Drug conjugates with improved drug delivery and internalization efficiency

Abstract

This invention relates to a unit drug conjugate wherein a binding group capable of binding to another drug conjugate is further linked to a unit drug conjugate in which a target substance and a drug specifically binding to a target cell are linked together. When the unit drug conjugate according to the invention is administered sequentially in vivo, the complex forms clusters through in vivo cross-linking, and these clusters promote endocytosis of the drug conjugate, thereby significantly enhancing the cellular internalization of the drug contained in the drug conjugate.

Description

Technical Field

[0001] This invention relates to a drug conjugate having improved drug delivery and cell internalization efficiency, and more specifically, to a unit drug conjugate wherein a binding group capable of binding to another drug conjugate is additionally linked to a drug conjugate wherein a target substance and a drug specifically bound to a target cell are linked together. Background Technology

[0002] Endocytosis is a general term defining the process by which a cell introduces selected extracellular species, such as molecules, viruses, particles, and microorganisms, and targets them to specific organelles within the cytoplasm. Endocytosis is generally divided into phagocytosis and pinocytosis. Pinocytosis can be further divided into macropinocytosis, clathrin-mediated endocytosis, caverin-mediated endocytosis, and clathrin- and caverin-independent endocytosis.

[0003] Endocytosis can depend on the size of endocytic vesicles, the nature of the endocytic cargo (ligands, receptors, and lipids), and the mechanism of vesicle formation (Conner SD et al., Nature 2003; 422: 37-44). For example, macropinocytosis (>1 μm) mainly delivers extracellular substances into the cell via plasma membrane invagination, while clathrin-mediated endocytosis (~120 nm), caverin-mediated endocytosis (~60 nm), and clathrin- and caverin-independent endocytosis (~90 nm) introduce extracellular substances into the cell via intracellular vesicles.

[0004] In molecular biology fields such as virology, or drug and gene delivery, the endocytosis of extracellular substances plays a crucial role in the targeted internalization of drugs that can specifically bind to molecules (e.g., receptors) widely expressed on the cell membrane. In particular, as a method for effectively internalizing drugs into target cells, efficient endocytosis can be induced by introducing molecules capable of specifically binding to molecules (e.g., receptors) widely expressed on the cell membrane (Marsh M. et al., Cell 2006; 124: 729-40; Smith AE et al., Science 2004; 304: 237-42). In this case, the drug-bound receptor is collected at the invagination site via surface diffusion. If this collection is not successful, endocytosis will not occur effectively for a short period.

[0005] Angiogenesis refers to the process by which new capillaries form from existing microvessels, and it is known to play a vital role in routine physiological processes such as embryogenesis, wound healing, and the female reproductive cycle. However, diseases can arise from the failure of autoregulation in angiogenesis and abnormal vascular growth. For example, abnormally excessive angiogenesis is known to play a key role in the growth or metastasis of tumors or cancers, as well as in diseases such as diabetic retinopathy, age-related macular degeneration, rheumatoid arthritis, endometriosis, psoriasis, or chronic inflammation. Angiogenesis is also associated with coronary artery disease, stroke, myocardial infarction, ulcers, or delayed wound healing.

[0006] Among these diseases, tumors, especially malignant tumors (cancer), refer to diseases in which the cells that make up the body divide irregularly due to internal or external factors, thus causing the cells to lose control of the body and proliferate unintentionally. Furthermore, tumors invade surrounding tissues through blood vessels, lymphatic vessels, etc., and metastasize to other organs. Angiogenesis is one of the important mechanisms in tumorigenesis.

[0007] Thus, angiogenesis-related diseases present a serious condition, and various therapeutic agents have been developed to treat them. However, these therapeutic agents often fail to demonstrate adequate therapeutic efficacy because the growth factors and signaling mechanisms that primarily promote angiogenesis are diverse. In such cases, resistance to the therapeutic agents is likely to develop due to repeated administration of excessive doses, and significant therapeutic effects cannot be expected.

[0008] Furthermore, since the target receptors for therapeutic agents targeting angiogenesis-related diseases are located in vascular endothelial cells, the binding between the target-specific substance and the receptor can dissociate rapidly due to factors such as blood pressure, blood flow, and vascular permeability, thereby releasing the therapeutic agent. Therefore, to achieve long-term therapeutic effects, the therapeutic agent is repeatedly administered at high doses, causing various side effects.

[0009] Meanwhile, unlike existing treatments, targeted cancer therapy, which has recently gained attention, is a treatment approach that targets tumor-specific biomarkers. Specific examples include targeted anticancer drug therapy, therapeutic radioisotope therapy, and photodynamic therapy. Because drugs used in targeted cancer therapy are delivered by targeting tumor-specific biomarkers, damage to normal cells is minimized while inducing tumor cell death. For drugs used in this type of targeted cancer therapy to exhibit effective anticancer effects, they must possess high selectivity and binding affinity for tumor cells and be efficiently delivered to tumor cells through internalization.

[0010] To improve drug internalization within cells, a ligand comprising a multimeric compound capable of binding to multiple target receptors simultaneously has been developed (Wu Z. et al., J Nucl Med 2007; 48: 1536-1544). However, due to the location specificity of vascular endothelial cells (blood pressure, blood flow, vascular permeability, etc.), the ligand's endocytosis in vivo is limited, resulting in low efficacy in internalizing drugs into cells.

[0011] Therefore, the inventors noted that in the case of conventional drug conjugates used for the diagnosis or treatment of various diseases, such as those related to angiogenesis, the internalization efficiency of the drug is very low due to limited endocytosis in vivo. It was anticipated that if the cellular internalization of drug conjugates via endocytosis were improved, the sensitivity and accuracy of diagnosis could be significantly improved, and the targeted therapeutic efficacy of the drug could be significantly enhanced. To this end, the inventors made considerable efforts to develop a novel drug conjugate and drug delivery platform, and discovered that when a binding group is additionally introduced into a drug conjugate in which the target substance is linked to the drug, an interaction can be induced between the binding group and another binding group introduced into another drug conjugate. Each drug conjugate can bind to target cells, and cross-linking between drug conjugates in vivo can be induced to form clusters, thereby activating endocytosis and significantly improving the cellular internalization of the drug contained in the drug conjugate. Based on this discovery, the present invention was completed.

[0012] [Existing technical documents]

[0013] [Patent Literature]

[0014] (Patent Document 1) PCT / KR2011 / 003801

[0015] [Non-patent literature]

[0016] (Non-patent literature 1) Conner SD et al., Nature 2003; 422: 37-44;

[0017] (Non-patent literature 2) Marsh M. et al., Cell 2006; 124: 729-40;

[0018] (Non-patent literature 3) Smith AE et al., Science 2004; 304: 237-42;

[0019] (Non-patent literature 4) Wu Z. et al., J Nucl Med 2007;48:1536-1544. Summary of the Invention

[0020] One object of the present invention is to provide a novel drug conjugate that has improved drug delivery efficiency due to a significant increase in cell internalization.

[0021] To achieve the above objectives, the present invention provides a unit drug conjugate wherein a binding group capable of binding to another drug conjugate is further connected to a drug conjugate wherein a target substance and a drug specifically binding to a target cell are linked together.

[0022] The present invention also provides a drug conjugate, comprising: the unit drug conjugate (first unit drug conjugate); and another drug conjugate (second unit drug conjugate), the drug conjugate being capable of binding to the unit drug conjugate via a binding group.

[0023] The present invention also provides a pharmaceutical composition for treating various diseases, preferably a pharmaceutical composition for treating angiogenesis-related diseases, the pharmaceutical composition comprising the pharmaceutical conjugate.

[0024] The present invention also provides a composition for diagnosing various diseases, preferably a composition for diagnosing angiogenesis-related diseases, the composition comprising the drug conjugate.

[0025] The present invention also provides a pharmaceutical composition for diagnosing and treating various diseases, preferably a pharmaceutical composition for diagnosing and treating angiogenesis-related diseases, the pharmaceutical composition comprising the pharmaceutical conjugate.

