Conjugates and Photoimmunotherapy

Abstract

To provide an agent useful for photoimmunotherapy.SOLUTION: A complex has one or more antibody molecules, a particle with an average particle size of 100 nm or less, and one or more photosensitive portions. In the complex, the particle with the average particle size of 100 nm or less is bound to the one or more antibody molecules. At least one of the antibody molecule and the particle is bound to the one or more photosensitive portions. The photosensitive portion is a portion that increases hydrophobicity upon irradiation with a light beam of 500-900 nm in wavelength or that includes a phthalocyanine skeleton.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a conjugate and photoimmunotherapy using the same. [Background technology]

[0002] Near-infrared photoimmunotherapy (NIR-PIT) has attracted attention as an innovative cancer treatment (see, for example, Patent Documents 1 and 2 and Non-Patent Document 1). NIR-PIT is a treatment method that involves preparing an antibody conjugate containing a near-infrared-sensitive photosensitizer for an antibody specific to an antigen on the surface of tumor cells, binding the antibody conjugate to tumor cells, and locally irradiating them with near-infrared light to selectively kill the tumor cells. The photosensitizer mainly used is a chemical species containing a phthalocyanine skeleton (e.g., so-called IR700 molecule, such as IRDye700DX). NIR-PIT is advantageous in terms of its high light penetration into the patient's body and its minimal invasiveness. However, further improvements in the tumor cell killing effect and imaging performance of NIR-PIT are desired.

[0003] Previously, the mechanism of cell death caused by photosensitizers was thought to be primarily oxidative stress due to free radicals. However, the detailed mechanism of tumor cell death by NIR-PIT was unclear. However, it has recently been elucidated that the mechanism of tumor therapeutic effect of NIR-PIT is that the photosensitizer in the antibody conjugate bound to the tumor cell surface becomes hydrophobic through a photochemical reaction, causing aggregation of the antibody conjugate, damaging the cell membrane of the tumor cell, which creates an osmotic pressure difference between the inside and outside of the cell, inducing cell death (Non-Patent Document 1).

[0004] In the fields of medicine and biology, imaging is an important technology that is applied to the creation of medical images, biosensors, etc. In recent years, imaging using quantum dots has attracted attention because of their advantages such as high luminescence quantum yield, wide absorption range, and resistance to bleaching. Patent Document 3 describes semiconductor nanoparticles with a core-shell structure having a core and a shell surrounding the core, in which the core is (AgIn) xZn 2(1-x) S2 (where x satisfies 0.4≦x≦0.95), the shell is made of ZnS or ZnO, and the shell surface has hydrophilic functional groups, and a fluorescent probe for labeling biological samples using the same is described. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Special Publication No. 2014-523907 [Patent Document 2] Special Publication No. 2019-218374 [Patent Document 3] Japanese Patent Application Laid-Open No. 2014-185224 [Non-patent literature]

[0006] [Non-Patent Document 1] The 40th Annual Meeting of the Japan Society for Laser Surgery and Medicine Award winning paper, "Elucidation of the mechanism of near-infrared photoimmunotherapy," Journal of the Japan Society for Laser Surgery and Medicine, Vol. 41, No. 2, 2020, pp. 104-109 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention provides an agent for photoimmunotherapy (PIT) with improved tumor cell killing effect and imaging performance, and a method for treating tumors by PIT using the agent. [Means for solving the problem]

[0008] The present inventors have found that a complex containing particles with an average particle size of 100 nm or less in addition to an antibody molecule and a photosensitizer is excellent in tumor imaging and PIT therapeutic effects.

[0009] Thus, the present invention provides the following: [1] A complex comprising an antibody molecule and a particle having an average particle diameter of 100 nm or less bound to the antibody molecule, wherein at least one of the antibody molecule and the particle is bound to a moiety whose hydrophobicity increases upon irradiation with light having a wavelength of 500 to 900 nm. [2] A conjugate comprising antibody molecules and particles having an average particle size of 100 nm or less bound to the antibody molecules, wherein at least one of the antibody molecules and the particles is bound to a moiety containing a phthalocyanine skeleton. (Hereinafter, the above-mentioned "moiety whose hydrophobicity increases upon irradiation with light having a wavelength of 500 to 900 nm" and "moiety containing a phthalocyanine skeleton" are collectively referred to as "photosensitive moiety.") [3] The complex according to [1] or [2], wherein the particle is a magnetic particle or a semiconductor particle. [4] The conjugate according to [1] or [3], wherein the antibody molecule is bound to a site whose hydrophobicity increases upon irradiation with light having a wavelength of 500 to 900 nm. [5] The conjugate described in [2] or [3], wherein the antibody molecule is bound to a site containing the phthalocyanine skeleton. [6] The complex according to [2], [3] or [5], wherein the moiety containing a phthalocyanine skeleton is a compound represented by the following formula (Ia): [ka] During the ceremony: L is a direct bond or a linker; Q is a reactive group for forming a bond with the antibody molecule or the particle; R 2 , R 3 , R 7 , and R 8 are each independently selected from substituted or unsubstituted alkyl and substituted or unsubstituted aryl; R 4 , R 5 , R 6 , R 9 , R 10 , and R 11are, when present, each independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkanoyl, substituted or unsubstituted alkoxycarbonyl, substituted or unsubstituted alkylcarbamoyl, and chelating ligands, wherein R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 at least one of which contains a water-soluble group; R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , and R 23 are each independently selected from hydrogen, halogen, substituted or unsubstituted alkylthio, substituted or unsubstituted alkylamino, and substituted or unsubstituted alkoxy; or i) R 13 and R 14 and the carbon to which they are attached, ii) R 17 and R 18 and the carbon to which they are attached, and iii) R 21 and R 22 and the carbon to which they are attached form a fused ring; and X 2 and X 3 are each independently a C1-C bond with or without a heteroatom between the carbon-carbon bonds. 10 It is alkylene. [7] The conjugate of [6], wherein the compound represented by formula (Ia) is a compound represented by formula (Ib): [ka] During the ceremony, X 1 and X 4 are each independently a C1-C substituted or unsubstituted heteroatom-interposed group. 10 alkylene; and 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 is as defined above. [8] The complex of [6], wherein the compound represented by formula (Ia) is a compound represented by formula (II): [ka] [9] The composite according to any one of [3] to [8], wherein the magnetic particles contain an iron oxide or a gadolinium compound.

[10] The composite according to any one of [3] to [8], wherein the semiconductor particles are quantum dots.

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

[10] , wherein the particles have an average particle size of 1 to 50 nm.

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

[11] , wherein the number of antibody molecules bound to each particle is 1 to 20 per particle.

[13] A composition comprising the complex according to any one of [1] to

[12] .

[14] The conjugate according to any one of [1] to

[12] , for use in treating a tumor by photoimmunotherapy.

[15] The complex according to

[14] , further for use in tumor imaging.

[16] The composition described in

[13] for use in treating tumors by photoimmunotherapy.

[17] The composition described in

[16] , further for use in tumor imaging.

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

[12] in the manufacture of a tumor therapeutic agent by photoimmunotherapy.

[19] Use of the conjugate described in

[18] , wherein the tumor therapeutic agent is further used for tumor imaging.

[20] A method for treating a tumor, comprising: A step of administering the complex according to any one of [1] to

[12] or the composition according to

[13] to a patient; and irradiating the patient with light having a wavelength of 500 to 900 nm; A method comprising:

[21] The method described in

[20] , further comprising a step of imaging the tumor in a patient to which the complex or composition has been administered prior to the step of irradiating with light.

[22] A tumor therapeutic agent for photoimmunotherapy, comprising the conjugate according to any one of [1] to

[12] .

