A drug delivery method using light, and a targeted integrated complex.

Abstract

This target accumulation type complex has a structure in which a near-infrared photosensitive substance and a drug are linked to a carrier. By using the target accumulation type complex and a light delivery drug therapeutic method using the target accumulation type complex, it is possible to deliver a drug to an affected site to provide a cure. After the target accumulation type complex is administered, an affected site is irradiated with light, whereby a drug is released from the target accumulation type complex at the affected site. As a result, the drug exhibits an effect on cells that exist at an affected site but do not have antigen molecules on the surfaces and cannot be treated by conventional ADC. The light delivery drug therapeutic method using the target accumulation type complex is a novel therapeutic method that not only can treat a wide range of diseases such as cancer, inflammatory diseases, infectious diseases, and organ-specific diseases, but also can reduce drug toxicity.

Description

[Technical Field]

[0001] This invention relates to a targeted accumulation complex that delivers a drug to target cells or organs and then releases the drug using light. Specifically, it relates to a complex in which a drug and a photosensitive substance are bound to a specific binding substance such as an antibody, or a substance that accumulates in tissues or organs, or a carrier substance that can be administered to a living organism, and to the same complex and its applications. The targeted accumulation complex of this invention specifically binds to or circulates near the target, and the drug can be released at the light-irradiated site by near-infrared (NIR) light irradiation. This provides a time- and spatially controllable drug delivery technology that exerts its action and effect locally, including not only the cells to which the specific binding substance binds or the vicinity where it circulates, but also the surrounding tissue. [Background technology]

[0002] Generally, drugs are administered through methods such as oral, intravenous, intramuscular, and subcutaneous injection, allowing them to reach target organs and cells via the body's circulation and exert their effects. Because administered drugs are distributed and diluted throughout the body via circulation, a certain concentration or higher is required to exert their effects at the target site, and this often leads to side effects in areas other than the target site. Many drugs have had to be abandoned due to these side effects (toxicity). To avoid this, drug delivery systems (DDS) and target protein-specific antibodies have been developed.

[0003] Drug delivery systems (DDS) are drug delivery systems that control the pharmacokinetics of drugs in the body by controlling sustained release, absorption, and targeting. For example, DDS technology that encapsulates drugs in polymer micelles or liposomes to serve as drug carriers is well known, but it has drawbacks such as low targeting ability, interaction between intravenously administered liposomes and blood components leading to leakage of the encapsulated drug or disintegration of the liposomes, and rapid elimination from the bloodstream due to capture by the reticuloendothelial tissue.

[0004] More recently, caged compounds have been discovered in which drugs are protected (modified) with a protecting group that can be deprotected by light, allowing the active substance to be generated by light irradiation at any location inside or outside of tissues (Patent Documents 1 and 2). However, since short-wavelength ultraviolet light is often used to deprotect these caged compounds, there are drawbacks such as cell damage, limited range of use in living organisms, and lack of target specificity.

[0005] Furthermore, as an example of controlling targeting, antibody-drug conjugates (ADCs), in which a drug with cytotoxic activity is bound to an antibody via a linker, have been developed and are already being used in cancer treatment. ADCs not only exert a selective effect on lesions through the synergistic effect of the biological activity of the antibody drug and the effect of the drug delivered by the antibody, but they can also deliver the therapeutic agent only to the lesion site, thus expanding the safety margin and are expected to be next-generation antibody drugs (Non-Patent Literature 1). The mechanism of action is as follows: The antibody portion of the ADC binds to an antigen on the cell surface and is then taken up into the cell by internalization. The internalized ADC has its linker cleaved within the cell by the action of lysosomes, and a small molecule drug is released. The released small molecule drug exerts its therapeutic effect within the cell and acts. By binding a compound to an antibody and delivering the drug to the cell to which the antibody is bound, and specifically removing the cell, the drug can be selectively delivered to target cells. Therefore, even smaller amounts of the compound can be effective in target cells, and the impact on normal cells is minimal, making it possible to achieve a wider therapeutic dose range compared to conventional chemotherapy. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] International Publication No. 2000 / 031588 [Patent Document 2] International Publication No. 2006 / 110804 [Patent Document 3] U.S. Patent No. 7,091,186 [Patent Document 4] U.S. Patent No. 7,223,837 [Patent Document 5] U.S. Patent No. 7,553,816 [Patent Document 6] U.S. Patent No. 7,659,241 [Patent Document 7] U.S. Patent No. 7,989,598 [Patent Document 8] U.S. Patent No. 8,163,888 [Patent Document 9] U.S. Patent No. 8,198,417 [Patent Document 10] U.S. Patent No. 8,236,319 [Patent Document 11] U.S. Patent No. 8,563,509 [Patent Document 12] U.S. Patent No. 6,214,345 [Patent Document 13] U.S. Patent No. 4,563,304 [Patent Document 14] International Publication No. 2009 / 134976 [Patent Document 15] International Publication No. 2009 / 134977 [Patent Document 16] International Publication No. 2012 / 177837 [Patent Document 17] International Publication No. 2005 / 099689 [Non-Patent Document]

[0007] [Non-Patent Document l] Beck A. et al., Nat. Rev. Drug Discov., 2017, vol.16(5), pp.315 - 337. [Non-Patent Document 2] Sato, K. et al., 2018, ACS Cent. Sci. Vol.4, pp.1559 - 1569. [Non-Patent Document 3] Greenberg, AK et al., 2002, Am. J. Respir. Cell Mol. Biol.Vol.27(3), pp.320-328. [Overview of the project] [Problems that the invention aims to solve]

[0008] Drug delivery systems (DDS) that use polymer micelles or liposomes to alter the physical properties of drugs, such as particle size and hydrophilicity, and DDS using caged compounds, have low target-specification, making it difficult to completely suppress side effects. Furthermore, DDS using caged compounds involves deprotection using ultraviolet light, which raises concerns about cell damage caused by UV radiation.

[0009] In contrast, antibody-based ADCs (antibody-drug conjugates) act selectively on target cells, making them a highly targeted and effective method compared to the aforementioned DDS (drug delivery systems). However, they require target molecules expressed on the cell surface, and if targets are not present throughout the cell surface of affected tissue, such as in inflammatory diseases or infections, their use will only yield limited results. Since ADCs are released after their linkers are cleaved by intracellular enzymes following cell uptake, they can only be expected to have limited effects when applied to inflammatory diseases or infections where inflammation is present throughout the tissue.

[0010] If it is possible to target molecules that cannot be targeted by conventional DDS, such as inflammatory proteins, inflammatory cytokines, proteins secreted by pathogens, and organ-specifically expressed molecules, in addition to substances that exhibit specific binding affinity like antibodies, then it will be possible to provide therapeutic drugs and treatment methods that are effective for a wider range of diseases. The objective of this invention is to provide drug conjugates and treatment methods that act on targets that could not be addressed by conventional DDS. [Means for solving the problem]

