Compound containing complex of cell membrane-penetrating polycationic isopeptide and antibody as active ingredient and pharmaceutical composition containing same

Abstract

PROBLEM TO BE SOLVED: The purpose of the present invention is to provide a compound capable of safely and efficiently introducing into cells an antibody drug capable of inhibiting proliferation of cancer cells. SOLUTION: According to the present invention, the proliferation of cancer cells is suppressed by introducing an antibody that binds specifically to IL-6 or a receptor thereof into cells using the PIECE method and specifically suppressing the autocrine intracellular IL-6 signaling pathway. This configuration makes it possible to target a pathogenic signaling pathway localized in a cell, and to provide a novel and practical technique for intracellular delivery of antibody drugs and cancer treatment methods that overcomes the application limits of conventional antibody drugs.

Description

Compound containing a complex of cell membrane-permeable polycation isopeptide and antibody as an active ingredient, and pharmaceutical composition containing the same

[0001] The present invention relates to a technology for intracellular delivery of antibodies, and in particular to a compound having as an active ingredient a complex of a cell membrane-permeable polycationic isopeptide (PIECE) and an antibody, and a pharmaceutical composition containing the same.

[0002] It is known that abnormal activation or inhibition of intracellular and extracellular protein-protein interactions, i.e., disruption of signal transduction, is the trigger for various diseases including cancer. These interactions are fundamental to life activities and are precisely regulated, so disruption of this balance directly leads to the onset of various diseases.

[0003] Antibody drugs have been researched and developed for many years as a means of inhibiting such abnormal protein-protein interactions. Antibodies can specifically recognize the three-dimensional structure of target proteins and bind strongly to them, making it possible to inhibit protein-protein interactions with high selectivity. Due to these properties, antibodies have already been put into practical use as therapeutic drugs for many diseases, and have been established as a treatment modality with relatively few side effects.

[0004] However, because antibodies are macromolecules with a molecular weight of approximately 150,000, they cannot pass through cell membranes on their own, and so their targets are currently limited to molecules present outside the cell or on the cell surface. On the other hand, many disease-causing molecules are present inside the cell, and if they could be directly targeted, it is expected that the range of application of antibody drugs will be greatly expanded and their therapeutic effects will be dramatically improved.

[0005] In response to this situation, various methods for introducing antibodies into cells have been investigated in recent years. Representative examples include a technique for encapsulating antibodies in nanoparticles made of lipids or polymers, and a technique for introducing antibodies by binding them to cell membrane-permeable peptides (CPPs). Furthermore, there are known examples where excellent intracellular delivery has been achieved by supporting CPPs on the surface of nanoparticles. Among these, positively charged polycationic CPPs are widely used as a means for introducing macromolecules such as antibodies into cells.

[0006] However, conventional intracellular delivery techniques using polycationic CPPs have several technical limitations. While CPP uptake is primarily via endocytosis, many of the delivered antibodies remain in endosomes and may be degraded before reaching the cytoplasm. For this reason, attempts have been made to design and synthesize CPPs with improved endosomal escape properties and enhanced stability against degradative enzymes. However, these often require complex organic synthesis, and the increased production costs pose a barrier to practical application. Furthermore, cytotoxicity of polycationic CPPs themselves has been reported, necessitating careful consideration of safety.

[0007] Against this background, there is a need to develop a new intracellular delivery technology for antibody drugs that combines efficient cytoplasmic delivery, safety, and ease of production.

[0008] WO2005 / 032593 A1 Patent No. 7123414

[0009] Niamsuphap, S. et al. Targeting the undruggable: emerging technologies in antibody delivery against intracellular targets. Expert Opin Drug Deliv 17, 1189-1211 (2020).Goswami, R., Jeon, T., Nagaraj, H., Zhai, S. & Rotello, V.M. Accessing Intracellular Targets through Nanocarrier-Mediated Cytosolic Protein Delivery. Trends Pharmacol. Sci. 41, 743-754 (2020).Guidotti, G., Brambilla, L. & Rossi, D. Cell-penetrating peptides: from basic research to clinics. Trends Pharmacol. Sci. 38, 406-424 (2017).Akishiba, M. et al. Cytosolic antibody delivery by lipid-sensitive endosomolytic peptide. Nat. Chem. 9, 751-761 (2017).Szabo, R. et al. Cellular Uptake Mechanism of Cationic Branched Polypeptides with Poly[l-Lys] Backbone. ACS Comb Sci 19, 246-254 (2017).Dougherty, P.G., Sahni, A. & Pei, D. Understanding cell penetration of cyclic peptides. Chem. Rev. 119, 10241-10287 (2019).Park, S.E., Sajid, M.I., Parang, K. & Tiwari,R.K. Cyclic cell-penetrating peptides as efficient intracellular drug delivery tools. Mol. Pharm. 16, 3727-3743 (2019).Sakamoto, K. et al. Optimizing Charge Switching in Membrane Lytic Peptides for Endosomal Release of Biomacromolecules. Angew. Chem. Int. Ed. Engl. (2020).Arafiles, J.V.V. & Futaki, S. Chemical passports to cross biological borders. Nat. Chem. 13, 517-519 (2021).Iwata, T. et al. Liquid Droplet Formation and Facile Cytosolic Translocation of IgG in the Presence of Attenuated Cationic Amphiphilic Lytic Peptides. Angew. Chem. Int. Ed. Engl. 60, 19804-19812 (2021).Mandal, S., Mann, G., Satish, G. & Brik, A. Enhanced Live-Cell Delivery of Synthetic Proteins Assisted by Cell-Penetrating Peptides Fused to DABCYL. Angew. Chem. Int. Ed. Engl. 60, 7333-7343 (2021).Schneider, A.F.L., Kithil, M., Cardoso, M.C., Lehmann, M. & Hackenberger, C.P.R. Cellular uptake of large biomolecules enabled by cell-surface-reactive cell-penetrating peptide additives. Nat. Chem. 13,530-539 (2021).Tietz, O., Cortezon-Tamarit, F., Chalk, R., Able, S. & Vallis, K.A. Tricyclic cell-penetrating peptides for efficient delivery of functional antibodies into cancer cells. Nat. Chem. 14, 284-293 (2022).Sun, Y. et al. Phase-separating peptides for direct cytosolic delivery and redox-activated release of macromolecular therapeutics. Nat. Chem. 14, 274-283 (2022).Takeuchi, Y. et al. First direct evidence for direct cell-membrane penetrations of polycationic homopoly(amino acid)s produced by bacteria. Commun. Biol. 5, 1132 (2022).Kaneda, K. et al. Cell-penetrating activity of a short-chain epsilon-poly-l-alpha-lysine. J. Biosci. Bioeng. 138, 249-253 (2024).Taniguchi, K. et al. A gp130-Src-YAP module links inflammation to epithelial regeneration. Nature 519, 57-62 (2015).Taniguchi, K. et al. YAP-IL-6ST autoregulatory loop activated on APC loss controls colonic tumorigenesis. Proc. Natl. Acad. Sci. U. S. A. 114, 1643-1648 (2017).Grivennikov, S.I., Greten, F.R. & Karin,M. Immunity, inflammation, and cancer. Cell 140, 883-99 (2010). Taniguchi, K. & Karin, M. NF-kappaB, inflammation, immunity and cancer: coming of age. Nat. Rev. Immunol. 18, 309-324 (2018). ,

