Complexes for chemokinetic therapy of tumors

Abstract

A complex for chemokinetic therapy of a tumor, the complex comprising a nanoparticle, and a target recognition molecule bound to the nanoparticle, the nanoparticle encapsulating iron oxide and having a hydrophilic polymer on a surface, the target recognition molecule being capable of binding to a target molecule of a tumor cell.

Description

Technical Field

[0001] This invention relates to a complex for chemokinetic therapy of tumors. Background Technology

[0002] Chemodynamic therapy (CDT) is a treatment method that uses a continuous chemical process within the tumor microenvironment (TME) to generate reactive oxygen species (ROS) from endogenous H₂O₂, thereby damaging tumor cells and inducing cell death. The ROS generation in CDT is primarily based on the Fenton reaction, and Fe is mainly used to promote this reaction. 2+ Based nanomaterials. Additionally, Cu for Fenton-like reaction-based CDT was developed. 2+ Transition metal-based nanomaterials. However, since TME is weakly acidic, it is not suitable for Fe alloys requiring strong acidity (pH 2–4). 2+ The conditions are unsuitable for the Fenton reaction of the cells. In addition, the production of hydrogen peroxide (H2O2) is insufficient compared with normal cells, so the therapeutic efficiency of existing CDT may not be sufficient.

[0003] Patent document 1 describes the use of polydopamine iron nanoparticles filled with artesunate coated with fibronectin for CDT; in these particles, fibronectin enhances the biocompatibility and targeting of the nanomaterials, and the chemotherapeutic agent artesunate promotes tumor cell apoptosis and reacts with ferrous ions to promote ROS generation.

[0004] Patent document 2 describes a magnetic nanozyme (Fc-MBL-rGO-Fe3O4) obtained by modifying the surface of reduced graphene oxide-supported magnetite nanozyme (rGO-Fe3O4) with mannose lectin (Fc-MBL) and then using CDT and photothermal therapy (PTT) to exert an antibacterial effect.

[0005] Non-Patent Literature 1 describes a Gd2O3 / CuS nanoparticle, wherein the CuS nanoparticle has a PTT effect caused by near-infrared light absorption and a CDT effect caused by ROS generation, and the PTT effect caused by CuS promotes the generation of ROS. The CuS nanoparticle is modified with Gd2O3 for magnetic imaging and Cy5.5 for fluorescence imaging, and the binding of RGD peptide endows it with selective recognition ability for tumor cells.

[0006] Near-infrared photoimmunotherapy (NIR-PIT) is a treatment method that involves preparing an antibody complex containing a photosensitizing substance that reacts with near-infrared light, along with an antibody specific to antigens on the surface of tumor cells. This antibody complex binds to the tumor cells, and the cells are then locally irradiated with near-infrared light, thereby selectively killing the tumor cells. The photosensitizing substance primarily uses chemical species containing a phthalocyanine backbone (e.g., so-called IR700 molecules such as IRDye700DX) (see Patent Documents 3 and 4 and Non-Patent Document 2). The mechanism of NIR-PIT is to induce cell death within a very short time (2–6 minutes) without mitochondrial damage, thus differing from existing phototherapy methods that utilize ROS-induced cell death generated by light irradiation. In recent years, the mechanism of tumor treatment based on NIR-PIT has been elucidated as follows: Light irradiation causes the antibody complex bound to the surface of tumor cells to aggregate, thereby damaging the cell membrane of the tumor cells and creating an osmotic pressure difference between the inside and outside of the cell, inducing cell death (Non-Patent Document 2). Patent document 5 describes a complex comprising an antibody molecule bound to a photosensitive substance and a magnetic or semiconductor particle bound to the antibody molecule, which can be used for NIR-PIT and tumor imaging, wherein the photosensitive substance comprises a phthalocyanine backbone.

[0007] Existing technical documents

[0008] Patent documents

[0009] Patent Document 1: Chinese Patent Publication No. 115089560

[0010] Patent Document 2: Chinese Patent Publication No. 115944727

[0011] Patent Document 3: Japanese Patent Publication No. 2014-523907

[0012] Patent Document 4: Japanese Patent Publication No. 2019-218374

[0013] Patent Document 5: International Publication No. 2022 / 054798

[0014] Non-patent literature

[0015] Non-patent literature 1: ACS Applied Materials & Interfaces, 2022, 14: 34365-34376

[0016] Non-Patent Literature 2: Award-winning paper from the 40th Japan Society for Laser Medicine Congress, "Elucidation of the Mechanism of Near-Infrared Photoimmunotherapy", Japanese Journal of Laser Medicine, 2020, Vol. 41, No. 2, pp. 104-109. Summary of the Invention

[0017] The present invention provides a nanoparticle composite for chemokinetic therapy (CDT) of tumors, and a method for treating tumors by using CDT with the composite.

[0018] The inventors have discovered that a nanoparticle composite formed by combining a target recognition molecule with nanoparticles containing iron oxide can exert a CDT-based tumor treatment effect through light irradiation.

[0019] Therefore, the present invention includes the following embodiments.

[0020] [1] A complex for chemokinetic therapy of tumors,

[0021] The complex comprises nanoparticles and target recognition molecules bound to the nanoparticles.

[0022] The aforementioned nanoparticles contain iron oxide and have a hydrophilic polymer on their surface.

[0023] The aforementioned target recognition molecules can bind to the target molecules of tumor cells.

[0024] [2] According to the complex described in [1], wherein the above-mentioned chemokinetic therapy is based on a photoFenton reaction generated by near-infrared light irradiation.

[0025] [3] The complex described in [1] or [2] is further used in photothermal therapy.

[0026] [4] The composite according to any one of [1] to [3] is further used for tumor imaging.

[0027] [5] The composite according to any one of [1] to [4] further comprises a photosensitive region that is bound to the above-mentioned nanoparticles or the above-mentioned target recognition molecules, the photosensitive region comprising a photosensitive group having a maximum absorption wavelength of 500 to 1500 nm and one or more hydrophilic functional groups connected or coordinated to the photosensitive group.

[0028] [6] According to the complex described in [5], wherein the photosensitive site binds to the target recognition molecule described above.

[0029] [7] The complex described in [5] or [6] may be further used in photoimmunotherapy.

[0030] [8] The complex according to any one of [1] to [7], wherein the target recognition molecule is an antibody.

[0031] [9] The composite according to any one of [1] to [8], wherein the hydrophilic polymer is a polysaccharide.

[0032]

[10] According to the complex described in [9], wherein the polysaccharide is dextran.

[0033]

[11] The composite according to any one of [1] to

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

[0034]

[12] The composite according to any one of [1] to

[11] , wherein the number of bindings of the target recognition molecule to the nanoparticle is 1 to 20 per nanoparticle.

[0035]

[13] The complex according to any one of [1] to

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

[0036]

[14] A composition for chemokinetic therapy of tumors, comprising any one of [1] to

[13] .

[0037]

[15] The composition according to

[14] further contains hydrogen peroxide.

[0038]

[16] The composition according to

[14] or

[15] further contains a biological reducing agent.

[0039]

[17] The composition according to any one of

[14] to

[16] is further used in photothermal therapy.

[0040]

[18] The composition according to any one of

[14] to

[17] is further used for tumor imaging.

[0041]

[19] The composition according to any one of

[14] to

[18] , wherein the composite has a photosensitive region comprising a photosensitive group having a maximum absorption wavelength of 500 to 1500 nm and one or more hydrophilic functional groups connected to or coordinated with the photosensitive group.

[0042]

[20] The composition according to

[19] is further used in photoimmunotherapy.

[0043]

[21] Use of any one of [1] to

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

[0044]

[22] According to the application described in

[21] , the above-mentioned tumor therapeutic agent is further used in photothermal therapy.

[0045]

[23] According to the application described in

[21] or

[22] , wherein the above-mentioned tumor therapeutic agent is further used for tumor imaging.

[0046]

[24] The application according to any one of

[21] to

[23] , wherein the composite has a photosensitive region comprising a photosensitive group having a maximum absorption wavelength of 500 to 1500 nm and one or more hydrophilic functional groups connected to or coordinated with the photosensitive group.

[0047]

[25] According to the application described in

[24] , the above-mentioned tumor therapeutic agent is further used for photoimmunotherapy.

[0048]

[26] The application according to any one of

[21] to

[25] , wherein a composition containing the above-described complex and hydrogen peroxide is used.

[0049]

[27] The application according to any one of

[21] to

[26] , wherein a composition containing the above-described complex and a reducing substance in vivo is used.

[0050]

[28] One method is to treat tumors using chemokinetics, including:

[0051] The procedure of administering the complex described in any one of [1] to

[13] to a patient requiring chemokinetic therapy for tumors, and

[0052] The procedure of irradiating the patient with light of wavelength 500-1500nm.

[0053]

[29] According to the method described in

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

[0054]

[30] The method according to

[28] or

[29] , wherein the patient is further given an in vivo reducing agent together with the above-described complex.

[0055] The composite of the present invention exerts a CDT-based tumor treatment effect through light irradiation. Furthermore, when the composite of the present invention further has a photosensitive site, it exerts a tumor treatment effect based on photothermal therapy (PTT) and near-infrared photoimmunotherapy (NIR-PIT) upon light irradiation, thus allowing the combined effects of these treatments to be enjoyed through the use of this composite. Additionally, the composite of the present invention can be used for tumor imaging, enabling the observation of target tumors located within a patient's body, or the optimization of light irradiation for CDT. Attached Figure Description

[0056] Figure 1 These are the ZETA potentials of Nanomag-D-spio and Nanomag-D-spio-pan-IR700.

[0057] Figure 2Images (top) and measurements (bottom) of Nanomag-D-spio and Nanomag-D-spio-pan-IR700 observed using atomic force microscopy (AFM), as well as inferred shapes (center image).

[0058] Figure 3 The magnetic susceptibility of Nanomag-D-spio and Nanomag-D-spio-pan-IR700 is based on VSM measurements.

[0059] Figure 4 This refers to the 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 added pan-IR700. Nanomag-D-Spio-pan-IR700: Cells with added Nanomag-D-Spio-pan-IR700. Pan blocking: Cells with added pan-IR700 or Nanomag-D-Spio-pan-IR700 in the presence of an EGFR inhibitor. B: Number of IR700-labeled cells in EGFR-expressing (EGFR-positive) cells (MDAMB468 and A431) with added Nanomag-D-Spio-pan-IR700 or Nanomag-D-Spio-cont-IR700 (cont: ligand other than EGFR ligand (IgG antibody)).

[0060] Figure 5 The fluorescence intensity is that of Nanomag-D-Spio-pan-IR700. A: in SDS, and B: in serum. A t-test was performed (*: p < 0.001, ns: no significant difference, vs. control, n = 3).

[0061] Figure 6 These are fluorescence images of cells treated with Nanomag-D-Spio-pan-IR700. Hoechst: Hearst staining; Lyso tracer: lysosomal staining; IR700: IR700 labeling; Merge: overlay image. DIC: bright-field image based on optical microscopy. White bar: 30 μm.

