Antihepatitis B virus agents that target the host factor LIPG

Abstract

An anti-HBV agent according to the present invention contains, as an active ingredient, a substance which binds to LIPG and inhibits the functions of LIPG. The inventors of the present application have found that LIPG is a factor that plays an important role in the adhesion and uptake of HBV, and has a function as a cofactor, which supports the intracellular invasion of HBV independently of NTCP, and also contributes to the proliferation of HBV in cells. The LIPG-binding substance exhibits an anti-HBV action by: binding, when on the surface of hepatocytes, to LIPG to inhibit the cell adhesion and uptake of HBV; and binding, when delivered into the hepatocytes, to LIPG present in cells to inhibit any step in the intracellular proliferation of HBV.

Description

[Technical Field]

[0001] This invention relates to an anti-hepatitis B virus agent that targets the host factor LIPG. [Background technology]

[0002] Hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC), and it is estimated that 250 million people are chronic carriers (Non-Patent Literature 1). HBV is a DNA virus and has a 3.2kb-long incomplete circular double-stranded DNA (relaxed circular genome) in which open reading frames overlap (Non-Patent Literature 2).

[0003] Hepatitis B virus control agents, such as entecavir, adefovir, and tenofovir, have been developed primarily as nucleoside analogs to inhibit the reverse transcription step, with research and development focusing on controlling HBs antigen production and HBV DNA levels. In other words, these are almost entirely classified as reverse transcription step inhibitors of pregenome HBV information, and are responsible for suppressing viral replication after infection is complete. Recently, the development of drugs aimed at inhibiting nucleocapsid formation has also shown promise. As advocated by the WHO, vaccine development is underway to control the spread of infection through mother-to-child transmission as part of the HBV eradication effort, and some success is being achieved. To aim for more proactive infection inhibition in terms of controlling the spread of infection, it was necessary to identify target molecules during viral infection. However, to date, the identification of specific infection receptors for HBV has not been successful.

[0004] In 2012, NTCP(Na) was identified as a specific and functional receptor for HBV. +The taurocholate cotransporting polypeptide (bile acid transporter) was identified (Non-Patent Literature 3), and based on this information, numerous drug candidates that inhibit the system utilizing NTCP as an infection target receptor were immediately investigated, leading to active screening research and development of infection and entry inhibitors (Non-Patent Literature 4-6). Furthermore, very recently, it has been reported that EGFR, as a co-factor of NTCP, enhances susceptibility to HBV infection and promotes its movement from infection into cells (Non-Patent Literature 7). In this study, HBV infection in in vitro cell systems was attenuated even with the administration of the cancer treatment agent gefitinib, which targets EGFR.

[0005] On the other hand, it has been suggested that NTCP is necessary for infection to occur, but not sufficient. It has also been shown that HBV can enter HepaRG cells via a pathway mediated by caveolin-1 (Non-Patent Literature 8), but another study using HepG2-NTCP cells suggested that caveolin-1 does not affect HBV entry (Non-Patent Literature 9), and that entry occurs via clathrin-dependent endocytosis (Non-Patent Literature 10). Although several receptors and cofactors necessary for the establishment of hepatitis C virus (HCV) infection have been identified (Non-Patent Literature 11), the factors necessary for HBV adhesion and entry have not yet been fully elucidated.

[0006] Therefore, Hashimoto et al. established an HC1 cell line in which HBV infection is maintained at a very low rate (about 1 in 3000 cells) and performed single-cell transcriptome analysis (Nx1-Seq) to identify four host factors highly expressed in HBV-infected cells (Non-Patent Literature 12). One of these, endothelial lipase (LIPG; Lipase G, Endothlial Type; NCBI Gene ID: 9388), is a member of the triglyceride lipase family and is mainly expressed in vascular endothelial cells (Non-Patent Literature 13), as well as in tissues such as the liver, kidney, lung, testes, and placenta (Non-Patent Literature 14). LIPG has been reported to contribute to various functions in the body, including the metabolism of high-density lipoprotein (HDL), cytokine expression, lipid formation, and the onset and progression of cancer (Non-Patent Literature 15-18). It has also been reported to contribute to the intracellular uptake of lipoproteins as a bridging molecule between lipoproteins and HSPG (Non-Patent Literature 19).

[0007] However, HBV does not infect cells using only NTCP. While cell entry inhibitors targeting NTCP are being developed for HBV, other infection pathways remain unknown, and corresponding inhibitors have not yet been researched or developed. As a result, HBV can still infect cells freely. There is an urgent need to elucidate the infection pathways and develop inhibitors. In other words, infection occurs even in cells that do not express NTCP, and it is presumed that unknown infection receptors exist. In the case of HCV, several infection receptors such as HSPG, hCD81, DC-SIGN, LDLR, SR-BI, CLDN-1, OCLN, and NPC1L1 have been identified, and this information is contributing to drug development along with the advancement of virology.

[0008] HSPG (Heparan sulfate proteoglycan), which is being discussed as a receptor during HCV infection, may also be utilized in HBV infection and is recognized as a receptor with relatively low affinity rather than a specific receptor (Non-Patent Literature 20). It is possible that molecules associated with HSPG function as the receptor itself. Several candidates have been proposed as the core protein of HSPG, but so far only Glypican-5 has been identified (Non-Patent Literature 21), and its true nature remains unclear. However, the mechanism of HBV cell entry is gradually being elucidated. Since NTCP expression suppression can also be caused by the addition of diurnally fluctuating bile acids (CDCA), elucidating infection routes other than NTCP is important.

[0009] LIPG is one of the molecules identified as functioning to maintain the HBV gene. Kaneko et al., one of the inventors of this application, discovered four host genes involved in HBV maintenance in infected cells and filed a patent application (Patent Document 1). The LIPG gene is among the four genes described. Patent Document 1 confirms the HBV-cccDNA reduction effect of GSK264220A, a known LIPG inhibitor, along with a gene expression repression system using shRNA. However, a LIPG inhibitor applicable to HBV-infected patients has not yet been developed. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] WO 2017 / 082202 A1 [Non-patent literature]

[0011] [Non-Patent Document 1] Thomas, DL., N. Engl. J. Med., 380:2041-2050, 2019 [Non-Patent Document 2] Sukuda, S. Watashi K., Antiviral Res, 104925, 2020 [Non-licensed document 3] Yan, H. et al., eLife, 1: e00049, 2012

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed Document 8

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

Non-licensed Document 15

Non-licensed Document 16

Non-licensed Document 17

Non-licensed Document 18

Non-licensed Document 19

Non-licensed Document 20

Non-licensed Document 21

Non-licensed Document 22

Non-licensed Document 23

Non-licensed Document 24

[0012] Since knockdown of LIPG and LIPG inhibitors significantly reduce the amount of HBV DNA and HBV cccDNA in host cells, LIPG is a promising target for HBV treatment. Therefore, in order to completely eliminate HBV from liver cells and prevent or treat hepatitis B, it is highly desirable to elucidate the HBV maintenance mechanism, including the behavior of cccDNA, and to find a new anti-HBV drug that targets LIPG for HBV treatment. The present invention aims to provide a novel anti-HBV drug that targets LIPG. [Means for solving the problem]

[0013] There have been reports that EL / LIPG and HSPG interact (Non-Patent Document 19), suggesting that LIPG may function as a cofactor involved in infection. When the LIPG gene was identified as a gene necessary for HBV maintenance, a decrease in HBV-DNA / HBV-cccDNA was confirmed in a system where LIPG expression was suppressed using the shRNA method (Patent Document 1). However, subsequent investigations revealed that, conversely, forced expression of LIPG increased the amount of HBV-DNA. From this, we suspected involvement as a receptor and found that it could be discussed as a new receptor candidate. Furthermore, we constructed a screening system for infection inhibitors targeting LIPG and discovered that HBV infection could occur even in cells with extremely low NTCP expression if LIPG was expressed. The possibility that LIPG contributes to HBV infection by exerting enzymatic function has already been confirmed by the inhibitory effect of GSK264220A, but there is also the possibility of functions other than direct enzymatic activity contributing to the infection. Generally, inhibitors that target host factors are required to have few side effects, so having prior information on side effects when inhibiting the target suggests the validity of the inhibitor development process. LIPG (endothelial lipase)-KO mice were reported in 2011, and it was reported that they increased HDL, known as good cholesterol, but other phenotypes were not described (Hara, T. et al., J. Lipid Res., 52: 57-67 (2011)). More recently, mAbs targeting LIPG (endothelial lipase) have been created and are already in the clinical trial stage, but no particular side effects have been reported (LeLay, JE. Et al., Sci. Transl. Med., 13:eabb0602 (2021)). Considering these factors, it is suggested that targeting LIPG is reasonable as it is associated with few serious side effects.

[0014] As a result of diligent research, the inventors of this application have found that LIPG increases HBV adhesion to cells and entry independently of NTCP, that LIPG, HSPG on the cell membrane surface, and HBV particles bind to each other, leading to uptake by hepatocytes via caveolin-dependent endocytosis, and that LIPG increases Caveolin-1 expression, thereby increasing HBV entry in a Caveolin-1-dependent manner. Aiming to develop peptide drugs that can help cure hepatitis B virus, the inventors further diligently researched and developed a polypeptide (LIPG-binding peptide) that can inhibit HBV adhesion to host cells via LIPG by binding to LIPG. Further intensive research revealed, surprisingly, that delivering LIPG-binding peptides, which inhibit the adhesion and invasion of HBV into hepatocytes on the surface of hepatocytes, into hepatocytes resulted in a far stronger anti-HBV effect compared to cases where delivery into hepatocytes was not performed. The present invention, completed through the diligent research described above, encompasses the following aspects.

[0015] [1] An anti-hepatitis B virus agent containing as an active ingredient a substance that binds to LIPG and inhibits the function of LIPG. [2] The anti-hepatitis B virus agent according to [1], wherein the inhibition of LIPG function is at least one selected from inhibition of LIPG binding to N4BP1 and inhibition of LIPG-mediated suppression of N4BP1 RNase activity. [3] The substance is an anti-hepatitis B virus agent according to [1] or [2], which has the effect of inhibiting intracellular entry of hepatitis B virus via LIPG. [4] The anti-hepatitis B virus agent according to [3], wherein the substance binds to the heparin-binding domain of LIPG and inhibits the binding of LIPG and heparan sulfate proteoglycan to hepatitis B virus, or the binding of the LIPG-heparan sulfate proteoglycan complex to hepatitis B virus. [5] The anti-hepatitis B virus agent according to any one of [1] to [4], wherein the substance is at least one polypeptide selected from the polypeptides listed in (1) to (30) below. (1) A polypeptide of the sequence NCRHNTAG (sequence number 25). (2) A polypeptide of the sequence NARHNTAG (sequence number 26). (3) A polypeptide with the sequence LNCRDNTR (sequence number 23). (4) A polypeptide of the sequence LNARDNTR (sequence number 24). (5) A polypeptide of the sequence LNCRDNTRPVMSAMTC (sequence number 1). (6) A polypeptide with the sequence IRNVNHSDH (sequence number 9). (7) A polypeptide with the sequence LNVGYVFYP (sequence number 18). (8) A polypeptide with the sequence SLYTGFRAH (sequence number 15). (9) A polypeptide with the sequence RCRQSWNTM (sequence number 14). (10) Polypeptide of the sequence GADLRRGCC (sequence number 11). (11) A polypeptide of the sequence RKSGGVCLNCRHNTAG (sequence number 21). (12) A polypeptide with the sequence LNCRHNTAGRHCHYCK (sequence number 22). (13) A polypeptide having the amino acid sequence shown in SEQ ID NO: 28. (14) A polypeptide comprising a partial sequence of the polypeptide of (13), the polypeptide comprising the region of amino acids 370-377 in SEQ ID NO: 28. (15) A polypeptide that is 95% or more identical to SEQ ID NO: 28, characterized by an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in a region other than the region of amino acids 370 to 377 in the amino acid sequence shown in SEQ ID NO: 28. (16) A polypeptide comprising a partial sequence of the polypeptide of (15), the polypeptide comprising the region of amino acids 370 to 377. (17) A polypeptide with the sequence LCECRDVLSCYYITDT (sequence number 4). (18) A polypeptide with the sequence CPCVNGATRHRPTSLC (sequence number 8). (19) A polypeptide with the sequence LVPWLRYAY (sequence number 17). (20) A polypeptide of the sequence NXRHNTAG (SEQ ID NO: 75) (where X is an amino acid other than cysteine ​​and alanine). (21) A polypeptide of the sequence LNXRDNTR (sequence number 76) (where X is an amino acid other than cysteine ​​and alanine). (22) A polypeptide of the sequence LNCRHNTAG (sequence number 77). (23) A polypeptide with the sequence SNCRINTFRTVPIEQK (sequence number 78). (24) Polypeptide of the sequence SNCRINTFR (sequence number 79). (25) Polypeptide of the sequence TSNCRINTFR (sequence number 80). (26) A polypeptide having the amino acid sequence shown in SEQ ID NO: 82. (27) A polypeptide comprising a partial sequence of the polypeptide of (26), the polypeptide comprising the region of amino acids 456 to 464 in SEQ ID NO: 82. (28) A polypeptide that has 95% or more identity with SEQ ID NO: 82, represented by an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in a region other than the region of amino acids 456 to 464 in the amino acid sequence shown in SEQ ID NO: 82. (29) A polypeptide comprising a partial sequence of the polypeptide of (28), the polypeptide comprising the region of amino acids 456 to 464. (30) A polypeptide having 80% or more but less than 100% identity with any of (1) to (14) and (17) to (27). [6] The hepatitis B virus agent according to [5], wherein the polypeptide of (15) has 98% or more identity with SEQ ID NO: 28, and the polypeptide of (28) has 98% or more identity with SEQ ID NO: 82. [7] The anti-hepatitis B virus agent according to [5], comprising at least one polypeptide selected from (1) to (13) and (22) to (26) above as an active ingredient. [8] The anti-hepatitis B virus agent according to [5], comprising at least one polypeptide selected from (1) to (12) and (17) to (25) above as an active ingredient. [9] The anti-hepatitis B virus agent according to any one of [5] to [8], wherein the polypeptide is linked to a carrier molecule for delivery into hepatocytes.

[10] The anti-hepatitis B virus agent according to [9], wherein the carrier molecule is an antibody, antibody fragment, or single-chain antibody that binds to an asialoclycoprotein receptor.

[11] The antibody, antibody fragment, or single-chain antibody, A heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NOs. 83, 89, 95, or 101, or an amino acid sequence in which some residues are substituted and which has 80% or more identity with the amino acid sequence, A heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NOs. 84, 90, 96, or 102, or an amino acid sequence in which some residues are substituted and which has 80% or more identity with the amino acid sequence, A heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NOs. 85, 91, 97, or 103, or an amino acid sequence in which some residues are substituted and which has 80% or more identity with the amino acid sequence, A light chain CDR1 comprising the amino acid sequence shown in SEQ ID NOs. 86, 92, 98, or 104, or an amino acid sequence in which some residues are substituted and which has 80% or more identity with the amino acid sequence, A light chain CDR2 comprising the amino acid sequence shown in SEQ ID NOs. 87, 93, 99, or 105, or an amino acid sequence in which some residues are substituted and which has 80% or more identity with the amino acid sequence, A light chain CDR3 containing the amino acid sequence shown in SEQ ID NOs: 88, 94, 100, or 106, or an amino acid sequence in which some residues are substituted and which has 80% or more identity with the amino acid sequence. The anti-hepatitis B virus agent according to

[10] , having the properties of

[10] .

[12] The anti-hepatitis B virus agent according to

[10] or

[11] , wherein the antibody, antibody fragment, or single-chain antibody is linked to the polypeptide via a cleavage sequence that is cleaved by an endogenous enzyme.

[13] The polypeptide is in a form linked to a cell membrane permeability promoting molecule, as described in any one of [5] to

[12] , an anti-hepatitis B virus agent.

[14] The anti-hepatitis B virus agent according to

[13] , wherein the cell membrane permeability promoting molecule is a polypeptide having the amino acid sequence shown in SEQ ID NO: 107 or 108.

[15] The polypeptide is in a form linked to a nuclear localization signal, as described in any one of [5] to

[14] , an anti-hepatitis B virus agent.

[16] Use of at least one polypeptide selected from the polypeptides listed below (1) to (30) as a LIPG-conjugated peptide. (1) A polypeptide of the sequence NCRHNTAG (sequence number 25). (2) A polypeptide of the sequence NARHNTAG (sequence number 26). (3) A polypeptide with the sequence LNCRDNTR (sequence number 23). (4) A polypeptide of the sequence LNARDNTR (sequence number 24). (5) A polypeptide of the sequence LNCRDNTRPVMSAMTC (sequence number 1). (6) A polypeptide with the sequence IRNVNHSDH (sequence number 9). (7) A polypeptide with the sequence LNVGYVFYP (sequence number 18). (8) A polypeptide with the sequence SLYTGFRAH (sequence number 15). (9) A polypeptide with the sequence RCRQSWNTM (sequence number 14). (10) Polypeptide of the sequence GADLRRGCC (sequence number 11). (11) A polypeptide of the sequence RKSGGVCLNCRHNTAG (sequence number 21). (12) A polypeptide with the sequence LNCRHNTAGRHCHYCK (sequence number 22). (13) A polypeptide having the amino acid sequence shown in SEQ ID NO: 28. (14) A polypeptide comprising a partial sequence of the polypeptide of (13), the polypeptide comprising the region of amino acids 370-377 in SEQ ID NO: 28. (15) A polypeptide that is 95% or more identical to SEQ ID NO: 28, characterized by an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in a region other than the region of amino acids 370 to 377 in the amino acid sequence shown in SEQ ID NO: 28. (16) A polypeptide comprising a partial sequence of the polypeptide of (15), the polypeptide comprising the region of amino acids 370 to 377. (17) A polypeptide with the sequence LCECRDVLSCYYITDT (sequence number 4). (18) A polypeptide with the sequence CPCVNGATRHRPTSLC (sequence number 8). (19) A polypeptide with the sequence LVPWLRYAY (sequence number 17). (20) A polypeptide of the sequence NXRHNTAG (SEQ ID NO: 75) (where X is an amino acid other than cysteine ​​and alanine). (21) A polypeptide of the sequence LNXRDNTR (sequence number 76) (where X is an amino acid other than cysteine ​​and alanine). (22) A polypeptide of the sequence LNCRHNTAG (sequence number 77). (23) A polypeptide with the sequence SNCRINTFRTVPIEQK (sequence number 78). (24) Polypeptide of the sequence SNCRINTFR (sequence number 79). (25) Polypeptide of the sequence TSNCRINTFR (sequence number 80). (26) A polypeptide having the amino acid sequence shown in SEQ ID NO: 82. (27) A polypeptide comprising a partial sequence of the polypeptide of (26), the polypeptide comprising the region of amino acids 456 to 464 in SEQ ID NO: 82. (28) A polypeptide that has 95% or more identity with SEQ ID NO: 82, represented by an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in a region other than the region of amino acids 456 to 464 in the amino acid sequence shown in SEQ ID NO: 82. (29) A polypeptide comprising a partial sequence of the polypeptide of (28), the polypeptide comprising the region of amino acids 456 to 464. (30) A polypeptide having 80% or more but less than 100% identity with any of (1) to (14) and (17) to (27).

[17] A LIPG-binding peptide comprising at least one polypeptide selected from the polypeptides listed below (1) to (30). (1) A polypeptide of the sequence NCRHNTAG (sequence number 25). (2) A polypeptide of the sequence NARHNTAG (sequence number 26). (3) A polypeptide with the sequence LNCRDNTR (sequence number 23). (4) A polypeptide of the sequence LNARDNTR (sequence number 24). (5) A polypeptide of the sequence LNCRDNTRPVMSAMTC (sequence number 1). (6) A polypeptide with the sequence IRNVNHSDH (sequence number 9). (7) A polypeptide with the sequence LNVGYVFYP (sequence number 18). (8) A polypeptide with the sequence SLYTGFRAH (sequence number 15). (9) A polypeptide with the sequence RCRQSWNTM (sequence number 14). (10) Polypeptide of the sequence GADLRRGCC (sequence number 11). (11) A polypeptide of the sequence RKSGGVCLNCRHNTAG (sequence number 21). (12) A polypeptide with the sequence LNCRHNTAGRHCHYCK (sequence number 22). (13) A polypeptide having the amino acid sequence shown in SEQ ID NO: 28. (14) A polypeptide comprising a partial sequence of the polypeptide of (13), the polypeptide comprising the region of amino acids 370-377 in SEQ ID NO: 28. (15) A polypeptide that is 95% or more identical to SEQ ID NO: 28, characterized by an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in a region other than the region of amino acids 370 to 377 in the amino acid sequence shown in SEQ ID NO: 28. (16) A polypeptide comprising a partial sequence of the polypeptide of (15), the polypeptide comprising the region of amino acids 370 to 377. (17) A polypeptide with the sequence LCECRDVLSCYYITDT (sequence number 4). (18) A polypeptide with the sequence CPCVNGATRHRPTSLC (sequence number 8). (19) A polypeptide with the sequence LVPWLRYAY (sequence number 17). (20) A polypeptide of the sequence NXRHNTAG (SEQ ID NO: 75) (where X is an amino acid other than cysteine ​​and alanine). (21) A polypeptide of the sequence LNXRDNTR (sequence number 76) (where X is an amino acid other than cysteine ​​and alanine). (22) A polypeptide of the sequence LNCRHNTAG (sequence number 77). (23) A polypeptide with the sequence SNCRINTFRTVPIEQK (sequence number 78). (24) Polypeptide of the sequence SNCRINTFR (sequence number 79). (25) Polypeptide of the sequence TSNCRINTFR (sequence number 80). (26) A polypeptide having the amino acid sequence shown in SEQ ID NO: 82. (27) A polypeptide comprising a partial sequence of the polypeptide of (26), the polypeptide comprising the region of amino acids 456 to 464 in SEQ ID NO: 82. (28) A polypeptide that has 95% or more identity with SEQ ID NO: 82, represented by an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in a region other than the region of amino acids 456 to 464 in the amino acid sequence shown in SEQ ID NO: 82. (29) A polypeptide comprising a partial sequence of the polypeptide of (28), the polypeptide comprising the region of amino acids 456 to 464. (30) A polypeptide having 80% or more but less than 100% identity with any of (1) to (14) and (17) to (27). [Effects of the Invention]

[0016] This invention provides the first anti-HBV agent targeting LIPG. The anti-HBV agent of this invention can reduce intracellular HBV DNA and cccDNA, thus offering the potential for a cure for hepatitis B. Substances that bind to and inhibit LIPG exert anti-HBV activity by inhibiting HBV's intracellular entry via LIPG, even when used without a hepatocyte delivery carrier molecule. However, a stronger anti-HBV effect can be obtained by using a hepatocyte delivery carrier molecule. The polypeptides listed in (1) to (12), (17) to (25), and the short-sized polypeptides in (14), (16), (29), and (30) can be easily prepared by chemical synthesis. Unlike manufacturing using genetic engineering technology, there is no risk of contamination with host cell-derived components, making them advantageous as pharmaceuticals. [Brief explanation of the drawing]

