Complex for tumor chemodynamic therapy

Abstract

This complex for use in tumor chemodynamic therapy comprises: nanoparticles that enclose iron oxide and include a hydrophilic polymer in a surface layer thereof; and target recognition molecules that bind to the nanoparticles and can bind to target molecules of the tumor cells.

Description

Conjugates for chemodynamic therapy of tumors - Patents.com

[0001] The present invention relates to a conjugate for the chemodynamic therapy of tumors.

[0002] Chemodynamic therapy (CDT) is a treatment that induces cell death by generating reactive oxygen species (ROS) from endogenous H2O2 through a continuous chemical process in the tumor microenvironment (TME), damaging tumor cells. ROS generation by CDT is basically based on the Fenton reaction, and CDT contains mainly Fe to promote this reaction. 2+ Cu-based nanomaterials have also been used for CDT based on Fenton-like reactions. 2+ However, because TME is weakly acidic, Fe, which requires a strong acidity (pH 2-4), is not suitable for nanomaterials based on transition metals. 2+ The therapeutic efficiency of conventional CDT is not necessarily sufficient because the conditions are unsuitable for the Fenton reaction and the production of hydrogen peroxide (H2O2) is poor compared to normal cells.

[0003] Patent Literature 1 describes the use of fibronectin-coated polydopamine iron nanoparticles loaded with artesunate for CDT, with fibronectin enhancing the biocompatibility and targeting of the nanomaterial, and the chemotherapeutic agent artesunate promoting tumor apoptosis and reacting with ferrous ions to promote ROS production. Patent Literature 2 describes the use of a magnetic nanoenzyme (Fc-MBL-rGO-Fe3O4) obtained by modifying the surface of a reduced graphene oxide-supported triiron tetroxide magnetic nanoenzyme (rGO-Fe3O4) with mannose lectin (Fc-MBL), which exhibits antibacterial effects when used in combination with CDT and photothermal therapy (PTT). Non-Patent Document 1 describes that CuS nanoparticles have a PTT effect due to near-infrared light absorption and a CDT effect due to ROS production, and that the PTT effect of CuS promotes the ROS production. It also describes GdO / CuS nanoparticles in which CuS nanoparticles are modified with GdO for magnetic imaging and the fluorophore Cy5.5 for fluorescent imaging, and are endowed with the ability to selectively recognize tumor cells by binding an RGD peptide.

[0004] Near-infrared photoimmunotherapy (NIR-PIT) is a treatment method that selectively kills tumor cells by preparing an antibody conjugate containing a near-infrared-sensitive photosensitizer against an antibody specific to an antigen on the tumor cell surface, binding the antibody conjugate to the tumor cells, and then locally irradiating them with near-infrared light. The photosensitizer mainly used is a chemical species containing a phthalocyanine skeleton (e.g., so-called IR700 molecule, such as IRDye700DX) (see Patent Documents 3 and 4 and Non-Patent Document 2). The mechanism of NIR-PIT differs from conventional phototherapy, where cell death is induced by ROS generated by light irradiation, because cell death occurs in an extremely short time (2-6 minutes) and no mitochondrial damage is observed. Recently, it has been elucidated that the mechanism of NIR-PIT's tumor therapeutic effect is that light irradiation causes aggregation of antibody conjugates bound to the tumor cell surface, damaging the tumor cell membrane, which then creates an osmotic pressure differential between the inside and outside of the cell, resulting in cell death (Non-Patent Document 2). Patent Document 5 describes that a complex containing an antibody molecule bound to a photosensitive substance containing a phthalocyanine skeleton and a magnetic particle or semiconductor particle bound to the antibody molecule can be used for NIR-PIT and tumor imaging.

[0005] Chinese Patent Publication No. 115089560 Chinese Patent Publication No. 115944727 Special Publication No. 2014-523907 Special Publication No. 2019-218374 International Publication No. 2022 / 054798

[0006] ACS Applied Materials & Interfaces, 2022, 14:34365-34376 Award-winning paper from the 40th Annual Meeting of the Japan Society for Laser Surgery and Medicine, "Elucidation of the mechanism of near-infrared photoimmunotherapy," Journal of the Japan Society for Laser Surgery and Medicine, Vol. 41, No. 2, 2020, pp. 104-109

[0007] The present invention provides nanoparticle conjugates for chemodynamic therapy (CDT) of tumors and methods of treating tumors by CDT using the conjugates.

[0008] The present inventors have found that a nanoparticle complex in which a target recognition molecule is conjugated to a nanoparticle containing iron oxide exhibits a tumor therapeutic effect by CDT when irradiated with light.

[0009] Therefore, the present invention includes the following embodiments. [1] A complex for use in chemodynamic therapy of tumors, the complex comprising: nanoparticles encapsulating iron oxide and having a hydrophilic polymer on their surface; and a target recognition molecule bound to the nanoparticles and capable of binding to a target molecule in tumor cells. [2] The complex of [1], wherein the chemodynamic therapy relies on a photo-Fenton reaction induced by near-infrared light irradiation. [3] The complex of [1] or [2], further for use in photothermal therapy. [4] The complex of any one of [1] to [3], further for use in tumor imaging. [5] The complex of any one of [1] to [4], further comprising a photosensitive moiety bound to the nanoparticle or the target recognition molecule, the photosensitive moiety comprising a photosensitive group having a maximum absorption wavelength in the range of 500 to 1500 nm and one or more hydrophilic functional groups linked to or coordinated to the photosensitive group. [6] The conjugate of [5], wherein the photosensitive site is bound to the target recognition molecule. [7] The conjugate of [5] or [6], further for use in photoimmunotherapy. [8] The conjugate of any one of [1] to [7], wherein the target recognition molecule is an antibody. [9] The conjugate of any one of [1] to [8], wherein the hydrophilic polymer is a polysaccharide.

[10] The conjugate of [9], wherein the polysaccharide is dextran.

[11] The conjugate of any one of [1] to

[10] , wherein the content of iron atoms in the conjugate is 30% by mass or more.

[12] The conjugate of any one of [1] to

[11] , wherein the number of the target recognition molecules bound to the nanoparticles is 1 to 20 per nanoparticle.

[13] The conjugate of any one of [1] to

[12] , wherein the target molecule on the tumor cell is epidermal growth factor receptor, EGF receptor family, or platelet-activating receptor.

[14] A composition for use in chemodynamic therapy of a tumor, comprising the complex of any one of [1] to

[13] .

[15] The composition of

[14] , further comprising hydrogen peroxide.

[16] The composition of

[14] or

[15] , further comprising a bioreductive substance.

[17] The composition of any one of

[14] to

[16] , further for use in photothermal therapy.

[18] The composition according to any one of

[14] to

[17] , further for use in tumor imaging.

[19] The composition according to any one of

[14] to

[18] , wherein the conjugate has a photosensitive moiety, the photosensitive moiety comprising a photosensitive group having a maximum absorption wavelength in the range of 500 to 1500 nm and one or more hydrophilic functional groups linked or coordinated to the photosensitive group.

[20] The composition according to

[19] , further for use in photoimmunotherapy.

[21] Use of the conjugate according to any one of [1] to

[13] in the manufacture of a tumor therapeutic agent for chemodynamic therapy of tumors.

[22] The use according to

[21] , wherein the tumor therapeutic agent is further used in photothermal therapy.

[23] The use according to

[21] or

[22] , wherein the tumor therapeutic agent is further used in tumor imaging.

[24] The use according to any one of

[21] to

[23] , wherein the conjugate has a photosensitive site, the photosensitive site comprising a photosensitive group having a maximum absorption wavelength in the range of 500 to 1500 nm, and one or more hydrophilic functional groups linked or coordinated to the photosensitive group.

[25] The use according to

[24] , wherein the tumor therapeutic agent is further used in photoimmunotherapy.

[26] The use according to any one of

[21] to

[25] , wherein a composition containing the conjugate and hydrogen peroxide is used.

[27] The use according to any one of

[21] to

[26] , wherein a composition containing the conjugate and a bioreductive substance is used.

[28] A method for treating a tumor by chemodynamic therapy, comprising the steps of administering the conjugate according to any one of [1] to

[13] to a patient in need of tumor chemodynamic therapy, and irradiating the patient with light having a wavelength of 500 to 1500 nm.

[29] The method of

[28] , wherein hydrogen peroxide is further administered to the patient together with the complex.

[30] The method of

[28] or

[29] , wherein a bioreductive substance is further administered to the patient together with the complex.

[0010] The conjugate of the present invention exerts a tumor therapeutic effect by CDT upon light irradiation. Furthermore, when the conjugate of the present invention further comprises a photosensitive site, it exerts a tumor therapeutic effect by photothermal therapy (PTT) or near-infrared photoimmunotherapy (NIR-PIT) in response to the light irradiation, and thus the use of the conjugate can enjoy the combined effects of these therapies. Furthermore, the conjugate of the present invention can be used for tumor imaging, thereby enabling observation of target tumors in the patient's body or optimization of light irradiation for CDT.