[0062] Figure 7 This refers to the absorbance of a methylene blue solution containing Nanomag-D-Spio. A: Change in absorbance caused by laser irradiation. B: Change in absorbance caused by heat treatment time in the presence of reduced glutathione.

[0063] Figure 8 This describes the heating behavior of the complex under laser irradiation. A: Temperature rise curve of the liquid containing the complex, error bar: SEM, t-test performed (*: p < 0.05, vs Pan-IR700, n = 3). B: Left: Infrared thermal image of the liquid containing Nanomag-D-Spio-pan-IR700 after laser irradiation; Right: Liquid temperature of the liquid containing Nanomag-D-Spio-pan-IR700 after laser irradiation.

[0064] Figure 9 These are fluorescence images of disease model cells (A431, MDAMB468, and PC-9) in in vitro spheroids treated with Nanomag-D-Spio-pan-IR700. Hoechst: Hurst staining; PI: pyridine iodide. Staining, IR700: IR700 label, Merge: overlay image. DIC: bright-field image based on optical microscopy. White rod: 30 μm.

[0065] Figure 10A This is a fluorescence image of disease model cells contained in in vitro spheroids treated with Nanomag-D-Spio-pan-IR700. The spheroids were prepared by mixing disease model cells with 3T3-RFP in a 1:1 (volume ratio). The disease model cells used were A431. 3T3-RFP was prepared by transfecting the gene for red fluorescent protein (RFP) into 3T3 cells. RFP: RFP marker, IR700: IR700 marker, SYTOX Blue: SYTOX. TM Blue staining, Merge: Overlay image. DIC: Bright-field image based on optical microscopy. White rod: 30 μm.

[0066] Figure 10B and Figure 10A Same. Disease model cells used are either MDAMB468 or PC-9.

[0067] Figure 11A This is the protocol for Experiment 10. Top: Timeline of administration of the complex and laser irradiation to xenograft model mice transplanted with A431, and tumor evaluation based on bioluminescent imaging (BLI). Bottom: Schematic diagram of the process of transplanting A431 into mice, administering the complex, and laser irradiation.

[0068] Figure 11B These are images of xenograft model mice that have been given the complex, as observed by luciferase assay.

[0069] Figure 11CThe changes in tumor size in xenograft model mice treated with the complex were evaluated using luciferase assay. The RLU of the region of interest in the tumor image at Day 0 was set to 100, and the change in tumor size was expressed as the RLU ratio. Error bars: SEM.

[0070] Figure 11D This represents the change in tumor size in xenograft model mice that received the complex. Tumor size was measured after tumor removal from the mice. Error bar: SEM.

[0071] Figure 12A This is the protocol for Experiment 11. Left: Schematic diagram of the process of transplanting spheroids into mice and laser irradiation. Right: Timeline of administering the complex and laser irradiation to xenograft model mice with transplanted spheroids, and tumor evaluation based on luciferase assay (BLI).

[0072] Figure 12B These are images of xenograft model mice that have been given the complex, as observed by luciferase assay.

[0073] Figure 12C The changes in tumor size in xenograft model mice treated with the complex were evaluated using luciferase assay. The RLU of the region of interest in the tumor image at Day 0 was set to 100, and the change in tumor size was expressed as the RLU ratio. Error bars: SEM.

[0074] Figure 12D This represents the change in tumor size in xenograft model mice that received the complex. Tumor size was measured after tumor removal from the mice and is represented as a relative value with the tumor size at Day 0 set to 100. Error bars: SEM.

[0075] Figure 13 This refers to the nuclear magnetic resonance intensity of the MRI image. The Dunnet test was performed on the control group (*: p < 0.0001, n = 5).

[0076] Figure 14 In the table, A represents the change in absorbance of a methylene blue solution containing Nanomag-D-Spio-Pan due to laser irradiation, and B represents the change in absorbance of a methylene blue solution containing Nanomag-D-Spio-Tra due to laser irradiation. Detailed Implementation

[0077] All patent documents, non-patent documents and other publications cited in this specification are incorporated herein by reference.

[0078] In this specification, "tumor" refers to all tumors, including benign and malignant tumors, as well as epithelial and non-epithelial tumors. Furthermore, its location (tissue and organ) is not particularly limited. Preferably, the tumor targeted in this invention is a malignant tumor such as carcinoma. Carcinoma can be a liquid tumor or a solid tumor, and can include all types of carcinoma such as epithelial carcinoma, adenocarcinoma, sarcoma, and malignant lymphoma. Examples of tumors include acute leukemia (acute lymphoblastic 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-type prolymphoblastic leukemia, large granular lymphocytic leukemia, adult T-cell leukemia, polycythemia vera, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, etc.; and fluid-filled tumors including fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, other sarcomas, synovial tumors, mesothelioma, Ewing's tumor, etc. Solid tumors including leiomyosarcoma, rhabdomyosarcoma, colorectal 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, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumors, bladder cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, etc. The tumors mentioned in this specification can be primary or recurrent tumors.

[0079] In this specification, "tumor lesion" or "lesion" refers to tumor tissue primarily containing tumor cells. This tumor tissue includes tissue composed entirely of tumor cells, as well as tissue where tumor cells are mixed with normal cells or normal tissue. In the case where normal cells or normal tissue are mixed in with the tumor tissue, the volume or number of tumor cells relative to the normal cells or normal tissue is not particularly limited.

[0080] In this specification, "antibody" refers to a polypeptide ligand containing 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, "antibody" in this specification includes any class of immunoglobulins such as IgG, IgA, IgD, IgE, IgM, and their subclasses, as well as their variants, and further includes chimeric antibodies such as humanized antibodies, and other immunoglobulin modifiers containing antigen recognition sites. In addition, "antibody" in this specification includes fragments or domains of immunoglobulins containing antigen recognition sites, such as Fab fragments, Fab' fragments, F(ab)' fragments, single-chain Fv ("scFv"), disulfide-stabilized Fv ("dsFv"), VHH (variable domain of heavy chain antibody), and VNAR (single variable new antigen receptor domain antibody).

[0081] The term "chemokinetic therapy" (CDT) in this specification refers to the following treatment: Reactive oxygen species (ROS), such as superoxide (·O), singlet oxygen (O), and hydroxyl radicals (·OH), are generated from H₂O₂ through a continuous chemical process within the tumor microenvironment (TME). Oxidative stress damages tumor cells, thereby inducing cell death. It is inferred that the ROS generation in CDT is essentially based on the Fenton reaction.

[0082] In this instruction manual, "photothermal therapy" (PTT) refers to the following treatment: irradiating cells with a photothermal material that generates heat upon light exposure, thereby killing the cells using the heat released by the photothermal material. The therapeutic effect of PTT depends on the difference between the upper limit of the survival temperature of cancer cells (approximately 42°C) and the upper limit of the survival temperature of normal cells (45°C).

[0083] The "Near-infrared immunotherapy" (NIR-PIT) described in this specification refers to the following treatment method: An antibody complex containing a photosensitive substance that reacts with near-infrared light is introduced into an antibody specific to antigens on the surface of tumor cells. This complex binds to the tumor cells and is then irradiated with near-infrared light. The photoresponsiveness of this photosensitive substance induces cell death. In recent years, the mechanism of NIR-PIT-based tumor treatment has been elucidated: the photosensitive substance contained in the antibody complex bound to the surface of tumor cells reacts with near-infrared light, resulting in the aggregation of the antibody complex, thereby damaging the cell membrane of the tumor cells. This creates an osmotic pressure difference between the inside and outside of the cell, inducing cell death (Non-Patent Literature 2).

[0084] 1. Complex

[0085] In one embodiment, the present invention provides a complex for use as a pharmaceutical agent in chemokinetic therapy (CDT) for tumors. The complex provided by the present invention comprises nanoparticles and a target recognition molecule bound to the nanoparticles, the target recognition molecule being capable of binding to target molecules on tumor cells.

[0086] 1.1. Nanoparticles

[0087] The nanoparticles used in the composite of the present invention are encapsulated with iron oxide and have a hydrophilic polymer on their surface. Because the nanoparticles have a hydrophilic polymer on their surface, a hydration layer is formed around them. This hydration layer prevents phagocytes from capturing and breaking down the nanoparticles (conferring concealment) by inhibiting the non-specific adsorption of proteins to the nanoparticles, thus improving the blood retention of the nanoparticles. Furthermore, the formation of the hydration layer by the nanoparticles promotes the introduction of the composite of the present invention into cells based on endocytosis. Examples of iron oxide encapsulated in the nanoparticles include FeO, Fe2O3, and Fe3O4. Fe2O3 or Fe3O4 is preferred. Examples of the hydrophilic polymer include polyacrylamide, poly(vinyl alcohol), polyethylene glycol, agarose, polysaccharides, glycoproteins, and proteins such as heparin and albumin. This allows for the absorption of water-soluble substances such as hydrogen peroxide and reducing agents in the body, or Fe... 2+ Plasma permeability is preferred, and polysaccharides are preferred. Examples of such polysaccharides include dextran, hyaluronic acid, alginate, and pullulan, with dextran being preferred. The hydrophilic polymer on the surface layer has a gel-like morphology.

[0088] The aforementioned nanoparticles may have reactive groups or linkers on their surface that enable them to bind to the target recognition molecules described later. For example, preferably, the nanoparticles have reactive functional groups such as amino or carboxyl groups, or protein-binding molecules such as avidin, streptavidin, or protein A on their surface.

[0089] The aforementioned nanoparticles are preferably particles with an average particle size of 500 nm or less. In this specification, "average particle size" refers to the average particle size measured using a dynamic light scattering method. The average particle size of the nanoparticles can be appropriately adjusted according to the type of cancer, taking into account factors such as the EPR effect (Enhanced Permeation and Retention Effect). For example, the average particle size of the nanoparticles is preferably 200 nm or less, more preferably 100 nm or less, and even more preferably 50 nm or less. There is no particular limitation on the lower limit of the average particle size of the nanoparticles; from the perspective of manufacturing feasibility, it is preferably 1 nm or more, more preferably 10 nm or more, and even more preferably 20 nm or more.

[0090] The iron oxide content in the nanoparticles used in the composite of the present invention is the iron atom content converted into the total mass of the composite of the present invention, preferably 30% by mass or more, more preferably 35% by mass or more, more preferably 80% by mass or less, and more preferably 70% by mass or less. If the iron atom content in the composite is too high, the water solubility or dispersibility of the composite tends to decrease. On the other hand, if the iron atom content in the composite is too low, the therapeutic effect based on CDT tends to decrease.

[0091] The nanoparticles used in the composites of this invention are commercially available. For example, a dispersion of dextran-coated superparamagnetic iron oxide particles is sold by Micromod as part of the Nanomag-D-spio series.