[0017] [Figure 1]Knockdown of LIPG expression negatively regulates the HBV life cycle. (A) qRT-PCR analysis results of LIPG mRNA in HC1 cells transfected with Cont shRNA and HC1 cells transfected with LIPG shRNA. Results were normalized by ACTB values. (B) qPCR analysis results of HBV-DNA in HC1 cells transfected with Cont shRNA and HC1 cells transfected with LIPG shRNA, 7 days after HBV infection. (C) qPCR analysis results of cccDNA in HC1 cells transfected with Cont shRNA and HC1 cells transfected with LIPG shRNA, 7 days after HBV infection. (D) Schematic diagram of the experimental design. (E) qRT-PCR analysis results of LIPG mRNA in PHH cells transfected with Cont shRNA and PHH cells transfected with LIPG shRNA. Results were normalized by ACTB results. (F) qPCR analysis results of HBV-DNA in PHH cells transfected with Cont shRNA and PHH cells transfected with LIPG shRNA (2 weeks after HBV infection). (G) Results of qPCR analysis of cccDNA in PHH cells transfected with Cont shRNA and PHH cells transfected with LIPG shRNA two weeks after HBV infection. (H) Results of qRT-PCR analysis of pgRNA transcripts in PHH cells transfected with Cont shRNA and PHH cells transfected with LIPG shRNA two weeks after HBV infection. Results were normalized by ACTB results. These data represent the mean ± SEM of three independent experiments. Statistical testing was performed using unpaired two-sided t-tests. ****p<0.0001, ***p<0.001, **p<0.01. [Figure 2]LIPG positively regulates the HBV life cycle in HepG2-NTCP cells. (A) qRT-PCR analysis of LIPG mRNA in HepG2, HepG2-NTCP-C4, HepG2.2.15, Huh1, Huh6, Huh7, T5B, HC1, and PHH cells. Results were normalized by ACTB results. (B) Immunoblot analysis of FLAG-tagged LIPG and β-actin in HepG2-NTCP-C4 cells (Control, HepG2-NTCP-C4 cells with empty vector) and HepG2-NTCP-C4-LIPG cells (LIPG-FLAG). (C) Schematic diagram of the experimental design. (D) qPCR analysis of HBV-DNA 3 hours after inoculation with HBV at 4°C in the presence or absence of heparin and heparanase in HepG2-NTCP-C4 cells (Cont) and HepG2-NTCP-C4-LIPG cells (LIPG). (E) qPCR analysis of HBV-DNA 12 hours after re-culturing at 37°C in HepG2-NTCP-C4 cells and HepG2-NTCP-C4-LIPG cells. (F) qPCR analysis of HBV-DNA 3 days after re-culturing at 37°C in HepG2-NTCP-C4 cells and HepG2-NTCP-C4-LIPG cells. (G) qPCR analysis of cccDNA 3 days after re-culturing at 37°C in HepG2-NTCP-C4 cells and HepG2-NTCP-C4-LIPG cells. (H) In HepG2-NTCP-C4 cells and LIPG-overexpressing HepG2-NTCP-C4 cells, HBV-DNA was analyzed by qPCR 3 hours after inoculation with HBV at 4°C in the presence of a LIPG inhibitor. (I) qPCR analysis of HBV-DNA 12 hours after re-culturing HepG2-NTCP-C4 cells and LIPG-overexpressing HepG2-NTCP-C4 cells at 37°C. These data show the mean ± SEM of three independent experiments (A, F, G, H, I) or two independent experiments (D, E). Statistical testing was performed using two-way ANOVA and Tukey's multiple comparison test. ****p<0.0001, ***p<0.001, *p<0.05, no significant difference (ns). [Figure 3]Knockdown of LIPG reduces HBV attachment, uptake, and replication in HepG2-NTCP-C4 cells. (A) qRT-PCR analysis of LIPG mRNA in HepG2-NTCP-C4 cells transfected with Cont shRNA and HepG2-NTCP-C4 cells transfected with LIPG shRNA. Results were normalized by ACTB results. (B) qPCR analysis of HBV-DNA was performed in HepG2-NTCP-C4 cells transfected with Cont shRNA and HepG2-NTCP-C4 cells transfected with LIPG shRNA 3 hours after inoculation with HBV at 4°C. (C) qPCR analysis of HBV-DNA in HepG2-NTCP-C4 cells transfected with Cont shRNA and HepG2-NTCP-C4 cells transfected with LIPG shRNA after re-culturing at 37°C. (D) qPCR analysis of HBV-DNA was performed 3 days after re-culturing at 37°C in HepG2-NTCP-C4 cells transfected with Cont shRNA and HepG2-NTCP-C4 cells transfected with LIPG shRNA. (E) qPCR analysis of cccDNA 3 days after re-culturing at 37°C in HepG2-NTCP-C4 cells transfected with Cont shRNA and HepG2-NTCP-C4 cells transfected with LIPG shRNA. These data represent the mean ± SEM of two or three independent experiments. Statistical testing was performed using unpaired two-sided t-tests. ***p<0.001, *p<0.05. [Figure 4]RD cells do not support HBV uptake and replication. (A) Immunoblot analysis of LIPG and β-actin in HBV-infected Huh7 cells (Huh7-Control + HBV), HBV-infected Huh7-LIPG cells (Huh7-LIPG-FLAG + HBV), HBV-uninoculated RD cells (RD), and HBV-inoculated RD cells (RD + HBV). (B) qPCR analysis of HBV-DNA in HBV-infected Huh7 cells, HBV-infected Huh7-LIPG cells, HBV-uninoculated RD cells, and HBV-inoculated RD cells 12 hours after HBV inoculation. (C) qPCR analysis of cccDNA in HBV-infected Huh7 cells, HBV-infected Huh7-LIPG cells, HBV-uninoculated RD cells, and HBV-inoculated RD cells 3 days after HBV inoculation. These data represent the mean ± SEM of three independent experiments. [Figure 5]LIPG supports the HBV life cycle in Huh7 cells in an NTCP-independent manner. (A) Immunoblot analysis of Flag-tagged LIPG and β-actin in Huh7 cells (Control), Huh7-LIPG cells (LIPG-FLAG), HepG2 cells (Control), HepG2-LIPG cells (LIPG-FLAG), HepG2-NTCP-C4 cells (Control), and HepG2-NTCP-C4-LIPG cells (LIPG-FLAG). (B) qPCR analysis of cccDNA in Huh7 cells (Cont) and Huh7-LIPG cells (LIPG) 24 hours and 72 hours after HBV infection. (C) qPCR analysis of cccDNA in HepG2 cells (Cont) and HepG2-LIPG cells (LIPG) 24 hours and 72 hours after HBV infection. (D) qPCR analysis of cccDNA in HepG2-NTCP-C4 cells (Cont) and HepG2-NTCP-C4-LIPG cells (LIPG) 24 and 72 hours after HBV infection. (E) qRT-PCR analysis of LIPG mRNA in Huh7, Huh7-LIPG, and HepG2-NTCP-C4 cells. Results were normalized by 18S rRNA values. (F) Immunoblot analysis of NTCP and β-actin in Huh7-WT and Huh7-NTCP-KO cells (under control or LIPG overexpression). (G) qPCR analysis of HBV-DNA in Huh7-WT and Huh7-NTCP-KO cells (under control or LIPG overexpression, 3 hours after HBV inoculation at 4°C). (H) qPCR analysis of HBV-DNA in Huh7-WT and Huh7-NTCP-KO cells (12 hours after re-incubation at 37°C under control or LIPG overexpression). (I) qPCR analysis of HBV-DNA in Huh7-WT and Huh7-NTCP-KO cells (3 days after re-incubation at 37°C under control or LIPG overexpression). (J) qPCR analysis of cccDNA in Huh7-WT and Huh7-NTCP-KO cells (3 days after re-incubation at 37°C under control or LIPG overexpression).(K) In Huh7-WT and Huh7-NTCP-KO cells, pgRNA transcription was analyzed by qRT-PCR 3 days after re-culturing at 37°C under control or LIPG overexpression. Results were normalized by ACTB results. These data represent the mean ± SEM of three independent experiments (G, I, J, K) or two independent experiments (B, C, D, H). Statistical tests were performed using two-way ANOVA and Tukey's multiple comparison test. *p<0.05, no significant difference (ns). [Figure 6]LIPG contributes to caveolae-dependent HBV uptake. (A) Immunoblot analysis of Flag-LIPG, Caveolin-1, and β-actin in Huh7 cells (Control) and Huh7-LIPG cells (LIPG-FLAG). (B) Immunoblot analysis of Caveolin-1 and β-actin in Huh7-Cont cells and Huh7-LIPG cells. (C) Analysis of HBV-DNA in Huh7 cells (Cont) and Huh7-LIPG cells (LIPG) by qPCR. (D) qPCR analysis of HBV-DNA in Huh7 cells (Cont) and Huh7-LIPG cells (LIPG) 12 hours after re-culturing at 37°C under Cont siRNA transfection or LIPG siRNA transfection. (E) qPCR analysis of cccDNA in Huh7 cells (Cont) and Huh7-LIPG cells (LIPG) 3 days after re-culturing at 37°C, with and without Cont siRNA. (F) qPCR analysis of cccDNA in HBV-infected Huh7-LIPG cells 3 days after chlorpromazine (CPZ) treatment. (G) qPCR analysis of cccDNA in HBV-infected Huh7-LIPG cells 3 days after 5-(N-ethyl-N-isopropyl)amyloride (EIPA) treatment. (H) qPCR analysis of cccDNA in HBV-infected Huh7-LIPG cells 3 days after LIPG inhibitor treatment. These data represent the mean ± SEM of two or three independent experiments. Statistical tests were performed using two-way ANOVA and Tukey's multiple comparison tests (C, D, E), or unpaired two-sided t-tests (F, G, H). ***p<0.001, **p<0.01, *p<0.05, no significant difference (ns). [Figure 7]LIPG enhances Vimentin expression. (A) Immunoblotting analysis of FLAG-LIPG, Vimentin, and β-actin in Huh7 cells (Control) and Huh7-LIPG cells (LIPG-FLAG). (B) Immunoblotting analysis of FLAG (LIPG), Vimentin, Pan-cadherin, E-cadherin, and β-actin in Huh7 cells (Control), Huh7-LIPG cells (LIPG-FLAG), HepG2 cells (Control), HepG2-LIPG cells (LIPG-FLAG), HepG2-NTCP-C4 cells (Control), and HepG2-NTCP-C4-LIPG cells (LIPG-FLAG). [Figure 8] LIPG does not affect clathrin-dependent viral endocytosis. (A) GLuc activity (multiple indications (MOI) 1.0) in Huh7 cells (Cont) and Huh7-LIPG cells (LIPG) 3 days after HCV infection. (B) qRT-PCR analysis of EMCV in Huh7 cells (Cont) and Huh7-LIPG cells (LIPG) 12 hours after EMCV infection (MOI 1.0). Results were normalized by ACTB values. (C) Immunoblotting analysis of LIPG, Influenza A H1N1, Influenza A nucleoprotein (NP), and β-actin in Huh7 cells (Cont) and Huh7-LIPG cells (LIPG) 12 hours after flu infection (MOI 1.0). These data show the mean ± SEM of three independent experiments. Statistical tests were performed using unpaired two-sided t-tests. ****p<0.0001, no statistically significant difference (ns). [Figure 9] Design of a DNA library for IVV screening. Starting from the 5' upstream, the RNA polymerase promoter SP6 and the 5' untranslated (UTR) region containing a portion of the tobacco mosaic virus omega sequence as a translation enhancer were followed by the addition of green fluorescent protein (GFP). The peptide portion consisted of a random peptide sequence corresponding to 16 or 9 amino acids. Flag tags, his tags, and a tail were further added. [Figure 10] Schematic diagram of the binding of endothelial lipase LIPG, heparan sulfate proteoglycan, and HBV virus. This study revealed that endothelial lipase LIPG and heparan sulfate proteoglycan (HSPG) play an important role in HBV entry. It is also known that LIPG and HSPG bind to each other, and that LIPG is homodimer bound to heparin (Gastroenterology 2014; 147: 48, and PLoS ONE, 2013, 8(3): e55716). It is thought that some peptides that bind to the LIPG-HSPG binding region may have activity that inhibits HBV entry. [Figure 11] Design of biotinylated LIPG as a bait. Two heparin-binding domains of LIPG were linked in series, and a biotinylated sequence was added at the end via a flexible glycine-serine linker. [Figure 12] Expression vector construction for the biotinylated LIPG heparin binding site. After constructing a vector by adding a biotinylated sequence, a Flag-tag sequence, and a His-tag sequence to the full-length LIPG gene, Fragment 1, Fragment 2, Fragment 3, and Fragment 4 were created using this vector as material. Finally, the complete bait gene was constructed using these four fragments by in-fusion cloning. [Figure 13] Schematic diagram of the peptide selection experiment for LIPG binding. After binding the LIPG dimer of the heparin-binding region to the sensor chip SA, the IVV of a random peptide was bound to it. Unbound peptides were thoroughly washed with buffer, and then heparin was added to competitively elute the LIPG-binding peptides. The recovered IVV of the random peptide was recovered as DNA by RT-PCR and sent to the next round. The amino acid sequence of the random peptide was determined by sequencing the DNA from the final round. [Figure 14] Sensorgrams of via cores in a peptide selection experiment for LIPG. The sensorgrams of via cores from rounds 1 through 4 of the LIPG peptide selection experiment are presented as a single figure. [Figure 15]PCR cycles and DNA band intensity in RT-PCR for peptide selection experiments involving LIPG binding. 5 μl of PCR product was subjected to 1.3% agarose gel electrophoresis, and the PCR cycle was determined by measuring ethidium bromide absorption using a molecular imager FX (Bio-rad). [Figure 16] A pull-down assay of LIPH4 peptide against LIPG was performed. Full-length LIPG (500 amino acids) was used as the bait for the pull-down assay. A plasmid was created by adding a secretion signal to the LIPG-binding peptide and transfecting 293T cells. The culture supernatant was directly added to the bait, the peptide was pulled down, and the results were compared with the control without the bait. [Figure 17] Anti-HBV activity of LIPG-binding peptides in HepG2-NTCP-C4 cells. A: Assay time schedule. B: Effect of LIPG-binding peptides on HBV-DNA copy number. C: Effect of LIPG-binding peptides on cccDNA copy number. [Figure 18] Anti-HBV activity of LIPG-binding peptides in PXB cells. A: Effect of LIPG-binding peptides on the copy number of HBV-DNA. B: Effect of LIPG-binding peptides on the copy number of cccDNA. [Figure 19] Anti-HBV activity of LIPG-binding peptides in PXB cells. A: Assay time schedule. B: Effect of LIPH4-23s on HBV-DNA copy number. C: Effect of LIPH4-23s on cccDNA copy number. D: Effect of LIPH4-NTNBs on HBV-DNA copy number. E: Effect of LIPH4-NTNBs on cccDNA copy number. [Figure 20] The copy number ratios of two LIPG-binding peptides, LIPH4-NTNBsA and LIPH4-NTNBs, at different concentrations of HBV-DNA (A) and cccDNA (B) in PXB cells. The amino acid sequence of LIPH4-NTNBsA is the same as that of LIPH4-NTNBs, but with the second cysteine ​​replaced by alanine. [Figure 21]Anti-HBV activity of full-length Netrin-1 in PXB cells and the effects of Netrin-1 gene knockdown using shRNA on HBV. A: Effect of Netrin-1 on the copy number of HBV-DNA. B: Effect of Netrin-1 on the copy number of cccDNA. C: Effect of Netrin-1 gene knockdown using shRNA on the copy number of HBV-DNA. D: Effect of Netrin-1 gene knockdown using shRNA on the copy number of cccDNA. [Figure 22-1] The effects of LIPG on promoting HBV cell entry and LIPG-binding peptides and Netrin-1 on inhibiting HBV cell entry. A: PXB cells were cultured for 3 hours with purified FLAG-LIPG, then cultured for another 30 minutes with TMR (tetramethylrhodamine)-labeled preS1 peptide, and observed using a confocal microscope. The fluorescence of the preS1 peptide was detected and standardized using the fluorescence intensity of DAPI. *p<0.05. B: PXB cells were treated with 100nM LIPG-binding peptide LIPH4-23S or LIPH4-NTNBS for 15 hours, then cultured for 3 hours with purified FLAG-LIPG. Subsequently, the cells were cultured for another 30 minutes with TMR-labeled preS1 peptide, and observed using a confocal microscope. The fluorescence of the preS1 peptide was detected and standardized using the fluorescence intensity of DAPI. *p<0.05. C: PXB cells were cultured with Netrin-1 for 3 hours, then TMR-fluorescently labeled preS1 peptide was added and cultured for a further 30 minutes. The cells were then observed using a confocal microscope. The fluorescence of the preS1 peptide was detected and normalized using the fluorescence intensity of DAPI. *p<0.05. D: PXB cells were cultured with Netrin-1 for 3 hours, then FLAG-LIPG was added and cultured for a further 3 hours. Subsequently, TMR-fluorescently labeled preS1 peptide was added and cultured for a further 30 minutes. The cells were then observed using a confocal microscope. The fluorescence of the preS1 peptide was detected and normalized using the fluorescence intensity of DAPI. [Figure 22-2]The study investigated the promotion of HBV cell entry by LIPG and the inhibitory effects of LIPG-binding peptides and Netrin-1 on HBV cell entry. E: Biotin-labeled heparan sulfate was added to streptavidin-coated 96-well plates and allowed to bind by standing overnight at 4°C. After washing the plates with TBS, FLAG-LIPG was added to 10, 25, and 50 fmol per well and reacted at room temperature for 2 hours. After washing, LIPG-binding peptides LIPH4-NTNBS, LIPH4-23S, LIPH4-NTNBSA, and LIPH4-23SA were added to 10 or 100 nmol per well and treated at room temperature for 1 hour, followed by washing. 0.1 nmol of preS1-TMR was then added to each well and treated at room temperature for another hour. After washing these plates, the fluorescence of TMR was measured using a plate reader. [Figure 23] Copy number of HBV-DNA in the liver of HBV-infected PXB mice administered the test substance (LIPH4-NTNBs). [Figure 24] Copy number of cccDNA in the liver of HBV-infected PXB mice administered the test substance (LIPH4-NTNBs). [Figure 25] Histopathological examination (HBsAg immunostaining) of liver tissue from HBV-infected PXB mice administered with the test substance (LIPH4-NTNBs). A: LIPH4-NTNBs 0.1 mg, B: control (PBS). [Figure 26] Histopathological examination (HBcAg immunostaining) of liver tissue from HBV-infected PXB mice administered with the test substance (LIPH4-NTNBs). A: LIPH4-NTNBs 0.1 mg, B: control (PBS). [Figure 27] Design of biotinylated asialocryprotein receptors. [Figure 28] Cloning sequences of the extracellular domains of ASGR1 and ASGR2, respectively. [Figure 29] A construct for a biotinylated asialocrycoprotein receptor. [Figure 30]Antibody structure. (Left) Basic structure of an IgG antibody. It consists of two H chains and two L chains linked by disulfide bonds, and is divided into a variable region (VH, VL) which is the antigen-binding site, and a constant region (CH, CL) which is the rest of the antibody. The Fc region of the constant region is the Fc receptor binding site. (Right) Complementarity determining region (CDR) located in the variable region. The rest of the region is the framework region. [Figure 31] Structure of a single-chain antibody (scFv). The C-terminus of the VH chain and the N-terminus of the VL chain are linked via a peptide linker. [Figure 32] A scheme for creating a cDNA library of mouse single-chain antibodies. [Figure 33] A scheme for creating a cDNA library of human single-chain antibodies. [Figure 34] Selection experiment of single-chain antibodies that bind to ASGR using the IVV method. [Figure 35] Pull-down assay using ASGR-conjugated single-chain antibody (1) [Figure 36] Pull-down assay using ASGR-conjugated single-chain antibody (2) [Figure 37] The hypothesized intracellular mechanism of the fusion of a LIPG-binding peptide and an anti-asiaroglycoprotein receptor antibody. A possible intracellular binding partner for LIPG is, for example, N4BP1. [Figure 38] A construct of a fusion of a LIPG-binding peptide and an anti-asialoclycoprotein receptor antibody. [Figure 39] Comparison of the sequences of LIPG-binding peptides used to create fusions with anti-asialocrypoprotein receptor antibodies. All peptides contain NCRXNT (SEQ ID NO: 136, X = D, H, or I). [Figure 40] Pull-down assay for LIPG using N4BP1 [Figure 41A] Anti-HBV activity of intracellularly delivered LIPG-binding peptides in PXB cells. Assay time schedule. [Figure 41B]Anti-HBV activity of intracellularly delivered LIPG-binding peptides in PXB cells. Effects of 10M-L23N, 10M-L23SN, 10M-L23SN-S39, and 10M-LNTSN on the copy number of HBV-DNA. [Figure 41C] Anti-HBV activity of intracellularly delivered LIPG-binding peptides in PXB cells. Effects of 10M-L23N, 10M-L23SN, 10M-L23SN-S39, and 10M-LNTSN on the copy number of cccDNA. [Figure 42] Anti-HBV activity of intrahepatic delivery LIPG-binding peptides in PXB cells (evaluated in the presence of 4% PEG). (A) Assay time schedule. (B) Effects of intrahepatic delivery LIPG-binding peptides 10M-LN4SN, 10M-LN4N, 10M-LLNTSN, and 10M-LLNTSN-S39 on HBV-DNA copy number. (C) Effects of intrahepatic delivery LIPG-binding peptides 10M-LN4SN, 10M-LN4N, 10M-LLNTSN, and 10M-LLNTSN-S39 on cccDNA copy number. [Figure 43] Inhibition of N4BP1 RNase activity by LIPG and rescue of that inhibition by LIPH-NTNBs. [Figure 44] Sample administration schedule in human liver cell chimeric mouse experiments. [Figure 45] Mouse weight changes. Comparison of 10M-LNTSN and control (PBS). [Figure 46] Measurement of HBsAg in the blood. Comparison of 10M-LNTSN and control (PBS). **p<0.01. [Figure 47] Measurement of HBeAg in the blood. Comparison of 10M-LNTSN with control (PBS). *p<0.05, **p<0.01. [Figure 48] Measurement of HBcrAg in the blood. Comparison of 10M-LNTSN and control (PBS). [Figure 49] Measurement of HBV DNA in blood. Comparison of 10M-LNTSN and control (PBS). [Figure 50]Measurement of hAlb in the blood. Comparison of 10M-LNTSN and control (PBS). [Figure 51] Measurement of ALT levels in the blood. Comparison of 10M-LNTSN and control (PBS). [Figure 52] Measurement of HBV-DNA in the liver. Comparison of 10M-LNTSN with control (PBS). †p<0.1. [Figure 53] Measurement of cccDNA in the liver. Comparison of 10M-LNTSN and control (PBS). †p<0.1. [Modes for carrying out the invention]

[0018] In this invention, the term "anti-hepatitis B virus (anti-HBV)" encompasses the treatment of HBV infection, prevention of HBV infection, inhibition of HBV proliferation, treatment of hepatitis B, and prevention of hepatitis B. The anti-HBV agent of this invention, when administered to a patient with hepatitis B, can suppress the proliferation of HBV in the patient's body (liver) and treat hepatitis B. Furthermore, by administering the anti-HBV agent to an HBV carrier before the onset of hepatitis B, the proliferation of HBV in the carrier can be prevented, thereby preventing the onset of hepatitis B (prevention of hepatitis B). In other words, the re-entry (reinfection) of HBV particles into the cell after they have been secreted extracellularly from the liver cells of the patient or HBV carrier is inhibited by the anti-HBV agent of this invention, so the proliferation of HBV DNA in the patient or carrier's body is suppressed, resulting in therapeutic effects and preventive effects against hepatitis B.

[0019] The anti-HBV agent of the present invention contains as an active ingredient a substance that binds to LIPG and inhibits the function of LIPG (hereinafter sometimes referred to as a LIPG-binding substance; in particular, if the substance is a polypeptide, it may be referred to as a LIPG-binding peptide). The inventors of the present invention have revealed that LIPG is a factor that plays an important role in HBV adhesion and uptake, and that it functions as a cofactor that supports intracellular entry of HBV independently of NTCP, as well as contributing to HBV proliferation inside cells. The LIPG-binding substance exerts its anti-HBV effect by binding to LIPG on the surface of hepatocytes to inhibit cell adhesion and uptake of HBV, and when delivered into hepatocytes, by binding to LIPG present inside the cell and inhibiting one of the steps of intracellular proliferation of HBV. Focusing on the inhibition of cell adhesion and uptake, LIPG-binding substances can be described as substances that inhibit HBV's intracellular entry via LIPG by binding to LIPG. For example, such substances bind to the heparin-binding domain of LIPG and inhibit the binding of LIPG, HSPG, or the LIPG-HSPG complex to HBV. LIPG has two isoforms, but the heparin-binding domain is the region from amino acids 312 to 340 in the amino acid sequence of LIPG isoform 1 shown in SEQ ID NO. 30 (NCBI Accession No. NP_006024.1), and the region from amino acids 238 to 266 in the amino acid sequence of LIPG isoform 2 shown in SEQ ID NO. 138 (NCBI Accession No. NP_001294935.1). It was known that LIPG has the function of maintaining HBV genomic DNA and cccDNA in host cells (Patent Document 1), but it was completely unknown at which step of the HBV infection process LIPG functions and how. The inventors of this application were the first to demonstrate that LIPG, HSPG, and HBV particles bind to each other (Figure 10), and that substances that bind to the heparin-binding domain of LIPG can inhibit the binding of LIPG, HSPG, or the LIPG-HSPG complex to HBV, thereby inhibiting the entry of HBV into host cells and suppressing the proliferation of HBV within host cells (in the bodies of HBV-infected patients, especially in the liver). Embodiments of inhibiting the binding of LIPG, HSPG, and HBV on the cell surface (formation of a complex of the three) include inhibition of binding between LIPG and HBV, inhibition of binding between HSPG and HBV, and inhibition of binding between the LIPG-HSPG complex and HBV; however, the substance used as the active ingredient in this application may be any of these embodiments.

[0020] Examples of substances that bind to the heparin-binding domain of LIPG and inhibit the binding of LIPG, HSPG, or the LIPG-HSPG complex to HBV include antibodies that recognize and bind to the heparin-binding domain of LIPG, or their antigen-binding fragments or aptamers, and polypeptides that selectively bind to the heparin-binding domain of LIPG.

[0021] The inhibition of LIPG function by a LIPG-binding substance within hepatocytes may be at least one selected from, for example, inhibition of LIPG binding to N4BP1 and inhibition of LIPG-mediated suppression of N4BP1 RNase activity (see Example 20 below). The present inventors have also revealed for the first time that HBV proliferation is suppressed by LIPG inhibition within hepatocytes.

[0022] The anti-HBV agent of the present invention may contain, for example, at least one polypeptide (LIPG-bound peptide) selected from the polypeptides listed in (1) to (30) below as an active ingredient. The names in brackets [] are the names of the polypeptides used in the following examples. (1) Polypeptide of the sequence NCRHNTAG (sequence number 25). [LIPH4-NTNBs] (2) Polypeptide of the sequence NARHNTAG (sequence number 26). [LIPH4-NTNBsA] (3) Polypeptide of the sequence LNCRDNTR (sequence number 23). [LIPH4-23s] (4) Polypeptide of the sequence LNARDNTR (sequence number 24). [LIPH4-23SA] (5) Polypeptide of the sequence LNCRDNTRPVMSAMTC (sequence number 1). [LIPH4-23] (6) Polypeptide of the sequence IRNVNHSDH (sequence number 9). [LIPH4-74] (7) Polypeptide of the sequence LNVGYVFYP (sequence number 18). [LIPH4-15] (8) Polypeptide of the sequence SLYTGFRAH (sequence number 15). [LIPH4-2] (9) Polypeptide of the sequence RCRQSWNTM (sequence number 14). [LIPH4-14] (10) Polypeptide of the sequence GADLRRGCC (sequence number 11). [LIPH4-87] (11) Polypeptide of the sequence RKSGGVCLNCRHNTAG (sequence number 21). [LIPH4-NTNA] (12) Polypeptide of the sequence LNCRHNTAGRHCHYCK (sequence number 22). [LIPH4-NTNB] (13) A polypeptide having the amino acid sequence shown in SEQ ID NO: 28. (14) A polypeptide comprising a partial sequence of the polypeptide of (13), the polypeptide comprising the region of amino acids 370-377 in SEQ ID NO: 28. (15) A polypeptide that is 95% or more identical to SEQ ID NO: 28, characterized by an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in a region other than the region of amino acids 370 to 377 in the amino acid sequence shown in SEQ ID NO: 28. (16) A polypeptide comprising a partial sequence of the polypeptide of (15), the polypeptide comprising the region of amino acids 370 to 377. (17) Polypeptide of the sequence LCECRDVLSCYYITDT (sequence number 4). [LIPH4-90] (18) Polypeptide of the sequence CPCVNGATRHRPTSLC (sequence number 8). [LIPH4-16] (19) Polypeptide of the sequence LVPWLRYAY (sequence number 17). [LIPH4-12] (20) A polypeptide of the sequence NXRHNTAG (SEQ ID NO: 75) (where X is an amino acid other than cysteine ​​and alanine). (21) A polypeptide of the sequence LNXRDNTR (sequence number 76) (where X is an amino acid other than cysteine ​​and alanine). (22) Polypeptide of the sequence LNCRHNTAG (sequence number 77). [LIPH4-LNTNBs] (23) Polypeptide of the sequence SNCRINTFRTVPIEQK (sequence number 78). [LIPH4-N4BP1a] (24) Polypeptide of the sequence SNCRINTFR (sequence number 79). [LIPH4-N4BP1b] (25) Polypeptide of the sequence TSNCRINTFR (sequence number 80). [LIPH4-N4BP1c] (26) A polypeptide having the amino acid sequence shown in SEQ ID NO: 82. (27) A polypeptide comprising a partial sequence of the polypeptide of (26), the polypeptide comprising the region of amino acids 456 to 464 in SEQ ID NO: 82. (28) A polypeptide that has 95% or more identity with SEQ ID NO: 82, represented by an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in a region other than the region of amino acids 456 to 464 in the amino acid sequence shown in SEQ ID NO: 82. (29) A polypeptide comprising a partial sequence of the polypeptide of (28), the polypeptide comprising the region of amino acids 456 to 464. (30) A polypeptide having 80% or more but less than 100% identity with any of (1) to (14) and (17) to (27).

[0023] Polypeptides (5)-(10) and (17)-(19) were identified in the following examples as peptides that bind to LIPG by the IVV method, and their binding to LIPG was positively confirmed by a pull-down assay using full-length LIPG as bait (Figure 16). All of these polypeptides have the effect of reducing the copy number of HBV DNA and HBV cccDNA in HepG2-NTCP-C4 cells or PXB cells (Figures 17B-C, 18A-B). Among these, polypeptides (5)-(10) showed particularly high efficacy in reducing the cccDNA copy number in HBV-infected hepatocyte lines (Figures 17C, 18B).

