NOVEL LIVER CANCER THERAPEUTIC AGENT CONTAINING INHIBITOR INHIBITING INTERACTION BETWEEN PKCδ AND E-Syt1

Abstract

To provide a novel liver cancer therapeutic agent.SOLUTION: In the present invention, it was found that due to the interaction of E-Syt1 on ER with PKCδ in liver cancer cells, PKCδ is secreted from the liver cancer cells by pathways via vesicular transport, and that the secretion of PKCδ from liver cancer cells can be inhibited by inhibiting the interaction between PKCδ and E-Syt1, and it was also found that as a result, the proliferation of liver cancer cells is suppressed.SELECTED DRAWING: Figure 5c

Description

[Technical field]

[0001] The present invention relates to a novel therapeutic agent for liver cancer, which comprises an inhibitor of the interaction between protein kinase C delta (PKCδ) and extended-synaptotagmin 1 (E-Syt1). [Background technology]

[0002] In eukaryotes, protein secretion is a fundamental mechanism required for intercellular communication during development, maintenance of homeostasis, and the development and progression of cancer. A transmembrane mechanism is important for secretory proteins to move outside the cell. A general secretory protein has a signal peptide at the N-terminus, binds to a signal recognition particle, migrates through a translocon pore into the endoplasmic reticulum (ER), is transported to the Golgi apparatus, and is secreted outside the cell (ER-Golgi secretory pathway). In recent years, many cases of cytoplasmic protein secretion that do not involve the ER-Golgi secretory pathway have been reported, such as a pathway that directly passes through the plasma membrane (PM) (Non-Patent Document 1) and a pathway that passes through vesicle transport (Non-Patent Document 2), and it has been reported that various types of organelles such as autophagosomes, lysosomes, and ER-Golgi intermediate compartments (ERGIC) are involved (Non-Patent Document 3). These secretory mechanisms of cytoplasmic proteins have been studied in the cases of inflammatory diseases and neurodegenerative diseases, but it was not clear whether these mechanisms also apply to other diseases such as cancer.

[0003] ER is the largest organelle in the cell, synthesizing proteins with signal peptides at the N-terminus. It is widely known that the ER membrane is distributed throughout the cell, often forming contact sites near the membranes of different organelles, controlling organelle dynamics and triggering the biogenesis of organelles such as autophagosomes. It is believed that autophagosome formation occurs starting from the ER membrane at the ER-mitochondria or ER-PM contact sites. Interestingly, it has been reported that autophagosomes are involved in the secretion of IL-1β, a secretory protein lacking an N-terminal signal peptide, in inflammatory cells. Recent studies have reported that SEC22B, one of the ER-binding tethering factors involved in transport from the ER to the Golgi apparatus, is involved in the secretion mechanism of IL-1β associated with autophagy (Non-Patent Document 3). It has also been reported that SEC22B accumulates at the ER-PM contact site, and E-Syt1 is known to be a tethering factor at the ER-PM contact site together with STIM1 (Non-Patent Document 4). However, it was unclear whether secretion of cytoplasmic proteins involved the ER or ER-PM contact sites.

[0004] Many studies have reported on several cytoplasmic proteins, such as PKCδ and importin α1, that are actively secreted from cancer cell lines and their involvement in cancer proliferation (Patent Document 1, Non-Patent Documents 5 and 6). It has been suggested that the secretion of cytoplasmic proteins such as PKCδ begins in the cytoplasm, but it was not clear which intracellular membrane is involved in the secretion of cytoplasmic proteins. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent Publication No. 2021-046362 [Non-patent literature]

[0006] [Non-Patent Document 1] The Journal of biological chemistry 279, 6244-6251 (2004) [Non-Patent Document 2] eLife 4, e11205 (2015) [Non-Patent Document 3] The EMBO journal 36, 42-60 (2017) [Non-Patent Document 4] Frontiers in cell and developmental biology 9, 635518 (2021) [Non-Patent Document 5] Cancer research 81, 414-425 (2021) [Non-Patent Document 6] Scientific reports 6, 21410 (2016) [Non-Patent Document 7] The Journal of cell biology 196, 801-810 (2012) [Non-Patent Document 8] Molecular biology of the cell 27, 1188-1196 (2016) [Non-Patent Document 9] Proceedings of the National Academy of Sciences of the United States of America 104, 3823-3828 (2007) [Non-Patent Document 10] Cancer research 69, 8844-8852 (2009) [Non-Patent Document 11] Autophagy 15, 1682-1693 (2019) Summary of the Invention [Problem to be solved by the invention]

[0007] An objective of the present invention is to provide a novel therapeutic agent for liver cancer, the mechanism of action of which is to inhibit the extracellular secretion of PKCδ. [Means for solving the problem]

[0008] The inventors discovered that in hepatoma cells, E-Syt1 on the ER interacts with PKCδ, causing PKCδ to be secreted from hepatoma cells via a pathway via vesicular transport, and that inhibiting the interaction between PKCδ and E-Syt1 can inhibit the secretion of PKCδ from hepatoma cells, and further discovered that, as a result, the proliferation of hepatoma cells is suppressed.

[0009] Based on the above findings, the present invention has been completed.

[0010] That is, the present invention is as follows. [1] A therapeutic agent for liver cancer, comprising an inhibitor of the interaction between PKCδ and E-Syt1 in cells. [2] The therapeutic agent for liver cancer according to [1], wherein the inhibitor of the interaction is an anti-PKCδ antibody or an antigen-binding fragment thereof. [3] The liver cancer therapeutic agent described in [1] or [2], wherein the anti-PKCδ antibody or antigen-binding fragment thereof recognizes an epitope sequence contained in the amino acid sequence of amino acid numbers 601 to 676 in SEQ ID NO: 1 or an amino acid sequence at a position corresponding thereto, or an amino acid sequence having 90% or more identity to such an amino acid sequence. [4] The liver cancer therapeutic agent according to any one of [1] to [3], wherein the antigen-binding fragment is a Fab, a Fab', a F(ab')2, a scFab, a scFv, a diabody, a triabody, a minibody, or a nanobody. [5] The liver cancer therapeutic agent according to any one of [1] to [4], wherein the liver cancer is hepatocellular carcinoma. [6] The liver cancer therapeutic agent according to any one of [1] to [5], further comprising an agent for introducing the interaction inhibitor into cells. [7] PKCδ secretion inhibitors, including inhibitors of the interaction between PKCδ and E-Syt1 in cells. [8] A growth inhibitor for hepatocellular carcinoma cells, which contains an inhibitor of the interaction between PKCδ and E-Syt1 in cells. Effect of the Invention

[0011] INDUSTRIAL APPLICABILITY The present invention provides a novel therapeutic agent for liver cancer, which comprises an inhibitor of the interaction between PKCδ and E-Syt1. [Brief description of the drawings]