[0011] Zeta potential of Nanomag-D-spio and Nanomag-D-spio-pan-IR700. Images (top) of Nanomag-D-spio and Nanomag-D-spio-pan-IR700 observed with an atomic force microscope (AFM), measured size (bottom), and estimated shape (center picture). Magnetic susceptibility of Nanomag-D-spio and Nanomag-D-spio-pan-IR700 measured by VSM. Specific binding of Nanomag-D-Spio-pan-IR700 to EGFR. A: Number of IR700-labeled cells in EGFR-expressing (EGFR positive) cells (MDAMB468 and A431) and non-expressing (EGFR negative) cells (H661 and 3T3). Pan-IR700: Cells with pan-IR700 added. Nanomag-D-Spio-pan-IR700: Cells with Nanomag-D-Spio-pan-IR700 added. Pan blocking: Cells with pan-IR700 or Nanomag-D-Spio-pan-IR700 added in the presence of an EGFR inhibitor. B: Number of IR700-labeled cells in EGFR-expressing (EGFR positive) cells (MDAMB468 and A431) treated with Nanomag-D-Spio-pan-IR700 or Nanomag-D-Spio-cont-IR700 (cont; a ligand other than EGFR ligand (IgG antibody)). Fluorescence intensity of Nanomag-D-Spio-pan-IR700. A: in SDS, and B: in serum. T-test performed (*; p<0.001, ns; not significant, vs. control, n=3). Fluorescence image of cells treated with Nanomag-D-Spio-pan-IR700. Hoechst: Hoechst staining, Lyso tracker: lysosomal staining, IR700: IR700 labeling, Merge: overlaid image. DIC: bright-field image by optical microscope. White bar: 30 μm. Absorbance of methylene blue solution containing Nanomag-D-Spio. A: Change in absorbance due to laser irradiation. B: Change in absorbance depending on heat treatment time in the presence of reduced glutathione. Heat generation behavior of the complex in response to laser irradiation. A: Temperature rise curve of the solution containing the complex, error bar: SEM, t-test (*; p<0.05, vs. Pan-IR700, n=3) was performed.B: Left: Infrared thermography image of Nanomag-D-Spio-pan-IR700-containing solution after laser irradiation. Right: Liquid temperature of Nanomag-D-Spio-pan-IR700-containing solution after laser irradiation. Fluorescence images of in vitro disease model cells (A431, MDAMB468, and PC-9) administered with Nanomag-D-Spio-pan-IR700. Hoechst: Hoechst staining. PI: Pyridium iodide staining. IR700: IR700 labeling. Merge: Overlaid image. DIC: Bright-field image taken with an optical microscope. White bar: 30 μm. Fluorescence images of disease model cells contained in in vitro spheroids administered with Nanomag-D-Spio-pan-IR700. Spheroids were prepared by mixing disease model cells and 3T3-RFP at a 1:1 (volume ratio). A431 disease model cells were used. 3T3-RFP was prepared by transfecting 3T3 with the red fluorescent protein (RFP) gene. RFP: RFP labeling, IR700: IR700 labeling, SYTOX Blue: SYTOX. TMBlue staining, Marge: overlaid image. DIC: bright-field image obtained by optical microscope. White bar: 30 μm. Same as Figure 10A. MDAMB468 or PC-9 disease model cells were used. Protocol for Experiment 10. Top: Schedule for complex administration to A431-implanted xenograft model mice, laser irradiation, and tumor evaluation by luciferase assay (bioluminescent imaging; BLI). Bottom: Schematic diagram of the process of A431 implantation into mice, complex administration, and laser irradiation. Images of complex-administered xenograft model mice observed by luciferase assay. Changes in tumor size in complex-administered xenograft model mice evaluated by luciferase assay. The RLU in the region of interest of the tumor image on Day 0 is set to 100, and the changes in tumor size are expressed as the RLU ratio. Error bars: SEM. Changes in tumor size in complex-administered xenograft model mice. Tumor size was measured after tumor removal from the mice. Error bars: SEM. Protocol for Experiment 11. Left: Schematic of the process of spheroid transplantation into mice and laser irradiation. Right: Schedule of complex administration to spheroid-transplanted xenograft model mice, laser irradiation, and tumor evaluation by luciferase assay (BLI). Images of complex-administered xenograft model mice observed by luciferase assay. Changes in tumor size in complex-administered xenograft model mice evaluated by luciferase assay. The RLU in the region of interest of the tumor image on Day 0 is set to 100, and the changes in tumor size are expressed as the RLU ratio. Error bars: SEM. Changes in tumor size in complex-administered xenograft model mice. Tumor size was measured after tumor removal from the mice, and is expressed as a relative value, with the tumor size on Day 0 set to 100. Error bars: SEM. Nuclear magnetic resonance intensity of MRI images. A Dunnett's test was performed using a control group as a comparison group (*p<0.0001, n=5). A: Change in absorbance of a methylene blue solution containing Nanomag-D-Spio-Pan upon laser irradiation. B: Change in absorbance of a methylene blue solution containing Nanomag-D-Spio-Tra upon laser irradiation.

[0012] All patents, non-patent documents, and other publications cited herein are hereby incorporated by reference in their entirety.

[0013] As used herein, the term "tumor" refers to tumors in general, including benign and malignant tumors, as well as epithelial and non-epithelial tumors, and the site (tissue and organ) of occurrence is not particularly limited. Preferably, the tumor targeted by the present invention is a malignant tumor such as cancer. Cancer may be a liquid tumor or a solid tumor, and may include any type of cancer, such as epithelial cancer, adenocarcinoma, sarcoma, or malignant lymphoma. Examples of tumors include liquid tumors, including acute leukemia (acute lymphocytic leukemia, acute myeloid leukemia, myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, etc.), chronic leukemia (chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia, etc.), T-cell prolymphocytic leukemia, large granular lymphocytic leukemia, adult T-cell leukemia, polycythemia vera, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, etc.; as well as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, other sarcomas, synovial tumor, mesothelioma, Ewing's tumor, Heikin's tumor, etc. Solid tumors include leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, lung cancer, colorectal cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma (e.g., adenocarcinoma of the pancreas, colon, ovary, lung, breast, stomach, prostate, cervix, or esophagus), sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, bladder cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, etc. The tumor herein may be a primary tumor or a recurrent tumor.

[0014] As used herein, the term "tumor affected area" or "affected area" refers to tumor tissue in which tumor cells are primarily present. Such tumor tissue includes tissue consisting of tumor cells and tissue in which tumor cells are mixed with normal cells or normal tissue. When normal cells or normal tissue are mixed with the tumor tissue, the ratio of the volume or number of tumor cells to the normal cells or normal tissue is not particularly limited.

[0015] As used herein, the term "antibody" refers to a polypeptide ligand comprising at least one light chain variable region and / or heavy chain variable region that specifically recognizes and binds to an epitope of an antigen. For example, the term "antibody" as used herein includes immunoglobulins of any class, such as IgG, IgA, IgD, IgE, IgM, and subclasses thereof, as well as variants thereof, and further includes chimeric antibodies such as humanized antibodies, other modified immunoglobulins containing an antigen recognition site, and the like. Furthermore, the term "antibody" as used herein includes immunoglobulin fragments or domains containing an antigen recognition site, such as Fab fragments, Fab' fragments, F(ab)'2 fragments, single-chain Fvs ("scFvs"), disulfide-stabilized Fvs ("dsFvs"), VHHs (variable domains of heavy chain antibodies), and VNARs (single variable new antigen receptor domain antibodies).

[0016] "Chemodynamic therapy" (CDT) as used herein refers to a treatment method that induces cell death by generating reactive oxygen species (ROS), such as superoxide (.O), singlet oxygen (O), and hydroxyl radical (.OH), from H2O2 through a continuous chemical process in the tumor microenvironment (TME). These ROS damage tumor cells through oxidative stress, which is believed to be primarily based on the Fenton reaction.

[0017] Photothermal therapy (PTT) in this specification is a treatment method in which cells administered with a photothermal agent that generates heat when irradiated with light are irradiated with light, and the heat emitted from the photothermal agent kills the cells. The therapeutic effect of PTT depends on the difference between the upper temperature limit for survival of cancer cells (approximately 42°C) and that of normal cells (45°C).

[0018] As used herein, "near-infrared photoimmunotherapy" (NIR-PIT) refers to a treatment method in which an antibody conjugate containing a near-infrared-sensitive photosensitizer is introduced into an antibody specific to an antigen on the surface of a tumor cell, and the tumor cells are then irradiated with near-infrared light, inducing cell death through the photoresponse of the photosensitizer. Recently, it has been elucidated that the mechanism of the tumor treatment effect of NIR-PIT is that the photosensitizer contained in the antibody conjugate bound to the tumor cell surface reacts to near-infrared light, causing the antibody conjugate to aggregate, damaging the cell membrane of the tumor cell, which in turn creates an osmotic pressure difference between the inside and outside of the cell, inducing cell death (Non-Patent Document 2).

[0019] 1. Conjugate In one embodiment, the present invention provides a conjugate for use as a drug for tumor chemodynamic therapy (CDT). The conjugate provided by the present invention comprises a nanoparticle and a target recognition molecule bound to the nanoparticle, the target molecule being capable of binding to a target molecule in tumor cells.

[0020] 1.1. Nanoparticles The nanoparticles used in the composite of the present invention encapsulate iron oxide and have a hydrophilic polymer on the surface. Because the nanoparticles have a hydrophilic polymer on the surface, a hydration layer forms around them. This hydration layer inhibits nonspecific adsorption of proteins to the nanoparticles, thereby preventing capture and degradation of the nanoparticles by phagocytes (conferring stealth properties), thereby improving the retention of the nanoparticles in the blood. Furthermore, the formation of a hydration layer on the nanoparticles promotes the uptake of the composite of the present invention into cells via endocytosis. Examples of iron oxides encapsulated in the nanoparticles include FeO, Fe2O3, and Fe3O4. Preferred are Fe2O3 and Fe3O4. Examples of hydrophilic polymers include polyacrylamide, poly(vinyl alcohol), polyethylene glycol, agarose, polysaccharides, glycoproteins, and proteins such as heparin and albumin. Preferred are water-soluble substances such as hydrogen peroxide and bioreductive substances, and Fe 2+The hydrophilic polymer of the surface layer is a polysaccharide because it can transmit ions such as hydroxybenzoates, hydroxybenzoates, hydroxybenzoates, etc. Examples of the polysaccharide include dextran, hyaluronic acid, alginic acid, and pullulan, with dextran being preferred. The form of the hydrophilic polymer of the surface layer includes a gelled form.

[0021] The nanoparticles may have, on their surface, reactive groups or linkers that bind to target recognition molecules (described later). For example, the nanoparticles preferably have, on their surface, reactive functional groups such as amino groups or carboxy groups, or protein-binding molecules such as avidin, streptavidin, or protein A.

[0022] The nanoparticles preferably have an average particle diameter of 500 nm or less. The "average particle diameter" of particles in this specification refers to the average particle diameter measured by dynamic light scattering. The average particle diameter of the nanoparticles can be appropriately adjusted depending on the type of cancer, taking into account the EPR effect (enhanced permeation and retention effect) and the like. For example, the average particle diameter of the nanoparticles is preferably 200 nm or less, more preferably 100 nm or less, and even more preferably 50 nm or less. The lower limit of the average particle diameter of the nanoparticles is not particularly limited, but is preferably 1 nm or more, more preferably 10 nm or more, and even more preferably 20 nm or more, from the viewpoint of manufacturability.

[0023] The content of iron oxide contained in the nanoparticles used in the complex of the present invention is preferably 30% by mass or more, more preferably 35% by mass or more, and preferably 80% by mass or less, more preferably 70% by mass or less, calculated as the content of iron atoms in the total mass of the complex of the present invention. If the content of iron atoms in the complex is too high, the water solubility or dispersibility of the complex tends to decrease. On the other hand, if the content of iron atoms in the complex is too low, the therapeutic effect of CDT tends to decrease.

[0024] The nanoparticles used in the complex of the present invention can be purchased commercially. For example, a dispersion of dextran-coated superparamagnetic iron oxide particles is sold by Micromod as the Nanomag-D-spio series.

[0025] 1.2. Target Recognition Molecule The target recognition molecule contained in the conjugate of the present invention is a molecule that allows the conjugate to bind to tumor cells, which are the therapeutic target. The target recognition molecule recognizes and binds to a target molecule (receptor) present on tumor cells. Examples of target recognition molecules include small molecular weight compounds, peptides, antibodies, ligands such as antigen-binding fragments, aptamers, sugar chains, and podoplanin. Preferably, the target recognition molecule is an antibody. The type of antibody can be selected appropriately depending on the antigen (target molecule) present on the surface of tumor cells.

[0026] A preferred example of the antigen is a transmembrane protein present in tumor cells. An example of the transmembrane protein is a tumor-specific protein (also known in the art as a tumor-specific antigen) expressed on the surface of tumor cells. A tumor-specific protein is a protein that is unique to cancer cells or is more abundant in cancer cells than in other cells, such as normal cells.