[23] The tumor therapeutic agent according to

[22] , which is an agent for tumor imaging and tumor treatment by photoimmunotherapy. [Effects of the Invention]

[0010] The complex of the present invention has a high tumor cell killing effect and imaging performance, and can improve the efficacy of tumor treatment by PIT. [Brief explanation of the drawings]

[0011] [Figure 1] Fluorescence imaging of a tumor cell-injected mouse administered Nanomag-Pan-IR700. Fluorescence images of the entire mouse body are shown from before administration to 48 hours after administration. The arrow indicates the injection site of the tumor cells. [Figure 2] Fluorescence imaging of a mouse with tumor cells implanted and administered QDs800-Pan-IR700. Fluorescence images of the entire mouse body are shown from 1 minute (1 min) to 90 minutes (90 min) after administration. The dotted box indicates the location of tumor cell injection. [Figure 3] Fluorescence imaging of a mouse transplanted with tumor cells administered with the complex of Comparative Example 1. Fluorescence images of the whole body of the mouse are shown 6 hours, 1 day, and 3 days after administration. [Figure 4]In vitro inhibition of tumor cell survival by PIT. The horizontal axis of each graph represents the time from administration of the complex, and the vertical axis represents cell viability. [Figure 5] MRI imaging of tumor cell-injected mice administered Nanomag-Pan-IR700. MRI images are shown 9 and 24 hours after administration. The arrows indicate the location of tumor cell engraftment. DETAILED DESCRIPTION OF THE INVENTION

[0012] As used herein, the term "tumor" refers to tumors in general, including benign and malignant tumors, as well as epithelial and non-epithelial tumors, and the site (tissue and organ) of development is not particularly limited. Preferably, the tumor targeted by the present invention is a malignant tumor such as cancer. Cancer may be a liquid tumor or a solid tumor, and may include any type of cancer, such as epithelial cancer, adenocarcinoma, sarcoma, or malignant lymphoma. Examples of tumors include liquid tumors, including acute leukemia (acute lymphocytic leukemia, acute myeloid leukemia, myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia), chronic leukemia (chronic myeloid (granulocytic) leukemia, chronic lymphocytic leukemia, and hairy cell leukemia), T-cell prolymphocytic leukemia, large granular lymphocytic leukemia, adult T-cell leukemia, polycythemia vera, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease; as well as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, other sarcomas, synovial tumors, mesothelioma, Ewing's tumor, and hepatocellular carcinoma. Solid tumors include leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, lung cancer, colorectal cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma (e.g., adenocarcinoma of the pancreas, colon, ovary, lung, breast, stomach, prostate, cervix, or esophagus), sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, bladder cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, etc. The tumor herein may be a primary tumor or a recurrent tumor.

[0013] As used herein, the term "tumor affected area" or "affected area" refers to tumor tissue in which tumor cells are primarily present. Such tumor tissue includes tissue consisting of tumor cells and tissue in which tumor cells are mixed with normal cells or normal tissue. When normal cells or normal tissue are mixed with the tumor tissue, the ratio of the volume or number of tumor cells to the normal cells or normal tissue is not particularly limited.

[0014] As used herein, the term "antibody" refers to a polypeptide ligand comprising at least one light chain variable region and / or heavy chain variable region that specifically recognizes and binds to an antigen epitope. For example, the term "antibody" as used herein includes immunoglobulins of any class, such as IgG, IgA, IgD, IgE, IgM, and their subclasses, as well as variants thereof. It also includes chimeric antibodies such as humanized antibodies and other modified immunoglobulins that contain an antigen-recognition site. Furthermore, the term "antibody" as used herein includes immunoglobulin fragments or domains that contain an antigen-recognition site, such as Fab fragments, Fab' fragments, F(ab)' fragments, single-chain Fvs ("scFvs"), disulfide-stabilized Fvs ("dsFvs"), VHHs (variable domains of heavy chains of heavy chain antibodies), and VNARs (single variable new antigen receptor domain antibodies).

[0015] 1. Complex In one aspect, the present invention provides a conjugate for use as a drug for photoimmunotherapy (PIT). The conjugate provided by the present invention comprises an antibody molecule, a particle having an average particle size of 100 nm or less (hereinafter also referred to as a "core particle"), and a photosensitive moiety. In the conjugate, the core particle is bound to the antibody molecule, and at least one of the antibody molecule and the particle is bound to the photosensitive moiety.

[0016] 1.1.Antibody molecules The antibody molecule contained in the complex of the present invention is an antibody molecule that binds to target tumor cells. The type of antibody can be appropriately selected depending on the antigen present on the surface of the target tumor cells. Examples of such antigens include proteins, lipids, polysaccharides, and nucleic acids, and preferred examples include cell surface proteins present on the surface of the target tumor cells.

[0017] Examples of such cell surface proteins include tumor-specific proteins (also known in the art as tumor-specific antigens) that are expressed on the surface of tumor cells. Tumor-specific proteins are proteins that are unique to cancer cells or that are more abundant in cancer cells compared to other cells, such as normal cells.

[0018] Examples of tumor-specific proteins include members of the epidermal growth factor receptor (EGFR) family (e.g., HER1, 2, 3, and 4), members of cytokine receptors (e.g., CD20, CD25, IL-13R, CD5, CD52, etc.), etc. For example, HER2 is found primarily in breast cancer, and HER1 is found primarily in adenocarcinomas found in many organs, such as the pancreas, breast, prostate, and colon.

[0019] Specific examples of 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), which is associated with breast cancer, ovarian cancer, gastric cancer, and uterine cancer; HER-1 (e.g., GenBank accession numbers NM_005228 and NP_005219), which is associated with lung cancer, anal cancer, and glioma, as well as adenocarcinoma.

[0020] Other specific examples of tumor-specific proteins include CD52, which is associated with chronic lymphocytic leukemia (e.g., GenBank accession numbers AAH27495.1 and CAI15846.1); CD33, which is associated with acute myeloid leukemia (e.g., GenBank accession numbers NM_023068 and CAD36509.1); CD20, which is associated with non-Hodgkin's lymphoma (e.g., GenBank accession numbers NP_068769 and NP_031667); and the like.

[0021] Other specific examples of tumor-specific proteins include any of the various MAGE (melanoma-associated antigen E) proteins, 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 numbers U01873 and AAB60319); mutant ras; mutant p53 (e.g., GenBank accession numbers X54156, CAA38095, and AA494311); p97 melanoma antigen (e.g., GenBank accession numbers M12154 and AAA59992); and human milk fat associated with breast tumors. Examples of such antigens include human melanoma-associated antigens (HMFG) (e.g., GenBank accession numbers S56151 and AAB19771); any of the various BAGEs (human melanoma type B associated antigen E), including BAGE1 (e.g., GenBank accession number Q13072) and BAGE2 (e.g., GenBank accession numbers NM_182482 and NP_872288); gp100 associated with melanoma (e.g., GenBank accession numbers S73003 and AAC60634); the MART1 antigen associated with melanoma (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 GAGEs 2-6; various gangliosides; and CD25 (e.g., GenBank accession numbers NP_000408.1 and NM_000417.2).

[0022] Other specific examples of 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); CEA (carcinoembryonic antigen) associated with colorectal cancer (e.g., GenBank accession numbers X98311 and CAA66955); cancer antigen 125 (CA125, also known as mucin 16 or MUC16) associated with ovarian 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; PSA antigen associated with prostate cancer (e.g., GenBank accession numbers X14810 and CAA32915).

[0023] Other specific examples of tumor-specific proteins include PMSA (prostate membrane-specific antigen; e.g., GenBank accession numbers AAA60209 and AAB81971.1), which is associated with prostate cancer; NY-ESO-1 (e.g., GenBank accession numbers U87459 and AAB49693), which is 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)); protein 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)); Other specific examples of tumor-specific proteins include PD-L1, PD-L2, and the like, which are associated with immune checkpoints.

[0024] The names of the tumor-specific proteins described herein are according to the GenBank database of the National Center for Biotechnology Information (NCBI) ([www.ncbi.nlm.nih.gov / genbank / ]).

[0025] Examples of antibodies that may be included in the conjugates of the present invention include cetuximab, panitumumab, zalutumumab, nimotuzumab, trastuzumab, Ado-trastuzumab emtansine, tositumomab, rituximab, ibritumomab tiuxetan, daclizumab, gemtuzumab, alemtuzumab, CEA-scan Fab fragment, OC125 monoclonal antibody, ab75705, B72.3, bevacizumab, afatinib, axitinib, bosutinib, cabozantinib, ceritinib, crizotinib, dabrafenib, dasatinib, erlotinib, everolimus, ibrutinib, imatinib, lapatinib, lenvatinib, nilotinib, olaparib, palbociclib, pazopanib, pertuzumab, ramuciclib lumab, regorafenib, ruxolitinib, sorafenib, sunitinib, temsirolimus, trametinib, vandetanib, vemurafenib, vismodegib, basiliximab, ipilimumab, nivolumab, pembrolizumab, MPDL3280A, pidilizumab (CT-011), MK-3475, BMS-936559, MPDL3280A (atezolizumab), tremelimumab, IM P321, BMS-986016, LAG525, urelumab, PF-05082566, TRX518, MK-4166, dacetuzumab (SGN-40), lucatumumab (HCD122), SEA-CD40, CP-870, CP-893, MEDI6469, MEDI6383, MEDI4736, MOXR0916, AMP-224, PDR001, avelumab (MSB0010 718C), rHIgM12B7, urocupulumab, BKT140, varlilumab (CDX-1127), ARGX-110, MGA271, lirilumab (BMS-986015, IPH2101), IPH2201, AGX-115, emactuzumab, CC-90002, and MNRP1685A, and fragments containing the antigen recognition site thereof.