[0010] As mentioned above, antibody drugs are useful in that they can inhibit protein-protein interactions with high specificity. However, they cannot penetrate cell membranes and therefore cannot target disease-related factors present within cells, limiting their applicability to diseases such as cancer.

[0011] Furthermore, conventional antibody delivery technologies using cell-penetrating peptides (CPPs) have not yet resolved the problems of increased production costs due to complex organic synthesis processes and cytotoxicity, and significant barriers remain to their clinical application.

[0012] Therefore, an object of the present invention is to provide a compound that safely and efficiently introduces an antibody drug capable of suppressing the proliferation of cancer cells into cells.

[0013] To achieve the above-mentioned object, the present inventors focused on the fact that ε-poly-L-α-lysine (ε-PαL), a microbial-derived polycation isopeptide, is known to improve the biological membrane permeability of antibodies, and conceived the idea that intracellular delivery of antibodies, which has been difficult to achieve until now, could be achieved by applying a polycation modification method (PIECE method) using this microbial-derived cell membrane-permeable polycation isopeptide (PIECE) to antibody pharmaceuticals.

[0014] Meanwhile, the present inventors have discovered that in cancer cells, the inflammatory cytokine IL-6 interacts with receptors in intracellular organelles to activate signals without being secreted extracellularly, resulting in "autocrine intracellular IL-6 signaling." This novel intracellular signaling pathway is difficult to inhibit with neutralizing antibodies that target the extracellular domain, and no effective therapeutic method has yet been established.

[0015] Therefore, the inventors hypothesized that autocrine intracellular IL-6 signaling could be suppressed by introducing an antibody that specifically binds to IL-6 or its receptor into cells using the aforementioned PIECE method, and conducted extensive experiments based on this hypothesis. As a result, they succeeded in producing an antibody preparation in which ε-PαL was chemically conjugated with a monoclonal antibody (mAb) that specifically binds to IL-6 or its receptor (hereinafter referred to as an ε-PαL-mAb conjugate), thereby completing the present invention. Furthermore, to verify the versatility of the polycation modification technology using the PIECE method, they similarly modified mAbs against TNFα and IL-33, and obtained various antibody conjugate preparations.

[0016] The obtained antibody conjugate was confirmed to be capable of being taken up into cancer cells via the endocytic pathway, escaping from the endosome, and diffusing and localizing in the cytoplasm or nucleus. In particular, the conjugate of IL-6-related mAb with ε-PαL specifically inhibited the autocrine IL-6 signaling pathway, which is activated intracellularly without extracellular secretion, thereby significantly suppressing phosphorylation of the downstream transcription factor STAT3. It was suggested that blocking such intramolecular signaling inhibits cancer cell proliferation or induces cell death.

[0017] That is, according to the present invention, the following inventions are provided.

[0018] (1) General formula (1) (wherein n is an integer of 3 to 100) or a salt thereof.

[0019] (2) The linker is represented by the following formula (2): (wherein m is an integer of 1 to 30; X represents an oxygen atom or an imino group; Y represents an alkylene group having 1 to 6 carbon atoms which may be substituted with a halogen atom, a hydroxyl group, an amino group, a mercapto group, a carboxyl group or a sulfo group; and Z includes a group generated by a click reaction from a click functional group which is an azide group, an alkenyl group, a nitrile group, a thiol group, a maleimide group, an epoxide group, an aziridine group or a thiirane group), or a salt thereof, according to (1) above.

[0020] [Rule 26 Supplement 04.11.2025] (3) The above formula (1) is expressed as the following formula (3): The compound or salt thereof according to (1) above,

[0021] (4) The compound or salt thereof according to any one of (1) to (3) above, wherein ε-poly-L-α-lysine (ε-PαL) is a cell membrane-permeable polycation isopeptide (PIECE) derived from a microorganism.

[0022] (5) The compound or salt thereof according to any one of (1) to (4) above, wherein the antibody is an antibody against an inflammation-associated protein.

[0023] (6) The compound or salt thereof according to any one of (1) to (5) above, wherein the antibody is an antibody against IL-6 or its receptor.

[0024] (7) The compound or salt thereof according to any one of (1) to (6) above, wherein the antibody is tocilizumab, siltuximab, or sarilumab.

[0025] (8) An agent for suppressing phosphorylation of STAT3, comprising the compound according to (6) or (7) above or a salt thereof as an active ingredient.

[0026] (9) The compound or salt thereof according to (1) above, wherein the antibody is an antibody against TNFα.

[0027] (10) The compound or salt thereof according to (9) above, wherein the antibody is infliximab.

[0028] (11) The compound or salt thereof according to (1) above, wherein the antibody is an antibody against IL-33.

[0029] (12) The compound or salt thereof according to (11) above, wherein the antibody is etokimab.

[0030] (13) A pharmaceutical composition containing the compound or salt thereof according to any one of (1) to (12) above as an active ingredient.

[0031] (14) The pharmaceutical composition according to (13) above, wherein the pharmaceutical composition is an anticancer agent.

[0032] According to the present invention, by introducing an antibody that specifically binds to IL-6 or its receptor into cells using the aforementioned PIECE method, it is possible to suppress the autocrine intracellular IL-6 signaling pathway and simultaneously inhibit the proliferation of cancer cells. This configuration makes it possible to provide a novel and practical intracellular delivery technology for antibody drugs and a cancer treatment method that targets a pathogenic signaling pathway localized within cells and overcomes the application limitations of antibody drugs to date.

[0033] The compound can be prepared by the method described in Japanese Patent No. 7,123,414 filed by the present inventor, the entire disclosure of which is incorporated herein by reference. Features of the present invention other than those described above will be made clear in the description of the embodiments of the present invention below.