[0011] This invention relates to a method of treatment in which a targeted accumulation complex, in which a drug and a photosensitive substance are bound to a carrier, is administered, and the drug is released by irradiating the disease site with light. This invention relates to a novel drug delivery system that exerts an effect even on cells that do not express the target molecule by binding the drug to a target-directed molecule such as an antibody, or a molecule that accumulates at the disease site, and then releasing the drug by light after it has accumulated in the target tissue. Conventional ADCs use a complex in which a compound is bound to an antibody, and act specifically on target cells. As shown in the following examples, this invention can also use an antibody as a carrier, but its concept and target of treatment differ significantly from conventional ADCs. Therefore, in order to distinguish it from other antibody-drug complexes, the complex in which a drug and a photosensitive substance are bound to the carrier of this invention is referred to here as a targeted accumulation complex. Furthermore, since the drug is released by light irradiation, even when a substance that is not target-directed and circulates in the body, such as albumin, is used as a carrier, it is possible to act locally only at the site where light is irradiated. Because targeted accumulation complexes only exert their therapeutic effect upon light irradiation, they can be administered locally, potentially allowing the use of compounds that were previously unusable due to side effects. Specifically, the following targeted accumulation complexes are provided. (1) A target-accumulation complex having a carrier structure in which a near-infrared light-sensitive substance and a drug are linked. (2) The target-accumulating complex according to (1), characterized in that the carrier is a binding molecule that exhibits binding affinity to a target molecule, or a molecule that accumulates at a disease site. (3) The target accumulation complex according to (1) or (2), characterized in that the disease to be treated is one of cancer, inflammation, infection, collagen disease, or organ-specific disease. (4) The target accumulation complex according to (3), wherein the organ-specific disease is one of the following: heart disease, kidney disease, liver disease, lung disease, thyroid disease, digestive disease, or neuromuscular disease. (5) A target-accumulating complex according to any one of (2) to (4), characterized in that the target molecule is a molecule that is expressed on the cell surface in association with disease, or a molecule that is expressed in an organ-specific manner. (6) A target-accumulating complex according to any one of (2) to (5), characterized in that the binding molecule is an antibody, an antigen-binding antibody fragment of an antibody, or an aptamer. (7) A target-accumulation complex according to any one of (1) to (6), wherein the drug is at least one of the following: an anticancer agent, an anti-inflammatory agent, an antiviral agent, or an antibacterial agent. (8) A target-accumulating complex according to any one of (1) to (7), characterized in that the drug is bound via a linker, and the linker is either a cleavage-type or non-cleavage-type linker. (9) A target-accumulating complex according to any one of (1) to (8), characterized in that the near-infrared light-sensitive substance is a phthalocyanine dye. (10) The target-accumulating complex according to (9), characterized in that the phthalocyanine dye is IR700. (11) A pharmaceutical composition comprising any one of the target-accumulating complexes described in (1) to (10) as an active ingredient. (12) A drug delivery technology comprising a target-accumulated complex having a structure in which a near-infrared light-sensitive substance and a drug are linked to a pre-administered carrier, and the drug is released from its binding molecule by irradiation of the complex with near-infrared light. (13) A therapeutic method characterized by administering the pharmaceutical composition described in (11) to a subject having a disease, and irradiating it with near-infrared light after a predetermined time has elapsed to release the drug. (14) The treatment method according to (13), characterized in that the predetermined time is the time during which the targeted accumulation complex accumulates at the disease site. (15) The treatment method according to (13) or (14), characterized in that the irradiation of near-infrared light is performed by direct irradiation from outside the body or by using a device that is introduced into the diseased area and irradiates with near-infrared light. [Brief explanation of the drawing]

[0012] [Figure 1] A diagram showing the synthesis pathway of dexamethasone derivatives. [Figure 2]A diagram illustrating the synthesis process of IgG-DEX-IR700. (A) shows the changes in the antibody from the binding of dexamethasone to the synthesis of a dexamethasone derivative. (B) is a diagram confirming that IR700 has bound to the antibody-dexamethasone complex. [Figure 3] This figure shows that dexamethasone was released from IgG-DEX-IR700 while maintaining its therapeutic effect upon light irradiation. [Figure 4] This figure shows the results of analyzing the change in fluorescence intensity when the T-DM1-IR700 was irradiated with near-infrared light at varying energies. [Figure 5] A schematic diagram showing the structure of T-DM1 and a tubulin polymerization inhibitor with activity cleaved at the linker end. [Figure 6] This figure shows the results of mass spectrometry analysis of T-DM1-IR700, which was cut by near-infrared light irradiation. [Figure 7] This figure shows the results of analyzing the Area value by mass spectrometry after irradiating the T-DM1-IR700 with varying light energy. [Figure 8] This figure shows the results of mass spectrometry analysis of mouse antibodies (αMFc-NC-DM1) conjugated with IR700 and DM1, after irradiation with near-infrared light. (A) is the blank, (B) is T-DM1-IR700 irradiated at 16 J / cm2, (C) is αMFc-NC-DM1 without near-infrared light irradiation, and (D) is αMFc-NC-DM1 irradiated with near-infrared light at 16 J / cm2 and analyzed by mass spectrometry. [Figure 9] This figure shows that T-DM1-IR700 specifically binds to HER2-positive cells. [Figure 10] A schematic diagram illustrating an analytical method using a heterogeneous in vitro disease model system. [Figure 11] A figure illustrating the effects of target-accumulating complexes in heterogeneous in vitro disease model systems. [Figure 12] This figure shows that HER2-negative cells alone, to which antibodies do not bind, are ineffective against targeted accumulation complexes. [Figure 13]This figure shows that target-accumulating complexes are effective in in vitro disease model systems using various HER2-positive cells. [Figure 14] This figure illustrates the effects of target-accumulation complexes using an in vivo disease model system. [Modes for carrying out the invention]

[0013] The inventors have discovered that by combining an ADC with a photosensitive substance and irradiating it with light, it is possible to provide therapeutic effects not only on ADCs, which selectively bind to target cells and exert their effects, but also on diseased cells that do not express antigen molecules. Furthermore, building on this, by using not only antibodies but any substance as a carrier, and especially by conjugating a drug and a photosensitive substance to a molecule with high biostability, the drug can be released only at the light-irradiated site, and local drug action can be expected. By creating a targeted accumulation complex in which a drug and a photosensitive substance are conjugated to a highly biostable carrier, it becomes possible to apply it to a wider range of diseases than ADCs. In addition, because the targeted accumulation complex can act locally, it may be possible to use compounds that could not be used before due to side effects. In the following examples, a complex in which an antibody is linked to a drug and a near-infrared photosensitive substance is shown as a targeted accumulation complex, but any molecule with high biostability can be used, and antibodies or molecules that accumulate at disease sites can be used in particular.

[0014] The inventors of this invention analyzed the mechanism by which an antibody-IR700 complex, used in near-infrared photoimmunotherapy (NIR-PIT), a type of ADC, induces necrosis. They found and reported that a photochemical reaction caused by irradiation with near-infrared light cleaves the ligand of IR700 from the antibody-IR700 complex, resulting in a physical change in the antibody-IR700 complex and instantaneously inducing cell death (Non-Patent Literature 2). Since a structural change occurs in the antibody-IR700 complex due to a photochemical reaction, a complex to which a drug is bound to the antibody-IR700 complex may also undergo a structural change. Therefore, a targeted accumulation complex was created in which a drug was bound to the antibody-IR700 complex via a linker, and the physical changes of the structure due to light irradiation were analyzed. As a result, it was revealed that the linker was cleaved and the drug was released, thus completing the present invention. While NIR-PITs and ADCs act only on target cells, targeted accumulation complexes differ significantly in that they can act over a wider range of cells while maintaining target selectivity.

[0015] The diseases to which this invention can be applied are not limited to those shown in the following examples, but can be applied to any disease. Diseases in which cell surface markers have been identified, or diseases in which molecules that are specifically present or accumulate at the disease site have been identified, are particularly suitable targets. Since the drug can be accumulated at the disease site, a therapeutic effect targeting the diseased tissue or organ itself can be expected even if the target molecule is not expressed throughout the entire diseased tissue. Furthermore, it is also possible to release the drug only at the disease site by binding the drug and a photosensitive substance to a non-target-directing carrier and irradiating the disease site with light.

[0016] Examples of target diseases include, but are not limited to, infectious diseases, inflammatory diseases, collagen diseases, cancer, or organ-specific diseases. Conventional antibody drugs required the expression of the target molecule on the cell surface to be effective. However, targeted accumulation conjugates can use a wide range of substances as carriers, such as disease-specific molecules, molecules that bind to molecules expressed at the disease site, molecules that accumulate at the disease site, or substances that circulate in the body. ADCs use antibodies that bind to molecules expressed at the disease site, but the antigen is not always uniformly expressed at the disease site, leading to the problem of not being able to obtain therapeutic effect. Targeted accumulation conjugates are a groundbreaking drug and treatment method that differs from conventional antibody drugs in that the drug's therapeutic effect extends to cells that do not express the antigen, even if there are cells in the surrounding area that express even a small amount of the antigen molecule. Furthermore, since the effect is obtained even when using molecules that accumulate at the disease site, it can be used to treat a wider range of diseases. In addition, since the effect is obtained only in the area irradiated with light, it is possible to use substances that circulate in the body as carriers.

[0017] A targeted accumulation complex has a structure in which a drug and a photosensitive substance are bound to a carrier. Any substance can be used as the carrier, but as mentioned above, a substance with high biostability is preferred. In particular, a binding molecule that shows affinity to the target molecule, or a molecule that accumulates at the disease site, can be suitably used. Here, a binding molecule is a molecule that is expressed in diseased cells and shows affinity to molecules expressed on the cell membrane. The target molecule of the binding molecule may be a molecule expressed in association with the disease, or a molecule expressed in the diseased organ. Depending on the target disease, the molecules to which the drug and photosensitive substance are bound should be appropriately selected.