[0026] 1.2.Core particle Surprisingly, the conjugate of the present invention can enhance the tumor cell killing ability of the conjugate by binding the above-mentioned antibody molecule to the core particle. Furthermore, by selecting a magnetic particle or a fluorescent particle as the core particle, in vivo imaging becomes possible, enabling not only treatment of tumors in patients but also diagnosis (e.g., confirmation of the location of the conjugate or confirmation of the location of the tumor to which the conjugate is bound). In vivo imaging using this conjugate allows optimization of the timing of near-infrared irradiation for PIT, the three-dimensional irradiation position in the body, and the irradiation amount (irradiation time, dose), thereby enhancing the therapeutic effect of PIT.

[0027] The core particles used in the complex of the present invention have an average particle diameter of 100 nm or less. The "average particle diameter" of particles herein refers to the average particle diameter of 100 particles randomly measured in an electron microscope image. If the average particle diameter of the core particles exceeds 100 nm, it becomes difficult for the complex to selectively distribute to tumor cells. The average particle diameter of the core particles is preferably 70 nm or less, more preferably 50 nm or less, and even more preferably 25 nm or less. The lower limit of the average particle diameter of the core particles is not particularly limited, but is preferably 1 nm or more from the viewpoint of manufacturability.

[0028] Preferred examples of core particles used in the composites of the present invention include magnetic particles and semiconductor particles. Inorganic materials such as magnetic substances and semiconductors generally have a higher absorption rate for light with wavelengths of 500 to 900 nm than organic materials, and therefore generate more heat when irradiated with light with wavelengths of 500 to 900 nm. Particles containing iron oxide, manganese compounds, or gadolinium compounds are preferred as the magnetic particles, as these enable imaging by magnetic resonance imaging (MRI). Furthermore, considering binding to antibody molecules, the magnetic particles preferably have reactive functional groups such as amino groups and carboxy groups, or protein-binding molecules such as avidin, streptavidin, and protein A on their surfaces. Examples of such magnetic particles include dispersions of superparamagnetic iron oxide particles sold by Micromod as the Nanomag-D-spio series.

[0029] As the semiconductor particles, quantum dots are preferably used because they have high brightness and small particle diameter. Furthermore, considering that the semiconductor particles will be bound to antibody molecules, it is preferable that the semiconductor particles have reactive functional groups such as amino groups and carboxy groups, or protein-binding molecules such as avidin, streptavidin, and protein A on their surfaces. A preferred example of such quantum dots is semiconductor nanoparticles with a core-shell structure having a core and a shell surrounding the core, as disclosed in Patent Document 3, in which the core is (AgIn) x Zn 2(1-x) Examples of suitable quantum dots include semiconductor nanoparticles having a shell of ZnS or ZnO, where x is 0.4≦x≦0.95, preferably 0.8≦x≦0.9, and the shell has hydrophilic functional groups such as carboxyl groups, sulfo groups, or salts thereof on the surface of the shell. Another preferred example of quantum dots is CdSe quantum dots (e.g., amino-PEG-QDs800; ThermoFisher) with amino groups introduced onto the particle surface.

[0030] In the complex of the present invention, the number of antibody molecules bound to the core particle is preferably 1 or more per core particle, and preferably 20 or less, more preferably 16 or less, even more preferably 12 or less, and even more preferably 5 or less. If the number of antibody molecules bound to the core particle is too large, the size of the entire complex increases, which tends to make it difficult for the complex to selectively distribute to tumor cells. In addition, the lower limit of the number of antibody molecules bound to 1 mg of the complex of the present invention is preferably 1.0 × 10 -12 mol / mg, more preferably 5.0×10 -12 mol / mg, more preferably 1.0×10 -11 On the other hand, the upper limit of the number of antibody molecules bound in 1 mg of the complex of the present invention is preferably 1.0 × 10 -8 mol / mg, more preferably 5.0×10 -9 mol / mg, more preferably 1.0×10 -9 It is in mol / mg.

[0031] 1.3. Photosensitive areas Preferably, the conjugates of the present invention are used as drugs for near-infrared photoimmunotherapy (NIR-PIT). Therefore, the photosensitive moiety used in the conjugates of the present invention is preferably a near-infrared (NIR)-sensitive moiety. As shown in Non-Patent Document 1, the photosensitive moiety of the conjugates used in conventional NIR-PIT is thought to be hydrophobic upon NIR irradiation, causing aggregation of the conjugates, thereby damaging the cell membrane of the tumor cells and inducing cell death. In light of this mechanism, examples of photosensitive moieties used in the conjugates of the present invention include moieties that become more hydrophobic upon irradiation with NIR light, for example, light with a wavelength of 500 to 900 nm. For example, the photosensitive moiety may include a photosensitive group having a maximum absorption wavelength in the range of 500 to 900 nm and one or more hydrophilic functional groups linked or coordinated to the photosensitive group. When irradiated with light of the wavelength, the photosensitive group undergoes a photochemical reaction, dissociating the hydrophilic functional group or changing its structure, thereby increasing the hydrophobicity of the photosensitive moiety. Examples of hydrophilic functional groups contained in the photosensitive moiety include carboxylate (-CO2 - ) group, sulfonate (-SO3 - ) group, sulfonyl (-SO2 - ) group, sulfate (-SO4 -2 ) group, hydroxy (-OH) group, phosphate (-OPO3 -2 ) group, phosphonate (-PO3 -2 ) group, amino (-NH2) group, substituted or unsubstituted quaternary nitrogen (each with an optional counterion), etc. Examples of counterions include, but are not limited to, sodium, potassium, calcium, ammonium, organic amino salts, magnesium salts, etc. The photosensitive moiety may further have a reactive group or linker for binding to an antibody molecule or a core particle.

[0032] Another example of the photosensitive moiety used in the composite of the present invention is a moiety containing a phthalocyanine skeleton. Preferably, the photosensitive moiety used in the composite of the present invention is a moiety containing a phthalocyanine skeleton. Phthalocyanine is an azaporphyrin (i.e., C ) containing four benzoindole groups connected by nitrogen bridges in a 16-membered ring in which carbon and nitrogen atoms are arranged alternately. 32 H 16 N8). Phthalocyanines form stable chelates with metal and nonmetal cations, where the ring center is occupied by an ion (either diamagnetic or paramagnetic) that can bear one or two ligands. The ring periphery can be unsubstituted or substituted.

[0033] Preferably, the phthalocyanine used in the present invention is water-soluble and has at least one water-solubilizing moiety. Preferably, the water-solubilizing moiety of the phthalocyanine contains silicon. Preferably, the phthalocyanine has a core atom such as Si, Ge, Sn, or Al at the center of the ring.

[0034] The moiety containing the phthalocyanine skeleton used in the present invention preferably has a maximum absorption wavelength of 500 to 900 nm, more preferably 600 to 850 nm, and even more preferably 660 to 740 nm. The moiety containing the phthalocyanine skeleton preferably has one or more ligands containing a hydrophilic functional group. Examples of the hydrophilic functional group include carboxylate (-CO2 - ) group, sulfonate (-SO3 - ) group, sulfonyl (-SO2 - ) group, sulfate (-SO4 -2 ) group, hydroxy (-OH) group, phosphate (-OPO3 -2 ) group, phosphonate (-PO3 -2 ) group, amino (-NH2) group, substituted or unsubstituted quaternary nitrogen (each with an optional counterion), etc. Examples of counterions include, but are not limited to, sodium, potassium, calcium, ammonium, organic amino salts, magnesium salts, etc.

[0035] Preferably, the moiety containing a phthalocyanine skeleton used in the present invention contains a linker having a reactive group capable of forming a bond with an antibody molecule or a core particle. That is, it has a linker-phthalocyanine skeleton (LD) structure. Preferably, the moiety containing a phthalocyanine skeleton is bonded to an antibody molecule or a core particle via the linker substituted around the ring of the phthalocyanine skeleton.

[0036] In a preferred embodiment, the moiety containing a phthalocyanine skeleton used in the present invention is a compound represented by the following formula (Ia):

[0037] [ka]

[0038] During the ceremony, L is a direct bond or a linker; Q is a reactive group for forming a bond with the antibody molecule or core particle; R 2 , R 3 , R 7 , and R 8 are each independently selected from substituted or unsubstituted alkyl and substituted or unsubstituted aryl; R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 are, when present, each independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkanoyl, substituted or unsubstituted alkoxycarbonyl, substituted or unsubstituted alkylcarbamoyl, and chelating ligands, wherein R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 at least one of which contains a water-soluble group; R 12 , R 13, R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , and R 23 are each independently selected from hydrogen, halogen, substituted or unsubstituted alkylthio, substituted or unsubstituted alkylamino, and substituted or unsubstituted alkoxy; or i) R 13 and R 14 and the carbon to which they are attached, ii) R 17 and R 18 and the carbon to which they are attached, and iii) R 21 and R 22 and the carbon to which they are attached, at least one of which forms a fused ring; and X 2 and X 3 are each independently a C1-C bond with or without a heteroatom between the carbon-carbon bonds. 10 In the present specification, C1 to C 10 Alkylene means a methylene group and an alkylene group having 2 to 10 carbon atoms.