[0026] The present invention also provides a method for treating various diseases, the method comprising the step of administering the drug conjugate.

[0027] The present invention also provides a method for diagnosing various diseases, the method comprising the step of using the drug conjugate.

[0028] The present invention also provides the use of the drug conjugate for treating various diseases.

[0029] The present invention also provides the use of the drug conjugate for the diagnosis of various diseases.

[0030] The present invention also provides the use of the drug conjugate in the manufacture of medicaments for treating various diseases. Attached Figure Description

[0031] Figure 1A This is a schematic diagram illustrating the structure of a unit drug conjugate according to the present invention.

[0032] Figure 1B This is a schematic diagram illustrating the working principle of the present invention.

[0033] Figure 2The results of confirming crosslinking between unit drug conjugates in a cellular environment according to an example of the invention are shown.

[0034] Figure 3A A fluorescence image depicting the result of confirming the effect of cross-linking between conjugates promoting endocytosis according to an example of the invention.

[0035] Figure 3B The fluorescence intensity quantification results of a fluorescence image, illustrating the effect of promoting endocytosis by the formation of crosslinks between conjugates according to an example of the present invention, are shown.

[0036] Figure 3C The results of Z-stack analysis of fluorescence images, illustrating the effect of crosslinking between conjugates in confirming the promoting endocytosis effect according to an example of the present invention, are shown.

[0037] Figure 4 Results of an in vitro biodistribution experiment of a drug conjugate according to an example of the present invention are shown.

[0038] Figure 5 The results of a continuous dose examination of a unit drug conjugate according to an example of the invention are shown, illustrating the differences in retention in tumors based on a unit drug conjugate.

[0039] Figure 6 The results of an embodiment of the invention, demonstrating the effect of continuous administration of a unit drug conjugate on improving tumor retention, are shown. Detailed Implementation

[0040] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Generally, the nomenclature used in this specification is well-known and commonly used in the art.

[0041] In this invention, it can be found that when the in vivo binding of binding groups between unit drug conjugates in which the target substance and the drug are bound together is induced, the drug conjugates (complexes) bound to the target cells aggregate through cross-linking, thereby promoting the endocytosis of the drug conjugates and enhancing their internalization into cells, thereby increasing the intracellular concentration and retention of the drug.

[0042] Therefore, in one aspect, the present invention relates to a unit drug conjugate wherein a binding group capable of binding to another drug conjugate is additionally linked to a drug conjugate wherein a target substance and a drug specifically binding to a target cell are linked together.

[0043] In this invention, the targeting substance can bind specifically to a target that is specifically expressed or overexpressed in the target cells, and the drug can be a therapeutic or diagnostic drug, but is not limited thereto.

[0044] In this invention, the target substance and the drug can be connected together by a connector, but the target substance and the drug can also be directly connected together without a connector.

[0045] In this invention, the binding group can also be connected to the linker, but it can also be connected to other sites of the drug conjugate. In this invention, the binding group can be one or more.

[0046] In another aspect, the present invention relates to a drug conjugate comprising: the unit drug conjugate (first unit drug conjugate); and another drug conjugate (second unit drug conjugate) capable of binding to the unit drug conjugate via a binding group.

[0047] In this invention, when the binding group contained in the first unit drug conjugate is named the first binding group and the binding group contained in the second unit drug conjugate is named the second binding group, the first unit drug conjugate and the second unit drug conjugate are characterized in that the first binding group and the second binding group are bound to each other.

[0048] In this invention, the target substances introduced into the first unit drug conjugate and the target substances introduced into the second unit drug conjugate may be the same or different from each other, but all target substances can specifically bind to the target that is specifically expressed or overexpressed in the same target cell.

[0049] In this invention, the drug introduced into the first unit drug conjugate and the drug introduced into the second unit drug conjugate may be the same as or different from each other.

[0050] The present invention can also provide a multidrug conjugate comprising multiple unit drug conjugates, which can be linked together by one or more binding group pairs.

[0051] The present invention also provides a unit drug conjugate pair, comprising: the unit drug conjugate (first unit drug conjugate); and another drug conjugate (second unit drug conjugate), the drug conjugate having a second binding group capable of binding to a first binding group of the first unit drug conjugate.

[0052] The present invention also provides a plurality of unit drug conjugates, including: the unit drug conjugate (first unit drug conjugate); and another drug conjugate (second unit drug conjugate), the drug conjugate having a second binding group capable of binding to a first binding group of the first unit drug conjugate.

[0053] The second unit drug conjugate may be a unit drug conjugate wherein a second binding group capable of binding to the first binding group of the first unit drug conjugate is further linked to a drug conjugate in which a second target substance specifically bound to the target, which is specifically expressed or overexpressed in the target cells, is linked to a therapeutic or diagnostic drug.

[0054] The present invention also relates to a second unit drug conjugate, wherein a second binding group capable of binding to a first binding group of a first unit drug conjugate is further linked to a drug conjugate in which a second target substance specifically bound to a target expressed or overexpressed in a target cell is linked to a therapeutic or diagnostic drug.

[0055] Furthermore, the present invention relates to a drug conjugate formed by the binding between a first binding group of a first unit drug conjugate and a second binding group of a second unit drug conjugate.

[0056] When using the first and / or second unit drug conjugates according to the present invention, target cells, such as cancer cells, can be rapidly targeted. That is, when existing anticancer drugs have low tumor uptake pharmacokinetics, they inhibit normal cells and their efficacy is reduced due to their rapid in vivo metabolism; therefore, high doses must be administered. However, when using the first and / or second unit drug conjugates according to the present invention, there is an advantage that the unit drug conjugates are stable in the blood and can rapidly and selectively target tumor cell membrane proteins at concentrations with virtually no toxicity.

[0057] Furthermore, the use of the first and / or second unit drug conjugates according to the invention has the advantage of enhancing drug internalization in tumor cells. That is, while existing anticancer drugs rely solely on natural intracellular uptake mechanisms for cancer cell internalization, when the first and / or second unit drug conjugates according to the invention are used, cross-linking between the first and / or second unit drug conjugates occurs through in vivo intermolecular interactions, rapidly and selectively binding to target cells (e.g., cancer target sites), and multiple complexes formed through cross-linking (complexes between membrane proteins and the target substance) are grouped to form clusters. These clusters accelerate endocytosis and thus enhance drug internalization within cells. This demonstrates a remarkable effect in artificially enhancing known natural cell internalization.

[0058] In this invention, any substance can be used as the target substance introduced into the first unit drug conjugate (first target substance) and the target substance introduced into the second unit drug conjugate (second target substance) without limitation, as long as they are substances that can specifically bind to the target expressed or overexpressed in the target cells.

[0059] In this invention, "specific expression in target cells" means that the target is not expressed in normal cells, but is specifically expressed only in target cells, and "overexpression in target cells" means that the target is abnormally highly expressed in target cells compared with normal cells.

[0060] The first and second targeting substances can target the same or different targets, and even when they target the same target, they can be different substances.

[0061] For example, when using a first and a second targeting substance that specifically targets epidermal growth factor (EGF) expressed on the surface of cancer cells, both the first and the second targeting substances can be antibodies against the EGF receptor, or the first targeting substance can be an antibody against the EGF receptor and the second targeting substance can be EGF as a ligand for the EGF receptor.

[0062] Furthermore, even when both the first and second target substances are antibodies (preferably monoclonal antibodies) against the EGF receptor, they can be antibodies with different CDRs (complementarity-determining regions) or variable regions.

[0063] Substances that can bind specifically to a target expressed in a target cell can be selected from, but are not limited to, antibodies, aptamers, peptides such as ligands, carbohydrates, and small molecule compounds.

[0064] As used in this article, the term "antibody" includes not only the entire antibody form but also the antigen-binding fragment of the antibody molecule.