[0024] (11) and (12) are polypeptides composed of partial sequences of Netrin-1, and their anti-HBV activity was confirmed in experiments using HBV-infected PXB cells in Example 12 below (Figure 18). Both are as effective as polypeptides (5) to (10) in reducing HBV DNA and cccDNA, and can be preferably used as active ingredients in anti-HBV agents.

[0025] Polypeptides (1) to (4) are polypeptides designed based on polypeptides (5) and (12), which were particularly effective in reducing the copy number of HBV DNA and cccDNA in HBV-infected hepatocyte lines. (1) is a polypeptide composed of residues 2 to 9 of polypeptide (12), and (3) is a polypeptide composed of residues 1 to 8 of polypeptide (5). Both exhibit a very strong effect in reducing the copy number of HBV DNA and cccDNA in HBV-infected hepatocyte lines (Figure 19). Furthermore, (2) is a polypeptide in which the second cysteine ​​of polypeptide (1) is replaced with alanine, and (4) is a polypeptide in which the third cysteine ​​of polypeptide (3) is replaced with alanine. Both exhibit a copy number reduction effect of HBV DNA and cccDNA at the same level as the original polypeptides (2) and (4) (Figure 20).

[0026] (22) to (25) are LIPG-binding peptides that were additionally synthesized in Example 14 for intracellular delivery experiments. (22) consists of a partial sequence of Netrin-1 and is a polypeptide with a sequence obtained by extending the polypeptide LIPH4-NTNBs of (1) by one residue to the N-terminus. (23) to (25) are polypeptides consisting of a partial sequence of N4BP1, synthesized based on the homology between LIPH4-23 and N4BP1 (NEDD4-binding protein 1). All of them have the effect of reducing the copy number of HBV DNA and cccDNA (see Examples 18 and 19) and can be used as anti-HBV agents.

[0027] Polypeptides (20) and (21) are polypeptides obtained by substituting the second cysteine ​​of polypeptide (1) with an amino acid other than alanine, and polypeptides obtained by substituting the third cysteine ​​of polypeptide (3) with an amino acid other than alanine, respectively. From the data in Figure 20, since the anti-HBV activity of both NCRHNTAG (1) and LNCRDNTR (3) is not adversely affected by the substitution of cysteine ​​residues, it can be said that polypeptides with the sequence NXRHNTAG (SEQ ID NO: 75) and LNXRDNTR (SEQ ID NO: 76) (where X is an amino acid other than cysteine ​​and alanine) also have the same level of anti-HBV activity as polypeptides (1) to (4). Preferred examples of X include, but are not limited to, glycine, isoleucine, leucine, and valine.

[0028] The polypeptide (13) is a polypeptide composed of the full-length sequence of Netrin-1. The sequences shown in SEQ ID NOs. 27 and 28 are the nucleotide sequence of the coding region in the Netrin-1 gene sequence of NCBI Accession No. NM_004822.3 and the amino acid sequence of Netrin-1 encoded therein (NP_004813.2). Since full-length Netrin-1 can also reduce the copy number of HBV DNA and cccDNA in HBV-infected hepatocytes (Figure 21A, B), the polypeptide (13) is also useful as an anti-HBV agent.

[0029] The polypeptide of (14) is a polypeptide (Netrin-1 partial polypeptide) composed of a fragment (partial sequence) of Netrin-1, including the region of amino acids 370 to 377 in the amino acid sequence of Netrin-1 shown in SEQ ID NO: 28. This region of amino acids 370 to 377 is the same amino acid sequence as the amino acid sequence of (1) (SEQ ID NO: 25). As is clearly shown in the following examples, the polypeptide consisting of the amino acid sequence of (1) has anti-HBV activity (see Figure 19 and Example 13). Therefore, as long as it includes this region of amino acids 370 to 377 (amino acid sequence of SEQ ID NO: 25), the polypeptide composed of a fragment (partial sequence) of Netrin-1 can also exhibit anti-HBV activity and is useful as an anti-HBV agent. The polypeptide of (14) includes polypeptides composed of fragments of (13) that include the region of amino acids 362-377 in the amino acid sequence of SEQ ID NO: 28 (the amino acid sequence of SEQ ID NO: 21), and polypeptides composed of fragments of (13) that include the region of amino acids 369-384 in the amino acid sequence of SEQ ID NO: 28 (the amino acid sequence of SEQ ID NO: 22).

[0030] The polypeptide of (15) is a polypeptide composed of a mutant Netrin-1 sequence. Specifically, it is a polypeptide with an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in a region other than the amino acid region of 370-377 in the amino acid sequence of Netrin-1 shown in SEQ ID NO: 28, and has an identity of 95% or more with SEQ ID NO: 28. As long as it includes the amino acid region of 370-377 (the amino acid sequence of SEQ ID NO: 25), it can exhibit anti-HBV activity even if a small number of mutations are present in the amino acids in other regions, making it useful as an anti-HBV agent. The polypeptide of (15) includes those in which one or more amino acid substitutions, deletions, insertions, or additions are contained in a region other than the amino acid region of 362-377 in SEQ ID NO: 28, or in a region other than the amino acid region of 369-384. The higher the sequence identity between the polypeptide of (15) and the amino acid sequence of SEQ ID NO: 28, the better; for example, 98% or more, or more preferably 99% or more, is preferable.

[0031] Here, "identity" of amino acid sequences is expressed as a percentage obtained by aligning the two amino acid sequences to be compared so that as many amino acid residues as possible match, and then dividing the number of matching amino acid residues by the total number of amino acid residues. When aligning the sequences as described above, gaps are inserted into one or both of the two sequences being compared as needed. Such sequence alignment can be performed using well-known programs such as BLAST, FASTA, or CLUSTAL W. When gaps are inserted, the total number of amino acid residues is calculated by counting each gap as one amino acid residue. If the total number of amino acid residues counted in this way differs between the two sequences being compared, the identity (%) is calculated by dividing the number of matching amino acid residues by the total number of amino acid residues in the longer sequence.

[0032] Conservative substitutions, that is, substitutions with amino acids that have similar chemical properties, are highly likely to not impair the properties and activity of the protein. Amino acids with similar side chains have similar chemical properties. When amino acids are grouped by the similarity of their side chains, they can be classified into groups such as those with aliphatic side chains (glycine, alanine, valine, leucine, isoleucine), those with aliphatic hydroxyl side chains (serine, threonine), those with amide-containing side chains (asparagine, glutamine), those with aromatic side chains (phenylalanine, tyrosine, tryptophan), those with basic side chains (arginine, lysine, histidine), those with acidic side chains (aspartic acid, glutamic acid), and those with sulfur-containing side chains (cysteine, methionine). A substitution with another amino acid belonging to the same group is a conservative substitution. A typical example of the amino acid sequence in (15) is the amino acid sequence in which a conservative substitution is introduced in a region other than the region of amino acids 370-377 (NCRHNTAG) of SEQ ID NO: 28, but it is not limited to this.

[0033] The polypeptide of (16) is a fragment of the polypeptide of (15) composed of a mutant Netrin-1 sequence, and is a polypeptide (mutant Netrin-1 partial polypeptide) composed of a fragment containing the region of amino acids 370 to 377 (amino acid sequence of SEQ ID NO: 25) as described above. As long as it contains the region of amino acids 370 to 377 (amino acid sequence of SEQ ID NO: 25), the polypeptide fragment composed of the mutant Netrin-1 sequence can also exert anti-HBV activity and is therefore useful as an anti-HBV agent.

[0034] The polypeptide of (26) is a polypeptide composed of the full-length sequence of N4BP1. The sequences shown in SEQ ID NOs. 81 and 82 are the nucleotide sequence of the coding region in the N4BP1 gene sequence of NCBI Accession No. NM_153029.4 and the amino acid sequence of N4BP1 encoded therein (NP_694574.3). As shown in Example 17 below, full-length N4BP1 binds to LIPG, and since the polypeptides of (23) to (25), which are N4BP1 partial polypeptides containing the specified region, have anti-HBV activity, full-length N4BP1 can also be used as an anti-HBV agent.

[0035] The polypeptide (27) is a polypeptide (N4BP1 partial polypeptide) composed of a partial sequence of N4BP1, including the region of amino acids 456-464 in the amino acid sequence of N4BP1 shown in SEQ ID NO: 82. This region of amino acids 456-464 is a region common to polypeptides (23)-(25) composed of partial sequences of N4BP1, and is also the full length of polypeptide LIPH4-N4BP1b (SEQ ID NO: 79) of (24). The polypeptide (24) has anti-HBV activity as shown in the examples below (Figure 17), and therefore, as long as it includes this region of amino acids 456-464, polypeptides composed of partial sequences of N4BP1 can also exhibit anti-HBV activity and are useful as anti-HBV agents. The polypeptide in (27) includes polypeptides composed of an N4BP1 subsequence containing the region of amino acids 455-464 in the amino acid sequence of SEQ ID NO: 82 (SEQ ID NO: 80), and polypeptides composed of an N4BP subsequence containing the region of amino acids 456-471 in the same amino acid sequence (SEQ ID NO: 78). Furthermore, the polypeptides in (23)-(25) are specific examples of the polypeptide in (27).

[0036] The polypeptide of (28) is a polypeptide composed of a mutant N4BP1 sequence. Specifically, it is a polypeptide with an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in a region other than the amino acid region of 456-464 in the amino acid sequence of N4BP1 shown in SEQ ID NO: 82, and has an identity of 95% or more with SEQ ID NO: 82. As long as it includes this amino acid region of 456-464 (the amino acid sequence of SEQ ID NO: 79), it can exhibit anti-HBV activity even if a small number of mutations are present in the amino acids in other regions, making it useful as an anti-HBV agent. The polypeptide of (28) includes those in which one or more amino acid substitutions, deletions, insertions, or additions are included in a region other than the amino acid region of 455-464 in SEQ ID NO: 82, or in a region other than the amino acid region of 456-471. The higher the sequence identity between the polypeptide of (28) and the amino acid sequence of SEQ ID NO: 82, the better; for example, 98% or more, or more preferably 99% or more, is preferable.

[0037] The polypeptide of (29) is a fragment of the polypeptide of (28) composed of a mutant N4BP1 sequence, and is a polypeptide (mutant N4BP1 partial polypeptide) composed of a partial sequence of the mutant N4BP1 sequence that includes the region of amino acids 456 to 464 (amino acid sequence of SEQ ID NO: 79) as described above. As long as it includes the region of amino acids 456 to 464 (amino acid sequence of SEQ ID NO: 79), the polypeptide fragment composed of the mutant N4BP1 sequence can also exert anti-HBV activity and is therefore useful as an anti-HBV agent.

[0038] The polypeptide of (30) is a polypeptide that has more than 80% sequence identity with the original polypeptide, obtained by modifying some residues in one of the polypeptides of (1) to (14) and (17) to (27). It is well known in this field that even if only a very small number of residues are modified, the activity of the polypeptide can be maintained at the same level or better. Therefore, polypeptides modified by altering a small number of residues from polypeptides (1)-(14), (16)-(27), and (29) (for short polypeptides of less than 20 residues such as (1)-(12) and (17)-(25), and for short polypeptides of (14) and (27) of less than about 20 residues, for example, 1-4 residues, 1-3 residues, 1 or 2 residues, or 1 residue; for long polypeptides of (14) and (27), for example, 1-20 residues, 1-15 residues, 1-10 residues, or 1-several residues), which have an identity of 80% or more, for example, 85% or more, 90% or more, 95% or more, or 98% or more, can exhibit anti-HBV activity in the same way as the original polypeptide and are useful as anti-HBV agents. The modification of residues referred to here is substitution, deletion, insertion, or addition, and is typically substitution, especially conservative substitution.

[0039] The chain lengths of the Netrin-1 partial polypeptide of (14), the mutant Netrin-1 partial polypeptide of (16), the N4BP1 partial polypeptide of (27), and the mutant N4BP1 partial polypeptide of (29) are not particularly limited, but may be set to sizes of, for example, 100 residues or less, 80 residues or less, 70 residues or less, 60 residues or less, 50 residues or less, 40 residues or less, 30 residues or less, 25 residues or less, or 20 residues or less, for convenience in synthesis and delivery into hepatocytes.

[0040] Polypeptides (1)-(12) and (17)-(25) have short chain lengths and can be easily prepared by chemical synthesis. Specific examples of chemical synthesis methods include the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method). They can also be synthesized by conventional methods using various commercially available peptide synthesizers.

[0041] The polypeptide (Netrin-1) of (13) and the polypeptide (N4BP1) of (26) can be prepared by well-known genetic engineering techniques. Specifically, RNA can be extracted from cells expressing Netrin-1 or N4BP1 (e.g., human-derived cell lines), a cDNA encoding Netrin-1 or N4BP1 can be prepared by RT-PCR using this RNA as a template, the cDNA can be incorporated into a suitable expression vector and introduced into a suitable host cell, the polypeptide can be produced in the host cell, and then extracted and purified to prepare the polypeptide of (13) or (26). Primers used for RT-PCR can be designed based on the nucleotide sequence shown in SEQ ID NO. 27 (the coding region sequence of NCBI Accession No. NM_004822.3, which encodes the amino acid sequence shown in SEQ ID NO. 28) if it is a primer for Netrin-1, and based on SEQ ID NO. 81 (the coding region sequence of NCBI Accession No. NM_153029.4, which encodes the amino acid sequence shown in SEQ ID NO. 82) if it is a primer for N4BP1. Various expression vectors and host cell lines are known and readily available commercially. The polypeptides of (15) and (27) can be prepared by introducing mutations into the cDNA encoding Netrin-1 and the cDNA encoding N4BP1, respectively, which were prepared as described above. Various kits for introducing mutations are also commercially available.

[0042] Polypeptides (14), (16), (27), (29), and (30) with short chain lengths can be easily prepared by chemical synthesis. Polypeptides (14), (16), (27), (29), and (30) with chain lengths that are difficult to prepare by chemical synthesis can be prepared by synthesizing DNA encoding the target polypeptide by methods such as using a primer set inside the coding region during RT-PCR using RNA extracted from Netrin-1 expressing cells or N4BP1 expressing cells as a template (mutations can be introduced if desired), or, if Netrin-1 cDNA or N4BP1 cDNA has already been synthesized or cloned, amplifying the fragment by PCR using Netrin-1 cDNA or N4BP1 cDNA as a template (mutations can be introduced if desired), incorporating the DNA into a suitable expression vector, expressing the polypeptide in host cells, and then extracting and purifying it.

[0043] Polypeptides prepared by chemical synthesis are advantageous as pharmaceuticals because, unlike genetically modified polypeptides, they do not contain contaminants derived from host cells. Therefore, among polypeptides (1) to (30), polypeptides (1) to (12) and (17) to (25), as well as those with short sizes (within a few tens of residues) among (14), (16), (27), (29), and (30), can be preferably used as active ingredients for anti-HBV agents. Polypeptides (14) and (16) contain the region of amino acids 370 to 377 (amino acid sequence of SEQ ID NO: 25), and therefore have a size of 9 residues or more. Polypeptides (27) and (29) contain the region of amino acids 456 to 464 (amino acid sequence of SEQ ID NO: 79), and therefore have a size of 9 residues or more. Polypeptides corresponding to (14) and (16) with a size of 8 residues are identical to polypeptide (1). (11), (12), and (22) are partial polypeptides of Netrin-1 that include a specified region, and therefore correspond to specific examples of (14). (23) to (25) are partial polypeptides of N4BP1 that include a specified region, and therefore correspond to specific examples of (27).

[0044] In the field of peptide formulations, various methods are employed to improve the in vivo stability of peptides and increase their blood half-life, including the addition of polyethylene glycol (PEG) chains (e.g., Clin Nephrol. 2006 Mar;65(3):180-90. and Proc Natl Acad Sci USA. 2005 Sep 6;102(36):12962-7.), the addition of sugar chains mainly to the N-terminus or C-terminus (e.g., J Am Chem Soc. 2004 Nov 3;126(43):14013-22 and Angew Chem Int Ed Engl. 2004 Mar 12;43(12):1516-20), and the use of D-isomers for at least a portion of the amino acid residues (e.g., J Pharmacol Exp Ther. 2004 Jun;309(3):1190-7 and J Pharmacol Exp Ther. 2004 Techniques such as modifying and adding to the Fc region of the antibody (e.g., J. Immunol., 154 (10), 5590-5600 (1995), Nature, 332, 563-564 (1998), Nature, 332, 738-740 (1998), BioDrugs. 2008;22:11-26, etc.), amidating the C-terminus, and acetylating the N-terminus have been employed. The polypeptide used as the active ingredient in the anti-HBV agent of the present invention may be one to which such techniques have been applied. It is also possible to use a polypeptide modified with other polypeptides or molecules that have the effect of enhancing delivery to the liver as the active ingredient.

[0045] In the present invention, the terms "polypeptide of the sequence of SEQ ID NO: X," "polypeptide represented by SEQ ID NO: X," and "polypeptide composed of SEQ ID NO: X" (where SEQ ID NO: X is an amino acid sequence with N amino acid residues) encompass polypeptides having a structure in which other functional polypeptides, such as polypeptides that enhance the Fc region or delivery to the liver, are added to a polypeptide having the amino acid sequence shown in SEQ ID NO: X and having a total length of N residues (such polypeptides are conveniently referred to as polypeptide X). The terms "containing 'polypeptide of the sequence of SEQ ID NO: X,' 'polypeptide represented by SEQ ID NO: X,' or 'polypeptide composed of SEQ ID NO: X' as an active ingredient" encompass not only embodiments containing polypeptide X having a total length of N residues as an active ingredient, but also embodiments containing polypeptides having a structure in which other functional polypeptides are added to polypeptide X as an active ingredient. Any functional polypeptide may be added as long as it does not impair the anti-HBV activity of the polypeptide. Polypeptides having a structure with other functional polypeptides added can be produced by chemical synthesis if the functional polypeptide is short in size, or by genetic engineering if the functional polypeptide is long in size.

[0046] Furthermore, LIPG-binding peptides may have tag sequences such as Flag tags or His tags added to them for convenience of purification and detection. Such tag sequences can also be considered as examples of functional molecules. For example, in the single-chain antibody fusion prepared in the following examples, Flag tags and His tags are added to the C-terminus of each LIPG-binding peptide. However, the addition of such tag sequences is optional, and it is also possible to prepare the anti-HBV agent of the present invention using LIPG-binding peptides without added tag sequences.

[0047] One example of a functional molecule is a carrier molecule for delivery into hepatocytes. That is, the LIPG-binding peptide may be in a form linked to a carrier molecule for delivery into hepatocytes. As described in the examples below, the anti-HBV effect can be significantly enhanced by delivering the LIPG-binding peptide into hepatocytes. As carrier molecules for drugs to be delivered into hepatocytes, drug delivery systems (DDS) utilizing asialoglycoprotein receptors (ASGR) and ApoE receptors, which are abundant in hepatocytes, are known (Yuko Ito, STI Horizon, 2019, Vol.5, No.4, pp.21-25). In the present invention, carrier molecules that deliver drugs into hepatocytes using ASGR or ApoE receptors can also be preferably used.

[0048] Examples of drug delivery carriers utilizing ASGR include specific binding molecules that bind to ASGR (antibodies, antibody fragments, single-chain antibodies, or aptamers, etc.), particularly antibodies, antibody fragments, or single-chain antibodies (scFv) that bind to ASGR. As shown in Figure 27, the asialoglycoprotein receptor (ASGR) is a type II single-pass transmembrane protein with its N-terminus facing intracellularly and its C-terminal sugar recognition sites (CRDs) facing extracellularly. In human hepatocytes, the receptor is formed by heterooligomers of ASGR1 and ASGR2. Any ASGR-specific binding molecule used as a drug delivery carrier should bind to the extracellular domain of ASGR. Specific binding molecules that can bind to both ASGR1 and ASGR2 are particularly preferred, but specific binding molecules that bind to only one of them can also be used as drug delivery carriers. In the case of specific binding molecules that bind to only one of them, they may be used alone as a drug delivery carrier, or a combination of a specific binding molecule that binds to one and a specific binding molecule that binds to the other may be used. When combining them, one can prepare a LIPG-binding peptide linked to an ASGR1-specific binding molecule and a LIPG-binding peptide linked to an ASGR2-specific binding molecule, and then mix the two to use as an anti-HBV agent.

[0049] The nucleotide sequence of the coding region in the human ASGR1 gene sequence registered in GenBank as NM_001671.5 and the amino acid sequence of ASGR1 encoded therein are shown in SEQ ID NOs. 113 and 114, and the nucleotide sequence of the coding region in the human ASGR2 gene sequence registered as NM_001181.4 and the amino acid sequence of ASGR2 encoded therein are shown in SEQ ID NOs. 115 and 116. Amino acids 41-61 of SEQ ID NO. 114 (ASGR1) and amino acids 59-79 of SEQ ID NO. 116 (ASGR2) constitute the transmembrane domain, with the N-terminal side being the intracellular domain and the C-terminal side being the extracellular domain. In the preparation of anti-ASGR antibodies, antibody fragments, or single-chain antibodies, the region from amino acids 62-291 of ASGR1 and the region from amino acids 80-331 of ASGR2 can be used as antigens or immunogens, but it is more preferable to use these extracellular domains in heterooligomerized form. Hereafter, in this specification, the term "ASGR extracellular domain" includes hetero-oligomerized extracellular domains. When "hetero-oligomer" is used, it refers to the hetero-oligomerized ASGR extracellular domain.

[0050] Anti-ASGR polyclonal antibodies can be obtained by immunizing non-human animals with the ASGR extracellular domain as an immunogen, collecting blood, separating serum, and recovering and purifying antibodies that bind to the ASGR extracellular domain from the serum. Anti-ASGR monoclonal antibodies can be obtained by collecting antibody-producing cells such as spleen cells and lymphocytes from non-human animals immunized with the ASGR extracellular domain, fusing these with myeloma cells to prepare hybridomas, selecting hybridomas that produce antibodies that bind to the ASGR extracellular domain, and growing them to obtain anti-ASGR monoclonal antibodies from the culture supernatant.

[0051] Anti-ASGR antibody fragments can be obtained by treating anti-ASGR antibodies with proteolytic enzymes such as papain or pepsin. The definition of an antibody fragment is as described above and includes antibody fragments that maintain binding affinity to the antigen (ASGR extracellular domain), such as Fab, Fab', and F(ab')2.

[0052] Anti-ASGR scFv can be obtained, for example, by extracting mRNA from a hybridoma prepared as described above, preparing cDNA, amplifying the immunoglobulin H chain and L chain genes by PCR using primers specific to the immunoglobulin H chain and L chain, linking these with a linker, adding appropriate restriction enzyme sites, introducing them into a plasmid vector, transforming E. coli with the vector to express scFv, and recovering it from E. coli.

[0053] Furthermore, anti-ASGR scFv can be obtained from scFv libraries using techniques such as phage display and IVV.

[0054] In the phage display method, an scFv library is prepared, which is a library of phages in which scFv is displayed on the surface of the phage. For example, a naive scFv phage library can be prepared by the following procedure: mRNA is extracted from antibody-producing cells such as splenocytes or lymphocytes collected from healthy humans or non-human animals, cDNA is synthesized by reverse transcription, and cDNA encoding the region containing the heavy chain variable region (VH) (VH cDNA) and cDNA encoding the region containing the light chain variable region (VL cDNA) (VL cDNA) are comprehensively amplified by PCR. Next, the amplified VH cDNA and VL cDNA are randomly linked via a suitable linker (e.g., a linker with three GGGGS units) by conventional assembly PCR or fusion PCR to prepare cDNA encoding scFv (scFv cDNA). After incorporating the scFv cDNA into a phage plasmid vector to prepare a vector library, a library of phages in which each scFv is displayed on the surface is prepared by the phage display method. There are two types of plasmid vectors for phages: phage vectors that contain all the phage genes necessary for phage particle formation and can form phage particles on their own, and phagemide vectors that contain the g3p gene but not other phage protein genes and require helper phages for phage particle formation. Phagemide vectors are preferred. By introducing an scFv cDNA library prepared using a phagemide vector into E. coli and co-infecting the E. coli with helper phages, the individual vectors of the scFv cDNA library are packaged, and a library of phages that display the scFv expressed by the vector on their surface can be prepared. Alternatively, mutations may be randomly introduced into the prepared VH cDNA and VL cDNA or scFv cDNA to prepare a phage library of mutant scFv.

[0055] From the prepared library, phages displaying scFv that bind to the ASGR extracellular domain are selected (panning). This panning process can be carried out using a solid-phase support (chip, plate, magnetic beads, etc.) immobilized with the ASGR extracellular domain, and a support immobilized with heterooligomers is particularly preferred. The extracellular domains of ASGR1 (ASGR1ex) and ASGR2 (ASGR2ex) are biotinylated, and by contacting the biotinylated ASGR1ex and ASGR2ex with an avidin-coated support, ASGR1ex and ASGR2ex can be heterooligomerized on the support surface. The phage library is brought into contact with the ASGR extracellular domain-immobilized support, washed, and the phages bound to the support are recovered. The recovered phages are lysed, the packaged vector is recovered and reintroduced into E. coli, and helper phages are co-infected to re-form phage particles. These phage particles are brought into contact with the ASGR extracellular domain-immobilized support again. By performing this operation multiple times, phage clones that display scFv specific to the ASGR extracellular domain can be enriched.

[0056] Phages enriched through multiple rounds of panning can be obtained as candidate clones for anti-ASGR scFv, but clone selection may be performed to further narrow down the candidates. The scFv expression vector is recovered from the enriched phage and introduced into a suitable host cell such as E. coli to prepare host cell clones that express scFv. The reactivity to the ASGR extracellular domain and the sequence of the variable region of these clones are examined, and clones with high specific binding affinity to the ASGR extracellular domain are selected. Reactivity can be confirmed by an immunoassay such as ELISA using the ASGR extracellular domain, preferably a heterooligomer, as the antigen. Since there may be multiple clones with identical or very similar variable region sequences, clone duplication may be confirmed by the VH and VL nucleotide sequences and the clones may be grouped. Through these clone selection procedures, clones with high specific binding affinity to the ASGR extracellular domain can be selected, and the candidates can be further narrowed down.

[0057] scFv expression vectors are recovered from candidate clones, scFv cDNA is amplified from the scFv expression vectors, and the resulting cDNA is incorporated into a suitable plasmid expression vector to express scFv in a suitable host cell. This cDNA is then recovered and purified. The reactivity of the purified scFv with ASGR1ex and ASGR2ex or with heterooligomers is then confirmed to obtain scFv that specifically binds to the extracellular domain of ASGR.