[0039] In one embodiment, L is a linker. In one embodiment, the linker is a straight or branched chain, cyclic or heterocyclic, saturated or unsaturated chain having 1 to 60 atoms, e.g., 1 to 45 atoms or 1 to 25 atoms. In some cases, the atoms of the linker can be selected from C, N, P, O, and S. In one embodiment, L can have additional hydrogen atoms (in addition to the 1 to 60 atoms) to satisfy the valence. Generally, the linker can include an ether, a thioether, an amine, an ester, a carbamate, a urea, a thiourea, a carbonyl, an amide, a single bond, a double bond, a triple bond, an aromatic carbon-carbon bond, a phosphorus-oxygen bond, a phosphorus-sulfur bond, a nitrogen-nitrogen bond, a nitrogen-oxygen bond or a nitrogen-platinum bond, an aromatic bond, or a heteroaromatic bond, or any combination thereof.

[0040] In one embodiment, L is of the formula -R 1 -YX 1 -Y 1 -, where R 1 is a divalent group or a direct bond; Y and Y 1 are each independently selected from a direct bond, oxygen, substituted or unsubstituted nitrogen, and sulfur; and X 1 is a C1-C bond with or without a direct bond and a heteroatom intervening between the carbon-carbon bonds 10 Examples of such divalent groups include, but are not limited to, substituted or unsubstituted alkylene, substituted or unsubstituted alkyleneoxycarbonyl, substituted or unsubstituted alkylenecarbamoyl, substituted or unsubstituted alkylenesulfonyl, and substituted or unsubstituted arylene.

[0041] R 1 Specific examples include, but are not limited to, substituted or unsubstituted alkylene, substituted or unsubstituted alkyleneoxycarbonyl, substituted or unsubstituted alkylenecarbamoyl, substituted or unsubstituted alkylenesulfonyl, substituted or unsubstituted alkylenesulfonylcarbamoyl, substituted or unsubstituted arylene, substituted or unsubstituted arylenesulfonyl, substituted or unsubstituted aryleneoxycarbonyl, substituted or unsubstituted arylenecarbamoyl, substituted or unsubstituted arylenesulfonylcarbamoyl, substituted or unsubstituted carboxyalkyl, substituted or unsubstituted carbamoyl, carbonyl, substituted or unsubstituted heteroarylene, substituted or unsubstituted heteroaryleneoxycarbonyl, substituted or unsubstituted heteroarylenecarbamoyl, substituted or unsubstituted heteroarylenesulfonylcarbamoyl, substituted or unsubstituted sulfonylcarbamoyl, thiocarbonyl, sulfonyl, and sulfinyl. Preferably, the alkylene contained in the substituted or unsubstituted alkylene, the substituted or unsubstituted alkyleneoxycarbonyl, the substituted or unsubstituted alkylenecarbamoyl, the substituted or unsubstituted alkylenesulfonyl, and the substituted or unsubstituted alkylenesulfonylcarbamoyl is a C1-C alkylene having or without a heteroatom between the carbon-carbon bonds. 10 It is alkylene.

[0042] In one embodiment, Q comprises a reactive group for forming a bond with the antibody molecule or core particle. As used herein, a "reactive group" refers to a moiety on a compound that can chemically react with a functional group on a different material (e.g., an antibody molecule) to form a bond. Typically, the reactive group is an electrophile or nucleophile that can form a covalent bond through exposure to a corresponding functional group that is a nucleophile or electrophile, respectively.

[0043] In one embodiment, Q comprises a reactive group that is reactive with a carboxyl group, an amino group, or a thiol group on the target antibody molecule or core particle. Examples of suitable reactive groups include, but are not limited to, activated esters, acyl halides, alkyl halides, anhydrides, carboxylic acids, carbodiimides, carbonates, carbamates, haloacetamides (e.g., iodoacetamide), isocyanates, isothiocyanates, maleimides, NHS (N-hydroxysuccinimide) esters, phosphoramidites, platinum complexes, sulfonate esters, and thiocyanates. In one embodiment, the reactive group is a sulfhydryl-reactive chemical group, such as maleimide, haloacetyl, or pyridyl disulfide. In one embodiment, the reactive group is amine-reactive. In a preferred embodiment, the reactive group is an NHS ester.

[0044] In one embodiment, R 2 , R 3 , R 7 , and R 8 is each independently substituted or unsubstituted alkyl, for example, substituted or unsubstituted methyl, ethyl, or isopropyl.

[0045] In one embodiment, R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 At least one of R contains a water-soluble group. 4 , R 5 , R 6 , R 9 , R 10 , and R 11 In one embodiment, at least two of R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 At least one of R is alkyl substituted with a water-soluble group. 4 , R 5 , R 6 , R 9 , R 10 , and R 11 are each independently substituted or unsubstituted alkyl, at least one of which, preferably two or more of which is alkyl substituted with a water-soluble group. 4 , R 5 , R 6 , R 9 , R 10 , and R 11 are each independently substituted or unsubstituted alkyl, and R 4 , R 5 , and R 6 at least one of R is alkyl substituted with a water-soluble group, and R 9 , R 10 , and R 11 At least one of the groups is an alkyl substituted with a water-soluble group. As used herein, the term "water-soluble group" refers to a group containing one or more polar and / or ionic substituents that improve the overall solubility of the molecule in aqueous media. Examples of water-soluble groups include, but are not limited to, carboxylate (-CO2 - ) group, sulfonate (-SO3- ) group, sulfonyl (-SO2 - ) group, sulfate (-SO4 -2 ) group, hydroxy (-OH) group, phosphate (-OPO3 -2 ) group, phosphonate (-PO3 -2 ) group, amino (-NH2) group, and substituted or unsubstituted quaternary nitrogen (each with an optional counterion). Examples of suitable counterions include, but are not limited to, sodium, potassium, calcium, ammonium, organic amino salts, magnesium salts, etc. Preferably, the counterion is a biologically acceptable counterion.

[0046] R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 The nitrogen atom to which is attached can be trivalent or tetravalent.

[0047] In one embodiment, R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , and R 23 are each hydrogen.

[0048] In one embodiment, X 2 and X 3 are each independently a C1-C bond with or without a heteroatom between the carbon-carbon bonds. 10 In one embodiment, X is alkylene. 2 and / or X 3 The nitrogen attached to may be quaternized.

[0049] In a preferred embodiment, the moiety containing a phthalocyanine skeleton used in the present invention is a compound represented by formula (Ib):

[0050] [ka]

[0051] During the ceremony, X 1 and X 4 are each independently a C1-C bond with or without a heteroatom between the carbon-carbon bonds. 10 alkylene; and 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 is as defined above for formula (Ia).

[0052] In the compound of formula (Ib) above, the reactive group for forming a bond with an antibody molecule or a core particle is an NHS ester. In one embodiment, the reactivity of the NHS ester is determined by the X between the NHS ester and the carbamate functional group. 4 In one embodiment, the length of the alkylene group in X between the NHS ester and the carbamate functional group can be adjusted. 4 The length of the alkylene group in X is inversely proportional to the reactivity of the NHS ester. 4 is C5-alkylene. 4 is C3-alkylene. 1 is C6-alkylene. 1 is a C3-alkylene.

[0053] In one embodiment, the compound of Formula (Ia) or Formula (Ib) has a total charge of zero. This neutral charge can be achieved with one or more optional counterions, or quaternized nitrogens, in certain cases.

[0054] In one embodiment, the compound of Formula (Ia) or Formula (Ib) has sufficient solubility in aqueous solution such that after binding to a targeting antibody molecule or core particle, the targeting molecule or particle retains its solubility.

