Compounds containing a cell membrane-permeable polycationic isopeptide and antibody as active ingredients, and pharmaceutical compositions containing the same.

Abstract

The object of the present invention is to provide a compound that allows for the safe and efficient introduction of an antibody drug capable of suppressing the proliferation of cancer cells into cells. [Solution] By introducing an antibody that specifically binds to IL-6 or its receptor into cells using the PIECE method, the proliferation of cancer cells is suppressed by specifically inhibiting the autocrine intracellular IL-6 signaling pathway. This configuration makes it possible to target pathogenic signaling pathways localized within cells, overcoming the limitations of conventional antibody drugs and providing a novel and practical intracellular delivery technology for antibody drugs and a cancer treatment method.

Description

[Technical Field]

[0001] The present invention relates to intracellular delivery technology for antibodies, and more particularly to compounds comprising a complex of a cell membrane-permeable polycationic isopeptide (PIECE) and an antibody as an active ingredient, and to pharmaceutical compositions containing the same. [Background technology]

[0002] In various diseases, including cancer, it is known that abnormal activation or suppression of intracellular and extracellular protein-protein interactions, i.e., disruption of signal transduction, triggers the pathogenesis. These interactions form the foundation of life activities and are precisely controlled; therefore, an imbalance in them 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 characteristics, antibodies have already been put into practical use as therapeutic drugs for many diseases and have been established as a therapeutic 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 currently, their targets are limited to molecules located outside or on the cell surface. On the other hand, many of the molecules that cause diseases are located inside cells, and if these can be targeted directly, it is expected that the scope 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 techniques for encapsulating antibodies in nanoparticles made of lipids or polymers, and introduction techniques by binding with cell membrane-permeable peptides (CPPs). Furthermore, there are known examples of successful intracellular delivery by supporting CPPs on the surface of nanoparticles. Among these, positively charged polycationic CPPs are widely used as a means of introducing polymers such as antibodies into cells.

[0006] However, conventional intracellular delivery techniques using polycationic CPPs have several technical limitations. While CPP uptake primarily occurs via endocytosis, many of the introduced antibodies remain within endosomes and are degraded before reaching the cytoplasm. For this reason, attempts have been made to design and synthesize CPPs with improved escape properties from endosomes and stability against degrading enzymes. However, these often require complex organic synthesis, and the resulting increase in manufacturing costs is a barrier to practical application. Furthermore, cytotoxicity of polycationic CPPs themselves has been reported, requiring careful consideration of safety.

[0007] Against this backdrop, there is a need to develop new intracellular delivery technologies for antibody drugs that combine efficient delivery to the cytoplasm, safety, and ease of manufacturing. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] WO2005 / 032593 A1 [Patent Document 2] Patent No. 7123414 [Non-patent literature]

[0009] [Non-Patent Document 1] Niamsuphap, S. et al. Targeting the undruggable: emerging technologies in antibody delivery against intracellular targets. Expert Opin Drug Deliv 17, 1189-1211 (2020). [Non-licensed document 2] Goswami, R., Jeon, T., Nagaraj, H., Zhai, S. & Rotello, VM Accessing Intracellular Targets through Nanocarrier-Mediated Cytosolic Protein Delivery. Trends Pharmacol. Sci. 41, 743-754 (2020). [Non-licensed document 3] Guidotti, G., Brambilla, L. & Rossi, D. Cell-penetrating peptides: from basic research to clinics. Trends Pharmacol. Sci. 38, 406-424 (2017).

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Problems to be Solved by the Invention

[0010] As described above, while antibody drugs are useful in that they can inhibit protein - protein interactions with high specificity, they cannot penetrate cell membranes, so they cannot target disease - related factors present inside cells, and there is a problem that their scope of application for diseases such as cancer is limited.

[0011] Furthermore, in the conventional antibody delivery technology using cell - membrane - permeable peptides (CPPs), the problems of increased production costs due to complex organic synthesis processes and cytotoxicity have not been solved, and there are still significant barriers to clinical application.

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

Means for Solving the Problems

[0013] To achieve the above object, the present inventors focused on the fact that ε - poly - L - α - lysine (ε - PαL), a polycationic isopeptide derived from microorganisms, is known to improve the biomembrane permeability of antibodies, and conceived that by applying a polycation modification method (PIECE method) using this cell - membrane - permeable polycationic isopeptide (PIECE) derived from microorganisms to antibody drugs, it might be possible to achieve the intracellular delivery of antibodies, which has been difficult in the past.