[0092] 1.2. Target recognition molecules

[0093] The target recognition molecule contained in the complex of the present invention is a molecule for binding the complex to tumor cells, which are therapeutic targets. This target recognition molecule recognizes and binds to target molecules (receptors) present on the tumor cells. Examples of such target recognition molecules include low-molecular-weight compounds, peptides, antibodies, ligands such as antigen-binding fragments, aptamers, glycans, and flatfoot proteins. The target recognition molecule is preferably an antibody. The type of antibody can be appropriately selected based on the antigens (target molecules) present on the surface of the tumor cells.

[0094] Preferred examples of the aforementioned antigens include transmembrane proteins present in tumor cells. Examples of such transmembrane proteins include tumor-specific proteins expressed on the surface of tumor cells (also known as tumor-specific antigens in this art). Tumor-specific proteins are proteins inherent in cancer cells, or proteins that are more abundant in cancer cells compared to other cells such as normal cells.

[0095] Examples of the aforementioned tumor-specific proteins include members of the epidermal growth factor receptor (EGFR) family (e.g., HER1, 2, 3, and 4) and members of cytokine receptors (e.g., CD20, CD25, IL-13R, CD5, CD52, etc.).

[0096] Specific examples of the aforementioned tumor-specific proteins include HER-2 (human epidermal growth factor receptor 2, e.g., GenBank accession numbers M16789.1, M16790.1, M16791.1, M16792.1, and AAA58637) associated with breast cancer, ovarian cancer, gastric cancer, and uterine cancer; and HER-1 (e.g., GenBank accession numbers NM_005228 and NP_005219) associated with adenocarcinoma, excluding lung cancer, anal cancer, and glioma.

[0097] Other specific examples of the aforementioned tumor-specific proteins include CD52 associated with chronic lymphocytic leukemia (e.g., GenBank accession numbers AAH27495.1 and CAI15846.1); CD33 associated with acute medullary leukemia (e.g., GenBank accession numbers NM_023068 and CAD36509.1); and CD20 associated with non-Hodgkin's lymphoma (e.g., GenBank accession numbers NP_068769 and NP_031667).

[0098] Other specific examples of the aforementioned tumor-specific proteins include any of the various MAGEs (melanoma-associated antigen E) including MAGE1 (e.g., GenBank accession numbers M77481 and AAA03229), MAGE2 (e.g., GenBank accession numbers L18920 and AAA17729), MAGE3 (e.g., GenBank accession numbers U03735 and AAA17446), and MAGE4 (e.g., GenBank accession numbers D32075 and A06841.1); any of the various tyrosinases (e.g., GenBank accession number U01873 and AAB60319); variant ras; variant p53 (e.g., GenBank accession numbers X54156, CAA38095, and AA494311); p97 melanoma antigen (e.g., GenBank accession number M12154 and AAA59992); and those associated with breast tumors. Human milk fat globules (HMFG) (e.g., GenBank accession numbers S56151 and AAB19771); any of the various BAGEs (human melanoma-associated antigen E) including BAGE1 (e.g., GenBank accession number Q13072) and BAGE2 (e.g., GenBank accession numbers NM_182482 and NP_872288); melanoma-associated gp100 (e.g., GenBank accession number S73003 and AAC60634); melanoma-associated MART1 antigen (e.g., GenBank accession number NP_005502); any of the various GAGEs (G antigens) including GAGE1 (e.g., GenBank accession number Q13065) or any of GAGE2-6; various gangliosides; CD25 (e.g., GenBank accession numbers NP_000408.1 and NM_000417.2), etc.

[0099] Other specific examples of the aforementioned tumor-specific proteins include HPV16 / 18 and E6 / E7 antigens associated with cervical cancer (e.g., GenBank accession numbers NC_001526, FJ952142.1, ADB94605, ADB94606, and U89349); mucin (MUC1)-KLH antigen associated with breast cancer (e.g., GenBank accession numbers J03651 and AAA35756); and CEA (carcinoembryonic antigen) associated with colorectal cancer (e.g., Gen...). The following are associated with cancers: GenBank accession numbers X98311 and CAA66955; cancer antigen 125 (also known as CA125, mucin 16, or MUC16) associated with ovarian cancer and other cancers (e.g., GenBank accession numbers NM_024690 and NP_078966); alpha-fetoprotein (AFP) associated with liver cancer (e.g., GenBank accession numbers 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 numbers X14810 and CAA32915), etc.

[0100] Other specific examples of the aforementioned tumor-specific proteins include PMSA (prepericardium membrane-specific antigen; e.g., GenBank accession numbers AAA60209 and AAB81971.1) associated with prepericardium cancer; NY-ESO-1 (e.g., GenBank accession numbers U87459 and AAB49693) associated with melanoma, sarcoma, testicular cancer, and other cancers; hTERT (also known as telomerase) (e.g., GenBank accession numbers NM_198253 and NP_937983 (variant 1), NM_198255 and NP_937986 (variant 2)); and proteins. Enzyme 3 (e.g., GenBank accession numbers M29142, M75154, M96839, X55668, NM00277, M96628, X56606, CAA39943 and AAA36342); Wilms' tumor 1 (WT-1, e.g., GenBank accession numbers 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)), etc.

[0101] Other specific examples of the aforementioned tumor-specific proteins include PD-L1 and PD-L2, which are associated with immune checkpoints.

[0102] The names of tumor-specific proteins described in this specification are based on the GenBank database of the National Center for Biotechnology Information (NCBI) ([www.ncbi.nlm.nih.gov / genbank / ]).

[0103] Examples of antibodies belonging to the target recognition molecules that may be included in the complex of the present invention include cetuximab, panitumumab, zalumab, nimotuzumab, trastuzumab, trastuzumab emtansine, tosimotuzumab, rituximab, teimomab, dazumab, gemtuzumab, alenzab, 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, ramucirumab, regorafenib, ruxolitinib, and sorafenib. Fenni, Sunitinib, Tesirolimus, Trametinib, Vandetanib, Vemolfenib, Vemodil, Baliximab, Ipilimumab, Nivolumab, Pembrolizumab, MPDL3280A, Pildilizumab (CT-011), MK-3475, BMS-936559, MPDL3280A (Atezolizumab), Tesimimumab, IMP321, BMS-986016, LAG52 5. Urerucizumab, PF-05082566, TRX518, MK-4166, Dacitocilizumab (SGN-40), Lucamumab (HCD122), SEA-CD40, CP-870, CP-893, MEDI6469, MEDI6383, MEDI4736, MOXR0916, AMP-224, PDR001, Acimetidine (MSB00107) 18C), rHIgM12B7, urorubin, BKT140, vararizumab (CDX-1127), ARGX-110, MGA271, lirelizumab (BMS-986015, IPH2101), IPH2201, AGX-115, imatozumab, CC-90002 and MNRP1685A, and fragments containing their antigen recognition sites, but not limited to these.

[0104] In the composite of the present invention, the number of bindings of the target recognition molecule to the nanoparticle is one or more per nanoparticle, preferably 20 or less, more preferably 16 or less, further preferably 12 or less, and even more preferably 5 or less. If the number of bindings of the target recognition molecule to the nanoparticle is too large, the overall size of the composite becomes larger, thus there is a tendency for the composite to be difficult to selectively distribute on tumor cells. In addition, the lower limit of the number of bindings of the target recognition molecule in 1 mg of the composite of the present invention is preferably 1.0 × 10⁻⁶. -12 mol / mg, more preferably 5.0 × 10 mol / mg. -12 mol / mg, more preferably 1.0 × 10 mol / mg. -11 mol / mg. On the other hand, the upper limit of the number of the target-recognizing molecule bound in 1 mg of the complex of the present invention is preferably 1.0 × 10⁻⁶ mol / mg. -8 mol / mg, more preferably 5.0 × 10 mol / mg. -9 mol / mg, more preferably 1.0 × 10 mol / mg. -9 mol / mg.

[0105] 1.3. Photosensitive sites

[0106] The composite of the present invention may further have a photosensitive region. This photosensitive region is bound to the aforementioned nanoparticles or target recognition molecules in the composite of the present invention. The photosensitive region comprises a photosensitive group having a maximum absorption wavelength of 500–1500 nm and one or more hydrophilic functional groups connected to or coordinated with the photosensitive group. It is presumed that the hydrophobicity of this photosensitive region increases upon irradiation with near-infrared (NIR) light, for example, light with a wavelength of 500–1500 nm. More specifically, it is presumed that if light of this wavelength is irradiated, the hydrophilic functional group will dissociate or its structure will change through a photochemical reaction, thereby increasing the hydrophobicity of the photosensitive region.

[0107] As an example of the hydrophilic functional groups contained in the aforementioned photosensitive sites, the carboxylic acid group (-CO2) can be cited. - ), sulfonic acid group (-SO3) - ), sulfonyl (-SO2) - ), sulfate group (-SO4) -2 ), hydroxyl group (-OH), phosphate group (-OPO3) -2 ), phosphonate group (-PO3) -2 The photosensitive site may contain amino groups (-NH2), substituted or unsubstituted quaternary nitrogen groups (each with any counterion), but is not limited to these. Examples of counterions are not limited and may include sodium, potassium, calcium, ammonium, organic amino acids, magnesium, etc. The photosensitive site may further have reactive groups or linkers for binding with the aforementioned target recognition molecules or nanoparticles.

[0108] As an example of the photosensitive site used in the composite of the present invention, a site comprising a phthalocyanine backbone can be cited. Phthalocyanine is an azaporphyrin (i.e., C463-C ... 32 H 16 (N8). Phthalocyanines form stable chelates with metal cations and non-metal cations, in which the ring center is occupied by an element capable of retaining one or two ligands. The area around the ring can be unsubstituted or substituted.

[0109] Preferably, the portion containing the phthalocyanine framework used in this invention is water-soluble and has at least one water-soluble portion. Preferably, the water-soluble portion containing the phthalocyanine framework contains silicon. Preferably, the phthalocyanine framework has a core element such as Si, Ge, Sn, or Al at the center of the ring.

[0110] The phthalocyanine skeleton-containing portion used in this invention preferably has a maximum absorption wavelength of 500–1500 nm, more preferably 600–850 nm, and even more preferably 660–740 nm. Furthermore, the phthalocyanine skeleton-containing portion preferably has one or more ligands containing hydrophilic functional groups. Examples of such hydrophilic functional groups include carboxylic acid groups (-CO2). - ), sulfonic acid group (-SO3) - ), sulfonyl (-SO2) - ), sulfate group (-SO4) -2 ), hydroxyl group (-OH), phosphate group (-OPO3) -2 ), phosphonate group (-PO3) -2 ), amino groups (-NH2), substituted or unsubstituted quaternary nitrogens (each with any counterion), etc., but not limited to these. Examples of counterions are not limited and can include sodium, potassium, calcium, ammonium, organic amino acids, magnesium, etc.

[0111] Preferably, the phthalocyanine backbone-containing site used in this invention comprises a linker having a reactive group capable of forming a bond with the aforementioned target recognition molecule or nanoparticle. That is, it has a structure having a linker-phthalocyanine backbone portion (LD). Preferably, the phthalocyanine backbone-containing site binds to the target recognition molecule or nanoparticle through the linker substituted around the ring of the phthalocyanine backbone.

