HTLV-1 cell-infected cell-cell contact infection inhibitor, therapeutic drug for HTLV-1 infectious disease, therapeutic drug for HTLV-1-associated myelopathy (ham / TSP)

Abstract

To provide a means for treating HTLV-1-associated myelopathy (HAM / TSP) and HTLV-1 infectious diseases by identifying important molecules on the side of infected cells and on the side of non-infected cells in the cell-cell contact infection of human T cell keukemia-1 virus (HTLV-1) and inhibiting the HTLV-1 cell-cell contact infection by inhibiting the mutual action thereof.SOLUTION: The HTLV-1 cell-cell contact infection inhibitor contains a substance inhibiting the interaction between N-acetyllactosamine (LacNAc) and galectin-3 (Gal-3).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a therapeutic agent for HTLV-1-associated myelopathy (HAM / TSP), a disease caused by human T-cell leukemia virus type 1 (HTLV-1), a therapeutic agent for HTLV-1 infection including the condition of HTLV-1-infected individuals including asymptomatic HTLV-1 carriers and complications caused by HTLV-1, or an inhibitor of HTLV-1 cell-to-infected cell contact infection, which is mechanistically named. More specifically, the present invention relates to a substance and compound that inhibits HTLV-1-infected cell-to-uninfected cell contact infection, which is the mode of proliferation of HTLV-1 provirus or infected cells in HTLV-1-infected individuals. [Background technology]

[0002] The retrovirus HTLV-1, which causes the intractable rare diseases adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy / tropical spastic paraparesis (HAM / TSP, hereafter referred to simply as "HAM"), is integrated as a provirus into the DNA of human CD4+ T lymphocytes in HTLV-1-infected individuals and causes an increase in the number of infected cells, i.e., the HTLV-1 proviral load, in the body through two main mechanisms: intracellular cell division or clonal expansion, and HTLV-1 cell-to-cell contact infection. It can also be transmitted between individuals by mother-to-child transmission via breast milk and horizontal transmission via semen and blood.

[0003] Including asymptomatic carriers, there are an estimated 1.08 million HTLV-1-infected individuals in Japan and approximately 20 million worldwide. ATL, in which infected cells undergo genetic mutation and tumorigenesis leading to clonal proliferation, and HAM, which causes inflammation due to the infiltration of infected cells into the spinal cord, are estimated to occur in approximately 1-6% of these infected individuals. However, there is currently no cure, and the prognosis for ATL is poor. While asymptomatic carriers have not been treated to reduce the HTLV-1 proviral load, it is known that there are high-risk carriers who progress to ATL. However, there has been little development or clinical trial of therapeutic drugs for HTLV-1 infection or HAM, and only a few developments for ATL have been conducted in Japan.

[0004] For ATL, the monoclonal antibody mogalizumab (trade name Poteligeo, Kyowa Kirin Co., Ltd.) is available on the market, which targets the surface marker CCR4, which is expressed on approximately 60-70% of HTLV-1-infected cells. However, clonal replacement occurs during treatment, making it incurable. Clinical trials investigating the efficacy of Poteligeo for HAM were conducted in recent years, but although an initial reduction in the number of infected cells was observed, the number quickly returned to normal, preventing its commercial launch. Similar results are expected as long as the surface marker of HTLV-1-infected cells is targeted for treatment. For HAM, oral steroids and IFN-α injections are available as symptomatic treatments to suppress inflammation, but the effect on reducing the amount of HTLV-1 provirus is small, and the current situation is that the condition is maintained.

[0005] The present inventors previously discovered that tyrosine kinase inhibitors (TKIs) specifically induce cell death in HTLV-1-infected cells and demonstrated their effectiveness as anti-HTLV-1 drugs and HAM treatments (see, for example, Patent Document 1). Because these drugs target ABL tyrosine kinase, an important molecule in the HTLV-1 infection-specific signaling pathway, rather than a surface marker, they are unrelated to the clonal replacement phenomenon and may be able to continuously reduce the number of HTLV-1-infected cells as long as administration is continued.

[0006] However, as mentioned above, there are two mechanisms by which the number of infected cells (HTLV-1 proviral load) increases in the body of infected individuals: infected cell division and contact infection between infected and uninfected cells. While TKIs can block the former by specifically inducing cell death in HTLV-1-infected cells, they are unable to adequately address the latter. Therefore, the development of drugs that inhibit cell-to-cell contact infection of HTLV-1 is needed. However, the viral receptor on uninfected cells has been found to be a ubiquitous molecular aggregate, making it difficult to target therapeutically.

[0007] A new structure of cell-to-cell contact infection has been identified: a biofilm-like extracellular virus structure consisting of various extracellular matrices, BST-2 / tetherin, Gal-3, CD62, and the O-glycan sialyl Lewis X exists around HTLV-1-infected CD4+ T cells; virus particles attach to this structure and move to uninfected cells, thereby spreading the infection (Patent Document 2, Non-Patent Document 1).

[0008] Galectin-3 (Gal-3) is a protein with a unique carbohydrate-recognition domain consisting of approximately 130 amino acids that binds to β-galactoside. Gal-3 is highly expressed in HTLV-1-infected T cells through transactivation by Tax, but is weakly constitutively expressed in uninfected T cells. Gal-3 is known to interact with the membrane protein-associated N-acetyllactosamine (Galβ1,4GlcNAc, LacNAc) in a ligand-receptor relationship, forming a lattice between Gal-3 pentamer and LacNAc. However, the highly expressed carbohydrate chains on HTLV-1-infected cells were unknown, and the interaction between LacNAc and Gal-3 on uninfected cells (hereinafter sometimes referred to as the LacNAc-Gal-3 axis) was not known to be involved in HTLV-1-infected cell-to-cell contact infection until the present inventors discovered this.