[0012] [Figure 1a] Figure 1 shows that BioID screening identified E-Syt1 as a membrane protein that interacts with cytoplasmic PKCδ. Figure 1a shows the results of two-dimensional image conversion analysis (2DICAL) of biotinylated proteins purified from membrane fractions of two types of HepG2 cells (clone E5 or clone A8) stably expressing PKCδ-BioID2. Untreated cells not expressing PKCδ-BioID2 were compared with doxycycline-treated cells, and 360 counts of 2DICAL were calculated. Specificity was defined as a >3-fold increase in the spectral counts of both clones E5 and A8. [Figure 1b] Figure 1b is a photograph showing the results of immunoblot analysis of biotinylated proteins purified with streptavidin from cell lysates of clone E5 or clone A8 (n=3; independent experiments). Cells were treated with 1 μg / mL doxycycline (Dox) to induce PKCδ-BioID2 expression. Actin was used as a loading control. [Figure 1c] Figure 1c shows confocal micrographs showing colocalization of PKCδ and E-Syt1 in HepG2 cells. Photographs are representative of three independent experiments. Scale bar: 10 μm. Inset: enlarged photograph of white box. [Figure 1d]Figure 1d shows HepG2 cells stably expressing doxycycline-inducible PKCδ-GFP incubated with 0.5 μg / mL doxycycline for 24 h, then fixed and stained with Sec61β as an ER marker, and the stained cells were imaged by super-resolution microscopy (3D SIM). Planar images (xy) and 3D reconstructed images (inset: enlarged photographs in white boxes; yz) are shown. Arrowheads indicate colocalization of PKCδ-GFP and the ER. The photographs are representative of three independent experiments. [Figure 1e] Figure 1e shows the interaction between PKCδ and E-Syt1 in HepG2 and AGS cells detected by confocal microscopy. The cells were fixed and reacted with a combination of mouse anti-PKCδ and rabbit anti-E-Syt1 antibodies (PKCδ×E-Syt1), a combination of mouse IgG and rabbit IgG (control×control), or a combination of mouse anti-PKCδ and rabbit anti-PKCδ antibodies (PKCδ×PKCδ) and then subjected to Duolink In Situ Proximity Ligation Assay (PLA). In the right graph, data are shown as mean±sd (HepG2 cells: n=68, AGS cells: n=80). *: p<0.0001 (two-tailed Mann-Whitney test). The photographs are representative of three independent experiments. Scale bar: 10 μm. [Figure 1f] Figure 1f shows the interaction of PKCδ and Sec61β (ER marker) in HepG2 cells (control), E-Syt1 knockout HepG2 cells (E-Syt1 KO), and AGS cells detected by confocal microscopy. Each cell was fixed and reacted with a combination of mouse anti-PKCδ and rabbit anti-Sec61β antibodies (PKCδ×Sec61β), and Duolink In Situ Proximity Ligation Assay (PLA) was performed. In the right graph, data are shown as mean±sd (HepG2 cells (control): n=64, E-Syt1 knockout HepG2 cells: n=61, AGS cells (WT): n=67). *: p<0.0001 (two-tailed Mann-Whitney test). Photographs are representative of three independent experiments. Scale bar: 10 μm. [Figure 2a] Figure 2 shows that E-Syt1 is required for PKCδ secretion. Figure 2a shows immunoblot analysis of control or E-Syt1 knockout HepG2 cells (E-Syt1 KO) and lysates (n=3 independent experiments). Relative signal density was quantified, and data are shown as mean±sd in the right graph. *: p<0.0001 (two-tailed Student's t-test). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Ponceau-S staining were used as loading controls for lysates or media. [Figure 2b] Figure 2b shows the results of treating doxycycline-induced HepG2 cells with scrambled (Scr) siRNA or E-Syt1 siRNA for 24 h and measuring PKCδ secretion by HiBiT extracellular assay (n=4, independent experiments). Secretion was measured after re-incubation with medium containing 0.5 μg / mL doxycycline for 24 h. Data are shown as mean ± SD. *: p=0.0053 (Welch's t-test). [Figure 2c] Figure 2c shows schematic diagrams of human wild-type PKCδ (WT) and partially deleted mutants of PKCδ (Δ451-676 and Δ601-676). These PKCδ constructs were fused with BioID2 and HA-epitope tags. [Figure 2d] Figure 2d shows the results of immunoblot analysis of lysates (input) and streptavidin-purified biotinylated proteins from doxycycline-induced HepG2 cells transfected with an empty vector (empty) or vectors containing human wild-type PKCδ (WT) or partial deletion mutants of PKCδ (Δ451-676 and Δ601-676) fused to BioID2 and an HA-epitope tag (n=3, independent experiments). Cells were treated with 1 μg / mL doxycycline for 24 hours. Representative blot photographs are shown. Actin was used as a loading control. [Figure 2e](e) Confocal micrographs of doxycycline-inducible HepG2 cells transfected with PKCδ-BioID2-HA vector containing WT or a partial deletion mutant of PKCδ (Δ601-676). Photographs are representative of two independent experiments. Scale bar: 10 μm. [Figure 2f] Figure 2f shows the results of immunoblot analysis of lysates and medium from doxycycline-induced HepG2 cells transfected with PKCδ-BioID2-HA vector containing WT or a partial deletion mutant of PKCδ (Δ601-676) (n=3 independent experiments). Cells were treated with 1 μg / mL doxycycline for 24 h. Actin was used as a loading control. [Figure 3a] Figure 3 shows that autophagy-related proteins are utilized for the secretion of PKCδ. Figure 3a shows the secretion of PKCδ in doxycycline-induced HepG2 cells measured by HiBiT extracellular assay. Cells were preincubated for 24 hours in 10% FBS-containing medium supplemented with 0.5 μg / mL doxycycline, and stimulated for 6 hours in 10% FBS-containing medium or EBSS medium (n>3, independent experiments). Data are shown as mean ± sd. *: p<0.0001 (two-tailed Student's t-test). [Figure 3b] Figure 3b shows the results of flow cytometry analysis of doxycycline-treated HepG2 cells incubated for 6 hours in 10% FBS-containing medium, 0.1% FBS-containing medium, or EBSS medium, and is representative of three independent experiments. [Figure 3c]Figure 3c shows the results of measuring the secretion of PKCδ in doxycycline-induced HepG2 cells treated for 48 h with siRNA (2 μM) of scramble (Scr), ATG5, ATG7, ATG16L1, p62, LC3B or PKCδ (as a control) using the HiBiT extracellular assay (n=3 independent experiments). Luminescence was measured after re-incubation for 24 h in medium containing 0.5 μg / mL doxycycline. Data are shown as mean ± sd. *: p<0.001, **: p<0.0292, ***: p<0.0022, ****: p<0.0001 (ANOVA). [Figure 3d] (d) Immunoblot analysis of lysates and media from doxycycline-induced HepG2 cells treated with scrambled (Scr), ATG5 or LC3B siRNA (2 μM) for 48 h. Photographs are representative of three independent experiments. GAPDH and Ponceau-S staining were used as loading controls for lysates and media, respectively. [Figure 3e] Figure 3e shows confocal micrographs showing the colocalization of intracellular PKCδ and LC3B in HepG2 cells cultured in 10% FBS-containing medium. The photographs are representative of three independent experiments. Scale bar: 10 μm. Inset: enlarged photograph of the white box. Arrows indicate the colocalization of PKCδ and LC3B. [Figure 3f] (f) Confocal micrograph of HepG2 cells expressing GFP and mCherry-LC3B cultured in EBSS medium for 3 h. The photograph is representative of three independent experiments. Scale bar: 10 μm. Inset: enlarged photograph of the white box. [Figure 3g] Figure 3g is a photograph showing the results of immunoblot analysis of biotinylated proteins purified with streptavidin in clone E5 cells (n=3, independent experiments). Cells were treated with medium containing 1 μg / mL doxycycline. [Figure 3h]Figure 3h shows confocal microscopy images of the interaction of PKCδ and LC3B (autophagosomal marker) in HepG2 and AGS cells. Each cell was fixed and reacted with a combination of mouse anti-PKCδ and rabbit anti-LC3B antibodies (PKCδ × LC3B), and Duolink In Situ PLA was performed. In the graph on the right, data are shown as mean ± sd (HepG2 cells: n = 20, AGS cells: n = 20). *: p < 0.0001 (two-tailed Mann-Whitney test). The photographs are representative of three independent experiments. Scale bar: 10 μm. [Figure 4a] Figure 4 shows that PKCδ is secreted via SEC22B+ vesicles. Figure 4a shows the results of measuring PKCδ secretion in doxycycline-inducible HepG2 cells treated with scrambled (Scr) or SEC22B siRNA (2 μM) for 48 h using the HiBiT extracellular assay (n=3 independent experiments). Luminescence was measured after re-incubation with medium containing 0.5 μg / mL doxycycline for 24 h. Data are shown as mean ± sd. *: p<0.0223 (two-tailed Student's t-test). [Figure 4b] Figure 4b shows confocal micrographs to demonstrate the close proximity of PKCδ and SEC22B in HepG2 (control), E-Syt1 KO HepG2 (E-Syt1 KO), or AGS cells. Each cell was fixed and reacted with a combination of mouse anti-PKCδ and rabbit anti-SEC22B antibodies (PKCδ x SEC22B), and Duolink In Situ PLA was performed. In the right panel, data are shown as mean ± sd (HepG2 control cells: n = 75, E-Syt1 KO HepG2 cells: n = 95, AGS cells: n = 89). *: p < 0.0001 (two-tailed Mann-Whitney test). Photographs are representative of three independent experiments. Scale bar: 10 μm. [Figure 4c]Figure 4c is an electron micrograph of a HepG2 cell. The left panel shows the presence of PKCδ (arrow) within SEC22B+ vesicles (arrowheads) around the PM, and the right panel shows the moment of vesicle-PM fusion leading to PKCδ secretion. Pictures are representative of two independent experiments. Scale bar: 100 nm. [Figure 4d] Figure 4d shows the secretion of PKCδ measured by HiBiT extracellular assay in doxycycline-induced HepG2 cells treated with 2 μM siRNA for scramble (Scr), SNAP23, STX3, STX4, or STX3 and STX4 (mixture) for 48 h (n>3, independent experiments). Luminescence was measured after re-incubation with medium containing 0.5 μg / mL doxycycline for 24 h. Data are shown as mean ± sd. *: p<0.0001 (ANOVA). [Figure 4e] Figure 4e shows a photograph of cancer tissue from a patient with hepatocellular carcinoma (HCC). Duolink In Situ PLA shows the cancer cell-specific colocalization of PKCδ and SEC22B (middle and right panels). Cancer (T) and non-cancer (NT) were defined by evaluating hematoxylin-eosin staining of each cancer section (left panel). The photographs are representative of two independent experiments on sections from five patients with HCC. Scale bar: 50 μm. [Figure 4f] Figure 4f shows a schematic diagram of the role of E-Syt1 at the ER-PM contact site in vesicle-mediated secretion of cytoplasmic proteins. The import of PKCδ into SEC22B+ vesicles is dependent on the expression of E-Syt1 and autophagy-related proteins. In hepatoma cells, E-Syt1 forms a special complex in cytoplasmic protein secretion at the ER-PM contact site. [Figure 5a] Figure 5 shows the inhibitory effect on HCC cell proliferation by targeting the interaction between PKCδ and E-Syt1. Figure 5a shows a photograph showing the delivery of anti-PKCδ antibody (C-20) into HepG2 cells using Ab(antibody)-DeliverIN transfection reagent (DeliverIN is a drug delivery system). Scale bar: 10 μm. [Figure 5b] Figure 5b shows confocal micrographs of HepG2 cells treated with DeliverIN and control IgG or anti-PKCδ antibody (C-20). Each cell was fixed and reacted with a combination of mouse anti-PKCδ antibody and rabbit anti-E-Syt1 antibody (PKCδ×E-Syt1), and Duolink In Situ PLA was performed. In the right panel, data are shown as mean±sd (n=91 treated with DeliverIN and control IgG, n=98 treated with DeliverIN and anti-PKCδ antibody). *: p<0.0001 (two-tailed Student's t-test). Photographs are representative of three independent experiments. Scale bar: 10 μm. [Figure 5c] Figure 5c shows the results of measuring PKCδ secretion in doxycycline-induced HepG2 cells treated with DeliverIN and control IgG or anti-PKCδ antibody (C-20) for 24 hours using the HiBiT extracellular assay (n=4, independent experiments). 0.5μg / mL doxycycline was added at the same time as DeliverIN and antibody treatment. Data are shown as mean±sd. *:p<0.0001 (two-tailed Student's t-test). [Figure 5d] Figures 5d-g show the results of a proliferation assay by counting the number of cells in cell lines treated with DeliverIN and control IgG or anti-PKCδ antibody (C-20) for 48 hours. A hemocytometer was used to count cells after trypan blue staining to remove dead cells, and each plot shows the average of four fields of view. Similar experiments were independently repeated three or more times. Data are shown as mean ± SD. Figure 5d shows the results of the same experiment using HepG2 cells. *: p = 0.0047 (Welch's t-test). [Figure 5e] Figure 5e shows the results of the proliferation assay using Hep3B cells. *: p=0.0129 (Welch's t-test). [Figure 5f] Figure 5f shows the results of the proliferation assay using HuH7 cells. *: p=0.0153 (Welch's t-test). [Figure 5g]Figure 5g shows the results of the proliferation assay using AGS cells, which do not secrete PKCδ. ns: no significant difference. [Figure 5h] Figure 5h shows photographs of 3D multicellular spheroids of HepG2 cells treated with control IgG or anti-PKCδ antibody (C-20) in the presence / absence of DeliverIN. Scale bar: 20 μm. [Figure 5i] In Fig. 5i, the number of 3D multicellular spheroids of HepG2 cells was counted based on the size >25 μm. Three independent experiments were performed. Data are shown as mean ± SD. *: p < 0.002 (ANOVA). ns: no significant difference. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] One embodiment of the present invention is a therapeutic agent for liver cancer, which comprises an inhibitor of the interaction between PKCδ and E-Syt1 in cells.

