WO2025154925A1 - Polypeptide drug conjugate binding to il13ra2 and use thereof - Google Patents

Abstract

The present invention relates to a polypeptide drug conjugate binding to IL13Ra2 and use thereof, and provides: a polypeptide comprising ligands specifically binding to IL13Ra2 and temperature-sensitive atypical domains; and a polypeptide-drug conjugate in which an apoptotic drug is bound to the polypeptide. More specifically, a polypeptide, in which IL13Ra2 binding ligands and temperature-sensitive atypical domains are repeatedly linked, binds to IL13Ra2 very selectively, and a polypeptide-drug conjugate, in which an apoptotic drug is bound to the polypeptide, exhibits greatly enhanced anticancer and anti-tumor activities compared with unbound drugs or existing anticancer agents, and was identified to have an effect of inhibiting anticancer agent resistance of cancer cells retaining resistance to the anticancer agent temozolomide. Therefore, the polypeptide-drug conjugate can be provided as an effective anticancer therapeutic agent for the treatment of tumors overexpressing IL13Ra2 and as a pharmaceutical composition for inhibiting drug resistance of cancer cells showing drug resistance to anticancer agents.

Description

Polypeptide drug conjugates binding to IL13Ra2 and uses thereof

The present invention relates to a polypeptide drug conjugate binding to interleukin 13 receptor subunit alpha 2 (IL13Ra2) and uses thereof, and more particularly, to a polypeptide-drug conjugate in which an IL13Ra2 binding ligand and a polypeptide comprising an atypical domain having temperature-sensitive properties are covalently bonded to an apoptotic drug, and medical uses thereof.

Glioblastoma is a rare disease that occurs in the brain of adults. It is a highly malignant disease with an average survival period of less than 15 months and a 5-year survival rate of less than 7%. Diffuse intrinsic pontine glioma mainly occurs in the pons of children. The average age at diagnosis is 6.1 years, the average survival period after diagnosis is 13.2 months, and the 5-year survival rate is less than 2%. It is a rare pediatric disease for which neurosurgery is completely impossible and there is no treatment.

Combination chemoradiation therapy using radiation and temozolomide simultaneously is the standard treatment for brain tumors such as glioblastoma or diffuse intrinsic pontine glioma, but the standard treatment is not only nonspecific and cannot distinguish between cancer cells and normal cells, but also induces resistance to temozolomide through the drug release mechanism mediated by P-glycoprotein.

Topotecan or panobinostat, which are used in chemotherapy for brain tumors, can also inhibit the proliferation of cancer cells or induce apoptosis, but because they lack cell selectivity, they have the side effect of killing not only cancer cells but also normal cells. 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 attempted to treat brain tumor patients. However, because there is a special cell membrane structure called the blood-brain barrier with very low drug permeability, anticancer drugs cannot penetrate into the tumor tissue, resulting in very low anticancer cure rates.

Convection-enhanced delivery has been developed to deliver high concentrations of drugs to brain tumor tissues by avoiding the blood-brain barrier. By applying convection-enhanced delivery, anticancer drugs such as 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 that they remain inside the brain tumor is very short, less than 3 hours, and therefore the anticancer efficacy of the injected drugs is very limited.

IL13Ra2 is specifically overexpressed in the cell walls of tumor cells of brain tumors including glioblastoma and diffuse intrinsic glioma, breast cancer, and pancreatic cancer, whereas IL13Ra1 is expressed in both cancer cells and normal cells.

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 by 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 avidity for receptors.

Against this technical backdrop, in order to solve the above-mentioned problems, an innovative anticancer drug development strategy is needed to produce a multivalent polypeptide with increased selectivity and binding affinity for the IL13Ra2 receptor, selectively deliver drugs to cancer cells of tumors in which IL13Ra2 is specifically expressed, overcome anticancer drug resistance, and selectively modify anticancer drugs so that the anticancer drugs remain inside the tumor for a long period of time and maintain a continuous anticancer effect.

The purpose of the present invention is to provide a ligand that specifically binds to IL13Ra2, which comprises any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 4, and a polypeptide that specifically binds to IL13Ra2, in which the ligand and a temperature-sensitive atypical domain comprising an amino acid sequence represented by SEQ ID NO: 5 are repeatedly linked.

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, a ligand-fluorophore conjugate or a polypeptide-fluorophore conjugate in which a fluorescent substance is bound to the ligand or the polypeptide.

In addition, another object of the present invention is to provide a composition for detecting IL13Ra2, a composition for diagnosing cancer in which IL13Ra2 is overexpressed, or a composition for imaging cancer cells in which IL13Ra2 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.

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.

In addition, another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer in which IL13Ra2 is overexpressed, comprising the ligand-drug conjugate or the polypeptide-drug conjugate as an active ingredient.

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.

To achieve the above object, the present invention provides a ligand that specifically binds to IL13Ra2, comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 4.

In addition, the present invention provides a polypeptide that specifically binds to IL13Ra2, wherein a temperature-sensitive atypical domain consisting of the ligand and an amino acid sequence represented by SEQ ID NO: 5 is repeatedly linked.

In addition, the present invention provides a polynucleotide encoding the ligand or the polypeptide, a recombinant vector comprising the polynucleotide, and an isolated transformant transformed with the recombinant vector.

In addition, the present invention provides a ligand-biotin conjugate in which biotin is bound to the ligand.

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

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

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

In addition, the present invention provides a composition for detecting IL13Ra2 comprising the ligand, polypeptide, ligand-biotin conjugate, polypeptide-biotin conjugate, ligand-fluorophore conjugate or polypeptide-fluorophore conjugate as an active ingredient.

In addition, the present invention provides a composition for diagnosing cancer in which IL13Ra2 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.

In addition, the present invention provides a composition for imaging cancer cells overexpressing IL13Ra2, 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.

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

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

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer in which IL13Ra2 is overexpressed, comprising the ligand-drug conjugate or the polypeptide-drug conjugate as an active ingredient.

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.

The present invention relates to a polypeptide drug conjugate binding to IL13Ra2 and a use thereof. According to the present invention, a polypeptide-drug conjugate comprising an IL13Ra2 binding ligand and a temperature-sensitive atypical domain exhibits significantly enhanced cancer cell killing activity compared to unbound exatecan, SN38, and the existing anticancer agent irinotecan, and not only exhibits potent antitumor efficacy in a triple-negative breast cancer animal model, but also has an anticancer effect of inhibiting tumor growth and prolonging animal survival compared to the small molecule anticancer agent topotecan in an orthotopic brain tumor animal model. Accordingly, the polypeptide-drug conjugate can be provided as an effective anticancer therapeutic agent for the treatment of cancer.

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.