[0009] It has been reported that the metabolic LacNAc biosynthesis inhibitor, peracetylated 4-fluorinated glucosamine analog (2-acetamido-1,3,6-tri-O-acetyl-4-deoxy-4-fluoroglucopyranose; 4-F-GlcNAc), inhibits the production of the LacNAc precursor uridine diphosphate-N-acetylgulucosamine (UDP-GlcNAc) in leukocytes, thereby reducing LacNAc expression on leukocytes (Patent Document 3, Non-Patent Document 2). Furthermore, the Gal-3 inhibitor GB1107 (3,4-dichlorophenyl 3-deoxy-3-[4(3,4,5-trifluorophenyl)-1H-1,2,3-triazol-1-yl]-1-thio-α-D-galactopyranoside) has been developed as a therapeutic agent for idiopathic pulmonary fibrosis (Patent Documents 4 and 5). However, there have been no reports on the clinical application of these drugs as drugs that inhibit the LacNAc-Gal-3 axis, or on their use as therapeutic agents targeting HTLV-1 infection. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] International Publication No. 2018 / 025923 [Patent Document 2] International Publication No. 2011 / 070545 [Patent Document 3] International Publication No. 03 / 093410 [Patent Document 4] International Publication No. 2016 / 120403 [Patent Document 5] International Publication No. 2020 / 248068 [Non-patent literature]

[0011] [Non-Patent Document 1] Pais-Correia AM., et al. Nat Med. 2010;16(1): 83-9. doi: 10.1038 / nm.2065. [Non-patent document 2] Barthel SR, et al. J Biol Chem. 2011;286(24): 21717-31. Epub 2011 / 04 / 16. doi: 10.1074 / jbc.M110.194597. Summary of the Invention [Problem to be solved by the invention]

[0012] The object of the present invention is to identify important molecules on the infected and non-infected cell sides in HTLV-1 cell-to-cell contact infection, and to inhibit HTLV-1 cell-to-cell contact infection by blocking their interaction, thereby suppressing the growth of HTLV-1 infected cells or HTLV-1 proviral load, thereby providing a means for treating HAM and HTLV-1 infections. [Means for solving the problem]

[0013] To achieve the above objective, the present inventors performed comprehensive glycan analysis of CD4+ T cell membrane proteins derived from HAM patients using a lectin array and identified LacNAc as a glycan significantly overexpressed on the membrane surface of HTLV-1-infected CD4+ T cells. Hypothesizing that LacNAc may be involved in cell-to-cell contact infection, the present inventors focused on the receptor-ligand relationship between LacNAc, which is highly expressed on infected cells, and Gal-3, a LacNAc-specific receptor known to be constitutively expressed on uninfected cells. Therefore, in an HTLV-1 infectivity assay, HTLV-1 infectivity was inhibited by treating infected cells with LacNAc-specific lectins STL, UDA, and LEL or the LacNAc biosynthesis metabolic inhibitor 4-F-GlcNAc, or by treating uninfected cells with the Gal-3 inhibitor GB1107, which targets the LacNAc ligand Gal-3. Furthermore, in cell-cell conjugate formation assays, 4-F-GlcNAc treatment inhibited conjugate formation. The inhibition of cell-cell contact infection and conjugate formation were strongly correlated, suggesting that once conjugates are formed, infection is irreversible, i.e., conjugate formation and cell-cell contact infection are an inseparable process. The present inventors have thus demonstrated that HTLV-1 cell-to-cell infection involves a receptor-ligand interaction (LacNAc-Gal-3 axis) between the disaccharide sugar chain LacNAc, which is highly expressed on HTLV-1-infected CD4+ T cells, and Gal-3 on uninfected CD4+ T cells, which is the target. They have also demonstrated that the mechanism of action of the agents of the present invention is to inhibit this interaction, thereby inhibiting conjugate formation and thereby inhibiting HTLV-1 cell-to-cell contact infection, thereby completing the present invention.

[0014] That is, the present invention is as follows. [Item 1] An inhibitor of human T-cell leukemia virus type 1 (HTLV-1) cell-to-cell contact infection, containing a substance that inhibits the receptor-ligand interaction between N-acetyllactosamine (LacNAc) and galectin-3 (Gal-3). [Item 2] A substance that inhibits the receptor-ligand interaction between LacNAc and Gal-3 is (a) a substance that inhibits the expression of LacNAc on the membrane surface, (b) a substance that binds to LacNAc and inhibits the binding of LacNAc to Gal-3; (c) a substance that binds to Gal-3 and inhibits the binding of LacNAc to Gal-3, or (d) Substances that inhibit Gal-3 expression Item 2. The inhibitor according to Item 1, [Item 3] The substance (a) is contained, and the substance is (a1) an inhibitor of UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 2 (B3GNT2) or beta-1,4-galactosyltransferase 1 (B4GALT1), or (a2) Metabolic LacNAc biosynthesis inhibitors Item 3. The inhibitor according to Item 2, wherein [Item 4] The inhibitor according to Item 3, which contains the inhibitor (a2) and is peracetylated 4-fluoroglucosamine (4-F-GlcNAc). [Item 5] The inhibitor according to Item 2, which contains the substance (b) and is Urtica dioica agglutinin (UDA), potato lectin (Solanum Tuberosum (Potato) lectin: STL), or tomato lectin (Lycopersicon Esculentum (Tomato) lectin: LEL). [Item 6] The inhibitor according to Item 1, which contains the substance (c) above, and the substance is selected from the group consisting of GB1107, TD139, GB1211, GCS-100, and GR-MD-02 (belapectin). [Item 7] The inhibitor according to any one of Items 1 to 6, which is used for the treatment of HTLV-1 associated myelopathy (HAM / TSP) and HTLV-1 infection. [Item 8] A method for inhibiting HTLV-1 cell-to-cell contact infection in an HTLV-1-infected individual, comprising administering to the infected individual an effective amount of a substance that inhibits the receptor-ligand interaction between LacNAc and Gal-3. [Effects of the Invention]

[0015] According to the present invention, by inhibiting the receptor-ligand interaction between LacNAc and Gal-3, cell-to-cell contact infection of HTLV-1 can be inhibited, and an increase in the number of infected cells (HTLV-1 proviral load) can be suppressed, which is useful for the treatment of HTLV-1-associated myelopathy (HAM) and HTLV-1 infection. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 shows the results of comprehensive glycan analysis (glycome by lectin array) of membrane proteins of CD4-positive T cells derived from HAM patients, asymptomatic carriers (AC), and non-infected individuals (NC).