[0014] On the other hand, the present inventors found that there is "autocrine intracellular IL - 6 signaling" in which the inflammatory cytokine IL - 6 interacts with receptors in intracellular organelles to activate signals without being secreted extracellularly in cancer cells. Such a novel intracellular signaling pathway is difficult to inhibit with neutralizing antibodies targeting the extracellular region, and currently no effective treatment means have 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 diligently conducted experiments based on this hypothesis. As a result, they succeeded in producing an antibody preparation (hereinafter referred to as ε-PαL-mAb conjugate) by chemically conjugating ε-PαL with a monoclonal antibody (mAb) that specifically binds to IL-6 or its receptor, thus completing the present invention. Furthermore, in order to verify the versatility of the polycation modification technology by the PIECE method, mAbs for TNFα and IL-33 were similarly modified to obtain various antibody conjugate preparations.

[0016] The obtained antibody conjugates were confirmed to be able to be taken up into cancer cells via the endocytosis pathway, escape from endosomes, and diffuse and localize in the cytoplasm or nucleus. In particular, the conjugate of an IL-6-related mAb and ε-PαL specifically inhibited the autocrine IL-6 signaling pathway, which is activated intracellularly without extracellular secretion, and thereby significantly suppressed the phosphorylation of the downstream transcription factor STAT3. This suggests that blocking of intramolecular signals suppresses cancer cell proliferation or induces cell death.

[0017] In other words, the present invention provides the following invention.

[0018] (1) General formula (1) [ka] (In the formula, n is an integer between 3 and 100) A compound or salt thereof, represented by the formula.

[0019] (2) The linker is given by the following equation (2) [ka] (In the formula, m is an integer between 1 and 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; (Z includes groups produced by a click reaction from click functional groups which are azide, alkenyl, nitrile, thiol, maleimide, epoxide, aziridine, or thiirane groups.) A compound or salt thereof according to (1) above, comprising a group represented by .

[0020] (3) The above equation (1) becomes the following equation (3) [ka] ...(3) The compound or salt thereof described in (1) above, represented by [the formula shown].

[0021] (4) ε-poly-L-α-lysine (ε-PαL) is a cell membrane-permeable polycationic isopeptide (PIECE) derived from microorganisms, and is a compound or salt thereof as described in any one of (1) to (3) above.

[0022] (5) The antibody is an antibody against inflammation-related proteins, and is a compound or salt thereof as described in any one of (1) to (4) above.

[0023] (6) The antibody is an antibody against IL-6 or its receptor, and is a compound or salt thereof as described in any one of (1) to (5) above.

[0024] (7) The antibody is tocilizumab, siltuximab, or sarilumab, and is a compound or salt thereof as described in any one of (1) to (6) above.

[0025] (8) A STAT3 phosphorylation inhibitor containing the compound or salt thereof described in (6) or (7) above as an active ingredient.

[0026] (9) The antibody is the compound described in (1) above or a salt thereof, which is an antibody against TNFα.

[0027] (10) The antibody is infliximab, the compound described in (9) above, or a salt thereof.

[0028] (11) The antibody is the compound described in (1) above or a salt thereof, which is an antibody against IL-33.

[0029] (12) The antibody is etokimab, the compound described in (11) above, or a salt thereof.

[0030] (13) A pharmaceutical composition comprising any one of the compounds described in (1) to (12) above, or a salt thereof, as an active ingredient.

[0031] (14) The pharmaceutical composition described above is an anticancer agent, as described in (13).

[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 suppress the proliferation of cancer cells. With this configuration, it is possible to target pathogenic signaling pathways localized within cells and overcome the limitations of conventional antibody drugs, providing a novel and practical intracellular delivery technology for antibody drugs and a cancer treatment method.

[0033] The aforementioned compounds can be prepared by the method described in Patent No. 7123414 of the present inventors, and by reference thereto the entire description of the patent specification is incorporated into this specification. Further features of the present invention will be revealed in the description of embodiments of the present invention below. [Brief explanation of the drawing]

[0034] [Figure 1] Figures 1a-c show the structures of ε-PαL, ε-PαL-PEG-azide, and DBCO-PEG-NHS, and Figure 1d shows the structure of the ε-PαL-antibody conjugate prepared using these. [Figure 2] Figure 2a shows the effects of IL-6 knockdown (left) and IL-6 neutralizing antibody (right) on cell proliferation, while Figure 2b is a schematic diagram of conventional autocrine signaling (left) and autocrine intracellular IL-6 signaling (right). [Figure 3] Figures 3a and 3b show the results of SDS-PAGE analysis performed under non-reducing and reducing conditions for ε-PαL modifiers of TCZ, SLX, and cIgG, respectively, while Figures 3c and 3d show the results of similar analysis under non-reducing and reducing conditions for modifiers of IFX and ETK. [Figure 4] Figure 4a shows the cell morphology of K562 cells without antibody treatment, and Figure 4b shows the cell morphology when various antibodies (TCZ, SLX, cIgG, IFX, ETK) modified with ε-PαL were added, respectively. [Figure 5] Figures 5a to 5e show the results of cell viability (WST assay) evaluated after adding various antibodies (TCZ, SLX, cIgG, IFX, ETK) that were either untreated or modified with ε-PαL to K562 cells. [Figure 6] Figure 6a shows the cell morphology of HeLa cells without antibody treatment, and Figure 6b shows the cell morphology when various antibodies (TCZ, SLX, cIgG, IFX, ETK) modified with ε-PαL were added, respectively. [Figure 7]Figures 7a-7e show the results of cell viability (WST assay) evaluated after adding various antibodies (TCZ, SLX, cIgG, IFX, ETK) that were either untreated or modified with ε-PαL to HeLa cells. [Figure 8] Figure 8 shows the results of apoptosis and cell death analysis of K562 cells. Figure 8a shows the control, and Figures 8b-d show the results with untreated cells or cells treated with various antibodies (TCZ, SLX, cIgG) modified with ε-PαL. [Figure 9] Figure 9 shows the results of Western blotting analysis of the STAT3 phosphorylation state after adding various antibodies (TCZ, SLX, cIgG) to K562 cells, either untreated or modified with ε-PαL. [Modes for carrying out the invention]