[0065] The entire antibody has a structure with two full-length light chains and two full-length heavy chains, which are linked to the heavy chains by disulfide bonds. The constant regions of each heavy chain have gamma(γ), mu(m), alpha(α), delta(δ), and epsilon(ε) types, and subclasses of gamma1(γ1), gamma2(γ2), gamma3(γ3), gamma4(γ4), alpha1(α1), and alpha2(α2). The constant regions of each light chain have kappa(κ) and lambda(λ) types.

[0066] As used in this article, the terminology " An antibody's antigen-binding fragment, or "antibody fragment," refers to a fragment that retains its antigen-binding function and includes Fab, F(ab′), F(ab′)2, and Fv. Within an antibody fragment, Fab has a structure with a heavy chain variable region and a light chain variable region, a light chain constant region, and a first heavy chain constant region (CH1), and Fab has one antigen-binding site. Fab' differs from Fab in that Fab' has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. The F(ab′)2 antibody is generated through disulfide bonds formed between cysteine ​​residues in the hinge region of the Fab' fragment. The *v* is the smallest antibody fragment containing only heavy and light chain variable regions. Double-chain *Fv* has a structure in which the heavy and light chain variable regions are linked together non-covalently, while single-chain *Fv* (scFv) typically comprises heavy and light chain variable regions covalently linked to each other via polypeptide linkers or directly linked at the C-terminus, forming a dimer structure as seen in double-chain *Fv*. These antibody fragments can be obtained using proteases (e.g., whole antibodies can be restricted-digested with papain to obtain the Fab fragment, and digested with pepsin to obtain the F(ab′)2 fragment) or generated through recombination techniques.

[0067] The “Fv” fragment is an antibody fragment containing the complete antibody recognition and binding site. This region contains a dimer, such as scFv, which consists of a heavy chain variable region and a light chain variable region that are substantially tightly covalently linked to each other.

[0068] The “Fab” fragment includes the variable and constant regions of the light chain, as well as the variable and first constant region (CHI) of the heavy chain. F(ab′)2 antibody fragments typically consist of a pair of Fab fragments covalently linked by a hinge cysteine ​​residue located between them near their carboxyl terminus.

[0069] A "single-chain Fv" or "scFv" antibody fragment includes the VH and VL regions of the antibody, wherein these regions are present in a single polypeptide chain. To enable the scFv to form the desired structure for antigen binding, the Fv polypeptide may also include a polypeptide linker between the VH and VL regions.

[0070] In this invention, the target substance of the drug conjugate refers to a substance that can bind to a target that is specifically expressed or overexpressed in target cells. Examples include, but are not limited to, RGD (arginine(R)-glycine(G)-aspartic acid(D)) peptide that specifically binds to integrin αvβ3, glutamate-urea-lysine (GUL) motif that binds to prostate-specific membrane antigen (PSMA), EGF that binds to EGF receptor, and VEGF-A or VEGF-B that bind to vascular endothelial growth factor (VEGF) receptor.

[0071] As mentioned above, a target that is specifically expressed or overexpressed in target cells refers to a protein that is specifically expressed or overexpressed in a specific disease compared to normal cells. Examples of such proteins include, but are not limited to, receptors or tumor-specific antigens.

[0072] Examples of targets specifically expressed in target cells include, but are not limited to, integrins such as integrin αvβ3, prostate-specific membrane antigen (PSMA), CD3, CD4, CD6, CD11a, CD19, CD20, CD22, CD30, CD33, CD38, CD40, CD52, CD62, CD79b, CD80, CGRP, OX-40, CTLA4, 4-1BB, PD-1, EGF receptor, TNF receptors such as TNF (tumor necrosis factor)-α, Fc receptor, folate receptor, GD2, HER2, Her2 / neu, HER3, HER4, VEGF receptor, interferon receptor, IgE receptor, IGF-1 receptor, interleukin-2 receptor, interleukin-5 receptor, interleukin-6 receptor, interleukin-17 receptor A, interleukin-31 receptor, interleukin-36 receptor, B7-H3, and CCR4.

[0073] For example, it could be an RGD (arginine(R)-glycine(G)-aspartic acid(D)) peptide that specifically binds to integrin αvβ3, a glutamic acid-urea-lysine (GUL) motif that targets prostate-specific membrane antigen (PSMA), an antibody, an aptamer, a small molecule compound, or a peptide that targets a tumor, but is not limited thereto.

[0074] In one embodiment of the invention, the RGD peptide used is a targeting substance that specifically binds to integrin αvβ3, a membrane protein involved in tumor angiogenesis, and is based on three amino acids (arginine-glycine-aspartic acid), and can be used as a targeting substance for the diagnosis or treatment of tumors. Meanwhile, the glutamate-urea-lysine motif, which can be used in another embodiment, can be a targeting substance that targets PSMA, a known biomarker for prostate cancer, but is not limited thereto.

[0075] In this invention, target cells are cells used for the diagnosis or treatment of diseases, preferably angiogenesis-related diseases, or for the diagnosis or treatment of prostate cancer. Examples of such cells include tumor cells, cells that cause atherosclerosis, cells that cause myocardial infarction, etc.

[0076] In this invention, the drugs contained in the first unit drug conjugate and the second unit drug conjugate can be the same drug or different drugs.

[0077] The term "medicine" encompasses both diagnostic and therapeutic drugs. Diagnostic drugs may be, but are not limited to, fluorescent dyes or diagnostic gamma / positron emission radioisotopes. Therapeutic drugs may be selected from, but are not limited to, substances such as photosensitizers for photodynamic therapy, boron (10B)-containing molecules for boron neutron capture therapy, alpha / beta radiation-emitting therapeutic radioisotopes for nuclear medicine therapy, and anticancer drugs for chemotherapy. The drug may be a small molecule compound, a synthetic drug, a peptide, a protein, or an antibody.

[0078] For example, the drug may be selected from at least one of the following: maytansine, auristatin, aminopterin, actinomycin, bleomycin, tamethasone, camptothecin, N8-acetylsemine, 1-(2-chloroethyl)-1,2-dimethylsulfonylhydrazine, esperamycin, etoposide, 6-mercaptopurine, sea haretoxin, trichothecene, spiculomycin, paclitaxel, taxane, paclitaxel, docetaxel, methotrexate, vincristine, vinblastine, doxorubicin, melphalan, mitomycin A, mitomycin C, chlorambucil, pyruvic acid, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosourea, cisplatin, carboplatin, mitomycin, dacarbazine, procarbazine, topotecan. Nitrogen mustard, cyclophosphamide, 5-fluorouracil, dichloroethylnitrosourea (BCNU), irinotecan, demethoxydaunorubicin, daunorubicin, actinomycin D, procainamide, mitoxantrone, asparaginase, vinorelbine, carmustine, lomustine, busulfan, triamcinolone, dacarbazine, teniposide, 9-aminocamptothecin, cristatol, trimethoprim, mycophenolic acid, thiazolinone, ribavirin, 5-ethynyl-1-beta-dribofuranosylimidazole-4-carboxamide (EICAR), deferoxamine, fluorouridine, deoxyfluorouridine, raltitrexed, cytarabine C) Cytosine arabinoside, fludarabine, tamoxifen, raloxifene, megestrol acetate, goserelin, leuprorelin acetate, flutamide, bicalutamide, EB1089, CB1093, KH1060, verteporfin, phthalocyanine, photosensitizer Pe4, demethoxy-rubigin A, interferon-α, interferon-γ, tumor necrosis factor, gemcitabine, velcade, revamid, thalamid, lovastatin, 1-methyl-4-phenylpyridinium ion, astrococcalin, bleomycin, bleomycin A2, bleomycin B2, pepromycin, epirubicin, pirarubicin, zorubicin, mitoxantrone, verapamil, carotenoids, temozolomide, nucleases, toxins from bacteria or animals / plants, radioactive isotopes, and fluorescent dyes, but not limited to these.

[0079] For example, the radioactive isotope may be an isotope that emits gamma rays or positrons to provide diagnostic images, or an isotope that emits beta rays or alpha rays to provide therapeutic effects, and may be, for example, 11 C 18 F, 99 mTc, 188 Re、 125 / 123 / 124 / 131 I, 89 Zr、 64 / 67 Cu、 68 Ga、 177 Lu、 90 Y、 225 Ac or 211 At, but not limited to.