[0058] The IVV method (Nemoto, N. et al., FEBS Lett., 414:405-408, 1997; Miyamoto-Sato, E. et al., Nucleic Acids Res., 28:1176-1182, 2000; WO 03 / 106675 A1) is a technology developed by Yanagawa, one of the inventors of this application, and his collaborators. In the IVV method, puromycin, a type of antibiotic, is bound to the 3' end of mRNA via a PEG (polyethylene glycol) spacer, and a cell-free translation reaction is performed using this as a template. This results in the formation of an mRNA-protein ligated molecule (in vitro virus; IVV) in which a protein molecule and the mRNA molecule encoding it are covalently linked via puromycin. From the IVV library thus constructed, IVV containing proteins that bind to bait (proteins, peptides, antigens, etc.) can be extracted in vitro. The gene (mRNA) linked to it can then be amplified by reverse transcription PCR, and its base sequence can be deciphered using a DNA sequencer, allowing for easy identification of interacting proteins, peptides, and antibodies. The method for eluting and recovering IVV bound to bait is generally competitive elution with free bait. However, if competitive elution is not possible, a PEG spacer with a photocleavable 2-nitrobenzyl linker can be used, and the spacer can be cleaved by irradiation with 365 nm UV light to elute and recover the mRNA portion (Doi, N. et al., J. Biotechnol., 131:231-239, 2007).

[0059] The details of the method for producing anti-ASGR scFv using the IVV method are described in the following examples. A brief explanation follows.

[0060] After preparing the scFv cDNA library as described above, mRNA is synthesized from the cDNA library by reverse transcription, and puromycin is ligated to the 3' end of the mRNA via a PEG spacer to create an scFv mRNA-puromycin library. Using this as a template, a cell-free translation reaction is performed to construct a library of mRNA-scFv ligatures (IVV) in which scFv molecules and the mRNA molecules encoding them are covalently linked via puromycin.

[0061] From the scFv IVV library, scFv cells that bind to the ASGR extracellular domain are selected. This selection step can also be performed using a solid-phase support (chip, plate, magnetic beads, etc.) immobilized with the ASGR extracellular domain, preferably a heterooligomer, as bait, similar to the phage display method. The scFv IVV library and the ASGR extracellular domain-immobilized support are brought into contact, washed, and then the IVV bound to the support is eluted and recovered. IVV can be eluted and recovered by competitive elution using ASGR1ex or ASGR2ex. If a PEG spacer with a 2-nitrobenzyl linker introduced is used as the PEG spacer, it is also possible to cleave the spacer by UV irradiation and then elute and recover the mRNA portion of IVV.

[0062] Reverse transcription PCR is performed using the recovered IVV or mRNA portion as a template to prepare an scFv cDNA library after the first round of selection. An IVV library can then be prepared again from this cDNA library, and the second round of selection can be performed. It is preferable to perform multiple rounds of selection while sequentially increasing the selection pressure (contact time between the IVV library and the bait, amount of bait immobilized on the carrier, etc.).

[0063] The scFv coding region is amplified from each scFv cDNA library obtained in subsequent rounds, cloned into a suitable plasmid vector, and a library of vectors incorporating the selected scFv cDNA is prepared. This vector is introduced into a suitable host cell such as E. coli to obtain a library of selected scFv clones. The obtained clone library is evaluated for reactivity to the ASGR extracellular domain, the variable region sequence is analyzed, and grouping is performed, similar to clone selection by phage display. Vectors are recovered from the selected clones to prepare scFv, and by finally confirming the reactivity to ASGR1ex and ASGR2ex or to heterooligomers, scFv with high specific binding affinity to the ASGR extracellular domain can be obtained.

[0064] Particularly preferred examples of anti-ASGR antibodies, antibody fragments, or scFvs include: A heavy chain CDR1 containing the amino acid sequence shown in SEQ ID NOs: 83, 89, 95, or 101, A heavy chain CDR2 containing the amino acid sequence shown in SEQ ID NOs: 84, 90, 96, or 102, A heavy chain CDR3 containing the amino acid sequence shown in SEQ ID NOs. 85, 91, 97, or 103, A light chain CDR1 containing the amino acid sequence shown in SEQ ID NOs: 86, 92, 98, or 104, A light chain CDR2 containing the amino acid sequence shown in SEQ ID NOs: 87, 93, 99, or 105, Light chain CDR3 containing the amino acid sequence shown in SEQ ID NOs. 88, 94, 100, or 106 Examples include antibodies, antibody fragments, or scFv having the following CDR sequences. These CDR sequences are based on the CDR sequences of anti-ASGR scFv obtained in the following examples. In the following examples, anti-ASGR scFv having heavy chain CDR1-3 of the amino acid sequence shown in SEQ ID NOs. 83-85 and light chain CDR1-3 of the amino acid sequence shown in SEQ ID NOs. 86-88, anti-ASGR scFv having heavy chain CDR1-3 of the amino acid sequence shown in SEQ ID NOs. 89-91 and light chain CDR1-3 of the amino acid sequence shown in SEQ ID NOs. 92-94, anti-ASGR scFv having heavy chain CDR1-3 of the amino acid sequence shown in SEQ ID NOs. 95-97 and light chain CDR1-3 of the amino acid sequence shown in SEQ ID NOs. 98-100, and anti-ASGR scFv having heavy chain CDR1-3 of the amino acid sequence shown in SEQ ID NOs. 101-103 and light chain CDR1-3 of the amino acid sequence shown in SEQ ID NOs. 104-106 were obtained.

[0065] Furthermore, the CDR sequence is not limited to the specific amino acid sequence described above. It may also be an antibody or the like having a CDR containing an amino acid sequence in which some bases are substituted in the above amino acid sequence, resulting in an amino acid sequence that is 80% or more identical to the original amino acid sequence. For example, the heavy chain and light chain CDR1 may allow for the substitution of one residue, the heavy chain and light chain CDR2 may allow for the substitution of 1 to 3, 1 to 2, or 1 residue, and the heavy chain and light chain CDR3 may allow for the substitution of 1 to 2 or 1 residue. Antibodies, antibody fragments, or scFvs having such modified CDRs can also be used as antibodies, antibody fragments, or scFvs that specifically bind to ASGR.

[0066] Antibodies and the like having a predetermined CDR sequence can be easily prepared, for example, by introducing mutations into the CDR region of the VH and VL genes of any cloned antibody or the scFv gene encoding any scFv to modify it to encode the above amino acid sequence. Any antibody and any scFv that serve as the base to which the above CDR sequence is introduced may be an anti-ASGR antibody and an anti-ASGR scFv having a CDR sequence different from the above, or an antibody and scFv against other antigens.

[0067] Anti-ASGR antibodies, antibody fragments, or scFv having a CDR or modified CDR containing the specific amino acid sequence described above are excellent not only as delivery carriers for the anti-HVB agent of the present invention, but also as delivery carriers for various drugs to be delivered into hepatocytes. These anti-ASGR antibodies, antibody fragments, or scFv can be used as drug delivery carriers into hepatocytes, and the drugs to be delivered into hepatocytes can be complexed with the carrier using techniques known in the pharmaceutical field to form a pharmaceutical composition. The anti-ASGR antibody may be a human antibody, a humanized antibody, a human-non-human animal chimeric antibody, or a non-human animal antibody. However, when used as a delivery carrier for human pharmaceuticals, human antibodies, humanized antibodies, or chimeric antibodies, particularly human antibodies or humanized antibodies, and especially human antibodies, are preferred. Similarly, anti-ASGR scFv antibodies may be derived from human antibodies, humanized antibodies, human-non-human animal chimeric antibodies, or non-human animal antibodies. However, when used as a carrier for delivering human pharmaceuticals, antibodies derived from human antibodies, humanized antibodies, or chimeric antibodies are preferred, particularly those derived from human antibodies or humanized antibodies, and especially those derived from human antibodies.

[0068] When linking a carrier molecule for intrahepatic delivery, such as an anti-ASGR antibody, to a LIPG-binding peptide, it may be linked to either end of the LIPG-binding peptide. If one or more other functional molecules are used, they should be linked to the LIPG-binding peptide in a manner that allows them to function appropriately in conjunction with those other functional molecules. Preferably, the intrahepatic delivery carrier molecule is linked to the LIPG-binding peptide via a cleavage sequence that is cleaved by an endogenous enzyme present in the cells of a patient receiving the anti-HBV agent of the present invention. An example of a cleavage sequence is the sequence RVRR (SEQ ID NO: 111), which is recognized and cleaved by Furin, but the cleavage sequence is not limited to this.

[0069] Another example of a functional molecule is a cell membrane permeability-promoting molecule. That is, LIPG-binding peptides may exist in a form linked to a cell membrane permeability-promoting molecule. A cell membrane permeability-promoting molecule is a molecule that promotes the release of LIPG-binding peptides, which have been taken into cells by endocytosis, from within endosomes through the membrane into the cytoplasm. Specific examples of molecules that promote cell membrane permeability include S19 (Sudo, K. et al., J. Control. Release, 255:1-11, 2017, WO 2016 / 199674 A1), a partial peptide of the protein syncytin 1, which is involved in cell fusion during human placenta formation, developed by Doi et al. Subsequent studies have led to the development of 28-residue syncytin 1 partial peptides S28 (PFVIGAGVLGALGTGIGGITTSTQFYYK, SEQ ID NO: 107) and 39-residue syncytin 1 partial peptides S39 (PFVIGAGVLGALGTGIGGITTSTQFYYKLSQELNGDMER, SEQ ID NO: 108), which are peptides that can function even more efficiently than S19 as membrane permeability-promoting peptides. These peptides, particularly S28 and S39, are cell membrane permeability-promoting molecules that can be preferably used in the present invention, but the usable cell membrane permeability-promoting molecules are not limited to these.

[0070] The cell membrane permeability-promoting molecule may be linked to either end of the LIPG-binding peptide. However, when linking the carrier molecule for intrahepatocyte delivery and the cleavage sequence to the LIPG-binding peptide, they should be linked to the end of the LIPG-binding peptide that is closer to the cleavage sequence, or to the end opposite to the carrier molecule.

[0071] As a further example of a functional molecule, nuclear localization signals can be cited. That is, LIPG-binding peptides may be in a form linked to nuclear localization signals. Various nuclear localization signals are known, and any of them may be used. In the following example, PAAKRVKLD (SEQ ID NO: 110) is used as the nuclear localization signal, but the example is not limited to this specific case. The nuclear localization signal may be linked to either end of the LIPG-binding peptide, but when linking a carrier molecule for intracellular delivery and a cleavage sequence to the LIPG-binding peptide, it should be linked to the LIPG-binding peptide side of the cleavage sequence, or to the end opposite the carrier molecule. When linking a cell membrane permeability-promoting molecule and a nuclear localization signal to the LIPG-binding peptide, they may be linked to both ends respectively, or both may be linked to one end. In the latter case, either may be placed at the very end. When linking a delivery carrier + cleavage sequence, a cell membrane permeability-promoting molecule, and a nuclear localization signal to the LIPG-binding peptide, the delivery carrier + cleavage sequence is linked to one end, and the cell membrane permeability-promoting molecule and nuclear localization signal are linked to the other end.

[0072] The primary target population for the anti-HBV agent of the present invention is HBV-infected patients, including hepatitis B patients and HBV-infected patients who have not developed hepatitis B (HBV carriers). Patients are typically mammals, particularly humans, but are not limited to these.

[0073] The dosage of the anti-HBV agent of the present invention should be an amount that provides an anti-HBV effect in the target patient. The effective dose can be appropriately selected according to the patient's symptoms, viral load, age, weight, etc. Although not particularly limited, the dosage of the anti-HBV agent of the present invention may be approximately 1 μg to 10,000 mg per kg of body weight per day, for example, approximately 100 μg to 1,000 mg, as the amount of active ingredient per day for the target patient. The amount of active ingredient referred to here is the amount of the polypeptide portion consisting of the amino acid sequences of (1) to (30) when polypeptides (1) to (30) are used as the active ingredient, and does not include the amount of the other functional polypeptide when polypeptides with added functional polypeptides are used. Furthermore, when administering multiple types of polypeptides, the amount of active ingredient is the total amount. Administration may be once a day or divided into several doses. Administration may be daily or every few days.

[0074] The anti-HBV agent of the present invention may be administered orally or parenterally, but generally parenteral administration such as intramuscular, subcutaneous, intravenous, or intra-arterial administration is preferred.

[0075] The active ingredient of the anti-HBV agent of the present invention can be formulated by appropriately mixing it with pharmaceutically acceptable additives such as carriers, diluents, excipients, binders, lubricants, disintegrants, sweeteners, suspending agents, emulsifiers, colorants, flavoring agents, and stabilizers, suitable for each route of administration. Formulations can include oral preparations such as tablets, capsules, granules, powders, and syrups, as well as parenteral preparations such as inhalants, injections, suppositories, and liquids. Formulation methods and usable additives are well known in the field of pharmaceutical formulations, and any of these methods and additives can be used.

[0076] An anti-HBV agent containing two or more polypeptides from (1) to (30) as active ingredients may be a combination preparation containing all two or more polypeptides in the same formulation, or it may include a combination of single agents containing each polypeptide individually. In the embodiment including a combination of single agents, the single agents are usually administered simultaneously or sequentially, but it is also acceptable to administer each single agent at an appropriate interval. [Examples]

[0077] The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

[0078] [Example 1]: Knockdown of LIPG expression negatively controls HBV infection. The effects of knockdown of LIPG expression on HBV infection were investigated. The cells, viruses, reagents, etc. used in the experiments of Examples 1-4 are as follows. HC1 cells (derived from human hepatocellular carcinoma), HepG2 cells (derived from human hepatocellular carcinoma), HepG2-NTCP-C4 cells (HepG2 cells overexpressing NTCP, the receptor for HBV, Iwamoto, M. et al. Biochem. Biophys. Res. Commun. 443:808-813, 2014), HepG2.2.15 cells (HepG cells with the HBV genome incorporated), Huh1 cells (derived from human hepatocellular carcinoma), Huh6 cells (derived from human hepatoblastoma), Huh7 cells (derived from human hepatocellular carcinoma), Huh7.5 cells (derived from Huh7), T5B cells (derived from normal human hepatocytes), and RD cells (derived from human rhabdomyosarcoma) were treated with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA) and 1% L-glutamine (Thermo Fisher Scientific). The cells were maintained at 5% CO2 and 37°C using DMEM (Thermo Fisher Scientific) supplemented with 1% penicillin / streptomycin (Thermo Fisher Scientific). PHH cells (primary human hepatocytes) were purchased from PhoenixBio (Hiroshima Prefecture). Heparin and heparanase were purchased from R&D systems (Minneapolis, MN). LIPG inhibitor (GSK-264220A) was purchased from Tocris Bioscience (Minneapolis). Chlorpromazine (CPZ) and 5-(N-ethyl-N-isopropyl)amiloride (EIPA) were purchased from Sigma-Aldrich (St. Louis, MO). Primary antibodies against DYKDDDDK(FLAG)(#14793)(SEQ ID NO: 74), Caveolin-1(#3238), Vimentin(#5741), Pan-cadherin(#4068), E-cadherin(#3195), and β-actin(#4970) were obtained from Cell Signaling Technology (Beverly, MA), while NTCP(ab131084) and LIPG(ab24447) were obtained from abcam (Cambridge, UK).The HBV used in infection experiments with PHH (primary human hepatocytes) was purchased from PhoenixBio. For infection experiments with cultured cells other than PHH cells, HBV derived from HepG2.2.15 cells was used. The culture medium of HepG2.2.15 cells was filtered through a 0.45 μm filter and precipitated with 10% PEG8000. HCV was grown from the culture medium of Huh7.5 cells transfected with pHJ3-5 / GLuc2A21 (Shimakami, T. et al. Sci. Rep. 4, 4688, 2014). Myocarditis virus (EMCV) was provided by Dr. Mitsutoshi Yoneyama (Medical Mycology Research Center, Chiba University, Japan). Influenza virus A / WSN / 33 strain was provided by Dr. Masayoshi Enami (Department of Molecular Genetics, Kanazawa University). The LIPG-target shRNA (target sequence: 5'-TTACACGGATGCG-GTCAATAA-3', SEQ ID NO: 73) cloned into the lentivirus-based pLKO.1-puro expression vector used for LIPG knockdown was purchased from Sigma. An empty vector was used for the control shRNA (Cont shRNA). Lentiviral particles were generated by co-transfection of packaging cells (293FT cells) with a packaging plasmid. Constitutive LIPG knockdown cells were selected using a puromycin-selectable marker.

[0079] Quantitative analysis of HBV-DNA and cccDNA by qPCR was performed as follows. Total DNA was extracted from cells using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). HBV-DNA was quantified by qPCR analysis as previously reported (Honda, M. et al. J. Infect. Dis., 213, 1096-1106, 2016). The extracted DNA (50 ng) was treated with 10U Plasmid safe DNase I (Epicentre, Madison, WI) at 37°C for 60 minutes, followed by treatment at 70°C for 30 minutes to inactivate the DNase. cccDNA was quantified by qPCR analysis as previously reported (Honda, M. et al. J. Infect. Dis., 213, 1096-1106, 2016). Quantitative RT-PCR was performed as follows. Total RNA was extracted using the GenElute™ Mammalian Total RNA Miniprep Kit (Sigma-Aldrich), and cDNA was synthesized using the High Capacity cDNA reverse transcription kit (Applied Biosystems, Carlsbad, CA). RT-PCR was performed using the 7500 Real Time PCR System (Applied Biosystems). 5'-GCTCTGTATCGGGAGGCCTTA-3' (SEQ ID NO: 31) and 5'-TGAGTGCTGTATGGTGAGGAGAA-3' (SEQ ID NO: 32) were used as primers for pregenomic RNA, and 5'-FAM-AGTCTCCGGAACATT-MGB-3' (SEQ ID NO: 33) was used as the probe. Primer pairs and probes for LIPG, NTCP, 18S rRNA, and ACTB were obtained from the TaqMan assay reagents library.

[0080] SDS-PAGE and immunoblotting were performed as follows: Cells were washed with phosphate-buffered saline (PBS) and lysed in RIPA Lysis Buffer (EMD Millipore, Burlington, MA) containing complete protease inhibitor cocktail (Roche Applied Science). The membrane was blocked with Blocking One solution (Nacalai Tesque, Kyoto). Western blotting was performed using a standard method. ChemiDoc Imaging Systems (Bio-Rad, Hercules, CA) was used for detection.

[0081] Statistical analysis was performed as follows. Unless otherwise specified, all intergroup comparisons were performed using one-way or two-way ANOVA or two-sided t-tests. Prism 7 software (GraphPad Software, La Jolla, CA) was used for calculations.

[0082] When HC1 cells established from human hepatocellular carcinoma were infected with a lentivirus expressing shRNA for the LIPG gene (LIPG shRNA), LIPG constitutive knockdown cells were created. As a result, LIPG expression was significantly reduced in these cells compared to control cells (Figure 1A). Furthermore, when these cells were infected with HBV, HBV DNA (Figure 1B) and cccDNA (Figure 1C) were significantly reduced in the LIPG constitutive knockdown cells. In particular, cccDNA was remarkably reduced (Figure 1C). Next, LIPG shRNA was introduced into primary human hepatocytes (PHH) to establish LIPG constitutive knockdown PHH cells. When these cells were also infected with HBV (Figure 1D), similar to HC1 cells, LIPG constitutive knockdown PHH cells showed significantly reduced LIPG expression (Figure 1E), HBV DNA (Figure 1F), cccDNA (Figure 1G), and pregenomic RNA (pgRNA) transcription (Figure 1H) compared to control cells.

[0083] These results indicate that knockdown of the LIPG gene using shRNA reduces the amount of HBV infection. Furthermore, these results suggest that LIPG is involved in HBV infection.

[0084] [Example 2]: LIPG positively controls HBV infection in HepG2-NTCP cells. Next, we investigated the relationship between LIPG expression levels and HBV infection. First, we established a cell line that stably expresses Flag-tagged LIPG. Using the LIPG cDNA plasmid as a template, we performed PCR amplification using the forward primer 5'-TTAGAATTCATGAGCAACTCCGTTC-3' (with EcoRI recognition site, SEQ ID NO: 34) and the reverse primer 5'-AATTCTAGATCATCTGCTCATCTGTTGTTGTGGGAAGCTCCACAGTG-3' (with XbaI recognition site, SEQ ID NO: 35) to create a lentiviral transfer plasmid encoding the LIPG gene (SEQ ID NO: 29). The PCR product was digested with EcoRI and XbaI and ligated to a similarly digested pLVSIN-CMV Pur Vector (Takara Bio, Otsu, Japan) to obtain a pLVSIN-CMV-Flag-tagged LIPG Vector. pLVSIN-CMV Pur Vector or pLVSIN-CMV-Flag-tagged LIPG Vector was transfected into Lenti-X 293T cells (Takara Bio) with Lentiviral High Titer Packaging Mix (Takara Bio) using FuGENE-HD transfection reagent (Promega, Madison, WI) to generate viral particles. After 72 hours, the supernatant was collected, filtered through a 0.22 μm syringe filter, and introduced into the cells. 72 hours after introduction, these cells were treated with puromycin (5 mg / mL) for antibiotic selection. To avoid clonal bias, an antibiotic-resistant bulk cell population was used in the experiment.

[0085] When comparing LIPG expression levels in various hepatocyte cell lines, similar levels of LIPG expression were observed among HC1 cells (KM), PHH cells, and HepG2-NTCP-C4 cells (Figure 2A). HepG2-NTCP-C4 cells overexpressing FLAG-LIPG (HepG2-NTCP-C4-LIPG cells) were established (Figure 2B), and assays were performed on HBV adhesion to and uptake of these cells. As control compounds, heparin, which has been reported to inhibit HBV adhesion (Schulze, A. et al. Hepatology 46:1759-1768, 2007), and heparanase, an endo-β-D-glucuronidase that cleaves the heparan sulfate side chain of heparan sulfate proteoglycan (HSPG), were used (Bame, KJ. ​​Glycobiology, 11:91R-98R, 2001). Cells (HepG2-NTCP-C4-LIPG cells and control HepG2-NTCP-C4 cells) were pretreated with these compounds for 1 hour, then treated with HBV at 4°C for 3 hours to allow adhesion to the cells, washed off, and cultured at 37°C for 12 hours to detect uptake (Figure 2C). In addition, cells were cultured in a normal culture medium for a further 3 days after HBV inoculation, and intracellular HBV DNA and cccDNA were detected (Figure 2C). HBV adhesion was detected by qPCR of HBV-DNA 3 hours after HBV inoculation at 4°C, in or out of the presence of the compounds (heparin, heparanase). In HepG2-NTCP-C4-LIPG cells, HBV adhesion (Figure 2D), uptake (Figure 2E), and replication of HBV DNA and cccDNA (Figures 2F and G) were all increased compared to control HepG2-NTCP-C4 cells. Furthermore, this increase was reduced to the same level as the control group by adding heparin or treating with heparanase.

[0086] Next, we investigated the effects of a LIPG inhibitor (GSK-264220A) on HBV adhesion and uptake. The LIPG inhibitor inhibited HBV adhesion and uptake in a concentration-dependent manner in HepG2-NTCP-C4-LIPG cells (Figure 2H and I). This did not affect control HepG2-NTCP-C4 cells. Furthermore, when LIPG constitutive knockdown HepG2-NTCP-C4 cells were created by introducing LIPH shRNA, HBV adhesion, uptake, and replication were significantly reduced in these cells (Figure 3).

[0087] These results indicate that LIPG contributes to HBV infection. Interestingly, in RD cells, which are not hepatocyte lines, overexpression of LIPG did not result in HBV uptake or replication (Figure 4).

[0088] We investigated whether the enhancement of HBV infection by LIPG is NTCP-dependent. Using a lentiviral expression system, we established Huh7 cells, HepG2 cells, and HepG2-NTCP-C4 cells that constitutively overexpress FLAG-LIPG (Figure 5A). When these cells were infected with HBV, the amount of cccDNA was measured after 24 or 72 hours. Huh7 cells maintained HBV replication due to FLAG-LIPG expression (Figure 5B), but no effect of LIPG overexpression on HBV replication was observed in HepG2 cells (Figure 5C). HepG2-NTCP-C4 cells maintained HBV replication even in the control group, but this was further enhanced by LIPG overexpression (Figure 5D). When we examined the NTCP expression levels of each cell type, Huh7 cells and Huh7-LIPG cells showed significantly lower levels compared to HepG2-NTCP-C4 cells (Figure 5E).

[0089] Therefore, to completely eliminate the effects of NTCP in Huh7 cells and Huh7-LIPG cells, NTCP was knocked out of Huh7 cells using CRISPR-Cas9 gene editing technology. The establishment of NTCP KO cell lines was performed as follows: sgRNA CRISPR / Cas9 lentivirus was produced by co-introducing an NTCP-targeting sgRNA expression vector, a non-targeting control (abm, Richmond, Canada), and a third-generation packaging system mix (abm) into Lenti-X 293T cells using FuGENE-HD transfection reagent. After 72 hours, the supernatant was collected, filtered through a 0.22 μm syringe filter, and introduced into Huh7 cells. 72 hours after introduction, the cells were treated with puromycin (5 mg / mL) for antibiotic selection. To avoid clonal bias, an antibiotic-resistant bulk cell population was used in the experiment.

[0090] NTCP was detected in Huh7 cells introduced with a non-target single guide RNA (sgRNA) containing a scrambled sequence, but not in NTCP-KO cells introduced with NTCP-specific sgRNA (Figure 5F). These cells were forced to express LIPG, and adhesion and uptake assays were performed using the method described above. In both Huh7-WT cells and Huh7-NTCP-KO cells, overexpression of LIPG increased HBV adhesion to cells (Figure 5G), uptake (Figure 5H), and replication (HBV-DNA, cccDNA, pgRNA transcripts) (Figures 5I, J, K) to the same levels.

[0091] These results indicate that LIPG promotes HBV adhesion and uptake independently of NTCP.

[0092] [Example 3]: LIPG contributes to caveolae-dependent HBV uptake. Next, we investigated whether the increase in HBV uptake by LIPG was clathrin-dependent or caveola-dependent. Overexpression of LIPG in Huh7 cells promoted Caveolin-1 protein expression (Figure 6A). Therefore, we knocked down Caveolin-1 using Caveolin-1-specific siRNA and investigated how the increase in HBV adhesion, uptake, and replication mediated by LIPG was affected. Caveolin-1-specific siRNA and negative control siRNA were obtained from Sigma-Aldrich. Lipofectamine RNAiMAX Transfection Reagent (Invitrogen) was used for siRNA transfection. Caveolin-1 protein expression was detected in Huh7-Cont cells and Huh7-LIPG cells introduced with control siRNA, but it was significantly reduced in these cells with Caveolin-1 knocked down (Figure 6B). Knockdown of Caveolin-1 suppressed the increase in HBV adhesion due to LIPG overexpression (Figure 6C), and the increase in uptake and replication (cccDNA) was blocked to the same level as the control (Figures 6D and E). Furthermore, neither the clathrin endocytosis inhibitor chlorpromazine (CPZ) nor the macropinocytosis inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA) affected the increase in LIPG-mediated HBV replication in Huh7-LIPG cells (Figures 6F and G), whereas LIPG inhibitors reduced HBV replication in Huh7-LIPG cells (Figure 6H).