[0014] Protein kinase C (PKC) is a serine-threonine kinase that phosphorylates the hydroxyl groups of serine and threonine residues in proteins. PKC requires diacylglycerol (DAG) and calcium ions (CA) for its activation. 2+ There are two types of PKC isozymes: conventional PKC isozymes (α, βI, βII, γ) that require DAG for activation, and novel PKC isozymes (δ, ε, θ, η) that require only DAG for activation. PKCδ, a novel PKC isozyme, is an intracellular signaling kinase of approximately 78 kilodaltons and is known to be expressed in various cells. In the present invention, PKCδ is preferably human PKCδ, and an example thereof includes one having the amino acid sequence shown in SEQ ID NO:1.

[0015] E-Syt1 is a protein involved in the intracellular transport of lipids, etc. It is also localized on the endoplasmic reticulum (ER) membrane and is known as a tethering factor involved in the binding between the ER and the plasma membrane. In the present invention, E-Syt1 is preferably human E-Syt1, and an example thereof is one having the amino acid sequence shown in SEQ ID NO:2.

[0016] The method for measuring the interaction between PKCδ and E-Syt1 is not particularly limited as long as it allows the measurement of the interaction. For example, the method can be based on immunoblotting, immunofluorescence analysis, PLA analysis, or the like.

[0017] Inhibition of the interaction between PKCδ and E-Syt1 may mean, for example, that the interaction between PKCδ and E-Syt1 when an inhibitor is added is reduced by 50% or more, 70% or more, or 90% or more, compared to when no inhibitor is added.

[0018] The inhibitor of the interaction between PKCδ and E-Syt1 is not particularly limited as long as it is introduced into a cell and inhibits the interaction between PKCδ and E-Syt1 within the cell, and may be an antibody or antigen-binding fragment, a peptide, a compound, a nucleic acid, etc.

[0019] When the interaction inhibitor is an antibody or an antigen-binding fragment thereof, it may be, for example, an antibody or an antigen-binding fragment thereof that targets PKCδ, or an antibody or an antigen-binding fragment thereof that targets E-Syt1, but it is preferably an antibody or an antigen-binding fragment thereof that targets PKCδ.