The polypeptide-biotin conjugate according to the present invention, in which a polypeptide and biotin are bound, bind very selectively to IL13Ra2 compared to IL13Ra1, and thus can be utilized to specifically detect cells expressing IL13Ra2.

The ligand-fluorescent substance conjugate according to the present invention can be utilized as a cancer diagnostic agent for diagnosing cancer in which IL13Ra2 is overexpressed.

Figure 1 is a high pressure liquid chromatography analysis graph of the ligands represented by sequence number 1 (A), sequence number 2 (B), sequence number 3 (C), and sequence number 4 (D).

Figure 2 shows the structures of ligand (SEQ ID NO: 1)-biotin conjugate (A), ligand (SEQ ID NO: 2)-biotin conjugate (B), ligand (SEQ ID NO: 3)-biotin conjugate (C), ligand (SEQ ID NO: 4)-biotin conjugate (D), and maleimide-biotin (E).

Figure 3 is a graph showing the determination of the equilibrium dissociation constant (Kd) between IL13Ra2 and a ligand (SEQ ID NO: 3) (A) and a ligand (SEQ ID NO: 4) (B), and a graph showing the analysis of the normalized detection signal (C).

Figure 4 is a graph showing the determination of equilibrium dissociation constants (Kd) between IL13Ra2 and a polypeptide (SEQ ID NO: 6) (A), IL13Ra2 and a polypeptide (SEQ ID NO: 7) (B), a polypeptide (SEQ ID NO: 8) (C), a polypeptide (SEQ ID NO: 9) (D), a polypeptide (SEQ ID NO: 10) (E), a polypeptide (SEQ ID NO: 11) (F), and a polypeptide (SEQ ID NO: 12) (G).

Figure 5 is a graph created by converting the absorbance of the Figure 4 graph into a standardized detection signal.

Figure 6 is a graph showing the determination of the equilibrium dissociation constant (Kd) between IL13Ra1 and a polypeptide (SEQ ID NO: 6) (A), a polypeptide (SEQ ID NO: 8) (B), or a polypeptide (SEQ ID NO: 10) (C), and a graph created by converting absorbance into a standardized detection signal (D).

Figure 7 shows the structures and fluorescence images of ligand (SEQ ID NO: 1)-AZDye549 conjugate (A), ligand (SEQ ID NO: 2)-AZDye549 conjugate (B), ligand (SEQ ID NO: 3)-AZDye549 conjugate (C), and ligand (SEQ ID NO: 4)-AZDye549 conjugate (D), and the absorbance spectra (E) of the conjugates.

Figure 8 shows a polypeptide (SEQ ID NO: 6)-AZDye647 conjugate (A), a polypeptide (SEQ ID NO: 10)-AZDye647 conjugate (B), the structure of AZDye647 (C), a photograph of the internalization of the polypeptide (SEQ ID NO: 6)-AZDye647 conjugate into SF8628 cells (D), and the UV absorbance spectra of the conjugates (E).

Figure 9 shows the synthetic processes of MPA-SN3, MEC-SN38, MPA-exatecan, and MEC-exatecan.

Figure 10 is a synthetic process of MVCP-exatecan and MVCP-doxorubicin.

Figure 11 is a graph of cell death of polypeptide (SEQ ID NO: 10)-MPA-SN38 (A), polypeptide (SEQ ID NO: 10)-MEC-SN38 (B), polypeptide (SEQ ID NO: 10)-MPA-exatecan (C), polypeptide (SEQ ID NO: 10)-MEC-exatecan (D), polypeptide (SEQ ID NO: 10)-MVCP-exatecan (E), and polypeptide (SEQ ID NO: 10)-MVCP-doxorubicin (F) in disseminated intrinsic glioma SF8628 cells.

Figure 12 shows that polypeptide (SEQ ID NO: 6)-MPA-SN38 overcomes anticancer drug resistance in human glioblastoma T98G cells resistant to temozolomide.

Figure 13 shows the tumor volume (A), body weight (B), weight of the excised tumor (C), and photograph (D) of the size of the excised tumor in an anti-tumor test of the polypeptide (SEQ ID NO: 10)-MPA-SN38 conjugate and the polypeptide (SEQ ID NO: 10)-MPA-exatecan conjugate using a subcutaneous transplantation triple-negative breast cancer animal model.

Figure 14 is a photograph monitoring the bioluminescence changes over time after administration of the negative control group (A), topotecan (B), and polypeptide (SEQ ID NO: 6)-MPA-SN38 conjugate (C) in an orthotopic transplantation glioblastoma animal model.

Figure 15 is a graph analyzing the inhibition of tumor proliferation (A), body weight (B), and survival rate (C) according to administration of the negative control group, topotecan, and polypeptide (SEQ ID NO: 5)-MPA-SN38 conjugate in an anti-tumor test using an orthotopic glioblastoma animal model.

The present invention has been completed by deriving a ligand that selectively binds to IL13Ra2, preparing a polypeptide comprising the ligand and a temperature-sensitive atypical domain, and preparing a polypeptide-drug conjugate in which the polypeptide and an apoptotic drug are conjugated to selectively kill cancer cells overexpressing IL13Ra2, overcome P-glycoprotein-mediated drug resistance, and sustainably maintain anticancer efficacy inside a tumor, thereby confirming overcoming anticancer drug resistance, cancer cell killing efficacy (in vitro), and in vivo anti-tumor efficacy (in vivo).

The present invention provides a ligand that specifically binds to IL13Ra2, comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 4.

Specifically, the ligand that specifically binds to IL13Ra2 has an amino acid sequence represented by the general formula (1) X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11 (general formula 1), wherein 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 amino acids are substituted, deleted, and/or added without abolishing the IL13Ra2 binding ability of the ligand, and X1 is R, K, H, N, or Q, X2 is R, K, H, N, or Q, X3 is A, G, V, L, or I, X4 is F, Y, or W, X5 is R, K, H, N, or Q, X6 is D, E, A, G, or V, X7 is A, G, or V, and X8 is R, K, H, N or Q, X9 can be F, Y, or W, X10 can be A, G, V, N, or Q, and X11 can be C or S.

More specifically, X1 may be R or K, X2 may be R or K, X3 may be V or L, X4 may be F or Y, X5 may be R or K, X6 may be D or E, X7 may be A or G, X8 may be R or K, X9 may be F or Y, X10 may be N or Q, and X11 may be C.

In addition, the present invention provides a polypeptide that specifically binds to IL13Ra2, wherein a temperature-sensitive atypical domain consisting of the ligand and an amino acid sequence represented by SEQ ID NO: 5 is repeatedly linked.

Specifically, the polypeptide may have an amino acid sequence represented by the following general formula (2) M[(IL13Ra2 binding ligand)(VGVPG)uCGVPG(VGVPG)v(IL13Ra2 binding ligand)(VGVPG)w]x(IL13Ra2 binding ligand)(VGVPG)yCGVPG(VGVPG)zW (general formula 2).