[0017] Membrane proteins from four samples each of HAM, AC, and NC were extracted by salting out and labeled with the fluorescent dye Cy3. 100 μl of each sample was placed in one well at various concentrations (2,000, 1,000, 500, 250, 125, 62.5, and 31.25 ng / ml) and incubated at 4°C for 17 hours. Raw images acquired with a GlycoStation Reader 1200 were quantified into signal intensities using GlycoStation Tools 2.0 software (all from GPBioscience KK., Tokyo; now transferred to Glycotechnica KK, Ltd., Yokohama). Comparable data from the low-intensity dataset were used to interpolate the saturated portion of the high-intensity dataset using the least-squares method (Kuno A. Journal of Proteomics & Bioinformatics. 1(2): 68-72. (2008)). The dynamic range of the lectin array was integrated in this way, and the integrated net lectin signal intensity was used for analysis. Furthermore, the normalized lectin signal intensity was calculated using the following formula and compared. Mean Netlectin signal intensity = (total of 45 net signal strengths) / 45 = {sum of 45 (raw signal intensity - background)} / 45 Normalized signal intensity for each lectin = (Net signal intensity of each lectin) / (Average net lectin signal intensity) × 100 The intensity of lectin signals indicates the expression of glycans with specific affinity. For comparison of the HAM, AC, and NC groups, normalized data were used for a sample with a protein concentration of 250 ng / ml, in which the signal intensity was not saturated for any of the lectins. The horizontal axis of the figure shows the normalized Cy3-labeled lectin signal intensity. The vertical axis shows the lectins loaded onto LecChip ver1.0 (GPBioscience). The black, gray, and white bars show the lectin signal intensity of CD4+ T cell membrane proteins derived from negative controls (NC), asymptomatic carriers (AC), and HAM patients, respectively. Comparison between HAM and NC patients by Student's t-test with Bonferroni correction showed significant differences (*: P < 0.0167, †: P < 0.0033).

[0018] Only LEL, STL, and UDA (shown within the solid line box) showed significantly high signal intensity in HAM. These are chitin-binding lectin hevein family members with disaccharide specificity for N-acetyllactosamine (LacNAc). This suggests that LacNAc sugar chains are significantly overexpressed on CD4+ T cell membrane proteins in HAM patients.

[0019] [Figure 2] FIG. 1 shows the relationship between LacNAc expression, LacNAc biosynthetic enzyme B3GNT2 gene expression, and HTLV-1 proviral load in CD4-positive T cells derived from HAM patients, asymptomatic carriers, and uninfected individuals.

[0020] a. Verification of LacNAc by enzyme-linked lectin assay (ELLA) Eight CD4+ T cell membrane protein samples from HAM, AC, and NC were prepared at a membrane protein concentration of 333.33 μg / ml and diluted 1:20 with PBS. 50 μl of each sample was added to an STL-immobilized microwell strip (AlerCHECK, Inc., Springvale, MA, USA) and incubated at room temperature for 30 minutes with shaking. Endogenous avidin / biotin was blocked with an Avidin / biotin Blocking Kit (Thermo Fisher Scientific KK., Tokyo). 100 μl of 2.5 μM biotinylated UDA (EY Laboratories) was added, sealed, and incubated at room temperature for 1 hour with shaking. 50 μl of streptavidin-Alexa488 conjugate (Thermo Fisher Scientific) diluted to 5 μM in 1% BSA-PBS was added to each well, sealed, and incubated at 37°C for 1 hour with shaking. Fluorescence intensity was measured at Ex 490 / Em 525 nm using a Tecan Infinite M200 plate reader (Tecan Japan Co., Ltd., Tokyo, Japan). A 1:5 dilution series starting at 4.0 μM of the standard substance N-acetyllactosamine (LacNAc) (Calbiochem, Sigma-Aldrich Japan KK., Tokyo) was used to generate a standard curve. The LacNAc concentration in the samples was determined using the measured fluorescence intensity and the standard curve, and then back-calculated from the dilution factor. Samples were measured in triplicate in three independent experiments. Comparisons between HAM or AC samples and NC samples were performed using the Mann-Whitney U test with Bonferroni correction. P < 0.0167 was considered significant, and ns indicated no significant difference. Significant differences were observed between HAM and NC, with the mean LacNAc concentration increasing in the order HAM > AC > NC. This confirms the lectin array results using the ELLA method with STL-coated plates.

[0021] b. Expression of B3GNT2 mRNA, the enzyme responsible for LacNAc biosynthesis Using 10 RNA samples from CD4+ T cells from HAM, AC, and NC, the expression of mRNA variant 1 (NCBI Reference Sequence: NM_006577.6) of the B3GNT2 gene (Gene ID: 10678) (Togayachi A. Proc Natl Acad Sci U S A. 104(40):15829-34.(2007)), which is thought to be the enzyme responsible for LacNAc biosynthesis, was examined using the qRT-PCR ΔΔCt method. The primers were: B3GNT2 mRNA forward, 5'-ACTCGGGGAGGTTAAAGACC-3' (1515-1534) (SEQ ID NO: 1); B3GNT2 mRNA reverse, 5'- GCCACAGACTGTCCTGGTATCT-3' (1589-1610) (SEQ ID NO: 2); GAPDH mRNA transcript variant 2 forward, 5'-GACTAACCCTGCGCTCCTG-3' (133-151) (SEQ ID NO: 3); and GAPDH mRNA transcript variant 2 reverse, 5'-GCCCAATACGACCAAATCAG-3' (268-249) (SEQ ID NO: 4). The sequence numbers correspond to the reference sequences (B3GNT2 mRNA: NM_007313.2; and GAPDH (glyceraldehyde-3-phosphate dehydrogenase), transcript variant 2, mRNA: NM_001256799.2). GAPDH mRNA was used as an endogenous control gene. The amplification cycles were 50°C for 2 min (1 cycle); 95°C for 10 min (1 cycle); and 40 cycles of 95°C for 15 sec and 60°C for 1 min. Comparisons between HAM or AC samples and NC samples were performed using the Mann-Whitney U test with Bonferroni correction. *: significant at P <0.0167; ns: not significant. There was no significant difference between AC and NC, but B3GNT2 gene expression was significantly higher in HAM than in NC. It was suggested that high expression of LacNAc was caused by high expression of the B3GNT2 gene, an enzyme involved in the LacNAc biosynthesis, in HTLV-1-infected cells in HAM peripheral blood.