[0055] In a preferred embodiment, the moiety containing a phthalocyanine skeleton used in the present invention is IR700 NHS ester, for example, IRDye 700DX NHS ester (LiCor 929-70010, 929-70011). In a preferred embodiment, the moiety containing a phthalocyanine skeleton is a compound represented by the following formula (II):

[0056] [ka]

[0057] For the purposes of the present invention, the photosensitive moiety described above is included in the conjugate of the present invention in a configuration where it is bound to a target antibody molecule or core particle via its reactive group. For example, the compounds represented by formula (Ia), (Ib), or (II) above, "IR700," "IRDye 700DX," or variations thereof, which may be included in the conjugate of the present invention, refer to these compounds in a configuration where they are bound to a target antibody molecule or core particle via their reactive group. In general, IR700 has several favorable chemical properties. Amino-reactive IR700 is relatively hydrophilic and can be covalently bound to antibodies using an NHS ester of IR700. Typically, IR700 is a hematoporphyrin derivative, Photofrin® (1.2 x 10 at 630 nm). 3 M -1 cm -1 ), meta-tetrahydroxyphenyl chlorin; Foscan® (2.2 × 10 at 652 nm) 4 M -1 cm-1 ), and mono-L-aspartyl chlorin e6; NPe6 / Laserphyrin® (4.0 × 10 at 654 nm) 4 M -1 cm -1 ) with an extinction coefficient (2.1 × 10 at absorption maximum at 689 nm) that is over five times higher than conventional photosensitizers such as 5 M -1 cm -1 )

[0058] The moieties containing the phthalocyanine skeleton used in the present invention, such as compounds represented by the above formula (Ia), (Ib), or (II), can be prepared using commercially available starting materials. For example, the skeleton can be synthesized by condensation of two or more different diiminoisoindolines. Synthetic strategies using different dinitriles or diiminoisoindolines can lead to phthalocyanines with various degrees of substitution and / or positional isomers. An exemplary synthetic scheme for producing the phthalocyanine skeleton is described in U.S. Pat. No. 7,005,518.

[0059] In the conjugate of the present invention, the above-mentioned photosensitive moiety may be bound to at least one of the antibody molecule and the core particle, but is preferably bound to the antibody molecule. Furthermore, the conjugate of the present invention may contain one or more of the above-mentioned photosensitive moieties, and these photosensitive moieties may have the same or different structures.

[0060] 2. Manufacturing method of the composite In producing the conjugate of the present invention, it is preferable to first synthesize an antibody molecule and / or core particle having a photosensitive site. The photosensitive site can be bound to the antibody molecule or core particle by known means. For example, specific techniques for conjugating a moiety containing a phthalocyanine skeleton to an antibody molecule include the methods disclosed in Patent Document 1 or the below-described Example 1. More specifically, an aqueous phosphate solution containing an antibody molecule and a phthalocyanine compound represented by the above formula (Ia) having an NHS ester at the reactive group Q (for example, the above-described IR700 NHS ester) may be incubated at room temperature, and the target antibody-photosensitive moiety conjugate may be purified from the reaction solution by column purification or the like. When bonding a moiety containing a phthalocyanine skeleton to a core particle, for example, a compound represented by the above formula (Ia) having an NHS ester as the reactive group Q can be reacted with a core particle having an amino group or a protein molecule such as avidin, streptavidin, or protein A introduced onto the particle surface in the same manner as above. The number of photosensitive moieties bound to an antibody molecule per antibody molecule is preferably 1 or more, more preferably 2 or more, and preferably 5 or less, more preferably 4 or less. The number of photosensitive moieties bound to a core particle per core particle is preferably 1 or more, and preferably 80 or less, more preferably 50 or less, and even more preferably 20 or less.

[0061] Next, the conjugate of the present invention can be produced by binding the antibody molecule to the core particle. For example, the conjugate of the present invention can be produced by mixing an antibody molecule biotinylated by a conventional method with core particles on whose surface a biotin-binding substance has been introduced, thereby binding the antibody molecule to the core particle. Examples of the biotin-binding substance include proteins such as avidin and streptavidin. Preferably, 1 to 5, more preferably 1 to 4, and particularly preferably 1 to 3 biotin-binding substances are introduced per core particle. Alternatively, as shown in the Examples below, a carbodiimide compound such as EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and an N-hydroxysuccinimide derivative such as Sulfo-NHS can be used to crosslink the antibody molecule and the core particle. In this case, the amounts of antibody molecules and core particles charged are set so that the number of antibody molecules bound per core particle is the desired number (preferably 1 or more and preferably 20 or less, more preferably 16 or less, even more preferably 12 or less, and even more preferably 5 or less). The amounts may also be set so that the number of antibody molecules bound per mg of core particle is the desired number. Specifically, the lower limit of the amount of antibody molecules charged per mg of core particle is preferably 1.0 × 10 -12 mol / mg, more preferably 5.0×10 -12 mol / mg, more preferably 1.0×10 -11 On the other hand, the upper limit of the amount of the antibody molecules to be charged per 1 mg of core particles is preferably 1.0 × 10 -8 mol / mg, more preferably 5.0×10 -9 mol / mg, more preferably 1.0×10 -9 However, the bond between the antibody molecule and the core particle is not limited to a covalent bond, and may also include bonds based on hydrogen bonds, ionic bonds, hydrophobic interactions, or combinations of these.

[0062] 2. Composition containing the complex In one aspect, the present invention provides a composition containing the above-described conjugate of the present invention. In a preferred embodiment, the composition containing the conjugate of the present invention (hereinafter also referred to as the composition of the present invention) is used as a pharmaceutical composition for photoimmunotherapy (PIT).

[0063] In one embodiment, the composition of the present invention contains the conjugate of the present invention and a pharmaceutically acceptable carrier or excipient. Examples of pharmaceutically acceptable carriers include, but are not limited to, water, oil, buffers, phosphate-buffered saline, and other diluents for injections. Examples of excipients include, but are not limited to, starch, glucose, lactose, dextrose, carboxymethylcellulose, glycerol, propylene glycol, water, and ethanol. If necessary, the composition of the present invention may contain lubricants, binders, wetting agents, emulsifiers, pH adjusters, isotonicity agents, buffers, antioxidants, suspending agents, solubility enhancers, preservatives, chelating agents, and other pharmaceutically acceptable substances. Pharmaceutically acceptable carriers, excipients, and the like are known in the art (see, for example, Remington's Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition, 1995).

[0064] The compositions of the present invention may be in liquid form, such as a solution, suspension, syrup, etc.; or in solid form, such as a powder, pill, tablet, capsule, transdermal patch, inhalant, suppository, etc. Alternatively, the compositions of the present invention may be in lyophilized form and may be reconstituted with a pharmaceutically acceptable carrier (e.g., a diluent for injection) before use and administered. These liquid or solid compositions can be prepared according to conventional methods. In one embodiment, the composition of the present invention is a single-dose formulation containing a carrier or excipient that contains the conjugate of the present invention. In another embodiment, the composition of the present invention is a two-dose formulation that separately contains the conjugate of the present invention and a diluent or the like to be administered together.

[0065] In a preferred embodiment, the composition of the present invention is a pharmaceutical composition. The form of the pharmaceutical composition may depend on its administration regimen. The administration regimen of the pharmaceutical composition can be appropriately designed depending on the type and condition of the target tumor, and the species, age, condition, etc. of the patient. The pharmaceutical composition may be an oral or parenteral formulation, for example, an injection, an oral agent, or an external agent. The pharmaceutical composition may be configured for single or multiple administration. The content of the conjugate of the present invention in the pharmaceutical composition can be appropriately designed depending on the form of the pharmaceutical composition, the dose to be administered to the patient, etc.

[0066] The pharmaceutical composition is adjusted to a pH range compatible with the animal body, for example, pH 5 or higher, preferably pH 5.5 or higher and pH 10 or lower, preferably pH 8 or lower, more preferably pH 7.3 or lower, or preferably pH 5.5 to 10, more preferably pH 5.5 to 8, even more preferably pH 5.5 to 7.3. The pH of the composition can be adjusted using the above-mentioned pH adjusters, buffer solutions, etc.

[0067] In one embodiment, the dosage of the conjugate of the present invention in the pharmaceutical composition may be in the range of 0.01 mg to 9000 mg, for example, in the case of an injectable formulation. In one embodiment, the single-dose dosage of the pharmaceutical composition may be in the range of 0.5 mL to 1000 mL, for example, in the case of an injectable formulation. In one embodiment, the pharmaceutical composition is an injectable formulation, the single-dose volume is 1 to 5 mL, and the single-dose volume contains 0.1 mg to 5000 mg of the conjugate of the present invention.

[0068] 3. Treatment of tumors using the complex of the present invention In another aspect, the present invention relates to the use of the conjugate of the present invention or a composition of the present invention containing the conjugate for the treatment of tumors by photoimmunotherapy (PIT). In one embodiment, the present invention provides the conjugate or composition of the present invention for use in the treatment of tumors by PIT. In another embodiment, the present invention provides the use of the conjugate or composition of the present invention in the manufacture of a tumor therapeutic agent by PIT. In another embodiment, the present invention provides a method for treating tumors by PIT using the conjugate or composition of the present invention. The method for treating tumors according to the present invention (hereinafter also referred to as the treatment method of the present invention) comprises the steps of administering the conjugate or composition of the present invention to a patient and irradiating the patient with near-infrared (NIR) rays. Therefore, the PIT used in the present invention is specifically near-infrared photoimmunotherapy (NIR-PIT). In this specification, the term "method for treating tumors" can be interpreted as "method for killing tumors."