[0035] One embodiment of the present invention will be described below with reference to the drawings.

[0036] (Background leading to the present invention) As mentioned above, intracellular and extracellular signal transduction is transmitted through protein interactions and plays a crucial role as the basis of life phenomena. Furthermore, since signal transduction is regulated by increases or decreases in protein-protein interactions, abnormalities in these interactions can lead to the onset of disease.

[0037] Generally, protein-protein interactions are broad and shallow. Therefore, it can be difficult for small molecules with a molecular weight of 1,000 or less to inhibit these interactions. On the other hand, antibodies with a large molecular weight (approximately 150,000) recognize the three-dimensional structure of target proteins and bind specifically, making them extremely effective as inhibitors of protein-protein interactions. For this reason, antibody drugs, which utilize antibodies as direct disease treatments, are attracting attention as a modality with fewer side effects.

[0038] However, because antibodies are macromolecules, they cannot penetrate the cell membranes of animal cells. Therefore, their targets are limited to extracellular or surface molecules, and their effectiveness as therapeutic agents is limited. On the other hand, since many disease-causing target molecules exist inside cells, developing practical delivery technologies to deliver antibodies into cells could dramatically improve the usefulness of antibody drugs.

[0039] Therefore, many methods are currently being attempted to deliver proteins and antibodies into cells, such as encapsulating them in nanoparticles made of lipids or polymers, or cross-linking (conjugating) them with cell membrane-permeable peptides (CPPs) (Non-Patent Documents 1, 2). Furthermore, there are examples of successful intracellular delivery by supporting CPPs on the surface of nanoparticles (Patent Document 1). Amphiphilic CPPs have also been reported (Non-Patent Document 3), but polycationic CPPs are often used as a tool for delivering biomacromolecules such as proteins into animal cells, and generally, the higher the polycationicity, the higher the cell membrane permeability (Non-Patent Documents 3-14).

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

[0041] CPP-cargoes taken into cells via endocytosis are trapped within endosomal granules and ultimately degraded by lysosomes. Therefore, for cargoes to exert their physiological functions within cells, they need to escape from endosomes. On the other hand, CPP-cargoes taken in by direct translocation of the cell membrane are expected to exert their functions rapidly after being taken into the cell. Generally, small molecule compounds bound to conventional cationic CPPs are taken into cells via both pathways, but conjugates with biomacromolecules such as proteins and antibodies are taken in only via the endocytosis pathway.

[0042] Currently, there is growing interest in the logical design and synthesis of polycationic polyphosphates (CPPs) that can effectively escape from endosomes and withstand peptide-degrading enzymes (Non-Patent Documents 12-14). However, the high synthesis costs associated with their complex organic synthesis make the practical use of CPPs difficult. Furthermore, the toxicity of polycationic CPPs to animal cells is another important issue that needs to be addressed (Non-Patent Document 7).