[0093] These results indicate that LIPG is involved in caveolae-dependent HBV uptake and promotes HBV replication. Furthermore, in Huh7 cells overexpressing LIPG, not only Caveolin-1 but also Vimentin expression was increased (Figure 7). Vimentin is known to play an important role in the adhesion, entry, and replication of human papillomavirus (HPV), influenza A virus (IAV), and dengue virus (DENV) (Ramos, I. et al. Int. J. Mol. Sci. 21: 4675, 2020), suggesting that the increase in Vimentin expression due to LIPG may contribute to increased HBV adhesion, entry, and replication. Recently, it has been reported that E-cadherin controls HBV entry by influencing the distribution of NTCP (Hu, Q. et al. Front. Cell Infect. Microbiol. 10:74, 2020), but our results showed that LIPG overexpression did not affect E-cadherin expression (Figure 7). Furthermore, E-cadherin protein expression was much weaker in HepG2-NTCP-C4 cells than in Huh7 cells (Figure 7), suggesting that E-cadherin is not involved in the increased adhesion, uptake, and replication of HBV by LIPG.

[0094] [Example 4]: LIPG does not affect clathrin-dependent viral endocytosis. Furthermore, to investigate the effect of LIPG on viral uptake, we examined the replication of hepatitis C virus (HCV), myocarditis virus (EMCV), and influenza virus (Flu), which enter cells in a clathrin-dependent manner, in Huh7-LIPG cells. Overexpression of LIPG tended to reduce the replication of HCV (Figure 8A) and EMCV (Figure 8B), but had no effect on Flu replication (Figure 8C). These results indicate that LIPG does not affect clathrin-dependent endocytosis.

[0095] The results above reveal that LIPG is a factor that plays an important role in HBV adhesion and uptake, and that it functions as a cofactor that supports intracellular entry of HBV independently of NTCP. In other words, it is suggested that HBV is taken up into hepatocytes by caveolin-dependent endocytosis after binding to the LIPG-HSPG complex on the cell membrane surface. This pathway is consistent with the action of LIPG in the uptake of HDL into cells (Gerold, G. Cold Spring Harb. Perspect Med. 10, a036830, 2020), and it is thought that HBV enters cells nonspecifically by utilizing this pathway.

[0096] Next, using the in vitro virus (IVV) method (Nemoto, N. et al., FEBS Lett., 414:405-408, 1997; Miyamoto-Sato, E. et al., Nucleic Acids Res., 28:1176-1182, 2000; WO 03 / 106675 A1), the interaction between the endothelial lipases LIPG and HSPG was inhibited, and as a result, peptides that inhibit HBV entry into cells were identified in a random peptide library (size 10). 13 We aimed to develop peptide drugs that could help cure hepatitis B virus by searching for individual samples. Furthermore, to understand the role of LIPG in hepatitis B virus entry, we aimed to elucidate the mechanism of action of LIPG in curing hepatitis B.

[0097] [Example 5]: Preparation of a DNA library (5-1) DNA Library Design The design of the DNA library for IVV screening is shown in Figure 9. In the IVV method, puromycin, a type of antibiotic, is attached to the 3' end of mRNA via a PEG (polyethylene glycol) spacer. This is then used as a template for cell-free translation, forming IVVs, simple mRNA-protein ligatures in which the protein and mRNA are covalently linked via puromycin (Miyamoto-Sato, E. et al., Nucleic Acids Res., 31: e78, 2003). After in vitro, IVVs containing proteins that bind to bait (proteins, peptides, antigens, etc.) are selected from the constructed IVV library. The gene (mRNA) linked to these IVVs is then amplified by reverse transcription and PCR, and its base sequence is deciphered using a DNA sequencer, allowing for easy identification of interacting proteins, peptides, and antibodies.

[0098] (5-2) Synthesis of DNA libraries A solution containing a green fluorescent protein (GFP) sequence DNA was mixed with 1 μl of 10× KOD plus buffer (TOYOBO), 10 μl of 2 mM dNTPs (TOYOBO), 4 μl of 25 mM MgSO4, 3 μl of forward primer: GSP6-GFP-F (10 pmol / μl), 3 μl of reverse primer: GFP-R (10 pmol / μl), and 2 μl of KOD plus polymerase (TOYOBO). RNase-free water was added to this mixture to make a total volume of 100 μl. This mixture was placed in one tube, and a total of 300 μl (3 minutes in the tube) was subjected to PCR. The PCR reaction was performed at 94°C for 5 minutes, followed by 16 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 68°C for 2 minutes, and then at 68°C for 5 minutes. The cDNA library was purified using the Wizard SV Gel PCR Clean-Up System (Promega) and recovered as a 50 μl GSP6-GFP-DNA solution.

[0099] 1 μl of DNA solution containing an Atail sequence, 10 μl of 10× KOD plus buffer (TOYOBO), 10 μl of 2 mM dNTPs (TOYOBO), 4 μl of 25 mM MgSO4, 3 μl of forward primer: Flag-His-F (10 pmol / μl), 3 μl of reverse primer: Atail (R) (10 pmol / μl), and 2 μl of KOD plus polymerase (TOYOBO) were mixed with RNase-free water to make a total volume of 100 μl. This mixture was placed in one tube, and a total of 300 μl (3 tubes) was used for PCR reaction. PCR was performed at 94°C for 5 minutes, followed by 16 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 68°C for 2 minutes, and then at 68°C for 5 minutes. The cDNA library was purified using the Wizard SV Gel PCR Clean-Up System (Promega) and recovered as a 50 μl Flag-His Atail-DNA solution.

[0100] 1 μl of Flag-His Atail-DNA solution, 10 μl of 10×KOD plus buffer (TOYOBO), 10 μl of 2 mM dNTPs (TOYOBO), 4 μl of 25 mM MgSO4, 3 μl of forward primer: 16NNS-F or 9NNS-F (10 pmol / μl), 3 μl of reverse primer: Atail (R) (10 pmol / μl), and 2 μl of KOD plus polymerase (TOYOBO) were mixed with RNase-free water to make a total volume of 100 μl. This was placed in one tube, and a total of 300 μl (3 tubes) was used for PCR reaction. PCR was performed by reacting at 94°C for 5 minutes, then repeating 12 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 68°C for 2 minutes, followed by reacting at 68°C for 5 minutes. The cDNA library was purified using the Wizard SV Gel PCR Clean-Up System (Promega) and recovered as 50 μl of 16NNS Atail-DNA solution or 9NNS Atail-DNA solution.

[0101] 1 μl of GSP6-GFP-DNA solution, 1 μl of 16NNS Atail-DNA solution or 9NNS Atail-DNA solution, 10 μl of 10×KOD plus buffer (TOYOBO), 10 μl of 2 mM dNTPs (TOYOBO), 4 μl of 25 mM MgSO4, 3 μl of forward primer: GSP6-GFP-F (10 pmol / μl), 3 μl of reverse primer: Atail (R) (10 pmol / μl), and 2 μl of KOD plus polymerase (TOYOBO) were mixed with RNase-free water to make a total volume of 100 μl. This mixture was placed in one tube, and a total of 300 μl (3 tubes) was used for overlap PCR. PCR was performed at 94°C for 5 minutes, followed by 12 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 68°C for 2 minutes, and then at 68°C for 5 minutes. The cDNA libraries were purified using the Wizard SV Gel PCR Clean-Up System (Promega) and recovered as 50 μl cDNA libraries 16NNSLib and 9NNSLib.

[0102] [Table 1]

[0103] [Example 6]: Preparation of IVV library (6-1) Library transcription 2 pmol each of cDNA libraries 16NNSLib and 9NNSLib were mixed with 8 μl of 5×SP6 buffer, 2 μl of ATP (100 mM), 2 μl of CTP (100 mM), 2 μl of UTP (100 mM), 4 μl of GTP (10 mM), 5 μl of cap analog (m7G(5')PPP(5')G) (Thermo Fisher Scientific) (40 mM), 4 μl of enzyme mix SP6RNA polymerase (Promega), and RNase-free water to a total volume of 40 μl. After reacting at 37°C for 3 hours, 10 μl of RQ1 RNase-Free DNase (Promega) was added, and the mixture was reacted at 37°C for another hour. RNA was purified using the RNeasy Mini kit (Qiagen). Specifically, RNase-free water is added to the transfer reaction solution to make a total volume of 100 μl, 350 μl of RLT buffer (Qiagen), 3.5 μl of 2-mercaptoethanol, and 250 μl of (100%) ethanol are added, and the mixture is placed on an RNeasy mini-spin column. After centrifugation at 25°C, 12000 rpm for 15 seconds, the discharged solution is removed. 500 μl of RPE buffer (Qiagen) is added to the same column, and after centrifugation at 25°C, 12000 rpm for 15 seconds, the discharged solution is removed. Another 500 μl of RPE buffer (Qiagen) is added to the same column, and after centrifugation at 25°C, 12000 rpm for 2 minutes, the discharged solution is removed. The column is then replaced with a new tube, and centrifuged at 25°C, 12000 rpm for 1 minute. The column is then replaced with a new tube again, and 33 μl of RNase-free water is added to the column. After standing at room temperature for 10 minutes, the mixture is centrifugated at 25°C, 13200 rpm. The RNA solution was collected by centrifugation at rpm for 1 minute.

[0104] (6-2) Ligation with PEG spacers 32 μl of RNA solution, 5 μl of T4 ligation 10× buffer, 1 μl of 0.1 M DTT, 0.5 μl of 40 mM ATP, 5 μl of 100% DMSO, 1 μl of 0.1% BSA, 1 μl of RNase inhibitor (TOYOBO), 0.5 μl of puromycin-bound polyethylene glycol photocleaved linker molecule (10 nmol), 1 μl of polyethylene glycol (PEG) 2000 (Nippon Oil & Fats) (30 nmol), and 3 μl of T4 RNA ligase (Takara) (40 U / μl) were mixed and reacted at 15°C for 15 hours under light-shielding conditions. The resulting PEG-RNA bound to the spacer molecule was purified using the RNeasy Mini kit (Qiagen).

[0105] (6-3) Preparation of IVV library 10 pmol of PEG-RNA, 20 μl of wheat germ extract (ZoeGene), 2 μl of creatine kinase (40 μg / μl) (ZoeGene), 3.2 μl of RNase inhibitor (TOYOBO), and 20 μl of 5× translation buffer (ZoeGene) were mixed with RNase-free water to a total volume of 100 μl. The mixture was reacted at 26°C for 1 hour under light-shielding conditions to perform translation and prepare an IVV library.

[0106] [Example 7]: Preparation of biotinylated LIPG Figure 10 shows a schematic diagram of the binding of endothelial lipase LIPG to heparan sulfate proteoglycan and HBV virus. Figure 11 shows the design of the biotinylated LIPG bait. Results from Examples 1-4 suggest that HBV binds to the LIPG-HSPG complex on the cell membrane surface and is taken up by hepatocytes via caveolin-dependent endocytosis. However, since LIPG has been reported to bind to heparin as a homodimer (Razzaghi et al., PLoS ONE, 2013, 8(3), e55716), the biotinylated LIPG was designed with two heparin-binding domains (RKNRCNSIGYNAKKMRNKRNSKMYLKTRA, SEQ ID NO: 42) of LIPG (SEQ ID NO: 30) linked in series.

[0107] (7-1) Construction of an expression vector for the biotinylated LIPG heparin binding site First, we created a LIPG-BioFLAGHis-pcDNA vector by adding biotinylation sequences, flag-tags, and His-tag sequences to the full-length LIPG gene.

[0108] 0.5 μl of LIPG expression vector (ORIGENE) pCMV6-LIPG (1 ng / μl), 12.5 μl of KAPA HiFi HS RM, 0.75 μl of 10 μM LIPG-ifpc-F, and 0.75 μl of 10 μM LIPG-ifbio-R were mixed with RNase-free water to a total volume of 25 μl, and a PCR reaction was performed. The PCR was performed at 95°C for 5 minutes, followed by 25 cycles of 98°C for 20 seconds, 60°C for 15 seconds, and 72°C for 1 minute, and then at 72°C for 1 minute. After confirming the DNA band (1533 bp) by agarose gel electrophoresis of the PCR product, it was purified using the Wizard SV Gel PCR Clean-Up System (Promega) and recovered as a 30 μl DNA solution to obtain LIPG-ifpcB.

[0109] BioEase TM A bio-tag (Nucleic Acids Research, 2009, Vol. 37, No. 8, page e64) consisting of the gene (72 amino acids) of the oxaloacetate decarboxylase α subunit of Klebsiella pneumoniae was prepared by PCR using F-Bio primers and R-Bio primers from a plasmid. A flag-tag and a his-tag were then added to this bio-tag. 2.37 μl of bio-tag, 40 μl of 10× KOD plus buffer (TOYOBO), 40 μl of 2 mM dNTPs (TOYOBO), 16 μl of 25 mM MgSO4, 12 μl of F-Bio (10 pmol / μl), 12 μl of Bio-Flag-Histag A stop (10 pmol / μl), and 8 μl of KOD plus polymerase (TOYOBO) were mixed with RNase-free water to a total volume of 200 μl, and the mixture was subjected to PCR. PCR was performed by reacting at 94°C for 5 minutes, followed by 20 or 25 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 68°C for 2 minutes, and then reacting at 68°C for 5 minutes. After confirming the DNA band (264 bp) by agarose gel electrophoresis, the PCR product was purified using the Wizard SV Gel PCR Clean-Up System (Promega) and recovered as a 50 μl DNA solution to obtain Bio-Flag-His.

[0110] 1 μl of LIPG-ifpcB solution, 1 μl of Bio-Flag-His solution, 10 μl of 10×KOD plus buffer (TOYOBO), 10 μl of 2 mM dNTPs (TOYOBO), 4 μl of 25 mM MgSO4, 3 μl of LIPG-ifpc-F (10 pmol / μl), 3 μl of Flag-Histag A stop (10 pmol / μl), and 2 μl of KOD plus polymerase (TOYOBO) were mixed with RNase-free water to make a total volume of 100 μl, which was placed in one tube. A total of 300 μl (3 tubes) was used for overlap PCR reaction. PCR was performed at 94°C for 5 minutes, followed by 12 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 68°C for 2 minutes, and then at 68°C for 5 minutes. The cDNA library was purified using the Wizard SV Gel PCR Clean-Up System (Promega), recovered as a 50 μl DNA solution, and obtained LIPG-BioFlagHis.

[0111] The LIPG-BioFlagHis was introduced into the pcDNA 3.3 vector using the TOPO cloning kit (Invitorogen) according to the procedure. After confirming that the obtained clones had the correct sequence, the colonies were inoculated and cultured at 37°C for 16 hours. PureYield was extracted from the bacterial pellet. TM Plasmid LIPG-BioFlagHis-pcDNA was purified using the Plasmid Maxiprep System (Promega).

[0112] Fragment 2 was created from the vector LIPG-BioFlagHis-pcDNA containing the LIPG gene (Figure 12). 0.5 μl of LIPG-BioFlagHis-pcDNA (1 ng / μl), 12.5 μl of KAPA HiFi HS RM, 0.75 μl of 10 μM LIPG-hb-F2, and 0.75 μl of 10 μM LIPG-hb-if-R2 were mixed with RNase-free water to a total volume of 25 μl, and then subjected to PCR. PCR was performed at 95°C for 5 minutes, followed by 25 cycles of 98°C for 20 seconds, 60°C for 15 seconds, and 72°C for 1 minute, and then at 72°C for 1 minute. After confirming the DNA band (102 bp) of the PCR product by agarose gel electrophoresis, the product was purified using the Wizard SV Gel PCR Clean-Up System (Promega), and recovered as a 30 μl DNA solution to obtain LIPGhb-2 (Fragment 2).

[0113] Fragment 1 was prepared from Fragment 2. 0.5 μl of LIPGhb-2, 12.5 μl of KAPA HiFi HS RM, 0.75 μl of 10 μM LIPG-hb-ifpc-F1, and 0.75 μl of 10 μM LIPG-hb-if-R1 were mixed with RNase-free water to a total volume of 25 μl, and the PCR reaction was carried out. The PCR reaction was performed at 95°C for 5 minutes, followed by 25 cycles of 98°C for 20 seconds, 60°C for 15 seconds, and 72°C for 1 minute, and then at 72°C for 1 minute. After confirming the DNA band (117 bp) by agarose gel electrophoresis of the PCR product, it was purified using the Wizard SV Gel PCR Clean-Up System (Promega) and recovered as a 30 μl DNA solution to obtain LIPGhb-1 (Fragment 1).

[0114] 0.5 μl of Linker, 12.5 μl of KAPA HiFi HS RM, 0.75 μl of 10 μM GSlinker-F1, and 0.75 μl of 10 μM GSlinker-ifbio-R1 were mixed with RNase-free water to a total volume of 25 μl, and the PCR reaction was carried out. The PCR reaction was performed at 95°C for 5 minutes, followed by 25 cycles of 98°C for 20 seconds, 60°C for 15 seconds, and 72°C for 1 minute, and then at 72°C for 1 minute. After confirming the DNA band (75 bp) of the PCR product by agarose gel electrophoresis, the product was purified using the Wizard SV Gel PCR Clean-Up System (Promega) and recovered as a 30 μl DNA solution to obtain GSlink-ifBio (Fragment 3).

[0115] 0.5 μl of LIPG-BioFLAGHis-pcDNA, 10 μl of KAPA HiFi HS RM, 0.6 μl of 10 μM F-Bio, and 0.6 μl of 10 μM pcDNA-KATG-inv were mixed with RNase-free water to a total volume of 20 μl, and the PCR reaction was carried out. The PCR reaction was performed at 95°C for 3 minutes, followed by 25 cycles of 98°C for 20 seconds, 60°C for 15 seconds, and 72°C for 3 minutes, and then at 72°C for 1 minute. After confirming the DNA band (5662 bp) by agarose gel electrophoresis of the PCR product, it was purified using the Wizard SV Gel PCR Clean-Up System (Promega) and recovered as a 30 μl DNA solution to obtain KATG-BFH-inv (Fragment 4).

[0116] 1.0 μl of LIPGhb-1 (Fragment 1), 1.0 μl of LIPGhb-2 (Fragment 2), 1.0 μl of GSlink-ifBio (Fragment 3), 1.0 μl of KATG-BFH-inv (Fragment 4), and 1.0 μl of 5x in fusion HD Enzyme premix (Takara) were mixed and reacted at 50°C for 15 minutes. 2.5 μl was transformed into One Shot TOP10 competent cells and cultured overnight at 37°C to obtain clones. Sequence analysis of the clones confirmed that they were the plasmid shown in Figure 12, LIPG-HBSx2-GS-BioFLAGHis-pcDNA.

[0117] [Table 2]

[0118] (7-2) Expression and purification of the heparin-binding site of biotinylated LIPG Human embryonic kidney cells (293T cells) were cultured in 10% FCS (Nichirei) and DMEM (1.0 g / l Glucose) with L-Gln and Sodium Pyruvate, liquid (Nacalai) medium. 24 hours before transfection, the cells were plated in a 1:2 ratio in 6 cm petri dishes. To each petri dish, plasmid DNA LIPG-HBSx2-GS-BioFLAGHis-pcDNA was placed in 500 μl of Opti-MEM I, to which 20 μl of Lipofectamine 2000, which had been placed in 500 μl of Opti-MEM I and left at room temperature for 5 minutes, was gently added. The mixture was left at room temperature for 20 minutes, then added to the petri dishes containing the 293T cells, and cultured at 37°C under 5% CO2 conditions for 1 day.

[0119] After removing the culture medium from the petri dish, the cells were washed with 2 ml of cold PBS. 800 μl of RIPA buffer (25 mM Tris·HCl, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, pH 7.6), 16 μl of protease inhibitor for animal cell extracts (Cat#25955-11) x50 (4-(2-aminoethyl) fluoride, benzenesulfonyl hydrochloride (AEBSF), aprotinin, E-64, leupeptin hemisulfate monohydrate, bestatin, pepstatin A), and 8 μl of 0.1 M PMSF were added to the cells, and the cells were left on ice for 15 minutes. The cells were detached with a cell scraper, collected in a tube, homogenized with a 21G needle, and left on ice for 60 minutes. The cells were centrifuged at 12,300 rpm for 30 minutes at 4°C, and the supernatant was transferred to a new tube to obtain the cell fraction. A total of two 6cm petri dishes were transfected to obtain the cell fraction. This was used to purify the biotinylated LIPG heparin binding site for fixation. 500 μl of TALON magnetic beads (Clontech) (50% slurry) were washed three times with 5.0 ml of TBS buffer (Nacalai), and 8.0 ml of the above cell fraction was added. The mixture was rotated and stirred overnight at 4°C using a mini-disc rotor (Bio Craft). The beads were washed three times with 5.0 ml of TBS buffer. 500 μl of TBS containing 500 mM imidazole was added, and the mixture was rotated and stirred at 4°C for 1 hour using a mini-disc rotor, after which the supernatant was collected (elution fraction E1). 250 μl of TBS containing 500 mM imidazole was added to the remaining beads, and elution fraction E2 was recovered using the same procedure. To 15 μl of the elution fraction E1, sample buffer LDS (4x) and 6.8 μl of 0.2 mM DTT were added, and the mixture was heated at 70°C for 10 minutes before being subjected to SDS-PAGE. SDS-PAGE was performed on a 4-12% Bis-Tris NuPAGE gel using MES electrophoresis buffer (Invitrogen) at 200 V, 400 mA, for 35 minutes. The gel after electrophoresis was collected in SimplyBlue. TM Staining with SafeStain (Invitorogen) allowed us to purify the biotinylated LIPG heparin-binding site as a band with a molecular weight of 17,508 Da.

[0120] [Example 8]: Selection of peptides to bind to LIPG The selection experiment for peptides that bind to LIPG was carried out using the procedure shown in (Figure 13). (8-1) Immobilization of LIPG For the viacore, a viacore 3000 system was used, and biotinylated LIPG was immobilized on the sensor chip SA. Flow was performed at 10 μl / min using buffer HBS-P (10 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 0.005% Tween-20). Pretreatment for immobilization was performed by repeatedly injecting 10 μl of a solution containing 50 mM NaOH and 1 M NaCl into flow cells 1-4 three times. Biotinylated LIPG, HBSx2-GS-BioFLAGHis (0.7 nM), was immobilized into flow cells 1-4. Flow was performed at 20 μl / min using buffer HBS-P. When 100 μl was manually injected, a total of 807 RU were bound to flow cells 1-4. An extra wash was performed using buffer HBS-P at 10 μl / min, 50% isopropanol, 50 mM NaOH, and 1 M NaCl to clean the sensor tip.

[0121] (8-2) Selection of peptides that bind to LIPG 100 μl of the prepared IVV library was mixed with 4 μl of 0.5 M EDTA to a final concentration of 20 mM, and the mixture was stirred at room temperature for 20 minutes. 100 μl of the IVV library solution was added to a 1 ml Sephadex G200 (Amersham Biosciences) gel, which had been swollen and equilibrated with HBS-P, packed into a column (Bio-Rad). Two drops of the solution were collected into a 96-well plate, and the IVV fractions from wells 1 to 10 were collected. Fluorescence was detected using a Multi-detection Microplate Reader POWERSCAN HT at an excitation wavelength of 485 nm and an fluorescence wavelength of 528 nm. The eluted IVV fractions from wells 3 to 6 were collected. 100 μl of Strep Magne Sphae Paramagnetic Part (9013-20-1) was washed three times with 500 μl of HBS-P, and approximately 200 μl of the IVV fraction was added. The mixture was rotated and stirred at room temperature for 20 minutes, and the supernatant was injected into via cores (Figure 14). Selection in the via cores was performed with buffer HBS-P at 40 μl / min. After binding for 240 seconds and dissociation for 5000 seconds, the via cores were recovered using the via core recovery method and eluted with 7 μl of 10 μM Heparin Sodium Salt (17513-41, Nakarai). The selective pressure for the selection experiment was increased stepwise with each round, as shown in Table 3.

[0122] [Table 3]

[0123] (8-3) Recovery of cDNA library by RT-PCR 7 μl of eluate collected in the selection experiment, 20 μl of 5×RT buffer (TOYOBO), 10 μl of (10 mM) dNTPs (TOYOBO), and 5 μl of reverse primer: Atail (10 pmol / μl) were mixed with RNase-free water to make a total volume of 90 μl. The mixture was reacted at 65°C for 9 minutes, then immediately cooled on ice and allowed to stand for 2 minutes. After that, 5 μl of ReverTra Ace (TOYOBO) and 5 μl of RNase inhibitor (TOYOBO) were added, and the reverse transcription reaction was carried out at 50°C for 30 minutes and then at 99°C for 5 minutes. 100 μl of the reverse transcription reaction solution, 100 μl of 10×KOD plus buffer (TOYOBO), 100 μl of 2 mM dNTPs (TOYOBO), 40 μl of 25 mM MgSO4, 30 μl of forward primer: GSP6omega F (10 pmol / μl), 30 μl of reverse primer: Atail (10 pmol / μl), and 20 μl of KOD plus polymerase (TOYOBO) were mixed with RNase-free water to make a total volume of 1000 μl for the PCR reaction. The PCR reaction was performed at 94°C for 5 minutes, followed by 20 to 40 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 68°C for 2 minutes, and then at 68°C for 5 minutes. The electrophoresis results after the PCR reaction are shown in Figure 15.

[0124] [Example 9]: Cloning and sequencing (9-1) Cloning and DNA sequencing In-fusion cloning from peptide libraries (LIPG) Create insert for library A 4-round selection experiment was performed on the LIPH-4 library. 1 μl of LIPH-4, 100 μl of 10× KOD plus buffer (TOYOBO), 100 μl of 2 mM dNTPs (TOYOBO), 40 μl of 25 mM MgSO4, 30 μl of GFP-F in (10 pmol / μl), 30 μl of His-Atail-R in (10 pmol / μl), and 20 μl of KOD plus polymerase (TOYOBO) were mixed with RNase-free water to a total volume of 1000 μl for PCR. The PCR reaction was performed at 94°C for 5 minutes, followed by 8 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 68°C for 2 minutes, and then at 68°C for 5 minutes. The cDNA library was purified using the Wizard SV Gel PCR Clean-Up System (Promega) and recovered as a 50 μl DNA solution to obtain GFP-LIPH4 in.

[0125] 8 μl of KIgk-GFP-FlagHis, 80 μl of 10×KOD plus buffer (TOYOBO), 80 μl of 2 mM dNTPs (TOYOBO), 32 μl of 25 mM MgSO4, 24 μl of Atail-F iv (10 pmol / μl), 30 μl of 3.3KIgk-GFP-Riv (10 pmol / μl), and 16 μl of KOD plus polymerase (TOYOBO) were mixed with RNase-free water to a total volume of 800 μl, and the PCR reaction was carried out. PCR was performed at 94°C for 2 minutes, followed by 25 cycles of 98°C for 10 seconds and 68°C for 5 minutes and 35 seconds. The cDNA library was purified using the Wizard SV Gel PCR Clean-Up System (Promega), and recovered as a 40 μl DNA solution to obtain the KIgk-GFP-Atail 3.3 vector.