[0022] c. HTLV-1 proviral load (PVL) measurement by absolute real-time PCR using TaqMan probes. b. The same samples as in b. were used in 10 AC and 10 PBMC samples from HAM patients, and HTLV-1 proviral load was measured using the standard method (Nagai M. J Neurovirol.4(6): 586-93.(1998)). Triplicate measurements were performed in three independent experiments, and comparisons between two groups were performed using the Mann-Whitney U test with Bonferroni correction. *: P <0.05 was considered significant. HTLV-1 proviral load was significantly higher in HAM patient samples than in AC samples.

[0023] d. Correlation between HTLV-1 proviral load and LacNAc biosynthetic enzyme B3GNT2 mRNA gene expression in HAM and AC patients ○: Individual data for HAM patients; △: Individual data for AC. Pearson's correlation coefficient and P value are shown in the figure. Pearson's correlation analysis was performed. *: P <0.05 indicates a significant difference. The straight line in the figure is a regression line. No significant correlation was observed between HTLV-1 PVL and the mRNA expression level of the LacNAc biosynthetic enzyme gene B3GNT2 in either HAM patients or AC. However, in all HTLV-1-infected individuals (HAM patients and AC combined), a significant moderate correlation was observed between HTLV-1 PVL and B3GNT2 mRNA expression level, with a Pearson's correlation coefficient of R = 0.54 (P = 0.013). This suggests that HTLV-1 infection activates the transcription of B3GNT2 mRNA.

[0024] [Figure 3] Flow cytometry analysis showing LacNAc expression in HTLV-1-infected and -uninfected cell lines. a) FACS histogram of STL staining. b) FACS histogram of UDA staining. c) Immunohistochemistry of UDA staining.

[0025] HTLV-1-infected human T cell lines (HUT102, C91 / PL, and MT-2) and HTLV-1-uninfected human T cell lines (Jurkat and Molt-4) were stained with specific lectins. LacNAc glycans were stained with either STL-DyLight488 conjugate (EY Labs) diluted 1:100 in staining buffer or a combination of biotinylated UDA (EY Laboratories) diluted 1:200 and Streptavidin-Alexa Fluor 488 conjugate (EY Laboratories). Each staining was performed without permeabilization and incubated for 15 minutes at room temperature. Comparison of LacNAc expression between specific lectin-stained (STL or UDA) and unstained samples was performed using the cell lines. The relative LacNAc expression ratio on the cell lines was calculated using the following formula: Relative cell surface LacNAc expression in cell lines = (MFI of specific lectin-stained cells) / (MFI of unstained cells) In addition, a portion of the UDA-stained sample was mounted on a glass slide for flow cytometry, and each stain was added with DAPI. Images were taken under the same conditions using a Leica SP8 laser scanning microscope at 1,890x magnification. All flow cytometry analyses were performed using a CytoFLEX flow cytometer and CytoExpert software version 2.0 (Beckman Coulter, Inc., Brea, CA, USA).

[0026] The stained / unstained MFI ratio was positive in both infected and uninfected cells, indicating that the cancer cell lines were positive for LacNAc expression, unlike normal lymphocytes, which do not express LacNAc. Although the STL signal-MFI ratio was not significantly different between HTLV-1-infected and uninfected cells, the UDA signal, which indicates LacNAc, showed a higher MFI ratio in HTLV-1-infected cells than in uninfected cells by flow cytometry. The same was true for UDA immunofluorescence microscopy. STL is a lectin more specific for Poly-LacNAc than for LacNAc. The findings in a–c suggest that specific lectin staining of cell lines, including uninfected cell lines, demonstrates the presence of LacNAc on the cell surface, that infected cell lines express higher levels, and that high expression of LacNAc is more closely related to HTLV-1 infection than Poly-LacNAc.

[0027] [Figure 4] Figure 1 shows the results of flow cytometry analysis of LacNAc expression on peripheral blood mononuclear cells derived from HAM patients. ad: The relationship between HTLV-1-infected cell markers CADM1 / TSLC1 and LacNAc expression (stained with specific lectins STL and UDA). e, f: The relationship between the expression level of HTLV-1-infected cell markers CADM1 / TSLC1 and LacNAc expression (stained with specific lectins STL and UDA).

[0028] PBMCs from seven HAM patients were stained with mouse monoclonal anti-CADM1 antibody (kindly provided by Professor Kazuhiro Morishita, Department of Tumor Biochemistry, Faculty of Medicine, University of Miyazaki), goat anti-mouse IgG1-PE (Southern Biotech, Birmingham, AL, USA), mouse monoclonal anti-human CD4 antibody (DAKO), goat anti-mouse IgG1-PC5 (CD4-PC5), and specific lectin staining for LacNAc glycans with either STL-DyLight 488 conjugate (EY Labs) diluted 1:100 in staining buffer or a combination of biotinylated UDA (EY Laboratories) diluted 1:200 and Streptavidin-Alexa Fluor 488 conjugate (EY Laboratories) diluted 1:200. Each staining was performed without permeabilization and incubated for 15 min at room temperature. To compare LacNAc expression between HTLV-1-infected and non-infected cells in clinical specimens, the relative LacNAc expression ratio on HTLV-1-infected cells was calculated using PBMCs from HTLV-1-infected individuals using the following formula: Relative expression ratio of cell surface LacNAc in HTLV-1-infected cells = (MFI of CADM1+ cells) / (MFI of CADM- cells) (MFI: mean fluorescent intensity) To compare LacNAc expression between samples stained with specific lectins (STL or UDA) and unstained samples, the relative LacNAc expression ratio on the cell lines was calculated using the following formula: Relative cell surface LacNAc expression in cell lines = (MFI of specific lectin-stained cells) / (MFI of unstained cells) Comparison of the relative LacNAc expression ratio between CADM1+ and CADM1- cells was performed using a paired t-test (P < 0.05).