More specifically, in the general formula (2), the temperature-responsive atypical domain is composed of an amino acid sequence represented by SEQ ID NO: 5, (VGVPG), 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: 5. Even more specifically, the temperature-responsive atypical domain is composed of an amino acid sequence represented by SEQ ID NO: 5, (VGVPG), 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-responsive atypical domain is composed of an amino acid sequence represented by SEQ ID NO: 5, (VGVPG), wherein u can be 5, v can be 4, w can be 10, x can be 3, y can be 5, and z can be 4.

Preferably, the polypeptide may be composed of any one amino acid sequence selected from the group consisting of SEQ ID NO: 6 to SEQ ID NO: 12, but is not limited thereto.

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

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 an appropriate primer. 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 AppliedBiosystems). 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 therefrom. The resulting transformant is cultured under an appropriate medium and conditions so that the DNA sequence is 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.

In the present invention, the ligand or polypeptide is a concept including its functional variants. “Functional variant” means all similar sequences in which some amino acids are substituted at amino acid positions that do not affect the property of the ligand or polypeptide of the present invention that specifically binds to IL13Ra2.

Additionally, the present invention provides a polynucleotide encoding the polypeptide.

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

The above polynucleotide comprises not only a nucleotide sequence encoding the above polypeptide, but also a complementary sequence thereto. The complementary sequence includes not only a perfectly complementary sequence, but also a substantially complementary sequence.

In addition, the polynucleotide may be modified. The modification includes addition, deletion, or non-conservative or conservative substitution of nucleotides. The polynucleotide encoding the amino acid sequence is also interpreted to include a nucleotide sequence that exhibits substantial identity to the nucleotide sequence. The 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.

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

In addition, the present invention provides an isolated transformant transformed with the recombinant vector.

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

In the present invention, the “recombinant vector” means 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 the 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 control expression, a selective marker, and a polynucleotide encoding the peptide of the present invention operably linked to an expression control sequence. The transformant may be one transformed by the recombinant vector.

Preferably, the transformant can be obtained by introducing a recombinant vector comprising 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, a gene gun, and other known methods for introducing nucleic acids into cells.

In addition, the present invention provides a ligand-biotin conjugate in which biotin is bound to the ligand.

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

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

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

Preferably, the fluorescent substance may be AZDye594 or AZDye647, but is not limited thereto.

In addition, the present invention provides a composition for detecting IL13Ra2 comprising the ligand, polypeptide, ligand-biotin conjugate, polypeptide-biotin conjugate, ligand-fluorophore conjugate or polypeptide-fluorophore conjugate as an active ingredient.

In addition, the present invention provides a composition for diagnosing cancer in which IL13Ra2 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.

Preferably, the cancer in which IL13Ra2 is overexpressed may be, but is not limited to, 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.

In the present invention, "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.

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.

In addition, the present invention provides a composition for imaging cancer cells overexpressing IL13Ra2, 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.

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.

More preferably, the fluorescent substance may be, but is not limited to, AZDye594 or AZDye647.

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.

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

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

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.

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

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer in which IL13Ra2 is overexpressed, comprising the ligand-drug conjugate or the polypeptide-drug conjugate as an active ingredient.

Preferably, the cancer in which IL13Ra2 is overexpressed may be, but is not limited to, 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.

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.

The pharmaceutical composition of the present invention can be manufactured using pharmaceutically suitable and physiologically acceptable auxiliary agents in addition to the effective 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 effective ingredient for administration. In the composition formulated as a liquid solution, acceptable pharmaceutical carriers are sterile and biocompatible, and can be used by mixing one or more of saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents as needed. 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.

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 active compounds, 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 means 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 age, weight, general health condition, sex and diet of the patient, the time of administration, the route of administration and the secretion rate of the composition, the treatment period, and drugs used simultaneously.

Hereinafter, the present invention will be described in detail based on examples that do not limit the present invention. It should be noted that the following examples of the present invention are intended only to concretize the present invention and do not limit or restrict the scope of the rights of the present invention. Therefore, it is interpreted that what can be easily inferred by a specialist in the technical field to which the present invention belongs from the detailed description and examples of the present invention falls within the scope of the rights of the present invention.

< Example 1> IL13Ra2 Synthesis of ligands and High pressure liquid chromatography ( HPLC ) analyze

The amino acid sequences of ligand (SEQ ID NO: 1), ligand (SEQ ID NO: 2), ligand (SEQ ID NO: 3), and ligand (SEQ ID NO: 4) that specifically bind to IL13Ra2 are shown in Table 1.

Sequence number Amino acid sequence 1 KKLFREGRFC 2 (Head-to-tail cyclic)KKLFREGRFC 3 KKLFREGRYNC 4 RKLFREGRYNC

Ligands KKLFREGRFC represented by SEQ ID NO: 1, (Head-to-tail cyclic)KKLFREGRFC represented by SEQ ID NO: 2, KKLFREGRYNC represented by SEQ ID NO: 3, and RKLFREGRYNC represented by SEQ ID NO: 4 were synthesized by solid-phase peptide synthesis.

The purity was analyzed using high pressure liquid chromatography, and the molecular weight was measured using the MALDI-TOF method, which was then compared with the theoretically predicted molecular weight.

As a result, the purities of ligand (SEQ ID NO: 1), ligand (SEQ ID NO: 2), ligand (SEQ ID NO: 3), and ligand (SEQ ID NO: 4) were 94.2%, 91.9%, 87.9%, and 91.7%, respectively, as shown in Fig. 1.

As shown in Table 2, the actual molecular weights were 1284.0 kDa, 1397.9 kDa, 1414.6 kDa, and 1439.7 kDa.

Sequence number Theoretical molecular weight ( Da ) Actual molecular weight ( Da ) 1 1,283.6 1,284.0 2 1,397.7 1,397.9 3 1,413.7 1,414.6 4 1,441.7 1,439.7

< Example 2> IL13Ra2 Combine Polypeptide preparation

1. IL13ra2 Combine Polypeptide Gene Cloning

The amino acid sequences of the polypeptides represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12 and the genes of the polypeptides represented by SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19 were compiled into a sequence list.

The gene of the polypeptide was synthesized by continuous solid-phase synthesis and ligated into linear pET26b(+) to generate novel expression vectors pET26b(+)-1, pET26b(+)-2, pET26b(+)-3, pET26b(+)-4, pET26b(+)-5, pET26b(+)-6 and pET26b(+)-7, which were then transfected into E. coli by heat shock method. After transformation into the expression strain, it was stored at -80℃.