[0080] For example, the fluorescent dye may be fluorescein isothiocyanate (FITC), tetramethylrhodamine (TRITC), alexa fluor series or cyan (Cy) series fluorescent dyes, but is not limited thereto.

[0081] Preferably, the drugs contained in the first and second unit drug conjugates according to the invention are different from each other. As described above, when the drugs contained in the first and second unit drug conjugates are different from each other, the advantage is that combination therapy can be tailored through the loading and delivery of multiple drugs. That is, cancer resistance to existing anticancer agents shortens the duration of anticancer agent use and eventually leads to patients discontinuing treatment; however, by introducing multiple drugs into the first and second unit drug conjugates for the purpose of diagnosing and treating cancer, tailored treatment and combination therapy corresponding to drug resistance can be achieved.

[0082] In this invention, the first and second binding groups can be used without limitation, as long as they are binding groups that can bind in vivo. The binding groups can be binding groups that bind via click reactions, binding groups that bind via host-guest chemical interactions, or avidin-biotin binding groups, but are not limited to these.

[0083] In this invention, binding can be achieved through click reactions, host-guest interactions, or avidin-biotin binding, but is not limited thereto. It will be apparent to those skilled in the art that any binding can be used without limitation, as long as in vivo cross-linking can occur to induce an interaction between two or more unit drug conjugates.

[0084] Specifically, the binding group includes binding groups used in click reactions. Among various types of click reactions, copper-free click reactions are known to be reactions in which binding occurs for a short time even in aqueous environments such as in vivo environments (Jewett JCet al. Chem. Soc. Rev. 2010; 39: 1272-1279). In some embodiments, the binding group that binds via a click reaction can be, but is not limited to, an azide-adibenzocyclooctyne (ADIBO) binding group, a trans-cyclooctene (TCO)-tetraazine binding group, or an alkyne-cyclopentadienone binding group.

[0085] In this invention, the host-guest interaction is a non-covalent bond between a host and a guest compound exhibiting high binding strength (Yu G. et al., Theranostics. 2019; 9: 3047-3074). In particular, the host-guest interaction is suitable for this invention because it exhibits high binding strength between compounds even in environments similar to the in vivo environment. In one embodiment, the binding group binding via the host-guest interaction can be a cucurbituril-adamantane or a cyclodextrin-amino acid binding group, but is not limited thereto.

[0086] The binding group can also use the avidin-biotin interaction. The avidin-biotin interaction is a selective binding and one of the strong non-covalent interactions that exist in nature. Its advantage is that it has a stronger binding strength than antibody-antigen binding (Jain A. et al. J Control Release. 2017; 245: 27-40).

[0087] In this invention, a first target substance and a drug contained in a first unit drug conjugate, and / or a second target substance and a drug contained in a second unit drug conjugate, can be connected together by a connector, or directly connected without a connector.

[0088] Furthermore, the first target substance and the first binding group contained in the first unit drug conjugate, and / or the second target substance and the second binding group contained in the second unit drug conjugate, can be linked together by a linker, or directly linked without a linker.

[0089] In a preferred embodiment, such as Figure 1A As shown, the first target substance, drug, and first binding group contained in the first unit drug conjugate are linked together by a linker (linking group) having three or more functional groups, but are not limited thereto. The first target substance, drug, and first binding group can be linked together by a linker or can be directly linked together.

[0090] In this invention, the linker can be an amino acid, a hydrocarbon, or a PEG chain, but is not limited thereto. That is, any substance can be used as the linker without limitation, as long as it is a substance known in the art that has atomic or molecular groups and other functional groups suitable for linking the target substance and the drug together and additionally attaching a binding group thereto.

[0091] In another aspect, the present invention relates to a drug conjugate comprising: the unit drug conjugate (first unit drug conjugate); and another drug conjugate (second unit drug conjugate) capable of binding to the unit drug conjugate via binding groups (first binding group and second binding group).

[0092] In this invention, binding can be achieved through click reactions, host-guest interactions, or avidin-biotin binding, but is not limited to these methods.

[0093] In this invention, the binding via click reaction can be an azide-ADIBO binding, a TCO-tetraazine binding, or an alkyne-cyclopentadienone binding, but is not limited to these.

[0094] In this invention, the binding through host-guest interaction can be cucurbituril-adamantane binding or cyclodextrin-amino acid binding, but is not limited to these.

[0095] In another aspect, the present invention relates to a pharmaceutical composition comprising the unit drug conjugate for treating angiogenesis-related diseases.

[0096] The pharmaceutical composition may include two or more different unit drug conjugates. The term "two or more different unit drug conjugates" can mean that the binding groups that function to enable the interaction between the drug conjugates constituting the individual unit drug conjugates are different. Furthermore, this can mean that the drugs bonded to the plurality of unit drug conjugates comprising two or more unit drug conjugates are different from each other.

[0097] In this invention, the first unit drug conjugate and the second unit drug conjugate can be administered simultaneously, but preferably, they can be administered sequentially. In a preferred embodiment of the invention, the first unit drug conjugate is administered before the second unit drug conjugate, and the second unit drug conjugate is administered after the first unit drug conjugate reaches and binds to the target cells.

[0098] In one embodiment, the second unit of the drug conjugate is administered 1 to 600 minutes, preferably 5 to 480 minutes, more preferably 10 to 300 minutes, and most preferably 30 to 240 minutes after administration of the first unit of the drug conjugate.

[0099] The amount of the second unit drug conjugate can be the same as the amount of the first unit drug conjugate. Meanwhile, each first unit drug conjugate may use one or more unit drug conjugates; for example, each first unit drug conjugate may use 1 to 10 types, preferably 1 to 5 types, but not limited thereto.

[0100] In this invention, angiogenesis-related diseases may be selected from, but are not limited to, benign tumors, malignant tumors (cancer), diabetic retinopathy, age-related macular degeneration, rheumatoid arthritis, endometriosis, psoriasis, chronic inflammation, coronary artery disease, atherosclerosis, stroke, ulcers, and myocardial infarction.

[0101] In another aspect, the present invention relates to a composition comprising the unit drug conjugate for treating angiogenesis-related diseases.

[0102] In this invention, angiogenesis-related diseases may be selected from, but are not limited to, benign tumors, malignant tumors (cancer), diabetic retinopathy, age-related macular degeneration, rheumatoid arthritis, endometriosis, psoriasis, chronic inflammation, coronary artery disease, atherosclerosis, stroke, ulcers, and myocardial infarction.

[0103] In another aspect, the present invention relates to a composition comprising the unit drug conjugate for the diagnosis and treatment of angiogenesis-related diseases, enabling simultaneous diagnosis and treatment.

[0104] In this invention, the first unit drug conjugate may be administered before the second unit drug conjugate. The drug contained in the first unit drug conjugate may be a diagnostic drug, and the drug contained in the second unit drug conjugate may be a therapeutic drug.

[0105] In this invention, the first unit drug conjugate may be administered before the second unit drug conjugate. The drug contained in the first unit drug conjugate may be a therapeutic drug, and the drug contained in the second unit drug conjugate may be a diagnostic drug.

[0106] In this invention, angiogenesis-related diseases may be selected from, but are not limited to, benign tumors, malignant tumors (cancer), diabetic retinopathy, age-related macular degeneration, rheumatoid arthritis, endometriosis, psoriasis, chronic inflammation, coronary artery disease, atherosclerosis, stroke, ulcers, and myocardial infarction.

[0107] In this invention, the pharmaceutical composition can be prepared in any dosage form selected from, but not limited to, injections, oral formulations, repair agents, solutions, capsules, granules, tablets, powders, sprays, ointments, gels, formulations for mucosal administration, and suppositories. These dosage forms can be prepared by conventional methods for formulation in this art, or by methods disclosed in Remington's Pharmaceutical Science (latest version), Mack Publishing Company, Easton PA, and can be prepared in different forms depending on the specific disease or ingredient. However, the above description is exemplary, and the dosage forms applied in this invention are not limited to those described above.