[0034] Figures 1a-c show the structures of ε-PαL, ε-PαL-PEG-azide, and DBCO-PEG-NHS, and Figure 1d shows the structure of an ε-PαL-antibody conjugate prepared using these conjugates. Figure 2a shows the effects of IL-6 knockdown (left) and IL-6 neutralizing antibody (right) on cell proliferation. Figure 2b shows a schematic diagram of conventional autocrine signaling (left) and autocrine intracellular IL-6 signaling (right). Figures 3a and 3b show the results of SDS-PAGE analysis of ε-PαL modified with TCZ, SLX, and cIgG under non-reducing and reducing conditions, respectively. Figures 3c and 3d show the results of similar analysis of modified with IFX and ETK under non-reducing and reducing conditions. Figure 4a shows the cell morphology of K562 cells without the addition of antibody, and Figure 4b shows the cell morphology after the addition of various antibodies (TCZ, SLX, cIgG, IFX, ETK) either untreated or modified with ε-PαL. Figures 5a-e show the results of cell viability (WST assay) assessed after the addition of various antibodies (TCZ, SLX, cIgG, IFX, ETK) either untreated or modified with ε-PαL. Figure 6a shows the cell morphology of HeLa cells without the addition of antibody, and Figure 6b shows the cell morphology after the addition of various antibodies (TCZ, SLX, cIgG, IFX, ETK) either untreated or modified with ε-PαL. Figures 7a-e show the results of cell viability (WST assay) assessed after addition of various antibodies (TCZ, SLX, cIgG, IFX, and ETK) untreated or modified with ε-PαL to HeLa cells. Figure 8 shows the results of analyzing apoptosis and cell death in K562 cells, with Figure 8a showing the control and Figures 8b-d showing the results after addition of various antibodies (TCZ, SLX, and cIgG) untreated or modified with ε-PαL. Figure 9 shows the results of Western blot analysis of the phosphorylation state of STAT3 after addition of various antibodies (TCZ, SLX, and cIgG) untreated or modified with ε-PαL to K562 cells.

[0035] Hereinafter, an embodiment of the present invention will be described with reference to the drawings.

[0036] (Background to the Invention) As described above, intracellular and extracellular signal transduction is mediated by protein interactions and plays an important role as the basis of life phenomena. Signal transduction is regulated by increases or decreases in protein-protein interactions, and abnormalities in signal transduction can lead to the onset of disease.

[0037] Generally, protein-protein interactions occur over a wide and shallow surface. This makes it difficult for small molecules with a molecular weight of 1,000 or less to inhibit such interactions. On the other hand, large-molecular-weight antibodies (approximately 150,000) recognize and specifically bind to the three-dimensional structure of target proteins, making them extremely effective as inhibitors of protein-protein interactions. For this reason, antibody drugs, which use antibodies as direct therapeutic agents for diseases, are attracting attention as a modality with few side effects.

[0038] However, because antibodies are macromolecules that cannot penetrate the cell membrane of animal cells, their targets are limited to molecules outside the cell or on the cell surface, limiting their effectiveness as therapeutic agents. On the other hand, since many target molecules that cause diseases are also present inside cells, the usefulness of antibody drugs could be dramatically improved if a practical delivery technology for delivering antibodies into cells could be developed.

[0039] Therefore, currently, many attempts have been made to deliver proteins and antibodies into cells by encapsulating them in nanoparticles made of lipids or polymers, or by crosslinking (conjugating) them with cell membrane-penetrating peptides (CPPs) (Non-Patent Documents 1 and 2). Furthermore, there have been examples of successful intracellular delivery achieved by supporting CPPs on the surface of nanoparticles (Patent Document 1). While amphiphilic CPPs have also been reported (Non-Patent Document 3), polycationic CPPs are often used as tools for delivering biopolymers such as proteins into animal cells, and generally, the higher the polycationicity, the higher the cell membrane permeability (Non-Patent Documents 3 to 14).

[0040] The substances transported into cells by CPPs are called cargos, and the pathways by which polycationic CPP-cargo conjugates (CPP / cargo) are transported into cells are broadly divided into two: an energy-independent pathway via direct cell membrane permeation and an energy-dependent pathway via endocytosis / macropinocytosis (Non-Patent Document 3). In both pathways, binding of CPP-cargo to anionic components such as heparan sulfate proteoglycans on the cell membrane is important for its uptake into cells.

[0041] CPP-cargo taken up into cells via endocytosis is trapped in endosomal granules and ultimately degraded in lysosomes. Therefore, to exert its physiological function within the cell, the cargo must escape from the endosome. On the other hand, CPP-cargo taken up by direct permeation through the cell membrane is expected to rapidly exert its function after being taken up into the cell. In general, low molecular weight compounds conjugated to conventional cationic CPPs are taken up into cells via both pathways, but conjugates with biopolymers such as proteins and antibodies are taken up only via the endocytic pathway.

[0042] Currently, the rational design and synthesis of CPPs that can effectively escape from endosomes and are resistant to peptidolytic enzymes have attracted attention (Non-Patent Documents 12-14). However, the high synthesis costs associated with the complex organic synthesis make the practical use of CPPs difficult. Furthermore, the toxicity of polycationic CPPs to animal cells is an important issue that needs to be resolved (Non-Patent Document 7).

[0043] Under these circumstances, a "polycation modification method (PIECE method) using microbially derived cell membrane-permeable polycation isopeptides (PIECE)" has been reported (Patent Document 2, Non-Patent Document 15). One PIECE, ε-poly-L-α-lysine (ε-PαL) (Fig. 1a), is a polymer in which the amino acid L-lysine is linearly linked via isopeptide bonds, and is a natural amino acid polymer produced by actinomycetes. Each monomer unit contains one amino group (-NH ２ ), and under physiological conditions (around pH 7),３ ＋ ) and exhibits a positively charged cationic property. Therefore, the longer the polymer chain length, the more positive charges there are, and the molecule as a whole exhibits polycationic properties. ε-PαL exhibits cell membrane permeability similar to conventional polycationic CPPs (Patent Document 2, Non-Patent Document 15), and the longer the polymer chain length, the better the cell membrane permeability. In fact, the cell membrane permeability exhibited by ε-PαL with 25-35 residues (n=25-35) is superior to that of ε-PαL with 5-14 residues (n=5-14) (Non-Patent Document 16).