[0020] The sequence of the epitope recognized by the anti-PKCδ antibody or the antigen-binding fragment thereof is not particularly limited, so long as it targets PKCδ in cells and inhibits the interaction between PKCδ and E-Syt1. The epitope recognized by the anti-PKCδ antibody or the antigen-binding fragment thereof is preferably an amino acid sequence at the C-terminus of PKCδ. For example, the antibody may recognize an epitope sequence contained in the amino acid sequence of amino acid numbers 601 to 676 in SEQ ID NO: 1 or an amino acid sequence at a position corresponding thereto, or an amino acid sequence having 90% or more, preferably 95% or more, more preferably 98% or more identity with these amino acid sequences, or may recognize an epitope sequence consisting of these amino acid sequences. The position corresponding to the amino acid sequence of amino acid numbers 601 to 676 in SEQ ID NO: 1 refers to the position corresponding to 601 to 676 in SEQ ID NO: 1 in PKCδ that does not have SEQ ID NO: 1.

[0021] The anti-PKCδ antibody may be either a polyclonal antibody or a monoclonal antibody, but is preferably a monoclonal antibody from the viewpoint of stability of the therapeutic effect. Furthermore, in order to use the anti-PKCδ antibody in human therapy, it is preferable that the anti-PKCδ antibody is a chimeric antibody, a humanized antibody, or a fully human antibody from the viewpoint of reducing antigenicity. For example, the animal from which the antibody is derived may be a human, a mouse, a rat, a rabbit, a monkey, etc., or may be a camelid such as a dromedary, a Bactrian camel, a llama, an alpaca, a guanaco, or a vicuna, or a cartilaginous fish such as a shark or a chimaera.

[0022] The antibody used may be a commercially available antibody, or an antibody prepared by a method known to those skilled in the art may be used. For example, in the case of a monoclonal antibody, a method for producing an antibody includes immunizing an animal such as a mouse with PKCδ as an antigen, recovering cells that produce an antibody against the PKCδ antigen protein, fusing the recovered cells with homogeneous or heterogeneous myeloma cells, selecting hybridoma cells that produce an anti-PKCδ monoclonal antibody, and culturing the hybridoma cells. It can be obtained from the culture supernatant. Furthermore, the hybridoma cells can be further modified to obtain chimeric or humanized antibodies. Specifically, for example, a desired antibody can be obtained by manipulating a gene extracted from the hybridoma cells by replacing a portion of the gene encoding an Fc region with a gene encoding a human Fc region using a gene recombination technique known to those skilled in the art.

[0023] A fully human anti-PKCδ antibody can be obtained from the culture supernatant of hybridoma cells by immunizing a genetically modified mouse capable of producing human antibodies with PKCδ as an antigen, recovering anti-PKCδ antibody-producing cells obtained from the genetically modified mouse, fusing the cells with myeloma cells, and selecting hybridoma cells that produce anti-PKCδ antibodies. Furthermore, a fully human anti-PKCδ antibody can also be prepared by using a phage display method, which is a method known to those skilled in the art.

[0024] Since the interaction inhibitor is introduced into cells and inhibits the interaction between PKCδ and E-Syt1 within the cells, it is preferable that the interaction inhibitor has a small molecular weight so that it can be easily introduced into cells.

[0025] When the interaction inhibitor is an antigen-binding fragment, examples thereof include Fab, Fab', F(ab')2, scFab, scFv, diabody, triabody, minibody, nanobody, etc. Any of the antigen-binding fragments can be produced by utilizing gene modification techniques known to those skilled in the art. For example, the antigen-binding fragments may be obtained from transgenic animals having genes capable of producing these antigen-binding fragments. The antigen-binding fragments may be VHH (variable domain of heavy chain of heavy chain antibody) derived from camelized animals, or VNAR (variable domain of new antigen receptor) derived from cartilaginous fish.

[0026] When the interaction inhibitor is a peptide, it may be, for example, a peptide of an interaction site of PKCδ or E-Syt1. As long as it can bind to the interaction site and inhibit the interaction between PKCδ and E-Syt1, the length of the peptide is not particularly limited, but the upper limit may be, for example, 300 amino acids or less, 200 amino acids or less, 100 amino acids or less, 50 amino acids or less, 30 amino acids or less, or 20 amino acids or less, and the lower limit may be, for example, 10 amino acids or more, 20 amino acids or more, 30 amino acids or more, or 50 amino acids or more. In addition, the upper and lower limits of these lengths may be arbitrarily combined, for example, 10 amino acids or more and 100 amino acids or less. For example, a peptide having a part of the amino acid sequence of amino acid numbers 601 to 676 in SEQ ID NO: 1 can be mentioned.

[0027] In addition, when the interaction inhibitor is a compound, it is preferably, for example, a medium molecular weight compound or a low molecular weight compound. A commercially available compound may be used as the compound, but a compound prepared by a method known to those skilled in the art may also be used.

[0028] Furthermore, when the interaction inhibitor is a nucleic acid, examples of the inhibitor include siRNA, miRNA, antisense nucleic acid, etc. that target PKCδ or E-Syt1. Commercially available nucleic acids may be used as the nucleic acid, but nucleic acids prepared by methods known to those skilled in the art may also be used.

[0029] Screening for an interaction inhibitor can be carried out, for example, by a binding assay.

[0030] The cells into which the interaction inhibitor is introduced are preferably hepatoma cells.

[0031] Examples of liver cancer to be treated include, but are not limited to, hepatocellular carcinoma, cholangiocarcinoma, mixed liver cancer, metastatic liver cancer, hepatoblastoma, fibrolamellar HCC, etc. Furthermore, liver cancer is not particularly limited in terms of specific disease site, disease stage, etc. in the liver, and includes any disease site, disease stage, etc.

[0032] The liver cancer to be treated is a type of cancer in which PKCδ interacts with E-Syt1 on the ER in the cells, resulting in the extracellular secretion of PKCδ. This extracellular secretion of PKCδ is preferably associated with an autophagy inducer, such as ATG5, ATG7, ATG16L1, LC3B, p62, etc. In addition, the extracellular secretion of PKCδ is preferably carried out via a pathway involving vesicles expressing SEC22B, in which SEC22B on the vesicles interacts with SNAP23, STX3, STX4, etc. directly beneath the cell membrane, resulting in the extracellular secretion of PKCδ that has been taken up into the vesicles.

[0033] The means for introducing the interaction inhibitor into cells is not particularly limited as long as the interaction inhibitor can be introduced into the target cells, but for example, an introduction agent can be used to introduce the interaction inhibitor into the cells. Examples of the introduction agent include lipid-based agents, such as liposomes. For liver-specific delivery, the agent may be a lipid carrier such as liposomes having a cell surface antigen specifically expressed in liver tissue bound to its surface.

[0034] The liver cancer therapeutic agent of this embodiment may contain other active ingredients and pharmacologically acceptable carriers.

[0035] The content of the interaction inhibitor in the liver cancer therapeutic drug is not particularly limited as long as it can inhibit the interaction between PKCδ and E-Syt1 in cells and treat liver cancer, and may be, for example, 1 ng / mL to 10 μg / mL.

[0036] The liver cancer therapeutic agent of the present invention may be formulated in any dosage form, such as a liquid, suspension, or injection.

[0037] The mode of administration is not particularly limited, but examples include local administration to the affected area or its surroundings by injection or the like, or intravenous or intraarterial injection.

[0038] Examples of other active ingredients include immunostimulants such as cytokines, chemotherapeutic agents, etc. These other active ingredients can be used appropriately in an appropriate amount.

[0039] Examples of pharmacologically acceptable carriers include solvents, distilled water, physiological saline, diluents, surfactants, stabilizers, solubilizers, suspending agents, emulsifiers, buffers, preservatives, etc. Furthermore, additives such as preservatives, antioxidants, colorants, adsorbents, wetting agents, etc. can be used as necessary. These carriers can be used appropriately and in appropriate amounts.

[0040] The subject of administration is a mammalian animal suffering from liver cancer, preferably a human suffering from liver cancer. The liver cancer patient is preferably a liver cancer patient whose blood PKCδ level is significantly increased compared to that of a healthy person. Therefore, before administering the liver cancer therapeutic agent of the present invention, the amount of PKCδ may be measured, and if the amount of PKCδ in blood is significantly increased compared to that of a healthy person, it may be decided to administer the drug.