[0069] The patient to whom the complex or composition of the present invention is administered is a patient in need of tumor treatment. Such patients include humans and non-human animals bearing tumors. Non-human animals include, but are not limited to, non-human mammals such as mice, rats, hamsters, rabbits, pigs, goats, dogs, cats, sheep, cows, and horses. In addition, such patients may or may not have received other tumor treatments (surgery, chemotherapy, radiation therapy, etc.).

[0070] The antibody molecule contained in the conjugate of the present invention is selected depending on the type of tumor to be targeted. The antibody molecule can specifically bind to an antigen present on the surface of the target tumor cells, preferably a tumor-specific protein expressed on the surface of the tumor cells. Appropriate selection of the antibody molecule enables the accumulation of the conjugate of the present invention in the target tumor. The cell surface antigen, e.g., tumor-specific protein, of various tumors to be targeted can be determined according to known information. Those skilled in the art can select an antibody molecule specific to the target antigen on the tumor to be treated.

[0071] 3.1. Administration of the Complex The administration regimen (e.g., administration route, dose, frequency, etc.) of the complex or composition of the present invention can be appropriately determined depending on the type and condition of the target tumor, and the species, age, condition, etc. of the patient. Examples of administration routes include local administration to the tumor-affected area by injection, catheter, spray, application, patch, suppository, etc., and systemic administration by infusion, oral administration, intraperitoneal administration, intravenous injection, etc. Preferably, the complex or composition of the present invention is administered locally. In one embodiment, the complex or composition of the present invention is administered intravenously. In one embodiment, the complex or composition of the present invention is administered directly to the target tumor-affected area using a syringe, etc., or injected via a catheter. The complex or composition of the present invention may be used alone to treat tumors, but may also be used in combination with other drugs or therapies, such as chemotherapy.

[0072] The conjugate or composition of the present invention may be administered to a patient in an effective amount. The term "effective amount" refers to an amount that allows the conjugate of the present invention to accumulate in a target tumor of the patient in an amount sufficient to exert the therapeutic effect of PIT. Preferably, the term "effective amount" refers to an amount that exerts the therapeutic effect of PIT in the patient while minimizing or keeping side effects to a tolerable level for the patient.

[0073] The dose of the complex of the present invention administered to a patient can be determined appropriately depending on the type and condition (location, volume, etc.) of the target tumor, the species, age, condition, and administration route of the patient, the form of the composition containing the complex, etc. For example, the dose of the complex of the present invention can be set according to the tumor volume. The tumor volume (V) can be determined, for example, by measuring the short diameter (W) and long diameter (L) of the tumor and calculating the tumor volume (V) using the formula: V = (W 2 × L) / 2. Alternatively, the dose can be adjusted depending on the degree of accumulation of the complex of the present invention in the target tumor, as measured by imaging, as described below. The dose of the complex of the present invention to be administered to humans can be determined based on the dose to be administered to mice. For example, the effective dose of the complex of the present invention when administered to humans can be determined as 5 to 10 times the effective dose in mice.

[0074] In one example, when a composition containing the complex of the present invention is injected into the tumor site of an adult (60 kg), the single dose (injection amount) of the composition is usually 1 to 5 mL. In another example, when a composition containing the complex of the present invention is injected into the tumor site of an adult (60 kg), the single dose (injection amount) of the complex is 0.01 mg to 20 mg / kg (body weight).

[0075] The dosage and frequency of administration of the complex of the present invention can be increased or decreased depending on the therapeutic effect of the tumor. The therapeutic effect of the tumor can be evaluated by common methods for evaluating tumor treatment, such as the rate of tumor tissue shrinkage. In one embodiment, the complex of the present invention is administered once at the above-mentioned dosage. In another embodiment, the complex of the present invention is administered multiple times. In the case of multiple administrations, the above-mentioned dosage may be administered repeatedly, or the dosage may be increased or decreased depending on the therapeutic effect of the tumor. In one embodiment, the second or subsequent administration can be performed after the previous dose has been cleared from the patient. In another embodiment, the complex of the present invention can be repeatedly administered once a week, once every two weeks, once a month, or less frequently. In another embodiment, the complex of the present invention can be administered again one week, two weeks, three weeks, four weeks, two months, six months, one year, or more after the previous administration if the target tumor remains.

[0076] 3.2. Photoimmunotherapy Following administration of the complex or composition of the present invention to a patient, the patient is irradiated with NIR. Preferably, NIR is applied locally to tumor cells or tumor-affected areas to which the complex of the present invention is bound. The complex of the present invention exposed to NIR induces a photochemical reaction, which kills the tumor cells to which it is bound. The complex of the present invention administered to a patient specifically binds to the target tumor cells via the antibody molecule. Therefore, the present invention achieves selective killing of target tumor cells.

[0077] The wavelength of the light beam to be irradiated is preferably 500 to 900 nm, more preferably 600 to 850 nm, and even more preferably 660 to 740 nm.

[0078] The timing of irradiation can be determined at any time after administration of the complex of the present invention, for example, between 30 minutes and 96 hours after administration, preferably between 30 minutes and 48 hours, between 30 minutes and 24 hours, between 1 hour and 48 hours, or between 1 hour and 24 hours. The irradiation time can be appropriately determined within a range of 5 seconds to 72 hours. Irradiation can be performed once or multiple times so that the cumulative irradiation time per administration of the complex of the present invention is within the above-mentioned range. The time per irradiation can be appropriately determined, and can be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or 55 seconds, or 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 minutes, or 10, 20, 30, 40, 50, or 60 minutes, or 1 or 2 hours. In one embodiment, the dose of NIR delivered to the patient is preferably 1 J / cm 2 More than 5J / cm 2 More preferably, 10 J / cm 2 or more, and preferably 1000 J / cm 2 Less than or equal to 500 J / cm 2 or less, more preferably 100 J / cm 2 or less, more preferably 50 J / cm 2 For example, 1 to 1000 J / cm 2 , 1~500J / cm 2 , 5~200J / cm 2 , 10~100J / cm 2 , or 10 to 50 J / cm 2 The range is.

[0079] Irradiation can be performed once or multiple times per administration of the complex of the present invention. Therefore, irradiation may be completed in one session or repeated over several days. When irradiation is performed multiple times, the conditions for each irradiation may be the same or different. The dose, conditions, or method of irradiation can be changed depending on the type and condition of the tumor.

[0080] Imaging When the core particle of the complex of the present invention is a magnetic particle or a fluorescent particle (e.g., a semiconductor particle), biological imaging becomes possible. In one embodiment, the complex or composition of the present invention is used for imaging of a patient or a tumor. For example, the complex or composition of the present invention administered to a patient is used for imaging of the tumor before tumor treatment. Tumor imaging makes it possible to confirm the location of the complex in the patient's body or the location of the tumor to which the complex is bound, i.e., makes it possible to diagnose the patient. This allows the timing of light irradiation for PIT, the three-dimensional irradiation position in the body, and the irradiation amount (irradiation time, dose) to be optimized, thereby improving the therapeutic effect of PIT.

[0081] Imaging methods include MRI and fluorescent imaging. MRI imaging is possible when magnetic particles containing MRI contrast agents such as iron oxide or gadolinium compounds are used as core particles. MRI imaging can be performed using conventional methods. Alternatively, fluorescent imaging is possible when fluorescent particles are used as core particles. In fluorescent imaging, excitation light is irradiated to excite the fluorescent particles. The wavelength of the irradiated excitation light can be selected appropriately depending on the fluorescent particles used. Fluorescence emitted from the fluorescent particles is detected by a detector. Detectors are not particularly limited, but include CCD cameras, optical CT devices, endoscopes, fundus cameras, and the like. Examples of fluorescent particles include semiconductor nanoparticles disclosed in Patent Document 3 and CdSe-based quantum dots with amino groups introduced onto the particle surface. From the perspective of simplicity, in the present invention, it is preferable to use a composite containing a magnetic particle as the core particle and perform MRI imaging.

[0082] 3.4.Other Methods The PIT and imaging techniques using the complex or composition of the present invention described above can be applied not only in vivo but also in vitro. For example, administration of the complex or composition of the present invention and NIR irradiation to not only tumors present in a patient's body but also cultured tumor cells or cultured tissues containing tumor cells can reduce or inhibit tumor cell proliferation. The administration method of the complex or composition of the present invention and the conditions for NIR irradiation can be appropriately modified depending on the state of the target cells or tissues. For example, the complex or composition of the present invention can be directly administered to tumor cells in culture. The dose of the complex or composition or the conditions for NIR irradiation can be selected to be milder than those for administration or irradiation to a patient. [Example]

[0083] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to these examples in any way.