[0027] Examples of such tumor-specific proteins include members of the epidermal growth factor receptor (EGFR) family (e.g., HER1, 2, 3, and 4), members of cytokine receptors (e.g., CD20, CD25, IL-13R, CD5, CD52, etc.), and the like.

[0028] Specific examples of such tumor-specific proteins include HER-2 (human epidermal growth factor receptor 2, e.g., GenBank Accession Nos. M16789.1, M16790.1, M16791.1, M16792.1, and AAA58637), which is associated with breast cancer, ovarian cancer, gastric cancer, and uterine cancer; and HER-1 (e.g., GenBank Accession Nos. NM_005228 and NP_005219), which is associated with lung cancer, anal cancer, and glioma, as well as adenocarcinoma.

[0029] Other specific examples of tumor-specific proteins include CD52, which is associated with chronic lymphocytic leukemia (e.g., GenBank accession numbers AAH27495.1 and CAI15846.1); CD33, which is associated with acute myeloid leukemia (e.g., GenBank accession numbers NM_023068 and CAD36509.1); and CD20, which is associated with non-Hodgkin's lymphoma (e.g., GenBank accession numbers NP_068769 and NP_031667).

[0030] Other specific examples of the tumor-specific proteins include MAGE1 (e.g., GenBank Accession Nos. M77481 and AAA03229), MAGE2 (e.g., GenBank Accession Nos. L18920 and AAA17729), MAGE3 (e.g., GenBank Accession Nos. U03735 and AAA17446), and MAGE4 (e.g., GenBank Accession Nos. D32075 and A06841.1). any of various MAGE (melanoma-associated antigen E); any of various tyrosinases (e.g., GenBank Accession Nos. U01873 and AAB60319); mutant ras; mutant p53 (e.g., GenBank Accession Nos. X54156, CAA38095, and AA494311); p97 melanoma antigen (e.g., GenBank Accession Nos. M12154 and AAA59992); human breast cancer associated with breast tumors Fat globules (HMFG) (e.g., GenBank Accession Nos. S56151 and AAB19771); any of the various BAGEs (human melanoma type B associated antigen E), including BAGE1 (e.g., GenBank Accession No. Q13072) and BAGE2 (e.g., GenBank Accession Nos. NM_182482 and NP_872288); gp100 associated with melanoma (e.g., GenBank Accession Nos. S73003 and AAB19771); AC60634); the MART1 antigen associated with melanoma (e.g., GenBank Accession No. NP_005502); any of the various GAGEs (G antigens), including GAGE1 (e.g., GenBank Accession No. Q13065), or any of GAGEs 2-6; various gangliosides; CD25 (e.g., GenBank Accession Nos. NP_000408.1 and NM_000417.2), and the like.

[0031] Other specific examples of the tumor-specific proteins include HPV16 / 18 and E6 / E7 antigens associated with cervical cancer (e.g., GenBank Accession Nos. NC_001526, FJ952142.1, ADB94605, ADB94606, and U89349); mucin (MUC1)-KLH antigen associated with breast cancer (e.g., GenBank Accession Nos. J03651 and AAA35756); CEA (carcinoembryonic antigen) associated with colorectal cancer (e.g., GenBank Accession Nos. GenBank Accession Nos. NM_024690 and NP_078966); alpha-fetoprotein (AFP), associated with liver cancer (e.g., GenBank Accession Nos. NM_001134 and NP_001125); Lewis Y antigen, associated with colorectal cancer, biliary tract cancer, breast cancer, small cell lung cancer, and other cancers; tumor-associated glycoprotein 72 (TAG72), associated with adenocarcinoma; and PSA antigen, associated with prostate cancer (e.g., GenBank Accession Nos. X14810 and CAA32915).

[0032] Other specific examples of the tumor-specific proteins include PMSA (prostate membrane-specific antigen; e.g., GenBank Accession Nos. AAA60209 and AAB81971.1), which is associated with prostate cancer; NY-ESO-1 (e.g., GenBank Accession Nos. U87459 and AAB49693), which is associated with melanoma, sarcoma, testicular cancer, and other cancers; hTERT (also known as telomerase) (e.g., GenBank Accession Nos. NM_198253 and NP_937983 (variant 1), NM_198255 and NP_937986 (variant 2)); protein Wilms' tumor 1 (WT-1, e.g., GenBank Accession Nos. NM_000378 and NP_000369 (variant A), NM_024424 and NP_077742 (variant B), NM_024425 and NP_077743 (variant C), and NM_024426 and NP_077744 (variant D)); and the like.

[0033] Other specific examples of the tumor-specific protein include PD-L1 and PD-L2, which are associated with immune checkpoints.

[0034] The names of the tumor-specific proteins described herein are according to the GenBank database of the National Center for Biotechnology Information (NCBI) ([www.ncbi.nlm.nih.gov / genbank / ]).

[0035] Examples of antibodies that can be included in the conjugate of the present invention as the target recognition molecule include cetuximab, panitumumab, zalutumumab, nimotuzumab, trastuzumab, Ado-trastuzumab emtansine, tositumomab, rituximab, ibritumomab tiuxetan, daclizumab, gemtuzumab, alemtuzumab, CEA-scan, Fab fragment, OC125 monoclonal antibody, ab75705, B72.3, bevacizumab, afatinib, axitinib, bosutinib, cabozantinib, ceritinib, crizotinib, dabrafenib, dasatinib, erlotinib, everolimus, ibrutinib, imatinib, lapatinib, lenvatinib, nilotinib, olaparib, palbociclib, pazopanib, pertuzumab, ramuciclib lumab, regorafenib, ruxolitinib, sorafenib, sunitinib, temsirolimus, trametinib, vandetanib, vemurafenib, vismodegib, basiliximab, ipilimumab, nivolumab, pembrolizumab, MPDL3280A, pidilizumab (CT-011), MK-3475, BMS-936559, MPDL3280A (atezolizumab), tremelimumab, IM P321, BMS-986016, LAG525, urelumab, PF-05082566, TRX518, MK-4166, dacetuzumab (SGN-40), lucatumumab (HCD122), SEA-CD40, CP-870, CP-893, MEDI6469, MEDI6383, MEDI4736, MOXR0916, AMP-224, PDR001, avelumab (MSB0010 718C), rHIgM12B7, urocupulumab, BKT140, varlilumab (CDX-1127), ARGX-110, MGA271, lirilumab (BMS-986015, IPH2101), IPH2201, AGX-115, emactuzumab, CC-90002, and MNRP1685A, and fragments comprising the antigen recognition site thereof.

[0036] In the complex of the present invention, the number of target recognition molecules bound to the nanoparticles is preferably 1 or more per particle, and preferably 20 or less, more preferably 16 or less, even more preferably 12 or less, and even more preferably 5 or less. If the number of target recognition molecules bound to the nanoparticles is too large, the size of the entire complex increases, which tends to make it difficult for the complex to selectively distribute to tumor cells. In addition, the lower limit of the number of target recognition molecules bound to 1 mg of the complex of the present invention is preferably 1.0 × 10 -12 mol / mg, more preferably 5.0×10 -12 mol / mg, more preferably 1.0×10 -11 On the other hand, the upper limit of the number of the target recognition molecules bound to 1 mg of the complex of the present invention is preferably 1.0 × 10 -8 mol / mg, more preferably 5.0×10 -9 mol / mg, more preferably 1.0×10 -9 mol / mg.

[0037] 1.3. Photosensitive Moiety The conjugate of the present invention may further have a photosensitive moiety. The photosensitive moiety is bound to the nanoparticle or target recognition molecule in the conjugate of the present invention. The photosensitive moiety comprises a photosensitive group having a maximum absorption wavelength in the range of 500 to 1500 nm and one or more hydrophilic functional groups linked or coordinated to the photosensitive group. The photosensitive moiety is presumed to become more hydrophobic upon irradiation with near-infrared light (NIR), for example, light with a wavelength of 500 to 1500 nm. More specifically, upon irradiation with light of this wavelength, the hydrophilic functional group is presumed to dissociate or change structure due to a photochemical reaction of the photosensitive group, thereby increasing the hydrophobicity of the photosensitive moiety.

[0038] Examples of hydrophilic functional groups contained in the photosensitive moiety include carboxylate (-CO2 - ) group, sulfonate (—SO3 - ) group, sulfonyl (—SO2 - ) group, sulfate (-SO4 -2 ) group, hydroxy (-OH) group, phosphate (-OPO3 -2 ) group, phosphonate (-PO-2 Examples of the photosensitive moiety include, but are not limited to, a substituted or unsubstituted quaternary nitrogen (each having an optional counterion), an amino (—NH2) group, and the like. Examples of the counterion include, but are not limited to, sodium, potassium, calcium, ammonium, organic amino acids, magnesium, and the like. The photosensitive moiety may further have a reactive group or linker for binding to the target recognition molecule or nanoparticle.

[0039] An example of a photosensitive moiety for use in the conjugates of the present invention is a moiety containing a phthalocyanine skeleton. Phthalocyanines are azaporphyrins (i.e., C ) containing four benzoindole groups connected by nitrogen bridges in a 16-membered ring of alternating carbon and nitrogen atoms. 32 H 16 N8). Phthalocyanines form stable chelates with metal and nonmetal cations, where the ring center is occupied by an element that can bear one or two ligands. The ring periphery can be unsubstituted or substituted.

[0040] Preferably, the moiety containing a phthalocyanine skeleton used in the present invention is water-soluble and has at least one water-solubilizing portion. Preferably, the water-solubilizing portion of the moiety containing a phthalocyanine skeleton contains silicon. Preferably, the phthalocyanine skeleton has a core element such as Si, Ge, Sn, or Al at the ring center.

[0041] The moiety containing the phthalocyanine skeleton used in the present invention preferably has a maximum absorption wavelength in the range of 500 to 1500 nm, more preferably 600 to 850 nm, and even more preferably 660 to 740 nm. The moiety containing the phthalocyanine skeleton preferably has one or more ligands containing a hydrophilic functional group. Examples of the hydrophilic functional group include carboxylate (-CO2 - ) group, sulfonate (—SO3 - ) group, sulfonyl (—SO2 - ) group, sulfate (-SO4 -2 ) group, hydroxy (-OH) group, phosphate (-OPO3 -2 ) group, phosphonate (-PO-2 ) group, amino (—NH2) group, substituted or unsubstituted quaternary nitrogen (each with an optional counterion), etc. Examples of counterions include, but are not limited to, sodium, potassium, calcium, ammonium, organic amino acids, magnesium, etc.

[0042] Preferably, the moiety containing a phthalocyanine skeleton used in the present invention contains a linker having a reactive group capable of forming a bond with the target recognition molecule or nanoparticle. That is, it has a structure of linker-phthalocyanine skeleton moiety (L-D). Preferably, the moiety containing a phthalocyanine skeleton is bonded to the target recognition molecule or nanoparticle via the linker substituted around the ring of the phthalocyanine skeleton.