[0112] In a preferred embodiment, the phthalocyanine skeleton-containing portion used in this invention is a compound represented by the following formula (Ia):

[0113]

[0114] In the formula,

[0115] L stands for direct bonding or connector;

[0116] Q is a reactive group used to form bonds with the target recognition molecules or nanoparticles mentioned above;

[0117] R 2 R 3 R 7 and R 8 Each is independently selected from substituted or unsubstituted alkyl and substituted or unsubstituted aryl groups.

[0118] R 4 R 5 R 6 R 9 R 10 and R 11 When present, each is independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkanoyl, substituted or unsubstituted alkoxycarbonyl, substituted or unsubstituted alkylcarbamoyl, and chelate ligand, wherein R 4 R 5 R 6 R 9 R 10 and R 11 At least one of them contains a water-soluble group.

[0119] 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 Each is 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 The carbon atoms bonded to them, ii)R 17 and R 18 The carbons bonded to them, and iii)R 21 and R 22 At least one of the carbons bonded to them forms a fused ring; and

[0120] X 2 and X 3 Each is independently a C1 to C2 bond with or without heteroatoms between carbon-carbon bonds. 10 Alkylene. It should be noted that in this specification, C1 to C2... 10 Alkylenes refer to methylene groups and alkylene groups with 2 to 10 carbon atoms.

[0121] In one embodiment, L is a linker. In one embodiment, the linker is a straight or branched, cyclic or heterocyclic, saturated or unsaturated chain having 1 to 60 atoms, for example 1 to 45 atoms or 1 to 25 atoms. In some cases, the atoms of the linker may be selected from C, N, P, O and S. In one embodiment, L may have an additional hydrogen atom satisfying a valence (based on the aforementioned 1 to 60 atoms). Generally, the linker may comprise ethers, thioethers, amines, esters, carbamates, ureas, thioureas, carbonyl groups, amides, single bonds, double bonds, triple bonds, aromatic carbon-carbon bonds, phosphorus-oxygen bonds, phosphorus-sulfur bonds, nitrogen-nitrogen bonds, nitrogen-oxygen bonds, nitrogen-platinum bonds, aromatic bonds or heteroaromatic bonds, or any combination thereof.

[0122] In one implementation, L is derived from equation -R 1 -YX 1 -Y 1 - indicates that, in the formula, R 1 It is a divalent group or a direct bond; Y and Y 1 Each is independently selected from directly bonded, oxygen, substituted or unsubstituted nitrogen and sulfur; and X 1 Selected from C1 to C1 carbons that are directly bonded or have heteroatoms sandwiched between carbon-carbon bonds, or have no heteroatoms sandwiched between carbon-carbon bonds. 10 Alkylene. Examples of this divalent group include, but are not limited to, substituted or unsubstituted alkylene, substituted or unsubstituted alkoxycarbonyl, substituted or unsubstituted alkylenecarbamoyl, substituted or unsubstituted alkylenesulfonyl and substituted or unsubstituted aryl.

[0123] As R 1 Detailed examples may include, but are not limited to, substituted or unsubstituted alkylene, substituted or unsubstituted alkoxycarbonyl, oxycarbonylamino, substituted or unsubstituted alkylene carbamoyl, substituted or unsubstituted alkylene sulfonyl, substituted or unsubstituted alkylene sulfonyl carbamoyl, substituted or unsubstituted aryl, substituted or unsubstituted arylsulfonyl, substituted or unsubstituted aryloxycarbonyl, substituted or unsubstituted aryloxycarbamoyl, substituted or unsubstituted arylsulfonyl carbamoyl, substituted or unsubstituted carboxylalkyl, substituted or unsubstituted carbamoyl, carbonyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroaryloxycarbonyl, substituted or unsubstituted heteroaryl carbamoyl, substituted or unsubstituted heteroarylsulfonyl carbamoyl, substituted or unsubstituted sulfonyl carbamoyl, thiocarbonyl, sulfonyl, and sulfinyl.

[0124] Preferably, the alkylene group contained in the above-mentioned substituted or unsubstituted alkylene group, the above-mentioned substituted or unsubstituted alkoxycarbonyl group, the above-mentioned substituted or unsubstituted alkylene carbamoyl group, the above-mentioned substituted or unsubstituted alkylene sulfonyl group, and the above-mentioned substituted or unsubstituted alkylene sulfonyl carbamoyl group is a C1-C1 group with or without heteroatoms between carbon-carbon bonds. 10 Alkylene.

[0125] In one embodiment, Q includes a reactive group for forming a bond with the target recognition molecule or nanoparticle described above. As used in this specification, a "reactive group" refers to a portion of a compound capable of chemically reacting with functional groups on different materials (e.g., target recognition molecules) to form a bond. Typically, the reactive group is an electrophile or nucleophile that forms a covalent bond by exposing the corresponding functional group, which is a nucleophile or electrophile, respectively.

[0126] In one embodiment, Q comprises a reactive group that reacts with a carboxyl, amino, or thiol group on the bound target recognition molecule or nanoparticle. Examples of preferred reactive groups, without limitation, include active esters, acyl halides, alkyl halides, anhydrides, carboxylic acids, carbodiimides, carboxylic esters, carbamates, haloacetamides (e.g., iodoacetamide), isocyanates, isothiocyanates, maleimides, NHS (N-hydroxysuccinimide) esters, phosphoramides, platinum complexes, sulfonates, and thiocyanates. In one embodiment, the reactive group is a mercapto-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.

[0127] In one implementation, R 2 R 3 R 7 and R 8 Each is independently a substituted or unsubstituted alkyl group, such as substituted or unsubstituted methyl, ethyl or isopropyl.

[0128] In one implementation, R 4 R 5 R 6 R 9 R 10 and R 11 At least one of them contains a water-soluble group. In some cases, R 4 R 5 R 6 R 9 R 10 and R 11 At least two of them contain water-soluble groups; otherwise, three or more contain water-soluble groups. In one embodiment, R4 R 5 R 6 R 9 R 10 and R 11 At least one of them is an alkyl group substituted with a water-soluble group. In one embodiment, R 4 R 5 R 6 R 9 R 10 and R 11 Each is independently a substituted or unsubstituted alkyl group, wherein at least one, preferably two or more, is an alkyl group substituted with a water-soluble group. In a preferred embodiment, R 4 R 5 R 6 R 9 R 10 and R 11 Each is independently a substituted or unsubstituted alkyl group, R 4 R 5 and R 6 At least one of them is an alkyl group substituted with a water-soluble group, and R 9 R 10 and R 11 At least one of them is an alkyl group substituted with a water-soluble group.

[0129] In this specification, "water-soluble group" refers to a group that improves the overall solubility of the molecule in an aqueous medium and contains one or more polar and / or ionic substituents. Examples of such a water-soluble group are not limited, but include the carboxylate group (-CO2). - ), sulfonic acid group (-SO3) - ), sulfonyl (-SO2) - ), sulfate group (-SO4) -2 ), hydroxyl group (-OH), phosphate group (-OPO3) -2 ), phosphonate group (-PO3) -2 The counterions are amino groups (-NH2) and substituted or unsubstituted quaternary nitrogen groups (each having any counterion). Examples of preferred counterions are not limited and include sodium, potassium, calcium, ammonium, organic amino acids, magnesium, etc. The counterions are preferably biologically acceptable.

[0130] R 4 R 5 R 6 R 9 R 10 and R 11 The bonded nitrogen atom can be trivalent or tetravalent.

[0131] In one implementation, R12 R 13 R 14 R 15 R 16 R 17 R 18 R 19 R 20 R 21 R 22 and R 23 They are hydrogen.

[0132] In one implementation, X 2 and X 3 Each is independently a C1 to C2 bond with or without heteroatoms between carbon-carbon bonds. 10 Alkylene. In one embodiment, it is added to X. 2 and / or X 3 Nitrogen can be quaternized.

[0133] In a preferred embodiment, the phthalocyanine skeleton-containing portion used in this invention is a compound represented by formula (Ib):

[0134]

[0135] In the formula,

[0136] X 1 and X 4 Each is independently a C1 to C2 bond with or without heteroatoms between carbon-carbon bonds. 10 alkylene, and

[0137] R 2 R 3 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 As defined in equation (Ia) above.

[0138] In the compound of formula (Ib) above, the reactive group used to form a bond with the target recognition molecule or nanoparticle is an NHS ester. In one embodiment, the reactivity of the NHS ester can be modified by changing the X group located between the NHS ester and the urethane functional group. 4The length of the alkylene group is adjusted. In one embodiment, the X-axis between the NHS ester and the carbamate functional group is... 4 The length of the alkylene group is inversely proportional to the reactivity of the NHS ester. In one embodiment, X 4 It is a C5-alkylene group. In another embodiment, X 4 It is a C3-alkylene group. In one embodiment, X 1 It is a C6-alkylene group. In another embodiment, X 1 It is a C3-alkylene group.

[0139] In one embodiment, the total charge of the compound of formula (Ia) or (Ib) is zero. This neutral charge may, under certain conditions, be obtained together with one or more arbitrary counterions or quaternary ammonium ions.

[0140] In one embodiment, after the compound of formula (Ia) or (Ib) binds to the target recognition molecule or nanoparticle described above, the molecule or particle retains its solubility and thus has sufficient solubility in aqueous solution.

[0141] In a preferred embodiment, the phthalocyanine skeleton-containing site used in this invention is an IR700 NHS ester, such as IRDye 700DX NHS ester (LI-COR Biosciences, P / N 929-70010 or 929-70011). In a preferred embodiment, the phthalocyanine skeleton-containing site is a compound represented by the following formula (II).

[0142]

[0143] The aforementioned photosensitizing site is preferably included in the composite of the present invention in a configuration in which the reactive group is bonded to the aforementioned target recognition molecule or nanoparticle. For example, compounds represented by formulas (Ia), (Ib), or (II), "IR700," "IRDye 700DX," or variations thereof may be included in the composite of the present invention in a configuration in which the reactive group is bonded to the aforementioned target recognition molecule or nanoparticle. Generally, IR700 has some preferred chemical properties. Amino-reactive IR700, for example, IR700 NHS ester or compounds of formula (II) are relatively hydrophilic and can be covalently bonded to the target recognition molecule using NHS ester. Typically, IR700 is combined with the hematoporphyrin derivative Photofrin (registered trademark) (1.2 × 10⁻⁶ at 630 nm). 3 M -1 cm -1 m-Tetrahydroxychlorophenol Foscan (registered trademark) (2.2 × 10⁻⁶ at 652 nm) 4 M-1 cm -1 ) and mono-L-asparagine dihydroporphyrin e6; NPe6 / Laserphyrin (registered trademark) (4.0 × 10 at 654 nm) 4 M -1 cm -1 Compared to existing photosensitizers, this one has a five-fold higher absorption coefficient (maximum absorption at 689 nm is 2.1 × 10⁻⁶). 5 M -1 cm -1 ).