[0126] 0.08 μl of GFP-LIPH4, 0.14 μl of KIgk-GFP-Atail 3.3 vector, and 1.0 μl of 5x in-fusion HD Enzyme premix (Takara) were mixed, and RNase-free water was added to make a total volume of 5 μl. The mixture was reacted at 50°C for 15 minutes. 2.5 μl of the reaction solution was transformed into One Shot TOP10 competent cells and cultured overnight at 37°C to obtain clones. Sequence analysis of the obtained clones was performed using Eurofins DNA sequencing service and ValueRead premix.

[0127] [Table 4] (9-2) Sequence analysis of libraries Sequence analysis of the library revealed eight clones with 16 amino acid residues and twelve clones with 9 amino acid residues, as shown in Table 5.

[0128] [Table 5]

[0129] [Example 10]: Evaluation of clone activity (10-1) Protein preparation In-frame clones were inoculated from a master plate into LB medium containing 20 μg / ml carbenicillin and incubated at 37°C for 16 hours. PureYield was extracted from the bacterial pellet. TM Plasmids were purified using the Plasmid Miniprep System (Promega).

[0130] (10-2) Protein preparation Human embryonic kidney cells (293T cells) were cultured in 10% FCS (Nichirei) and DMEM (1.0 g / l Glucose) with L-Gln and Sodium Pyruvate, liquid (Nacalai) medium. 24 hours before transfection, the cells were plated in a 1:2 ratio in 6 cm Petri dishes. To each 6 cm Petri dish, 8 μg of plasmid DNA was added to 500 μl of Opti-MEM I, and 20 μl of Lipofectamine 2000 (which had been added to 500 μl of Opti-MEM I and left at room temperature for 5 minutes) was gently added. The mixture was left at room temperature for 20 minutes before being added to the 6 cm Petri dish containing the 293T cells. The mixture was then cultured at 37°C under 5% CO2 conditions for 4-6 days.

[0131] (10-3) Pull-down experiment The resin (Streptavidin MagneSphere Paramagnetic Particles) (Promega) was washed using a MagneSphere Technology Magnetic Separation Stand (tweleve-position). 40 μl of resin was placed in a 1.5 ml tube, the solution was removed, and the tube was washed three times with 1 ml of PBS. The resin was transferred to a new tube, LIPG-BioFLAGHis 0.5 nmol (109 μl) or, without bait, 109 μl of PBS was added, and the tube was rotated and mixed with a mini-disc rotor (Bio Craft) and bound at 4°C for 1 hour. After removing the solution, the tube was washed three times with 1 ml of TBST (Tween20 0.05%). The resin was transferred to a new tube, blocked at 4°C for 1 hour with 1 ml of ELISA BSA buffer (Nacalai Tesque Co., Ltd. TBS, 0.05%, Tween 20, 1% BSA), and washed three times with 1 ml of ELISA BSA buffer. The resin was transferred to a new tube, and 1000 μl of culture supernatant expressing Kigk-LIPH4 was mixed with 40 μl of resin treated with LIPG-BioFLAGHis or without bait. The mixture was then rotated and stirred using a mini disc rotor (Bio Craft) and allowed to fuse at 4°C for 1 hour and 30 minutes. After removing the solution, the resin was washed three times with 500 μl of TBST (Tween20 0.1%), and the recovered resin was Western-blotted.

[0132] Water, sample buffer LDS (4x), and 0.2 mM DTT were added to each resin, and after heating at 70°C for 10 minutes, the samples were subjected to SDS-PAGE. SDS-PAGE was performed using 4-12% Bis-Tris Gel, NuPAGE MES SDS electrophoresis buffer (Invitrogen) at 200V, 400mA, for 35 minutes, followed by transfer using Mini Format, 0.2 μm PVDF, Single application (BIORAD) Trans-Blot Turbo. The membranes were blocked with Blocking One Buffer:TBST (1:9) and reacted with anti-Flag-HRP (Sigma: A8592) diluted 2:3000 with Blocking One Buffer:TBST (1:9). Detection was performed using ECL (Enhanced ChemiLuminescence) with ChemiDoc (BIORAD).

[0133] A pull-down assay (Figure 16) was performed on full-length LIPG (500 amino acids) using the LIPH4 peptide. Of the 20 LIPH4 clones evaluated after 4 rounds of selection experiments, 9 (KIgk-LIPH4-23 (SEQ ID NO: 1), KIgk-LIPH4-90 (SEQ ID NO: 4), KIgk-LIPH4-16 (SEQ ID NO: 8), KIgk-LIPH4-74 (SEQ ID NO: 9), KIgk-LIPH4-87 (SEQ ID NO: 11), KIgk-LIPH4-14 (SEQ ID NO: 14), KIgk-LIPH4-2 (SEQ ID NO: 15), KIgk-LIPH4-12 (SEQ ID NO: 17), KIgk-LIPH4-15 (SEQ ID NO: 18)) were positive.

[0134] (10-4)Cell culture For culturing HepG2-NTCP-C4 cells, HepG2-NTCP-C4-Dox-inducible DOCK11-Halo cells (HepG2-NTCP-C4 cells that express Halo-tagged DOCK11 using a Tet expression induction system), HepG2.2.15, HepG292 (HepG2 cells that stably express a helper plasmid lacking the packaging signal (ε) located upstream of the HBV core), and Huh7-NTCP-YFP cells (Huh7 cells that stably express YFP-fused NTCP), a culture medium consisting of DMEM (Thermo Fisher Scientific) supplemented with 10% FBS (Thermo Fisher Scientific), 1% L-glutamine (Thermo Fisher Scientific), and 1% penicillin / streptomycin (Thermo Fisher Scientific) was used, and the cells were cultured at 37°C in a 5% CO2 incubator.

[0135] (10-5) Infection experiments using PXB cells For the source medium, a PXB-cell culture medium (PhoenixBio) was prepared using HBV-infected chimeric mouse serum (PhoenixBio) at a final concentration of 5 genome equivalent / cell, DMSO at a final concentration of 2%, and PEG 8000 solution at a final concentration of 4%. Various synthesized peptides were prepared at different concentrations and added to this medium, along with the PXB cells (PhoenixBio). After standing in a 37°C, 5% CO2 incubator for 20-28 hours, the medium was removed and the wells were washed once with PBS(-). The PXB-cell culture medium was then changed by adding DMSO at a final concentration of 2% and various synthesized peptides at different concentrations to the PXB cells. This medium change was performed 1, 5, and 9 days after infection, and the samples were collected on day 14.

[0136] (10-6) Determination of HBV-DNA and cccDNA For DNA extraction from samples, use Smitest. TMEX-R&D (Medical & Biological Laboratories) was used. Quantitative PCR for HBV-DNA was performed using extracted DNA as a template. For cccDNA detection, quantitative PCR was performed using DNA treated with T5 Exonuclease (New England Biolabs) as a template. The T5 Exonuclease treatment was carried out at 37°C for 30 minutes, followed by DNase inactivation at 80°C for 15 minutes. The following primer set was used for detection. Forward primer: 5'-ACTCACCAACCTCCTGTCCT-3' (SEQ ID NO: 61) Reverse primer: 5'-GACAAACGGGCAACATACCT-3' (SEQ ID NO: 62) TaqMan probe: 5'-(FAM)-TATCGCTGGATGTGTCTGCGGCGT-(TAMRA)-3'(Sequence ID 63)

[0137] (10-7) Infection experiments using HBV derived from HepG2.2.15: Cells were infected with HBV derived from HepG2.2.15 at a dose equivalent to 20,000 genomes per cell. 12–15 days after infection, the cells were transfected with Lipofectamine 3000 using plasmids encoding various peptides. Samples were collected 3–5 days after transfection, and HBV-DNA, cccDNA, pgRNA, etc., were evaluated.

[0138] [Example 11]: Anti-HBV activity of screened LIPG-binding peptides (11-1) Anti-HBV activity of LIPG-binding peptide in HepG2-NTCP-C4 cells The anti-HBV activity of the LIPG peptide in HepG2-NTCP-C4 cells was evaluated for clones that were positive in pull-down experiments against full-length LIPG (Figure 17). The plasmid containing the peptide was tagged with a secretion signal. Therefore, it is thought that transfection of cells with the plasmid causes the peptide to be secreted extracellularly and bind to extracellular LIPG. When KIgk-LIPH4-23 was added, the copy number of HBV-DNA decreased to near zero. In addition, all other clones that were positive in the pull-down experiment also showed a decrease in the copy number of HBV DNA compared to the control. Furthermore, the copy number of cccDNA was decreased in most clones.

[0139] [Example 12]: Anti-HBV activity of LIPG-binding peptide in PXB cells PXB cells, which are fresh human hepatocytes isolated from PXB mice (human hepatocyte chimeric mice), are human hepatocytes that maintain high levels of human-type liver function and are susceptible to persistent hepatitis B virus (HBV) infection. Therefore, the anti-HBV activity of LIPG-binding peptides was evaluated using PXB cells. Since the LIPG-binding peptides were added from outside the cell, they were chemically synthesized (Eurofins Genomics, Inc.). Clones that showed good results in Examples 10 and 11 (KIgk-LIPH4-23, KIgk-LIPH4-74, KIgk-LIPH4-15, KIgk-LIPH4-2, KIgk-LIPH4-14, KIgk-LIPH4-87) were selected, and the peptides were chemically synthesized. Furthermore, homology searches were performed on LIPH4-23 (SEQ ID NO: 1), which showed remarkable anti-HBV activity in HepG2-NTCP-C4 cells, using BLASTP, and the results showed high homology with netrin (Netrin-1, Netrin-3). Therefore, LIPH4-NTNA and LIPH4-NTNB, consisting of 16 amino acids and focusing on sequences common to netrin, were also chemically synthesized. The chemically synthesized peptides were dissolved in DMSO and subjected to an HBV assay in PXB cells.

[0140] [Table 6]

[0141] Figure 18 shows the anti-HBV activity of LIPG-binding peptides in PXB cells. LIPH4-23, which showed significant anti-HBV activity in HepG2-NTCP-C4 cells, and LIPH4-NTNB, which contains the Netrin sequence, showed overall high anti-HBV activity. Therefore, peptides with a reduced number of residues to 8 amino acids (LIPH4-23s and LIPH4-NTNBs) were chemically synthesized. As a result, the peptides became soluble in PBS. Figure 19 shows the anti-HBV activity of these two LIPG-binding peptides in PXB cells. As a result, it was revealed that LIPH4-23s and LIPH4-NTNBs containing the Netrin-1 sequence significantly reduced the copy number of HBV-DNA and cccDNA, exhibiting very high anti-HBV activity.

[0142] The amino acid sequence of LIPH4-NTNBs contains a cysteine ​​residue (C) second from the N-terminus. Since cysteine ​​residues can form dimers, we chemically synthesized a peptide (LIPH4-NTNBsA) in which the cysteine ​​residue was replaced with an alanine residue to investigate its anti-HBV activity (Figure 20). Figure 20 shows the ratio of copy numbers of the two LIPG-binding peptides, LIPH4-NTNBsA and LIPH4-NTNBs, at various concentrations of HBV-DNA and cccDNA in PXB cells (Figures 20A and 20B). LIPH4-NTNBsA showed equivalent or superior activity compared to LIPH4-NTNBs at both HBV-DNA and cccDNA copy numbers. As a result, it was found that the cysteine ​​residue does not affect anti-HBV activity.

[0143] [Table 7]

[0144] Next, we investigated the anti-HBV activity of full-length Netrin-1 in PXB cells. The full-length human recombinant Netrin-1 (molecular weight 67.5 kDa, SEQ ID NO: 28), consisting of 604 amino acid residues, was purchased from R&D Systems in the United States. For Netrin-1 gene knockdown, CCGG GACTGCGATTCCTACTGCAAGctcgagcTTGCAGTAGGAATCGCAGTCT TTTTTG The shRNA sequence of (Sequence ID 64) was used. The culture conditions for the HBV assay were the same as in Figure 19A. As shown in Figures 21A and 21B, full-length Netrin-1 significantly reduced the copy number of HBV-DNA and cccDNA compared to the control at a concentration of 100 nM. Furthermore, knockdown of the Netrin-1 gene using shRNA significantly increased the copy number of HBV-DNA and cccDNA compared to the control (Figures 21C and 21D).

[0145] The above results suggest that LIPG-binding peptides LIPH4-NTNBs containing the Netrin-1 sequence and full-length Netrin-1 inhibit HBV from entering cells. To further clarify this, we investigated cell adhesion and entry using the Pre-S1 domain, which is the human hepatocyte recognition site for HBV and a region important for HBV infection. The preS1 region is known to be important for HBV infection of hepatocytes (Iwamoto, M. et al., Pro. Natl. Acad. Sci. USA, 116, 8487-8492, 2019). Therefore, we created a preS1 peptide (SEQ ID NO: 66) by modifying the C-terminus of 2-48 residues of the preS1 region (SEQ ID NO: 65) with TAMRA and the N-terminus with a myristoyl group. Using this preS1 peptide and PXB cells (primary human hepatocytes), we investigated the effects of LIPG, LIPG-binding peptides, and full-length Netrin-1 on HBV adhesion or entry. When PXB cells were treated with FLAG-tagged LIPG (FLAG-LIPG), intracellular preS1 peptide levels increased in a FLAG-LIPG concentration-dependent manner (Figure 22A). This result confirmed that LIPG promotes HBV adhesion and entry.

[0146] Next, we investigated the effects of LIPG-binding peptides LIPH4-23S and LIPH4-NTNBS on HBV adhesion and entry via LIPG. When PXB cells pre-treated with LIPG-binding peptides were treated with FLAG-LIPG, we examined the uptake of preS1 into cells and found that the uptake of preS1 peptides was suppressed by the LIPG-binding peptides (Figure 22B).

[0147] These results indicate that LIPG-binding peptides inhibit HBV adhesion and entry via LIPG by binding to LIPG. Since LIPG-binding peptides bind to both LIPG and HSPG, it is suggested that inhibiting the binding of LIPG to HSPG inhibits HBV adhesion. Furthermore, while LIPG is suggested to contribute to HBV caveolae-dependent endocytosis (see Example 3), HBV is known to first bind to polysaccharides containing HSPG on the cell surface, become concentrated, and then bind to receptors such as NTCP to enter the cell (Watashi, K., Wakita, T. Cold Spring Harb. Perspect. Med., 5:a021378, 2015). Therefore, it is suggested that LIPG-binding peptides inhibit the binding of HSPG to HBV, thereby potentially reducing clathrin-dependent endocytosis.

[0148] Since the LIPG-binding peptide LIPH4-23S has homology to the Netrin-1 sequence, and LIPH4-NTNBS mimics the Netrin-1 sequence, we then investigated the effect of Netrin-1 on HBV entry. When PXB cells were treated with Netrin-1 and the uptake of preS1 peptide into cells was examined, preS1 peptide entry was suppressed in a Netrin-1 concentration-dependent manner (Figure 22C).

[0149] Furthermore, when PXB cells treated with Netrin-1 were further treated with FLAG-LIPG and preS1 peptide uptake was examined, Netrin-1 suppressed the promotion of preS1 peptide uptake by FLAG-LIPG (Figure 22D). In addition, by immobilizing biotin-labeled heparan sulfate on streptavidin-coated plates and adding the preS1 peptide, the binding of the preS1 region of HBV to heparan sulfate present on the cell surface was examined. The fluorescence of TMR detected by the addition of the preS1 peptide increased, indicating that the preS1 region has the ability to bind to heparan sulfate even on its own (Figure 22E). Furthermore, this binding was promoted in a concentration-dependent manner with FLAG-LIPG, suggesting that LIPG promotes the binding of preS1 to heparan sulfate (Figure 22E). Furthermore, LIPG-promoted binding was also inhibited in a concentration-dependent manner by Netrin-1, the LIPG-binding peptides LIPH4-NTNBS and LIPH4-23S, and their derivatives LIPH4-NTNBSA and LIPH4-23SA, in which the cysteine ​​was replaced with alanine (Figure 22E).

[0150] These results indicate that LIPG promotes HBV uptake into hepatocytes via HSPG, while LIPG-binding peptides and Netrin-1 inhibit HBV uptake into hepatocytes. Since the LIPG-mediated uptake-promoting effect of Netrin-1 is negated, it is possible that the binding of LIPG to HSPG (heparan sulfate proteoglycan) is competitively inhibited by Netrin-1 present on the surface of liver tissue in living organisms.

[0151] [Example 13]: Efficacy study using HBV-infected PXB mice The following experiment was conducted to confirm the efficacy of the test substance (LIPH4-NTNBs, SEQ ID NO: 25) after infecting PXB mice with HBV.

[0152] (13-1) Test substance, solvent, and preparation method Drug A: LIPH4-NTNBs 4mg Properties: powder Quantity: 288 mg (24 mg x 12 tablets) Storage: -80°C Preparation method: Dissolve 24 mg in 1800 μL of PBS and administer at 300 μL / rat (4 mg / rat)

[0153] Drug B: LIPH4-NTNBs 2mg Appearance: Powder Quantity: 144mg (12 mg x 12 tablets) Storage: -80°C Preparation method: Dissolve 12mg in 1800 μl of PBS and administer at 300 μL / rat (2mg / rat)

[0154] Drug C: LIPH4-NTNBs 0.1mg Appearance: Powder Quantity: 7.2mg (0.6 mg x 12 tablets) Storage: -80°C Preparation method: Dissolve 0.6mg in 1800 μl of PBS and administer at 300 μL / rat (0.1mg / rat)

[0155] Solvent Name: PBS Source: Thermo Fisher Scientific (Life) 10010049 PBS pH 7.4 Storage: Refrigeration

[0156] (13-2) Virus used The virus used was HBV distributed by Phoenix Bio Co., Ltd. Virus name: Hepatitis B Virus Strain name: PBB004 (Genotype C) BSL: Two Virus titer: .1E + 09 copies / mL (2 tubes of 10 μL each) Storage: Store in an ultra-low temperature freezer Preparation method: Thaw one tube of the cryopreserved virus solution and prepare it to 1.0E + 06 copies / mL using physiological saline (Otsuka Pharmaceutical Co., Ltd.).

[0157] (13-3) Animals used Animal species: Mouse Strain: PXB mouse (a mouse in which human hepatocytes have been introduced into cDNA-uPAwild / + / SCID mice, and which has a predicted human hepatocyte replacement rate of 70% or higher, calculated based on the mouse blood h-alb concentration) Source: Phoenix Bio Co., Ltd. Gender: Male Age: 12 weeks or older (at time of arrival) More than 9 weeks after human liver cell transplantation. Number of animals used: 30 animals Reason for animal species selection: It is commonly used as an HBV infection model.

[0158] (13-4) Animal management conditions 13-4.1 Rearing conditions (SOP / Environment / 504) Room temperature 24 ± 3℃, humidity 50 ± 20%, ventilation (10-25 times / hour), lighting 12 hours (8:00-20:00)

[0159] 13-4.2 Feed (SOP / Feeding / 206, SOP / Feeding / 512) The animals were given free access to a feed mixture of MF (Oriental Yeast Co., Ltd.) and PS-A (Oriental Yeast Co., Ltd.) in a 2:1 ratio.

[0160] 13-4.3 Drinking water (SOP / Animal Rearing / 206, SOP / Animal Rearing / 512) Drinking water prepared by adding sodium hypochlorite (final concentration: 14.4 ppm) to autoclaved tap water was available for free consumption.

[0161] 13-4.4 Cage (SOP / Animal / 301) Use autoclaved cages. Keep 1 to 5 animals per cage. Cage changes, feeding (replenishment), and water bottle changes should be performed simultaneously to reduce the risk of microbial contamination.

[0162] 13-4.5 Quarantine and habituation (SOP / Animals / 301) From the time of animal arrival until the end of quarantine and acclimation, the general condition is observed daily. Also, the body weight is measured on the arrival day and at the end of quarantine and acclimation.

[0163] 13-4.6 Grouping (SOP / Test / 002) Blood sampling is performed at 6 weeks after virus inoculation, and based on the amount of HBV DNA in the blood, mice are allocated to each group at 8 weeks after virus inoculation (the first administration day of the test substance).

[0164] 13-4.7 Animal Identification (SOP / Test / 001) Individual identification is performed on the dorsal tail using oil-based ink. A label with the test number, animal number, test period, test group, name of the test responsible person, etc. is attached to the cage.

[0165] (13-5) Test Group Composition and Administration Schedule As shown in Table 8 below.

[0166]

Table 8

[0167] Virus Inoculation HBV is inoculated into the mouse tail vein at 1.0E+05 copies / 100 μL / body.

[0168] Administration of the Test Substance From 8 weeks after virus inoculation (Day 0) until the day before sample collection (Day 27), it is intraperitoneally administered to mice at 300 μL / body once every 2 - 3 days (Monday, Wednesday, Friday).

[0169] Necropsy After euthanasia on Day 28, the liver is collected and weighed. For HBV DNA and cccDNA in the liver, a part is cryopreserved and the rest is fixed in formalin for histopathological examination. Also, the lungs, spleen, and kidneys are fixed in formalin.

[0170] Measurement of HBV DNA in the Liver DNA was extracted from RNAlater - immersed liver samples using DNeasy® Blood & Tissue Kits (Qiagen K.K., Tokyo), and the DNA was dissolved in nuclease - free water. After measuring the DNA concentration with a BioPhotometer® 6131 (Eppendorf AG), the final concentration was adjusted to 20 ng / μL using nuclease - free water. The PCR reaction mixture was prepared using 5 μL of the dissolved DNA stock solution or diluted DNA and TaqMan® Fast Advanced Master Mix. Also, for PCR reaction and analysis, a CFX96 Touch TM Real - Time PCR Detection System was used. The PCR reaction was carried out as follows: 50°C for 2 minutes → 95°C for 20 seconds → (95°C for 3 seconds → 60°C for 32 seconds) × 53 cycles. The HBV DNA concentration in the liver was calculated as the average of 2 wells. The sequences of the primers and probes used are shown in Table 9 below. The lower limit of detection by this quantification method is 50 copies / 100 ng DNA. Serum obtained from HBV - infected PXB mice was used as the HBV DNA standard. The HBV DNA concentration contained in this serum was quantified by digital PCR.

[0171]

Table 9

[0172] Measurement of cccDNA 5 μL of the DNA stock solution or diluted DNA purified during the measurement of HBV DNA in the liver was used to prepare a reaction mixture with TaqMan® Fast Advanced Master Mix. Also, for PCR reaction and analysis, a CFX96 Touch TMA Real-Time PCR Detection System was used. The PCR reaction was performed in 50°C for 2 minutes, followed by 95°C for 20 seconds, then (95°C for 3 seconds, followed by 60°C for 32 seconds) for 55 cycles. Liver HBV cccDNA concentration was calculated as the average of two wells. The sequences of the primers (Takara Bio Inc., Shiga) and probes (Takara Bio Inc.) used are shown in Table 10 below. The detection limit for this quantitative method is 1.0 × 10⁻⁶. 2 The DNA count was copies / 100 ng. Additionally, a plasmid containing the entire HBV genome sequence was used as the HBV cccDNA standard.

[0173] [Table 10]

[0174] Histopathological examination (HBsAg immunostaining, HBcAg immunostaining) Liver tissue was fixed in 10% neutral buffered formalin solution, then replaced with 70% ethanol. These samples were sent to the Nara Institute of Pathology (Nara) to prepare paraffin-embedded blocks using standard methods, after which thin sections were obtained.

[0175] Paraffin sections were deparaffinized and then subjected to antigen retrieval by microwave. Primary antibodies (HBsAg (anti-HBsAg antibody, Code: OBT0990, Bio-Rad AbD Serotec Limited, Oxford, UK) or HBcAg (anti-HBcAg antibody, Code: PAB14506, Abnova, Taipei City, Taiwa)) were reacted overnight at 4°C. The primary antibodies were reacted with the biotin-avidin-peroxidase complex and then stained with DAB. After staining the cell nuclei with hematoxylin, these sections were dehydrated, cleared, and mounted. Subsequently, microscopic examination was performed using an optical microscope at Hamli Corporation.

[0176] (13-6) Observation and Measurement Items Measurements of HBV-DNA (Figure 23) in liver tissue obtained by autopsy revealed that LIPH4-NTNBs 4 mg, LIPH4-NTNBs 2 mg, and LIPH4-NTNBs 0.1 mg all reduced the copy number of HBV DNA compared to the control group. Similarly, measurements of ccc DNA (Figure 24) in liver tissue showed that LIPH4-NTNBs 4 mg, LIPH4-NTNBs 2 mg, and LIPH4-NTNBs 0.1 mg all significantly reduced the copy number of HBV DNA compared to the control group.

[0177] Microscopic images of HBsAg immunohistopathological examination are shown in Figure 25. The LIPH4-NTNBs 0.1 mg dose group showed a clear reduction in antigen staining compared to the control (PBS) dose group. Similarly, microscopic images of HBcAg immunohistopathological examination are shown in Figure 26. For this antigen as well, the LIPH4-NTNBs 0.1 mg dose group showed a clear reduction in antigen staining compared to the control (PBS) dose group.

[0178] Based on these results, LIPG-binding peptides (LIPH4-NTNBs) were shown to have a clear anti-HBV effect in animal experiments using human hepatocyte chimeric mice infected with HBV, by reducing HBV-DNA and cccDNA in the liver. Furthermore, since LIPG-binding peptides (LIPH4-NTNBs) were administered after sufficient HBV infection in both in vitro and in vivo assay systems, it is highly probable that LIPG-binding peptides (LIPH4-NTNBs) inhibit the reinfection process in which HBV particles are secreted outside the cell and then re-enter the cell.

[0179] [Example 14]: Further preparation of LIPG-bound peptides LIPH4-23 (SEQ ID NO: 1), which showed remarkable anti-HBV activity in HepG2-NTCP-C4 cells in Example 11, was found to have homology with N4BP1 (NEDD4 Binding Protein 1, SEQ ID NO: 82) through a sequence portion shared with netrin, as revealed by further data analysis. Therefore, three N4BP1 partial peptides, LIPH4-N4BP1a, LIPH4-N4BP1b, and LIPH4-N4BP1c, were chemically synthesized, focusing on the sequence shared with N4BP1. In addition, LIPH4-NTNBs, a peptide containing a netrin partial sequence that showed very high anti-HBV activity in Example 12 and whose in vivo anti-HBV effect was confirmed in Example 13, were chemically synthesized as LIPH4-LNTNBs, a netrin partial peptide with one residue extended to the N-terminus.

[0180] In the following LIPG-binding peptide intracellular delivery experiments, seven LIPG-binding peptides were used, including LIPH4-23s.