[0029] The relative cell surface LacNAc expression ratios in CADM1+ CD4+ cells were 1.47-fold and 1.99-fold higher by STL staining and UDA staining, respectively (Figure 4a and c). In a study of seven cases, the relative cell surface LacNAc expression ratios were significantly higher in CADM1+ cells than in CADM1- cells for both stainings (P = 0.018, b and d). This suggests that the LacNAc staining levels and CADM1 staining levels, i.e., the LacNAc expression level and the HTLV-1 infection level, are parallel. This supports a strong correlation between HTLV-1 PVL and B3GNT2 mRNA expression levels. Conversely, LacNAc expression by either STL or UDA specific lectin staining was not clearly expressed in CADM1- CD4+ T cells, i.e., normal CD4+ T cells. This is consistent with the previous report that N-acetyllactosamine is not expressed in normal CD4+ T cells (Campos L. Eur J Cancer 28(1):37-41. (1992)).

[0030] [Figure 5] This figure shows the evaluation of candidate compounds (STL, UDA, 4-F-GlcNAC, GB1107) as inhibitors of HTLV-1 cell-to-cell contact infection by cell viability assay. The figure shows the cell viability 48 hours after treatment of HTLV-1-infected cell lines (C91 / PL, MT-2) and uninfected cell lines (Jurkat, Molt-4) with various drug treatment concentrations of candidate compounds (a. STL, b. UDA, c. 4-F-GlcNAc, d. GB1107) as inhibitors of HTLV-1 cell-to-cell contact infection.

[0031] HTLV-1-infected and non-infected cell lines derived from human CD4+ T cells were cultured in RPMI1640 medium at 5 × 10 3Cells were seeded in triplicate (three wells per cell) onto 96-well polystyrene plates at a density of 1000 cells / well. They were cultured for 48 hours in the presence or absence of various concentrations of the LacNAc-specific lectin STL, UDA, the LacNAc biosynthetic metabolic inhibitor 4-F-GlcNAc, and the Gal-3 inhibitor GB1107, a specific ligand for LacNAc, in a 5% CO2 incubator at 37°C. Cell viability was then assayed using the XTT method (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) using the XTT Cell Proliferation Kit II (Roche Diagnostics KK, Tokyo). The XTT method utilizes a bioreduction reaction in which a tetrazolium salt is cleaved to a formazan dye only in viable cells, resulting in a colorimetric assay based on the correlation between viable cell number and the dye. The cells were measured using a plate reader with a maximum absorption wavelength of 492 nm and a reference wavelength of 650 nm. The cell viability (%) following drug treatment was calculated using the following formula: Cell viability = {(number of viable cells under drug treatment) / (number of viable cells without treatment)} x 100 (%) Cell viability was calculated from three independent experiments. For each cell line, a paired t-test (Bonferroni correction) was performed to compare cell viability at each concentration with that without drug treatment (0 μM). *: P < 0.0167, †: P < 0.003 was significant. The horizontal axis of each graph represents the drug treatment concentration (μM), and the vertical axis represents cell viability (%). The black circle, open circle, gray diamond, and open diamond markers represent C91 / PL, MT-2, Jurkat, and Molt-4, respectively.

[0032] When cells were treated with the LacNAc-specific lectins STL and UDA, HTLV-1-infected cell lines (C91 / PL, MT-2) showed cytotoxicity at 5 μM, as shown in a and b. Treatment with both STL and UDA at 5 μM significantly reduced cell viability in both HTLV-1-infected cell lines compared to untreated (0 μM). Treatment with 0.1 μM and 1 μM did not show any significant difference. Furthermore, as shown in (c), 4-F-GlcNAc was less cytotoxic to HTLV-1-infected cell lines (C91 / PL, MT-2) than STL or UDA, with no cytotoxicity observed up to 100 μM. In this experiment (d), GB1107 targets both transactivated Gal-3 in infected cells and Gal-3 constitutively expressed in uninfected CD4+ T cells. Significant cytotoxicity was observed at concentrations above 10 μM in HTLV-1-infected cell lines (C91 / PL, MT-2) and the uninfected Jurkat cell line, and at 100 μM in the uninfected Molt-4 cell line. These results determined the drug concentrations examined in Figure 6.

[0033] [Figure 6] Figure 1 shows the evaluation of HTLV-1 cell-to-cell contact infection inhibitors (STL, UDA, 4-F-GlcNAC, and GB1107) using an HTLV-1 infectivity assay. a) 2 μM STL, b) 0.75 μM UDA, c) 100 μM 4-F-GlcNAc, d) 100 μM GB1107.

[0034] HTLV-1-infectivity assays were performed using HTLV-1-infected cell lines (C91 / PL, MT-2) co-cultured with a reporter plasmid-transfected uninfected cell line (Jul) to assess HTLV-1 cell-cell contact infection. HTLV-1-infected cell lines were treated with the LacNAc-specific lectin (a) 2 μM STL, (b) 0.75 μM UDA, or the peracetylated fluorinated glucosamine analog (c) 100 μM 4-F-GlcNAc for 24 hours. The results of HTLV-1 infectivity assays were also performed using uninfected cell lines (reporter cells Jul) treated with the Gal-3 inhibitor (d) 100 μM GB1107 for 24 hours. The vertical axis indicates calculated HTLV-1 infectivity, and the horizontal axis indicates the drug concentration used to treat the Tax donor cells or Jul acceptor cells used in the assay. Uninfected cells (Jurkat and Molt-4) on the horizontal axis served as negative controls for Tax donor cells. Black and white bars indicate the calculated mean HTLV-1 infectivity of drug-treated and drug-control cell lysates, respectively. Error bars indicate standard deviation. *: P < 0.05; †: P < 0.01, based on a paired t-test.