[0043] Under these circumstances, a "polycation modification method using cell membrane-permeable polycation isopeptides derived from microorganisms (PIECE method)" has been reported (Patent Document 2, Non-Patent Document 15). One of the PIECEs, ε-poly-L-α-lysine (ε-PαL) (Figure 1a), is a polymer in which the amino acid L-lysine is linked in a linear chain by isopeptide bonds, and is a natural amino acid polymer produced by actinomycetes. Each monomer unit has one amino group (-NH2), and under physiological conditions (around pH 7), it undergoes protonation (-NH3). + ) is positively charged and exhibits cationic properties. Therefore, the longer the polymer chain length, the more positive charges it has, and thus the molecule as a whole exhibits polycationic properties. Like conventional polycationic CPP, ε-PαL exhibits cell membrane permeability (Patent Document 2, Non-Patent Document 15), and the longer its polymer chain length, the better its 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, it is expected that longer-chain ε-PαL will exhibit even better cell membrane permeability due to improved polycationicity. However, currently, ε-PαL with 36 or more residues (n>36) has not been found in nature, and no chemically synthesized versions have been reported. ε-PαL not only exhibits excellent cell membrane permeability but is also resistant to peptide-degrading enzymes due to its isopeptide structure. Even more interestingly, ε-PαL does not show toxicity to animal cells, making it a novel cell membrane permeable peptide that simultaneously improves upon the various weaknesses of conventional CPPs. Furthermore, ε-PαL-PEG-azide (Figure 1b), a derivative of ε-PαL, can be directly used for conjugation with biomolecules via click chemistry. Therefore, by introducing DBCO groups to the surface of biomacromolecules such as antibodies using dibenzocyclooctyne-PEG-N-hydroxysuccinimide ester (DBCO-PEG-NHS ester) (Figure 1c), polycation-modified antibodies with ε-PαL (ε-PαL-antibody conjugates) (Figure 1d) can be easily prepared.

[0045] The ε-PαL-antibody conjugate is taken up into animal cells via endocytosis, but escapes from endosomes and diffuses to localize in the cytoplasm and nucleus (Non-Patent Literature 15). In other words, it is an innovative intracellular antibody delivery technology that allows antibodies to fully exert their function after being delivered into animal cells.

[0046] Therefore, the inventors of this invention conceived a new technology in which a monoclonal antibody (mAb) that specifically binds to inflammation-related proteins is conjugated with ε-PαL (ε-PαL-mAb conjugate), and 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 uses the immune system to respond to pathogens such as bacteria, viruses, and fungi, as well as foreign substances, harmful stimuli, and trauma. It is a beneficial response that eliminates pathogens and foreign substances and repairs and regenerates tissue. Inflammation is divided into two types: acute inflammation, which ends in a few days to a few weeks, and chronic inflammation, which lasts for several months to several years, and their roles differ greatly. Generally, acute inflammation is a "good inflammation" that eliminates pathogens and foreign substances and induces tissue repair and regeneration. In contrast, chronic inflammation is a "bad inflammation" that is involved in the onset and progression of almost all diseases, including cancer, neurodegenerative diseases, and diabetes. Acute inflammation caused by tissue damage promotes tissue repair and regeneration, but the signaling pathways important for tissue repair and regeneration were unknown. Furthermore, it has been revealed that the inflammatory cytokine IL-6, induced by inflammation, activates a novel signaling pathway called the Src family kinase (SFK)-YAP pathway, in addition to the conventionally known JAK-STAT3 pathway, thereby promoting intestinal regeneration, and that the SFK-YAP pathway is a novel therapeutic target for colorectal cancer (Non-Patent Literature 17, 18). In cancer, acute inflammation has been confirmed to suppress cancer, and BCG (attenuated bovine tuberculosis) intravesical instillation therapy for bladder cancer is still used clinically. On the other hand, chronic inflammation contributes to the development and progression of many types of cancer, and in recent years, the mechanisms by which inflammation promotes cancer have gradually become clearer (Non-Patent Literature 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, and while IL-6 family cytokines promote tissue repair and regeneration, they are also the most important players in promoting cancer through inflammation, exhibiting a dual nature (Non-Patent Literature 20).

[0048] In 2011, Dr. Weinberg, who discovered the oncogene RAS and the tumor suppressor gene RB, added inflammation as one of the hallmarks of cancer. However, as of 2024, cancer therapies targeting inflammation have not been clinically applied due to side effects such as immunosuppression and suppression of tissue repair and regeneration. Historically, research on cancer and inflammation has focused on cases where there is clear infection (e.g., Helicobacter pylori, hepatitis viruses, human papillomavirus) and associated inflammation. Inflammatory carcinogenesis, involving inflammation caused by such infections, is thought to account for about 20% of cancers, and these cancers can be prevented or detected early through vaccination. On the other hand, in the remaining 80% of cancers where infection or inflammation is not clear, "tumor-induced inflammation," which is induced by gene mutations in cancer cells that precede the expression of inflammatory cytokines and chemokines such as IL-6 and act autocrine or paracrinely in the tumor microenvironment, is also important. In short, inflammation is thought to play a crucial role in almost 100% of cancers, but as mentioned above, cancer therapies targeting 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 treatment using IL-6 or IL-6 receptor neutralizing antibodies has not shown efficacy in human clinical trials or mouse models, and the reasons for this are not fully understood.