[0181] [Table 11]

[0182] [Example 15]: Preparation of a single-chain antibody that binds to ASGR Most proteins in the blood are glycoproteins with sialic acid attached, but as they degrade, they become asialoglycoproteins, which are mainly broken down in the liver. For this reason, the liver has asialoglycoprotein receptors (ASGRs) that specifically recognize and take up asialoglycoproteins in the blood. Anti-ASGR antibodies are also thought to bind to the receptor and be taken up into liver cells in a similar manner. In order to conduct experiments to selectively deliver LIPG-binding peptides into liver cells, we prepared anti-ASGR single-chain antibodies using the IVV method as intrahepatic delivery carriers.

[0183] (15-1) Preparation of biotinylated ASGR First, we created a biotinylated asialoglycoprotein receptor (ASGR) to serve as the antigen. As shown in Figure 27, the asialoglycoprotein receptor (ASGR) is a type II single-pass transmembrane protein with its N-terminus oriented intracellularly and its sugar recognition sites (CRDs) oriented extracellularly. To present this on the ViaCore sensor chip, we created a construct by removing the transmembrane domain and adding biotinylated sequences. Human hepatocytes contain both ASGR1 (SEQ ID NO: 114) and ASGR2 (SEQ ID NO: 116) receptors, which form heterooligomers. Therefore, we created biotinylated ASGR1ex and biotinylated ASGR2ex by adding biotin to the extracellular domains of each receptor.

[0184] (15-1-i) Construction of a biotin ASGR expression vector We created pcDNA3.3 TOPO HisBioFLAG-ASGR1ex vector and pcDNA3.3 TOPO HisBioFLAG-ASGR2ex vector by adding a His-tag, biotinylation sequence, Flag-tag, and sequence to the extracellular domain of ASGR.

[0185] As shown in Figure 28, the extracellular domains of ASGR1 and ASGR2 were cloned. A PCR reaction was performed using 1 μl of cDNA Library, Human Liver (1 ng / μl) (Takara Bio), 10 μl of KAPA HiFi HS RM, 0.6 μl of 10 μM ASGR1-ex-if-F1 or 0.6 μl of ASGR2-ex-if-F1, and 0.6 μl of 10 μM ASGR1-if-R2 or 0.6 μl of ASGR2-if-R2. RNase-free water was added to the mixture to a total volume of 20 μl. The PCR reaction was carried out at 95°C for 5 minutes, followed by 25 cycles of 98°C for 20 seconds, 60°C for 15 seconds, and 72°C for 1 minute, followed by 72°C for 1 minute. After confirming the DNA band by agarose gel electrophoresis, the PCR product was purified using the Wizard SV Gel PCR Clean-Up System (Promega) and recovered as a 30 μl DNA solution to obtain ASGR1-if or ASGR2-if.

[0186] To create expression plasmids of the construct shown in Figure 29, ASGR1-if or ASGR2-if was infusion cloned using the vector pcDNA3.3 TOPO KHisBioFlag, which contains the kozak sequence, His tag sequence, biotinylated sequence, and Flag tag sequence. 1.0 μl of pcDNA3.3 TOPO KHisBioFlagINV, 1.0 μl of ASGR1-if or ASGR2-if, and 1.0 μl of 5x in fusion HD Enzyme premix (Takara) were mixed with RNase-free water to a total volume of 5 μl, and the mixture was reacted at 50°C for 15 minutes. 2.5 μl was transformed into One Shot TOP10 competent cells and cultured overnight at 37°C to obtain clones. Sequence analysis of the clones confirmed that they were either plasmids pcDNA3.3 TOPO KHisBioFlag-ASGR1ex or pcDNA3.3 TOPO KHisBioFlag-ASGR2ex.

[0187] [Table 12]

[0188] (15-1-ii) Expression and purification of biotinylated extracellular domain ASGR1ex or biotinylated extracellular domain ASGR2ex Human embryonic kidney cells (293T cells) were transfected with either the plasmid pcDNA3.3 TOPO KHisBioFlag-ASGR1ex or pcDNA3.3 TOPO KHisBioFlag-ASGR2ex. After culturing at 37°C in 5% CO2 for 48 hours, the resulting cells were extracted with cell lysis buffer (Tris-HCl pH 7.4 25 mM, NaCl 137 mM, KCl 2.68 mM, Triton X-100 1%). M2 agarose beads (ANTI-FLAG® M2 Affinity Gel, Sigma, A2220) washed with TBS were added to the resulting cell extract and mixed at 4°C for 16 hours. After washing three times with TBST (10x TBS-t 1% Tween-20, Nakarai, 12749-21), competitive elution was performed with 150 μg / mL 3x FLAG peptide (Sigma, F4799) diluted with TBS.

[0189] 20 μL of SA beads (Streptavidin MagneSphere® Paramagnetic Particles, Promega) were washed three times with 200 μL of TBST (10x TBS-T 1% Tween-20, Nakalai), and the purified FLAG protein was added. After mixing by swishing at room temperature for 1 hour, the mixture was washed three times with 200 μL of TBST. The beads were suspended in 10 μL of TBS and treated at 70°C for 10 min. Detection by SDS-PAGE and Western blotting using Flag antibodies revealed that KHisBioFlag-ASGR1ex was identified as a band with a molecular weight of 35,597 Da, and KHisBioFlag-ASGR2ex was identified as a band with a molecular weight of 35,708 Da.

[0190] Cell extracts of HisBioFLAG-ASGR1ex obtained by a similar method were bound to SA beads, washed, and then replaced with EKMax buffer (500 mM Tris-HCl, pH 8.0, 10 mM CaCl2, 1% Tween-20). TMEnterokinase (Thermo Fisher Scientific) was added, and EKMax digestion was performed by rotating at 37°C O / N (16.5h). SA beads were adsorbed using a magnetic stand, and the supernatant was collected. Pre-treated EK Away resin was added to the collected supernatant, and EKMax was removed by rotating at rt for 15 min. The supernatant was collected by centrifugation (5000 rcf 2 min x2) and detected by SDS-PAGE and Western blotting using a flag antibody, confirming it as a band of ASGR1ex with a molecular weight of 26394 Da.

[0191] (15-2) Preparation of a single-chain antibody cDNA library (15-2-i) DNA Library Design Antibody antigen recognition is carried out by the N-terminal domains of the H and L chains, which are called variable regions. On the other hand, the other domains are constant for each class and are called constant regions (Figure 30 left). Both the VH and VL chains are composed of three CDRs (Complementarity-determining regions) and four FRs (Framework regions) (Figure 30 right). CDRs consist of diverse amino acid sequences for each antibody, enabling binding to various antigens. As an artificial antibody fragment, there is a single-chain antibody (scFv) in which the VH and VL chains are linked by a peptide linker (Figure 31). To prevent the 15-amino acid peptide linker from interfering with the association between the domains of the VH and VL chains, a linker consisting of three repeats of a GGGGS unit containing glycine and hydrophilic serine (SEQ ID NO: 112) is often used, as it is a sequence that does not force a secondary structure.

[0192] (15-3) Preparation of a mouse-derived single-chain antibody cDNA library Mouse-derived single-chain antibody cDNA libraries were prepared using mouse spleen Poly A+ RNA as the starting material, as shown in Figure 32. Preparation of heavy chain DNA solution, light chain DNA solution, and unified heavy and light chain PCR were performed. (Tabata, N., et al., Nucleic Acids Res., 37, e64, 2009).

[0193] (15-3-i) Preparation of H-chain DNA solution To prepare the single-chain antibody cDNA library, the H-chain DNA solution was prepared first. 11 μl of mouse spleen Poly A+ RNA (5 μg / μl) (DEPC-treated water) (CLONTECH) diluted 100-fold with RNase-free water was mixed with 22 μl of 5×RT buffer (TOYOBO), 11 μl of (10 mM) dNTPs (TOYOBO), 27.5 μl of forward primer MulgG1 / 2 (1 pmol / μl), and 27.5 μl of forward primer MulgG3 (1 pmol / μl). The mixture was reacted at 65°C for 9 minutes, then immediately cooled to 4°C and left to stand for 2 minutes. After that, 5.5 μl of ReverTra Ace (TOYOBO) and 5.5 μl of RNase inhibitor (TOYOBO) were added, and the mixture was reacted at 50°C for 30 minutes and then at 99°C for 5 minutes to synthesize cDNA-H. To 5 μl of cDNA-H solution, 2.5 μl each of the HB primers indicated on the HB primers (1 pmol / μl), 2.5 μl of 10× PCR buffer (TOYOBO), 1.25 μl of forward primer MulgG1 / 2 (1 pmol / μl), 1.25 μl of forward primer MulgG3 (1 pmol / μl), 0.25 μl of KOD DASH polymerase (TOYOBO), and RNase-free water were added to make a total volume of 25 μl, and PCR was performed in each case. PCR was performed at 96°C for 5 minutes, followed by 25 cycles of 96°C for 30 seconds, 50°C for 30 seconds, and 72°C for 1 minute, followed by 72°C for 5 minutes. The amplified genes were examined by 2% agarose gel electrophoresis to identify bands of 500-900 bp, and then treated with phenol / chloroform. Specifically, equal amounts of phenol:chloroform:isoamyl alcohol (25:24:1) were added and thoroughly mixed. The mixture was then centrifuged at 13,200 rpm for 5 minutes at 4°C, and only the aqueous layer was transferred to a new tube. The same amount of phenol:chloroform:isoamyl alcohol (25:24:1) was added again and thoroughly mixed. The mixture was then centrifuged at 13,200 rpm for 5 minutes at 4°C, and only the aqueous layer was transferred to a new tube. The resulting solution was precipitated with ethanol. After centrifuging and drying for approximately 15 minutes, each DNA (19 types) was dissolved in 20 μl of RNase-free water.To each of the 19 synthesized DNA solutions, 1 μl was added, along with 2 μl of the corresponding HB primer (10 pmol / μl) indicated for the HB primers, 10 μl of 10× PCR buffer (TOYOBO), 10 μl of (2 mM) dNTPs (TOYOBO), 2 μl of the VH forward primer mixture HF1:HF2:HF3:HF4 (1:1:1:1) indicated for the HF primers (10 pmol / μl), 0.5 μl of KOD DASH polymerase (TOYOBO), and RNase-free water, bringing the total volume to 100 μl. PCR was then performed on each of these solutions. PCR was carried out at 96°C for 5 minutes, followed by 20 cycles of 96°C for 30 seconds, 50°C for 30 seconds, and 72°C for 1 minute, followed by 72°C for 5 minutes. The amplified genes were examined by 2% agarose gel electrophoresis to identify bands of 500-900 bp, and then treated with phenol / chloroform and precipitated with ethanol. After centrifugation drying for approximately 15 minutes, each DNA (19 types) was dissolved in 10 μl of RNase-free water. The obtained 19 DNA types were subjected to 2% low-melting-point agarose gel (Sigma) electrophoresis, and each band was excised. Each DNA (19 types) was dissolved in 10 μl of RNase-free water. The absorption at 260 nm was measured for each DNA solution, and they were mixed in the ratio HB1:HB2:HB3:HB4:HB5:HB6:HB7:HB8:HB9:HB10:HB11:HB12:HB13:HB14:HB15:HB16:HB17:HB18:HB19 (8:9:4:4:12:4:1:4:12:4:4:2:2:4:4:8:6:1:1) to obtain a total of 0.5 pmol of H-chain DNA solution.

[0194] (15-3-ii) Preparation of light chain DNA solution 10 μl of mouse spleen Poly A+ RNA (5 μg / μl) (DEPC-treated water) (CLONTECH) diluted 100-fold with RNase-free water was mixed with 20 μl of 5×RT buffer (TOYOBO), 10 μl of (10 mM) dNTPs (TOYOBO), and 50 μl of forward primer MuCK (1 pmol / μl). The mixture was reacted at 65°C for 9 minutes, then immediately cooled to 4°C and left to stand for 2 minutes. After that, 5 μl of ReverTra Ace (TOYOBO) and 5 μl of RNase inhibitor (TOYOBO) were added, and the mixture was reacted at 50°C for 30 minutes and then at 99°C for 5 minutes to synthesize cDNA-L. To 5 μl of cDNA-L solution, 2.5 μl each of the LB primers (1 pmol / μl) indicated for LB primers, 2.5 μl of 10× PCR buffer (TOYOBO), 2.5 μl of forward primer MuCK (1 pmol / μl), 0.25 μl of KOD DASH polymerase (TOYOBO), and RNase-free water were added to make a total volume of 25 μl, and PCR reactions were carried out. PCR was performed at 96°C for 5 minutes, followed by 25 cycles of 96°C for 30 seconds, 48°C for 30 seconds, and 72°C for 1 minute, followed by 72°C for 5 minutes. The amplified genes were examined by 2% agarose gel electrophoresis to identify bands of 500-900 bp, and then treated with phenol / chloroform. The resulting solutions were precipitated with ethanol. After centrifugation for approximately 15 minutes, each DNA (18 types) was dissolved in 20 μl of RNase-free water. To each of the 18 synthesized DNA solutions, 1 μl was added, along with 2 μl of the corresponding LB primer (10 pmol / μl) indicated for the LB primers, 10 μl of 10× PCR buffer (TOYOBO), 10 μl of (2 mM) dNTPs (TOYOBO), 2 μl of the LH forward primer mixture LF1:LF2:LF3:LF4:LFλ (1:1:1:1) indicated for the LF primers (10 pmol / μl), 0.5 μl of KOD DASH polymerase (TOYOBO), and RNase-free water, bringing the total volume to 100 μl. PCR was then performed on each of these solutions. The PCR was carried out at 96°C for 5 minutes, followed by 20 cycles of 96°C for 30 seconds, 48°C for 30 seconds, and 72°C for 1 minute, and then at 72°C for 5 minutes.The amplified genes were subjected to 2% agarose gel electrophoresis to identify bands of 500-900 bp, followed by phenol / chloroform treatment and ethanol precipitation. After centrifugation drying for approximately 15 minutes, each DNA (18 types) was dissolved in 10 μl of RNase-free water. The resulting 18 DNA types were subjected to 2% low-melting-point agarose gel (Sigma) electrophoresis, and the respective bands were excised. Each DNA (18 types) was dissolved in 10 μl of RNase-free water. The absorption at 260 nm was measured for each DNA solution, and they were mixed in the ratio LB1:LB2:LB3:LB4:LB5:LB6:LB7:LB8:LB9:LB10:LB11:LB12:LB13:LB14:LB15:LB16:LB17:LBλ (2:4:8:8:8:16:12:4:4:8:16:16:12:4:4:2:2:1) to obtain a total of 0.5 pmol of L-chain DNA solution.

[0195] (15-3-iii) PCR for unifying the H and L chains A total volume of 45.75 μl was added to a solution of synthesized heavy DNA (H-chain) solution, light DNA (L-chain) solution, 0.5 μl of 5'UTR (1 pmol / μl), 0.5 μl of McD-Linker+ (1 pmol / μl), 0.5 μl of McD 3'UTR (His Tag) (1 pmol / μl), 5 μl of 10× PCR buffer (TOYOBO), 5 μl of (2 mM) dNTPs (TOYOBO), 0.25 μl of KOD DASH polymerase (TOYOBO), and RNase-free water, and the PCR reaction was carried out. The PCR reaction was performed at 96°C for 5 minutes, then at 96°C for 30 seconds followed by a 5-minute slope to 58°C, followed by 30 seconds at 58°C and 1 minute at 72°C, repeated 10 times, and then at 72°C for 5 minutes. Next, 45.75 μl of PCR reaction solution, 2 μl of McD-F (1 pmol / μl), 2 μl of McD-R (His Tag) (1 pmol / μl), and 0.25 μl of KOD DASH polymerase (TOYOBO) were added, and the PCR reaction was continued. The PCR was performed at 96°C for 5 minutes, followed by 15 cycles of 96°C for 30 seconds, 58°C for 30 seconds, and 72°C for 1 minute, and then at 72°C for 5 minutes. The obtained DNA was subjected to 1% low-melting-point agarose gel (Sigma) electrophoresis, and each band was excised. The DNA was dissolved in 10 μl of RNase-free water to obtain a mouse-derived single-strand antibody library DNA.

[0196] (15-4) Preparation of a human single-chain antibody cDNA library As shown in Figure 33, a single-chain antibody cDNA library was prepared from human bone marrow-derived lymphocytes using human bone marrow-derived Poly A+ RNA as the starting material. Similar to the mouse study, a human single-chain antibody cDNA library for IVV selection experiments was created by preparing a heavy chain DNA solution and a light chain DNA solution using multiple specific primers, and then performing unified PCR of the heavy and light chains. (J.Mol.Biol., 222,581-597,1991; Nat.Biotechnol.,23,344-348,2005; Antibody Engineering, Springer Lab.Manual,93-108,2001).

[0197] Furthermore, random mutations were introduced into the prepared mouse heavy chain DNA and mouse light chain DNA, or human heavy chain DNA and human light chain DNA, using Mutazyme II via error-prone PCR. Unified heavy chain and light chain PCR was performed using the mouse mutant heavy chain DNA and mouse mutant light chain DNA to create a cDNA library of mouse mutant single-chain antibodies. Unified heavy chain and light chain PCR was performed using human mutant heavy chain DNA and human mutant light chain DNA to create a cDNA library of human mutant single-chain antibodies.

[0198] (15-5) Selection of single-chain antibodies that bind to ASGR The selection experiment for single-chain antibodies that bind to ASGR was carried out using the procedure shown in Figure 34. (15-5-i) Fixation of ASGR Viacore was immobilized onto the sensor chip SA using the Viacore 3000 system. Flow was performed at 10 μl / min using buffer HBS-P (10 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 0.005% Tween-20). Pretreatment for immobilization was performed by repeatedly injecting 10 μl of a solution containing 50 mM NaOH and 1 M NaCl into flow cells 1-4 three times. First, the bait was immobilized in flow cells 1-4 using the biotinylated extracellular domain ASGR1ex. Flow was performed at 20 μl / min using buffer HBS-P. The amount of bait immobilized is shown in Table 13. Subsequently, the bait was immobilized in flow cells 3-4 using the biotinylated extracellular domain ASGR2ex. Flow was performed at 20 μl / min using buffer HBS-P. The amount of bait immobilized is shown in Table 7. An extra wash was performed using buffer HBS-P at 10 μl / min, 50% isopropanol, 50 mM NaOH, and 1 M NaCl to clean the sensor tip.

[0199] (15-5-ii) Selection of single-chain antibodies that bind to ASGR 100 μl of the prepared single-chain antibody IVV library was mixed with 4 μl of 0.5 M EDTA to a final concentration of 20 mM, and the mixture was rotated and stirred at room temperature for 20 minutes. 100 μl of the IVV library solution was added to a 1 ml Sephadex G200 (Amersham Biosciences) gel, which had been swollen and equilibrated with HBS-P, packed into a column (Bio-Rad). Two drops of the solution were collected into a 96-well plate, and the eluted IVV fractions from wells 1 to 10 were collected. Fluorescence was detected using a Multi-detection Microplate Reader POWERSCAN HT at an excitation wavelength of 485 nm and an fluorescence wavelength of 528 nm. The eluted IVV fractions from wells 3 to 7 were collected. 100 μl of Strep Magne Sphae Paramagnetic Part (9013-20-1) was washed three times with 500 μl of HBS-P, and approximately 200 μl of the IVV fraction was added. The mixture was rotated and stirred at room temperature for 20 minutes, and the supernatant was injected into a via core. The selection conditions in the via core are shown in Table 13. As shown in Figure 34, in the selection experiments of rounds 1 to 3, after the washing process was completed, the sensor tip was removed from the machine, 55 μl of RNase-free water was gently added to the gold film of the sensor tip, and 365 nm UV light was irradiated for 20 minutes to cut the spacer and elute and recover the mRNA portion. In the selection experiments of rounds 4 to 5, after the washing process was completed, competitive elution was performed using ASGR1ex to recover the IVV fraction.

[0200] (15-5-iii) Recovery of cDNA library by RT-PCR 55 μl of the solution recovered in the selection experiment, 20 μl of 5×RT buffer (TOYOBO), 10 μl of (10 mM) dNTPs (TOYOBO), and 5 μl of reverse primer: Flag His tag A02 (10 pmol / μl) were mixed with RNase-free water to make a total volume of 90 μl. The mixture was reacted at 65°C for 9 minutes, then immediately cooled on ice and allowed to stand for 2 minutes. After that, 5 μl of ReverTra Ace (TOYOBO) and 5 μl of RNase inhibitor (TOYOBO) were added, and the reverse transcription reaction was carried out at 50°C for 30 minutes and then at 99°C for 5 minutes. 100 μl of the reverse transcription reaction solution, 100 μl of 10× KOD plus buffer (TOYOBO), 100 μl of 2 mM dNTPs (TOYOBO), 40 μl of 25 mM MgSO4, 30 μl of forward primer: SP6 Omega (10 pmol / μl), 30 μl of reverse primer: Flag His tag A02 (10 pmol / μl), and 20 μl of KOD plus polymerase (TOYOBO) were mixed with RNase-free water to make a total volume of 1000 μl. 10 tubes were then filled with 100 μl of this solution and subjected to PCR. PCR was performed at 94°C for 5 minutes, followed by 20 to 40 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 68°C for 2 minutes, and then at 68°C for 5 minutes.

[0201] Using via cores, the selection pressure in the selection experiment was gradually increased round by round, as shown in Table 13.

[0202] [Table 13]

[0203] (15-6) Cloning and sequencing (15-6-i) Cloning and nucleotide sequences For in-fusion cloning, a library insert was prepared from a single-chain antibody library that binds to ASGR. After 3 rounds of selection experiments, 1 μl of the library was mixed with 100 μl of 10× KOD plus buffer (TOYOBO), 100 μl of 2 mM dNTPs (TOYOBO), 40 μl of 25 mM MgSO4, 30 μl of T7-long-F in (10 pmol / μl), 30 μl of Flag (Histag) in R (10 pmol / μl), and 20 μl of KOD plus polymerase (TOYOBO). RNase-free water was added to make a total volume of 1000 μl, and the mixture was subjected to PCR. PCR was performed at 94°C for 5 minutes, followed by 8 cycles of 94°C for 30 seconds, 58°C for 30 seconds, and 68°C for 2 minutes, and then at 68°C for 5 minutes. The cDNA library was purified using the Wizard SV Gel PCR Clean-Up System (Promega), and recovered as a 50 μl DNA solution to obtain T7-ASGR3.

[0204] 8 μl of pcDNA 3.3 vector containing the kozak-Igk-T7 tag sequence, His sequence, and stop codon, 80 μl of 10× KOD plus buffer (TOYOBO), 80 μl of 2 mM dNTPs (TOYOBO), 32 μl of 25 mM MgSO4, 24 μl of His-stop-topo F (10 pmol / μl), 24 μl of Igk-T7-R (10 pmol / μl), and 16 μl of KOD plus polymerase (TOYOBO) were mixed with RNase-free water to a total volume of 800 μl for PCR reaction. PCR was performed by reacting at 94°C for 2 minutes, followed by 25 cycles of 98°C for 10 seconds and 68°C for 5 minutes and 11 seconds. The cDNA library was purified using the Wizard SV Gel PCR Clean-Up System (Promega), recovered as a 40 μl DNA solution, and obtained the KIgk-stop 3.3 vector.

[0205] 0.08 μl of T7-ASGR3, 0.14 μl of KIgk-stop 3.3 vector, and 1.0 μl of 5x in fusion HD Enzyme premix (Takara) were mixed, and RNase-free water was added to make a total volume of 5 μl. The mixture was reacted at 50°C for 15 minutes. 2.5 μl of the reaction solution was transformed into One Shot TOP10 competent cells and cultured overnight at 37°C to obtain KIgk-ASGR3 clones. Sequence analysis of the obtained clones was performed using Eurofins DNA sequencing service and ValueRead premix.

[0206] [Table 14]

[0207] (15-6-ii) Sequence analysis of libraries As a result of the library sequence analysis, clones of ASGR-conjugated single-chain antibodies, ASGR3-10M, ASGR3-39D, ASGR4-70D, and ASGR5-24M, were obtained from rounds 3, 4, and 5, respectively, as shown in Table 15.

[0208] [Table 15]

[0209] (15-7) Evaluation of clone activity (15-7-i) Protein preparation Clones were inoculated from a master plate into LB medium containing 20 μg / ml carbenicillin and incubated at 37°C for 16 hours. Plasmids were purified from the bacterial pellet using the PureLink HiPure Plasmid Maxiprep Kit (Invitrogen).

[0210] (14-7-ii) Protein preparation Human embryonic kidney cells (293T cells) were cultured in 10% FCS (Nichirei) and DMEM (1.0 g / l Glucose) with L-Gln and Sodium Pyruvate, liquid (Nacalai) medium. 24 hours before transfection, the cells were plated in a 1:2 ratio in 6 cm Petri dishes. To each 6 cm Petri dish, 8 μg of plasmid DNA was added in 500 μl of Opti-MEM I, to which 20 μl of Lipofectamine 2000 or 20 μl of PEI-MAX (added to 500 μl of Opti-MEM I and left at room temperature for 5 minutes) was gently added. The mixture was left at room temperature for 20 minutes before being added to the 6 cm Petri dish of 293T cells. The mixture was then cultured at 37°C under 5% CO2 conditions for 4-6 days.

[0211] (15-7-iii) Pull-down experiment Washing of the resin (Streptavidin MagneSphere Paramagnetic Particles) (Promega) was performed using a MagneSphere Technology Magnetic Separation Stand (tweleve-position). 40 μl of resin was transferred to a 1.5 ml tube, the solution was removed, and the tube was washed three times with 1 ml of PBS. The resin was transferred to a new tube, and an equal volume of PBS was added for biotinylated extracellular domain ASGR1ex or biotinylated extracellular domain ASGR2ex or no bait. The tubes were rotated and mixed with a mini-disc rotor (Bio Craft) and allowed to bind at 4°C for 1 hour. After removing the solution, the tubes were washed three times with 1 ml of TBST (Tween 20 0.05%). The resin was transferred to a new tube, blocked at 4°C for 1 hour with 1 ml of ELISA BSA buffer (Nacalai Tesque Co., Ltd. TBS, 0.05%, Tween 20, 1% BSA), and washed three times with 1 ml of ELISA BSA buffer. The resin was transferred to a new tube, and 1000 μl each of culture supernatant expressing ASGR3-10M, ASGR3-39D, ASGR4-70D, or ASGR5-24M was mixed with 40 μl of resin treated with biotinylated extracellular domain ASGR1ex, biotinylated extracellular domain ASGR2ex, or without bait. The mixture was then rotated and mixed using a mini-disc rotor (Bio Craft) and allowed to fuse at 4°C for 1 hour and 30 minutes. After removing the solution, the resin was washed three times with 500 μl of TBST (Tween20 0.1%), and the recovered resin was Western-blotted.

[0212] Water, sample buffer LDS (4x), and 0.2 mM DTT were added to each resin, and after heating at 70°C for 10 minutes, the samples were subjected to SDS-PAGE. SDS-PAGE was performed using 4-12% Bis-Tris Gel, NuPAGE MES SDS electrophoresis buffer (Invitrogen) at 200V, 400mA, for 35 minutes, followed by transfer using Mini Format, 0.2 μm PVDF, Single application (BIORAD) Trans-Blot Turbo. The membranes were blocked with Blocking One Buffer:TBST (1:9) and reacted with anti-Flag-HRP (Sigma: A8592) diluted 2:3000 with Blocking One Buffer:TBST (1:9). Detection was performed using ECL (Enhanced ChemiLuminescence) with ChemiDoc (BIORAD).