[0144] The phthalocyanine skeleton-containing sites used in this invention, such as those represented by formulas (Ia), (Ib), or (II) above, can be prepared using commercially available starting materials. For example, the skeleton can be synthesized by the condensation of two or more different diiminoisoindolines. Various degrees of substitution and / or positional isomers of phthalocyanines can be derived using different dinitrile or diiminoisoindoline synthetic strategies. An exemplary synthetic scheme for generating the phthalocyanine skeleton is described in U.S. Patent No. 7,005,518.

[0145] In the composite of the present invention, the aforementioned photosensitive site only needs to bind to the aforementioned target recognition molecule or nanoparticle, preferably to the aforementioned target recognition molecule. The target recognition molecule is preferably an antibody. Furthermore, the composite of the present invention may contain one or more of the aforementioned photosensitive sites, and these photosensitive sites may have the same or different structures.

[0146] 1.4. Efficacy

[0147] The composite of the present invention is used for CDT (cancer-induced tumor therapy). Specifically, the composite of the present invention exerts a CDT-based therapeutic effect on tumors through photoirradiation. It is believed that the CDT-based therapeutic effect of the composite of the present invention is due to the Fenton reaction of the iron oxide nanoparticles in the composite induced by photoirradiation. Therefore, CDT using the composite of the present invention is based on the photo-Fenton reaction.

[0148] Furthermore, the composite of the present invention generates heat upon irradiation with light, such as NIR (e.g., light with wavelengths of 500–1500 nm), and can exert a PTT-based therapeutic effect. Additionally, because the composite of the present invention contains nanoparticles that are magnetic particles containing iron oxide, it is capable of imaging within living organisms.

[0149] Furthermore, when the composite of the present invention has a photosensitive site, it can exert a therapeutic effect based on photoimmunotherapy, specifically NIR-PIT. It is believed that the NIR-PIT-based therapeutic effect of the composite of the present invention is due to the photosensitive site of the composite, which binds to tumor cells, becoming hydrophobic upon NIR irradiation, causing aggregation of the composite, thereby damaging the cell membrane of the tumor cells and inducing cell death.

[0150] Therefore, the composite of the present invention enables combined treatment with CDT and PTT. Furthermore, when the composite of the present invention has a photosensitive site, it enables combined treatment with CDT and NIR-PIT, or further combined treatment with CDT, NIR-PIT, and PTT. Furthermore, the composite of the present invention can be used not only for tumor treatment in patients but also for diagnosis (e.g., to confirm the location of the composite by using in vivo imaging of the composite, or to image the tumor to which the composite is bound).

[0151] 2. Manufacturing method of the composite

[0152] The composite of the present invention can be manufactured by binding the aforementioned nanoparticles to the aforementioned target recognition molecule. The binding of the target recognition molecule to the nanoparticles can be carried out by known methods. When the composite of the present invention has a photosensitive site, it is preferable first to synthesize a target recognition molecule and / or nanoparticles having the photosensitive site. The binding of the photosensitive site to the target recognition molecule or nanoparticles can be carried out by known methods.

[0153] For example, as a specific method for binding the aforementioned photosensitive site to the antibody, the method disclosed in Patent Document 5 or Experiment 1 described later can be cited. As a more specific example, the target antibody-photosensitive site conjugate can be purified from the reaction solution by incubating an aqueous phosphate solution containing the antibody and a phthalocyanine compound represented by the above formula (Ia) having an NHS ester in the reactive group Q at room temperature, or by column purification or the like.

[0154] When the above-mentioned photosensitive site is combined with the above-mentioned nanoparticles, for example, the compound represented by the above formula (Ia) having NHS ester in the reactive group Q can be reacted with nanoparticles on which protein molecules such as amino groups or avidin, streptavidin, and protein A have been introduced onto the particle surface by the same method as described above.

[0155] The number of bindings of the aforementioned photosensitive site to the aforementioned target recognition molecule is one or more per target recognition molecule (e.g., antibody), preferably two or more, and more preferably five or less, more preferably four or less. Furthermore, the number of bindings of the aforementioned photosensitive site to the aforementioned nanoparticle is one or more per nanoparticle, preferably 80 or less, more preferably 50 or less, and even more preferably 20 or less.

[0156] The composite of the present invention can be manufactured by binding the aforementioned target recognition molecules to the aforementioned nanoparticles. For example, the nanoparticles are bound to the target recognition molecules by mixing a target recognition molecule that has been biotinylated using conventional methods with nanoparticles to which a biotin-binding substance has been introduced onto the particle surface. Examples of biotin-binding substances include proteins such as avidin and streptavidin. Preferably, 1 to 5, more preferably 1 to 4, and particularly preferably 1 to 3 of the biotin-binding substance are introduced per nanoparticle. Alternatively, the antibody can be cross-linked with the nanoparticles using carbodiimide compounds such as EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and N-hydroxyhydroxysuccinimide derivatives such as Sulfo-NHS. In this case, the amount of target recognition molecules and nanoparticles introduced is set such that the number of target recognition molecules bound per nanoparticle is a desired number (preferably 1 or more, more preferably 20 or less, more preferably 16 or less, more preferably 12 or less, and even more preferably 5 or less). Furthermore, the dosage can be set such that the number of target recognition molecules bound per 1 mg of nanoparticles is the desired amount. Specifically, the lower limit of the dosage of target recognition molecules per 1 mg of nanoparticles is preferably 1.0 × 10⁻⁶. -12 mol / mg, more preferably 5.0 × 10 mol / mg. -12 mol / mg, more preferably 1.0 × 10 mol / mg. -11 mol / mg. On the other hand, the upper limit of the amount of target recognition molecules introduced per 1 mg of nanoparticles is preferably 1.0 × 10⁻⁶ mol / mg. -8 mol / mg, more preferably 5.0 × 10 mol / mg. -9 mol / mg, more preferably 1.0 × 10 mol / mg. -9 mol / mg. The binding of the target recognition molecule to the nanoparticle is not limited to covalent bonding, but may also include bonding using hydrogen bonds, ionic bonds, hydrophobic interactions, or combinations thereof.

[0157] 3. Compositions containing complexes

[0158] In one embodiment, the present invention provides a composition comprising the above-described composite of the present invention. The composition comprising the composite 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 combined CDT and PTT treatment. Furthermore, when the composite of the present invention has a photosensitive site, the composition of the present invention is used as a pharmaceutical composition for combined CDT and NIR-PIT treatment, or for combined CDT and NIR-PIT and PTT treatment. In another embodiment, the composition of the present invention, in addition to being used for CDT-based, or further NIR-PIT and / or PTT-based tumor treatment, can also be used as a pharmaceutical composition for in vivo imaging (e.g., tumor imaging).

[0159] In one embodiment, the composition of the present invention comprises the complex of the present invention and a pharmaceutically acceptable carrier or excipient. Examples of pharmaceutically acceptable carriers are not limited and include water, oil, buffers, phosphate-buffered saline, other diluents for injection, etc. Examples of excipients are not limited and include starch, glucose, lactose, dextran, carboxymethyl cellulose, glycerol, propylene glycol, water, and ethanol, etc. The composition of the present invention may, as needed, contain lubricants, binders, wetting agents, emulsifiers, pH adjusters, isotonic agents, buffers, antioxidants, suspending agents, solubilizers, preservatives, chelating agents, and other pharmaceutically acceptable substances. Pharmaceutically acceptable carriers, excipients, etc., are well known in the art (e.g., see Remington's Pharmaceutical Sciences, by EW Martin, Mack Publishing Co., Easton, Pa., 19th Edition, 1995).

[0160] In one embodiment, the composition of the present invention may contain hydrogen peroxide. Hydrogen peroxide may be included in the composition of the present invention as a substrate for the Fenton reaction to supplement the hydrogen peroxide produced intracellularly. Hydrogen peroxide can react with Fe... 3+ Reduced to Fe 2+ Oxygen is produced. Fe 2+ It can function as a substrate for the Fenton reaction, and the generated oxygen is also expected to be used for hyperoxia therapy of cancer.

[0161] In one embodiment, the composition of the present invention may contain a biological reducing agent. Examples of such biological reducing agents include reduced glutathione, catalase, peroxidase, cytochrome peroxidase, and glutathione peroxidase. This biological reducing agent is used to reduce Fe... 3+Reduced to Fe 2+ It is used because the reducing substances in the organism cause the reduction of reactive oxygen species (ROS) or the decomposition of hydrogen peroxide, so it is used when the above-mentioned purposes are achieved.

[0162] The compositions of the present invention may be in liquid form, such as a dispersion, or in solid form, such as powders, pills, tablets, capsules, transdermal patches, inhalants, suppositories, etc. Alternatively, the compositions of the present invention may be administered by reconstitution using a pharmaceutically acceptable carrier (e.g., a diluent for injection). These liquid or solid compositions may be prepared using conventional methods. In one embodiment, the composition of the present invention is a single-dosage formulation comprising a carrier or excipient containing the complex of the present invention. In another embodiment, the composition of the present invention is a two-dosage formulation comprising the complex of the present invention and a diluent, etc., administered together. Therefore, in one embodiment, the composition of the present invention comprises a kit containing the complex of the present invention. In one embodiment, the kit contains the complex of the present invention and hydrogen peroxide and / or an in vivo reducing agent.

[0163] The composition of the present invention can be morphologically determined according to its administration plan. The administration plan can be appropriately designed based on the type and state of the tumor targeted for treatment, as well as the patient's type, age, and condition. The composition can be an oral or non-oral formulation, for example, an injection, an oral dose, or a topical application. The composition can be configured for single or multiple administration. The content of the complex of the present invention in the composition can be appropriately designed based on the composition's morphology, the dosage for the patient, etc.

[0164] The composition of the present invention can be adjusted to a pH range suitable for animal bodies, 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, and even more preferably pH 5.5 to 7.3. The pH of the composition can be adjusted using the pH adjuster, buffer solution, etc. described above.

[0165] 4. Tumor treatment using the composite of the present invention

[0166] As described above, the composite of the present invention, or the composition of the present invention containing the composite, is used in CDT-based tumor treatment. In one embodiment, the present invention provides a method for treating tumors using the composite or composition of the present invention via CDT. The method for treating tumors of the present invention (hereinafter also referred to as the treatment method of the present invention) includes the steps of administering the composite or composition of the present invention to a patient and irradiating the patient with near-infrared radiation (NIR). It should be noted that, in this specification, "method for treating tumors" can be translated as "method for killing tumors".

[0167] As described above, the composite and composition of the present invention enable combined treatment with CDT and PTT. Furthermore, when the composite of the present invention has a photosensitive site, it can further enable combined treatment with NIR-PIT. Therefore, in one embodiment, the treatment method for tumors using the composite and composition of the present invention is a method of treating tumors by combining CDT and PTT. In another embodiment, the treatment method for tumors using the composite and composition of the present invention is a method of treating tumors by combining CDT and NIR-PIT, or by combining CDT with NIR-PIT and PTT.