[0108] In this invention, the pharmaceutical composition may further comprise acceptable excipients, and the adjuvant may be, for example, a carrier. Pharmaceutically acceptable carriers that can be used are one or more of physiological saline, sterile water, Ringer's solution, buffered saline, glucose solution, maltodextrin solution, glycerol, ethanol, and mixtures thereof. Other conventional additives such as antioxidants, buffers, and antibacterial agents may be added as needed. Furthermore, the pharmaceutical composition may be formulated into injectable dosage forms, such as aqueous solutions, suspensions, or emulsions; pills; capsules; granules; or tablets by additionally adding diluents, dispersants, surfactants, binders, and lubricants. However, the above description is exemplary, and the adjuvants or carriers that can be used in this invention are not limited to those described above.

[0109] The compositions of the present invention can be administered parenterally (e.g., intravenously, subcutaneously, orally, intraperitoneally, or locally) according to desired methods, and their dosage can vary depending on the patient's weight, age, sex, health condition and dietary habits, duration of administration, route of administration, excretion rate, and severity of disease. In this case, the dosing regimen and dosage will vary according to the patient's age, weight, and response. An appropriate dosing regimen and dosage should be determined by those skilled in the art taking these factors into account.

[0110] The unit drug conjugate is composed of a target substance, a binding group, a drug, and a linker, such as Figure 1A As shown in the figure, the functions of each component are summarized below.

[0111] Targeted substances

[0112] A targeting substance is a substance that can bind to a target that is specifically expressed or overexpressed in target cells. For example, a targeting substance is a ligand that specifically binds to an integrin receptor contained in tumor angiogenesis, a ligand that binds to a receptor contained in PSMA, a ligand that binds to an EGF receptor, a ligand that binds to a vascular endothelial growth factor (VEGF) receptor, etc., but is not limited thereto.

[0113] Binding groups

[0114] A binding group is a functional group that induces cross-linking between different unit drug conjugates in vivo, and can spontaneously bind to each other by covalent or non-covalent bonds when brought close together. Examples of binding include, but are not limited to, click reaction binding, host-guest interaction binding, or avidin-biotin binding.

[0115] The binding group used for binding via click reaction can be, but is not limited to, azide-ADIBO, TCO-tetraazine, or alkynyl-cyclopentadienone.

[0116] The binding group used for binding via host-guest interaction can be, but is not limited to, cucurbituril-adamantane or cyclodextrin-amino acid.

[0117] The avidin-biotin binding group can be avidin-biotin, but is not limited to this. There are many ways to link multiple unit drug conjugates together via binding groups, and some of these implementation schemes will now be described in detail.

[0118] According to the present invention, one or more second-unit drug conjugates may be combined with a first-unit drug conjugate.

[0119] When a second unit drug conjugate binds to a first unit drug conjugate, in vivo cross-linking can be achieved through click reaction binding between binding groups, binding between binding groups through host-guest interactions, or avidin-biotin binding between binding groups. For example, when the binding groups used for binding between unit drug conjugates are designated as "A" and "a", the unit drug conjugates may each include one "A" and one "a". When multiple second unit drug conjugates bind to a first unit drug conjugate, the binding group structure of the first unit drug conjugate can be more diverse than that of a single second unit drug conjugate. Specifically, in the binding method selected for in vivo cross-linking, the binding groups introduced into the unit drug conjugates may be designated as "A" and "a". In this case, the first unit drug conjugate may include one or more "A"s as binding groups, and the unit drug conjugate may bind to multiple second unit drug conjugates including "a". In this context, the multiple "A"s contained in the unit drug conjugate can be linked to the unit drug conjugate in various forms. Specifically, as shown below, the multiple "A"s can be linked in a straight-chain, cyclic, branched, or other forms, but are not limited to these. In this context, "A" refers to a binding group that can interact as described in the binding groups above, and specific examples include: azide-ADIBO, TCO-tetraazine, alkyne-cyclopentadienone, cucurbituril-adamantane, cyclodextrin-amino acid, avidin-biotin, etc. The number of binding groups can be from 1 to 30. Figure 1A As shown, "Y" is the connector. It is a binding group. Below are exemplary representations of straight-chain, cyclic, and branched structures, but the invention is not limited thereto.

[0120] straight chain

[0121]

[0122] ring

[0123]

[0124] branch

[0125]

[0126] drug

[0127] In this invention, the term "drug" is a concept that includes both diagnostic drugs and therapeutic drugs. The diagnostic drug may be characterized as a fluorescent dye or a diagnostic gamma-ray / positron emission radioisotope, but is not limited thereto. The therapeutic drug may be selected from, but is not limited to, photosensitizers for photodynamic therapy, boron (10B)-containing molecules for boron neutron capture therapy, alpha / beta radiation radiotherapeutic radioisotopes for nuclear medicine therapy, and anticancer drugs for anticancer chemotherapy.

[0128] Connector

[0129] The term "linker" refers to a substance having atomic or molecular groups and other functional groups for linking a target substance to a drug, and additionally, a binding group. The linker may be, but is not limited to, amino acids, hydrocarbons, or PEG chains. Specifically, the linker may include: an amino acid chain having carboxylic acids and amines as functional groups; a hydrocarbon chain having an amine (-NH2), carboxylic acid (-COOH), sulfonic acid (-SH), alcohol (-OH), or halogen group (-Br, Cl, I, etc.) in at least one or more residues; or a PEG chain as a functional group for linking components together.

[0130] In another aspect, the present invention relates to a kit for diagnosing or treating angiogenesis-related diseases, the kit comprising one or more of the aforementioned unit drug conjugates.

[0131] In another aspect, the present invention relates to the use of the drug conjugate for the treatment of angiogenesis-related diseases.

[0132] In another aspect, the present invention relates to the use of the drug conjugate in the manufacture of a medicament for treating angiogenesis-related diseases.

[0133] In another aspect, the present invention relates to the use of the drug conjugate for the diagnosis of angiogenesis-related diseases.

[0134] In another aspect, the present invention relates to the use of the drug conjugate in the manufacture of a medicament for diagnosing angiogenesis-related diseases.

[0135] In another aspect, the present invention relates to the use of the drug conjugate for the treatment and diagnosis of angiogenesis-related diseases.

[0136] In another aspect, the present invention relates to the use of the drug conjugate in the manufacture of medicaments for the treatment and diagnosis of angiogenesis-related diseases.

[0137] The unit drug conjugates according to the present invention can be used in a variety of diagnostic and therapeutic methods such as fluorescence image-guided surgery, nuclear medicine diagnostics (nuclear imaging), photodynamic therapy (PDT), boron neutron capture therapy (BNCT), radioimmunotherapy, and targeted anticancer chemotherapy.

[0138] Therefore, in another aspect, the present invention relates to a method for treating angiogenesis-related diseases, comprising the following steps:

[0139] (a) Administration of a first unit of the drug conjugate to a subject requiring treatment for angiogenesis-related diseases; and

[0140] (b) Administer a second unit of the drug conjugate to the subject.

[0141] Therefore, in another aspect, the present invention relates to a method for diagnosing angiogenesis-related diseases, comprising the following steps:

[0142] (a) administering the first unit of the drug conjugate to a subject requiring diagnosis of angiogenesis-related disease; and

[0143] (b) Administer a second unit of the drug conjugate to the subject.

[0144] Therefore, in another aspect, the present invention relates to a method for diagnosing and treating angiogenesis-related diseases, comprising the following steps:

[0145] (a) Administration of the first unit of the drug conjugate to a subject requiring diagnosis and treatment of angiogenesis-related diseases; and

[0146] (b) Administer a second unit of the drug conjugate to the subject.

[0147] In summary, the drug conjugates according to the present invention have the following characteristics.