[0044] Therefore, longer ε-PαLs are expected to exhibit even better cell membrane permeability due to their improved polycationic nature. However, ε-PαLs with 36 or more residues (n>36) have not yet been found in nature, and no chemically synthesized versions have been reported. ε-PαL not only exhibits excellent cell membrane permeability but also is resistant to peptidases due to its isopeptide structure. Interestingly, ε-PαL is non-toxic to animal cells, making it a novel cell membrane-permeable peptide that simultaneously overcomes the various weaknesses of conventional CPPs. Furthermore, the ε-PαL derivative, ε-PαL-PEG-azide (Figure 1b), can be directly conjugated with biopolymers via click chemistry. Therefore, by introducing DBCO groups onto the surface of a biopolymer such as an antibody using dibenzocyclooctyne-PEG-N-hydroxysuccinimide ester (DBCO-PEG-NHS ester) (Fig. 1c), it is possible to easily prepare an antibody modified with a polycation (ε-PαL) (ε-PαL-antibody conjugate) (Fig. 1d).

[0045] The ε-PαL-antibody conjugate is taken up into animal cells by endocytosis, but escapes from the endosome and diffusely localizes in the cytoplasm and nucleus (Non-Patent Document 15). In other words, this is an innovative intracellular antibody delivery technology that enables the antibody to fully exert its function after delivery into animal cells.

[0046] Therefore, the inventors created a conjugate of ε-PαL with a monoclonal antibody (mAb) that specifically binds to an inflammation-related protein (ε-PαL-mAb conjugate), and conceived a new technology in which the ε-PαL-mAb conjugate is delivered into cancer cells to suppress cell proliferation, thereby completing the present invention.

[0047] Inflammation is an important biological defense mechanism that utilizes the immune system to respond to pathogens such as bacteria, viruses, and fungi, foreign substances, harmful stimuli, and trauma. It is a beneficial response that eliminates pathogens and foreign substances and repairs and regenerates tissues. Inflammation can be divided into two types: acute inflammation, which lasts for days to weeks, and chronic inflammation, which lasts for months to years. The roles of these two types are significantly different. Generally, acute inflammation is "good inflammation," which eliminates pathogens and foreign substances and induces tissue repair and regeneration. In contrast, chronic inflammation is "bad inflammation," which is involved in the onset and progression of almost all diseases, including cancer, neurodegenerative diseases, and diabetes. Acute inflammation induced by tissue injury promotes tissue repair and regeneration, but the signaling pathways important for tissue repair and regeneration remain unknown. Furthermore, it has been shown that the inflammation-induced inflammatory cytokine IL-6 activates a new signaling pathway, the Src family kinase (SFK)-YAP pathway, in addition to the previously known JAK-STAT3 pathway, promoting intestinal regeneration, and that the SFK-YAP pathway is a novel therapeutic target for colorectal cancer (Non-Patent Documents 17, 18). Acute inflammation has been shown to suppress cancer, and intravesical BCG (attenuated Mycobacterium bovis) therapy for bladder cancer is currently being used clinically. Meanwhile, chronic inflammation contributes to the development and progression of many types of cancer, and the mechanisms by which inflammation promotes cancer have been gradually revealed in recent years (Non-Patent Document 19). It has been reported that during inflammation, inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor (TNF) are produced in large quantities by immune cells. IL-6 family cytokines have a dual nature: they promote tissue repair and regeneration, while at the same time they are the most important players in the promotion of cancer through inflammation (Non-patent Document 20).

[0048] In 2011, Dr. Weinberg, who discovered the oncogene RAS and the tumor suppressor gene RB, added inflammation to the hallmarks of cancer (Hallmarks of Cancer). However, as of 2024, cancer treatments targeting inflammation have not yet been clinically applied due to side effects such as immunosuppression and inhibition of tissue repair and regeneration. Historically, research on cancer and inflammation has focused on cases of obvious infection (e.g., Helicobacter pylori, hepatitis virus, human papillomavirus) and the associated inflammation. Inflammatory carcinogenesis, which involves inflammation due to such infections, is thought to account for approximately 20% of cancers, and these cancers can be prevented or detected early with vaccines. However, even in the remaining 80% of cancers where infection or inflammation is not clearly present, "tumor-initiating inflammation" is important, as it is induced by genetic mutations in cancer cells, which significantly alter the gene expression profile of the cancer cells themselves, leading to the expression of inflammatory cytokines and chemokines such as IL-6, which act in an autocrine or paracrine manner in the tumor microenvironment. In other words, inflammation is thought to play an important role in almost 100% of cancers, but as mentioned above, cancer treatments that target inflammation have not been clinically applied due to side effects such as immunosuppression and inhibition of tissue repair and regeneration. In fact, in vivo cancer treatments using neutralizing antibodies to IL-6 or IL-6 receptors have not shown efficacy in either human clinical trials or mouse models, and the reasons for this are not fully understood.

[0049] The present inventors have been conducting research into inflammation and cancer, focusing on IL-6, particularly autocrine IL-6 in cancer cells. During this research, we discovered that suppression of IL-6 or IL-6 receptor expression in cancer cells using siRNA / shRNA strongly inhibits cancer cell proliferation and induces cell death, whereas neutralizing antibodies to IL-6 or IL-6 receptor have no effect on cancer cell proliferation or cell death (Figure 2a). The mechanism underlying this finding is not that the autocrine IL-6 signaling pathway in cancer cells is previously thought to be a pathway in which IL-6 is first released outside the cell to stimulate the receptor, but rather that IL-6 can activate signals at the intracellular ER / Golgi and endosome levels without being secreted outside the cell (Figure 2b). We named this novel IL-6 signaling pathway "autocrine intracellular IL-6 signaling." We hypothesized that if this signaling pathway could be suppressed specifically in cancer cells, a new cancer therapy targeting IL-6 could be developed without causing immunosuppression. In other words, we anticipated that by introducing antibodies that specifically bind to IL-6 or the IL-6 receptor into cells using the PIECE method described above, we could suppress the autocrine intracellular IL-6 signaling pathway and inhibit cancer cell proliferation.

[0050] (Definitions) Unless otherwise specified, terms used in this specification have the meanings that are commonly understood in the art. In addition, the following definitions apply to the entire specification.

[0051] As used herein, the term "PIECE" refers to a microbially derived, cell membrane-permeable polycationic isopeptide entering cells. As used herein, PIECE is ε-poly-L-α-lysine (ε-PαL).

[0052] As used herein, the "PIECE method" refers to a technique for conjugating a microbially derived polycationic isopeptide with a target molecule and introducing the target molecule into a cell. Conjugation in the PIECE method can be preferably performed by a click reaction, which proceeds highly efficiently and selectively under physiological conditions. For example, by using ε-PαL-PEG-azide, a derivative of ε-PαL, as the polycationic isopeptide, and dibenzocyclooctyne-PEG-N-hydroxysuccinimide ester (DBCO-PEG-NHS ester), which is reactive with the target molecule, it is possible to specifically and efficiently conjugate the two via an azide-alkyne cycloaddition reaction.