[0018] Suitable binding molecules include antibodies and aptamers, but antibodies are preferred. The antibody only needs to specifically recognize the antigen's epitope. It doesn't have to be an intact antibody; it can also contain functional fragments of the antibody, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-stabilized V-region fragments (dsFv), rIgG fragments, or peptides containing CDRs. It just needs to contain the antibody portion capable of binding to the epitope. These functional fragments of antibodies that bind to the antigen are called antigen-binding antibody fragments. Antibodies also include those produced by genetic engineering, such as single-chain antibodies (scFv), low-molecular-weight antibodies (Fv-Clasp), bispecific antibodies, chimeric antibodies such as humanized antibodies, and humanized CDR-transplanted antibodies. All of these binding molecules can be produced using known methods.

[0019] As mentioned above, target molecules for binding molecules such as antibodies include molecules expressed on the cell surface. Examples of target molecules are not limited to molecules expressed on the cell surface in relation to disease, but can be any molecule expressed at the disease site. In other words, the target molecule may be a molecule that causes the disease, a molecule expressed in relation to the disease, or a molecule expressed at the disease site even if it is not directly related to the disease. Alternatively, it may be a molecule derived from a pathogen. Specifically, target molecules for binding molecules include so-called cancer antigens that are specifically expressed on the cell surface of cancer cells, surface antigens and inflammatory proteins that are expressed on specific immune cells in collagen diseases, and, in the case of infectious diseases, pathogen-derived molecules that are expressed on the cell surface from pathogens such as viruses, bacteria, fungi, and parasites, or substances secreted by pathogens. Alternatively, it may be a host cell molecule expressed on the cell surface due to a disease caused by a pathogen. Examples of target molecules include cell surface molecules composed of peptides, proteins, lipids, polysaccharides, proteoglycans, lipopolysaccharides, nucleic acids, etc.

[0020] Furthermore, the drug and photosensitive substance may be conjugated to molecules that accumulate at the disease site. Examples of molecules that accumulate at the disease site include, in the case of inflammatory diseases, cytokines, chemokines, growth factors (FGF, EGF, BTC, PEDF, SCF, HER2, HER3), angiogenic factors (VEGF, PIGF), growth factors (LIF, OSM, TGF, BMP, IGF, CNTF), chemical mediators that act as inflammatory mediators (bradykinin, serotonin, histamine), prostanoids (prostaglandins, leukotrienes), thallium chloride, citrate, etc.; in the case of kidney diseases, creatinine, albumin, low molecular weight albumin, peptides, MAG3 (mercaptoacetylglycylglycylglycine), diethylenetriamine compounds such as DTPA (diethylenetriaminepentaacetic acid), DMSA (dimercaptosuccinate); and in the case of myelogenic diseases, ferritin, phosphates, Ca compounds, bisphosphonates. Examples of substances that may be involved in bleeding include sphingate compounds, transferrin, HMDP (hydroxymethylenediphosphonate), MDP (methylenediphosphonic acid), platelets and coagulation factors (I-XIII) at the bleeding site, thrombin, growth factors, inflammatory substances and cytokines (chemical mediators, vasoconstrictors), and in the case of brain and nerve diseases, cholinesterase, dopamine, L-dopa, serotonin, acetylcholine, adrenaline, GABA, endorphins, enkephalin, glutamic acid, aspartic acid, histamine, vasopressin, vasoactive intestinal peptide (VIP), carnosine, bradykinin, cholecystokinin, bombesin, somatostatin, corticotropin-releasing factor, neurotensin, adenosine, ECD (cysteine ​​nail dimer), HMPAO (hexamethylpropyleneamine oxime), IMP, iomagene, and ioflupane.For eye diseases, VEGF and Rho are used; for lung diseases, surfactant (surfacten), KL-6, SP-A, SP-D, phosphatidylcholine, choline, sphingosine, ACE, and MAA (MAA coarse aggregated albumin); for thyroid diseases, iodine and sodium pertechnetate; for heart diseases, cardiac myosin, MIBG (metaiodobenzylguanidine), BMIPP (β-methyl-p-iodophenylpentadecanoate), tetrophosmin, MIBI (methoxyisobutylisonitrile), and thallium chloride; for liver diseases, tin colloid, GSA, and PMT; for lymph node diseases, tin colloid and phutenic acid are used; and for digestive diseases, proton pump targets, tin colloid, pertechnetate, HSA-D, PMT, and GSA are used. Substances that accumulate in each organ can also be used as carriers. Substances other than those exemplified here, such as those used in nuclear medicine to bind radioactive isotopes like technetium as tracers, can be suitably used as carriers. Any substance known to accumulate at disease sites, other than those exemplified above, can be used as a carrier to bind a drug and a photosensitive substance for treatment.

[0021] As described above, targeted accumulation complexes can also be used to treat organ-specific diseases such as heart disease, kidney disease, liver disease, lung disease, thyroid disease, digestive disease, and neuromuscular disease. By attaching a photosensitive substance to a binding molecule that binds to molecules specifically expressed in organs such as the heart and kidneys, or to a molecule that accumulates specifically in organs, and administering the drug used to treat each disease, it is possible to accumulate the drug specifically in the diseased organ and perform treatment. Since the objective is to accumulate the targeted accumulation complex in the diseased organ, antibodies that bind to organ-specific molecules, such as cardiac troponin in the heart, aquaporin in the kidneys, ACE receptors in the lungs, TTF-1 in the thyroid, neuropeptides, neurotransmitters, myosin in neuromuscular diseases, transferrin, ferritin in the bone marrow, and incretins, gastrin, and glp-1 in the digestive system, can also be used, even if they are not related to the disease.

[0022] Furthermore, molecules known to be expressed and accumulate in association with disease, such as α-synuclein and β-amyloid, which are known to accumulate in the nervous system in neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease, can be targeted and bound to. For heart diseases such as heart failure and myocardial infarction, molecules that bind to β1 receptors, AT1 (angiotensin receptor), and CRHR2 (corticotropin-releasing hormone receptor 2), which are highly expressed in the heart, can be targeted. For kidney diseases, molecules that bind to (pro)renin receptors, which are highly expressed in the kidneys, can be targeted. For liver diseases, molecules that bind to asialoglycoprotein receptors, which are highly expressed in the liver, can be targeted.

[0023] Furthermore, non-target-directed substances can also be used as carriers. By binding a drug and a photosensitive substance to a carrier, circulating it throughout the body, and irradiating the disease site with light, the drug can act selectively on the disease site. Any substance can be used as a non-target-directed carrier, such as compounds, peptides, proteins, lipids, polysaccharides, proteoglycans, lipopolysaccharides, nucleic acids, extracellular vesicles, exosomes, or biocompatible nanomaterials, but it is preferable that the substance has biological stability. For example, even if a drug and a photosensitive substance are bound to a highly biologically stable substance such as albumin or dextran, the drug will not be released unless light is irradiated, so even if it circulates in the blood, the drug will not cause side effects. By irradiating the disease site with light, the drug is released and exerts its effect only at the disease site.

[0024] Furthermore, a substance possessing biological stability here refers to a molecule that is physically stable for the time between administration and light irradiation. The time between administration and light irradiation cannot be determined definitively, as the time required for the accumulation of the target-accumulating complex varies depending on the target cells, tissues, and organs, and multiple light irradiations may be performed depending on the disease. However, any compound with a blood half-life of 10 minutes to 6 months, preferably 1 hour to 12 weeks, is acceptable.

[0025] If the binding molecule is an antibody, already approved antibody drugs may be used. More than 70 antibody drugs have already been approved in Japan, the United States, and Europe, and these antibody drugs can be used by binding them with drugs or photosensitive substances. Furthermore, targeted accumulation conjugates may be manufactured using antibody drugs that will be approved in the future. Some of the major antibody drugs that are binding molecules and have been approved for use in cancer include rituximab, ofalmumab, and obinutuzumab, which target CD20; trastuzumab and pertuzumab, which target HER2; cetuximab, panitumumab, and nesitumumab, which target EGFR; alemtuzumab, which targets CD52; denosumab, which targets RANKL; ipilimumab, which targets CTLA-4; mogalizumab, which targets CCR4; ramucirumab, which targets VEGFR2; nivolumumab and pembrolizumab, which target PD-1; blinatumomab, which targets CD19 / CD3; dinutuximab, which targets GD2; daratumumab, which targets CD38; and elotuzumab, which targets SLAMF7, but are not limited to these. The targeted accumulation conjugates utilizing the antibody drugs described above are effective in treating cancer, particularly solid tumors. Solid tumors generally exhibit heterogeneity, where cells expressing the target molecule and cells that do not express the target molecule coexist within a single tumor. While conventional antibody drugs are effective only against cells expressing the target molecule, targeted accumulation conjugates are effective against cancer cells surrounding the cells expressing the target molecule. Conventional drug delivery systems (DDS) and antibody-drug conjugates (ADCs) could not treat heterogeneous tumors, but targeted accumulation conjugates are effective in treating heterogeneous tumors as well.