[0168] The patient given the composite or composition of the present invention is a patient who requires CDT for the treatment of a tumor. In one embodiment, the patient is a patient who requires combined CDT and NIR-PIT for the treatment of a tumor. Examples of such patients include humans and non-human animals with tumors to be treated with CDT. Examples of non-human animals include non-human mammals such as mice, rats, hamsters, rabbits, pigs, goats, dogs, cats, sheep, cattle, horses, etc., but are not limited to these. Furthermore, the patient may or may not have experience with other treatments for the tumor (surgery, radiation therapy, phototherapy, chemotherapy using agents other than the composite or composition of the present invention, etc.).

[0169] The target recognition molecule included in the complex of the present invention is selected according to the type of tumor that is the therapeutic target. By appropriately selecting the target recognition molecule, the complex of the present invention can aggregate toward the therapeutic target tumor. This target recognition molecule is preferably an antibody that can specifically bind to antigens present on the surface of the target tumor cells, preferably tumor-specific proteins expressed on the surface of the tumor cells. The cell surface antigens of the target tumor, such as tumor-specific proteins, that should serve as the target recognition molecule can be determined based on known information. Those skilled in the art can select antibodies that are specific to the target antigens on the therapeutic target tumor.

[0170] 4.1. Administration of the complex

[0171] The administration schedule (e.g., administration route, dosage, frequency, etc.) of the composite or composition of the present invention can be appropriately determined according to the type and condition of the tumor targeted for treatment, and the species, age, and condition of the patient. Examples of administration routes include local administration to the tumor patient via injection, catheter, spray, application, patch, suppository, etc.; and systemic administration via infusion, oral administration, intraperitoneal administration, intravenous injection, etc. Local administration of the composite or composition of the present invention is preferred. In one embodiment, the composite or composition of the present invention is administered intravenously. In another embodiment, the composite or composition of the present invention is administered directly to the affected area of ​​the tumor using a syringe or the like, or via catheter injection. For treating tumors, the composite or composition of the present invention can be used alone or in combination with other agents or therapies, such as surgery, radiation therapy, phototherapy, chemotherapy using agents other than the composite or composition of the present invention, etc.

[0172] The complex or composition of the present invention can be administered to a patient only in an effective amount. "Effective amount" refers to the amount of the complex of the present invention that is sufficient to exert a therapeutic effect on the tumor targeting the patient's tumor. "Effective amount" preferably refers to the amount that exerts a therapeutic effect on the tumor targeting the patient while minimizing or allowing side effects on the patient.

[0173] The dosage of the complex or composition of the present invention administered to a patient can be appropriately determined based on the type and condition (location, volume, etc.) of the target tumor, as well as the patient's species, age, condition, route of administration, and the morphology of the composition containing the complex. For example, the dosage of the complex of the present invention can be set based on the tumor volume. The tumor volume (V) can be measured, for example, by measuring the minor axis (W) and major axis (L) of the tumor and substituting them into the formula: V = (W... 2 The dosage can be calculated by (×L) / 2. Alternatively, the dosage can be adjusted based on the degree of tumor aggregation of the complex of the present invention as measured by imaging, as described later. The dosage of the complex of the present invention administered to humans can be determined based on the dosage administered to mice. For example, the effective dosage of the complex of the present invention administered to humans can be determined to be 5 to 10 times the effective dosage in mice.

[0174] In one embodiment, the single dose of the complex or composition of the present invention, for example, in the case of local injection, is only required to be in the range of 0.01 mg to 9000 mg, based on the amount of the complex of the present invention. In one embodiment, the single dose of the composition of the present invention, for example, in the case of an injectable formulation, is only required to be 0.5 mL to 1000 mL. In one embodiment, the composition of the present invention is an injectable formulation, and the single dose is 1 to 5 mL, which contains 0.1 mg to 5000 mg of the complex of the present invention.

[0175] In one embodiment, when the composite or composition of the present invention is injected into a tumor site in an adult (60 kg), the single dose (injection volume) is 0.01 mg to 20 mg / kg (body weight) based on the amount of the composite of the present invention. In another embodiment, when the composition of the present invention is injected into a tumor site in an adult (60 kg), the single dose (injection volume) is typically 1 to 5 mL.

[0176] The dosage and frequency of administration of the complex or composition of the present invention can be increased or decreased according to the therapeutic effect on the tumor. The therapeutic effect on the tumor can be evaluated using general tumor treatment evaluation methods, such as the tumor shrinkage rate. In one embodiment, the complex or composition of the present invention is administered once at the dosage described above. In another embodiment, the complex or composition of the present invention is administered multiple times. In the case of multiple administrations, the dosage described above can be repeatedly administered, or the dosage can be increased or decreased according to the therapeutic effect on the tumor. In one embodiment, subsequent administrations can be performed after the dosage of the previous administration has been cleared from the patient. In another embodiment, the complex or composition of the present invention can be repeatedly administered at a frequency of once a week, once every two weeks, once a month, or less. In another embodiment, if residual tumor of the therapeutic target remains after the previous administration, the complex or composition of the present invention can be administered again after 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 6 months, 1 year, or longer.

[0177] In a preferred embodiment, in the treatment method of the present invention, in addition to the composite of the present invention, hydrogen peroxide may be further administered to the patient. For example, hydrogen peroxide water may be administered to the patient intravenously. Alternatively, the composition of the present invention containing hydrogen peroxide described above may simply be administered to the patient. In another preferred embodiment, in the treatment method of the present invention, in addition to the composite of the present invention, an in vivo reducing agent may be further administered to the patient. An example of an in vivo reducing agent is as described above. Alternatively, the composition of the present invention containing an in vivo reducing agent described above may simply be administered to the patient. The hydrogen peroxide and the in vivo reducing agent may be used together.

[0178] The route and frequency of administration of hydrogen peroxide and the in vivo reducing agent can be appropriately determined in the same manner as with the complex or composition of the present invention. For example, hydrogen peroxide or the in vivo reducing agent can be administered to the patient together with the complex or composition of the present invention. The dosage of hydrogen peroxide and the in vivo reducing agent can be appropriately set according to the patient. The dosage of hydrogen peroxide and the in vivo reducing agent is preferably set to an amount that can enhance the therapeutic effect of the tumor based on the complex or composition of the present invention, and to minimize or minimize any side effects of administering the hydrogen peroxide and the in vivo reducing agent to the patient.

[0179] 4.2.Light irradiation

[0180] Following administration of the composite or composition of the present invention to a patient, the patient is then subjected to light irradiation. NIR irradiation is preferred. NIR irradiation is also preferred on the tumor cells bound to the composite or on the affected tumor site. The light-irradiated composite of the present invention exerts a CDT-based therapeutic effect on tumors, or further exerts a PTT-based therapeutic effect on tumors. When the composite of the present invention has a photosensitive site, the NIR-irradiated composite further exerts a NIR-PIT-based therapeutic effect on tumors. That is, the NIR-irradiated composite induces a photochemical reaction, killing the tumor cells it binds to.

[0181] The wavelength of the irradiated light is preferably 500–1500 nm, more preferably 600–850 nm, and even more preferably 660–740 nm. The timing of irradiation can be determined at any time after the composite of the present invention has been applied. For example, it can be determined as any time within 30 minutes to 96 hours after application, preferably 30 minutes to 48 hours, 30 minutes to 24 hours, 1 hour to 48 hours, or 1 hour to 24 hours after application. The irradiation time can be appropriately determined within the range of 5 seconds to 72 hours. Irradiation can be performed once or multiple times such that the cumulative irradiation time for each application of the composite of the present invention falls within the above-mentioned range. The duration of each irradiation session can be appropriately determined, for example, it can be 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.

[0182] In one embodiment, the preferred dose of irradiation to the patient is 1 J / cm². 2 The above, more preferably 5J / cm 2 The above is further preferred to be 10 J / cm. 2 The above, and preferably 1000 J / cm 2 The following is more preferably 500 J / cm 2 Hereinafter, 100 J / cm is further preferred. 2 The following is a further preferred value: 50 J / cm 2 The following, for example, are 1 to 1000 J / cm³ 2 1~500J / cm 2 5~200J / cm 2 10~100J / cm 2 Or 10-50 J / cm 2 The range.

[0183] In contrast to a single administration of the composite of the present invention, one or multiple irradiations can be performed. Therefore, irradiation can be completed in one session or repeated over several days. In the case of multiple irradiations, the conditions for each irradiation can be the same or different. The dosage, conditions, or method of irradiation can be varied depending on the type and state of the tumor.

[0184] 4.3. and combined therapy

[0185] The aforementioned light irradiation enables the composite of the present invention to exert a PTT-based tumor treatment effect. That is, light irradiation of the patient causes the composite of the present invention to generate heat, thereby killing tumor cells. Therefore, in a preferred embodiment, the treatment method of the present invention is to treat tumors using the composite or composition of the present invention in combination with CDT and PTT. Furthermore, when the composite of the present invention has photosensitive sites, the aforementioned light irradiation enables the composite of the present invention to exert a NIR-PIT-based tumor treatment effect. That is, NIR irradiation of the patient causes the photosensitive sites of the composite that bind to tumor cells to become hydrophobic, thereby causing the aggregation of the composite, which in turn damages the cell membrane of the tumor cells and kills the tumor cells. Therefore, in a preferred embodiment, the treatment method of the present invention is to treat tumors using the composite or composition of the present invention in combination with CDT and NIR-PIT, or in combination with CDT, NIR-PIT, and PTT.

[0186] The complex of the present invention, administered to the patient, specifically binds to the tumor cells of the therapeutic target via a target recognition molecule. The complex of the present invention, which specifically binds to tumor cells, kills the bound tumor cells through the mechanisms described above, such as CDT, or NIR-PIT and / or PTT. Therefore, the present invention enables the selective killing of tumor cells of the therapeutic target.

[0187] 4.4. Imaging

[0188] Because the nanoparticles contained in the composite of the present invention are magnetic particles containing iron oxide, they can be used for bioimaging using MRI. In one embodiment, the composite or composition of the present invention is used for imaging a patient or tumor. For example, the composite or composition of the present invention administered to a patient is used for tumor imaging before CDT treatment of the tumor. Tumor imaging can confirm the location of the composite in the patient's body or the location of the tumor to which the composite is bound, i.e., patient diagnosis is possible. Therefore, the timing of light irradiation for CDT, or further for NIR-PIT and / or PTT, the three-dimensional irradiation location and dose (irradiation time, dose) in the biological body can be optimized, improving the tumor treatment efficacy based on the composite of the present invention. MRI-based imaging of the patient or tumor can be performed using conventional methods.

[0189] 4.5. Other methods

[0190] The CDT and imaging methods using the composites or compositions of the present invention described above can be applied not only in vivo but also in vitro. For example, by administering and irradiating the composites or compositions of the present invention not only to tumors present in a patient's body but also to cultured tumor cells or cultured tissues containing tumor cells, the proliferation of tumor cells can be reduced or inhibited. The method of administering the composites or compositions of the present invention and the conditions of light irradiation can be appropriately varied according to the state of the cells or tissues being treated. For example, the composites or compositions of the present invention can be administered directly to tumor cells in a culture. The amount of the composites or compositions administered or the conditions of light irradiation can be selected to be milder than those for administration or irradiation to a patient.