[0043] In a preferred embodiment, the moiety containing a phthalocyanine skeleton used in the present invention is a compound represented by the following formula (Ia):

[0044]

[0045] wherein L is a direct bond or a linker; Q is a reactive group for forming a bond with the target recognition molecule or nanoparticle; R 2 , R 3 , R 7 , and R 8 are each independently selected from substituted or unsubstituted alkyl and substituted or unsubstituted aryl; R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 are, when present, each independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkanoyl, substituted or unsubstituted alkoxycarbonyl, substituted or unsubstituted alkylcarbamoyl, and chelating ligands, wherein R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 at least one of R contains a water-soluble group; 12 , R13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , and R 23 are each independently selected from hydrogen, halogen, substituted or unsubstituted alkylthio, substituted or unsubstituted alkylamino, and substituted or unsubstituted alkoxy; or i) R 13 and R 14 and the carbon to which they are attached, ii) R 17 and R 18 and the carbon to which they are attached, and iii) R 21 and R 22 and the carbon to which they are attached, at least one of which forms a fused ring; and X 2 and X 3 are each independently a C to C bond with or without a heteroatom between the carbon-carbon bonds. 10 In this specification, C1 to C 10 Alkylene means a methylene group and an alkylene group having 2 to 10 carbon atoms.

[0046] In one embodiment, L is a linker. In one embodiment, the linker is a straight or branched chain, cyclic or heterocyclic, saturated or unsaturated chain having 1 to 60 atoms, e.g., 1 to 45 atoms or 1 to 25 atoms. In some cases, the atoms of the linker can be selected from C, N, P, O, and S. In one embodiment, L can have additional hydrogen atoms (in addition to the 1 to 60 atoms) to satisfy the valence. In general, the linker can include an ether, a thioether, an amine, an ester, a carbamate, a urea, a thiourea, a carbonyl, an amide, a single bond, a double bond, a triple bond, an aromatic carbon-carbon bond, a phosphorus-oxygen bond, a phosphorus-sulfur bond, a nitrogen-nitrogen bond, a nitrogen-oxygen bond, a nitrogen-platinum bond, an aromatic bond, or a heteroaromatic bond, or any combination thereof.

[0047] In one embodiment, L is a group of formula -R1 -Y-X 1 -Y 1 -, where R 1 is a divalent group or a direct bond; Y and Y 1 are each independently selected from a direct bond, oxygen, substituted or unsubstituted nitrogen, and sulfur; and X 1 is a direct bond and a C-C bond with or without a heteroatom intervening between the carbon-carbon bonds. 10 Examples of such divalent groups include, but are not limited to, substituted or unsubstituted alkylene, substituted or unsubstituted alkyleneoxycarbonyl, substituted or unsubstituted alkylenecarbamoyl, substituted or unsubstituted alkylenesulfonyl, and substituted or unsubstituted arylene.

[0048] R 1 Specific examples include, but are not limited to, substituted or unsubstituted alkylene, substituted or unsubstituted alkyleneoxycarbonyl, oxycarbonylamino, substituted or unsubstituted alkylenecarbamoyl, substituted or unsubstituted alkylenesulfonyl, substituted or unsubstituted alkylenesulfonylcarbamoyl, substituted or unsubstituted arylene, substituted or unsubstituted arylenesulfonyl, substituted or unsubstituted aryleneoxycarbonyl, substituted or unsubstituted arylenecarbamoyl, substituted or unsubstituted arylenesulfonylcarbamoyl, substituted or unsubstituted carboxyalkyl, substituted or unsubstituted carbamoyl, carbonyl, substituted or unsubstituted heteroarylene, substituted or unsubstituted heteroaryleneoxycarbonyl, substituted or unsubstituted heteroarylenecarbamoyl, substituted or unsubstituted heteroarylenesulfonylcarbamoyl, substituted or unsubstituted sulfonylcarbamoyl, thiocarbonyl, sulfonyl, and sulfinyl.

[0049] Preferably, the alkylene contained in the substituted or unsubstituted alkylene, the substituted or unsubstituted alkyleneoxycarbonyl, the substituted or unsubstituted alkylenecarbamoyl, the substituted or unsubstituted alkylenesulfonyl, and the substituted or unsubstituted alkylenesulfonylcarbamoyl is a C-C alkylene having or without a heteroatom between the carbon-carbon bonds.10 It is alkylene.

[0050] In one embodiment, Q comprises a reactive group for forming a bond with the target recognition molecule or nanoparticle. As used herein, a "reactive group" refers to a moiety on a compound that can chemically react with a functional group on a different material (e.g., a target recognition molecule) to form a bond. Typically, the reactive group is an electrophile or nucleophile that can form a covalent bond through exposure to a corresponding functional group that is a nucleophile or electrophile, respectively.

[0051] In one embodiment, Q comprises a reactive group that is reactive with a carboxyl group, amino group, or thiol group on the target recognition molecule or nanoparticle to which it is attached. Examples of suitable reactive groups include, but are not limited to, activated esters, acyl halides, alkyl halides, anhydrides, carboxylic acids, carbodiimides, carbonates, carbamates, haloacetamides (e.g., iodoacetamide), isocyanates, isothiocyanates, maleimides, NHS (N-hydroxysuccinimide) esters, phosphoramidites, platinum complexes, sulfonate esters, and thiocyanates. In one embodiment, the reactive group is a sulfhydryl-reactive chemical group, such as maleimide, haloacetyl, or pyridyl disulfide. In one embodiment, the reactive group is amine-reactive. In a preferred embodiment, the reactive group is an NHS ester.

[0052] In one embodiment, R 2 , R 3 , R 7 , and R 8 is each independently substituted or unsubstituted alkyl, for example, substituted or unsubstituted methyl, ethyl, or isopropyl.

[0053] In one embodiment, R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 At least one of R 4 , R 5 , R 6, R 9 , R 10 , and R 11 In one embodiment, at least two of R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 At least one of R is alkyl substituted with a water-soluble group. 4 , R 5 , R 6 , R 9 , R 10 , and R 11 are each independently substituted or unsubstituted alkyl, at least one of which, preferably two or more of which is alkyl substituted with a water-soluble group. 4 , R 5 , R 6 , R 9 , R 10 , and R 11 are each independently substituted or unsubstituted alkyl, and R 4 , R 5 , and R 6 at least one of R is alkyl substituted with a water-soluble group, and R 9 , R 10 , and R 11 At least one of the groups is an alkyl substituted with a water-soluble group.

[0054] As used herein, the term "water-soluble group" refers to a group containing one or more polar and / or ionic substituents that improve the overall solubility of the molecule in aqueous media. Examples of water-soluble groups include, but are not limited to, carboxylate (-CO2 - ) group, sulfonate (—SO3 - ) group, sulfonyl (—SO2 - ) group, sulfate (-SO4 -2 ) group, hydroxy (-OH) group, phosphate (-OPO3 -2 ) group, phosphonate (-PO -2) groups, amino (—NH2) groups, and substituted or unsubstituted quaternary nitrogens, each with an optional counterion. Examples of suitable counterions include, but are not limited to, sodium, potassium, calcium, ammonium, organic amino salts, magnesium salts, and the like. Preferably, the counterion is a biologically acceptable counterion.

[0055] R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 The nitrogen atom to which is attached can be trivalent or tetravalent.

[0056] In one embodiment, R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , and R 23 are each hydrogen.

[0057] In one embodiment, X 2 and X 3 are each independently a C to C bond with or without a heteroatom between the carbon-carbon bonds. 10 In one embodiment, X is alkylene. 2 and / or X 3 The nitrogen attached to may be quaternized.

[0058] In a preferred embodiment, the moiety containing a phthalocyanine skeleton used in the present invention is a compound represented by formula (Ib):

[0059]

[0060] In the formula, 1 and X 4 are each independently a C to C bond with or without a heteroatom between the carbon-carbon bonds. 10 alkylene; and R 2 , R3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 16 , R 17 , R 18 , R 19 , X 2 , and X 3 is as defined above for formula (Ia).

[0061] In the compound of formula (Ib), the reactive group for forming a bond with the target recognition molecule or nanoparticle is an NHS ester. In one embodiment, the reactivity of the NHS ester is determined by the X between the NHS ester and the carbamate functional group. 4 In one embodiment, the length of the alkylene group in X between the NHS ester and the carbamate functional group can be adjusted. 4 The length of the alkylene group in X is inversely proportional to the reactivity of the NHS ester. 4 is C5-alkylene. 4 is C-alkylene. 1 is C-alkylene. 1 is C3-alkylene.

[0062] In one embodiment, the compounds of Formula (Ia) or Formula (Ib) have a total charge of zero. This neutral charge can be achieved with one or more optional counterions or quaternized nitrogens in certain cases.

[0063] In one embodiment, the compound of formula (Ia) or formula (Ib) has sufficient solubility in aqueous solution so that after binding to the target recognition molecule or nanoparticle, the molecule or particle retains its solubility.

[0064] In a preferred embodiment, the moiety containing a phthalocyanine skeleton used in the present invention is IR700 NHS ester, for example, IRDye 700DX NHS ester (LI-COR Biosciences, P / N 929-70010 or 929-70011). In a preferred embodiment, the moiety containing a phthalocyanine skeleton is a compound represented by the following formula (II):

[0065]

[0066] The photosensitive moiety is preferably contained in the conjugate of the present invention in a configuration in which it is bound to the target recognition molecule or nanoparticle via its reactive group. For example, the compounds represented by formula (Ia), (Ib), or (II), "IR700," "IRDye 700DX," or variations thereof, which may be contained in the conjugate of the present invention, refer to these compounds in a configuration in which they are bound to the target recognition molecule or nanoparticle via their reactive group. In general, IR700 has several favorable chemical properties. Amino-reactive IR700, for example, IR700 NHS ester or the compound of formula (II), is relatively hydrophilic and can be covalently bound to the target recognition molecule using the NHS ester. Typically, IR700 is a hematoporphyrin derivative, Photofrin® (1.2 x 10 at 630 nm). 3 M -1 cm -1 ), meta-tetrahydroxyphenyl chlorin; Foscan® (2.2 × 10 at 652 nm) 4 M -1 cm -1 ), and mono-L-aspartyl chlorin e6; NPe6 / Laserphyrin® (4.0 × 10 at 654 nm) 4 M -1 cm -1 ) with an extinction coefficient (2.1 × 10 at absorption maximum at 689 nm) that is more than five times higher than conventional photosensitizers such as 5 M -1 cm -1 )

[0067] The phthalocyanine skeleton-containing moieties used in the present invention, such as compounds represented by formula (Ia), (Ib), or (II), can be prepared using commercially available starting materials. For example, the skeleton can be synthesized by condensation of two or more different diiminoisoindolines. Synthetic strategies using different dinitriles or diiminoisoindolines can lead to phthalocyanines with various degrees of substitution and / or positional isomers. An exemplary synthetic scheme for producing the phthalocyanine skeleton is described in U.S. Pat. No. 7,005,518.