[0053] In the present invention, the conjugate of ε-PαL and an antibody is represented by the following general formula (1):

[0054] In general formula (1), n ​​may be any number from 3 to 100, preferably from 5 to 90, 10 to 80, 15 to 70, 20 to 60, 20 to 50, or 20 to 40, and more preferably from about 25 to 35. Although n cannot be generalized because it differs depending on the type of bacteria to be cultured and the culture conditions of the bacteria (e.g., pH, temperature, time, etc.), usually, when n is 15 or less, the cell membrane permeability of the antibody into which ε-PαL has been introduced is improved and the antibody can have high chemical stability.

[0055] In the present invention, the term "linker" refers to a divalent linking group capable of binding to other groups at both ends, which is used to link an antibody to an ε-PαL moiety. Its length and flexibility can be adjusted as desired depending on the purpose. The type of linker is not particularly limited, and it may be composed of linking groups known in the art, such as a linear alkylene group, a polyethylene glycol (PEG) chain, an aromatic group, an amide bond, an ester bond, a urethane bond, or a (thio)ether bond, or a combination thereof. Furthermore, linkers do not simply mediate binding; they may also possess a variety of properties, such as those that impart hydrophilicity or hydrophobicity, or those that contain specific reactive functional groups (e.g., azide groups, alkyne groups, NHS ester groups, etc.) and can be easily introduced using known reactions such as click chemistry. A specific example of a linker included in the above general formula (1) is represented by the following formula (2):

[0056] In the formula, m may be any number from 1 to 30, but is preferably about 2 to 20. When m is 2 or greater, the cell membrane permeability of the antibody into which ε-PαL has been introduced is improved, and the antibody can have high chemical stability. X represents an oxygen atom or an imino group. The imino group represented by X is represented by -NR-, where R is H or an appropriate substituent, for example, a lower alkyl group having 1 to 6 carbon atoms such as a methyl group or an ethyl group, an aryl group such as a phenyl group, or a heterocyclic group containing 1 to 3 heteroatoms such as a furyl group, a pyridyl group, or a thienyl group. Of these, R is preferably a 5- or 6-membered heterocyclic group. Examples of alkylene groups having 1 to 6 carbon atoms represented by Y include a methylene group, an ethylene group, a propylene group, an isopropylene group, a butylene group, an isobutylene group, a pentene group, and a hexene group. Of these, Y is preferably an alkylene group having 2 to 6 carbon atoms. The alkylene group may be substituted with a conventional substituent, examples of which include halogen atoms (e.g., bromine, chlorine, and fluorine), hydroxyl, amino, mercapto, carboxyl, and sulfo groups. The alkylene group may also contain a linker such as -O-, -S-, -SS-, or a silyl group. Z includes groups generated by a click reaction from a click functional group, such as an azide group, alkenyl group, nitrile group, thiol group, maleimide group, epoxide group, aziridine group, or thiirane group. Specific examples of click functional groups used in the present invention and the groups generated thereby are shown below. That is, the reaction of an azide group with an alkyne group generates a 1,2,3-triazole group. The reaction of an alkenyl group with a thiol group generates a thioether group, and the reaction of a nitrile group with an azide group generates a tetrazole group. The reaction of a thiol group with an epoxide group generates a β-hydroxythioether group, and the reaction of a maleimide group with a thiol group generates a thioether group. Furthermore, the ring-opening reaction of the aziridine group generates a β-amino group-containing addition group, and the nucleophilic ring-opening reaction of the thiirane group generates a β-thio group-containing addition group.

[0057] Examples of pharmacologically acceptable salts of general formula (1) include salts with inorganic acids such as hydrochloric acid, sulfuric acid, and phosphoric acid; salts with organic acids such as oxalic acid, maleic acid, succinic acid, and acetic acid; salts with inorganic bases such as sodium and potassium; and salts with organic bases such as dimethylamine and triethylamine.

[0058] ε-poly-L-α-lysine (ε-PαL) may be obtained by any method, such as a chemical reaction, such as fermentation or chemical synthesis, or may be obtained by chemical synthesis from lysine. An example of such ε-PαL is that obtained by culturing Streptomyces albulus subsp. lysinopolymerus, as described in Japanese Patent No. 1245361, in a medium (e.g., 5 wt% glucose, 0.5 wt% yeast extract, 1 wt% ammonium sulfate, 0.08 wt% dipotassium hydrogen phosphate, 0.136 wt% potassium dihydrogen phosphate, 0.05 wt% magnesium sulfate heptahydrate, 0.004 wt% zinc sulfate heptahydrate, 0.03 wt% ferrous sulfate heptahydrate, adjusted to pH 6.8), and then isolating and collecting ε-polylysine from the resulting culture. Furthermore, ε-PαL may be obtained by any method, and can also be obtained by the method described in the following non-patent document. For example, γ-poly-L-diaminobutanoic acid, an ε-PαL analogue that is expected to have cell membrane permeability similar to ε-PαL, has been described in the paper (Takehara, M., Saimura, M., Inaba, H. & Hirohara, H. Poly(gamma-L-diaminobutanoic acid), a novel poly(amino acid), copreduced with poly(epsilon-L-lysine) by two strains of Streptomyces celluloflavus. FEMS Microbiol Lett 286, 110-7). (2008). γ-Poly-D-diaminobutanoic acid, an ε-PαL analog, can be produced by referring to the paper (Ohkuma, H., Tenmyo, O., Konishi, M., Oki, T. & Kawaguchi, H. BMY-28190, a novel antibiotic complex. J. Antibiot (Tokyo) 41, 849-54 (1988)).Furthermore, β-poly-L-diaminopropionic acid, an ε-PαL analog, was prepared as described in the paper (Xia, J., Xu, H., Feng, X., Xu, Z. & Chi, B. Poly(L-diaminopropionic acid), a novel non-proteinic amino acid oligomer co-produced with poly(epsilon-L-lysine) by Streptomyces albulus PD-1. Appl Microbiol Biotechnol 97, 7597-605 (2013)). or (Xu, Z. et al. Systematic unraveling of the biosynthesis of poly (L-diaminopropionic acid) in Streptomyces albulus PD-1. Sci Rep 5, 17400 (2015).