[0041] The dosage of the hepatic cancer therapeutic agent may be an amount in which the interaction inhibitor, which is an active ingredient, can inhibit the interaction between PKCδ and E-Syt1 in cells. The dosage can be appropriately adjusted depending on the age, sex, weight, symptoms, therapeutic effect, area of ​​the treatment site, administration method, etc. of the subject, but for example, in the case of an average human weighing about 60 kg, the dosage is preferably about 0.01 mg to 5000 mg per day, more preferably about 0.1 mg to 500 mg. The total dosage per day may be a single dosage or a divided dosage.

[0042] Regarding the therapeutic effect, in the case of in vivo analysis, it can be judged that the therapeutic effect has been achieved when it is confirmed that the proliferation of liver cancer cells has been suppressed, the number of liver cancer cells has decreased, the size of liver cancer has decreased, etc., as a result of treatment with a liver cancer therapeutic drug, compared to before treatment with the liver cancer therapeutic drug or compared to a control not treated with the liver cancer therapeutic drug. In this case, the analytical method is not particularly limited, and can be performed by a method known to those skilled in the art.

[0043] In addition, in the case of cell biological analysis, the therapeutic effect of a therapeutic drug in vivo can be predicted by comparing a sample after treatment with a therapeutic drug with a sample before treatment with the therapeutic drug. The cell biological analysis is not particularly limited, but examples thereof include cell proliferation assay, cell mass (spheroid) formation assay, Western blotting, etc., and can be performed by methods known to those skilled in the art.

[0044] Another embodiment of the present invention is a PKCδ secretion inhibitor, which comprises an inhibitor of the interaction between PKCδ and E-Syt1 in cells.

[0045] With regard to PKCδ secretion inhibitors, the descriptions regarding inhibitors of interaction between PKCδ and E-Syt1, the descriptions regarding cells into which the interaction inhibitor is introduced, the descriptions regarding liver cancer, the descriptions regarding the means of introducing into cells, the descriptions regarding the content of the inhibitor of interaction between PKCδ and E-Syt1, the dosage form, the mode of administration, other ingredients, the subjects to be administered, the dosage, etc. can be referenced from the corresponding descriptions in the above liver cancer therapeutic drugs.

[0046] The analysis of the PKCδ secretion inhibitory effect can be determined to have a PKCδ secretion inhibitory effect, for example, when the amount of PKCδ secreted from liver cancer cells is reduced after treatment with a PKCδ secretion inhibitor, as compared to before treatment with the PKCδ secretion inhibitor or compared to a control that was not treated with the PKCδ secretion inhibitor. In this case, the analysis method is not particularly limited, and can be performed by a method known to those skilled in the art.

[0047] Another embodiment of the present invention is an agent for suppressing the proliferation of hepatic cancer cells, comprising an inhibitor of the interaction between intracellular PKCδ and E-Syt1.

[0048] With regard to the proliferation inhibitor of liver cancer cells, the descriptions regarding the inhibitor of the interaction between PKCδ and E-Syt1, the descriptions regarding the cells into which the interaction inhibitor is introduced, the descriptions regarding liver cancer, the descriptions regarding the means of introducing it into cells, the descriptions regarding the content of the inhibitor of the interaction between PKCδ and E-Syt1, the dosage form, the mode of administration, other ingredients, the subjects to be administered, the dosage, etc. can be referenced from the corresponding descriptions in the above liver cancer therapeutic drugs.

[0049] The analysis of the hepatoma cell proliferation inhibitory effect can be determined, for example, when the amount of PKCδ secreted from hepatoma cells is decreased after treatment with a hepatoma cell proliferation inhibitor, as compared to before treatment with the hepatoma cell proliferation inhibitor or compared to a control that was not treated with the hepatoma cell proliferation inhibitor. In this case, the analysis method is not particularly limited, and can be performed by a method known to those skilled in the art. EXAMPLES

[0050] The examples are set forth for purposes of disclosure and are not intended to limit the scope of the invention.

[0051] <Plasmid> pcDNA3.1-mCherry-hLC3B (plasmid no. 40827) and pEGFP-C1-EGFP-ESYT1 (plasmid no. 66830) were purchased from Addgene. BioID2-HA was cloned from MCS-BioID2-HA plasmid 1 (plasmid no. 74224) and a 13×GS linker was attached to the C-terminus of PKCδ to generate a pTRE vector containing PKCδ-13×GS-BioID2. For the preparation of the truncated PKCδ-13×GS-BioID2 plasmid, inverse PCR was performed using the KOD-Plus-mutagenesis kit (TOYOBO) to generate a pTRE vector containing PKCδ (Δ451-676)-13×GS-BioID2 and a pTRE vector containing PKCδ (Δ601-676)-13×GS-BioID2. For C-terminal GFP fusions, PKCδ (WT) and PKCδ (Δ601-676) were grown and cloned into pTRE using the DNA Assembly kit (NEB). For C-terminal HiBiT fusions, the HiBiT sequence (5'-GTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGC-3' (SEQ ID NO: 3)) was grown and cloned into pTRE under license from Promega. Cloning was performed into the TRE-PKCδ-13×GS vector and the pTRE-importinα1-13×GS vector.

[0052] <Cell culture> The human hepatoma cell line HepG2 and the human gastric cancer cell line AGS were obtained from the Japan Research Bioresource Collection (Osaka, Japan) in 2017. HepG2 Tet-On® Advanced cells were purchased from Takara (Shiga, Japan) in 2018. HepG2 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM; Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Sigma). AGS cells were maintained in RPMI1640 (Sigma) supplemented with 10% FBS. HepG2 Tet-On® Advanced cells were maintained in α-MEM (Nacalai, Kyoto, Japan) supplemented with 0.1 mM NEAA, 500 g / mL G418, and 10% Tet system approved FBS (Takara). For the low-nutrient (starvation) condition, cells were washed and resuspended in Earle's Balanced Salt Solution (EBSS; Sigma) as specified. The cells were cultured for 1 h. The cell lines were monitored periodically for mycoplasma (4A Biotech Co.). The cell lines were confirmed by short tandem repeat profiling every 6 months. The cells used in the experiments had been passaged less than 10 times after thawing.

[0053] <Creation of doxycycline-inducible cell lines> Doxycycline-inducible PKCδ(WT)-13×GS-BioID2-HA, PKCδ(Δ451-676)-13×GS-BioID2-HA, PKCδ(Δ601-676)-13×GS-BioID2-HA, PKCδ(WT)-13×GS-EGFP, PKCδ(Δ601-676)-13×GS-EGFP, PKCδ(wt)-13×GS-HiBiT and importin α1-13×GS-HiBiT-containing TRE-inducible HepG2 cells were generated by the following method. HepG2 Tet-On® Advanced cells were cultured in 6-well plates and transfected with 2.5 μg of each pTRE-based plasmid using 5 μL of Lipofectamin 3000 in 250 μL of Opti-MEM. Two days later, the cells were incubated in medium containing 2 μg / mL puromycin and induced for expression with 1 μg / mL doxycycline and confirmed by immunoblotting and confirmed by immunoblotting

[0054] <Knockout via CRISPR / Cas9> The human E-Syt1 gRNA: 5’-GATGCGCAGGAACGGCGCCATGG-3’ (SEQ ID NO: 4) was designed with CRISPRdirect (https: / / crispr.dbcls.jp). The gRNA was cloned into the lentiviral lentiCRISPR v2 vector. The construct was confirmed by DNA sequencing and confirmed by DNA sequencing