[0026] When the target is cancer, a cytotoxic drug is linked to the antibody, which is a binding molecule, as a payload. The drug may also be linked to the antibody in the form of a prodrug. Examples of cytotoxic drugs include alkylating agents, platinum-based drugs, antimetabolites, topoisomerase inhibitors, microtubule polymerization inhibitors, and microtubule depolymerization inhibitors. Target-accumulation conjugates selectively bind to cells in which the target molecule is present on the cell surface, and the drug's effect extends only to the surrounding cells. Therefore, even cytotoxic drugs have fewer side effects and are effective even against cancer cells that do not express the target molecule on their cell surface.

[0027] In addition, among approved antibody drugs, those targeting diseases other than cancer include collagen diseases, infectious diseases, and inflammatory diseases. Major antibody drugs for these diseases include tocilizumab and sarilumab, which target IL-6R, for rheumatoid arthritis (a collagen disease); efalizumab, which targets CD11, and brodalumab, which targets IL-17R, for psoriasis (an inflammatory disease); vedolizumab, which targets α4β7 integrin, for Crohn's disease; and dupilumab, which targets IL-4Rα, for atopic dermatitis. Regarding infectious diseases, examples include bezlotoxumab, which targets Clostridium difficile toxin B, for Clostridium difficile infection; oviltoxaximab and laxibakumab, which target Bacillus ancilasis toxin, for inhaled anthrax; and casilivimab and imdevimab, for COVID-19. Furthermore, it goes without saying that we can not only utilize existing antibody drugs, but also create novel antibody drugs and construct target-accumulated conjugates.

[0028] In the case of collagen diseases, steroids or similar drugs can be linked to the carrier as a payload, and in the case of inflammatory diseases, anti-inflammatory drugs can be linked as drugs. Alternatively, compounds already used to treat each disease can be linked as payloads. Specific steroids include, but are not limited to, dexamethasone acetate, prednisolone, hydrocortisone acetate, prednisolone acetate, prednisolone valerate, triamcinolone acetonide, clobetasone butyrate, hydrocortisone butyrate, dexamethasone propionate, dexamethasone valerate, halcinonide, betamethasone valerate, beclomethasone propionate, fluocinolone acetonide, mometasone furoate, betamethasone propionate butyrate, fluocinonide, betamethasone dipropionate, clobetasol propionate, and diflorasone acetate. For example, for rheumatoid arthritis, in addition to steroids, non-steroidal anti-inflammatory drugs (NSAIDs) and disease-modifying antirheumatic drugs (DMARDs) can be used as payloads. For psoriasis, steroids, JAK inhibitors, and PDE4 inhibitors can be used. For Crohn's disease, anti-inflammatory drugs, steroids, and immunomodulators can be used as payloads. For infectious diseases, antibiotics, antibacterial agents, antiviral agents, and other drugs used to treat each specific infection can be loaded as payloads.

[0029] Examples of collagen diseases that may be treatable with targeted accumulation complexes include systemic lupus erythematosus, rheumatic fever, scleroderma, dermatomyositis, polymyositis, polyarteritis nodosa, rheumatoid arthritis, Sjögren's syndrome, mixed connective tissue disease (MCTD), granulomatosis with polyangiitis (Wegener's granulomatosis), eosinophilic granulomatosis with polyangiitis (Churg-Strauss syndrome), microscopic polyangiitis, Takayasu's arteritis (aortitis syndrome), giant cell arteritis (temporal arteritis), polymyalgia rheumatica, eosinophilic fasciitis, adult-onset Still's disease, ankylosing spondylitis, psoriatic arthritis, relapsing polychondritis, Behçet's disease, and sarcoidosis.

[0030] Examples of viruses that can cause infectious diseases include various coronaviruses, specifically SARS-CoV-2, SARS-CoV, and MERS-CoV. Other examples include Ebola virus, Zika virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, human immunodeficiency virus, human T-cell leukemia virus, herpes simplex virus type 1, herpes simplex virus type 2, varicella-zoster virus, cytomegalovirus, human herpesvirus 6, human herpesvirus 7, Epstein-Barr virus, human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus), influenza virus, adenovirus, norovirus, rotavirus, respiratory syncytial virus (RSV), measles virus, mumps virus, rhinovirus, dengue virus, papillomavirus, poliovirus, and rabies virus.

[0031] Examples of bacteria that can cause infectious diseases include pathogenic Escherichia coli, Shigella bacteria (such as Shigella dysenteriae, S. frexneri, S. sonnei, etc.), Salmonella bacteria (such as Salmonella typh, S. paratyphi-A, S. schottmuelleri, S. typhimurium, S. enteritidis, etc.), Enterobacter bacteria (such as Enterobacter aerogenes, E. cloacae, etc.), Klebsiella bacteria (such as Klebsiella pneumoniae, K. oxytoca, etc.), Proteus bacteria (such as Proteus mirabilis, P. vulgaris, etc.), Yersinia bacteria (such as Yersinia pestos, Y. enterocolitica, etc.), Vibrio bacteria (such as Vivrio chorelae, V. parahaemolytucs, etc.), Haemophilus bacteria (such as Haemophilus influenzae, H. parainfluenzae, H. ducreyi, etc.), Pseudomonas bacteria (such as Pseudomonas aeruginosa, P. capacia, P. putida, etc.), Acinetobacter bacteria (such as Acinetobacter calcoaceticus, A. baumannii, A. lowffii, etc.), Legionella bacteria (such as Legionella pneumophila, etc.), Bordetella bacteria (such as Bordetella. melitensis, B. abortus, B. suis, etc.), Francisella tularensis, Bacteroides bacteria (such as Bacteroides fragilis, B. melaninogenicus, etc.), Neisseria bacteria (such as Neisseria gonorrhoeae, N. meningitidis, etc.), Staphylococcus bacteria (such as Staphylococcus aureus, S. epidermidis, S. saprophyticus, etc.), Enterococcus bacteria (such as Enterococcus faecalis, E. faecium, E. avium, etc.), Bacillus bacteria (such as Bacillus subtiris, B.anthracis, B. cereus, etc.), Clostridium bacteria (such as Clostridium difficiole, C.botulinum, C. perfringens, C. tetani, etc.), Corynebacterium bacteria (Corynebacterium diphtheirae, etc.), Mycobacterium tuberculosis, M. bovis, M. leprae, M. avium, M. intracellulare, M. kansasii, M. ulcerans, etc.), mycoplasma, Borrelia recurrentis, B. burgdoferi, etc., Treponema pallidum, Campylobacter coli, C. jejuni, C. fetus, etc., Helicobacter pylori, H. heilmannii, etc.), Rickettsia prowazekil, R. mooseri, R. Examples include bacteria of the genus Chlamydia (e.g., *Tsutsugamushi*), Chlamydia (e.g., *Chlamydia trachoma*, *C. psittaci*), and Listeria (e.g., *Listeria monocytogenes*).

[0032] Furthermore, examples of fungi that can cause infections include Candida species (Candida albicans, C. krusei, C. glabrata, C. tropicalis, etc.), Cryptococcus neoformans, Aspergillus species (Aspergillus fumigatus, A. niger, etc.), Mucorales species (Mucor circinelloides, Lichtheimia corymbifera, Rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, and Coccidioides imitis. Examples include *Immitis* and *Histoplasma capsulatum*.

[0033] Examples of parasites that can cause infectious diseases include Entamoeba histolytica parasites, Balantidium colonis, Naegleria fowleri, Acanthamoeba species, Giardia lamblia, Cryptosporidium spp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Ancylostoma braziliense.

[0034] For use in treating infectious diseases, a target-accumulation complex can be created by linking a drug and a near-infrared light-sensitive molecule to a binding molecule that specifically binds to molecules that appear on the cell surface in association with the disease, or substances secreted by pathogens. If infected cells can be specifically killed, it may be possible to completely eliminate the virus from the body of virus carriers such as hepatitis B virus, hepatitis C virus, human immunodeficiency virus, and human T-cell leukemia virus.

[0035] The payload may consist of one drug or two or more drugs. When multiple drugs are used as the payload, targeted accumulation complexes, each carrying a drug individually, may be mixed and used, or multiple drugs may be linked to a single carrier. Generally, a higher drug-antibody ratio (DAR) is desirable for the number of drug molecules linked to the binding molecule, but for targeted accumulation complexes, a drug-carrier ratio (equivalent to DAR) of 1 to 10 is sufficient. Unlike existing ADCs, targeted accumulation complexes are applicable not only to cancer but also to a wide range of diseases such as inflammatory diseases, collagen diseases, and infectious diseases. Therefore, the payloads carried are diverse, and the amount of drug carried is expected to differ for each disease, so it is desirable to optimize them as appropriate.