[0084] Example 1 Preparation of the Complex 1) Synthesis of panitumumab conjugated with IRDye 700DX (IR700) Two mg (13.6 nmol) of the human monoclonal antibody panitumumab was incubated with 133.6 μg (68.4 nmol, 5 mmol / L DMSO) of IRDye 700DX NHS Ester (LI-COR Biosciences) in 0.2 mol / L NaHPO (pH 8.5) for 30–120 min at room temperature. The mixture was purified on a Sephadex G50 column (PD-10; GE Healthcare, Piscataway, NJ). Protein concentration was determined by measuring absorbance at 595 nm with a Coomassie Plus Protein Assay Kit (Pierce Biotechnology, Rockford, IL) using a UV-Vis system (8453 Value system; Agilent Technologies, Palo Alto, CA). IR700 concentration was measured by UV-Vis system (Shimadzu UV-VIS). There were approximately three molecules of IR700 per molecule of panitumumab. Hereafter, panitumumab bound to IR700 will be referred to as Pan-IR700.

[0085] 2) Biotinylation of Pan-IR700 5.69 mg of (+)-biotin N-hydroxysuccinimide ester (Sigma-Aldrich, hereafter 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 placed in a microtube, to which 8 μL of the previously prepared Biotin-NHS DMSO solution ([Biotin-NHS] / [Pan-IR700] = 10) was added and 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 with Dulbecco's Phosphate Buffered Saline (Wako Pure Chemical Industries, hereafter referred to as D-PBS) to obtain biotinylated Pan-IR700. Hereinafter, biotinylated Pan-IR700 will be referred to as Pan-IR700-Biotin.

[0086] 3) Preparation of a composite of Pan-IR700 and magnetic particles 120 mg (5 mg / mL, 24 mL) of Nanomag-D-Spio 79-19-201 (Micromod, streptavidin surface-modified magnetic particles, particle size 20 nm) was placed in a 50 mL tube, and 1.8 mg (1.9 mg / mL, 947 μL) of Pan-IR700-Biotin was added and stirred at room temperature for 60 minutes. 18 μg (0.1 mg / mL, 180 μL) of biotin (Wako Pure Chemical Industries, Ltd.) was then added and stirred at room temperature for 30 minutes. In this way, a dispersion containing a complex of Pan-IR700 and magnetic particles was obtained. 1 mL of the dispersion was passed through an MS-columns (Miltenyi Biotec) column mounted on a magnetic stand, and the complex bound to the magnetic particles was adsorbed onto the column. Then, 2 mL of D-PBS was passed through the column. This procedure was repeated four times. The fourth filtrate was irradiated with excitation light at 676 nm and fluorescence measurement at 700 nm confirmed that no unreacted Pan-IR700-Biotin was detected. In this way, a complex of Pan-IR700 and magnetic particles was produced. Hereafter, the resulting complex is referred to as Nanomag-Pan-IR700. In Nanomag-D-Spio 79-19-201, the particle concentration is 8.0 × 10 14 pieces / mL(1.6×10 14 The amount of streptavidin bound was 1.5 μg / mg. From this, it was calculated that an average of approximately 1.1 streptavidin (tetramer) molecules bound per magnetic particle in Nanomag-D-Spio 79-19-201. Furthermore, in the synthesis of Nanomag-Pan-IR700, the amount of Pan-IR700-Biotin used was 3.5 times (1.0 × 10 per mg of magnetic particles) in molar ratio to the streptavidin (tetramer) in Nanomag-D-Spio 79-19-201. -10 mol).

[0087] 4) Preparation of complexes of Pan-IR700 and quantum dots 1.0 mg of Pan-IR700 (2.0 mg / mL, 500 μL) and 0.4 mg of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (ThermoFisher, final concentration 2 mM) were placed in a microtube and reacted in 0.1 M MES buffer (pH 4.7). 1.1 mg of sulfo-NHS (ThermoFisher, final concentration 5 mM) was then added and reacted at room temperature for 15 minutes. Excess EDC and sulfo-NHS were then removed using ultrafiltration (Merck, Amicon Ultra 100 kDa, 14,000 × g, 10 min) to obtain sulfo-NHS ester-conjugated Pan-IR700. To the resulting compound, 170 μL of CdSe quantum dots, amino-PEG-QDs800 (ThermoFisher, particle size 20 nm, 8 μM), was added and allowed to stand overnight at room temperature. Finally, excess Pan-IR700 was removed using ultrafiltration (Pall, Nanosep 300 kDa, 1000 × g, 30 min). In this way, a composite of Pan-IR700 and quantum dots was produced. Hereafter, the resulting composite is referred to as QDs800-Pan-IR700. The particle concentration of amino-PEG-QDs800 was 8 μM, and the amount of Pan-IR700 reacted with the quantum dots was approximately 4.9 particles per quantum dot particle.

[0088] Comparative Example 1: Preparation of a complex of Pan-IR700 and magnetic particles (particle size >100 nm) Three tubes were each filled with 10 mg (10 mg / mL, 1 mL) of Magnosphere SS015 streptavidin (JSR Life Sciences, particle size 150 nm), a paramagnetic iron oxide particle. 68, 135, and 405 μg (1.9 mg / mL) of Pan-IR700-Biotin were added to each tube and stirred at room temperature for 60 minutes to produce three complexes with different Pan-IR700 coverage. These complexes are hereafter referred to as Magnosphere-Pan-IR700-A, Magnosphere-Pan-IR700-B, and Magnosphere-Pan-IR700-C, respectively. In the synthesis of Magnosphere-Pan-IR700-A to -C, the amount of Pan-IR700-Biotin used was 4.5 × 10 per 1 mg of magnetic particles. -11 mol, 9.0 × 10 -11 mol, 2.7 × 10 -10 It was mol.

[0089] Example 2 Evaluation of biodistribution of the complex 6 x 10 cells (hereinafter referred to as "A431-Luc-GFP") transfected with luciferase and green fluorescent protein into A431 (human epidermal carcinoma-derived cells) expressing epidermal growth factor receptor (EGFR). 6 100 μL of cells were subcutaneously injected into the right groin of female homozygous athymic nude mice and allowed to engraft for 5 days.

[0090] Five days later, the mice were anesthetized, and Nanomag-Pan-IR700 prepared in Example 1 was administered into the tail vein at 111.1 μL / body (30 μg / body equivalent antibody, 13.3 mg / body equivalent particle). Fluorescence observation of the whole body of the mouse was performed at a wavelength of 700 nm using a Pearl Trilogy (LI-COR) from before administration until 48 hours after administration to examine the distribution of IR700 in the body. Fluorescence imaging of the whole body of the mouse is shown in Figure 1. Increased fluorescence was observed in the tumor area (arrow in the figure), indicating accumulation of the complex in the affected area. Similarly, QDs800-Pan-IR700 produced in Example 1 was administered into the tail vein of mice at 30 μg / body equivalent in antibody, and fluorescence observation was performed at 800 nm to examine the distribution of IR700 in the body. Fluorescence imaging of the whole mouse body is shown in Figure 2. Increased fluorescence was observed in the tumor area (enclosed by a dotted line in the figure), indicating accumulation of the complex in the affected area.

[0091] Similarly, Magnosphere-Pan-IR700-A, Magnosphere-Pan-IR700-B, and Magnosphere-Pan-IR700-C prepared in Comparative Example 1 were each intra-arterially administered to mice at 100 μg / body equivalent of antibody, and the biodistribution of IR700 was examined by fluorescence observation. Magnosphere-Pan-IR700-C was also administered to mice via the tail vein at 200 μg / body equivalent of antibody, and the biodistribution of IR700 was examined by fluorescence observation. As shown in Figure 3, no accumulation of the complex in tumor sites was observed.

[0092] Example 3 In vitro PIT using the complex A431-Luc-GFP cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin in tissue culture flasks in a humidified incubator at 37°C under an atmosphere of 95% air and 5% carbon dioxide. 2 × 10 cells were cultured. 5 The cells were seeded in the four corners and center of a 12-well plate with 300 μL of RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin, and incubated for 6 hours.

[0093] Nanomag-Pan-IR700, QDots-Pan-IR700, and Pan-IR700 prepared in Example 1 were added to the prepared culture at 10 μg / mL antibody equivalent, and the mixture was incubated at 37° C. for 24 hours. After that, the cells were washed with 500 μL of phosphate buffered saline (PBS), and the culture medium was replaced with phenol red-free medium.