[0055] <Immunoprecipitation of biotinylated proteins> Biotinylated proteins were isolated by a method using BioID2 known to those skilled in the art. Specifically, it was as follows. TRE-inducible HepG2 cells containing the BioID2 construct were seeded in two 100 cm 2The cells were cultured in a dish. Biotin (50 μM) and 1 μg / mL doxycycline were reacted for 24 hours before cell lysis. The cells were washed with PBS, collected with a cell scraper, and resuspended in 1 mL of lysis buffer (50 mM Tris-HCl [pH 7.6], 150 mM NaCl, 1 mM PMSF, 1 mM DTT, 10 μg / mL aprotinin, 1 μg / mL leupeptin, 1 μg / mL pepstatin A, and 1% NP-40). After centrifugation at 20,000 g for 15 min, the supernatant was used as the cell lysate for the following procedures. The protein concentration of the cell lysate was measured using a bicinchoninic acid (BCA) protein assay kit (Thermo). The cell lysate was incubated overnight at 4°C with Dynabeads MyOne streptavidin C1 beads (Thermo) that had been prewashed twice with the same buffer. Beads were washed twice with wash buffer 1 (2% SDS), three times with wash buffer 2 (1% TritonX-100, 0.1% deoxycholate, 500 mM NaCl, 1 mM EDTA, 50 mM HEPES, pH 7.5), and three times with wash buffer 3 (50 mM Tris pH 7.4). For mass spectrometry, beads were incubated in 2% sodium deoxycholate. For immunoblotting, beads were incubated in sodium dodecyl sulfate (SDS) sampling buffer and boiled at 100 °C for 10 min.

[0056] <Mass spectrometry> To identify the isolated proteins, liquid chromatography and mass spectrometry two-dimensional image transformation analysis (2DICAL) shotgun proteomic analysis was performed using methods known to those skilled in the art.

[0057] <siRNAによるノックダウン> Knockdown was performed using On-TARGETplus siRNA SMARTpool (Dharmacon GE). The following siRNAs were used: E-Syt-1 (Dharmacon, Catalog #: L-010652-00), PKCδ (Dharmacon, Catalog #: L-003524-00), ATG5 (Dharmacon, Catalog #: L-004374-00), ATG7 (Dharmacon, Catalog #: L-020112-00), ATG16L1 (Dharmacon, Catalog #: L-021033-01), and p62 / SQSTM1 (Dharmacon, Catalog #: L-021033-01). , Catalog #: L-010230-00), LC3B (Dharmacon, Catalog #: L-012846-00), SEC22B (Dharmacon, Catalog #: L-011963-00), SNAP23 (Dharmacon, Catalog #: L-017545-00), STX3 (Dharmacon, Catalog #: L-015401-00), and STX4 (Dharmacon, Catalog #: L-016256-00). Cells were transfected using Lipofectamine RNAiMAX (Thermo, Rockford, IL, USA).

[0058] <Reagents> The reagents used were phorbol 12-myristate 13-acetate (PMA; Abcam, Cambridge, UK), doxycycline (Sigma), LY2109761 (MedChemExpress, NJ, USA), chloroquine (CQ; FUJIFILM, Tokyo, Japan), and bafilomycin A1 (Baf A 1; InvivoGen, San Diego, CA, USA) was used.

[0059] <Preparation of secreted proteins> Cells were maintained under physiological medium conditions (10% FBS). After incubation, culture supernatants (containing secreted proteins) were collected and centrifuged sequentially, first at 300 g for 5 min to remove contaminating cells, and then at 1000 g for 10 min to remove cell debris. Finally, the medium was passed through a Millex 0.22 μm GV filter (Merck Millipore, Massachusetts, USA).

[0060] <Cell-based HiBiT assay> Extracellular localization of HiBiT fusion proteins was assessed using the Nano-Glo HiBiT Extracellular Detection System (Promega) according to the manufacturer's instructions. Tet-On HepG2 cells were plated in 96-well plates and treated with doxycycline for the indicated times. Nano-Glo HiBiT Extracellular Detection reagents were added to all wells and incubated for 5 min before reading on an Infinite 200PRO plate reader (Tecan, Kanagawa, Japan).

[0061] <Cell fractionation and immunoblotting> Membrane fractions were extracted using the Qproteome cell compartment kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions with minor modifications. Membrane fractions included endosomes, membrane compartment organelles such as mitochondria and endoplasmic reticulum (ER), and cell surface proteins. Cell lysates were prepared by harvesting cells and resuspending them in lysis buffer with or without phosphatase inhibitors (10 mM NaF and 1 mM Na3VO4). The supernatant was isolated by centrifugation and used as the cell lysate. Equal amounts of protein were subjected to 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with ESYT1 (ATLAS HPA016858; 1:1,000), HA (Roche 3F10; 1:500), LC3B (MBL 4E12; 1:1,000), PKCδ (Abcam ab181076; 1:2,000), ATG5 (Abcam ab108327; 1:1,000), GAPDH (Sigma MAB374; 1:2,000), and actin (Thermo MS1295; 1:2,000), and the signals were visualized by enhanced chemiluminescence (E The measurements were performed using a fluoroscopy (CL, Thermo Fisher Scientific).

[0062] <Flow cytometry> Cells were dissociated with Accutase (Thermo Fisher Scientific) and pelleted by centrifugation at 500 × g for 5 min at 4 °C. Cell suspensions were incubated with 7-aminoactinomycin D (7-AAD, 1:2,000 dilution; Thermo Fisher Scientific) in phosphate-buffered saline (PBS) containing 0.1% FBS for 30 min at 4 °C. Flow cytometric analysis was performed using a MACS Quant (Miltenyi Biotec, North Rhine-Westphalia, Germany). An experiment was conducted.

[0063] <Immunofluorescence analysis> Cultured cells grown on glass cover slips were fixed with 4% paraformaldehyde for 15 minutes at room temperature. Subsequently, the cells were permeabilized with PBS containing 0.1% Triton X-100 and 1% bovine serum albumin (BSA) for 5 minutes. The cells were washed three times with PBS, and the following antibodies were added and incubated overnight at 4°C: mouse anti-PKCδ monoclonal antibody (BD Bioscience 610398; 1:500), rabbit anti-E-Syt1 monoclonal antibody (ATLAS HPA016858; 1:200), rat anti-HA monoclonal antibody (Roche 3F10; 1:500), rabbit anti-SEC61B monoclonal antibody (CST 14648; 1:200), rabbit anti-Calnexin monoclonal antibody (CST 2679; 1:200), rabbit anti-PDI monoclonal antibody (CST3501; 1:200), and rabbit anti-LC3B monoclonal antibody (MBL 4E12; 1:100). After washing three times with PBS, Alexa Fluor 488-conjugated, Al exa Fluor 594-conjugated secondary antibody, or Alexa Fluor 647-conjugated secondary antibody and 4’,6-diamidino-2-phenylindole (DAPI) for nuclear detection were used to incubate for 30 minutes at room temperature. The cells were observed by confocal laser fluorescence microscopy (LSM880, Zeiss, Germany) or fluorescence microscopy (BZ-X800; Keyence, Osaka, Japan).

[0064] <Duolink In Situ Proximity Ligation Assay (PLA)> Cells were fixed with 4% paraformaldehyde for 15 minutes and permeabilized with 0.1% Triton X-100 for 5 minutes. After blocking, the cells were incubated with the following mouse antibodies: PKCδ (BD The sections were incubated for 30 min with antibodies against the following antibodies: LC3B (MBL 4E12; 1:1,000), importinα1 (BD Bioscience 610485; 1:200), and rabbit antibodies: PKCδ (santacruz sc937; 1:200), E-Syt1 (ATLAS HPA016858; 1:200), Sec61B (CST 14648; 1:200), and SEC22B (Abcam ab181076; 1:200). Fluorescent signals were detected with Duolink In Situ PLA probes according to the manufacturer's instructions and visualized with a Zeiss LSM 880 laser microscope and ImageJ software (National All data were processed for quantification using the National Institutes of Health.