[0036] Furthermore, as shown in the following examples, the method of the present invention is a therapeutic method that can be applied locally. Therefore, it may be possible to use drugs whose development was abandoned due to off-target effects or systemic toxicity. In addition, by acting locally, it becomes possible to use lower doses than before, thus reducing side effects.

[0037] The drug is attached to the binding molecule using linkers or spacers by known methods (Patent Documents 3-16). Examples of linkers / spacers include maleimidocaproyl, maleimidocaproyl-polyethylene 20 glycol (MC(PEG)6-OH), p-aminobenzylcarbomer (PAB), valine-citrulline (vc), N-methyl-valinecitrulline, N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl 4-(2-pyridyldithio)2-sulfobutanoate (sulfo-SPDB), N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), 2-iminothiolane, and acetylsuccinic anhydride.

[0038] Furthermore, existing ADCs typically link drugs to antibodies via coupling reactions using lysine or cysteine. To produce more homogeneous ADCs, techniques such as selective biocoupling reactions using non-natural amino acids, introduction of free cysteine ​​through genetic modification (THIOMAB method), methods that expose aldehydes from the N-terminus or sequences containing free cysteine ​​to perform coupling reactions (SMARTag method), and enzyme-based ligation methods can also be applied.

[0039] Targeted accumulation complexes include not only drugs that act on the disease but also near-infrared photosensitizers. To efficiently induce photochemical reactions, high-energy photons, i.e., short-wavelength light, are necessary. However, to prevent nonspecific damage to DNA, wavelengths longer than ultraviolet light must be used. Therefore, near-infrared photosensitizers are usually selected. Furthermore, to be used as a targeted accumulation complex by linking to carriers, particularly antibodies or other specifically binding molecules, the molecules must be small. Phthalocyanine-based photosensitizers are compounds that meet these requirements.

[0040] As the phthalocyanine dye, those having an absorption peak at 600 nm to 950 nm, preferably 660 nm to 740 nm, and more preferably 680 nm to 720 nm can be suitably used (Patent Document 17). A particularly preferred phthalocyanine dye is IR700 (IRDye® 700DX, LI-COR Biosciences). Using the NHS ester of IR700, it can be conjugated to specific binding molecules such as antibodies by covalent bonding.

[0041] The targeted accumulation complex can be administered locally or systemically by any method. Specifically, it can be administered intramuscularly by injection into the affected area, subcutaneously, intradermally, intravenously, or intraperitoneally, or parenterally by inhalation, ointment, patch, application, nasal drops, or eye drops, but oral administration is also possible.

[0042] Parenteral formulations typically include injectable fluids containing pharmaceutically acceptable fluids and physiologically acceptable fluids, such as water, physiological saline, equilibrium salt solutions, aqueous dextrose, or glycerol as the vehicle. Non-toxic solid carriers for solid compositions (e.g., in powder, pill, tablet, or capsule form) may include, for example, pharmaceutical-grade mannitol, lactose, starch, or magnesium stearate. In addition to a biologically neutral carrier, the administered pharmaceutical composition may contain small amounts of non-toxic auxiliary substances, such as humectants or emulsifiers, preservatives, and pH buffers, for example, sodium acetate or sorbitan monolaurate.

[0043] The effective therapeutic dose of a targeted accumulation complex depends on the target disease and the compound used as the drug, but can be appropriately set according to the disease and symptoms. For example, it can be set between 0.1 mg and 1000 mg per 60 kg of body weight. The dosage also differs depending on the method of administration, such as intravenous, local, or intraperitoneal administration. Furthermore, the administration method, such as daily administration or single dose, can be selected according to the disease and symptoms. In addition, it can be administered in the presence of other therapeutic agents.

[0044] Near-infrared light irradiation can be achieved by using LEDs, LED lasers, or filtered light, and irradiating with an appropriate wavelength and therapeutic dose. The therapeutic dose should be between 1 and 1000 J / cm². -2 The irradiation time can be appropriately determined between 5 seconds and 1 hour. While irradiation can be performed directly from outside the body, devices such as light guide catheters, endoscopic light guide fibers, puncture irradiation fibers, vascular light guide catheters, drain-type light guide devices, or implantable, patch-type, or bracelet-type light irradiation devices can be used for irradiation after being introduced into the diseased area. When a targeted accumulation complex is administered systemically by intravenous injection as a pharmaceutical composition, the time required for the targeted accumulation complex to accumulate in the lesion should be considered, and near-infrared light should be irradiated after accumulation in the lesion. The time required for the targeted accumulation complex to accumulate in the lesion varies depending on the carrier used, but is typically between 5 minutes and 48 hours. In the case of local administration, light irradiation can be performed in a shorter time after administration than in the case of systemic administration.

[0045] Furthermore, instead of a single irradiation, multiple irradiations may be performed. In the case of multiple irradiations, there are no particular restrictions on the interval between irradiations. For example, multiple irradiations can be performed on the same day with predetermined intervals of 5 minutes to 10 hours, or irradiations can be performed daily, every other day, or every few days to several weeks. Various irradiation schedules can be set. In addition, there are no particular restrictions on the administration schedule when setting a multiple irradiation schedule, but if time has passed since the previous administration of the targeted accumulation complex, it may be determined considering the pharmacokinetics of the targeted accumulation complex, such as administering it again before irradiation.

[0046] The present invention will be described in detail below with reference to examples, but is not limited thereto. [Example 1] 1. Creation of a target-accumulation complex by conjugating mouse IgG with dexamethasone (DEX) and IR700. We prepared targeted accumulation complexes by conjugating dexamethasone and IR700 to an antibody and investigated their effects. First, a dexamethasone derivative containing an SH group was synthesized, conjugated to an antibody, and finally, IR700 was conjugated to create a targeted accumulation complex. First, the method for synthesizing the dexamethasone derivative is explained (Figure 1).

[0047] [Synthesis of dexamethasone derivatives] 1st process Dexamethasone (400.4 mg), N-(tert-butoxycarbonyl)-N-methyl-L-alanine (225.9 mg), and DMAP (68.4 mg) were suspended in methylene chloride (7.5 mL). EDC hydrochloride (536.9 mg) was added under ice bath and the mixture was stirred overnight at room temperature. Saturated ammonium chloride aqueous solution was added to the reaction mixture, and the separated organic phase was sequentially washed with 5% sodium bicarbonate solution and 5% saline solution. Anhydrous sodium sulfate was then added and the mixture was dried. After filtering off the drying agent, the solvent was removed by reduced pressure distillation, and the mixture was purified by column chromatography (support: SiO2, solvent: ethyl acetate / n-hexane = 0 / 100 → 75 / 25) to obtain intermediate product 1 (Int.1) (606.3 mg, quantitative) as a white solid. Confirmation was performed by mass spectrometry. ESI MS:m / z found 578.19(Calcd for C 31 H 44 FNO8[M+H] + 578.32)

[0048] 2nd process To a solution of intermediate product 1 (601.6 mg) in methylene chloride (9 mL), 4.5 mL of 4 M ethyl HCl was added under an ice bath. After stirring at room temperature for 30 minutes, the solvent was removed by distillation under reduced pressure to obtain intermediate product 2 (Int.2) (530.7 mg, crude yield 93%) as a white solid. Confirmation was made by mass spectrometry. ESI MS:m / z found 478.33(Calcd for C 26 H 36 FNO6[M+H] + 478.26)

[0049] 3rd process To a solution of intermediate product 2 (371.1 mg), 3-(triphenylmethylthio)propionic acid (256.7 mg), and HATU (330.7 mg) in DMF (7 mL), DIPEA (307 μL) was added under an ice bath, and the mixture was stirred at room temperature for 1 hour. The reaction solution that had been examined under the same conditions using intermediate product 2 (51.3 mg) was combined with this reaction solution, ethyl acetate and 5% aqueous sodium bicarbonate were added, and the mixture was separated by liquid-liquid extraction. The organic phase was washed twice with 5% brine, then anhydrous sodium sulfate was added for drying. After filtering off the desiccant, the solvent was distilled off under reduced pressure, and the residue was purified by column chromatography (support: SiO2, solvent: ethyl acetate / n-hexane = 0 / 100 → 100 / 0) to obtain intermediate product 3 (653.2 mg, yield 98%) as a white solid. Confirmation was performed by mass spectrometry. ESI MS: m / z found 566.28 (Calcd for C 29 H 40 FNO7S [M+H-Tr] + 566.26)