2. IL13ra2 Combine Polypeptide Expression

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 the expression culture (4 L flasks containing 1 L of medium with 100 μg/mL ampicillin) was inoculated and cultured at 37°C with shaking. 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.

3. IL13Ra2 Combine Polypeptide Refined

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. NaCl (2.5 M) was added to the cell lysate, and IL13ra2 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 inverse transition cycling was performed to obtain high-purity IL13ra2 binding polypeptide.

The molecular weights were measured by the MALDI-TOF method and compared with the theoretically predicted molecular weights. As a result, as shown in Table 3, the actual molecular weights of the polypeptide (SEQ ID NO: 6), polypeptide (SEQ ID NO: 7), polypeptide (SEQ ID NO: 8), polypeptide (SEQ ID NO: 9), polypeptide (SEQ ID NO: 10), polypeptide (SEQ ID NO: 11), and polypeptide (SEQ ID NO: 12) were 37151.4 kDa, 37154.0 kDa, 38061.6 kDa, 37951.0 kDa, 38146.9 kDa, 37950.8 kDa, and 38174.9 kDa.

Sequence number Theoretical molecular weight ( Da ) Actual molecular weight ( Da ) 6 37,152.2 37,151.4 7 37,130.9 37,154.0 8 38,063.0 38,061.6 9 37,950.9 37,951.0 10 38,147.0 38,146.9 11 37,950.9 37,950.8 12 38,174.9 38,174.9

< Example 3> IL13Ra2 Binding ligand- Biotin Conjugate synthesis

The KKLFREGRFC ligand (2.5 mg, 1.9 μmol) represented by the sequence number 1 was placed in a round-bottom flask (10 mL), and DMF (3 mL) and sodium phosphate solution (1.0 mL) were added to dissolve it. Then, the maleimide-biotin solution (25 mg/mL, 35.2 μL) was added, and the reaction mixture was stirred at room temperature for 2 hours. The ligand (SEQ ID NO: 1)-biotin conjugate was purified by the recrystallization method.

Ligand (SEQ ID NO: 2)-biotin conjugate, ligand (SEQ ID NO: 3)-biotin conjugate and ligand ((SEQ ID NO: 4)-biotin conjugate were synthesized using the same method as the synthesis of the above ligand (SEQ ID NO: 1)-biotin conjugate.

As a result, the structure of the synthesized IL13Ra2 binding ligand-biotin conjugate is as shown in Figure 2.

< Example 4> IL13Ra2 Combine Polypeptide - Biotin Conjugate synthesis

Polypeptide (SEQ ID NO: 6) (38.4 μM, 0.53 mL) and DMF (3.47 mL) were mixed in a round-bottom flask (20 mL), and maleimide-biotin solution (25 mg/mL, 153.3 μL) was added. The reaction mixture was stirred at room temperature for 4 h. The polypeptide (SEQ ID NO: 6)-biotin conjugate was purified by recrystallization.

By the same method as the synthesis of the above polypeptide (SEQ ID NO: 6)-biotin conjugate, the polypeptide (SEQ ID NO: 7)-biotin conjugate, the polypeptide (SEQ ID NO: 8)-biotin conjugate, the polypeptide (SEQ ID NO: 9)-biotin conjugate, the polypeptide (SEQ ID NO: 10)-biotin conjugate, the polypeptide (SEQ ID NO: 11)-biotin conjugate and the polypeptide (SEQ ID NO: 12)-biotin conjugate were synthesized.

< Example 5> IL13Ra2 With binding ligands With IL13Ra2 Equilibrium dissociation constant ( Kd ) decision

Il13Ra2 solution (2.5 μg/ml) was prepared using phosphate buffered saline and 100 μL was added to each 96-well plate (Nunc MaxiSorp™ flat-bottom plate). After 2 h, the IL13Ra2 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 h, the blocking solution was removed, washed twice with washing solution (380 μL), and then ligand (SEQ ID NO: 3)-biotin conjugate solution (100 μL) was added to the wells at concentrations of 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 h, the ligand (SEQ ID NO: 3)-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 h, the streptavidin-HRP solution was removed, washed three times with washing solution (380 μL), TMB Substrate solution (100 μL) was added, and the blue color was monitored. When the absorbance at 650 nm reached approximately 0.7, 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: 3)-biotin conjugate was analyzed to determine the equilibrium dissociation constant (Kd).

The equilibrium dissociation constant (Kd) of the ligand (SEQ ID NO: 4)-biotin conjugate was determined in the same manner as the equilibrium dissociation constant (Kd) of the ligand (SEQ ID NO: 3)-biotin conjugate was determined.

As a result, as shown in Fig. 3, the equilibrium dissociation constant (Kd) of the ligand (SEQ ID NO: 3)-biotin conjugate for IL13Ra2 was 575.4 nM, and the equilibrium dissociation constant (Kd) of the ligand (SEQ ID NO: 3)-biotin conjugate was 4,005 nM.

< Example 6> IL13Ra2 Combine Polypeptides and With IL13Ra2 Equilibrium dissociation constant ( Kd ) decision

A polypeptide solution (1.0 ug/ml) represented by the sequence number 6 was prepared using sodium phosphate solution and 100 μL was added to a 96-well plate. After 2 hours, the polypeptide solution was removed, washed twice with washing solution (380 μL), and blocking solution (200 μL) was added. After 1 hour, the blocking solution was removed, washed twice with washing solution (380 μL), and biotin-IL13Ra2 solution (100 μL) was added to the wells at concentrations of 400, 200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, 0.20, 0.1, and 0.00 nM. After 1 h, the biotin-IL13Ra2 solution was removed, washed three times with washing solution (380 μL), and streptavidin-HRP solution (100 μL) was added. After 1 h, the streptavidin-HRP solution was removed, washed three times with washing solution (380 μL), and TMB Substrate solution (100 μL) was added, and the blue color development was monitored. When the absorbance at 650 nm reached approximately 0.7, a sulfuric acid solution (0.5 M, 100 μL) was added, and the absorbance was measured at 450 nm. The absorbance values according to the concentration of biotin-IL13Ra2 were analyzed to determine the equilibrium dissociation constant (Kd).

The equilibrium dissociation constant (Kd) of the polypeptides represented by SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 and IL13Ra2 was determined in the same manner as that of determining the equilibrium dissociation constant (Kd) of the polypeptides represented by SEQ ID NO: 6 and IL13Ra2.

As a result, as shown in Fig. 4, the equilibrium dissociation constants (Kd) of IL13Ra2 and polypeptide (SEQ ID NO: 6), polypeptide (SEQ ID NO: 7), polypeptide (SEQ ID NO: 8), polypeptide (SEQ ID NO: 9), polypeptide (SEQ ID NO: 10), repeptide (SEQ ID NO: 11), and polypeptide (SEQ ID NO: 12) were 12.8 nM, 131.1 nM, 8.0 nM, 38.4 nM, 29.0 nM, 72.7 nM, and 20.3 nM, respectively.