[0035] HTLV-1-infected cells were cultured for 24 hours with or without the LacNAc-specific ligand STL (2 μM), UDA (0.75 μM), or the LacNAc biosynthesis inhibitor 4-F-GlcNAc (100 μM). Infected cells:Jul cells were cultured at a 1:1 ratio of 2 × 10 5 Jul cells were co-cultured at 2 ml / well for 48 hours, or Jul cells were cultured for 24 hours with or without the Gal-3 inhibitor GB1107 (100 μM). After 2 × 10 5After 48 hours of co-culture at 2 ml / well, luciferin was added to the cell lysate, and luciferase chemiluminescence was measured using the One-Glo luciferase assay system (Promega Corporation, Maddison, WI, USA) and a Tecan Infinite M200 spectrophotometer (Tecan Japan Co., Ltd., Tokyo). Results were compared with drug controls (no drug treatment) and Tax donor cell negative controls (donor cells were replaced with uninfected cells (Jurkat and Molt-4) instead of infected cells (C91 / PL, MT-2) and co-cultured with Jul cells). HTLV-1 infectivity of drug-treated cell lysates was calculated using the following formula: HTLV-1 infectivity = {(Luciferase chemiluminescence in cell lysate treated with drug) / (Luciferase chemiluminescence in cell lysate of drug control (no drug treatment))} x 100 (%) Triplicate measurements were performed in three independent experiments. This experimental system utilizes an HTLV-1-infected cell-to-uninfected cell contact infection system consisting of HTLV-1-infected cell lines (here, C91 / PL or MT-2) as Tax donor cells and uninfected Jul cells (Jurkat cells transfected with the pLTR-GL3 plasmid) as acceptor cells. HTLV-1 virus particles budded from the HTLV-1-infected cell lines bind to, fuse with, and enter the HTLV-1 viral receptor (a ubiquitous molecular complex present on normal cells) on the surface of uninfected Jul cells. Tax protein derived from the particles or reverse-transcribed and translated from viral RNA binds to the tax-responsive element (TRE) 21-base pair repeat sequence in the pLTR-GL3 plasmid in the acceptor cells. This results in expression of the reporter gene luciferase gene inserted downstream, allowing cell-to-cell contact infection to be assessed. This is an assay for determining HTLV-1 infectivity.

[0036] The pLTR-GL3 plasmid was constructed from pLTR-CAT (Fujisawa J., et al. Proc Natl Acad Sci USA 1985. 82(8): 2277-81. (1985)). A 636 base pair region (-342 to +294) spanning the 5' half of U3, R, and U5 of the HTLV-1 LTR region was amplified by PCR using the following primers: a forward primer with an XhoI linker and a reverse primer with a HindIII linker: Forward: 5'- CTCGAG ATGAGCCCCAAATATCCCCCGG-3' (SEQ ID NO: 5); Reverse: 5'- AAGCTT AATGAAAGGGAAAGGGGTG GAACT-3' (SEQ ID NO: 6) The underlined parts are linker sequences. The amplified fragment was digested with restriction enzymes XhoI and HindIII, and the resulting XhoI-HindIII fragment was inserted into the pGL2-Basic vector plasmid (GenBank: X65323, Promega Corporation, Madison, WI, USA) to create the pLTR-Luc plasmid. The reporter firefly luciferase gene and transcription termination signal were replaced with the corresponding sequences from the pGL3-Basic vector (GenBank: U47295, Promega Corporation, Madison, WI, USA) to create the pLTR-GL3 plasmid.

[0037] In each plot, the two black bars on the left side of the graph show drug controls (no drug treatment) and the two black bars on the right side show Tax donor cell controls, which showed no effect on HTLV-1 infectivity. Furthermore, the two white bars on the right side show drug-treated uninfected cells, which showed no difference in infectivity compared to the drug controls. These experimental systems were considered appropriate for evaluating HTLV-1 infectivity and presented no problems. Treatment of HTLV-1-infected cell lines (a) with 2 μM STL reduced the infectivity to 84.7±3.7% and 83.3±5.9%, respectively, for C91 / PL and MT-2, resulting in a 15.3% and 16.7% inhibition of infection. In contrast, treatment with 0.75 μM UDA (b) reduced the infectivity to 28.1±4.9% and 37.8±6.1%, respectively, for C91 / PL and MT-2, resulting in a significant 71.9% and 62.2% inhibition of infection. Treatment with 100 μM 4-F-GlcNAc, an inhibitor of LacNAc biosynthesis metabolism (c), reduced the infectivity to 53.8±4.9% and 27.9±2.4%, respectively, resulting in a 46.2% and 72.1% inhibition of infection. When acceptor Jul cells were treated with 100 μM of the Gal-3 inhibitor GB1107 (d), the infectivity of Tax donor cells C91 / PL and MT-2 was 0.46±0.03 and 0.54±0.02%, respectively, demonstrating a 99.5% inhibitory effect on infection in both cases, and infectivity was almost completely eliminated in both cases. Oral administration of GB1107 to patients also appears to induce cell death in HTLV-1-infected CD4+ T cells, the Tax donor cells, as shown in Figure 5. This suggests that GB1107 may be effective at concentrations lower than 100 μM.

[0038] These results suggest that the LacNAc-specific lectins (STL, UDA), the LacNAc biosynthesis metabolic inhibitor 4-F-GlcNAc, and the Gal-3 inhibitor GB1107 all inhibited HTLV-1 infectivity, demonstrating their potential as therapeutic agents for HTLV-1 infection and HAM. In summary, these results suggest that the mechanism of cell-to-cell contact spread of infection involves the interaction of LacNAc on HTLV-1-infected CD4+ T cells with its in vivo ligand, Gal-3, which has a strong affinity for LacNAc on uninfected CD4+ T cells, i.e., the LacNAc-Gal-3 receptor-ligand interaction (LacNAc-Gal-3 axis) and that inhibition of these molecules inhibits cell-to-cell infection.