[0059] The compound represented by general formula (1) or a salt thereof can be conveniently produced using a linker conventionally used in the field of click chemistry. A linker is, in essence, a group that can be present to connect an antibody and ε-PαL, and any group may be used as long as it does not interfere with the objectives of the present invention. For example, a conjugate of ε-PαL and an antibody via a linker is represented by the following formula (3): ...(3)

[0060] Other examples of linkers include those represented by any of the following formulae (4a) to (4d), in which an amino group, a carboxyl group, or a hydroxyl group in a compound serving as a linker reacts with a carboxyl group, a hydroxyl group, or an amino group in an antibody to form a covalent bond, and the triple bond in the compound reacts with a click functional group in ε-PαL-PEG-azide, an ε-PαL derivative, to form a covalent bond. The types of such groups and methods for introducing them into antibodies (amidation reaction, esterification reaction, Fisgen reaction, etc.) have been well established in the field of click chemistry, and these methods may also be used in the present invention.

[0061]

[0062]

[0063]

[0064]

[0065] When introducing ε-PαL into an antibody, a click chemistry intermediary compound such as DBCO (dibenzocyclooctyne) may be used as an intermediary. That is, it is advantageous to introduce ε-PαL into a compound obtained by binding an antibody to a click chemistry intermediary compound such as DBCO.

[0066] As used herein, the term "antibody" is not particularly limited and includes any monoclonal antibody (mAb), and preferably refers to an antibody against an inflammation-related protein. More preferably, it is an antibody against IL-6 or its receptor, TNFα, or IL-33. Specific examples of antibodies include IL-6 receptor neutralizing antibodies (e.g., tocilizumab and sarilumab), IL-6 neutralizing antibodies (e.g., siltuximab), TNF-α neutralizing antibodies (e.g., infliximab, adalimumab, certolizumab, and golimumab), IL-1β neutralizing antibodies (e.g., canakinumab and anakinra), IL-17A neutralizing antibodies (e.g., secukinumab and ixekizumab), IL-33 neutralizing antibodies (e.g., ethoximab), IL-12 / IL-23 p40 neutralizing antibodies (e.g., ustekinumab), and IL-23 These include, but are not limited to, p19 neutralizing antibodies (e.g., guselkumab and risankizumab), IL-4Rα neutralizing antibodies (e.g., dupilumab), IFN-γ neutralizing antibodies (e.g., emapalumab), type I interferon receptor neutralizing antibodies (e.g., anifrolumab), VEGF neutralizing antibodies (e.g., bevacizumab), and EGF receptor neutralizing antibodies (e.g., cetuximab and panitumumab).

[0067] The compound or salt thereof of the present invention can be used for the treatment of animals or humans as a pharmaceutical composition containing the compound or salt thereof as an active ingredient. The pharmaceutical composition is preferably used as an anticancer agent and can target all types of cancer, including solid cancers and hematological tumors. In addition to its use as an anticancer agent, the pharmaceutical composition can also be used to treat autoimmune diseases (e.g., rheumatoid arthritis, systemic lupus erythematosus (SLE)), inflammatory diseases (e.g., ulcerative colitis, Crohn's disease), and allergic diseases.

[0068] (Reagents and Cell Lines Used) The various monoclonal antibodies (mAbs), reagents, cell lines and media used in the present invention are as follows.

[0069] Tocilizumab (TCZ), a mAb against IL-6 receptor, and siltuximab (SLX), a mAb against IL-6, were purchased from Selleck Biotech, Inc. (Kanagawa, Japan) or Bio X Cell, Inc. (New Hampshire, USA). RecombiMAb human IgG1 isotype control, anti-hen egg lysozyme (Bio X Cell) was used as a control antibody (control IgG: cIgG). Infliximab (IFX), a mAb against human TNFα, and ethoximab (ETK), a mAb against human IL-33, were also purchased from Bio X Cell.

[0070] DBCO-PEG4-NHS, used to introduce DBCO groups onto the antibody surface, was purchased from MedChemExpress (New Jersey, USA). A derivative (ε-PαL-PEG-azide), in which PEG and azide groups were added to ε-poly-L-α-lysine, a polycationic cell membrane-permeable peptide, was purchased from Micro-Bchem (Fukui, Japan).

[0071] Human cervical cancer cells (HeLa cells) and human chronic myeloid leukemia cells (K562 cells) used in the cell experiments were obtained from RIKEN BRC CELL BANK (Ibaraki Prefecture, Japan). Animal cells were cultured at 37°C and 5% CO ２HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). K562 cells were cultured in RPMI 1640 medium containing FBS. All media were supplemented with penicillin (100 U / ml) and streptomycin (100 μg / ml).

[0072] Example 1 Preparation of Various ε-PαL-mAb Conjugates In this example, various mAbs were modified with polycations by the PIECE method to prepare ε-PαL-mAb conjugates.

[0073] First, 10 mM NaH ２ P.O. ４ -Na ２ HPO ４ A reaction solution containing 1 M NaCl, 5% (w / v) glycerol, and each mAb (1 mg / mL, 6.7 μM) in a buffer solution (pH 7.4) was prepared, to which 33.5 μM DBCO-PEG4-NHS ester was added, followed by incubation at 4°C for 1 to 3 hours. Next, a 67 μM aqueous solution of ε-PαL-PEG-azide was added, followed by incubation at 4°C for 3 hours or longer. The resulting reaction solution was ultrafiltered (50 kDa NMWL) to remove unreacted ε-PαL-PEG-azide, and the buffer was then replaced with phosphate-buffered saline (PBS) using the same filter.

[0074] The modification states of the various ε-PαL-mAb conjugates obtained (ε-PαL-TCZ, ε-PαL-SLX, ε-PαL-cIgG, ε-PαL-IFX, and ε-PαL-ETK) were evaluated using polyacrylamide gel electrophoresis (SDS-PAGE).

[0075] First, analysis of ε-PαL-TCZ, ε-PαL-SLX, and ε-PαL-cIgG under non-reducing conditions revealed that the bands of each antibody shifted toward higher molecular weights compared to the unmodified antibody, confirming modification with ε-PαL (Fig. 3a). On the other hand, analysis under reducing conditions revealed unmodified bands in the heavy and light chains of each antibody, suggesting that ε-PαL was randomly modified to these chains (Fig. 3b). Similarly, analysis of ε-PαL-IFX and ε-PαL-ETK under non-reducing (Fig. 3c) and reducing (Fig. 3d) conditions revealed similar band shifts, confirming that each antibody was modified with ε-PαL.