Figure 5 is a graph drawn by converting the actual measured absorbance shown in Figure 4 into a standardized signal.

< Example 7> IL13Ra2 Combine Polypeptides and With IL13Ra1 Equilibrium dissociation constant ( Kd ) decision

A polypeptide solution (1.0 μg/ml) represented by SEQ ID NO: 8 was prepared using sodium phosphate solution and 100 μL was added to a 96-well plate. After 2 hours, the polypeptide solution was removed, washed twice with washing solution (380 μL), and blocking solution (200 μL) was added. After 1 hour, the blocking solution was removed, washed twice with washing solution (380 μL), and biotin-IL13Ra2 solution (100 μL) was added to the wells at concentrations of 400, 200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, 0.20, 0.1, and 0.00 nM. After 1 h, the biotin-IL13Ra1 solution was removed, washed three times with washing solution (380 μL), and streptavidin-HRP solution (100 μL) was added. After 1 h, the streptavidin-HRP solution was removed, washed three times with washing solution (380 μL), and TMB Substrate solution (100 μL) was added, and the blue color development was monitored. When the absorbance at 650 nm reached approximately 0.7, a sulfuric acid solution (0.5 M, 100 μL) was added, and the absorbance was measured at 450 nm. The absorbance values according to the concentration of biotin-IL13Ra2 were analyzed to determine the equilibrium dissociation constant (Kd).

The equilibrium dissociation constant (Kd) between the polypeptide represented by SEQ ID NO: 10 and IL13Ra1 was determined in the same manner as that between the polypeptide represented by SEQ ID NO: 8 and IL13Ra1.

As a result, as shown in FIG. 6(A), FIG. 6(B), and FIG. 6(C), the equilibrium dissociation constants (Kd) of IL13Ra1 and the polypeptide (SEQ ID NO: 6), the polypeptide (SEQ ID NO: 8), and the polypeptide (SEQ ID NO: 10) were 632.2 nM, 414.6 nM, and 4,660 nM, respectively.

As shown in Fig. 6(D), the actual absorbances shown in Figs. 6(A), 6(B), and 6(C) were converted into standardized signals to compare the binding avidity of the polypeptide (SEQ ID NO: 6), the polypeptide (SEQ ID NO: 8), and the polypeptide (SEQ ID NO: 10) to IL13Ra1 or IL13Ra2.

As shown in Table 4, the selectivity, affinity and binding avidity of the ligand (SEQ ID NO: 3), ligand (SEQ ID NO: 4), polypeptide (SEQ ID NO: 8) and polypeptide (SEQ ID NO: 10) for IL13Ra1 or IL13Ra2 were compared.

ligand or Polypeptide
Sequence number Kd for IL13Ra1
(nM) Kd for IL13Ra2
(nM) In IL13Ra2 Selectivity for Korea In IL13Ra1
About Korea
Increased binding force In IL13Ra2
About Korea
Increased binding force 3 966.7 (a) 575.4 (b) 1.68 (a/b) - - 8 414.6 (c) 8.00 (d) 51.8 (c/d) 2.33 (a/c) 71.9 (b/d) 4 2,777 (e) 4,005 (f) 0.69 (e/f) - - 10 4,660 (g) 29.0 (h) 153.7 (g/h) 0.60 (e/g) 138.1 (f/h)

< Example 8> L13Ra2 Binding ligand- AZDye549 Conjugate synthesis

KKLFREGRFC ligand (1.3 mg, 1.01 μmol) represented by SEQ ID NO: 1 was placed in a round-bottom flask (10 mL), and DMF (0.5 mL) and sodium phosphate solution (0.25 mL) were added to dissolve it. Then, maleimide-AZDye594 solution (2.82 mL, 35.9 μL) was added, and the reaction mixture was stirred at room temperature for 2 hours. The KKLFREGRFC (SEQ ID NO: 1)-AZDye594 conjugate was purified by recrystallization.

(Head-to-tail cyclic)KKLFREGRFC(SEQ ID NO: 2)-AZDye594 conjugate, KKLFREGRYNC(SEQ ID NO: 3)-AZDye594 conjugate, and RKLFREGRYNC((SEQ ID NO: 4)-AZDye594 conjugate were synthesized using the same method as the synthesis of the above KKLFREGRFC(SEQ ID NO: 1)-AZDye594 conjugate.

As a result, as shown in Fig. 7(E), the ligand-AZDye594 conjugates were confirmed to absorb UV light at 593 nm, which is the maximum absorption, and exhibited fluorescence.

< Example 9> L13Ra2 Combine Polypeptide - AZDye647 Conjugate synthesis

Polypeptide (SEQ ID NO: 10) (89.0 μM, 0.82 mL) and DMF (3.28 mL) were mixed in a round-bottom flask (20 mL), and maleimide-AZDye647 solution (5.0 mg/mL, 142.0 μL) was added. The reaction mixture was stirred at room temperature for 2 h. The polypeptide (SEQ ID NO: 10)-biotin conjugate was purified by recrystallization.

A polypeptide (SEQ ID NO: 6)-AZDye647 conjugate was synthesized in the same manner as the synthesis of the above polypeptide (SEQ ID NO: 10)-AZDye647 conjugate.

As a result, the polypeptide-AZDye647 conjugates as shown in Fig. 8(D) were confirmed to absorb UV light at 650 nm, which is the maximum absorption, and exhibited fluorescence.

As a result, as shown in Fig. 8(E), the polypeptide (SEQ ID NO: 6)-AZDye647 conjugate was internalized into the diffuse endogenous glioma SF3826 cells in a time- and concentration-dependent manner.

< Example 10> Maleimidopropionyl 38 ( MPA -SN38) Synthesis

MPA-SN38 having a structure similar to that in Fig. 9(A) was manufactured.

SN38 (2.0 g, 5.1 mmol), 3-Maleimidopropionic Acid [3-Maleimidopropionic Acid, 1.0 g, 6.6 mmol], dimethylaminopyridine [Dimethylaminopyridin, 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 h.

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

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

< Example 11> Malayimido Echil Cavamoyl SN38 ( Maleimidoethyl carbamoyl SN38, MEC -SN38) Synthesis

MEC-SN38 having a structure similar to that in Fig. 9(B) was manufactured.

1. 4- Nitrophenyl SN38 Carbonate (4- Nitrophenyl SN38 carbonate) synthesis

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 h.

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

2. Malayimido Echil Cavamoyl SN38 ( MEC -SN38) Synthesis

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 h.

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

< Example 12> Maleimidopropionyl Exatecan ( MPA - Exatecan ) Synthesis

MPA-exatecan having a structure similar to that in Figure 9(C) was manufactured.