[0148] First, the unit drug conjugate according to the invention can rapidly target the cells to which the unit drug conjugate acts. Typically, if a drug does not specifically bind to the target cells, or if its uptake by the target cells is slow, the problem arises that the drug inhibits the function of normal cells other than the target cells, and the efficacy of the drug is reduced due to metabolism in the body, leading to an increase in drug dosage. However, because the drug conjugate according to the invention introduces a targeting substance that specifically binds to the target cells, it exhibits high therapeutic efficacy even when administered at low doses, thereby preventing toxicity or side effects caused by high-dose administration.

[0149] Secondly, the drug conjugates according to the invention enhance cell internalization. Conventional tumor cell-targeting therapies rely solely on natural internalization uptake mechanisms for cell internalization, while the unit drug conjugates according to the invention introduce binding groups that induce in vivo cross-linking, thereby inducing in vivo binding between unit drug conjugates. In particular, in a sequential dosing method where the unit drug conjugate having the binding group is administered first, followed by another unit drug conjugate capable of binding to the binding group, the unit drug conjugate specifically binds to the target cell because each unit drug conjugate contains a targeting substance that specifically binds to the target cell. Multiple unit drug conjugates bound to these target cells cross-link in vivo and form clusters by inducing in vivo cross-linking between multiple formed complexes. These clusters accelerate endocytosis and promote drug internalization. Therefore, even when the drug is administered at low doses, it can be effectively delivered to the target cells. This is a method of artificially enhancing natural endocytosis. In this method, one or more identical or different binding groups (e.g., binding groups that bind via click reaction, binding groups that bind via host-guest interaction, etc.) can be introduced into a unit drug conjugate, and the unit drug conjugates with different binding groups introduced therein can interact with and bind to other unit drug conjugates. Therefore, clusters can be formed by inducing in vivo cross-linking between three or more different unit drug conjugates, and the drugs contained in the drug conjugates can also be three or more different drugs.

[0150] Finally, according to the present invention, multipurpose therapies and combination therapies can be tailored to specific needs. Since multiple unit drug conjugates can have different binding groups and different drugs, and multiple different unit drug conjugates can contain a variety of drugs, they can be used very effectively for multipurpose therapies and combination therapies. Furthermore, when one unit drug conjugate of the present invention contains a diagnostic drug and another unit drug conjugate contains a therapeutic drug, diagnosis and treatment can be performed simultaneously.

[0151] The invention will now be described in more detail with reference to embodiments. These embodiments are merely for the purpose of describing the invention in more detail, and it will be apparent to those skilled in the art that the scope of the invention should not be construed as limited to these embodiments.

[0152] Example 1. Production of drug conjugates

[0153] 1-1. Production of unit drug conjugate

[0154] In one embodiment of the invention, the selected target substance is the RGD peptide that binds to integrin αvβ3 in tumor angiogenesis, and its structure is shown below.

[0155] Peptide A

[0156]

[0157] Peptide B

[0158]

[0159] Specifically, peptide A is D-[c(RGDfK)]2 obtained by linking two cyclic RGDfK molecules to aspartic acid (D), and peptide B is Dc(RGDyK)-c(RGDfK) obtained by linking a cyclic RGDfK molecule and a tyrosine-containing cyclic RGDyK molecule to aspartic acid (D). These targeting substances were synthesized based on BC Lee et al., RSC Advances 2013; 3: 782-792.

[0160] The unit drug conjugates used in this invention are shown below. Each unit drug conjugate has a structure in which the target substance, drug, and binding group are linked together by a linker. Various methods for linking components together by linkers are known in the art; however, in this embodiment, amide bonds are used to produce the various drug conjugates.

[0161] Unit drug conjugate 1

[0162]

[0163] Targeting substance = peptide B, drug = FITC, binding group = azide

[0164] Unit drug conjugate 1-1

[0165]

[0166] Targeting substance = peptide B, drug = FITC, binding group = β-CD

[0167] Unit drug conjugate 2

[0168]

[0169] Target substance = peptide A, drug = TRITC, binding group = ADIBO

[0170] Unit drug conjugate 2-1

[0171]

[0172] Target substance = peptide A, drug = TRITC, binding group = adamantane

[0173] Unit drug conjugate 3

[0174]

[0175] Targeting substance = peptide B, drug = FITC, binding group = tetrazine

[0176] Unit drug conjugate 4

[0177]

[0178] Target substance = peptide A, drug = TRITC, binding group = TCO

[0179] Unit drug conjugate 5

[0180]

[0181] Targeting substance = RGD peptide A, drug = radioactive isotope iodine ( 123 I or 125 I), the binding group = azide

[0182] Unit drug conjugate 6

[0183]

[0184] Targeting substance = RGD peptide B, binding group = ADIBO

[0185] Unit drug conjugate 7

[0186]

[0187] Targeting substance = RGD peptide A, drug = iodine, binding group = azide

[0188] Unit drug conjugate 8

[0189]

[0190] Targeting substance = cyclic RGDyK, binding group =

[0191]

[0192] Unit drug conjugate 9

[0193]

[0194] Targeting substance = cyclic RGDyK, binding group =

[0195] Unit drug conjugates (9a-b)

[0196]

[0197] Targeting substance = cyclic RGDyK (9a), peptide A (9b), binding group =

[0198]

[0199] Unit drug conjugate 10

[0200]

[0201] Targeting substance = cyclic RGDyK, binding group =

[0202]

[0203] Unit drug conjugate 11

[0204]

[0205] Targeting substance = cyclic RGDfK, binding group = ADIBO

[0206] Unit drug conjugate 12

[0207]

[0208] Target substance = peptide A, drug = temozolomide, binding group = ADIBO

[0209] Unit drug conjugate (12a-c)

[0210]

[0211] Targeting substance = peptide A, drug = temozolomide (12a), 5-fluorouracil (12b), sodium borate (12c), binding group = ADIBO

[0212]

[0213] Drug conjugates 1-2: A combination of unit drug conjugates 1 and 2

[0214]

[0215] Drug conjugates 3-4: A combination of unit drug conjugates 3 and 4

[0216]

[0217] Drug conjugates 5-6: A combination of unit drug conjugates 5 and 6

[0218]

[0219] Drug conjugates 6-7: A combination of unit drug conjugates 6 and 7

[0220]

[0221] In another embodiment, a unit drug conjugate targeting PSMA was produced.

[0222] The selected target material is a glutamate-urea-lysine (GUL) motif that binds to PSMA, with the structure shown below. The target material was synthesized based on Maresca KPet al., J Med Chem 2009;52:347-357.

[0223] Peptide C

[0224]

[0225] Unit drug conjugate 13

[0226]

[0227] Target substance = peptide C, drug = FITC, binding group = azide

[0228] Unit drug conjugate 14

[0229]

[0230]

[0231] Target substance = peptide C, drug = TRITC, binding group = ADIBO

[0232] Unit drug conjugate 15

[0233]

[0234] Target substance = peptide C, drug = FITC, binding group = azide

[0235] Unit drug conjugate 16

[0236]

[0237]

[0238] Target substance = peptide C, drug = TRITC, binding group = ADIBO

[0239] Unit drug conjugate 17

[0240]

[0241] Target substance = peptide C, drug = FITC, binding group = azide

[0242] Unit drug conjugate 18

[0243]

[0244]

[0245] Target substance = peptide C, drug = TRITC, binding group = ADIBO

[0246] Example 2. Detection of crosslinking between unit drug conjugates

[0247] Unit drug conjugates 1 through 4 were used in the experiment to examine the time required for crosslinking and binding between unit drug conjugates to complete under in vitro experimental conditions. Unit drug conjugate 1 was dissolved in a mixture of water and PBS buffer (v / v = 1:1, 0.5 mL), and then unit drug conjugate 2 dissolved in the same solution (0.5 mL) was added. The reaction was carried out at room temperature for 40 minutes. After the reaction started, a small amount (0.1 mL) was collected from the reaction mixture every 10 minutes and analyzed by HPLC. The same experiment was performed on unit drug conjugates 3 and 4.