[0049] The inventors of this invention have previously conducted research on inflammation and cancer, focusing on IL-6, particularly autocrine IL-6 in cancer cells. In the course of this research, they found that while suppression of IL-6 and IL-6 receptor expression by siRNA / shRNA in cancer cells strongly induces inhibition of cancer cell proliferation and cell death, neutralizing antibodies against IL-6 and IL-6 receptors have no effect whatsoever on inhibiting cancer cell proliferation or cell death (Figure 2a). As a mechanism, they discovered that the autocrine IL-6 signaling pathway in cancer cells is not the previously thought-provoking "pathway where IL-6 is released extracellularly to stimulate the receptor," but rather "a pathway in which IL-6 can activate signals at the intracellular ER / Golgi and endosome levels without being secreted extracellularly" (Figure 2b). They named this novel IL-6 signaling pathway Autocrine intracellular IL-6 signaling, and hypothesized that if this signaling pathway could be specifically suppressed in cancer cells, a novel cancer treatment targeting IL-6 could be developed without causing immunosuppression. In other words, it was expected that introducing antibodies that specifically bind to IL-6 or the IL-6 receptor into cells using the PIECE method described above would suppress the autocrine intracellular IL-6 signaling pathway and inhibit the proliferation of cancer cells.

[0050] (definition) Unless otherwise specified, terms used herein shall have the meanings commonly understood in the art. The following definitions of terms applicable throughout this specification are provided below.

[0051] In this specification, the term "PIECE" refers to a cell membrane-permeable polycationic isopeptide (Polycationic isopeptide entering cells) derived from microorganisms. The PIECE used herein is ε-poly-L-α-lysine (ε-PαL).

[0052] In this specification, "PIECE method" refers to a technique for conjugating a polycationic isopeptide derived from a microorganism with a target molecule and introducing the target molecule into a cell. Conjugation in the PIECE method can preferably be carried out by a click reaction that proceeds 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 antibody is represented by the following general formula (1). [ka]

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

[0055] In the present invention, "linker" refers to a divalent linking group, whose ends are capable of binding to other groups, that is interposed to bind the antibody and the ε-PαL moiety, and whose length and flexibility can be arbitrarily adjusted according to the purpose. The type of linker is not particularly limited and may consist of linking groups known in the art, such as linear alkylene groups, polyethylene glycol (PEG) chains, aromatic groups, amide bonds, ester bonds, urethane bonds, (thio) ether bonds, or combinations thereof. Furthermore, linkers may not merely serve as mediators of binding, but may also possess diverse properties, such as conferring hydrophilicity, conferring hydrophobicity, or having specific reactive functional groups (e.g., azide groups, alkyne groups, NHS ester groups, etc.) that can be easily introduced using known reactions such as click chemistry. As one specific example, the linker included in the above general formula (1) is represented by the following formula (2). [ka]

[0056] In the formula, m may be any value from 1 to 30, but is preferably around 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 it may also have high chemical stability. X represents an oxygen atom or an imino group. The imino group represented by X is represented as -NR-, and R can be H or a suitable substituent, such as 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 fryle group, a pyridyl group, or a thienyl group. Among these, R is preferably a 5 to 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, a hexene group, etc. Among these, Y is preferably an alkylene group having 2 to 6 carbon atoms. The alkylene group may be substituted with a conventional substituent, such as halogen atoms (e.g., bromine, chlorine, fluorine, etc.), hydroxyl groups, amino groups, mercapto groups, carboxyl groups, sulfo groups, etc. The alkylene group may also contain linkers such as -O-, -S-, -SS-, silyl groups, etc. Z includes groups produced by a click reaction from a click functional group which is 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 produced thereby are shown below. Specifically, a 1,2,3-triazole group is produced by the reaction of an azide group with an alkyne group. A thioether group is produced by the reaction of an alkenyl group with a thiol group, and a tetrazole group is produced by the reaction of a nitrile group with an azide group. A β-hydroxythioether group is produced by the reaction of a thiol group with an epoxide group, and a thioether group is produced by the reaction of a maleimide group with a thiol group. Furthermore, a β-amino group-containing adduct is generated by the ring-opening reaction of the aziridine group, and a β-thio group-containing adduct is generated by the nucleophilic ring-opening reaction of the thiirane 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 chemical reaction method, such as fermentation or chemical synthesis, or it may be obtained by chemical synthesis from lysine. For example, ε-PαL can be obtained by culturing Streptomyces albulus subspecies lysinopolymeras, as described in Japanese Patent No. 1245361, in a culture medium (for example, 5% by weight glucose, 0.5% by weight yeast extract, 1% by weight ammonium sulfate, 0.08% by weight dipotassium hydrogen phosphate, 0.136% by weight potassium dihydrogen phosphate, 0.05% by weight magnesium sulfate heptahydrate, 0.004% by weight zinc sulfate heptahydrate, 0.03% by weight iron sulfate heptahydrate, adjusted to pH 6.8), and then separating and collecting ε-polylysine from the resulting culture. Furthermore, ε-PαL may be obtained by any method, and it can also be obtained by the method described in the following non-patent document. For example, γ-poly-L-diaminobutanoic acid, an ε-PαL analog that is expected to exhibit cell membrane permeability similar to ε-PαL, can be produced by referring to the paper (Takehara, M., Saimura, M., Inaba, H. & Hirohara, H. Poly(gamma-L-diaminobutanoic acid), a novel poly(amino acid), coproduced with poly(epsilon-L-lysine) by two strains of Streptomyces celluloflavus. FEMS Microbiol Lett 286, 110-7 (2008).). Furthermore, γ-poly-D-diaminobutanoic acid, an ε-PαL analog, can be manufactured by referring to the paper (Ohkuma, H., Tenmyo, O., Konishi, M., Oki, T. & Kawaguchi, H. BMY-28190, a novel antiviral antibiotic complex. J Antibiot (Tokyo) 41, 849-54 (1988).).Furthermore, β-poly-L-diaminopropionic acid, an ε-PαL analog, can be produced by referring to the following papers: (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 unravelling 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, which is a conventional method in the field of click chemistry. A linker is, in short, a group that can be interposed to conjugate the antibody and ε-PαL, and can be any group as long as it does not hinder the objective of the present invention. For example, the conjugate of ε-PαL and antibody via a linker is represented by the following formula (3). [ka] ...(3)