[0050] Step 4 To a solution of intermediate product 3 (216.3 mg) in methylene chloride (4 mL), TIS (270 μL) and TFA (2 mL) were added under an ice bath, and the mixture was stirred at the same temperature for 30 minutes. Then the solvent was distilled off under reduced pressure, and the residue was purified by column chromatography (ODS, acetonitrile / water = 0 / 100 → 100 / 0) to obtain the dexamethasone derivative (110.6 mg, 73%) as a white solid. Confirmation was performed by mass spectrometry. ESI MS: m / z found 566.24 (Calcd for C 29 H 40 FNO7S [M+H] + 566.26)

[0051] [Synthesis of maleimidated antibody] Next, an SMCC linker was attached to the antibody to maleimidate it, and modification was performed with the dexamethasone derivative. A 4.86 mg / mL mouse monoclonal antibody solution (205 μL, 1 mg) was transferred to a centrifugal filter unit (Amicon Ultra-4, cutoff 30K, Merck Millipore), and Milli-Q water was added to bring the volume to 4 mL. The solution was centrifuged at 4000 g at 4 °C for 15 minutes, and after removing the filtrate, Milli-Q water was added and the solution was centrifuged again at 4000 g at 4 °C for 15 minutes to remove sodium azide from the antibody solution. 500 μL of 0.1 M phosphate buffer (pH 7.4) was added to the recovered desalted antibody solution (280 μL), and then 213 μL of Milli-Q water was added. 7 μL of a separately prepared 10 mg / mL Sulfo-SMCC / DMSO solution was added to this solution, and the solution was incubated at 25 °C for 1 hour. The reaction solution was transferred to a centrifugal filter unit (Amicon Ultra-4, cutoff 30K, Merck Millipore), and Milli-Q water was added to bring the volume to 4 mL. The mixture was centrifuged at 4000 g at 4 °C for 15 minutes, and the filtrate was removed. Milli-Q water was then added, and the mixture was centrifuged again at 4000 g at 4 °C for 15 minutes to remove reagent residue from the antibody solution. The maleimidized antibody was recovered from the filter unit, and Milli-Q water was added to make a 500 μL solution. This solution was then used directly in the next reaction.

[0052] [Conjugation of dexamethasone derivatives to maleimidized antibodies] 500 μL of maleimidized antibody was mixed with 500 μL of 0.1 M phosphate buffer (pH 7.4). To this solution, 7 μL of a separately prepared 10 mg / mL dexamethasone derivative / DMSO solution was added, and the mixture was incubated at 25°C for 1 hour. The reaction solution was transferred to a centrifugal filter unit (Amicon Ultra-4, cutoff 30K, Merck Millipore), and Milli-Q water (containing 5% DMSO) was added to bring the volume to 4 mL. The mixture was centrifuged at 4000 g at 4°C for 15 minutes, the filtrate was removed, and Milli-Q water was added. The mixture was centrifuged again at 4000 g at 4°C for another 15 minutes. This centrifugal filtration procedure was repeated two more times to remove reagent residue from the antibody solution. The dexamethasone conjugate antibody was recovered from the filter unit, and Milli-Q water was added to make a 100 μL solution (antibody concentration 5.8 mg / mL, 580 μg).

[0053] The synthesis of the maleimidized antibody and the changes in the antibody due to the conjugation of a dexamethasone derivative to the maleimidized antibody are shown in LC / MS TIC (total ion chromatogram) (Figure 2(A)). When the antibody used (Figure 2(A) antibody) was maleimidized (Figure 2(A) after maleimidization) and then modified with dexamethasone (Figure 2(A) after DEX modification and two ultrafiltrations), a shift in the antibody retention time was observed in accordance with the reaction. The peak observed after dexamethasone modification (indicated by arrows in the figure) indicates a peak derived from the dexamethasone derivative to which an SH group has been added. By repeating ultrafiltration twice, the peak derived from the dexamethasone derivative disappears (Figure 2(A) after two ultrafiltrations), indicating that only the antibody with dexamethasone added has been purified.

[0054] [IR-700 Conjugation] IR700 was conjugated to the dexamethasone conjugate antibody synthesized and purified using the above process. 145 μL of 0.1 M phosphate buffer (pH 7.4) was added to the dexamethasone conjugate antibody (50 μL, 290 μg). 80 μL of Milli-Q water was added, followed by 14.5 μL of DMSO. 0.77 μL of a separately prepared 10 mM IR-700 NHS ester / DMSO solution was added and incubated at 25°C for 1 hour. The reaction solution was transferred to a centrifugal filter unit (Amicon Ultra-4, cutoff 30K, Merck Millipore), and Milli-Q water (containing 5% DMSO) was added to bring the volume to 4 mL. The mixture was centrifuged at 4000 g at 4°C for 15 minutes, the filtrate was removed, and then Milli-Q water was added and the mixture was centrifuged again at 4000 g at 4°C for 15 minutes. This centrifugal filtration procedure was repeated two more times to remove reagent residue from the antibody solution. The dexamethasone-IR-700 conjugate antibody was recovered from the filter unit, and Milli-Q water was added to make a 200 μL solution (antibody concentration 170 μg / mL, 34 μg).

[0055] Dexamethasone conjugate antibody (IgG-SMCC-DEX) and IgG, dexamethasone, and IR700 complex (IgG-SMCC-DEX-IR700) were identified by SDS-PAGE. Figure 2(B) shows the CBB stained image on the left and the image with IR700 visualized by fluorescence on the right. A fluorescent band was visible only in IgG-SMCC-DEX-IR700, confirming that IR700 is bound to the antibody-dexamethasone complex.

[0056] 2. Release of dexamethasone by light irradiation IgG-SMCC-DEX-IR700 was irradiated with light, and analysis was performed to determine whether dexamethasone was released. 16 J / cm² 2The IgG-SMCC-DEX-IR700 complex, either irradiated with near-infrared light or left unirradiated, was centrifuged using a centrifugal filter unit (3K filter, Amicon) to remove the antibody components. The membrane-permeated liquid was collected and analyzed using an evaluation system (Non-Patent Literature 3) that detects the phosphorylation of Rb protein altered by the addition of dexamethasone. It is known that the phosphorylation of Rb protein increases with the addition of dexamethasone, and this evaluation system evaluates the release of dexamethasone by bioassay using this system.

[0057] Human lung adenocarcinoma-derived cell line A549 cells 1 × 10 5 The cells were seeded in a 6-well plate, and the following day, 2 μl each of the fraction obtained by removing the antibody component from the IgG-SMCC-DEX-IR700 complex (either irradiated with light or not) was added, and the cells were cultured for 2 days. A negative control was used (no additive), and a positive control was used (2 × 10⁶ cells). -7 Dexamethasone was added to achieve a mass of M, and the cells were cultured for two days in the same manner. The cells were harvested and analyzed by Western blotting (Figure 3).

[0058] Western blotting was performed using antibodies to detect Rb protein (Cell Signaling Technology, Rb(4H1) mouse monoclonal antibody), to detect phosphorylated Rb protein (Cell Signaling Technology, Phospo-Rb(Ser807 / 811)(D20B12)XP rabbit monoclonal antibody), and to detect β-actin (Fujifilm Wako Pure Chemical Industries, Ltd., anti-β-actin monoclonal antibody) (Figure 3 left). After normalizing the protein amount with β-actin and comparing the amount of phosphorylated Rb protein, the result was 16 J / cm³. 2 When using a sample irradiated with near-infrared light, the positive control was 2 × 10⁻¹⁰. -7 Phosphorylated Rb protein levels were detected at levels comparable to those with M-dexamethasone (Figure 3, right). On the other hand, in the case of no near-infrared light irradiation (0 J / cm²), the levels were significantly higher. 2In the samples obtained from ), the amount of phosphorylated Rb protein was almost the same as in the control. This result indicates that light irradiation releases dexamethasone, i.e., the drug component, from the IgG-SMCC-DEX-IR700 complex while maintaining its efficacy. Here, the antibody component was removed after light irradiation for analysis, but this result indicates that in vivo, by accumulating antibodies on a target and releasing the drug by light irradiation, it is possible to treat surrounding cells and tissues to which the antibodies are bound with the drug.

[0059] [Example 2] 1. Effects of light irradiation on T-DM1 (trastuzumab-emtansine)-IR700 As demonstrated in Example 1 above, near-infrared light irradiation cleaves the drug from the carrier while maintaining its efficacy. To confirm this, we performed an analysis using an existing ADC (Automated Cell Derivative) system in which IR700 was coupled.

