 4)-MPA-SN38 in vivo, according to the same schedule as in Fig. 19, brain tumors were induced in BALB / c nude mice using the human glioblastoma U87-MG-Luc2 cell line, and polypeptide (SEQ ID NO: 4)-MPA-SN38 was administered intracerebral to evaluate the effect of polypeptide (SEQ ID NO: 4)-MPA-SN38 on brain tumors.

[0321] 2. Human glioblastoma cells are U87-MG-Luc2.

[0322] 3. The experimental animals were BALB / cSlc-nu / nu mice. The temperature of the breeding environment was 19.0-25.0℃, the relative humidity was 30.0-70.0%, the ventilation frequency was 10-15 times / hour, the lighting cycle was 12 hours (lights on at 7:00 AM - lights off at 7:00 PM), and the illuminance was 150-300 Lux. The breeding box was changed once a week, and the water bottle was changed at least twice a week.

[0323] 4. Tumor cell transplantation was performed two days after the final quarantine was completed. U87-MG-Luc2 cells in culture were harvested and 0.75 × 10 5cells / μL. The head of the test animal was fixed in a stereotaxic coordinate system, and the cell suspension was injected into the striatum (Striatum; Antero-Posterior from bregma: 0.6 mm; Medium-Lateral: 1.8 mm; Dorso-ventral from the skull surface: 3.5 mm) in the brain of the animal using a microinjector equipped with a Hamilton syringe (10 μL) at a dose of 2 μL (1.5 × 10 5 U87-MG-Luc2 cells were injected into the mouse (cells / animal).

[0324] 5. Seven days after tumor cell transplantation, at the first IVIS imaging, the animal with the weakest bioluminescence among the test animals transplanted with tumor cells was judged to be abnormal and excluded, and the animals were divided into three groups, six animals per group, to ensure that the luminescence values ​​were distributed as evenly as possible.

[0325] 6. Administration of polypeptide (SEQ ID NO: 4)-MPA-SN38: The head of the test animal was fixed in a stereotaxic coordinate system, and 10 μL / animal of the negative control substance, positive control substance, or polypeptide (SEQ ID NO: 4)-MPA-SN38 was injected three times using a microinjector equipped with a Hamilton syringe (10 μL, CED Needle) into the tumor implantation site in the animal's brain (Antero-Posterior from bregma: 0.6 mm; Medium-Lateral: 1.8 mm; Dorso-ventral from the skull surface: 3.5 mm). The injection rate was 1 μL / min, and the needle was left in place for 2 minutes after injection was completed and then removed.

[0326] 7. During the test period, general symptoms were observed once a day and the presence of moribund or dead animals was checked.

[0327] 8. Body weight was measured twice a week from the day of military separation.

[0328] 9. From the day of group separation until day 41, the growth of tumor cells was observed twice a week using IVIS. D-Luciferin (Cat. No.: 122799, PerkinElmer, USA) 100X stock (15 mg / ml) was diluted 100-fold using PBS to prepare D-Luciferin. D-Luciferin was administered intraperitoneally at a dose of 10 μL per 1 g of body weight to test animals transplanted with U-87 MG-Luc2 cells, and anesthesia was administered with Isoflurane 5 minutes later. Images were taken using IVIS (In Vivo Imaging System, IVIS Lumina X5 Imaging System, PerkinElmer, USA) 10 minutes after D-Luciferin administration. Tumor growth was expressed as the light intensity (protons / sec) in the ROI area, and bioluminescence was measured twice a week until the end of the experiment, and is shown in Figure 19.

[0329] 10. Survival rates were monitored for up to 65 days after military separation.

[0330] 11. All measurement results obtained in the experiment were statistically analyzed using SPSS (Version 27.0, IBM Corporation). The positive control group (G2) and the polypeptide (SEQ ID NO: 4)-MPA-SN38 administration group were subjected to Levene's equality of variance test and the independent t test was performed to verify significance.

[0331] 12. As a result, as shown in Figs. 20 and 21, in the negative control group, deaths occurred from the 39th day after tumor cell transplantation, and all animals died by the 42nd day. In the positive control group, deaths occurred from the 34th day after tumor cell transplantation, and all animals died by the 44th day. In the polypeptide (SEQ ID NO: 4)-MPA-SN38 administration group, deaths occurred from the 49th day after tumor cell transplantation, and a total of 3 test animals died by the 65th day, showing a survival rate of 50%. In the polypeptide (SEQ ID NO: 4)-MPA-SN38 administration group, a decrease in body weight was observed after three intracerebral administrations of the test substance, but it increased again thereafter. After the third administration, the increase was statistically significant compared to the negative control group and the positive control group topotecan administration group, which showed a decrease in body weight.

[0332] As a result, the anti-tumor efficacy of polypeptide (SEQ ID NO: 4)-MPA-SN38 was confirmed using an orthotopic glioblastoma model, as summarized in Table 8.