[0248] The analysis results are shown in Figure 2 It can be confirmed that drug conjugates 1-2, formed by crosslinking between unit drug conjugates 1 and 2, are generated 10 minutes after the start of the reaction, and this binding reaction ends at 40 minutes. Similarly, drug conjugates 3-4, formed by crosslinking between unit drug conjugates 3 and 4, are generated 10 minutes after the start of the reaction, and this binding reaction ends at 30 minutes. Therefore, experiments have demonstrated that crosslinking between unit drug conjugates can be performed in vitro under conditions similar to those in vivo, as originally designed, indicating that there are no difficulties in demonstrating the invention in subsequent in vitro experiments.

[0249] Example 3. Verification of in vivo binding affinity and intracellularization of unit drug conjugates

[0250] Fluorescence resonance energy transfer (FRET) is an analytical method for examining the interaction between two compounds by measuring the fluorescence resonance energy between two fluorescent dyes occurring at 1 nm to 10 nm. According to the present invention, the FRET method is used to measure the binding between unit drug conjugates and the intracellular internalization of unit drug conjugates. The conditions for the FRET experiment used to compare and confirm intracellular internalization are designed as shown in Table 1 below, and the unit drug conjugates used in this experiment are unit drug conjugates 1 and 2, and unit drug conjugates 3 and 4, respectively.

[0251] The post-targeting coupling described in Table 1 below refers to the cross-linking of unit drug conjugates prior to in vitro experiments. For example, it refers to the formation of drug conjugates 1-2 by binding azide (the binding group of unit drug conjugate 1) with ADIBO (the binding group of unit drug conjugate 2) before targeting. Post-targeting coupling refers to the technique according to the invention, in which, in the case of unit drug conjugates 1 and 2 as examples, unit drug conjugate 1 targets and binds to target cells, and then unit drug conjugate 2 targets and binds to the same target cells. At this time, unit drug conjugates 1 and 2, as a pair of unit drug conjugates, are cross-linked to each other through their binding groups.

[0252] [Table 1]

[0253]

[0254] 1×10 6 U-87MG cells (Korean cell line bank) were seeded in confocal culture dishes (SPL Life Sciences) and cultured at 37°C and 5% CO2. Cells were treated with the unit drug conjugate alone or together with a target competitor according to design conditions 1 to 3 described in Table 1 above, followed by induction of cell binding at 37°C for 2 hours. After the reaction, the cells were washed three times with 2 mL of PBS buffer and fixed in culture dishes with 4% paraformaldehyde for 1 hour at room temperature.

[0255] Fluorescence images were analyzed using a confocal laser scanning microscope (Nikon A1 Rsi) and Z-stack analysis was performed using NIS Elements Imaging software (version 5.01, NIKON).

[0256] Figure 3A and Figure 3B The results of fluorescence images under the experimental conditions described in Table 1 are shown. Figure 3A Fluorescence images are shown, and Figure 3BA graph illustrating the results of fluorescence intensity quantification of the fluorescence images is presented. First, as a result of treating cells individually with either drug conjugate 1 or 2 (which confirms the tumor cell uptake capacity of each drug conjugate), the wavelengths of the fluorescent dyes of each drug conjugate can be read in the FITC and TRITC channels, indicating that each drug conjugate unit is taken up into the target cells. As a result of treating cells with drug conjugate 1 together with a target competitor to confirm the selective binding affinity of the drug conjugate, no fluorescence signal was observed in the FITC channel, indicating that drug conjugate 1 binds selectively to the target cells. Similarly, as a result of treating cells with drug conjugate 2 together with a target competitor, no fluorescence signal was observed in the TRITC channel, indicating that drug conjugate 2 binds selectively to the target cells.

[0257] Meanwhile, in a control group of the present invention, prior to treatment of target cells, unit drug conjugates 1 and 2 were dissolved in ethanol and water (v / v = 1:1) and subjected to a binding reaction at room temperature for 40 minutes to synthesize drug conjugates 1-2 for post-conjugation targeting. Drug conjugates 1-2 were then isolated and used to treat cells. In an experimental group, target cells were sequentially treated using unit drug conjugates 1 and 2 for post-conjugation targeting. Subsequently, fluorescence wavelength analysis was performed in the FITC and TRITC channels, where the wavelengths of the fluorescent dyes of the individual unit drug conjugates can be read, and in the FRET channel, where the wavelengths of binding between the unit drug conjugates can be read.

[0258] The analysis results are shown in Figure 3A and Figure 3B In the case of drug conjugates 1-2 used for post-conjugation targeting, the fluorescence intensity measured in the FRET channel was quite low. However, when unit drug conjugate 1 was administered followed by unit drug conjugate 2 sequentially (i.e., post-targeting conjugate), the fluorescence intensity was four times higher than when cells were treated with drug conjugates 1-2 (i.e., post-conjugation targeting). This indicates that when unit drug conjugates capable of interacting with each other are administered sequentially, the endocytosis (internalization) of the compound (unit drug conjugate) can be significantly improved. Furthermore, Z-stack analysis, which allows for three-dimensional analysis of fluorescence images, confirmed that when cells were treated sequentially with unit drug conjugates (post-targeting conjugate), the unit drug conjugates were distributed in the cytoplasm within the cells. Figure 3C ).

[0259] The results of the experiment conducted under the same conditions as the above experiments, according to Experiment Example 2 in Table 1, are shown below. Figure 3BAt the bottom, results almost identical to those of Experimental Example 1 are shown. Therefore, it can be confirmed once again that when the unit drug conjugate is administered sequentially according to the invention (i.e., post-targeting conjugate), the effect of internalizing the compound into cells is significantly improved.

[0260] The principle of maximizing cellular internalization by sequentially introducing conjugates into cells, according to the invention, is applicable to different types of drugs contained in drug conjugates. In this case, the invention is particularly advantageous for combination therapies in which various drugs must be administered together, and the invention can be applied to multipurpose therapies because it can improve the endocytosis of various drugs used to treat serious diseases and complications.

[0261] Example 4. Detection of target cell binding affinity, stability, and in vivo cell internalization of unit drug conjugates

[0262] 4-1. Stability Test

[0263] The unit drug conjugates used in this experiment are unit drug conjugate 5 labeled with the radioactive isotope iodine, unit drug conjugate 6 not labeled with the radioactive isotope, and drug conjugates that are a combination of unit drug conjugates 5 and 6.

[0264] Stability testing of unit drug conjugate 5 and drug conjugate 5-6 was performed using Radio-TLC. Serum obtained by centrifuging human blood at 3,500 rpm for 5 minutes was used. 0.5 mL of serum was treated with unit drug conjugate 5 and drug conjugate 5-6 (3.7 MBq) labeled with radioisotopes, respectively. As a result of stability measurements taken by Radio-TLC (Bioscan) at five time points over 4 hours (10 min, 30 min, 60 min, 120 min, and 240 min) after treatment, both drug conjugates exhibited stability of over 90%, indicating that these drugs can be conjugated in subsequent in vivo binding experiments (data not shown).

[0265] 4-2. Binding affinity to target cells

[0266] The unit drug conjugates used in this experiment are unit drug conjugate 7, which introduces non-radioactive iodine, and drug conjugate 6-7, which is a combination of unit drug conjugates 6 and 7.

[0267] The target cell binding affinity of unit drug conjugate 7 and drug complex 6-7 was measured using U-87MG cells (Korean cell line bank) known to have high integrin αvβ3 expression. Cells were used with known competitive inhibitors... 125Cells were treated with Ic(RGDyV) (0.037 MBq) and then with individual units of drug conjugate 7 and drug conjugate 6-7 at different concentrations (0 to 5 nM) and cultured for 1 hour. Subsequently, the cell pellet was separated by centrifugation and washed three times with PBS to remove unbound drug conjugates, and then the radiation dose was measured using a gamma counter.

[0268] Measuring the binding affinity (IC50) of each drug conjugate 50 The results are shown in Table 2 below.