[0068] In the conjugate of the present invention, the photosensitive moiety may be bound to the target recognition molecule or nanoparticle, but is preferably bound to the target recognition molecule. Preferably, the target recognition molecule is an antibody. Furthermore, the conjugate of the present invention may contain one or more of the photosensitive moieties described above, and these photosensitive moieties may have the same or different structures.

[0069] 1.4. Efficacy The complex of the present invention is used for CDT of tumors. Specifically, the complex of the present invention exerts a tumor therapeutic effect by CDT when irradiated with light. The therapeutic effect of CDT of the complex of the present invention is thought to be due to the Fenton reaction that occurs when the complex is irradiated with light, using the iron oxide nanoparticles of the complex. Therefore, CDT by the complex of the present invention is due to the photo-Fenton reaction.

[0070] Furthermore, the complex of the present invention generates heat in response to light irradiation, for example, NIR irradiation (e.g., light with a wavelength of 500 to 1500 nm), and can exert a therapeutic effect by PTT. Furthermore, the complex of the present invention contains nanoparticles, which are magnetic particles containing iron oxide, and therefore enables in vivo imaging.

[0071] Furthermore, when the conjugate of the present invention has a photosensitive site, it can exhibit therapeutic effects through photoimmunotherapy, specifically NIR-PIT. The therapeutic effect of the conjugate of the present invention through NIR-PIT is thought to be due to the fact that the photosensitive site of the conjugate bound to tumor cells becomes hydrophobic upon NIR irradiation, causing aggregation of the conjugate, which damages the cell membrane of the tumor cells and induces cell death.

[0072] Therefore, the conjugate of the present invention enables combined treatment with CDT and PTT. Furthermore, when the conjugate of the present invention has a photosensitive moiety, combined treatment with CDT and NIR-PIT, or further combined treatment with CDT, NIR-PIT, and PTT, becomes possible. Furthermore, the conjugate of the present invention can be used not only for the treatment of tumors in patients but also for diagnosis (e.g., confirmation of the location of the conjugate by in vivo imaging using the conjugate, or imaging of tumors to which the conjugate has bound).

[0073] 2. Method for Producing the Conjugate The conjugate of the present invention can be produced by binding the nanoparticles and the target recognition molecules. The binding of the target recognition molecules and nanoparticles can be carried out by known means. When the conjugate of the present invention has a photosensitive site, it is preferable to first synthesize a target recognition molecule and / or nanoparticles having the photosensitive site. The binding of the photosensitive site and the target recognition molecule or nanoparticle can be carried out by known means.

[0074] For example, specific techniques for conjugating the photosensitive moiety and the antibody include the methods disclosed in Patent Document 5 or the below-described Experiment 1. A more specific example involves incubating an aqueous phosphate solution containing an antibody and a phthalocyanine compound represented by formula (Ia) having an NHS ester at the reactive group Q (for example, the above-mentioned IR700 NHS ester) at room temperature, and purifying the target antibody-photosensitive moiety conjugate from the reaction solution by column purification or the like.

[0075] When the photosensitive site is to be bonded to the nanoparticle, for example, the compound represented by formula (Ia) having an NHS ester as the reactive group Q can be reacted with nanoparticles having an amino group or a protein molecule such as avidin, streptavidin, or protein A introduced onto the particle surface in the same manner as described above.

[0076] The number of photosensitive moieties bound to the target recognition molecule per target recognition molecule (e.g., antibody) is preferably 1 or more, more preferably 2 or more, and preferably 5 or less, more preferably 4 or less. The number of photosensitive moieties bound to the nanoparticle per nanoparticle is preferably 1 or more, and preferably 80 or less, more preferably 50 or less, and even more preferably 20 or less.

[0077] The conjugate of the present invention can be produced by binding the target recognition molecule to the nanoparticle. For example, a target recognition molecule biotinylated by a conventional method is mixed with nanoparticles on whose surface a biotin-binding substance has been introduced, thereby binding the target recognition molecule to the nanoparticle. Examples of the biotin-binding substance include proteins such as avidin and streptavidin. Preferably, 1 to 5, more preferably 1 to 4, and particularly preferably 1 to 3 biotin-binding substances are introduced per nanoparticle. Alternatively, a carbodiimide compound such as EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and an N-hydroxysuccinimide derivative such as sulfo-NHS can be used to crosslink the antibody and nanoparticle. In this case, the amounts of target recognition molecule and nanoparticles charged are set so that the desired number of target recognition molecules bound per nanoparticle is achieved (preferably 1 or more and preferably 20 or less, more preferably 16 or less, even more preferably 12 or less, and even more preferably 5 or less). The amount of nanoparticles may be set so that the number of target recognition molecules bound per mg of nanoparticles is a desired number. Specifically, the lower limit of the amount of target recognition molecules per mg of nanoparticles is preferably 1.0 × 10 -12 mol / mg, more preferably 5.0×10 -12 mol / mg, more preferably 1.0×10 -11 On the other hand, the upper limit of the amount of target recognition molecules charged per 1 mg of nanoparticles is preferably 1.0 × 10 -8 mol / mg, more preferably 5.0×10 -9 mol / mg, more preferably 1.0×10 -9The bond between the target recognition molecule and the nanoparticle is not limited to a covalent bond, but may also include bonds based on hydrogen bonds, ionic bonds, hydrophobic interactions, or combinations thereof.

[0078] 3. Compositions Containing the Conjugate In one embodiment, the present invention provides a composition containing the conjugate of the present invention described above. The composition containing the conjugate of the present invention (hereinafter also referred to as the composition of the present invention) is used as a pharmaceutical composition for CDT. In a preferred embodiment, the composition of the present invention is used as a pharmaceutical composition for a combined treatment of CDT and PTT. Furthermore, when the conjugate of the present invention has a photosensitive site, the composition of the present invention is used as a pharmaceutical composition for a combined treatment of CDT and NIR-PIT, or for a combined treatment of CDT, NIR-PIT, and PTT. In another embodiment, the composition of the present invention is used as a pharmaceutical composition for performing in vivo imaging (e.g., tumor imaging) in addition to tumor treatment with CDT, or further with NIR-PIT and / or PTT.

[0079] In one embodiment, the composition of the present invention contains the conjugate of the present invention and a pharmaceutically acceptable carrier or excipient. Examples of pharmaceutically acceptable carriers include, but are not limited to, water, oil, buffers, phosphate-buffered saline, and other diluents for injections. Examples of excipients include, but are not limited to, starch, glucose, lactose, dextrose, carboxymethylcellulose, glycerol, propylene glycol, water, and ethanol. If necessary, the composition of the present invention may contain lubricants, binders, wetting agents, emulsifiers, pH adjusters, isotonicity agents, buffers, antioxidants, suspending agents, solubility enhancers, preservatives, chelating agents, and other pharmaceutically acceptable substances. Pharmaceutically acceptable carriers, excipients, and the like are known in the art (see, e.g., Remington's Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition, 1995).

[0080] In one embodiment, the composition of the present invention may contain hydrogen peroxide. Hydrogen peroxide may be contained in the composition of the present invention as a substrate for the Fenton reaction in order to supplement the hydrogen peroxide generated in cells. Hydrogen peroxide is a compound containing Fe 3+ Fe 2+ can be reduced to generate oxygen. 2+ It is expected that the oxygen generated from the oxidized carbon dioxide will be useful in hyperoxia therapy of cancer.

[0081] In one embodiment, the composition of the present invention may contain a bioreductive substance. Examples of the bioreductive substance include reduced glutathione, catalase, peroxidase, cytochrome peroxidase, and glutathione peroxidase. The bioreductive substance may be Fe 3+ Fe 2+ The bioreductive substance reduces reactive oxygen species (ROS) or decomposes hydrogen peroxide, and is therefore used when the above-mentioned purposes are required.

[0082] The composition of the present invention may be in a liquid form, such as a dispersion; or in a solid form, such as a powder, pill, tablet, capsule, transdermal patch, inhalant, or suppository. Alternatively, the composition of the present invention may be reconstituted with a pharmaceutically acceptable carrier (e.g., a diluent for injection) prior to use and administered. These liquid or solid compositions can be prepared according to conventional methods. In one embodiment, the composition of the present invention is a single-dose formulation containing a carrier or excipient containing the conjugate of the present invention. In another embodiment, the composition of the present invention is a two-dose formulation containing a separate conjugate of the present invention and a diluent or the like to be administered together. Accordingly, in one embodiment, the composition of the present invention encompasses a reagent kit containing the conjugate of the present invention. In one embodiment, the reagent kit contains hydrogen peroxide and / or a bioreductant together with the conjugate of the present invention.

[0083] The form of the composition of the present invention may depend on its administration regimen. The administration regimen of the composition can be appropriately designed depending on the type and condition of the tumor to be treated, and the species, age, condition, etc. of the patient. The composition may be an oral or parenteral formulation, for example, an injection, an oral agent, or an external agent. The composition may be configured for single or multiple administration. The content of the complex of the present invention in the composition can be appropriately designed depending on the form of the composition, the dose to be administered to the patient, etc.

[0084] The composition of the present invention is adjusted to a pH range compatible with the animal body, for example, pH 5 or higher, preferably pH 5.5 or higher, and pH 10 or lower, preferably pH 8 or lower, more preferably pH 7.3 or lower, or preferably pH 5.5 to 10, more preferably pH 5.5 to 8, even more preferably pH 5.5 to 7.3. The pH of the composition can be adjusted using the aforementioned pH adjuster, buffer solution, etc.

[0085] 4. Treatment of Tumors Using the Conjugate of the Present Invention As described above, the conjugate of the present invention or a composition of the present invention containing the conjugate is used for treating tumors with CDT. In one embodiment, the present invention provides a method for treating tumors with CDT using the conjugate or composition of the present invention. The method for treating tumors according to the present invention (hereinafter also referred to as the treatment method of the present invention) comprises the steps of administering the conjugate or composition of the present invention to a patient and irradiating the patient with near-infrared (NIR) radiation. In this specification, the term "method for treating tumors" can be interpreted as "method for killing tumors."

[0086] As described above, the conjugates and compositions of the present invention enable combined treatment with CDT and PTT, and when the conjugates of the present invention have a photosensitive moiety, they also enable combined treatment with NIR-PIT. Thus, in one embodiment, the method for treating tumors using the conjugates and compositions of the present invention is a method for treating tumors using CDT and PTT in combination. In another embodiment, the method for treating tumors using the conjugates and compositions of the present invention is a method for treating tumors using CDT and NIR-PIT in combination, or CDT, NIR-PIT, and PTT in combination.

[0087] The patient to whom the complex or composition of the present invention is administered is a patient in need of CDT for the treatment of a tumor. In one embodiment, the patient is a patient in need of combined treatment with CDT and NIR-PIT for the treatment of a tumor. Such patients include humans and non-human animals having tumors to be treated with CDT. Non-human animals include, but are not limited to, non-human mammals such as mice, rats, hamsters, rabbits, pigs, goats, dogs, cats, sheep, cows, and horses. In addition, the patient may or may not have received other treatments for the tumor (surgery, radiation therapy, phototherapy, chemotherapy using drugs other than the complex or composition of the present invention, etc.).