[0060] Furthermore, other linkers include, for example, those represented by any of the following formulas (4a) to (4d), in which an amino group, carboxyl group, or hydroxyl group in the compound that forms the basis of the linker reacts with a carboxyl group, hydroxyl group, or amino group in the antibody to form a covalent bond, and the triple bond in the compound reacts with a click functional group in ε-PαL-PEG-azide, which is 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 the present invention may follow those methods.

[0061] [ka]

[0062] [ka]

[0063] [ka]

[0064] [ka]

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

[0066] In this specification, "antibody" is not particularly limited, but includes any monoclonal antibody (mAb), and preferably means an antibody against inflammation-related proteins. 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 Examples of neutralizing antibodies 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., emaparmab), type I interferon receptor neutralizing antibodies (e.g., aniflorumab), VEGF neutralizing antibodies (e.g., bevacizumab), and EGF receptor neutralizing antibodies (e.g., cetuximab and panitumumab).

[0067] The compounds or salts thereof in the present invention can be used in the treatment of animals or humans as pharmaceutical compositions containing them as active ingredients. The pharmaceutical compositions are preferably used as anticancer agents and can target all types of cancer, including solid tumors and hematological malignancies. In addition to their use as anticancer agents, the pharmaceutical compositions can also be used in the treatment of autoimmune diseases (e.g., rheumatoid arthritis, systemic lupus erythematosus (SLE), etc.), inflammatory diseases (e.g., ulcerative colitis, Crohn's disease, etc.), and allergic diseases.

[0068] (Reagents and cell lines used) The various monoclonal antibodies (mAbs), reagents, cell lines, and culture media used in this invention are as follows.

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

[0070] DBCO-PEG4-NHS, used for introducing DBCO groups onto the antibody surface, was purchased from MedChemExpress (New Jersey, USA). The derivative (ε-PαL-PEG-azide), obtained by adding PEG and azide groups to the polycationic cell membrane-permeable peptide ε-poly-L-α-lysine, was purchased from MicrobeChem (Fukui Prefecture, Japan).

[0071] Human cervical cancer cells (HeLa cells) and human chronic myeloid leukemia cells (K562 cells) used in cell experiments were obtained from RIKEN BRC CELL BANK (Ibaraki Prefecture, Japan). Animal cells were cultured at 37°C under 5% CO2 conditions. For HeLa cell culture, Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) was used. For K562 cells, FBS-containing RPMI1640 medium was used. Penicillin (100 U / ml) and streptomycin (100 μg / ml) were added to all culture media.

[0072] (Example 1) Preparation of various ε-PαL-mAb conjugates In this example, various mAbs were subjected to polycation modification using the PIECE method to prepare ε-PαL-mAb conjugates.

[0073] First, a reaction solution was prepared containing 1 M NaCl, 5% (w / v) glycerol, and each mAb (1 mg / mL, 6.7 μM) in 10 mM NaH2PO4-Na2HPO4 buffer (pH 7.4). 33.5 μM DBCO-PEG4-NHS ester was added, and the mixture was incubated at 4°C for 1 to 3 hours. Next, a 67 μM aqueous solution of ε-PαL-PEG-azide was added, and the mixture was incubated at 4°C for at least 3 hours. 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 modified state of the various ε-PαL-mAb conjugates obtained (ε-PαL-TCZ, ε-PαL-SLX, ε-PαL-cIgG, ε-PαL-IFX, ε-PαL-ETK) was evaluated using polyacrylamide gel electrophoresis (SDS-PAGE).