[0076] Example 2 Cytotoxicity Test of Various ε-PαL-mAb Conjugates (Morphological Observation) In this example, various mAbs and their conjugates modified with ε-PαL were evaluated for cytotoxicity to K562 cells and HeLa cells by morphological observation.

[0077] In a 96-well plate, K562 cells (1 × 10 ４ cells / well) or HeLa cells (2.5 × 10 ３ Cells (cells / well) were seeded and cultured for 24 hours in a 37°C incubator. Subsequently, the following antibodies were added to each well at concentrations of 0, 2.5, 5, and 10 μM, respectively, and culture was continued for another 48 hours. The antibodies used were unmodified TCZ, SLX, cIgG, IFX, and ETK, as well as ε-PαL-modified antibodies: ε-PαL-TCZ, ε-PαL-SLX, ε-PαL-cIgG, ε-PαL-IFX, and ε-PαL-ETK. After culture, cells in each well were observed under a phase-contrast microscope (LASX, Leica Microsystems, Hesse, Germany). For HeLa cells, the medium was removed and the cells were observed under a phase-contrast microscope.

[0078] In K562 cells, the addition of unmodified TCZ, SLX, cIgG, IFX, or ETK at 10 μM did not significantly alter cell morphology. However, morphological abnormalities were observed in cells treated with ε-PαL-TCZ and ε-PαL-SLX at 5 μM or higher, with most cells exhibiting morphological abnormalities at 10 μM (Figures 4a and 3b). The control antibody, ε-PαL-cIgG, produced only a small number of abnormal cells, even at 10 μM, suggesting that intracellular delivery of TCZ and SLX exerts specific cytotoxic effects. Similarly, morphological abnormalities were observed in ε-PαL-IFX and ε-PαL-ETK at 5 μM or higher, with most cells exhibiting morphological abnormalities at 10 μM (Figures 4a and 3b).

[0079] In HeLa cells, no clear changes in cell morphology were observed when unmodified TCZ, SLX, cIgG, IFX, or ETK were added at 10 μM. In contrast, significant morphological abnormalities were observed when ε-PαL-TCZ was added at 10 μM (Fig. 6a, 5b). On the other hand, only a few cells showed abnormalities even when ε-PαL-SLX or the control antibody ε-PαL-cIgG were added at 10 μM, suggesting that intracellularly delivered TCZ also exhibits specific cytotoxicity in HeLa cells. Unlike K562 cells, ε-PαL-IFX and ε-PαL-ETK did not cause morphological abnormalities in HeLa cells, even when added at 10 μM (Fig. 6a, 5b).

[0080] Example 3 Cytotoxicity Test of Various ε-PαL-mAb Conjugates (WST Assay) In this example, the degree of cytotoxicity of various mAbs and their conjugates modified with ε-PαL against K562 cells and HeLa cells was quantitatively evaluated by WST assay.

[0081] K562 cells or HeLa cells (2 × 10 cells each) were cultured in a 384-well plate. ３ 100 cells / well) were seeded and incubated at 37°C in CO ２The cells were cultured in an incubator for 24 hours. After the culture, the following antibodies or antibody conjugates were added to each well at concentrations of 0 to 10 μM, and the culture was continued for another 48 hours. The antibodies used were unmodified TCZ, SLX, cIgG, IFX, and ETK, as well as ε-PαL-modified antibodies: ε-PαL-TCZ, ε-PαL-SLX, ε-PαL-cIgG, ε-PαL-IFX, and ε-PαL-ETK. Cell viability was measured using Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan), and the viability was calculated from the absorbance data obtained (n = 3). Graphs of cell viability and IC were generated using GraphPad Prism (ver. 8) (GraphPad Software, Inc., California, USA). ５０ The values ​​were calculated.

[0082] The results of the WST assay on K562 cells showed that the IC values ​​of ε-PαL-TCZ, ε-PαL-SLX, and ε-PαL-cIgG were ５０ The IC values ​​were 0.3 μM, 1.3 μM, and 5.0 μM, respectively (Fig. 5a-c). Concentration-dependent cytotoxicity was also observed for ε-PαL-cIgG, but the effect was slower than that of ε-PαL-TCZ and ε-PαL-SLX (Fig. 5c), suggesting that a different mechanism of action may be involved. Furthermore, the IC values ​​of ε-PαL-IFX and ε-PαL-ETK were ５０ The values ​​were 0.8 μM and 0.5 μM, respectively (FIGS. 5d and 5e), and both showed significant cytotoxicity against K562 cells.

[0083] In the WST assay in HeLa cells, the IC values ​​of ε-PαL-TCZ, ε-PαL-SLX, and ε-PαL-cIgG were ５０The IC values ​​were 5.9 μM, >10 μM, and 5.3 μM, respectively (Fig. 7a-c). ε-PαL-TCZ showed rapid cytotoxicity at concentrations of 2.5 μM or higher, and ε-PαL-SLX also showed rapid cytotoxicity at concentrations of 5 μM or higher (Fig. 7a, b). On the other hand, ε-PαL-cIgG showed a concentration-dependent, gradual toxicity (Fig. 7c), suggesting that its cytotoxicity may be expressed by a different mechanism from that of ε-PαL-TCZ and ε-PαL-SLX. Both ε-PαL-IFX and ε-PαL-ETK showed IC ５０ The values ​​exceeded 10 μM (FIGS. 7d ​​and 7e), and no clear cytotoxicity was observed against HeLa cells.

[0084] Example 4 Apoptosis-inducing activity of various ε-PαL-mAb conjugates In this example, the apoptosis-inducing activity of various mAbs and their conjugates modified with ε-PαL on K562 cells was quantitatively evaluated by flow cytometry.

[0085] K562 cells (5 × 10) were cultured in 8-well chamber slides. ４ 100 cells / well) were seeded and incubated at 37°C in CO ２ The cells were cultured in an incubator for 24 hours. After incubation, the cells were washed with medium, and the following antibodies or antibody conjugates were added to each well at a concentration of 10 μM. The cells were then cultured at 37°C for an additional 3 hours. The antibodies used were unmodified TCZ, SLX, and cIgG, as well as ε-PαL-modified antibodies ε-PαL-TCZ, ε-PαL-SLX, and ε-PαL-cIgG. An apoptosis detection kit was used for staining, and flow cytometry analysis was performed using a Spectral Cell Analyzer SA3800 (SONY, Tokyo, Japan).

[0086] When K562 cells were cultured with 10 μM of unmodified TCZ, SLX, or cIgG for 3 hours, almost no apoptotic or dead cells were observed (Fig. 8a, Fig. 8b-d, left), similar to the PBS-added control, and none of the antibodies exhibited apoptosis-inducing activity.