Exatecan mesylate (0.53 g, 1.0 mmol), N-Succinimidyl 3-maleimidopropionate (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.

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

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

< Example 13> Malaysian Island Etchil Cavamoyl Exatecan ( Maleimidoethyl carbamoyl exatecan, MEC - Exatecan ) Synthesis

MEC-exatecan having a structure similar to that in Figure 9(D) was manufactured.

1. N-(2-( Hydroxy ) Malaymidyl ethyl ) 4- Nitrophenyl Carbonate Synthesis

N-(2-Hydroxyethyl)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.

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

2. Malaysian Island Etchil Cavamoyl Exatecan ( Maleimidoethyl carbamoyl exatecan , MEC-exatecan) synthesis

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 h.

Water (100 mL) was slowly added to the reaction solution to form a solid, which was then filtered to recover the product.

< Example 14> Malaysian Caproyl -Balin- Citrulline -p- Aminobenzyl Cavamoyl Exatecan ( Maleimidocaproyl -L- valine -L- citrulline -p- aminobenzyl carbamoyl exatecan, MVCP - Exatecan ) Synthesis

MVCP-exatecan having a structure similar to that in Figure 10(A) was prepared.

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 h.

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

< Example 15> Malaysian Caproyl -Balin- Citrulline -p- Aminobenzyl Cavamoyl Doxorubicin ( Maleimidocaproyl -L- valine -L- citrulline -p- aminobenzyl carbamoyl doxorubicin, MVCP - Doxorubicin ) Synthesis

MVCP-doxorubicin having a structure similar to that in Figure 10(B) was prepared.

Doxorubicin hydrochloride (145.0 mg, 0.25 mmol), N,N-diisopropylethylamine (64.6 mg, 0.5 mmol), and maleidocaproyl-valine-citrulline-p-aminobenzylalcohol p-nitrophenyl carbonate (184.4 mg, 0.25 mmol) were treated in dimethylformamide (10 mL), and the mixture was stirred at room temperature for 4 h.

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

< Example 16> Ligands represented by sequence numbers 1 to 4 and MVCP - With exatecan Conjugate synthesis

The ligand represented by the sequence number 1 (4.8 mg, 3.7 μmol) and MVCP-exatecan (947.1 mM in Dimethylsulfoxide, 39.5 μL) were treated in dimethylformamide (Dimethylformamide, 2 mL), and the mixture was stirred at room temperature for 3 hours.

The ligand (SEQ ID NO: 1)-MVCP-exatecan conjugate was recovered by recrystallization.

Ligand (SEQ ID NO: 2)-MVCP-exatecan conjugate, Ligand (SEQ ID NO: 3)-MVCP-exatecan conjugate and Ligand (SEQ ID NO: 4)-MVCP-exatecan conjugate were synthesized by the same method as the synthesis of Ligand (SEQ ID NO: 1)-MVCP-exatecan conjugate.

< Example 17> Ligands represented by sequence numbers 1 to 4, MVCP - With doxorubicin Conjugate synthesis

The ligand represented by the sequence number 1 (3.2 mg, 2.49 μmol) and MVCP-doxorubicin (955.5 mM in Dimethylsulfoxide, 208.7 μL) were treated in dimethylformamide (Dimethylformamide, 1 mL), and the mixture was stirred at room temperature for 12 h.

The ligand (SEQ ID NO: 1)-MVCP-doxorubicin conjugate was recovered by recrystallization.

Ligand (SEQ ID NO: 2)-MVCP-doxorubicin conjugate, Ligand (SEQ ID NO: 3)-MVCP-doxorubicin conjugate and Ligand (SEQ ID NO: 4)-MVCP-doxorubicin conjugate were synthesized in the same manner as the synthesis of Ligand (SEQ ID NO: 1)-MVCP-doxorubicin conjugate.

< Example 18> represented by sequence number 6, sequence number 7, sequence number 9, sequence number 10 and sequence number 11. Polypeptides and MPA - Manufacture of conjugates with SN38

A polypeptide represented by the sequence number 6 (70.6 μM, 37 mL) and MPA-SN38 (6.185 mM in Dimethylsulfoxide, 1.86 mL) were treated in dimethylformamide (Dimethylformamide, 37 mL), and the mixture was stirred at room temperature for 1 hour.

The polypeptide (SEQ ID NO: 6)-MPA-SN38 conjugate was recovered by recrystallization.

Polypeptide (SEQ ID NO: 7)-MPA-SN3 conjugate, polypeptide (SEQ ID NO: 9)-MPA-SN3 conjugate, polypeptide (SEQ ID NO: 10)-MPA-SN3 conjugate and polypeptide (SEQ ID NO: 11)-MPA-SN3 conjugate were prepared by the same method as for the synthesis of polypeptide (SEQ ID NO: 6)-MPA-SN3 conjugate.

< Example 19> indicated by sequence number 10 Polypeptides and MEC - Manufacture of conjugates with SN38

A polypeptide represented by SEQ ID NO: 10 (88.9 μM, 375.0 μL) and MEC-SN38 (1.27 mM in Dimethylsulfoxide, 125.0 μL) were treated in dimethylformamide (Dimethylformamide, 2.25 mL), and the mixture was stirred at room temperature for 1 hour.

The polypeptide (SEQ ID NO: 10)-MEC-SN38 conjugate was recovered by recrystallization.

< Example 20> Sequence number 6 and sequence number 10 Displayed Polypeptides and MPA - With exatecan Manufacturing of the joint

A polypeptide represented by the sequence number 6 (20.2 μM, 3.5 mL) and MPA-exatecan (5.1 mM in Dimethylsulfoxide, 143 μL) were treated in dimethylformamide (Dimethylformamide, 6.0 mL), and the mixture was stirred at room temperature for 5 hours.

The polypeptide (SEQ ID NO: 6)-MPA-exatecan conjugate was recovered by recrystallization.

A polypeptide (SEQ ID NO: 10)-MPA-SN3 conjugate was prepared by the same method as for the synthesis of the polypeptide (SEQ ID NO: 6)-MPA-SN3 conjugate.

< Example 21> indicated by sequence number 10 Polypeptides and MEC - With exatecan Manufacturing of the joint

A polypeptide represented by the sequence number 10 (89.0 μM, 3.5 mL) and MEC-exatecan (2.23 mM in Dimethylsulfoxide, 167.9 μL) were treated in dimethylformamide (Dimethylformamide, 1.05 mL), and the mixture was stirred at room temperature for 2 hours.