[0075] First, in the analysis of ε-PαL-TCZ, ε-PαL-SLX, and ε-PαL-cIgG under non-reducing conditions, the bands of each antibody were shifted towards the high molecular weight side compared to the unmodified antibody, confirming modification by ε-PαL (Figure 3a). On the other hand, in the analysis under reducing conditions, unmodified bands were observed in the heavy and light chains of each antibody, suggesting that ε-PαL randomly modifies these chains (Figure 3b). Similarly, in the analysis of ε-PαL-IFX and ε-PαL-ETK under both non-reducing (Figure 3c) and reducing (Figure 3d) conditions, similar band shifts were observed under both conditions, confirming that each antibody is modified by ε-PαL.

[0076] (Example 2) Cytotoxicity tests (morphological observation) of various ε-PαL-mAb conjugates In this example, the presence or absence of cytotoxicity of various mAbs and their conjugates modified with ε-PαL was evaluated for K562 cells and HeLa cells by morphological observation.

[0077] In a 96-well plate, K562 cells (1 × 10⁶4 (cells / well) or HeLa cells (2.5 × 10⁶) 3 Cells were seeded (100 cells / well) and cultured in a 37°C incubator for 24 hours. 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, the cells in each well were observed using a phase-contrast microscope (LASX, Leica Microsystems, Hesse, Germany). For HeLa cells, the culture medium was removed and the cells were observed using a phase-contrast microscope.

[0078] In K562 cells, no significant changes in cell morphology were observed when unmodified TCZ, SLX, cIgG, IFX, or ETK were added at 10 μM. On the other hand, when ε-PαL-TCZ and ε-PαL-SLX were added at concentrations of 5 μM or higher, cells showing morphological abnormalities were observed, and at 10 μM, morphological abnormalities were observed in most cells (Figure 4a, 3b). Since only a small number of cells showed abnormalities even when the control antibody ε-PαL-cIgG was added at 10 μM, it was suggested that specific cytotoxicity is expressed by intracellular delivery of TCZ and SLX. Similarly, morphological abnormalities were observed with ε-PαL-IFX and ε-PαL-ETK at concentrations of 5 μM or higher, and at 10 μM, morphological abnormalities were observed in most cells (Figure 4a, 3b).

[0079] In HeLa cells, no clear changes in cell morphology were observed when 10 μM of unmodified TCZ, SLX, cIgG, IFX, or ETK was added. In contrast, significant morphological abnormalities were observed when 10 μM of ε-PαL-TCZ was added (Figure 6a, 5b). On the other hand, with ε-PαL-SLX and the control antibody ε-PαL-cIgG, only a very small number of cells showed abnormalities even when 10 μM was added, suggesting that TCZ delivered into cells exhibits specific cytotoxicity even in HeLa cells. Furthermore, unlike K562 cells, no morphological abnormalities were observed in HeLa cells with ε-PαL-IFX and ε-PαL-ETK even when 10 μM was added (Figure 6a, 5b).

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

[0081] Place K562 cells or HeLa cells (2 × 10⁶ cells each) in a 384-well plate. 3 Cells were seeded (10 cells / well) and cultured for 24 hours in a CO2 incubator set to 37°C. After culturing, the following antibodies or antibody conjugates were added to each well at concentrations of 0-10 μM, and culture was continued for a further 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 (Dojin Chemical Research Institute, Kumamoto Prefecture, Japan), and the viable cell rate was calculated from the obtained absorbance data (n=3). In addition, GraphPad Prism (ver.8) (GraphPad Software, California, USA) was used to create graphs of the viable cell rate and for IC analysis. 50 The value was calculated.

[0082] Results of the WST assay against K562 cells showed that the IC 50 values of ε-PαL-TCZ, ε-PαL-SLX, and ε-PαL-cIgG were 0.3 μM, 1.3 μM, and 5.0 μM, respectively (Figs. 5a - c). Although concentration-dependent cytotoxicity was also observed for ε-PαL-cIgG, its effect was slower compared to ε-PαL-TCZ and ε-PαL-SLX (Fig. 5c), suggesting that different mechanisms of action might be involved. Furthermore, the IC 50 values of ε-PαL-IFX and ε-PαL-ETK were 0.8 μM and 0.5 μM, respectively (Figs. 5d, e), and both showed significant cytotoxicity against K562 cells.

[0083] In the WST assay in HeLa cells, the IC 50 values of ε-PαL-TCZ, ε-PαL-SLX, and ε-PαL-cIgG were 5.9 μM, >10 μM, and 5.3 μM, respectively (Figs. 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 (Figs. 7a, b). On the other hand, since ε-PαL-cIgG showed mild toxicity in a concentration-dependent manner (Fig. 7c), it was suggested that it might exhibit cytotoxicity through a different mechanism of action from ε-PαL-TCZ and ε-PαL-SLX. For ε-PαL-IFX and ε-PαL-ETK, both had IC 50 values exceeding 10 μM (Figs. 7d, e), and no clear cytotoxicity was observed against HeLa cells.