[0065] <Immunoelectron microscopy analysis> Cells were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (PB) for 2 h at room temperature. Dehydration was performed with graded ethanol. Cells were embedded in LR-White (Oken, Tokyo, Japan). Samples were reacted with primary antibodies against PKCδ (BD Bioscience 610398; 1:500) and SEC22B (Abcam ab181076; 1:200), and reacted with anti-mouse IgG (H+L) secondary antibodies conjugated to 10 nm immunogold (bbi solutions, Crumlin, UK) or anti-rabbit IgG (H+L) secondary antibodies conjugated to 20 nm immunogold (bbi solutions). For transmission electron microscopy analysis, sections approximately 70 nm thick were cut with a diamond knife, stained with uranyl acetate and lead citrate solutions, and observed at 100 Kv with a JEOL JEM1400 Plus electron microscope (Tokyo, Japan).

[0066] <Proliferation assay> Cells were cultured in 96-well plates in medium alone, with isotype control IgG, or with anti-PKCδ antibody (santacruz sc937; 100 ng / mL, C-20). 100 μL (5 × 10 3 The cells were cultured at 3 × 100 cells / well. After incubation at 7°C for 24 or 48 h, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (Nacalai Tesque, Tokyo, Japan) was added to each well. After incubation for 1 h, the water-soluble formazan dye, 1-methoxy-5-methylphenazinium, produced by bioreduction in the presence of electron carriers, was measured at 450 nm using an Infinite 200PRO plate reader (Tecan).

[0067] <Spheroid formation assay> Cells were cultured in ultra-low attachment 96-well plates (Cornell) to form spheres. g) with isotype control IgG or with anti-PKCδ antibody (Santa Cruz Antibody) z sc937; 100ng / mL) together with Ab-DeliverIN (OZ Bio After 8 days of treatment, cell growth on the spheroids was observed and measured using a BZ-9000 fluorescence microscope. Spheroids with a major axis of 40 μm or more were randomly observed in multiple fields and counted.

[0068] <Clinical samples> Cancer specimens from HCC patients were obtained at the Jikei University Hospital. The protocol used in this study confirmed the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Ethics Committee of the Jikei University Hospital (Ethics Approval Numbers: 29-038(8654), 29-006(8622)).

[0069] <Statistical analysis> Statistical analysis was performed using Prism 8 software (Graphpad Inc.) using unpaired two-tailed Student's t test or Welch's t test, analysis of variance (ANOVA), and statistical analysis. VA) or Mann-Whitney test.

[0070] Example 1: BioID screening identifies E-Syt1 as a membrane protein that interacts with cytoplasmic PKCδ. Using hepatoma cultured cells that always secrete cytoplasmic proteins under normal culture conditions (Non-Patent Document 7), we explored the interaction between intracellular membrane proteins and secreted cytoplasmic proteins by the proximal-dependent biotinylation enzyme (BioID) method. Based on the fact that PKCδ has high specificity in hepatoma, PKCδ was used as a model for secreted cytoplasmic proteins (Non-Patent Document 5). To obtain biotinylated proteins that interact with cytoplasmic PKCδ, we established a hepatoma HepG2 cell line that stably expresses full-length PKCδ fused with proximal-dependent biotinylation enzyme 2 (BioID2) (Non-Patent Document 8), an improved promiscuous biotin ligase. By confirming active secretion under physiological culture conditions and the inhibitory effect of treatment with phorbol 12-myristate 13-acetate1, we confirmed the similarity of the PKCδ-BioID2 fusion to endogenous PKCδ. Interactome analysis of affinity-purified biotinylated proteins in membrane fractions (including PM and organelles but not nuclei) showed that several ER-associated proteins, such as extended-synaptotagmin 1 (E-Syt1), chloride channel CLIC like 1 (CLCC1), starch binding domain 1 (STBD1), and stromal interaction molecule 1 (STIM1), were highly expressed in both PKCδ-BioID2-expressing clones (A8 and E5) (Fig. 1a). Other proteins identified included a mitochondrial intermembrane protein (mitochondrial antiviral signaling protein: MAVS) and abundant cytoplasmic or nuclear proteins, such as (i) PRKAR2A, (ii) TPD52L2, and (iii) LBR. Among these proteins, E-Syt1, which had the highest abundance, is known to be a tethering factor at the ER-PM contact site together with STIM1. Many of the extended-synaptotagmins, such as E-Syt2 and E-Syt3, are associated with the cell membrane. PKCδ is distributed along the ER membrane, whereas E-Syt1 shows a different distribution, being localized not only at the ER-PM contact site but also in the cytoplasmic ER membrane (Non-Patent Document 9). The localization pattern of E-Syt1 allows cytoplasmic proteins to easily access the ER, and the results of the interactome analysis were verified by experiments such as immunoblotting of streptavidin-purified biotinylated proteins using PKCδ-BioID2-expressing HepG2 cell lines, colocalization analysis of conventional immunofluorescence observation, and proximity ligation assay (PLA) (Fig. 1b-d). To confirm the interaction between ER and PKCδ, we further performed colocalization analysis using Sec61β, an ER marker. Super-resolution imaging by structured illumination microscopy (SIM) revealed that some PKCδ is adjacent to the ER (Fig. 1e). Similar observations were also obtained by PLA analysis, suggesting that PKCδ interacts with the ER membrane (Fig. 1f). Importantly, PKCδ localization in the ER was significantly decreased by E-Syt1 deletion (Fig. 1f), suggesting that PKCδ localization in the ER is dependent on E-Syt1. Furthermore, when gastric cancer cell line AGS, which lacks the ability to secrete PKCδ, was used, colocalization of PKCδ with E-Syt1 or Sec61β was hardly observed (Fig. 1e). These results suggest that E-Syt1-mediated localization of PKCδ in the ER contributes to PKCδ secretion.

[0071] Example 2: E-Syt1 is required for secretion of PKCδ. Next, we investigated the importance of E-Syt1 in PKCδ secretion by E-Syt1 deficiency. Immunoblot analysis showed that the level of PKCδ was obviously decreased in the culture medium of E-Syt1 knockout cells (Fig. 2a), suggesting that E-Syt1 is required for PKCδ secretion. However, it was confirmed that the secretion of α-fetoprotein (AFP), a classical secretory protein specific to hepatocellular carcinoma, was not affected in E-Syt1 knockout cells (Fig. 2a), suggesting that the E-Syt1-mediated secretory pathway is different from the previously known ER-Golgi secretory pathway. To further confirm the secretion of PKCδ, we established a cell-based assay for quantification of extracellular PKCδ using the HiBiT system. A decrease in the chemiluminescence signal was observed in E-Syt1-deficient cells (Fig. 2b). To further investigate the effect of the interaction between PKCδ and E-Syt1 on PKCδ secretion, we created a mutant in which a portion of PKCδ fused to BioID2 was deleted to search for the site of PKCδ required for interaction with E-Syt1 (Fig. 2c). When a mutant in which a portion of the C-terminal side of PKCδ was deleted was used, it was shown that the interaction with E-Syt1 was weakened (Fig. 2d). In HepG2 cells expressing the Δ601-676 mutant (PKCδ represented by SEQ ID NO: 1 in which the amino acid sequence 601 to 676 was deleted), both the colocalization with the ER and the amount detected in the medium were clearly decreased (Fig. 2e-f). From these results, we concluded that the interaction of PKCδ with E-Syt1 in the ER is an important process in PKCδ secretion.