[0088] The target recognition molecule contained in the conjugate of the present invention is selected depending on the type of tumor to be treated. Appropriate selection of the target recognition molecule enables the accumulation of the conjugate of the present invention in the tumor to be treated. Preferably, the target recognition molecule is an antibody, which can specifically bind to an antigen present on the surface of the tumor cell of the target, preferably a tumor-specific protein expressed on the surface of the tumor cell. The tumor cell surface antigen, for example, a tumor-specific protein, to be targeted by the target recognition molecule can be determined according to known information. Those skilled in the art can select an antibody specific to a target antigen on the tumor to be treated.

[0089] 4.1. Administration of Conjugates The administration regimen (e.g., administration route, dose, frequency, etc.) of the conjugate or composition of the present invention can be appropriately determined depending on the type and condition of the tumor to be treated, as well as the species, age, and condition of the patient. Examples of administration routes include local administration to the tumor site via injection, catheter, spray, application, patch, suppository, etc., and systemic administration via infusion, oral administration, intraperitoneal injection, intravenous injection, etc. Preferably, the conjugate or composition of the present invention is administered locally. In one embodiment, the conjugate or composition of the present invention is administered intravenously. In one embodiment, the conjugate or composition of the present invention is administered directly to the tumor site using a syringe or the like, or injected via a catheter. The conjugate or composition of the present invention may be used alone for tumor treatment, or may be used in combination with other drugs or therapies, such as surgery, radiation therapy, phototherapy, or chemotherapy with drugs other than the conjugate or composition of the present invention.

[0090] The conjugate or composition of the present invention may be administered to a patient in an effective amount. The term "effective amount" refers to an amount that allows the conjugate of the present invention to accumulate in a therapeutic target tumor in a patient in an amount sufficient to exert a therapeutic effect on the tumor. Preferably, the term "effective amount" refers to an amount that exerts a therapeutic effect on the therapeutic target tumor in the patient while minimizing or keeping side effects to a tolerable level for the patient.

[0091] The dose of the complex or composition of the present invention administered to a patient can be determined appropriately depending on the type and condition (location, volume, etc.) of the target tumor, the species, age, condition, and administration route of the patient, the form of the composition containing the complex, etc. For example, the dose of the complex of the present invention can be set according to the tumor volume. The tumor volume (V) can be determined, for example, by measuring the short diameter (W) and long diameter (L) of the tumor and calculating the tumor volume (V) using the formula: V = (W 2× L) / 2. Alternatively, the dose can be adjusted depending on the level of accumulation of the complex of the present invention in tumors measured by imaging, as described below. The dose of the complex of the present invention to be administered to humans can be determined based on the dose to be administered to mice. For example, the effective dose when the complex of the present invention is administered to humans can be determined as 5 to 10 times the effective dose in mice.

[0092] In one embodiment, the single dose of the conjugate or composition of the present invention may be in the range of 0.01 mg to 9000 mg as the amount of the conjugate of the present invention, for example, in the case of local injection. In one embodiment, the single dose of the composition of the present invention may be in the range of 0.5 mL to 1000 mL, for example, in the case of an injectable formulation. In one embodiment, the composition of the present invention is an injectable formulation, and the single dose is 1 to 5 mL, and the single dose contains 0.1 mg to 5000 mg of the conjugate of the present invention.

[0093] In one embodiment, when the complex or composition of the present invention is injected into the affected area of ​​a tumor in an adult (60 kg), the single dose (injection amount) is 0.01 mg to 20 mg / kg (body weight) of the complex of the present invention. In another embodiment, when the composition of the present invention is injected into the affected area of ​​a tumor in an adult (60 kg), the single dose (injection amount) is usually 1 to 5 mL.

[0094] The dosage and frequency of administration of the complex or composition of the present invention can be increased or decreased depending on the therapeutic effect of the tumor. The therapeutic effect of the tumor can be evaluated by common methods for evaluating tumor treatment, such as the rate of tumor tissue shrinkage. In one embodiment, the complex or composition of the present invention is administered once at the aforementioned dosage. In another embodiment, the complex or composition of the present invention is administered multiple times. In the case of multiple administrations, the aforementioned dosage may be repeated, or the dosage may be increased or decreased depending on the therapeutic effect of the tumor. In one embodiment, the second or subsequent administration can be performed after the patient has cleared the previous dose. In another embodiment, the complex or composition of the present invention can be administered repeatedly once a week, once every two weeks, once a month, or less frequently. In another embodiment, the complex or composition of the present invention can be administered again one week, two weeks, three weeks, four weeks, two months, six months, one year, or more after the previous administration if the tumor targeted for treatment remains.

[0095] In a preferred embodiment, in the treatment method of the present invention, hydrogen peroxide is further administered to the patient in addition to the complex of the present invention. For example, hydrogen peroxide solution is administered to the patient by intravenous drip infusion as hydrogen peroxide. Alternatively, the above-mentioned composition of the present invention containing hydrogen peroxide may be administered to the patient. In another preferred embodiment, in the treatment method of the present invention, a bioreductive substance is further administered to the patient in addition to the complex of the present invention. Examples of bioreductive substances are as described above. Alternatively, the above-mentioned composition of the present invention containing a bioreductive substance may be administered to the patient. The hydrogen peroxide and the bioreductive substance may be used in combination.

[0096] The administration route and frequency of administration of hydrogen peroxide and a bioreductive substance can be determined appropriately, as with the complex or composition of the present invention. For example, hydrogen peroxide or a bioreductive substance can be administered to a patient together with the complex or composition of the present invention. The doses of hydrogen peroxide and a bioreductive substance can be determined appropriately for each patient. Preferably, the doses of hydrogen peroxide and a bioreductive substance are set to an amount that enhances the tumor therapeutic effect of the complex or composition of the present invention and minimizes or limits to an acceptable range the side effects of administering the hydrogen peroxide and a bioreductive substance to a patient.

[0097] 4.2. Light Irradiation Following administration of the conjugate or composition of the present invention to a patient, the patient is irradiated with light. Preferably, the patient is irradiated with NIR. Preferably, tumor cells or tumor-affected areas to which the conjugate of the present invention is bound are locally irradiated with NIR. The conjugate of the present invention irradiated with light exerts a tumor therapeutic effect by CDT, or further exerts a tumor therapeutic effect by PTT. If the conjugate of the present invention has a photosensitive site, the NIR-irradiated conjugate further exerts a tumor therapeutic effect by NIR-PIT. That is, the NIR-irradiated conjugate causes a photochemical reaction that kills the tumor cells to which it is bound.

[0098] The wavelength of the irradiated light is preferably 500 to 1500 nm, more preferably 600 to 850 nm, and even more preferably 660 to 740 nm. The timing of irradiation can be determined at any time after administration of the complex of the present invention. For example, it can be determined at any time between 30 minutes and 96 hours after administration, preferably between 30 minutes and 48 hours, 30 minutes and 24 hours, 1 hour and 48 hours, or 1 hour and 24 hours after administration. The irradiation time can be appropriately determined within a range of 5 seconds to 72 hours. Irradiation can be performed once or multiple times so that the cumulative irradiation time per administration of the complex of the present invention is within the above-mentioned range. The duration of each irradiation can be determined appropriately, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 55 seconds, or 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 minutes, or 10, 20, 30, 40, 50, or 60 minutes, or 1 or 2 hours.

[0099] In one embodiment, the dose delivered to the patient is preferably 1 J / cm 2 More preferably, 5 J / cm 2 More preferably, 10 J / cm 2 or more, and preferably 1000 J / cm 2 or less, more preferably 500 J / cm 2 More preferably, 100 J / cm or less 2 More preferably, 50 J / cm or less 2 For example, 1 to 1000 J / cm 2 , 1~500J / cm 2 , 5-200J / cm 2 , 10-100J / cm 2 , or 10 to 50 J / cm 2 The range is.

[0100] Irradiation can be performed once or multiple times for one administration of the complex of the present invention. Therefore, irradiation may be completed in one session or repeated over several days. When irradiation is performed multiple times, the conditions for each irradiation may be the same or different. The dose, conditions, or method of irradiation can be changed depending on the type and condition of the tumor.

[0101] 4.3. Combination Therapy The aforementioned light irradiation allows the conjugate of the present invention to exert the tumor therapeutic effect of PTT. That is, light irradiation of the patient causes the conjugate of the present invention to generate heat, killing tumor cells through the heat. Therefore, in a preferred embodiment, the treatment method of the present invention is a method of treating tumors using the conjugate or composition of the present invention in combination with CDT and PTT. Furthermore, when the conjugate of the present invention has a photosensitive moiety, the aforementioned light irradiation allows the conjugate of the present invention to exert the tumor therapeutic effect of NIR-PIT. That is, NIR irradiation of the patient hydrophobizes the photosensitive moiety of the conjugate bound to tumor cells, causing aggregation of the conjugate, which results in damage to the cell membrane of the tumor cells and killing them. Therefore, in a preferred embodiment, the treatment method of the present invention is a method of treating tumors using the conjugate or composition of the present invention in combination with CDT and NIR-PIT, or in combination with CDT, NIR-PIT, and PTT.

[0102] The conjugate of the present invention administered to a patient specifically binds to tumor cells as therapeutic targets via the target recognition molecule. The conjugate of the present invention that has specifically bound to tumor cells kills the tumor cells to which it has bound by the aforementioned CDT or NIR-PIT and / or PTT mechanisms. Therefore, the present invention achieves selective killing of tumor cells as therapeutic targets.

[0103] 4.4. Imaging Because the nanoparticles contained in the complex of the present invention are magnetic particles containing iron oxide, bioimaging by MRI is possible. In one embodiment, the complex or composition of the present invention is used for imaging of a patient or a tumor. For example, the complex or composition of the present invention administered to a patient is used for imaging of the tumor before tumor CDT treatment. Tumor imaging enables confirmation of the location of the complex within the patient's body or the location of the tumor to which the complex is bound, thereby enabling a diagnosis of the patient. This allows optimization of the timing of light irradiation for CDT, or further NIR-PIT and / or PTT, the three-dimensional irradiation position in the body, and the irradiation amount (irradiation time, dose), thereby enhancing the tumor treatment effect of the complex of the present invention. MRI imaging of a patient or tumor can be performed using conventional methods.

[0104] 4.5. Other Methods The CDT and imaging techniques using the complex or composition of the present invention described above can be applied not only in vivo but also in vitro. For example, by administering the complex or composition of the present invention to tumors present in a patient's body, as well as to cultured tumor cells or cultured tissues containing tumor cells, and irradiating them with light, it is possible to reduce or inhibit the proliferation of tumor cells. The administration method of the complex or composition of the present invention and the light irradiation conditions can be appropriately modified depending on the state of the target cells or tissues. For example, the complex or composition of the present invention can be directly administered to tumor cells in culture. The dose of the complex or composition or the light irradiation conditions can be selected to be milder than those for administration or irradiation to a patient.