[0039] [Figure 7]These are microscopic images showing the inhibitory effect of 4-F-GlcNAc in an HTLV-1 conjugate formation assay. HTLV-1-infected cell lines (C91 / PL, MT-2) (1 × 104 cells) were stained with the carbocyanine fluorescent dye DiO (green, Ex 484 / Em 501 nm) to label the cell membrane, and uninfected Jurkat cells (2 × 104 cells) were stained with DiI (red, Ex 550 / Em 565 nm) to assess conjugate formation. After 40 minutes of co-incubation in a 37°C, 5% CO2 incubator, overlaid images of DiO, DiI, and differential interference contrast were acquired using a BZ-X800 fluorescence confocal microscope with BZ-X800 Viewer software ver. 1.2.2 and BZ-X800 Analyzer software ver. 1.1.1 (Keyence Corporation, Osaka, Japan). a. Representative images showing the drug concentration-dependent inhibition of conjugate formation by 4-F-GlcNAc. b, c. Representative magnified images of conjugate formation, showing two and three conjugates, respectively. Note: DiO: 3,3'-dioctadecyloxacarbocyanine perchlorate, DiO18(3). Excitation wavelength (Ex) 484 / Emission wavelength (Em) 501 nm. DiI: 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, Ex 550 / Em 565 nm.

[0040] [Figure 8] Figure 1 shows the inhibitory effect (quantitative evaluation) of 4-F-GlcNAc in an HTLV-1 conjugate formation assay. a. Inhibition of conjugate formation frequency and conjugate formation rate by 4-F-GlcNAc. b. Correlation between the results of the HTLV-1 conjugate formation assay and the HTLV-1 infectivity assay.

[0041] Conjugate formation, where HTLV-1-infected cells (green) and uninfected cells (red) come into contact, was counted using ImageJ software. Fields containing up to 1,000 infected cells were counted, and the conjugate frequency (per 1,000 HTLV-1-infected cells) was defined by the following formula: Conjugate frequency ( / 1,000 HTLV-1-infected cells) = {(total number of DiO-stained cells involved in zygote formation) / (total number of DiO-stained cells in the field)} × 1,000 The effect of a drug on inhibiting zygote frequency was defined as the ratio of zygote frequency with drug treatment compared to zygote frequency without drug treatment, as follows: Conjugate formation rate(%) = {(Frequency of zygotes with drug treatment) / (Frequency of zygotes without drug treatment)} × 100 Three independent experiments were performed. Figure a shows a two-axis graph. The left vertical axis of the bar graph represents the mean conjugate frequency (1 / 1,000 HTLV-1-infected cells) with error bars indicating standard deviation. The right vertical axis represents a line graph, similarly showing the mean conjugate formation rate (%). The error bars of the line graph markers represent standard deviation. The 4-F-GlcNAc concentrations listed on the horizontal axis represent the concentration of 4-F-GlcNAc treated with HTLV-1-infected cells 48 hours before coculture with Jurkat cells. Black and white bars represent the conjugate frequencies between C91 / PL and Jurkat cells, and between MT-2 and Jurkat cells, respectively. Black circle (●) and diamond (◇) line graph markers represent the conjugate formation rates between C91 / PL and Jurkat cells, and between MT-2 and Jurkat cells, respectively. Results for each concentration of 4-F-GlcNAc treated cell were compared to the untreated control (0 μM) using a paired t-test with Bonferroni correction. *: P <0.025, †: P <0.005 indicates significance.

[0042] In C91 / PL and Jurkat mice, the conjugate frequency in the untreated control (4-F-GlcNAc) was 472.26 ± 17.00 ( / 1,000 cells), whereas the conjugate frequency decreased in a concentration-dependent manner to 385.51 ± 24.00 and 343.81 ± 15.63 ( / 1,000 cells) with 50 μM and 100 μM treatment, respectively. The conjugate formation rate, defined as 100% ± 3.60% for the untreated control, was 81.63 ± 5.08% (a decrease of 18.37%) with 50 μM treatment and 72.80 ± 3.19% (a decrease of 27.2%) with 100 μM treatment, demonstrating a concentration-dependent decrease in the conjugate formation rate. In MT-2 and Jurkat mice, the conjugate frequency in the untreated control was 411.05 ± 22.44 ( / 1,000 cells), whereas in the 50 μM and 100 μM treatments it was 346.21 ± 27.50 and 287.95 ± 16.24 ( / 1,000 cells), respectively, in a concentration-dependent manner. Similarly, the conjugate formation rate was significantly reduced in a concentration-dependent manner, from 100 ± 5.46% in the untreated control to 84.22 ± 6.69% (a 15.78% decrease) in the 50 μM treatment and 70.05 ± 3.95% (a 29.95% decrease) in the 100 μM treatment. These results indicate that inhibition of LacNAc biosynthesis by 4-F-GlcNAc inhibits HTLV-1 conjugate formation; in other words, inhibition of part of the LacNAc-Gal-3 axis inhibits HTLV-1 conjugate formation, i.e., cell-cell contact; conversely, LaNAc is required for conjugate formation, i.e., cell-cell contact.

[0043] Figure 6b shows the data from the HTLV-1 conjugation assay and the HTLV-1 infectivity assay plotted at the point where the cells and 4-F-GlcNAc treatment concentrations matched. In addition to the results of the HTLV-1 infectivity assay using C91 / PL or MT-2 cells treated with 100 μM 4-F-GlcNAc (Fig. 6c), the data from the HTLV-1 infectivity assay using C91 / PL or MT-2 cells treated with 50 μM 4-F-GlcNAc are also plotted. Filled circles (●), shaded circles (○), and open circles (○) represent the data for C91 / PL and Jurkat conjugation with 0 μM, 50 μM, and 100 μM 4-F-GlcNAc, respectively. Filled diamonds (◆), shaded diamonds, and open diamonds (◇) represent data for MT-2 and Jurkat conjugate formation following treatment with 0 μM, 50 μM, and 100 μM 4-F-GlcNAc, respectively. In the plots, "r = 0.93" represents the Spearman rank correlation coefficient for C91 / PL and Jurkat, and "r = 0.87" represents the Spearman rank correlation coefficient for MT-2 and Jurkat. The two straight lines represent the simple regression equation for the conjugate formation between C91 / PL and Jurkat (lower graph) and MT-2 and Jurkat (upper graph), respectively.