[0213] A pull-down assay using ASGR-conjugated single-chain antibody (Figures 35 and 36) was performed. Clones ASGR3-10M and ASGR3-39D, which underwent 3 rounds of selection testing, recognized the antigen more strongly than the control. ASGR3-10M recognized both antigens, while ASGR3-39D particularly strongly recognized the ASGR1 antigen. Clones ASGR4-70D and ASGR5-24M, which underwent 4 and 5 rounds of selection testing, recognized the antigen more strongly than the control. ASGR4-70D and ASGR5-24M particularly strongly recognized the ASGR1 antigen.

[0214] [Example 16]: Preparation of a LIPG-conjugated peptide-anti-ASGR single-chain antibody fusion Figure 37 shows a schematic diagram of a hepatocyte-selective delivery method for LIPG-binding peptides using an anti-ASGR single-chain antibody. The LIPG-binding peptide, linked to the anti-ASGR antibody, binds to the receptor and is then encapsulated in early endosomes via endocytosis. A cleavage sequence, which is cleaved by enzymes within the endosome, is incorporated upstream of the LIPG-binding peptide, causing it to dissociate. A membrane fusion-promoting peptide is added downstream of the LIPG-binding peptide, thereby releasing the peptide into the cytoplasm. For nucleus delivery of the LIPG-binding peptide, a nuclear localization signal is added.

[0215] In this example, a single-chain antibody-LIPG-conjugated peptide fusion with the structure shown in Figure 38 was prepared. Selection experiments using the IVV method yielded multiple LIPG-binding peptides and ASGR-binding single-chain antibodies. As shown in Figure 39, fusions were created using ASGR3-10M as the anti-ASGR single-chain antibody and LIPH4-23 (SEQ ID NO: 1), LIPH4-23s (SEQ ID NO: 23), Netrin-1 partial polypeptide LIPH4-NTNBs (SEQ ID NO: 25), Netrin-1 partial polypeptide LIPH4-LNTNBs (SEQ ID NO: 77), N4BP1 partial polypeptide LIPH4-N4BP1a (SEQ ID NO: 78), N4BP1 partial polypeptide LIPH4-N4BP1b (SEQ ID NO: 79), or N4BP1 partial polypeptide LIPH4-N4BP1c (SEQ ID NO: 80) as LIPG-binding peptides. Constructs were created by infusion cloning using the necessary functional peptides for the genes incorporated into the expression vectors. In this example, the sequence recognized by the endogenous protease Furin (RVRR, SEQ ID NO: 111) was used as the cleavage sequence, the membrane fusion-promoting peptide S28 (PFVIGAGVLGALGTGIGGITTSTQFYYK, SEQ ID NO: 107) or S39 (PFVIGAGVLGALGTGIGGITTSTQFYYKLSQELNGDMER, SEQ ID NO: 108) was used as the membrane translocation-promoting peptide, and PAAKRVKLD (SEQ ID NO: 110) was used as the nuclear localization signal. These constructed structures were designated as 10M-L23N, 10M-L23SN, 10M-L23SN-S39, 10M-LNTSN, 10M-LLNTSN, 10M-LLNTSN-S39, 10M-LN4N, 10M-LN4SN, or 10M-TLN4SN (Table 16). The amino acid sequences of the cleavage sequence + LIPG-binding peptide + S28 / S39 + nuclear localization signal portion of these constructs are shown in SEQ ID NOs. 127-135.

[0216] [Table 16]

[0217] [Example 17]: Pull-down assay of N4BP1 against full-length LIPG Since the LIPG-binding peptide showed homology to N4BP1, a pull-down assay was performed to investigate whether LIPG and N4BP1 bind to each other.

[0218] The resin (Streptavidin MagneSphere Paramagnetic Particles) (Promega) was washed using a MagneSphere Technology Magnetic Separation Stand (tweleve-position). 40 μl of resin was placed in a 1.5 ml tube, the solution was removed, and the tube was washed three times with 1 ml of PBS. The resin was transferred to a new tube, and an equal volume of PBS was added for biotinylated full-length LIPG or, without bait, an equal volume of PBS. The tube was rotated and mixed with a mini-disc rotor (Bio Craft) and allowed to bind at 4°C for 1 hour. After removing the solution, the tube was washed three times with 1 ml of TBST (Tween 20 0.05%). The resin was transferred to a new tube, blocked at 4°C for 1 hour with 1 ml of ELISA BSA buffer (Nacalai Tesque Co., Ltd. TBS, 0.05%, Tween 20, 1% BSA), and then washed three times with 1 ml of ELISA BSA buffer. The resin was transferred to a new tube, and 1000 μl of culture supernatant expressing N4BP1 was mixed with 40 μl of resin treated with biotinylated full-length LIPG or without bait. The mixture was rotated and stirred using a mini disc rotor (Bio Craft), and the mixture was allowed to fuse at 4°C for 1 hour and 30 minutes. After removing the solution, the resin was washed three times with 500 μl of TBST (Tween20 0.1%), and the recovered resin was Western-blotted.

[0219] Water, sample buffer LDS (4x), and 0.2 mM DTT were added to each resin, and after heating at 70°C for 10 minutes, the samples were subjected to SDS-PAGE. SDS-PAGE was performed using 4-12% Bis-Tris Gel, NuPAGE MES SDS electrophoresis buffer (Invitrogen) at 200V, 400mA, for 35 minutes, followed by transfer using Mini Format, 0.2 μm PVDF, Single application (BIORAD) Trans-Blot Turbo. The membranes were blocked with Blocking One Buffer:TBST (1:9) and reacted with T7-tag HRP conjugate (Novagen 69048) diluted 2:3000 with Blocking One Buffer:TBST (1:9). Detection was performed using ECL (Enhanced ChemiLuminescence) with ChemiDoc (BIORAD).

[0220] A pull-down assay (Figure 40) of N4BP1, which has homology to the LIPG-binding peptide, was performed and it was found that N4BP1 binds to LIPG.

[0221] [Example 18]: Anti-HBV activity of LIPG-binding peptide delivered into PXB cells - 1 The anti-HBV activity of LIPG-binding peptides delivered into cells was evaluated using PXB cells and anti-ASGR single-chain antibody-LIPG-binding peptide fusions. The fusions used were 10M-L23N, 10M-L23SN, 10M-L23SN-S39, and 10M-LNTSN, which were prepared in Example 16. The anti-HBV activity of the fusions was evaluated using the methods described in "(10-5) Infection experiment using PXB cells" and "(10-6) Quantification of HBV-DNA and cccDNA" according to the time schedule shown in Figure 41A.

[0222] Figures 41B and 41C show the anti-HBV activity of LIPG-binding peptides delivered into PXB cells. All four LIPG-binding peptides—10M-L23N, 10M-L23SN, 10M-L23SN-S39, and 10M-LNTSN—significantly reduced the copy number of HBV-DNA and cccDNA at the peptide concentrations (nM) shown, demonstrating very high anti-HBV activity.

[0223] 10M-L23SN and 10M-L23SN-S39 are fusion compounds prepared using the LIPG-binding peptide LIPH4-23s, while 10M-LNTSN is prepared using the LIPG-binding peptide LIPH4-NTNBs. The anti-HBV activity when LIPH4-23s and LIPH4-NTNBs were added alone to PXB cell culture medium without being bound to an intrahepatocyte delivery carrier is shown in Figures 19B-E, at addition concentrations of 1-100 μM. In contrast, with the fusion compounds 10M-L23SN-S39 and 10M-LNTSN, the copy number of HBV-DNA and cccDNA was significantly reduced at addition concentrations of approximately 2.5-25 nM (Figures 41B, C). From a comparison of these data, it became clear that delivering LIPG-binding peptides into hepatocytes increased anti-HBV activity by approximately 100-1000 times.

[0224] [Example 19]: Anti-HBV activity of LIPG-binding peptide delivered into PXB cells - 2 The anti-HBV activity of LIPG-binding peptides delivered into cells was evaluated using PXB cells and anti-ASGR single-chain antibody-LIPG-binding peptide fusions. Anti-HBV activity was evaluated in the presence of 4% PEG. In the presence of PEG, the copy number of HBV-DNA and cccDNA increases, resulting in a stricter anti-HBV evaluation than under the conditions of Example 18 without PEG. The fusions used were 10M-LN4SN, 10M-LN4N, 10M-LLNTSN, and 10M-LLNTSN-S39, which were prepared in Example 16. The anti-HBV activity of the fusions was evaluated using the methods described in "(10-5) Infection experiment using PXB cells" and "(10-6) Quantification of HBV-DNA and cccDNA" according to the time schedule shown in Figure 42A.

[0225] Figures 42B and 42C show the anti-HBV activity of LIPG-binding peptides delivered into PXB cells. Even under harsh conditions in the presence of PEG, all LIPG-binding peptides—10M-LN4SN, 10M-LN4N, 10M-LLNTSN, and 10M-LLNTSN-S39—significantly reduced the copy number of HBV-DNA and cccDNA, demonstrating very high anti-HBV activity.

[0226] [Example 20]: Mechanism of action of LIPG-binding peptide in cells As mentioned above, LIPH4-23, a LIPG-binding peptide obtained through IVV screening, showed homology not only to netrin but also to the sequence of N4BP1 (NEDD4 Binding Protein 1). Since N4BP1 has RNase activity (residues 617-769 of sequence number 82 are the RNase domain, and D623, D704, and D723 are catalytic sites), it is possible that in HBV-infected cells, N4BP1 binds to LIPG and suppresses the degradation of viral mRNA, thereby contributing to HBV maintenance.

[0227] Therefore, we in vitro investigated whether LIPG (500aa, MW: 56.8kDa) binds to N4BP1, inhibits the degradation of HBV pregenomic RNA, and promotes HBV proliferation, and whether LIPG-binding peptides bind to LIPG, release LIPG-mediated inhibition of N4BP1's RNase activity, promote the degradation of HBV pregenomic RNA, and thus suppress HBV proliferation. HBV L protein RNA of 1203 bases was used as the RNA. LIPH4-NTNBs (SEQ ID NO: 25), which have high homology to N4BP1, were used as the LIPG-binding peptide. First, N4BP1, LIPG, and LIPH4-NTNBs were pre-incubated at 37°C for 30 minutes, then RNA was added and incubated at 37°C for 15 minutes to allow the reaction to proceed. The concentrations were 20 nM RNA, 50 pM N4BP1, 50 pM LIPG, and 50 pM LIPH4-NTNBS. The buffer composition was 25 mM HEPES, 50 mM potassium acetate, 5 mM DTT, 5 mM magnesium acetate, and 0.2 U / ml RNase inhibitor (TOYOBO). The total volume of the reaction mixture was 10 μl.

[0228] Figure 43 shows the results of 2% TAE agarose gel electrophoresis (Buffer: TAE) and ethidium bromide staining after the reaction was completed. As shown in lane 1, it was confirmed that RNA was degraded by N4BP1. As shown in lane 2, in the presence of LIPG, LIPG bound to N4BP1 and suppressed RNA degradation. When the LIPG-binding peptide LIPH4-NTNBs was added, as shown in lane 3, LIPH4-NTNBs inhibited the binding of LIPG to N4BP1, thus rescuing RNA degradation by N4BP1. As shown in lane 4, RNA degradation by N4BP1 occurred in the absence of LIPG. Lanes 5-8 show that materials other than N4BP1 did not affect RNA degradation.

[0229] These results suggest that LIPG inhibits the RNase activity of N4BP1, thus facilitating the maintenance of HBV. However, inhibiting the binding of LIPG to N4BP1 by LIPG-binding peptides rescues the RNase activity of N4BP1, thereby suppressing HBV proliferation.

[0230] [Example 21]: Efficacy study of anti-ASGR antibody-LIPG-conjugated peptide fusion using HBV-infected PXB mice To confirm the efficacy of LIPG-binding peptides fused with intrahepatocyte delivery carriers, anti-HBV activity was evaluated by administering an anti-ASGR single-chain antibody-LIPG-binding peptide fusion to HBV-infected PXB mice. The fusion used was 10M-LNTSN, in which LIPH4-NTNBs (SEQ ID NO: 25) were fused to an anti-ASGR single-chain antibody.

[0231] (21-1) Test substance, solvent, and preparation method Drug A: 10M-LNTSN Properties: Dissolves in PBS Amount: 936μg / 21.6mL (78μg / 1800μL x 12 bottles) Storage: Refrigerate Preparation method: The stock solution of 78 μg / 1800 μL was diluted 1-fold and administered at a dose of 300 μL / animal (13 μg / animal).

[0232] Drug B: 10M-LNTSN Properties: Dissolves in PBS Amount: 936μg / 21.6mL (78μg / 1800μL x 12 bottles) Storage: Refrigerate Preparation method: Drug A was diluted 5-fold with PBS and administered to 300 μL / animal (2.6 μg / animal).

[0233] solvent Similar to Example 13, PBS was used.

[0234] (21-2) Virus used Similar to Example 13, HBV strain PBB004 (Genotype C) (viral titer: 1.1E+09 copies / mL) purchased from PhoenixBio Co., Ltd. was prepared to 1.0E+06 copies / mL with physiological saline and used.

[0235] (21-3) Animals used Similar to Example 13, PXB mice purchased from Phoenix Bio Co., Ltd. were used. Thirty mice were used in this example.

[0236] (21-4) Animal management conditions Same as Example 13.

[0237] (21-5) Study group composition and administration schedule As shown in Table 17 below.

[0238] [Table 17]

[0239] Virus inoculation, administration of test substance Same as Example 13.

[0240] Blood sample Approximately 70 μL of blood was collected from the subclavian vein once a week from week 6 of viral vaccination (two weeks before the first dose) and from day 0 (week 8 of viral vaccination) until day 21. At autopsy (day 28), the entire amount of blood that could be collected (more than 400 μL) was collected from the abdominal vena cava or heart under isoflurane inhalation anesthesia. After blood collection, 2 μL of blood was mixed with 200 μL of physiological saline to measure the blood h-Alb concentration, and the mixture was centrifuged at 390 × g at room temperature for 10 minutes. The supernatant was then placed in a 1.5 mL tube (INA·OPTIKA, RC-0150) and stored frozen (below -60°C) until sent to the analysis laboratory. The remaining blood was centrifuged at 6000 rpm for 15 minutes at 4°C to separate the serum (at time of blood collection (for HBV-related factor measurement): 30 μL (2 x 5 μL vials, 1 x 20 μL vial), at autopsy: 160 μL or more (2 x 5 μL vials, 1 x 20 μL vial, 1 remaining vial)), and stored frozen (-60°C or below) until sent to the analysis laboratory.

[0241] autopsy Same as Example 13.

[0242] Measurement of HBsAg, HBeAg, and HBcrAg Serum HBsAg concentration was measured by SRL Co., Ltd. (Tokyo). The measurement was performed using Lumipulse® PrestoII (Fujirebio Inc., Tokyo), which utilizes chemiluminescence enzyme immunoassay (CLEIA). The measurement range was 0.005 to 150 IU / mL. The dilution ratio of the sample used in this study was 30-fold, and the measurement range at this dilution ratio was 0.15 to 4500 IU / mL.

[0243] Serum HBsAg concentration was measured by SRL Co., Ltd. (Tokyo). The measurement was performed using Lumipulse® PrestoII, which utilizes CLEIA. The measurement range was 0.1 to 1590 COI. The dilution ratio of the sample used in this study was 30 times, and the measurement range at this dilution ratio was 3 to 47700 COI.

[0244] Serum HBc-rAg concentration was measured by SRL Co., Ltd. (Tokyo). The LUMIPULSE HBcrAg and LUMIPULSE F (Fujirebio Inc., Tokyo), utilizing CLEIA, were used for the measurements. The detection limit was 3.0 log U / mL. The dilution ratio of the sample used in this study was 300-fold, and the detection limit at this dilution ratio was 5.5 log U / mL.

[0245] Measurement of hAlb The following procedure was performed at Phoenix Bio Co., Ltd.: 2 μL of blood was diluted with 200 μL of physiological saline and centrifuged at 390 × g at room temperature for 10 minutes. Blood h-Alb concentration was measured using latex agglutination immunoturbidimetry (LZ test 'Eiken' U-ALB, Eiken Chemical Co., Ltd., Tokyo) with an automated analyzer BioMajesty™ (JCA-BM6050, JEOL, Tokyo).

[0246] ALT measurement The measurements were performed using serum collected at autopsy with PhoenixBio. Plasma ALT activity was measured using 10 μL of collected plasma. The target of measurement was diarylimidazole leuco dye (diarylimidazole leuco dye is colored blue by hydrogen peroxide and peroxidase generated by pyruvate oxidase), and a Drychem 7000 / NX500sV was used for the measurement.

[0247] Measurement of HBV-DNA in the blood This was conducted by Cubix Co., Ltd. via PhoenixBio Co., Ltd. The serum was mixed with HBV Pre-treatment Solution for HBV-DNA in Serum and heat-treated at 98°C for 5 minutes to inactivate HBV and expose HBV-DNA. The PCR reaction and analysis were performed using the KUBIX HBV qPCR Kit (KUBIX Corporation) and CFX96 Touch. TMA Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used. 10 μL of HBV 2× PCR Solution was mixed with 10 μL of a heat-treated standard or sample to be measured. The PCR reaction was performed at 95°C for 2 minutes → (95°C for 5 seconds → 54°C for 30 seconds) × 45 cycles. The blood HBV-DNA concentration was calculated as the average of two wells. The detection limit using this quantitative method is 4.0E+04 copies / mL. Serum obtained from HBV-infected PXB mice was used as the HBV-DNA standard. The HBV-DNA concentration in the serum was quantified by digital PCR. The primers and probes used were the same as those used for measuring liver HBV-DNA in Example 13. Serum obtained from HBV-infected PXB mice was used as the HBV-DNA standard.

[0248] Measurement of HBV-DNA in the liver Same as Example 13.

[0249] Measurement of cccDNA in the liver Same as Example 13.

[0250] Histopathological examination (HBsAg immunostaining, HBcAg immunostaining) Same as Example 13.

[0251] (21-6) Observation and measurement items The sample administration schedule shown in Figure 44 was followed. No abnormalities were observed in the 10M-LNTSN administration group compared to the control (PBS) in terms of mouse weight changes (Figure 45) and general mouse condition. Comparison of HBsAg (Figure 46), HBeAg (Figure 47), and HBcrAg (Figure 48) between 10M-LNTSN and the control (PBS) showed that 10M-LNTSN had an inhibitory effect in all cases. 10M-LNTSN also had an inhibitory effect on serum HBV DNA (Figure 49). Measurement of h-Alb (Figure 50) showed that sufficient human albumin levels were obtained in chimeric mice. This assay proved excellent for evaluation in human liver cells. Measurement of ALT (Figure 51) showed that 10M-LNTSN did not exacerbate hepatitis compared to the control (PBS).

[0252] Measurements of HBV-DNA (Figure 52) in liver tissue obtained by autopsy showed that 10M-LNTSN reduced the copy number of HBV-DNA compared to the control group. Similarly, measurements of cccDNA (Figure 53) in liver tissue showed that 10M-LNTSN reduced the copy number of HBV-DNA compared to the control group.

[0253] Based on these results, 10M-LNTSN significantly reduced HBV-DNA and cccDNA in the liver, as well as HBsAg and HBeAg in the blood, in animal experiments using HBV-infected human hepatocyte chimeric mice, compared to a control group without peptide supplementation, demonstrating a clear anti-HBV effect. As a result, it can be considered that proof of concept (POC) for 10M-LNTSN has been obtained in animal experiments using human liver chimeric mice.

[0254] The 10M-LNTSN used in Example 21 is a fusion compound using the LIPG-binding peptide LIPH4-NTNBs. In Example 13, experiments were conducted in which LIPH4-NTNBs were administered to PXB mice without being bound to an intracellular delivery carrier, and the results are shown in Figures 23-26. Comparing the measurement results of HBV DNA and cccDNA in the liver in Example 13 (Figures 23, 24) with those in Example 21 (Figures 52, 53), the fusion compound exhibited anti-HBV activity equivalent to that of the unfused LIPG-binding peptide at approximately 1 / 1000th of the dose (grams). From the comparison of these data, it became clear that delivering the LIPG-binding peptide into hepatocytes increased the in vivo anti-HBV activity by more than 1000 times.

Claims

1. An anti-hepatitis B virus agent comprising as an active ingredient a substance that binds to LIPG and inhibits the function of LIPG, wherein the substance is at least one polypeptide selected from the polypeptides listed below (22), (1) to (13), (15), (17) to (19), (23) to (26), and (28). (22) Polypeptide of the sequence LNCRHNTAG (sequence number 77). (1) A polypeptide of the sequence NCRHNTAG (sequence number 25). (2) A polypeptide of the sequence NARHNTAG (sequence number 26). (3) A polypeptide of the sequence LNCRDNTR (sequence number 23). (4) A polypeptide of the sequence LNARDNTR (sequence number 24). (5) A polypeptide of the sequence LNCRDNTRPVMSAMTC (sequence number 1). (6) A polypeptide with the sequence IRNVNHSDH (sequence number 9). (7) A polypeptide of the sequence LNVGYVFYP (sequence number 18). (8) A polypeptide of the sequence SLYTGFRAH (sequence number 15). (9) A polypeptide of the sequence RCRQSWNTM (sequence number 14). (10) Polypeptide of the sequence GADLRRGCC (sequence number 11). (11) A polypeptide of the sequence RKSGGVCLNCRHNTAG (SEQ ID NO: 21). (12) A polypeptide with the sequence LNCRHNTAGRHCHYCK (sequence number 22). (13) A polypeptide having the amino acid sequence shown in SEQ ID NO:

28. (15) A polypeptide that is 98% or more identical to SEQ ID NO: 28, characterized by an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in a region other than the region of amino acids 370 to 377 in the amino acid sequence shown in SEQ ID NO:

28. (17) A polypeptide of the sequence LCECRDVLSCYYITDT (sequence number 4). (18) A polypeptide with the sequence CPCVNGATRHRPTSLC (sequence number 8). (19) A polypeptide with the sequence LVPWLRYAY (sequence number 17). (23) A polypeptide with the sequence SNCRINTFRTVPIEQK (sequence number 78). (24) Polypeptide of the sequence SNCRINTFR (sequence number 79). (25) Polypeptide of the sequence TSNCRINTFR (sequence number 80). (26) A polypeptide having the amino acid sequence shown in SEQ ID NO:

82. (28) A polypeptide that is 98% or more identical to SEQ ID NO: 82, characterized in that one or more amino acids are substituted, deleted, inserted, or added in a region other than the region of amino acids 456 to 464 in the amino acid sequence shown in SEQ ID NO:

82.

2. The anti-hepatitis B virus agent according to claim 1, comprising at least one polypeptide selected from (22), (1) to (13) and (23) to (26) above as an active ingredient.

3. The anti-hepatitis B virus agent according to claim 1, comprising at least one polypeptide selected from (22), (1) to (12), (17) to (19) and (23) to (25) as an active ingredient.

4. The anti-hepatitis B virus agent according to claim 1, wherein the polypeptide is in a form linked to an antibody, antibody fragment, or single-chain antibody that binds to an asialoglycoprotein receptor.

5. The antibody, antibody fragment, or single-chain antibody, The heavy chain CDR1-3 of the amino acid sequences shown in SEQ ID NOs. 83-85 and the light chain CDR1-3 of the amino acid sequences shown in SEQ ID NOs. 86-88, respectively. The heavy chain CDR1-3 of the amino acid sequences shown in SEQ ID NOs. 89-91 and the light chain CDR1-3 of the amino acid sequences shown in SEQ ID NOs. 92-94, respectively. The heavy chain CDR1-3 of the amino acid sequences shown in SEQ ID NOs. 95-97 and the light chain CDR1-3 of the amino acid sequences shown in SEQ ID NOs. 98-100, respectively, or The heavy chain CDR1-3 of the amino acid sequences shown in SEQ ID NOs. 101-103 and the light chain CDR1-3 of the amino acid sequences shown in SEQ ID NOs. 104-106 The anti-hepatitis B virus agent according to claim 4, having the properties of the anti-hepatitis B virus agent according to claim 4.

6. The anti-hepatitis B virus agent according to claim 4 or 5, wherein the antibody, antibody fragment, or single-chain antibody is linked to the polypeptide via a cleavage sequence that is cleaved by an endogenous enzyme.

7. The anti-hepatitis B virus agent according to claim 1, wherein the polypeptide is linked to a cell membrane permeability promoting molecule which is a polypeptide having the amino acid sequence shown in SEQ ID NO: 107 or 108.

8. The anti-hepatitis B virus agent according to claim 1, wherein the polypeptide is in a form linked to a nuclear localization signal.

9. A LIPG-binding peptide comprising at least one polypeptide selected from the polypeptides listed below (22), (1) to (12), (15), (17) to (19), (23) to (25), and (28). (22) Polypeptide of the sequence LNCRHNTAG (sequence number 77). (1) A polypeptide of the sequence NCRHNTAG (sequence number 25). (2) A polypeptide of the sequence NARHNTAG (sequence number 26). (3) A polypeptide of the sequence LNCRDNTR (sequence number 23). (4) A polypeptide of the sequence LNARDNTR (sequence number 24). (5) A polypeptide of the sequence LNCRDNTRPVMSAMTC (sequence number 1). (6) A polypeptide with the sequence IRNVNHSDH (sequence number 9). (7) A polypeptide of the sequence LNVGYVFYP (sequence number 18). (8) A polypeptide of the sequence SLYTGFRAH (sequence number 15). (9) A polypeptide of the sequence RCRQSWNTM (sequence number 14). (10) Polypeptide of the sequence GADLRRGCC (sequence number 11). (11) A polypeptide of the sequence RKSGGVCLNCRHNTAG (SEQ ID NO: 21). (12) A polypeptide with the sequence LNCRHNTAGRHCHYCK (sequence number 22). (15) A polypeptide that is 98% or more identical to SEQ ID NO: 28, characterized by an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, or added in a region other than the region of amino acids 370 to 377 in the amino acid sequence shown in SEQ ID NO:

28. (17) A polypeptide of the sequence LCECRDVLSCYYITDT (sequence number 4). (18) A polypeptide with the sequence CPCVNGATRHRPTSLC (sequence number 8). (19) A polypeptide with the sequence LVPWLRYAY (sequence number 17). (23) A polypeptide with the sequence SNCRINTFRTVPIEQK (sequence number 78). (24) Polypeptide of the sequence SNCRINTFR (sequence number 79). (25) Polypeptide of the sequence TSNCRINTFR (sequence number 80). (28) A polypeptide that is 98% or more identical to SEQ ID NO: 82, characterized in that one or more amino acids are substituted, deleted, inserted, or added in a region other than the region of amino acids 456 to 464 in the amino acid sequence shown in SEQ ID NO: 82.

Images