[0072] Example 3: Autophagy-related proteins are utilized for the secretion of PKCδ. The present inventors have previously demonstrated that in hepatoma cells, the secretion of cytoplasmic proteins (such as PKCδ and importin α1) is promoted under nutrient-deficient conditions (Non-Patent Documents 5 and 6). It is also known that E-Syt1 acts as a tethering factor at the ER-PM contact site when autophagosomes are synthesized de novo. Thus, the present inventors hypothesized that the secretion of PKCδ is associated with autophagy. Indeed, when HepG2 cells were cultured in low-nutrient Earle's balanced salt solution, It was shown that when the cells were cultured under on (EBSS) culture conditions, the secretion of PKCδ was significantly increased (Figure 3a). At the same time, it was confirmed that PKCδ was not leaked due to membrane destruction during EBSS culture (Figure 3b). In general, autophagy is known to occur constantly in hepatoma cells under normal culture conditions (Non-Patent Document 10). We previously confirmed that normal cultured HepG2 cells were treated with an autophagy inhibitor (LY2109761) and that the secretion of PKCδ was reduced in a dose-dependent manner. On the other hand, we previously confirmed that other autophagy inhibitors (chloroquine and bafilomycin A1) did not affect the secretion of PKCδ. These results suggest that the mechanism of PKCδ secretion is mediated by common factors of autophagy at a relatively early stage, and branches off from autophagy toward degradation at the middle to late stages. Consistent with this suggestion, the secretion of PKCδ was significantly reduced in cells with knockdown of autophagy inducers (ATG5, ATG7, ATG16L1, p62, and LC3B) (Fig. 3c-d). In HepG2 cells, colocalization of PKCδ and LC3B was observed under both culture conditions with 10% fetal bovine serum (FBS) (Fig. 3e) and EBSS (Fig. 3f-g). We also confirmed that E-Syt1 and LC3B colocalized in EBSS-cultured cells (Fig. 3f), whereas colocalization of PKCδ and LC3B was not observed in AGS cells, which do not secrete PKCδ (Fig. 3h).

[0073] Example 4: PKCδ is SEC22B + Secreted via vesicles> Considering that a series of autophagy pathways are related to membrane trafficking, we previously verified that PKCδ secretion is involved in vesicle trafficking. As a result, PKCδ bound to intracellular membranes was detected by a biochemical protection assay using membrane fractions treated with protease K, which digests membrane-bound surface proteins. Evidence has also been accumulating that autophagosomes are involved in cytoplasmic protein secretion in immune cells. Furthermore, it is known that these secretory autophagosomes retain SEC22B as an R-SNARE on the membrane surface instead of STX17, which is necessary to promote autophagosome-lysosome fusion and cargo degradation (Non-Patent Document 11). Furthermore, it is generally known that SEC22B is a member of the ER-PM contact site. Therefore, we verified whether SEC22B is involved in the secretion of PKCδ by cancer cells. As a result, it was shown that PKCδ secretion was significantly reduced by knocking down SEC22B (Figure 4a), It was suggested that SEC22B is essential for PKCδ secretion. Close interaction between PKCδ and SEC22B was observed around the PM of HepG2 cells, but not in other cells that do not secrete PKCδ (e.g., E-Syt1 knockout HepG2 cells and AGS cells) (Fig. 4b), suggesting that the interaction between PKCδ and SEC22B is essential for PKCδ secretion. To visualize the location information of PKCδ and SEC22B in the cytoplasm, we performed observations using transmission electron microscopy (TEM). In HepG2 cells, SEC22B + It was observed that PKCδ was incorporated into the vesicles (Fig. 4c). + Many vesicles were observed around the PM, which correlated with the results of PLA analysis (Fig. 4c). + It was also observed that vesicles fused with the PM and secreted PKCδ (Fig. 4c). + These results suggest that vesicular trafficking is an important process for fusion to the PM for secretion of cytoplasmic proteins in cancer cells. +To identify factors that interact with vesicles, we performed RNA interference experiments targeting Q-SNAREs on the PM (Non-Patent Document 3). It was shown that when SNAP23, STX3, or STX4 was knocked down in HepG2 cells, secretion of PKCδ was significantly reduced (Figure 4d). In addition, in hepatoma cells, E-Syt1-SEC22B-SNAP23 / STX3,4-min was knocked down, and PKCδ secretion was significantly reduced (Figure 4c). It was confirmed that the secretory pathway can be extended to other cytoplasmic secretory proteins, importin α1 and nucleolin. Furthermore, to examine the specificity of this secretory pathway in vivo, we confirmed it using human liver cancer tissues, and found that the interaction between PKCδ and SEC22B was observed only in the cancer lesions, but not in normal tissues (Figure 4e). From these results, as shown in the schematic diagram in Figure 4f, it was concluded that SEC22B + It has been suggested that vesicles are important organelles for cytoplasmic protein secretion in hepatoma cells.

[0074] <Example 5: Inhibitory effect on HCC cell proliferation by targeting the interaction between PKCδ and E-Syt1> To investigate the effect of cytoplasmic protein secretion on cancer, anti-PKCδ antibody was delivered into cells using DeliverIN transfection reagent as a drug delivery system. In HepG2 cells in which anti-PKCδ antibody (C-20), which recognizes the C-terminus of PKCδ and inhibits PKCδ-ER interaction, was delivered into the cells, the interaction between PKCδ and E-Syt1 was inhibited, and PKCδ secretion was significantly suppressed (Figure 5a-c). In addition, in HepG2 cells treated with Ab alone, intracellular accumulation of anti-PKCδ antibody (C-20) was not observed. On the other hand, when anti-PKCδ antibody, which recognizes the N-terminus of PKCδ, was used, no change in PKCδ secretion was observed. In addition, intracellular delivery of anti-PKCδ antibody (C-20) significantly reduced the proliferation of liver cancer cell lines (HepG2 cells, Hep3B cells, and HuH7 cells), confirming the inhibition of cell proliferation. However, the inhibition of cancer cell proliferation could not be confirmed in AGS cells, which do not secrete PKCδ (Fig. 5d-g), suggesting that the secretion of PKCδ via E-Syt1 is related to cell proliferation. To further evaluate the effect of PKCδ in cancer formation, HepG2 cells were cultured in the presence of anti-PKCδ antibody (C-20) under 3D culture conditions. The results showed that the proliferation of HepG2 cells was not affected by the anti-PKCδ antibody (C-20) under normal culture conditions, and that there was no effect on spheroid formation. On the other hand, when anti-PKCδ antibody (C-20) was delivered intracellularly using DeliverIN, the number of spheroids was significantly reduced, suggesting that the combined use of anti-PKCδ antibody (C-20) and DeliverIN produces an anti-cancer effect (Figure 5h-i). These results suggest that targeting the interaction between PKCδ and E-Syt1 may be useful for the treatment of liver cancer.

Claims

1. A therapeutic agent for liver cancer, comprising an inhibitor of the interaction between PKCδ and E-Syt1 in cells.

2. The liver cancer therapeutic agent according to claim 1 , wherein the interaction inhibitor is an anti-PKCδ antibody or an antigen-binding fragment thereof.

3. The liver cancer therapeutic agent according to claim 1 or 2, wherein the anti-PKCδ antibody or its antigen-binding fragment recognizes an epitope sequence contained in the amino acid sequence of amino acid numbers 601 to 676 in SEQ ID NO: 1 or an amino acid sequence at a position corresponding thereto, or an amino acid sequence having 90% or more identity to said amino acid sequence.

4. The antigen-binding fragment may be Fab, Fab', F(ab') ２ 4. The liver cancer therapeutic agent according to claim 1, which is an scFab, an scFv, a diabody, a triabody, a minibody, or a nanobody.

5. The liver cancer therapeutic agent according to any one of claims 1 to 4, wherein the liver cancer is hepatocellular carcinoma.

6. The liver cancer therapeutic agent according to any one of claims 1 to 5, further comprising an agent for introducing the interaction inhibitor into cells.

7. A PKCδ secretion inhibitor, which comprises an inhibitor of the intracellular interaction between PKCδ and E-Syt1.

8. A proliferation inhibitor for hepatocellular carcinoma cells, comprising an inhibitor of the interaction between PKCδ and E-Syt1 in cells.

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