[0094] Next, near-infrared light with a wavelength of 690 nm was applied at 0.5 J / cm using an LED to the four corners of the 12-well plate containing the cultures. 2 (18mW / cm 2 ×30s), or 1J / cm 2 (18mW / cm 2After irradiation for 60 s (×60 s), 200 μL of luciferin was added. After irradiation, the luminescence of the culture was measured periodically using a plate reader, and the number of viable cells was calculated. The center of the 12-well plate was used as an unirradiated control. The cell viability was calculated from the luminescence intensity relative to the control. The results are shown in Figure 4. Both complexes reduced cell viability, but the complexes Nanomag-Pan-IR700 and QDots-Pan-IR700, which contain magnetic particles or quantum dots, significantly reduced cell viability compared to Pan-IR700, especially after 3 hours of irradiation.

[0095] Example 4 In vivo PIT by the complex MDA-MB-468 (human breast cancer-derived cells) expressing epidermal growth factor receptor (EGFR) were transfected with luciferase and green fluorescent protein (hereinafter referred to as "MDA-MB-468-Luc-GFP"). 7 Three female homozygous athymic nude mice were subcutaneously injected with 100 μL of 100 cells near the base of the right foot and allowed to engraft for three weeks. Two mice were then anesthetized, and Nanomag-Pan-IR700 prepared in Example 1 was administered to each mouse via the tail vein at 80 μL / body (equivalent to 30 μg / body of antibody). One day later, one of the mice was irradiated with a 690 nm laser at 100 J / cm. 2 (470mW / cm 2 ), and 200J / cm to the other mouse. 2 (470mW / cm 2 The tumor luciferase activity (luminescence intensity) before and after irradiation was measured using an IVIS imaging system (Perkin-Elmer). Similarly, another mouse was anesthetized, and Pan-IR700 prepared in Example 1 was administered into the tail vein at 60 μL / body (equivalent to 30 μg / body of antibody). One day later, the mouse was irradiated with a 690 nm laser at 200 J / cm. 2 (470mW / cm 2 ) and the luminescence intensity was measured before and after irradiation.

[0096] Table 1 shows the luminescence intensity after irradiation, with the luminescence intensity before irradiation set at 100. Note that the higher the luminescence intensity, the larger the tumor size. As is clear from Table 1, a significant tumor shrinkage was confirmed when using the magnetic particle-containing complex Nanomag-Pan-IR700.

[0097] [Table 1]

[0098] Example 5 Evaluation of the heat-generating behavior of the composite 1 μg of Nanomag-Pan-IR700 produced in Example 1 was placed in a microtube in terms of antibody, and PBS was added to prepare 50 μL of Nanomag-Pan-IR700 solution. 1 μg of Pan-IR700 produced in Example 1 was placed in a separate microtube in terms of antibody, and PBS was added to prepare 50 μL of Pan-IR700 solution. As a control, 50 μL of PBS was placed in a microtube. The above microtubes were left to stand on ice for a while, and then each microtube was irradiated with a 690 nm laser at 282 J / cm on ice. 2 (470mW / cm 2 The liquid temperature was measured before and after irradiation using a compact thermography camera FLIR C2.

[0099] The measurement results are shown in Table 2. As is clear from Table 2, a temperature rise of over 30°C was confirmed in the Nanomag-Pan-IR700 solution upon irradiation with near-infrared light. This heat generation was thought to be one of the factors that enhanced the effectiveness of PIT.

[0100] [Table 2]

[0101] Example 6 Imaging with the complex 1) MRI The MRI imaging performance of mice administered Nanomag-Pan-IR700 in Example 2 was evaluated. A 3T MRI-MRS 3000 (MR Solutions) was used as the MRI device, and measurements were performed using T1WI (TR 250 msec, TE 6.0 msec, FA 90 deg) and T2WI (TR 2000 msec, TE 69.0 msec, FA 90 deg). The MRI images taken are shown in Figure 5.

[0102] 2) Confocal microscopy As a model of normal non-target cells, 3T3-RFP cells were prepared by transfecting a mouse skin-derived fibroblast cell line with (EF1a)-Puro lentiviral particles (AMSBIO, Cambridge, MA, USA) that express the marker red fluorescent protein (RFP).

[0103] The 3T3-RFP cells were mixed with EGFR-expressing cells A431 (human epidermal carcinoma-derived cells), MDAMB468 (human breast carcinoma-derived cells), and PC9 (human lung carcinoma-derived cells). Each cell mixture was plated in a 12-well plate at 5 × 10 cells per well with 300 μL of RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. 4 The cells were seeded at 10 μg / mL of Nanomag-Pan-IR700 antibody and incubated for 24 hours. The cells were then incubated for 1 hour, washed with 500 μL of PBS, and the culture medium was replaced with phenol red-free medium. The cells were then irradiated with 690 nm near-infrared light at 4 J / cm using an LED. 2 (18mW / cm 2 The cells were irradiated with light (240 x 240 s) and the fluorescence of the cells was observed before and after irradiation using an A1R-s Confocal Microscope (Nikon).

[0104] Furthermore, necrotic cells were observed using Sytox blue (Thermo Fisher). As a result, cell death was confirmed in all cancer cells after irradiation, while normal cells (3T3-RFP cells) continued to show fluorescence (RFP expression) even after irradiation, confirming their survival.

Claims

1. A tumor therapeutic agent for photoimmunotherapy, comprising: Containing a complex, The complex comprises an antibody molecule and a particle having an average particle size of 1 nm to 100 nm bound to the antibody molecule, wherein: the particles are magnetic particles, at least one of the antibody molecule and the particle is bound to a photosensitive moiety that becomes more hydrophobic upon irradiation with light having a wavelength of 500 to 900 nm; the photosensitive moiety is a moiety containing a phthalocyanine skeleton having one or more water-soluble groups, The water-soluble group is selected from a carboxylate (-CO 2 - ) group, a sulfonate (-SO 3 - ) group, a sulfonyl (-SO 2 - ) group, a sulfate (-SO 4 -2 ) group, a hydroxy (-OH) group, a phosphate (-OPO 3 -2 ) group, a phosphonate (-PO 3 -2 ) group, an amino (-NH 2 ) group, and a substituted or unsubstituted quaternary nitrogen, each having a counterion; Tumor treatment agent.

2. The tumor therapeutic agent according to claim 1, wherein the antibody molecule is conjugated to the photosensitive moiety.

3. The tumor therapeutic agent according to claim 1 or 2, wherein the photosensitive moiety is a compound represented by the following formula (Ia): 【Chemistry 1】 During the ceremony, L is a direct bond or a linker; Q is a reactive group for forming a bond with the antibody molecule or the particle; R 2 , R 3 , R 7 , and R 8 are each independently selected from substituted or unsubstituted alkyl and substituted or unsubstituted aryl; R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 are, when present, each independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkanoyl, substituted or unsubstituted alkoxycarbonyl, substituted or unsubstituted alkylcarbamoyl, and chelating ligands, wherein R 4 , R 5 , R 6 , R 9 , R 10 , and R 11 at least one of which contains said water-soluble group; R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , and R 23 are each independently selected from hydrogen, halogen, substituted or unsubstituted alkylthio, substituted or unsubstituted alkylamino, and substituted or unsubstituted alkoxy; or i) R 13 and R 14 and the carbon to which they are attached, ii) R 17 and R 18 and the carbon to which they are attached, and iii) R 21 and R 22 and the carbon to which they are attached form a fused ring; and X 2 and X 3 each independently represents a C with or without a heteroatom between the carbon-carbon bonds; 1 ~C 10 It is alkylene.

4. The tumor therapeutic agent according to claim 3, wherein the compound represented by formula (Ia) is a compound represented by the following formula (Ib): 【Chemistry 2】 During the ceremony, X 1 and X 4 each independently represents a C which may or may not be interrupted by a heteroatom; 1 ~C 10 alkylene; and 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 is as defined above.

5. The tumor therapeutic agent according to claim 3, wherein the compound represented by formula (Ia) is a compound represented by the following formula (II): 【Transformation 3】

6. The tumor therapeutic agent according to any one of claims 1 to 5, wherein the magnetic particles comprise an iron oxide or a gadolinium compound.

7. The tumor therapeutic agent according to any one of claims 1 to 6, wherein the particles have an average particle size of 1 to 50 nm.

8. The tumor therapeutic agent according to any one of claims 1 to 7, wherein the number of antibody molecules bound to each of the particles is 1 to 20 per particle.

9. The tumor therapeutic agent according to any one of claims 1 to 8, which is an agent for tumor imaging and tumor treatment by photoimmunotherapy.

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