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

[0085] K562 cells (5×10 4Cells were seeded (10 cells / well) and cultured for 24 hours in a CO2 incubator set to 37°C. After culturing, the cells were washed with culture medium, and then the following antibodies or antibody conjugates were added to each well at a concentration of 10 μM, followed by further incubation at 37°C for 3 hours. The antibodies used were unmodified TCZ, SLX, cIgG, and 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 for 3 hours with 10 μM of unmodified TCZ, SLX, and cIgG added, almost no apoptotic or dead cells were observed, similar to the PBS-added control (Figure 8a, Figures 8b-d left), and none of the antibodies showed apoptosis-inducing activity.

[0087] On the other hand, when 10 μM of ε-PαL-TCZ, which was modified with ε-PαL, was added, approximately 68% of cells were apoptotic and 13% were dead (Figure 8b right). Furthermore, when ε-PαL-SLX was added, approximately 55% of cells were apoptotic and 23% were dead (Figure 8c right). In contrast, the proportion of apoptotic cells was extremely low with the control antibody ε-PαL-cIgG (Figure 8d right), and no nonspecific induction of cell death by ε-PαL modification itself was observed.

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

[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 status of STAT3 as an indicator to analyze the mechanism of apoptosis induction in K562 cells for various mAbs and antibody conjugates modified with ε-PαL. STAT3 is a transcription factor activated by the cytokine IL-6 and is involved in cell survival and proliferation. Therefore, suppression of STAT3 phosphorylation (activation) is a useful indicator suggesting that the antibody conjugate inhibits intracellular IL-6 signaling and induces apoptosis.

[0090] K562 cells (2 × 10⁶) in a 48-well plate 5 Seeds were seeded (1 cell / well) and incubated in a 37°C CO2 incubator for 24 hours. After changing the medium, 2×PBS (control), unmodified TCZ, SLX, cIgG, or ε-PαL-modified TCZ, ε-PαL-SLX, and ε-PαL-cIgG, respectively, were added to each well at a concentration of 1 μM and incubated for 6 hours.

[0091] After culturing, the cells were washed with 1×PBS and then lysed with RIPA buffer (containing a cOmplete protease inhibitor cocktail (Roche, Basel, Switzerland) and 1 mM Na3VO4). The mixture was centrifuged at 4°C and 20,000×g for 10 minutes, and the supernatant was heated at 98°C for 5 minutes with SDS sample buffer added. Subsequently, the sample was subjected to SDS-PAGE on a 10% acrylamide gel, and the gel was transferred to a PVDF membrane.

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

[0093] Western blotting was performed after adding 1 μM of PBS, unmodified TCZ, SLX, and cIgG and incubating for 6 hours. No significant changes were observed in STAT3 phosphorylation levels, and all showed signal intensity equivalent to the control (Figure 9, lanes 1, 2, 4, 6). On the other hand, when 1 μM of ε-PαL-TCZ was added, a significant decrease in phosphorylated STAT3 was observed, and STAT3 activation was suppressed (Figure 9, lane 5). Similarly, STAT3 phosphorylation was suppressed with ε-PαL-SLX (Figure 9, lane 7). In contrast, no change in STAT3 phosphorylation levels was observed with ε-PαL-cIgG (Figure 9, lane 3).

[0094] The results above clearly show that TCZ and SLX delivered into cells 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 within cells and supports the finding that intracellular delivery of antibodies by ε-PαL modification is effective.

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

Claims

1. General formula (1) 【Chemistry 1】 (In the formula, n is an integer between 3 and 100.) A compound or salt thereof, represented by the formula.

2. The linker is given by the following equation (2) 【Chemistry 2】 (wherein m is an integer between 1 and 30; X represents an oxygen atom or an imino group; Y represents a C1-C6 alkylene group which may be substituted with a halogen atom, a hydroxyl group, an amino group, a mercapto group, a carboxyl group, or a sulfo group; (Z includes groups produced by a click reaction from click functional groups which are azide, alkenyl, nitrile, thiol, maleimide, epoxide, aziridine, or thiirane groups.) A compound or salt thereof according to claim 1, comprising a group represented by [the group name].

3. The above equation (1) becomes the following equation (3) 【Transformation 3】 ・・・（３） A compound or salt thereof according to claim 1, represented as such.

4. The compound or salt thereof according to claim 1, wherein ε-poly-L-α-lysine (ε-PαL) is a cell membrane-permeable polycationic 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 containing the compound or a salt thereof described in claim 6 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 comprising the compound described in claim 1 or a salt thereof as an active ingredient.

14. The pharmaceutical composition according to claim 13, wherein the pharmaceutical composition is an anticancer agent.

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