[0105] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to these examples in any way.

[0106] Experiment 1: Preparation of antibody-nanoparticle conjugate Nanomag-D-Spio-Pan-IR700 1) Preparation of Pan-IR700 Two mg of the human monoclonal antibody panitumumab (hereinafter referred to as "Pan") was incubated with 133.6 μg of IRDye 700DX NHS Ester (hereinafter referred to as "IR700"; LI-COR Biosciences) in 0.2 mol / L NaHPO (pH 8.5) at room temperature for 30 to 120 minutes. The mixture was purified on a Sephadex G50 column (PD-10; GE Healthcare, Piscataway, NJ). Protein concentration was determined using a Coomassie Plus Protein Assay Kit (Pierce Biotechnology, Rockford, IL) by measuring absorbance at 595 nm with an ultraviolet-visible system (8453 Value system; Agilent Technologies, Palo Alto, CA). IR700 concentration was measured by absorbance with an ultraviolet-visible system (Shimadzu UV-VIS). There were approximately three molecules of IR700 per molecule of Pan. Hereinafter, Pan bound to IR700 will be referred to as Pan-IR700.

[0107] 2) Biotinylation of Pan-IR700 5.69 mg of (+)-biotin N-hydroxysuccinimide ester (Sigma-Aldrich, hereinafter also referred to as Biotin-NHS) was dissolved in 1 mL of DMSO (Sigma-Aldrich). 1 mL of the Pan-IR700 solution (2.0 mg / mL) was placed in a microtube, and 8 μL of the previously prepared Biotin-NHS DMSO solution ([Biotin-NHS] / [Pan-IR700]=10) was added, followed by standing at room temperature for 3 hours. Unreacted biotin was removed using an ultrafiltration filter (Amicon Ultra 100k), and the solution was adjusted to 1.9 mg / mL with Dulbecco's Phosphate Buffered Saline (manufactured by Wako Pure Chemical Industries, Ltd., hereinafter also referred to as D-PBS) to obtain biotinylated Pan-IR700. Hereinafter, biotinylated Pan-IR700 will be referred to as Pan-IR700-Biotin.

[0108] 3) Preparation of a Complex of Pan-IR700 and Magnetic Nanoparticles: 120 mg (5 mg / mL, 24 mL) of Nanomag-D-Spio 79-19-201 (Micromod, streptavidin surface-modified magnetic particles, particle size 20 nm, hereinafter also referred to as "Nanomag-D-Spio") was placed in a 50 mL tube, and 1.8 mg (1.9 mg / mL, 947 μL) of Pan-IR700-Biotin was added and stirred at room temperature for 60 minutes. Furthermore, 18 μg (0.1 mg / mL, 180 μL) of biotin (Wako Pure Chemical Industries, Ltd.) was added, and the mixture was stirred at room temperature for 30 minutes. In this way, a dispersion containing an antibody-nanoparticle complex (also referred to as "Nanomag-D-Spio-pan-IR700") in which Pan-IR700-Biotin and Nanomag-D-Spio were bound was obtained. 1 mL of the dispersion was passed through a column (MS-columns, manufactured by Miltenyi Biotec) installed on a magnetic stand. 2 mL of D-PBS was then passed through the column. The filtrate was irradiated with excitation light at a wavelength of 676 nm and subjected to fluorescence measurement at 700 nm, confirming that no unreacted Pan-IR700-Biotin was detected.

[0109] 4) Preparation of antibody-nanoparticle conjugate Nanomag-D-Spio-Cont An antibody-nanoparticle conjugate (Nanomag-D-Spio-Cont-IR700) was prepared by the same procedures as in 1) to 3) above, except that a ligand for a tyrosine kinase receptor not expressed in MDAMB468 or A431 was used in place of Pan.

[0110] 5) Preparation of antibody-nanoparticle conjugate Nanomag-D-Spio-Pan An antibody-nanoparticle conjugate (Nanomag-D-Spio-Pan) was prepared by the same procedure as in 3), except that biotinylated Pan (Pan-Biotin) obtained by the same method as in 2) above was used instead of Pan-IR700-Biotin.

[0111] Experiment 2: Evaluation of Nanomag-D-Spio-pan-IR700 The antibody-nanoparticle conjugate Nanomag-D-Spio-pan-IR700 produced in Experiment 1 and the nanoparticle material Nanomag-D-Spio were subjected to zeta potential measurement, atomic force microscope (AFM) observation, and vibrating sample magnetometer (VSM) measurement. The zeta potential was measured by diluting the dispersion of Nanomag-D-Spio-pan-IR700 and Nanomag-D-Spio (0.1 mg / ml KCl, pH 7, 25°C) and measuring the zeta potential with a Zetasizer. TM The zeta potential was measured using a microscope (Malvern Panalytical Ltd). The zeta potential measurement results are shown in Figure 1. The AFM image, as well as the size measurement results and estimated shape based on the AFM image, are shown in Figure 2. The VSM measurement results are shown in Figure 3. The AFM image shows that Nanomag-D-Spio was spherical, while Nanomag-D-Spio-pan-IR700 had an uneven shape.

[0112] Experiment 3: Cell Binding of Nanomag-D-Spio-pan-IR700 The cell binding of the antibody-nanoparticle conjugate produced in Experiment 1 was evaluated. Disease model cells MDAMB468 and A431, as well as normal cells H661 and 3T3, were used. MDAMB468 are human breast cancer cells expressing EGFR, A431 are human epidermoid carcinoma cells expressing EGFR, H661 are human lung epithelial-like cells not expressing EGFR, and 3T3 are mouse embryonic fibroblasts not expressing EGFR. RPMI1640 (basal medium) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin was used as the cell culture medium. Cells were seeded (1 x 10) onto culture plates. 5 The wells were incubated at 37°C and 5% CO2 for 24 hours. Next, the complex (pan-IR700 or Nanomag-D-Spio-pan-IR700, 10 μg / mL) was added to the wells and incubated at 37°C and 5% CO2 for 6 hours. The complex-free cells served as a control. The fluorescence intensity of the cells after incubation was measured by flow cytometry using IR700 (wavelength 698 nm) as a fluorescent label.

[0113] The results of measuring the fluorescence intensity are shown in Figure 4(A). In Figure 4(A), "Pan blocking" indicates the fluorescence intensity of cells incubated in the presence of an EGFR inhibitor (panitumumab, a tyrosine kinase inhibitor). Here, cells were seeded onto a culture plate as described above and incubated for 24 hours (37°C, 5% CO2 by volume). The EGFR inhibitor was then added to the well, and the complex was added to the well and incubated for 6 hours (37°C, 5% CO2 by volume). As shown in Figure 4(A), it was suggested that the EGFR-positive cells MDAMB468 and A431 specifically bind to pan-IR700 or Nanomag-D-Spio-pan-IR700.

[0114] Instead of PAN, an antibody (IgG antibody)-nanoparticle conjugate (Nanomag-D-spio-cont-IR700) conjugated with a ligand for a tyrosine kinase receptor not expressed in MDAMB468 or A431 was used. MDAMB468 and A431 were cultured in the presence of the conjugate using the same procedure as above, and the fluorescence intensity of the cells was measured by flow cytometry. The results of the fluorescence intensity measurements are shown in Figure 4(B). As shown in Figure 4(B), the fluorescence intensity of Nanomag-D-spio-cont-IR700 was not different from that of the control. The results in Figure 4(B) suggest that Nanomag-D-Spio-cont-IR700 does not bind to MDAMB468 or A431.

[0115] Experiment 4: Stability of Nanomag-D-Spio-pan-IR700 in Blood 100 mL of Nanomag-D-Spio-pan-IR700 solution (5 μg / mL) diluted with PBS, 100 mL of PBS-diluted sodium laurate (SDS) solution (1 wt%), or 400 mL of mouse serum were placed in a microtube and incubated for 6 hours. The fluorescence intensity of the solution after incubation was measured using IR700 (wavelength 698 nm) as the fluorescent label. Nanomag-D-Spio-pan-IR700 solution without SDS or serum served as a control. The measurement results are shown in Figures 5(A) and (B). The intensity of fluorescence derived from Nanomag-D-Spio-pan-IR700 was about 2.2 times that of the control in SDS, but was no different from the control in serum.

[0116] Experiment 5: Endocytic uptake of Nanomag-D-Spio-pan-IR700. A431 cells were seeded on a culture plate (1 x 10 5 The cells were cultured in the same culture medium as in Experiment 3 (cells / well) and incubated for 24 hours (37°C, 5% CO2 by volume). Nanomag-D-Spio-pan-IR700 was added to the wells at 10 μg / mL, and after 6 hours of incubation (37°C, 5% CO2 by volume), the cells were fluorescently stained (Hoechst stain, lysosomal stain (green)) and observed for fluorescence. Fluorescence images are shown in Figure 6. In Figure 6, "DIC" indicates a bright-field image taken by an optical microscope, "Hoechst," "Lyso tracker," and "IR700" indicate fluorescent images labeled with Hoechst stain, lysosomal stain, and IR700, respectively, and "Merge" indicates a superimposed image of each fluorescent image.

[0117] Experiment 6: Reaction of nanoparticles with methylene blue Nanomag-D-Spio (0.5 mg / mL) was placed in a microtube and heated at 37°C for 30 minutes in the presence or absence of reduced glutathione (GSH, 10 mM). After heating, methylene blue (10 μg / mL) was added to the tube with or without hydrogen peroxide (1 mM). The pH of the solution in the microtube was adjusted to 5.4 with PBS containing HCl, and the absorbance (500-800 nm) was measured. The solution was then irradiated with laser light (690 nm, 200 J / cm). 2 The absorbance was measured (500 to 800 nm). The results of the absorbance measurement are shown in FIG.

[0118] Similarly, Nanomag-D-Spio (20 μg / mL) and reduced glutathione (10 mM) were placed in a microtube and heated at 37°C for 0 to 120 minutes. Then, methylene blue (10 μg / mL) and hydrogen peroxide (20 μM) were added. The pH of the solution in the microtube was adjusted to 5.4 with PBS containing HCl, and the absorbance (500 to 800 nm) was measured. The results are shown in Figure 7(B).

[0119] Experiment 7: Heat generation behavior of antibody-nanoparticle complexes by near-infrared radiation. A solution containing Nanomag-D-Spio-pan-IR700 (0.1-10 mg / mL) or pan-IR700 (10 mg / mL) was placed in a microtube, and laser light (690 nm, 500 mW / cm 2 ) was irradiated, and the liquid temperature in the microtube was measured. Figure 8(A) shows the temperature rise curve of the complex-containing liquid versus the irradiation dose for each liquid. Figure 8(B) also shows an infrared thermography image of the Nanomag-D-Spio-pan-IR700-containing liquid after laser irradiation, and the liquid temperature measured based on the thermography. The power conversion efficiency (PCE) of the laser irradiation was measured and found to be 29.21%. These results demonstrate that the nanoparticles contained in the antibody-nanoparticle complex generate heat in response to near-infrared irradiation.