< Example 22> represented by sequence number 6, sequence number 8, sequence number 10 and sequence number 12. Polypeptides and MVCP - With exatecan Manufacturing of the joint

A polypeptide represented by the sequence number 6 (74.7 μM, 1.0 mL) and MVCP-exatecan (947.1 μM in Dimethylsulfoxide, 298.2 μL) were treated in dimethylformamide (Dimethylformamide, 3.0 mL), and the mixture was stirred at room temperature for 2 hours.

The polypeptide (SEQ ID NO: 6)-MVCP-exatecan conjugate was recovered by recrystallization.

Polypeptide (SEQ ID NO: 8)-MVCP-exatecan, polypeptide (SEQ ID NO: 10)-MVCP-exatecan and polypeptide (SEQ ID NO: 12)-MVCP-exatecan conjugates were prepared by the same method as for the synthesis of polypeptide (SEQ ID NO: 6)-MPA-SN3 conjugate.

< Example 23> Human Scattered Immanence Brain tumor Ligands for cells- MVCP - Exatecan Confirmation of cytotoxicity of the conjugate

The cytotoxicity of the ligand (SEQ ID NO: 1)-MVCP-exatecan conjugate was determined using human disseminated intraepithelial neoplasia SF8628 cells.

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.

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

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

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 0, 10, 25, 50, 100, 250, 500, 1000, 2500, 5000, 10000, and 25000 nM of irinotecan.

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 under conditions of 37°C, 95% relative humidity, and 5% CO2.

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

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

By the same method as that used to determine the cytotoxicity of the ligand (SEQ ID NO: 1)-MVCP-exatecan conjugate, the cytotoxicity of the ligand (SEQ ID NO: 2)-MVCP-exatecan conjugate, the ligand (SEQ ID NO: 3)-MVCP-exatecan conjugate, and the ligand (SEQ ID NO: 4)-MVCP-exatecan conjugate was determined.

As a result, the IC50 values of ligand-MVCP-exatecan conjugate (nM) and irinotecan (nM) for SF8628 cells were confirmed as shown in Table 5.

cancer cells SF8628 Anticancer drug Irinotecan Ligand (SEQ ID NO: 1)-MVCP-Exatecan Ligand (SEQ ID NO: 2)-MVCP-Exatecan Ligand (SEQ ID NO: 3)-MVCP-Exatecan Ligand (SEQ ID NO: 4)-MVCP-Exatecan IC50 (nM) 871.0 (a) 5.287 (b) 4.308 (c) 6.347 (d) 9.102 (e) Cell death efficiency (fold) 1.0 (a/a) 164.7 (a/b) 202.2 (a/c) 137.2 (a/d) 95.7 (a/e)

< Example 24> Temozolomide Confirmation of overcoming drug resistance in drug-resistant cancer cells

To confirm the ability of temozolomide to overcome anticancer drug resistance in cancer cells resistant to temozolomide, human glioblastoma T98G cells were used.

Cytotoxicity was confirmed using the same method as for confirming cytotoxicity of ligand-drug conjugates.

As a result, as shown in Fig. 12, the IC50 values of polypeptide (SEQ ID NO: 6)-MPA-SN38 and SN38 were 3.35 nM and 10.87 nM, respectively, and the polypeptide (SEQ ID NO: 6)-MPA-SN38 conjugate overcame the anticancer drug resistance of cancer cells.

< Example 25> For human cancer cells Polypeptide - Confirmation of cytotoxicity of drug conjugates

To determine the cytotoxicity of the polypeptide-drug conjugate against various types of tumors, breast cancer MDA-MB231 cells, pancreatic cancer PANC-1 cells, diffuse intrinsic glioma SF8628 cells, and glioblastoma U87-MG cells were used.

As a result, the (nM) IC50 values of the polypeptide-drug conjugates against breast cancer, pancreatic cancer, diffuse intrinsic glioma, and glioblastoma cells were confirmed as shown in Table 6.

Sequence number Linker-drug IC50 (nM) MDA -MB231 PANC -1 SF8628 U87-MG 6 MPA-SN38 5.415 2.370 1.654 14.2 MPA-Exatecan 12.50 1.911 7.679 16.73 MVCP-Exatecan - - 0.3839 0.8277 7 MPA-SN38 1.036 2.121 0.8904 5.921 8 MVCP-Exatecan 0.4899 1.647 0.3613 1.783 9 MPA-SN38 1.989 5.093 1.262 5.090 10 MPA-SN38 1.018 4.443 0.7817 3.241 MEC-SN38 - - 0.5784 - MPA-Exatecan - - 2.378 - MEC-Exatecan - - 0.7443 6.876 MVCP-Exatecan 0.5124 2.032 0.4173 1.946 MVCP-Doxorubicin - - 2.169 - 11 MPA-SN38 0.9861 3.183 0.8814 4.312 12 MVCP-Exatecan - - 0.3116 0.7143

< Example 26> In a mouse model of human triple-negative breast cancer Polypeptide (Sequence number 10)- MPA -SN38 and Polypeptide (Sequence number 10)- MPA - Exatecan Anticancer efficacy test of the conjugate

MDA-MB-231 cells ( ^1x106 cells) in normal growth status were subcutaneously inoculated into the flank of NSG mice (Athymic NCr-nu/nu, Koatech), and 12 days after cancer cell transplantation, when the tumor volume grew to approximately 100 ^mm3, the experimental animals were separated into groups such that the tumor volumes were uniformly distributed. On days 12, 14, 17, 20, 23, and 26 after cancer cell transplantation, 50 μL of polypeptide (SEQ ID NO: 10)-MPA-SN38 conjugate (262 μM based on SN38) or polypeptide (SEQ ID NO: 10)-MPA-exatecan conjugate (203 μM based on exatecan) was administered six times in total, and the negative control group was administered the same amount of sodium phosphate solution at the same time.

As a result, in the group administered with the polypeptide (SEQ ID NO: 10)-MPA-SN38 and the polypeptide (SEQ ID NO: 10)-MPA-exatecan conjugates as shown in Fig. 13, the tumor-bearing mice showed a decrease in tumors 5 days after the first administration, and compared to the negative control group, the tumor volume and tumor weight were statistically significantly reduced, but there was no statistically significant difference in body weight.

As a result, the anti-tumor efficacy of the polypeptide (SEQ ID NO: 10)-MPA-SN38 conjugate and the polypeptide (SEQ ID NO: 10)-MPA-exetecan conjugate was confirmed using the orthotopic glioblastoma model, as summarized in Table 7.

item Voice control group Polypeptide (SEQ ID NO: 10)-MPA-SN38 conjugate Polypeptide (SEQ ID NO: 10)-MPA-Exatecan conjugate Inhibition of tumor weight growth ( % ) 0 79.6 68.6 Inhibition of tumor volume growth ( % ) 0 78.6 70.0

< Example 27> In a mouse model of human glioblastoma Polypeptide (Sequence number 6) - Anticancer efficacy test of MPA-SN38 conjugate

1. To test the anti-tumor efficacy of the polypeptide (SEQ ID NO: 6)-MPA-SN38 in vivo, brain tumors were induced in BALB/c nude mice using the U87-MG-Luc2 cell line, and the polypeptide (SEQ ID NO: 6)-MPA-SN38 was administered intracerebral to evaluate the effect of the polypeptide (SEQ ID NO: 6)-MPA-SN38 on brain tumors.