[0191] Example

[0192] The present invention will be further described in detail below with reference to embodiments. However, the present invention is not limited to these embodiments or any other specific examples.

[0193] Experiment 1: Fabrication of the antibody-nanoparticle complex Nanomag-D-Spio-Pan-IR700

[0194] 1) Manufacturing of Pan-IR700

[0195] 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", manufactured by LI-COR Biosciences) in 0.2 mol / L Na2HPO4 (pH 8.5) at room temperature for 30–120 minutes. The mixture was purified using a Sephadex G50 column (PD-10: GE Healthcare, Piscataway, NJ). Protein concentration was determined using the CoomassiePlus Protein Assay Kit (Pierce Biotechnology, Rockford, IL) by measuring absorbance at 595 nm using a UV-Vis system (8453 Valuesystem: Agilent Technologies, Palo Alto, CA). The concentration of IR700 was determined by measuring absorbance using a UV-Vis system (Shimadzu UV-VIS). Approximately three molecules of IR700 were present per molecule of Pan. From now on, the Pan that incorporates IR700 will be referred to as Pan-IR700.

[0196] 2) Biotinylation of Pan-IR700

[0197] 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 Pan-IR700 solution (2.0 mg / mL) was collected into a microtube, and 8 μL of the pre-prepared Biotin-NHS DMSO solution was added ([Biotin-NHS] / [Pan-IR700] = 10). The solution was allowed to stand 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 using Duchenne phosphate-buffered saline (Wako Purified Pharmaceuticals, hereinafter also referred to as D-PBS) to obtain biotinylated Pan-IR700. Hereinafter, biotinylated Pan-IR700 will be referred to as Pan-IR700-Biotin.

[0198] 3) Fabrication of the Pan-IR700-magnetic nanoparticle composite

[0199] 120 mg (5 mg / mL, 24 mL) of Nanomag-D-Spio 79-19-201 (manufactured by Micromod, streptavidin-modified magnetic particles, 20 nm in diameter, hereinafter also referred to as "Nanomag-D-Spio") was collected in a 50 mL tube, and 1.8 mg (1.9 mg / mL, 947 μL) of Pan-IR700-Biotin was added. The mixture was stirred at room temperature for 60 minutes. Then, 18 μg (0.1 mg / mL, 180 μL) of biotin (manufactured by Wako Pure Chemicals Co., Ltd.) was added, and the mixture was stirred at room temperature for 30 minutes. This yielded a dispersion of an antibody-nanoparticle complex (also referred to as "Nanomag-D-Spio-pan-IR700") containing Pan-IR700-Biotin and Nanomag-D-Spio. 1 mL of this dispersion was passed through a column (MS-columns, Miltenyi Biotec Co., Ltd.) mounted on a magnetic rack. Then, 2 mL of D-PBS was passed through the column. The filtrate was irradiated with excitation light at a wavelength of 676 nm, and fluorescence was measured at 700 nm. The results confirmed that no unreacted Pan-IR700-Biotin was detected.

[0200] 4) Fabrication of the antibody-nanoparticle complex Nanomag-D-Spio-Cont

[0201] The antibody-nanoparticle complex (Nanomag-D-spio-cont-IR700) was manufactured by replacing Pan with a ligand of a tyrosine kinase receptor not expressed in MDAMB468 and A431, following the same steps as described in 1) to 3) above.

[0202] 5) Fabrication of the antibody-nanoparticle complex Nanomag-D-Spio-Pan

[0203] Instead of Pan-IR700-Biotin, the biotinylated Pan (Pan-Biotin) obtained by the same method as in 2) above was used. In addition, the antibody-nanoparticle complex (Nanomag-D-Spio-Pan) was manufactured by the same steps as in 3).

[0204] Experiment 2 Evaluation of Nanomag-D-Spio-pan-IR700

[0205] The antibody-nanoparticle complex Nanomag-D-Spio-pan-IR700 and the Nanomag-D-Spio nanoparticles prepared in Experiment 1 were subjected to ZETA potential measurement, atomic force microscopy (AFM) observation, and vibrating sample magnetometer (VSM) measurement. The ZETA potential was determined by diluting the dispersions of Nanomag-D-Spio-pan-IR700 and Nanomag-D-Spio (0.1 mg / ml KCl, pH 7, 25℃) using a Zetasizer. TM The ZETA potential was obtained by measuring it (by Malvern Panalytical Ltd). The measurement results of the ZETA potential are shown below. Figure 1 The AFM images and the dimensional measurements and inferred shapes based on the AFM images are shown in the figure. Figure 2 The results of the VSM measurement are shown in... Figure 3 According to AFM images, Nanomag-D-Spio is spherical, while Nanomag-D-Spio-pan-IR700 has an uneven shape.

[0206] Experiment 3 Cell binding of Nanomag-D-Spio-pan-IR700

[0207] The binding affinity of the antibody-nanoparticle complexes manufactured in Experiment 1 to cells was evaluated. MDAMB468 and A431 were used as disease model cells, and H661 and 3T3 were used as normal cells. MDAMB468 were EGFR-expressing human breast cancer cells, A431 were EGFR-expressing human epidermal carcinoma cells, H661 were EGFR-non-expressing human lung epithelial-like cells, and 3T3 were EGFR-non-expressing mouse embryonic fibroblasts. The culture medium used for cell culture was RPMI 1640 (basal medium) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. Cells were seeded onto culture plates (1×10⁻⁶ cells / cm²). 5 Cells were incubated in wells for 24 hours (37°C, 5% CO2). Next, the complex (pan-IR700 or Nanomag-D-Spio-pan-IR700, 10 μg / mL) was added to the wells and incubated for 6 hours (37°C, 5% CO2). Cells without the complex served as a control. IR700 (wavelength 698 nm) was used as a fluorescent label, and the fluorescence intensity of the incubated cells was measured by flow cytometry.

[0208] The results of the fluorescence intensity measurement are shown in Figure 4 (A). In Figure 4 In (A), "Pan blocking" refers to the fluorescence intensity of cells incubated in the presence of an EGFR inhibitor (panitumumab, a tyrosine kinase inhibitor). Here, as described above, cells were seeded onto culture plates and incubated for 24 hours (37°C, 5% CO2). Next, the EGFR inhibitor was placed into the wells, followed by the complex, and incubation for 6 hours (37°C, 5% CO2). Figure 4 As shown in (A), EGFR-positive cells MDAMB468 and A431 specifically bind to pan-IR700 or Nanomag-D-Spio-pan-IR700.

[0209] Instead of pan, an antibody (IgG antibody)-nanoparticle complex (Nanomag-D-spio-cont-IR700) was used to culture MDAMB468 and A431 cells in the presence of the complex, following the same steps as described above. The fluorescence intensity of the cells was then measured by flow cytometry. The results of the fluorescence intensity measurements are shown below. Figure 4 (B). For example Figure 4 As shown in (B), the fluorescence intensity of Nanomag-D-spio-cont-IR700 was not different from that of the control. According to... Figure 4 The results of (B) indicate that Nanomag-D-Spio-cont-IR700 does not bind to MDAMB468 and A431.

[0210] Experiment 4: Blood stability of Nanomag-D-Spio-pan-IR700

[0211] Place 100 mL of Nanomag-D-Spio-pan-IR700 solution (5 μg / mL) diluted in PBS and 100 mL of sodium dodecyl sulfate (SDS) solution (1% by weight) diluted in PBS or 400 mL of mouse serum into microtubes and incubate for 6 hours. Use IR700 (wavelength 698 nm) as a fluorescent label and measure the fluorescence intensity of the incubated solution. Use Nanomag-D-Spio-pan-IR700 solution without SDS or serum as a control. The results are shown below. Figure 5 (A) and (B). The fluorescence intensity from Nanomag-D-Spio-pan-IR700 was approximately 2.2 times that of the control in SDS, but there was no difference in serum compared to the control.

[0212] Experiment 5: Uptake of Nanomag-D-Spio-pan-IR700 via endocytosis

[0213] A431 cells were seeded onto culture plates (1×10⁶ cells per plate). 5 Cells / wells, culture medium same as in Experiment 3), incubated for 24 hours (37℃, 5% CO2). Nanomag-D-Spio-pan-IR700 was added to the wells at a concentration of 10 μg / mL, and after incubation for 6 hours (37℃, 5% CO2), the cells were stained with fluorescence (Hurster staining, lysosomal staining (green)) for fluorescence observation. Figure 6 The image shows a fluorescence pattern. Figure 6 In this text, "DIC" represents a bright-field image obtained using an optical microscope, "Hoechst", "Lyso tracer" and "IR700" represent Hoechst staining, lysosomal staining and fluorescence images labeled with IR700, respectively, and "Merge" represents a superimposed image of the various fluorescence images.

[0214] Experiment 6: Reaction of Nanoparticles with Methylene Blue

[0215] Nanomag-D-Spio (0.5 mg / mL) was placed in a microtube and heated at 37°C for 30 minutes, with or without reduced glutathione (GSH, 10 mM). Methylene blue (10 μg / mL) was added to the heated tube along with hydrogen peroxide (1 mM), or methylene blue (10 μg / mL) was added without hydrogen peroxide. The solution in the microtube was adjusted to pH 5.4 with PBS containing HCl, and the absorbance was measured (500–800 nm). The solution was then further irradiated with a laser (690 nm, 200 J / cm²). 2 The absorbance was measured (500–800 nm) over a period of 2 hours. The absorbance results are shown below. Figure 7 (A).

[0216] Similarly, Nanomag-D-Spio (20 μg / mL) and reduced glutathione (10 mM) were placed in microtubes, heated at 37°C for 0–120 minutes, and then methylene blue (10 μg / mL) and hydrogen peroxide (20 μM) were added. The solution in the microtubes was adjusted to pH 5.4 with PBS containing HCl, and the absorbance was measured (500–800 nm). The results are presented as follows. Figure 7 (B).

[0217] Experiment 7. Heating behavior of antibody-nanoparticle complex induced by near-infrared radiation

[0218] Place a liquid containing Nanomag-D-Spio-pan-IR700 (0.1–10 mg / mL) or pan-IR700 (10 mg / mL) into a microtube and irradiate with a laser (690 nm, 500 mW / cm²). 2 The liquid temperature inside the microtube was measured. The temperature rise curves of the complex-containing liquids relative to the irradiation dose are shown in the figure. Figure 8 (A). Additionally, infrared thermal imaging images of the liquid containing Nanomag-D-Spio-pan-IR700 after laser irradiation and the liquid temperature determined based on thermal imaging are shown in... Figure 8 (B). It should be noted that the power conversion efficiency (PCE) based on laser irradiation was measured, and the PCE result was 29.21%. Based on these results, it is indicated that the nanoparticles contained in the antibody-nanoparticle complex generate heat upon near-infrared irradiation.