[0060] T-DM1 (Kadcyla®, 1.0 mg, 6.6 nM) and IRDye700DX NHS (66.8 μg, 34.2 nM) were incubated in 0.1 M phosphate buffer (Na2HPO4, pH 8.5) at room temperature for 1 hour. Unreacted reagents and the T-DM1-IR700 complex were separated using a Sephadex G50 column (PD-10; GE Healthcare). Protein concentrations were measured by the CBB method to determine the concentration of the recovered T-DM1-IR700. The IR700 concentration was also determined by measuring the absorbance at 698 nm, and the number of fluorescent molecules bound to the antibody was confirmed. Here, three times the number of fluorescent molecules were bound to T-DM1, but the ratio of fluorescent molecules to ADC can be approximately 1 to 20.

[0061] 2 μg of the prepared T-DM1-IR700 was subjected to 0, 1, 4, 8, and 16 J / cm² emissions using an LED with an emission wavelength of 690 nm. 2Near-infrared light was irradiated, and the samples were analyzed by SDS-PAGE (Figure 4). Proteins were visualized by CBB staining (Figure 4 left), and IR700 was detected by fluorescence (Figure 4 right). Although the same amount of T-DM1-IR700 was electrophoresed in each lane (Figure 4 left), the fluorescence intensity decreased as the irradiated light energy increased, reaching 16 J / cm². 2 In samples irradiated with near-infrared light, almost no fluorescence was observed (Figure 4, right). The disappearance of fluorescence upon near-infrared light irradiation clearly indicates that a structural change has occurred in IR700, but it is unclear whether any changes have occurred in emtansine, the drug bound to the antibody by a linker. Therefore, we analyzed whether emtansine is affected by near-infrared light irradiation.

[0062] T-DM1 (Kadcyla) is a complex in which trastuzumab is linked to emtansine, a microtubule polymerization inhibitor derived from trastuzumab, by a non-cleaving SMCC linker (Figure 5). After trastuzumab binds to HER2 on the cell surface, T-DM1 is taken up into the cell by internalization. In the cell, emtansine is cleaved from the antibody portion of T-DM1 by enzymes, and it exerts its therapeutic effect as a microtubule polymerization inhibitor. If, in the case of the targeted accumulation complex T-DM1-IR700, no change occurs in the T-DM1 portion upon light irradiation, then T-DM1-IR700 will act selectively only on HER2-positive cells to which trastuzumab can bind, similar to Kadcyla. Furthermore, if light irradiation induces a structural change in the trastuzumab derivative, and causes a significant structural change, it may lose its effect as a microtubule polymerization inhibitor. Therefore, after irradiation with near-infrared light, we performed a mass spectrometry analysis to investigate whether the meitansine derivative, which is the drug, was cleaved from the antibody, and if so, whether the DM1 structure was maintained, that is, whether it was cleaved while maintaining its efficacy as a microtubule polymerization inhibitor.

[0063] T-DM1-IR700 with 0 or 16 J / cm² 2Near-infrared light was irradiated, and the antibody components were separated and removed by centrifugation. The filtrate was then analyzed by mass spectrometry. Mass spectrometry was performed using a QTRAP6500 (SCIEX) and an HPLC system Prominence (Shimadzu Corporation) under the following conditions. Detection: Cation mode Column: L-column2 ODS 3μm, 1.5×150mm Column temperature: 40℃ Mobile phase: A 0.1% formic acid, 5% acetonitrile :B 0.1% Formic acid, acetonitrile gradient JPEG0007839553000001.jpg26118 Monitoring time: 20 minutes Injection volume (μl): 10

[0064] The results are shown in Figure 6. T-DM1-IR700 irradiated with near-infrared light showed a peak at 485.2 m / z that was not present in the control sample that was not irradiated with near-infrared light (Figure 6, bottom, circled peak). Table 1 shows the molecular species generated by near-infrared light irradiation. In the molecular formula (Formula), molecules indicated by M and M1 are molecules in which the emtansine structure is preserved. The mass spectrometry results suggest that near-infrared light irradiation cleaves emtansine from the antibody while maintaining its efficacy.

[0065] [Table 1]

[0066] The above result is 16 J / cm 2 The following are the results of irradiating with near-infrared light. We analyzed the amount of light energy required to cleave and release the drug from the antibody by changing the irradiation intensity (Figure 7). 1, 4, 8, 16 J / cm² 2 Near-infrared light was irradiated, and mass spectrometry was performed to analyze the area value. The result was 4 J / cm². 2 Since the Area value remains almost unchanged under these conditions, it is clear that drug release occurs even at lower energy levels.

[0067] 2. Release of drugs from mouse antibodies To confirm that drug release due to light energy is not a phenomenon specific to T-DM1-IR700, IR700 was bound to αMFc-NC-DM1 (MORADEC LLC.), which was formed by conjugating DM1 to mouse IgG via a non-cleaving linker called SMCC, and analyzed. Near-infrared light was irradiated, and the analysis was performed by mass spectrometry under the same conditions as above (Figure 8).

[0068] Near-infrared light 16 J / cm 2 In αMFc-NC-DM1-IR700 irradiated with light (Figure 8(D)), a peak was observed at a different retention time compared to unirradiated αMFc-NC-DM1-IR700 (Figure 8(C)). Furthermore, the retention time of 13.68 minutes observed for the peak after light irradiation of αMFc-NC-DM1-IR700 was almost identical to the retention time of 13.58 minutes observed for the peak after light irradiation of T-DM1-IR700 (Figure 8(B)), suggesting that S-Me-DM1 was released in both cases. Figure 8(A) shows the blank.

[0069] 3. Examination using cell models Since light irradiation releases the drug from the antibody, it is assumed that the drug's effects can extend to target cells even if the antibody does not bind to those cells, provided the target cells are nearby. Therefore, we conducted an analysis using a model system simulating cancer.

[0070] As a heterogeneous cell model system, a system was created in which HER2-positive and HER2-negative cells were mixed, and the effects of target accumulation complexes were analyzed using T-DM1-IR700. The analysis was performed using HER2-positive cells 3T3 / HER2, which were mouse fibroblasts into which the HER2 gene was introduced, and MDA-MB-468 luc, which were HER2-negative human breast cancer cells into which luciferase was introduced.

[0071] First, we analyzed whether T-DM1-IR700 specifically binds to HER2-positive cells. 3T3 / HER2 cells (Figure 9 left) or MDA-MB-468 luc cells (Figure 9 right) were pre-blocked with 10 μg / ml T-DM1-IR700 or 100 μg / ml T-DM1, then 10 μg / ml T-DM1-IR700 was added, and analysis was performed by flow cytometry. As a result, specific binding of T-DM1-IR700 was observed in 3T3 / HER2 cells, while in MDA-MB-468 luc cells, no difference was observed between cells blocked with T-DM1 and cells incubated with T-DM1-IR700 alone, confirming that T-DM1-IR700 did not bind.

[0072] Next, we decided to create a cell model and investigate the effects of the target accumulation complex (Figure 10). Antigen-positive cells (HER2-positive cells in this case) and antigen-negative cells (HER2-negative cells) were placed in 12-well plates, 5 × 10⁶ each. 4 Mix the cells in a well and seed. After 24 hours, replace the medium with one containing T-DM1-IR700 or Tra-IR700 (trastuzumab-IR700), and incubate for 6 hours. Wash the cells with PBS, then light them using a 690 nm LED at 4 J / cm². 2 The samples were irradiated with near-infrared light, and luciferase activity was measured after 4 days of incubation.

[0073] 3T3 / HER2 cells and MDA-MB-468 luc cells were mixed and seeded as described above, and treated with T-DM1-IR700 at 1 μg / ml, 5 μg / ml, and 10 μg / ml, and with Tra-IR700 at 10 μg / ml (Figure 11). In T-DM1-IR700-treated cells, a decrease in luciferase activity was observed in near-infrared light-irradiated cells, even when treated at 1 μg / ml. In contrast, no decrease in luciferase activity was observed in Tra-IR700-treated cells. As shown in Figure 9, MDA-MB-468 luc cells into which luciferase has been introduced are HER2-negative, and T-DM1-IR700 does not bind to them. Therefore, the suppression of luciferase activity is considered to be the effect of DM1 on cells that do not express HER2. In Tra-IR700-treated cells, luciferase activity was increased in near-infrared light-irradiated cells compared to unirradiated cells. This indicates that while HER2-positive cells die, HER2-negative cells to which luciferase has been introduced remain unaffected.