[0087] On the other hand, when 10 μM of ε-PαL-TCZ (which had been modified with ε-PαL) was added, approximately 68% apoptotic cells and 13% dead cells were detected (Fig. 8b, right). Furthermore, the addition of ε-PαL-SLX resulted in approximately 55% apoptotic cells and 23% dead cells (Fig. 8c, right). In contrast, the percentage of apoptotic cells was extremely low with the control antibody ε-PαL-cIgG (Fig. 8d, right), indicating that the ε-PαL modification itself did not induce nonspecific cell death.

[0088] These results suggest that TCZ and SLX delivered into cells by ε-PαL induce apoptosis via their respective target pathways, causing cell death.

[0089] Example 5 Analysis of the Mechanism of Apoptosis Induction by Various ε-PαL-mAb Conjugates In this example, Western blotting analysis was performed using the phosphorylation state of STAT3 as an indicator to analyze the mechanism of apoptosis induction in K562 cells for various mAbs and their antibody conjugates modified with ε-PαL. STAT3 is a transcription factor activated by stimulation with the cytokine IL-6 and is involved in cell survival and proliferation. Therefore, inhibition of STAT3 phosphorylation (activation) is an effective indicator that suggests that the antibody conjugate inhibits intracellular IL-6 signaling and induces apoptosis.

[0090] K562 cells (2 × 10 ５ 100 cells / well) and incubated at 37°C in CO ２ After 24 hours of incubation in an incubator, 2x PBS (control), unmodified TCZ, SLX, or cIgG, or ε-PαL-modified ε-PαL-TCZ, ε-PαL-SLX, or ε-PαL-cIgG (each of which was modified with ε-PαL) was added to each well at a concentration of 1 μM, and the cells were incubated for 6 hours.

[0091] After incubation, the cells were washed with 1x PBS and then resuspended in RIPA buffer (cComplete protease inhibitor cocktail (Roche, Basel, Switzerland) and 1 mM Na ３ VO ４The samples were dissolved in a 10% acrylamide gel containing 1,000 mg of PEG-4000 (containing PEG-4000). The mixture was centrifuged at 20,000 x g for 10 minutes at 4°C, and the resulting supernatant was mixed with SDS sample buffer and heated at 98°C for 5 minutes. The samples were then subjected to SDS-PAGE on a 10% acrylamide gel, and the gel was transferred to a PVDF membrane.

[0092] The blots were treated with blocking buffer TBS-Tw (TBS containing 0.05% Tween 20 and 5% skim milk) for 1 hour at room temperature, then incubated with primary antibodies overnight at 4°C, followed by incubation with secondary antibodies for 1 hour at room temperature. The blots were treated with Chemi-Lumi One L or Chemi-Lumi One Super (Nacalai Tesque, Kyoto, Japan), and the resulting signals were detected using ChemiDoc Touch (BIO-RAD, California, USA). The primary antibodies used were phosphorylated STAT3 (Tyr705; Cell Signaling Technology, Part Number #9145, 1:1,000 dilution), STAT3 (Santa Cruz Biotechnology, Part Number SC-8019, 1:1,000 dilution), and GAPDH (Fujifilm Wako Pure Chemical Industries, Part Number 015-25473, 1:20,000 dilution). The secondary antibodies used were TidyBlot Western Blot Detection Reagent (BIO-RAD, 1:400 dilution) or HRP-conjugated secondary antibody (Jackson ImmunoResearch, Part Number AB_230734, 1:10,000 dilution).

[0093] Western blotting was performed after 6 hours of incubation with PBS, unmodified TCZ, SLX, or cIgG at 1 μM. No significant changes in STAT3 phosphorylation levels were observed, and the signal intensities were comparable to those of the control (Fig. 9, lanes 1, 2, 4, and 6). On the other hand, the addition of 1 μM ε-PαL-TCZ significantly reduced phosphorylated STAT3, indicating that STAT3 activation was suppressed (Fig. 9, lane 5). Similarly, ε-PαL-SLX also suppressed STAT3 phosphorylation (Fig. 9, lane 7). In contrast, ε-PαL-cIgG did not alter STAT3 phosphorylation (Fig. 9, lane 3).

[0094] These results demonstrate that TCZ and SLX delivered intracellularly by ε-PαL block the intracellular signaling pathway activated by IL-6 and suppress the phosphorylation (i.e., activation) of STAT3. This indicates that both antibodies functionally act on their targets intracellularly, supporting the effective intracellular delivery of antibodies via ε-PαL modification.

[0095] The present invention is not limited to the above-described embodiment, and various modifications and improvements are possible without departing from the spirit of the invention.

Claims

1. General formula (1) (wherein n is an integer of 3 to 100) or a salt thereof.

2. The linker is represented by the following formula (2): (wherein m is an integer of 1 to 30; X represents an oxygen atom or an imino group; Y represents an alkylene group having 1 to 6 carbon atoms which may be substituted with a halogen atom, a hydroxyl group, an amino group, a mercapto group, a carboxyl group, or a sulfo group; and Z includes a group generated by a click reaction from a click functional group which is an azide group, an alkenyl group, a nitrile group, a thiol group, a maleimide group, an epoxide group, an aziridine group, or a thiirane group), or a salt thereof according to claim 1.

3. [Rule 26 amendment 04.11.2025] The above formula (1) is The compound according to claim 1 or a salt thereof, 4. The compound or salt thereof according to claim 1, wherein ε-poly-L-α-lysine (ε-PαL) is a cell membrane-permeable polycation isopeptide (PIECE) derived from a microorganism.

5. The compound or salt thereof according to claim 1, wherein the antibody is an antibody against an inflammation-related protein.

6. The compound or salt thereof according to claim 1, wherein the antibody is an antibody against IL-6 or its receptor.

7. The compound or salt thereof according to claim 6, wherein the antibody is tocilizumab, siltuximab, or sarilumab.

8. A STAT3 phosphorylation inhibitor comprising the compound according to claim 6 or a salt thereof as an active ingredient.

9. The compound or salt thereof according to claim 1, wherein the antibody is an antibody against TNFα.

10. The compound or salt thereof according to claim 9, wherein the antibody is infliximab.

11. The compound or salt thereof according to claim 1, wherein the antibody is an antibody against IL-33.

12. The compound or salt thereof according to claim 11, wherein the antibody is etokimab.

13. A pharmaceutical composition containing the compound or salt thereof according to claim 1 as an active ingredient.

14. The pharmaceutical composition of claim 13, wherein the pharmaceutical composition is an anti-cancer agent.

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