[0269] [Table 2]

[0270]

[0271]

[0272] It can be confirmed that the IC50 of unit drug conjugate 7 is... 50 The concentration was 1.08 ± 0.08 nM, and the IC50 of drug conjugates 6-7 was... 50 The value was 0.52 ± 0.12 nM. This confirms that both molecules exhibit nanomolar (nM) level binding affinity, indicating that unit drug conjugate 7 and drug conjugate 6-7 have excellent binding affinity to target cells.

[0273] 4-3. Biodistribution experiment in tumor model mice

[0274] The unit drug conjugates used in this experiment are unit drug conjugate 5 labeled with the radioactive isotope iodine, unit drug conjugate 6 not labeled with the radioactive isotope, and drug conjugate 5-6 as a combination of unit drug conjugate 5 and unit drug conjugate 6.

[0275] In this experiment, 1×10 7 U-87MG cells were suspended in PBS and subcutaneously injected into mice with congenital thymus deficiency (7 weeks old, BLAB / c nude mice, OrientBio, male). After a growth period of about 2 weeks, models with tumor volumes of 0.4 cc to 0.5 cc were selected and used.

[0276] Units of drug conjugate 5 (18.5 MBq) and drug conjugate 5-6 (18.5 MBq) were injected into the tail veins of prepared nude mouse tumor models (n=3, respectively). Organs (blood, brain, heart, lung, liver, spleen, kidney, stomach, muscle, thigh, small intestine, large intestine, thyroid, and tumor) were extracted at 10 minutes, 30 minutes, 1 hour, and 2 hours, and radiation dose was measured using a gamma counter (PerkinElmer, Wellesley, MA, USA).

[0277] The results are shown in Figure 4 It can be confirmed that both drug conjugate 5 and drug conjugate 5-6 showed high uptake in the tumor at 15 minutes, but more than 50% of them were excreted from the tumor within 2 hours, mainly through the kidneys, while their uptake in other organs was not high.

[0278] 4-4. Compare the retention of a unit dose of drug conjugate in tumor cells in tumor model mice. improvement

[0279] In the same manner as described in Examples 4-3, 1×10 7 U-87MG cells were suspended in PBS and subcutaneously injected into male mice with congenital thymus deficiency (7 weeks old, BLAB / c nude mice, OrientBio). After a growth period of approximately 2 weeks, tumor models with a volume of 0.4 cc to 0.5 cc were selected and used in experiments. In vivo imaging was performed using SPECT / CT (NanoSPECT / CT, Bioscan Inc., Washington DC).

[0280] Unit drug conjugates 5 and 6, as well as drug conjugate 5-6, were used in the experiment. In the control group, drug conjugate 5-6 (18.5 MBq) (n=3) or unit drug conjugate 5 (18.5 MBq) (n=3) was injected. In the experimental group, unit drug conjugate 5 (18.5 MBq) was injected 15 minutes later, and unit drug conjugate 6 was injected at doses of 18 mg / kg, 1.8 mg / kg, and 0.18 mg / kg (for each dose, n=3). As described above, the experiment was conducted under a total of five conditions. Imaging was performed 2 hours after administration of each unit drug conjugate (or after the first administration in the case of sequential administration).

[0281] The results are shown in Figure 5 In the study, it was confirmed that in the control group, tumor uptake of drug conjugates 5-6 and unit drug conjugate 5 decreased to less than half after 2 hours, while in the experimental group treated sequentially with drug conjugates 5 and 6, over 60% of tumor uptake was maintained for 2 hours regardless of the dose of unit drug conjugate 6. Specifically, in the group treated sequentially with the minimum dose (0.18 mg / kg) of unit drug conjugate 6, over 95% of tumor uptake was maintained for up to 2 hours, thus demonstrating the most significant retention of the unit drug conjugate in the tumor.

[0282] 4-5. Comparison of the effects of tumor cell preservation in mice after 24 hours in tumor models

[0283] In Examples 4-4, the effect of sequential treatment with unit drug conjugates to improve retention in tumors over a short period of time was demonstrated. Therefore, experiments were conducted to observe this effect over a longer period. For this purpose, the same tumor model as described in Examples 4-3 was prepared, and on the one hand, unit drug conjugate 5 (18.5 MBq) was administered intravenously to a nude mouse tumor model injected with U-87MG cells (n=3). On the other hand, unit drug conjugate 5 (18.5 MBq) and unit drug conjugate 6 (0.18 mg / kg) were administered sequentially at 15-minute intervals (n=3). The retention and half-life of each unit drug conjugate in the tumor were compared by imaging at 1 hour, 2 hours, 4 hours, and 24 hours after administration. Furthermore, the tumors were excised at 10 minutes and 24 hours after administration of each unit drug conjugate, and the effect of improving retention in tumor cells was evaluated in vitro.

[0284] As a result, such Figure 6 As shown, it can be confirmed that when unit drug conjugate 5 is administered alone, its uptake decreases to less than half of the initial uptake within 4 hours. However, when units drug conjugates 5 and 6 are administered sequentially, its uptake in the tumor remains at approximately 60% of the initial uptake until 24 hours. It can be confirmed that when unit drug conjugate 5 is administered alone, its half-life is 80.7 minutes, while when units drug conjugates 5 and 6 are administered sequentially, its half-life is 213.4 minutes, indicating that the half-life is three times greater in the case of sequential administration.

[0285] Meanwhile, as a result of evaluating the retention of unit drug conjugates in tumor cells by extracting tumors 10 minutes and 24 hours after drug conjugate administration, it was confirmed that when unit drug conjugate 5 (18.5 MBq, n=3) was administered alone, its uptake in tumors remained at 10%, while when unit drug conjugate 5 (18.5 Mbq, n=3) and unit drug conjugate 6 (0.18 mg / kg) were administered sequentially, more than 50% of the initial uptake was maintained until 24 hours.

[0286] Based on the above results, it can be seen that the drug conjugates according to the present invention can exert a prolonged effect on tumor cells after internalization.

[0287] Industrial applicability

[0288] The drug conjugates according to the invention comprise a targeting substance that specifically binds to target cells, enabling them to rapidly target the target cells. Along with this interaction between the target cells and the targeting substance, binding groups that allow interaction between the drug conjugates are additionally introduced into the drug conjugates. Therefore, when the drug conjugates are injected sequentially, the binding groups bind to each other in vivo and induce in vivo cross-linking between the complexes (different drug conjugates binding to the target cells), thereby causing the drug conjugates to form a cluster. This clustering of drug conjugates on the surface of the target cells artificially enhances cell internalization induced by endocytosis, thereby maximizing therapeutic and / or diagnostic effects even when administered at low doses, and preventing side effects caused by high-dose administration. Furthermore, the drugs introduced into the drug conjugates and the different binding groups can be designed in different ways, thus facilitating the co-administration of diagnostic and therapeutic drugs or the co-administration of multiple drugs exhibiting synergistic effects.

[0289] Although the invention has been described in detail with reference to specific features, it will be apparent to those skilled in the art that this description is merely a description of preferred embodiments and does not limit the scope of the invention. Therefore, the essential scope of the invention will be defined by the appended claims and their equivalents.

Claims

1. A drug conjugate comprising a first unit drug conjugate and a second unit drug conjugate; in, The first unit drug conjugate is unit drug conjugate 1, and the second unit drug conjugate is unit drug conjugate 2; or Wherein, the first unit drug conjugate is unit drug conjugate 3, and the second unit drug conjugate is unit drug conjugate 4; or Wherein, the first unit drug conjugate is unit drug conjugate 5, and the second unit drug conjugate is unit drug conjugate 6; or Wherein, the first unit drug conjugate is unit drug conjugate 6, and the second unit drug conjugate is unit drug conjugate 7: [Unit drug conjugate 1] [Unit drug conjugate 2] [Unit drug conjugate 3] [Unit drug conjugate 4] [Unit drug conjugate 5] [Unit Drug Conjugate 6] [Unit Drug Conjugate 7] 。 2. Use of the drug conjugate according to claim 1 in the preparation of a medicament for treating angiogenesis-related malignancies.

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