[0074] MDA-MB-468 luc cells were seeded alone, treated with T-DM1-IR700, and irradiated with near-infrared light to confirm the effect of the target-accumulating complex (Figure 12). HER2-negative MDA-MB-468 luc cells were seeded alone, and after 24 hours, the medium was changed to one supplemented with T-DM1-IR700 or PBS as a control. After 6 hours, irradiation was performed using an LED with an emission wavelength of 690 nm at 4 J / cm². 2 Near-infrared light was irradiated onto the cells. After light irradiation, the cells were cultured for 4 days, and the luciferase activity of each group was analyzed (Figure 12).

[0075] In MDA-MB-468 luc cells, which are HER2-negative cells, luciferase activity remained unchanged and no cell death occurred, regardless of whether T-DM1-IR700 was administered or whether near-infrared light irradiation was performed. These results indicate that the presence of HER2-positive cells causes T-DM1-IR700 to bind to HER2-positive cells, and near-infrared light irradiation leads to the release of DM1, which then has an effect on HER2-negative cells.

[0076] We investigated whether similar phenomena occur not only in 3T3 / HER2 cells, which are cells into which the HER2 gene has been introduced, but also in cancer cell lines established from human tumors, using human HER2-positive cells (Figure 13). Using the human lung cancer cell line H2170 or the human breast cancer cell line SK-BR-3, we created a heterogeneous in vitro model system by co-culturing with MDA-MB-468 luc cells in the same manner as above, and analyzed the effects of T-DM1-IR700.

[0077] In all cell types, when T-DM1-IR700 was applied and irradiated with near-infrared light, a decrease in luciferase activity was observed, and HER2-negative MDA-MB-468 luc cells were found to be dead. This indicates that the effect of T-DM1-IR700 extends to cells to which the antibody portion, trastuzumab, does not bind. Comparing these results with the results of mass spectrometry, it can be concluded that DM1 is released from the antibody upon near-infrared light irradiation and exerts an effect on cells to which the antibody does not bind. These results indicate that the drug affects not only the cells to which the targeted accumulation complex directly binds, but also cells in the vicinity. These results clearly demonstrate that treatment with targeted accumulation complexes is a different treatment method from conventional ADCs and NIR-PIT.

[0078] In this study, the effect was investigated by attaching IR700 to T-DM1 (Kadcyla), which has DM1 bound to an antibody. However, it is obvious that IR700 can be attached to existing ADCs, such as Mylotarg (trademark, generic name: gemtuzumab ozogamicin) and Adcetris (trademark, generic name: pretuximab vedotin), which are already approved. Furthermore, IR700 may also be attached to ADCs developed in the future.

[0079] 4. Investigation using in vivo model systems While we have demonstrated the in vitro effects of target-accumulating complexes, we investigated whether they can be used in actual treatment using an in vivo model system. 3T3 / HER2 cells (5×10)6 (1 x 10) and MDA-MB-468 luc cells (1 x 10) 7 The cells were mixed with 150 μl of PBS and subcutaneously transplanted into both dorsal and rump sides of 8-10 week old nude mice (BALB / cSlc-nu / nu). Three days after tumor cell transplantation (one day before near-infrared light irradiation (Day-1)), T-DM1-IR700 was administered, and four days after tumor cell transplantation, near-infrared light irradiation was performed. The day irradiation began was designated as Day 0. T-DM1-IR700 was administered by tail injection at a dose of 3.6 μg per g of mouse body weight. Since this is a treatment using near-infrared light with an antibody drug, treatment using a target-specific complex is also called NIR-PIT. NIR-PIT was administered at 15 J / cm² on Day 0. 2 , 30 J / cm on Day 1 2 This was done by irradiating only the right side with a laser.

[0080] Therapeutic efficacy was assessed by measuring the estimated tumor volume and the luciferase activity of the tumor. The estimated tumor volume was calculated by measuring the longest and shortest diameters of the tumor (longest diameter × shortest diameter). 2 The calculation was performed using the formula (×1 / 2). Luciferase activity was measured using the IVIS® imaging system after intraperitoneal administration of D-luciferin (7.5 mg / ml, 200 μl). The unit of emission measured was radiance, and the analysis was performed using Living Image Software® (Figure 14).

[0081] As is evident from the images of mice in which luciferase activity was visualized, a clear decrease in luciferase activity was observed on the side irradiated with near-infrared light. As shown in the graph below, quantitative measurements also showed that from 1 day after near-infrared light irradiation (Day 1), both tumor volume and luciferase activity decreased on the side irradiated with near-infrared light (indicated as PIT in the figure) compared to the side that was not irradiated (indicated as ivonly in the figure), and a significant difference was observed 3 days after irradiation. The effect of the target-accumulating complex was also demonstrated in the in vivo model system.

[0082] The results above demonstrate that by using a targeted accumulation complex, which is a drug conjugate like an ADC further modified with IR700, it is possible to deliver drugs to disease sites via near-infrared light irradiation. The fact that dexamethasone and emtansine, compounds with different structures, were released from their carriers by near-infrared light while maintaining their efficacy indicates that other compounds can also be released by light irradiation and act locally. This makes it possible to attach drugs not only to substances with specific binding properties such as antibodies and aptamers, but also to substances with high blood stability that accumulate at disease sites, allowing for the delivery and release of drugs at high concentrations at the target site, thus making it applicable to a variety of diseases. In addition to antibodies, as with conventional ADCs, drugs can be attached to cytokines and albumin, and the drugs can be released and act at the disease site by light, making it possible to target diseases that were previously not suitable for DDS. The photopharmaceutical delivery technology using targeted, integrated complexes to which drugs and IR700 are bound to a specific binding substance is a completely different technology from conventional near-infrared photoimmunotherapy (NIR-PIT) and ADCs in that it also affects cells other than the target cells. [Industrial applicability]

[0083] The inventors created a targeted accumulation complex by conjugating an antibody with a drug and a photosensitive substance, and analyzed its effects and mechanism of action. As a result, they discovered that the drug's effects extend not only to the cells to which the antibody is bound, but also to surrounding cells that do not express the antigen, thus completing the present invention. Conventional NIR-PIT and ADC treatments are effective only on the cells to which the antibody is bound, but the targeted accumulation complex has the characteristic of also affecting cells that do not express the antigen.

[0084] Conventional antibody drugs are ineffective unless the target molecule is expressed on the cell surface, and many cases have been difficult to treat. However, targeted accumulation conjugates affect not only cells expressing the target molecule but also surrounding cells, so they can be expected to have a high therapeutic effect even in cases where conventional antibody drugs were ineffective. Furthermore, targeted accumulation conjugates can be produced not only by specific binding substances such as antibodies, but also by conjugating drugs and photosensitive substances to substances that accumulate at the disease site or substances with high blood stability, so they can be applied to a wider range of diseases compared to conventional ADCs.

Claims

1. In your career, Phthalocyanine dyes, which are near-infrared light-sensitive substances, Targeted accumulation complexes for inflammation, infection, collagen diseases, and organ-specific diseases, having a drug-linked structure.

2. The aforementioned carrier, The target-accumulation complex according to claim 1, characterized in that it is a binding molecule that exhibits binding affinity to a target molecule, or a molecule that accumulates at a disease site.

3. The aforementioned organ-specific disease is The target accumulation complex according to claim 1, which is any of the following: heart disease, kidney disease, liver disease, lung disease, thyroid disease, digestive disease, or neuromuscular disease.

4. The aforementioned target molecule The target-accumulating complex according to claim 2, characterized in that it is a molecule that is expressed on the cell surface in connection with disease, or a molecule that is expressed in an organ-specific manner.

5. The aforementioned binding molecule The target-accumulated complex according to claim 2 or 4, characterized in that it is an antibody, an antigen-binding antibody fragment, or an aptamer.

6. The aforementioned drug, A target-accumulating complex according to any one of claims 1 to 5, wherein the complex is at least one of an anti-inflammatory agent, an antiviral agent, or an antibacterial agent.

7. The aforementioned drugs are bound via a linker, The target-accumulation complex according to any one of claims 1 to 6, characterized in that the linker is a cleavage-type or non-cleavage-type linker.

8. The target-accumulating complex according to claim 1, characterized in that the phthalocyanine dye is IR700.

9. A pharmaceutical composition for inflammation, infection, or collagen disease, comprising a target-accumulation complex according to any one of claims 1 to 8 as an active ingredient.

10. A technology for delivering drugs (excluding delivery within the human body), A target-accumulation complex having a structure in which a phthalocyanine dye, a near-infrared light-sensitive substance, and the drug are linked to a pre-administered carrier. Photopharmaceutical delivery technology that releases drugs from their binding molecules by irradiating them with near-infrared light.

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