[0333] Item Negative control Topotecan polypeptide (SEQ ID NO: 4)-MPA-SN38 conjugate Dose (SN38 μg / time) 01.228 1.232 Number of administrations 333 Tumor growth inhibition (%) 023.19 5.9 Average survival 4242.565 days or more

[0334]

[0335] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. In other words, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A ligand that specifically binds to IL4Ra, comprising an amino acid sequence represented by sequence number 1 or sequence number 2.

2. A polypeptide that specifically binds to IL4Ra, wherein a temperature-sensitive atypical domain comprising the ligand of paragraph 1 and an amino acid sequence represented by sequence number 3 is repeatedly linked.

3. A polypeptide according to claim 2, characterized in that the polypeptide comprises any one amino acid sequence selected from the group consisting of SEQ ID NO: 4 to SEQ ID NO:

6.

4. A polynucleotide encoding the ligand of paragraph 1 or the polypeptide of paragraph 2.

5. A recombinant vector comprising the polynucleotide of clause 4.

6. A transformant isolated by transformation with the recombinant vector of Article 5.

7. A ligand-biotin conjugate in which biotin is bound to the ligand of clause 1.

8. A polypeptide-biotin conjugate in which biotin is bound to the polypeptide of clause 2.

9. A ligand-fluorescent substance conjugate in which a fluorescent substance is bound to the ligand of clause 1.

10. A polypeptide-fluorescent substance conjugate in which a fluorescent substance is bound to the polypeptide of clause 2.

11. A conjugate according to claim 9 or 10, characterized in that the fluorescent substance is AZDye647 or carboxyfluorescein.

12. A composition for detecting IL4Ra, comprising the ligand of claim 1, the polypeptide of claim 2, or the conjugate of any one of claims 7 to 10 as an active ingredient.

13. A composition for diagnosing cancer in which IL4Ra is overexpressed, comprising the ligand of claim 1, the polypeptide of claim 2, or the conjugate of any one of claims 7 to 10 as an active ingredient.

14. A composition for diagnosing cancer, characterized in that the cancer in which IL4Ra is overexpressed in claim 13 is glioblastoma, diffuse intrinsic pontine glioma, brain tumor, breast cancer, pancreatic cancer, liver cancer, bone cancer, ovarian cancer, biliary tract cancer, colon cancer, head and neck cancer, bladder cancer, stomach cancer, kidney cancer, uterine cancer, prostate cancer, spinal cord cancer, lung cancer, or skin cancer.

15. A composition for imaging IL4Ra-overexpressing cancer cells, comprising the ligand of claim 1, the polypeptide of claim 2, or the conjugate of any one of claims 7 to 10 as an active ingredient.

16. A ligand-drug conjugate in which a drug is bound to the ligand of clause 1.

17. A polypeptide-drug conjugate in which a drug is conjugated to the polypeptide of paragraph 2.

18. A conjugate according to claim 16 or 17, characterized in that the drug is an anticancer agent.

19. A conjugate according to claim 18, characterized in that the anticancer agent is at least one selected from the group consisting of Exatecan, DXD, deruxtecan, SN38, Topotecan, Doxorubicin, Monomethyl Auristatin E (MMAE), Monomethyl Auristatin F (MMAF), and Mal-PEG4-VA-PBD.

20. A conjugate according to claim 16 or 17, characterized in that the ligand or polypeptide is linked to a drug via a linker.

21. A conjugate according to claim 20, wherein the linker is 3-Maleimidopropionic Acid (MPA), 1-(2-Aminoethyl)maleimide, N-(2-hydroxyethyl)maleimide, 6-Maleimidohexanoic acid, succinic acid or maleidocaproyl-valine-citrulline-paranitroaminobenzoic acid (Mc-Val-Cit-Pab; MVCP).

22. A pharmaceutical composition for the prevention or treatment of cancer in which IL4Ra is overexpressed, comprising the ligand-drug conjugate of clause 16 or the polypeptide-drug conjugate of clause 17 as an active ingredient.

23. A pharmaceutical composition for preventing or treating cancer, characterized in that in claim 22, the cancer in which IL4Ra is overexpressed is glioblastoma, diffuse intrinsic pontine glioma, brain tumor, breast cancer, pancreatic cancer, liver cancer, bone cancer, ovarian cancer, biliary tract cancer, colon cancer, head and neck cancer, bladder cancer, stomach cancer, kidney cancer, uterine cancer, prostate cancer, spinal cord cancer, lung cancer, or skin cancer.

24. A pharmaceutical composition for suppressing resistance to temozolomide in a patient with a brain tumor, comprising the ligand-drug conjugate of clause 16 or the polypeptide-drug conjugate of clause 17 as an active ingredient.

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