[0120] Experiment 8: In vitro antitumor activity of antibody-nanoparticle conjugates. A431, MDAMB468, and PC-9 were used as disease model cells. A431 and MDAMB468 were disease model cells as described in Experiment 3, and PC-9 was a human lung adenocarcinoma cell line expressing EGFR. The cells were seeded (1 × 10) on a culture plate. 5 The cells / well and culture medium were the same as in Experiment 3), and the cells were incubated for 24 hours (37°C, 5% CO2 by volume). Nanomag-D-Spio-pan-IR700 was added to the wells at 10 μg / mL, and the cells were incubated for 24 hours (37°C, 5% CO2 by volume). The medium was replaced with PBS, and the cells were exposed to laser light (690 nm, 4 mJ / cm 2 ) was irradiated. The cells were fluorescently stained (Hoechst staining, pyridium iodide (PI) staining) before and after laser irradiation, and fluorescence observation was performed. Fluorescence images are shown in Figure 9. In Figure 9, "DIC" indicates a bright-field image taken by an optical microscope, "Hoechst" and "PI" indicate fluorescent images stained with Hoechst and PI, respectively, and "Merge" indicates a superimposed image of each fluorescent image.

[0121] Experiment 9 Antitumor activity of antibody-nanoparticle conjugates against in vitro spheroids Cells for laser light irradiation were prepared using the same procedure as in Experiment 8, except that spheroids prepared by mixing antigen-presenting cells (A431, MDAMB468, or PC-9) with non-antigen-presenting cells (3T3-RFP) were used instead of disease model cells. A431, MDAMB468, and PC-9 are the same disease model cells as those used in Experiment 8, and 3T3-RFP are cells prepared by transfecting the red fluorescent protein (RFP) gene as a reporter gene into the 3T3 cells used in Experiment 3. The spheroids were fluorescently stained (SYTOX) before and after laser light irradiation. TMThe spheroids were stained with 3T3-RFP (Blue staining) and observed for fluorescence. Fluorescence images are shown in Figures 10A and 10B. Figure 10A shows a fluorescence image of a spheroid in which A431 and 3T3-RFP were mixed at a 1 / 1 (volume ratio). Figure 10B shows fluorescence images of a spheroid in which MDAMB468 and 3T3-RFP were mixed at a 1 / 1 (volume ratio), and a spheroid in which PC-9 and 3T3-RFP were mixed at a 1 / 1 (volume ratio). In the figures, "DIC" indicates a bright-field image taken by an optical microscope, "RFP" indicates a fluorescence image in which the reporter RFP incorporated into 3T3-RFP is fluorescently labeled, "IR700" indicates a fluorescence image in which IR700 is fluorescently labeled, and "SYTOX Blue" indicates a fluorescence image in which SYTOX TM The figure shows a fluorescent image of blue staining, and "Marge" shows an overlaid image of each fluorescent image. It was shown that Nanomab-D-Spio-IR700 bound only to tumor cells, and that after laser light irradiation, the fluorescence from IR700 was quenched, while cell death of tumor cells bound to Nanomab-D-Spio-IR700 occurred.

[0122] Experiment 10: In vivo antitumor activity of antibody-nanoparticle conjugates. The in vivo antitumor activity of antibody-nanoparticle conjugates was evaluated according to the experimental protocol shown in Figure 11A. Disease model cells (A431, 6 x 10 6 Cells / 100 μL) were subcutaneously injected near the base of the right foot of female homozygous athymic nude mice (Day-10 in Figure 11A) to prepare xenograft model mice. Mice were anesthetized, and PBS or the complex (pan-IR700, Nanomag-D-Spio-pan, or Nanomag-D-Spio-pan-IR700 as controls) was administered into the tail vein at 80 μL / body (equivalent to 30 μg / body of antibody) (Day-1 in Figure 11A). Starting the next day (Day 0 in Figure 11A), the affected area was irradiated with laser light (690 nm, 200 mJ / cm) five times daily. 21 / time, (a) to (e) in Figure 11(A)). Tumor size after irradiation was evaluated by luciferase assay (bioluminescent imaging; BLI) at the timings indicated by "BLI" in Figure 11A (arrows in the figure). Figure 11B shows images of xenograft model mice observed by luciferase assay. Figure 11C is a graph showing the change in tumor size evaluated based on luciferase assay images (n = 6). The change in tumor size is expressed as the Relative Light Unit (RLU) ratio, with the RLU in the region of interest of the tumor image on Day 0 set to 100. Figure 11D is a graph showing the change in tumor volume measured after tumor removal (n = 10). All of the conjugates showed a tendency to reduce tumor volume in mice irradiated with laser light after administration of the conjugates, but the reduction in tumor volume in mice administered with Nanomag-D-Spio-pan-IR700 was particularly significant (Dunnett's test using Pan-IR700 + NIR-light as the control group, p<0.05).

[0123] Experiment 11: Antitumor activity of antibody-nanoparticle conjugates against in vivo spheroids. The antitumor activity of antibody-nanoparticle conjugates against in vivo spheroids was evaluated according to the experimental protocol shown in Figure 12A. Spheroids (6 x 10 cells) containing antigen-presenting cells (A431) and non-antigen-presenting cells (H661-luc-GFP) mixed at a 1 / 1 (volume ratio) were used. 6A spheroid solution (100 μL / 100 μL) was prepared. A431 cells were the same disease model cells used in Experiment 3, and H661-luc-GFP cells were the H661 cells used in Experiment 3 transfected with luciferase and green fluorescent protein (GFP) genes as reporter genes. The prepared spheroids were subcutaneously injected into the base of the two feet of female homozygous athymic nude mice (Figure 12A, Day-8) to prepare xenograft model mice. Mice were anesthetized, and PBS or the complex (pan-IR700, Nanomag-D-Spio-pan, or Nanomag-D-Spio-pan-IR700 as controls) was administered into the tail vein at 80 μL / body (equivalent to 30 μg / body of antibody) (Figure 12A, Day-1). From the next day (Day 0 in FIG. 12A ), the affected area near the base of the left foot was irradiated with laser light (690 nm, 200 mJ / cm ) five times a day. 2 1 / time). Tumor size after irradiation was evaluated by luciferase assay (BLI) at the timing indicated by "BLI" in Figure 12A (arrow in the figure). Figure 12B shows images of xenograft model mice observed by luciferase assay. Figure 12C is a graph showing the change in tumor size evaluated from the luciferase assay image (n = 6). The RLU in the region of interest of the tumor image on Day 0 is set to 100, and the change in tumor size is expressed as the RLU ratio. Figure 12D is a graph showing the change in tumor volume measured after tumor removal, and represents the relative value with the tumor size on Day 0 set to 100 (n = 6). Tumor volume at the laser light irradiation site in Nanomag-D-Spio-pan-IR700-administered mice was significantly reduced (Dunnett's test using Pan-IR700 + NIR-light as the comparison group, p < 0.05).

[0124] Experiment 12: Tumor imaging using antibody-nanoparticle conjugates. Using the same procedure as in Experiment 10, Nanomag-D-Spio-pan-IR700 was administered intravenously to the tail vein of A431 xenograft model mice, and tumors were imaged by MRI after administration. As a control, mice were used that received intravenous administration of PBS instead of Nanomag-D-Spio-pan-IR700. The nuclear magnetic resonance intensity (NMR) of the acquired MRI images is shown in Figure 13.

[0125] Experiment 13: Preparation of antibody-nanoparticle conjugate (Nanomag-D-Spio-Tra) An antibody-nanoparticle conjugate (Nanomag-D-Spio-Tra) was prepared using the same procedure as in Experiment 1-3), except that biotinylated human monoclonal antibody trastuzumab (Tra-Biotin), obtained by the same method as in Experiment 1-2), was used instead of Pan-IR700-Biotin.

[0126] Experiment 14: Reaction of nanoparticles with methylene blue Nanomag-D-Spio-Pan (0.02 mg / mL) or Nanomag-D-Spio-Tra (0.02 mg / mL) was placed in a microtube and heated at 37°C for 30 minutes in the presence or absence of reduced glutathione (GSH, 10 mM). After heating, methylene blue (10 μg / mL) was added to the tube with or without hydrogen peroxide (1 mM). The solution in the microtube was adjusted to pH 5.4 with PBS containing HCl, and the absorbance (500-800 nm) was measured. The solution was then irradiated with laser light (690 nm, 10 J / cm). 2 The nanoparticles were irradiated with UV light (UV, 3 hours) and the absorbance (500-800 nm) was measured. The absorbance measurement results are shown in Figure 14(A) (Nanomag-D-Spio-Pan) and Figure 14(B) (Nanomag-D-Spio-Tra). Table 1 shows the absorbance at a wavelength of 662 nm of the nanoparticle conjugates measured in Experiments 6 and 14.

[0127]

Claims

1. A complex for use in chemodynamic therapy of a tumor, the complex comprising: a nanoparticle encapsulating iron oxide and having a hydrophilic polymer on its surface; and a target recognition molecule bound to the nanoparticle, the target molecule being capable of binding to a target molecule in a tumor cell.

2. The complex according to claim 1, wherein the chemodynamic therapy is a photo-Fenton reaction induced by near-infrared light irradiation.

3. The complex of claim 1, further for use in photothermal therapy.

4. The conjugate of claim 1, further for use in tumor imaging.

5. The complex of claim 1, further comprising a photosensitive moiety bound to said nanoparticle or said target recognition molecule, said photosensitive moiety including a photosensitive group having a maximum absorption wavelength in the range of 500 to 1500 nm and one or more hydrophilic functional groups linked or coordinated to said photosensitive group.

6. The conjugate of claim 5, wherein said photosensitive moiety is bound to said target recognition molecule.

7. The conjugate of claim 5, further for use in photoimmunotherapy.

8. The conjugate of claim 1, wherein the target recognition molecule is an antibody.

9. The composite of claim 1, wherein said hydrophilic polymer is a polysaccharide.

10. The conjugate of claim 9, wherein said polysaccharide is dextran.

11. The complex according to claim 1, wherein the content of iron atoms in said complex is 30 mass % or more.

12. The complex according to claim 1, wherein the number of said target recognition molecules bound to said nanoparticles is 1 to 20 per nanoparticle.

13. The conjugate of claim 1, wherein the target molecule on a tumor cell is epidermal growth factor receptor, the EGF receptor family, or a platelet-activating receptor.

14. A composition for use in chemodynamic therapy of tumors, comprising a complex according to any one of claims 1 to 13.

15. The composition of claim 14, further comprising hydrogen peroxide.

16. The composition according to claim 14, further comprising a bioreductive substance.

17. The composition of claim 14, further for use in photothermal therapy.

18. The composition of claim 14, further for use in tumor imaging.

19. The composition of claim 14, wherein the complex has a photosensitive site, the photosensitive site comprising a photosensitive group having a maximum absorption wavelength in the range of 500 to 1500 nm and one or more hydrophilic functional groups linked or coordinated to the photosensitive group.

20. The composition of claim 19, further for use in photoimmunotherapy.