2. The glioblastoma cells are U87-MG-Luc2.

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 boxes were changed once a week, and the water bottles were changed at least twice a week.

4. Tumor cell transplantation was performed for 2 days from the day after the final quarantine was completed. U87-MG-Luc2 cells in culture were harvested and evenly suspended in PBS to prepare 0.75 × 10 ⁵ cells/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) of the animal brain using a microinjector equipped with a Hamilton syringe (10 μL). 2 μL (1.5 × 10 ⁵ cells/animal) of U87-MG-Luc2 cells.

5. Seven days after tumor cell transplantation, during the first IVIS photograph, the test animals that had the weakest bioluminescence among the tumor cell transplanted test animals were judged to be abnormal and excluded, and the animals were divided into three groups with six animals per group to ensure that the luminescence values were distributed as evenly as possible.

6. Administration of polypeptide (SEQ ID NO: 6)-MPA-SN38: The head of the test animal was fixed in a stereotaxic coordinate system, and 10 μL/animal of test substance 1, test substance 2, or positive control substance was injected 3 times using a microinjector equipped with a Hamilton syringe (10 μL, CED Needle) into the tumor implantation site in the brain of the animal (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 completion of injection and then removed.

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

8. Weight was measured twice a week from the day of military separation.

9. From the day of group separation to the end of the experiment, the growth of tumor cells was confirmed twice a week using IVIS. D-Luciferin (Cat. No.: 122799, PerkinElmer, U.S.A.) 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 the test animals transplanted with U87-MG-Luc2 cells, and anesthetized with Isoflurane 5 minutes later. Images were taken using IVIS (In Vivo Imaging System, IVIS Lumina X5 Imaging System, PerkinElmer, U.S.A.) 10 minutes after D-Luciferin administration. Tumor growth was expressed as the light intensity (protons/sec) for the ROI area, and bioluminescence was measured twice a week until the end of the experiment.

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

11. All measurement results obtained in the experiment were statistically analyzed using SPSS (Version 27.0, IBM Corporation, U.S.A.). The positive control group (G2) and the test substance administration groups (G3, G4) were subjected to Levene's equal variance test and the Independent t test was performed to test significance.

12. As a result, as shown in Figs. 14 and 15, in the negative control group, death occurred from the 39th day after tumor cell transplantation, and all animals died by the 42nd day. In the positive control group, death occurred from the 34th day after tumor cell transplantation, and all animals died by the 44th day. In the polypeptide (SEQ ID NO: 6)-MPA-SN38 administration group, death occurred from the 48th 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 test substance 2 administration group, a decrease in body weight was observed after three intracerebral administrations of the test substance in the polypeptide (SEQ ID NO: 6)-MPA-SN38 administration group after tumor cell administration, but it increased again thereafter, and compared to the negative control group where body weight decreased after the third administration of the test substance, the increase was statistically significantly higher. After the third administration of the positive control substance, the positive control group showed a decrease in body weight, and there was no statistically significant difference from the negative control group.

13. As a result, the anti-tumor efficacy of the polypeptide (SEQ ID NO: 6)-MPA-SN38 was confirmed using an orthotopic glioblastoma model, as summarized in Table 8.

item Voice control group Topotecan Polypeptide (SEQ ID NO: 6)-MPA-SN38 conjugate Capacity (SN38 μg /episode) 0 1.228 0.954 Number of doses 3 3 3 Inhibition of tumor growth ( % ) 0 23.1 94.9 Average survival days 42 42.5 65 days or more

While the specific parts of the present invention have been described in detail above, it is obvious to those skilled in the art that such specific description is merely a preferred embodiment and that the scope of the present invention is not limited thereby. In other words, the actual scope of the present invention is defined by the appended claims and their equivalents.

Claims (24)

A ligand that specifically binds to IL13Ra2, comprising any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 4. A polypeptide that specifically binds to IL13Ra2, wherein a temperature-sensitive atypical domain comprising a ligand of the first clause and an amino acid sequence represented by SEQ ID NO: 5 is repeatedly linked. 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: 6 to SEQ ID NO: 12. A polynucleotide encoding the ligand of claim 1 or the polypeptide of claim 2. A recombinant vector comprising the polynucleotide of claim 4. A transformant isolated by transformation with the recombinant vector of Article 5. A ligand-biotin conjugate in which biotin is bound to the ligand of claim 1. A polypeptide-biotin conjugate in which biotin is bound to the polypeptide of claim 2. A ligand-fluorescent substance conjugate in which a fluorescent substance is bound to the ligand of claim 1. A polypeptide-fluorescent substance conjugate in which a fluorescent substance is conjugated to the polypeptide of claim 2. A conjugate according to claim 9 or 10, characterized in that the fluorescent substance is AZDye594 or AZDye647. A composition for detecting IL13Ra2, 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. A composition for diagnosing cancer in which IL13Ra2 is overexpressed, comprising as an active ingredient the ligand of claim 1, the polypeptide of claim 2, or the conjugate of any one of claims 7 to 10. A composition for diagnosing cancer, characterized in that in claim 13, the cancer in which IL13Ra2 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. A composition for imaging cancer cells overexpressing IL13Ra2, comprising as an active ingredient the ligand of claim 1, the polypeptide of claim 2, or the conjugate of any one of claims 7 to 10. A ligand-drug conjugate in which a drug is bound to the ligand of claim 1. A polypeptide-drug conjugate comprising a drug conjugate to the polypeptide of claim 2. A conjugate according to claim 16 or 17, characterized in that the drug is an anticancer agent. 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. A conjugate according to claim 16 or 17, characterized in that the ligand or polypeptide is linked to a drug via a linker. 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 or maleidocaproyl-valine-citrulline-paranitroaminobenzoic acid (Mc-Val-Cit-Pab; MVCP). A pharmaceutical composition for the prevention or treatment of cancer in which IL13Ra2 is overexpressed, comprising the ligand-drug conjugate of claim 16 or the polypeptide-drug conjugate of claim 17 as an active ingredient. A pharmaceutical composition for preventing or treating cancer, characterized in that in claim 22, the cancer in which IL13Ra2 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. A pharmaceutical composition for suppressing resistance to temozolomide in a patient with a brain tumor, comprising the ligand-drug conjugate of claim 16 or the polypeptide-drug conjugate of claim 17 as an active ingredient.