[0219] Experiment 8. Antitumor activity of antibody-nanoparticle complex in vitro

[0220] A431, MDAMB468, and PC-9 cells were used as disease model cells. A431 and MDAMB468 were the disease model cells described in Experiment 3, and PC-9 was a human lung adenocarcinoma cell line expressing EGFR. Cells were seeded onto culture plates (1 × 10⁶ cells per cell line). 5 Cells / wells, culture medium same as in Experiment 3), incubated for 24 hours (37℃, 5% CO2). Nanomag-D-Spio-pan-IR700 was added to the wells at a concentration of 10 μg / mL, and incubated for 24 hours (37℃, 5% CO2). The culture medium was replaced with PBS, and the cells / wells were irradiated with a laser (690 nm, 4 mJ / cm²). 2 Cells were fluorescently stained before and after laser irradiation (Hurst staining, pyridine iodide staining). (PI) staining was performed for fluorescence observation. Figure 9 The image shows a fluorescence pattern. Figure 9 In this text, "DIC" represents a bright-field image obtained using an optical microscope, "Hoechst" and "PI" represent fluorescence images obtained by Hoechst staining and PI staining, respectively, and "Merge" represents a superimposed image of the fluorescence images.

[0221] Experiment 9 Antibody-nanoparticle complex antitumor activity against in vitro spheroids

[0222] 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. Otherwise, cells for laser irradiation were prepared using the same procedures as in Experiment 8. A431, MDAMB468, and PC-9 were the same disease model cells used in Experiment 8. 3T3-RFP cells were prepared by transfecting the red fluorescent protein (RFP) gene (as a reporter gene) into the 3T3 cells used in Experiment 3. The spheroids were stained with SYTOX before and after laser irradiation. TM (Blue staining) was performed for fluorescence observation. Figure 10A and Figure 10B The image shows a fluorescence pattern. Figure 10A The image shows a fluorescence image of a spherical body obtained by mixing A431 with 3T3-RFP in a 1 / 1 (volume ratio). Figure 10BThe figures show fluorescence images of spherical particles obtained by mixing MDAMB468 with 3T3-RFP in a 1 / 1 (volume ratio) ratio and spherical particles obtained by mixing PC-9 with 3T3-RFP in a 1 / 1 (volume ratio) ratio. In the figures, "DIC" represents a bright-field image obtained using an optical microscope, "RFP" represents a fluorescence image with reporter RFP taken up in 3T3-RFP as a fluorescent label, "IR700" represents a fluorescence image with IR700 as a fluorescent label, and "SYTOX Blue" represents SYTOX. TM The images are blue-stained fluorescence images, with "Merge" indicating a superimposed image of the individual fluorescence images. This shows that Nanomab-D-Spio-IR700 binds only to tumor cells, and that the fluorescence from IR700 is quenched after laser irradiation. Furthermore, tumor cells bound to Nanomab-D-Spio-IR700 undergo cell death.

[0223] Experiment 10: Antitumor activity of antibody-nanoparticle complex in vivo

[0224] according to Figure 11A The experimental protocol shown evaluates the antitumor activity of the antibody-nanoparticle complex in vivo. Disease model cells (A431, 6 × 10⁻⁶) were used. 6 (cell / 100μL) was subcutaneously injected into the right groin area of ​​female homozygous athymic nude mice. Figure 11A On Day 10, mice were prepared for xenograft modeling. Mice were anesthetized, and PBS or a complex (pan-IR700, Nanomag-D-Spio-pan, or Nanomag-D-Spio-pan-IR700 as controls) was administered intravenously via the tail vein at a rate of 80 μL / body (equivalent to 30 μg / body for antibody conversion). Figure 11A (Middle, Day-1). From the second day ( Figure 11A From Day 0 onwards, the affected area is irradiated with laser (690nm, 200mJ / cm²) 5 times daily. 2 / times, in Figure 11(A) (a) to (e)). In Figure 11A The timing indicated by "BLI" (arrow in the figure) is used to evaluate the size of the irradiated tumor by bioluminescent imaging (BLI). Figure 11B The image shown is of a xenograft model mouse as observed by luciferase detection. Figure 11CThis is a graph (n=6) representing changes in tumor size evaluated based on luciferase detection images. The relative light units (RLU) of the region of interest in the tumor image at Day 0 are set to 100, and the changes in tumor size are expressed as RLU ratios. Figure 11D This is a graph showing the change in tumor volume as measured after tumor removal (n=10). All complexes showed a trend of reducing tumor volume in laser-irradiated mice after administration of the complex, with mice given Nanomag-D-Spio-pan-IR700 showing a significant reduction in tumor volume (Dunnet test with Pan-IR700+NIR-light as the comparison group, p<0.05).

[0225] Experiment 11 Anti-tumor activity of antibody-nanoparticle complex against in vivo spheroids

[0226] according to Figure 12A The experimental protocol shown evaluates the antitumor activity of the antibody-nanoparticle complex against in vivo spheroids. Spheroids (6 × 10⁶ cells / year) were prepared by mixing antigen-presenting cells (A431) and non-antigen-presenting cells (H661-luc-GFP) at a 1:1 (volume ratio). 6 (cell / 100μL). A431 was the same disease model cell used in Experiment 3. H661-luc-GFP was a cell obtained by transfecting the luciferase gene (as a reporter gene) and the green fluorescent protein (GFP) gene into the H661 cells used in Experiment 3. The prepared spheroids were subcutaneously injected into the groin area on both sides of female homozygous athymic nude mice. Figure 12A (Day 8) Mice were prepared for xenograft modeling. Mice were anesthetized, and PBS or a complex (pan-IR700, Nanomag-D-Spio-pan, or Nanomag-D-Spio-pan-IR700 as controls) was administered intravenously via the tail vein at a rate of 80 μL / body (equivalent to 30 μg / body for antibody conversion). Figure 12A (Middle, Day-1). From the second day ( Figure 12A From Day 0 onwards, the affected area near the left groin will be irradiated with laser (690nm, 200mJ / cm²) 5 times daily. 2 / times). In Figure 12A The timing indicated by "BLI" (arrow in the figure) is used to evaluate the size of the irradiated tumor by luciferase assay (BLI). Figure 12B The image shows a xenograft model mouse as observed by luciferase detection. Figure 12CThis is a graph (n=6) showing the changes in tumor size as evaluated based on luciferase detection images. The RLU of the region of interest in the tumor image at Day 0 is set to 100, and the changes in tumor size are expressed as RLU ratios. Figure 12D This is a graph showing the change in tumor volume after tumor removal, with the tumor size at Day 0 set as 100 (n=6). In mice treated with Nanomag-D-Spio-pan-IR700, the tumor volume was significantly reduced at the laser irradiation site (Dunnet test with Pan-IR700+NIR-light as the comparison group, p<0.05).

[0227] Experiment 12 Tumor Imaging Using Antibody-Nanoparticle Complexes

[0228] Following the same procedures as in Experiment 10, Nanomag-D-Spio-pan-IR700 was administered intravenously via the tail vein to A431 xenograft model mice, and tumors were imaged using MRI. As a control, mice were administered PBS intravenously instead of Nanomag-D-Spio-pan-IR700. The magnetic resonance intensity of the obtained MRI images is shown below. Figure 13 .

[0229] Experiment 13 Fabrication of Antibody-Nanoparticle Complex (Nanomag-D-Spio-Tra)

[0230] The antibody-nanoparticle complex (Nanomag-D-Spio-Tra) was manufactured using the same steps as in Experiment 1, 3), instead of Pan-IR700-Biotin, with the biotinylated human monoclonal antibody trastuzumab (Tra-Biotin) obtained by the same method as in Experiment 1, 2).

[0231] Experiment 14: Reaction of Nanoparticles with Methylene Blue

[0232] Place Nanomag-D-Spio-Pan (0.02 mg / mL) or Nanomag-D-Spio-Tra (0.02 mg / mL) in a microtube and heat at 37°C for 30 minutes, with or without reduced glutathione (GSH, 10 mM). Add methylene blue (10 μg / mL) to the heated tube along with hydrogen peroxide (1 mM), or add methylene blue (10 μg / mL) without hydrogen peroxide. Adjust the pH of the solution in the microtube to 5.4 using PBS containing HCl and measure the absorbance (500–800 nm). Further irradiate the solution with a laser (690 nm, 10 J / cm²). 2 The absorbance was measured (500–800 nm) over a period of 3 hours. The absorbance results are shown below. Figure 14 (A) (Nanomag-D-Spio-Pan) and Figure 14 (B) (Nanomag-D-Spio-Tra). Additionally, Table 1 shows the absorbance of the nanoparticle composite at a wavelength of 662 nm as measured in Experiments 6 and 14.

[0233] [Table 1]

[0234]

Claims

1. A complex for chemokinetic therapy of tumors, The complex comprises nanoparticles and target recognition molecules bound to the nanoparticles. The nanoparticles contain iron oxide and have a hydrophilic polymer on their surface. The target recognition molecule can bind to the target molecules of tumor cells.

2. The composite according to claim 1, wherein, The chemokinetic therapy is based on a photo-Fenton reaction generated by near-infrared light irradiation.

3. The composite according to claim 1, further used in photothermal therapy.

4. The composite according to claim 1, further used for tumor imaging.

5. The composite according to claim 1, further comprising a photosensitive region bound to the nanoparticles or the target recognition molecule, the photosensitive region comprising a photosensitive group having a maximum absorption wavelength of 500-1500 nm and one or more hydrophilic functional groups connected or coordinated to the photosensitive group.

6. The composite according to claim 5, wherein, The photosensitive site binds to the target recognition molecule.

7. The complex according to claim 5, further used in photoimmunotherapy.

8. The composite according to claim 1, wherein, The target recognition molecule is an antibody.

9. The composite according to claim 1, wherein, The hydrophilic polymer is a polysaccharide.

10. The composite according to claim 9, wherein, The polysaccharide is dextran.

11. The composite according to claim 1, wherein, The iron content in the composite is 30% by mass or more.

12. The composite according to claim 1, wherein, The number of binding molecules to the nanoparticles is 1 to 20 per nanoparticle.

13. The composite according to claim 1, wherein, The target molecules on the tumor cells are epidermal growth factor receptor, EGF receptor family, or platelet-activating receptor.

14. A composition for chemokinetic therapy of tumors, comprising the complex according to any one of claims 1 to 13.

15. The composition according to claim 14, further comprising hydrogen peroxide.

16. The composition according to claim 14, further comprising an in vivo reducing substance.

17. The composition according to claim 14, further used in photothermal therapy.

18. The composition according to claim 14, further used for tumor imaging.

19. The composition according to claim 14, wherein, The composite has a photosensitive region comprising a photosensitive group having a maximum absorption wavelength of 500–1500 nm and one or more hydrophilic functional groups connected to or coordinated with the photosensitive group.

20. The composition according to claim 19, further used in photoimmunotherapy.

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