[0044] Correlation analysis of the results of the infectivity and conjugation assays using C91 / PL and Jurkat, and of the results of both assays using MT-2 and Jurkat revealed extremely strong correlations with Spearman's rank correlation coefficients of r = 0.93 (P < 0.01) and r = 0.87 (P < 0.05), respectively. This indicates that 4-F-GlcNAc inhibits infectivity and conjugation in a concentration-dependent manner in both infected and uninfected cell pairs.

[0045] Thus, the results of experiments using 4-F-GlcNAc, a drug that inhibits the LacNAc glycan, showed a strong correlation between the results of the HTLV-1 infectivity assay, which is completely unrelated to glycans, and the results of the HTLV-1 conjugate formation assay. This indicates that the LacNAc glycan, which is highly expressed on HTLV-1-infected CD4+ T cells, is clearly involved in HTLV-1 cell-to-cell contact infection and conjugate formation. Furthermore, the strong correlation between the results of the infectivity assay and the conjugation assay suggested that conjugation formation and contact infection are inseparable and irreversible processes, and that inhibition of the LacNAc-Gal-3 interaction (LacNAc-Gal-3 axis) inhibits infectivity through inhibition of conjugation formation. HTLV-1 conjugation formation inhibitors, i.e., HTLV-1 cell-cell contact infection inhibitors, inhibit infectivity and thereby suppress HTLV-1 proviral load, suggesting that they could be used clinically as therapeutic agents for HTLV-1 infection and HAM. DETAILED DESCRIPTION OF THE INVENTION

[0046] The present invention provides an inhibitor of HTLV-1 cell-to-cell contact infection (hereinafter also referred to as "the inhibitor of the present invention"), which contains a substance that inhibits the receptor-ligand interaction between LacNAc and Gal-3.

[0047] HTLV-1-infected CD4+ T cells highly express the LacNAc epitope on membrane surface protein-bound glycans. LacNAc has a β-1,4-linked structure between galactose and N-acetylglucosamine, and is added to the N-glycan terminus by the action of two glycosyltransferases: UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 2 (B3GNT2) and beta-1,4-galactosyltransferase 1 (B4GALT1). In this specification, the term also encompasses poly-LacNAc, which is a polymer of two or more LacNAc molecules.

[0048] In HTLV-1-infected CD4+ T cells, the Gal-3 gene is transactivated by the viral protein tax, resulting in high expression of the Gal-3 protein, which binds to β-galactoside. Gal-3 forms a pentamer and forms a lattice structure with LacNAc on the membrane surface. Gal-3 is weakly and constitutively expressed even in uninfected CD4+ T cells. When infected and uninfected cells form conjugates, the LacNAc on the infected cell irreversibly binds to Gal-3 on the uninfected cell due to the affinity between them. HTLV-1 virions colocalized with Gal-3 on the infected cell are transferred to uninfected cells, resulting in cell-to-cell contact infection.

[0049] As used herein, the term "LacNAc-Gal-3 axis" refers to the interaction between LacNAc on infected cells and Gal-3 on uninfected cells through cell-to-cell contact infection, based on the receptor-ligand relationship. Therefore, the term "substance that inhibits the LacNAc-Gal-3 axis" is not particularly limited as long as it blocks this interaction by some mechanism, and examples thereof include: (a) a substance that inhibits the expression of LacNAc on the membrane surface, (b) a substance that binds to LacNAc and inhibits the binding of LacNAc to Gal-3; (c) a substance that binds to Gal-3 and inhibits the binding of LacNAc to Gal-3, and (d) Substances that inhibit Gal-3 expression Examples include:

[0050] (a) Substances that inhibit the expression of LacNAc on the membrane surface The substance (a) is not particularly limited as long as it can inhibit the expression of LacNAc on membrane protein-bound sugar chains, and may inhibit any step in the biosynthetic pathway. For example, (a1) an inhibitor of either B3GNT2 or B4GALT1, two glycosyltransferases that catalyze the reaction of adding LacNAc to a sugar chain, preferably a B3GNT2 inhibitor; (a2) A drug that metabolically inhibits LacNAc biosynthesis, preferably a drug that reduces the production of UDP-GlcNAc, which is a substrate for LacNAc biosynthesis. These include, but are not limited to, the following:

[0051] Examples of inhibitors of B3GNT2 or B4GALT1 include substances that bind to these enzymes and inhibit their enzymatic activity. For example, the three-dimensional structures of B3GNT2 and B4GALT1 have been published in the NCBI and UniProtKB databases (UniProtKB accession Nos. Q9NY97 and P15291), and inhibitors can be searched for and identified using known methods such as docking. Alternatively, examples of substances that bind to these enzymes and inhibit their enzymatic activity include antibodies against the enzymes. The antibodies may be either polyclonal or monoclonal. These antibodies can be produced according to known methods for producing antibodies or antisera. The isotype of the antibody is not particularly limited, but is preferably IgG, IgM, or IgA, with IgG being particularly preferred. Furthermore, the antibody is not particularly limited as long as it has at least a complementarity-determining region (CDR) for specifically recognizing and binding to B3GNT2 or B4GALT1, and may be a complete antibody molecule, or may be, for example, a fragment such as Fab, Fab', or F(ab')2, a genetically engineered conjugate molecule such as scFv, scFv-Fc, a minibody, or a diabody, or a derivative thereof modified with a molecule having a protein-stabilizing effect such as polyethylene glycol (PEG).

[0052] Alternatively, in another embodiment, a substance that inhibits the expression of B3GNT2 or B4GALT1 enzyme proteins can be used as an inhibitor of these enzyme proteins. Such a substance may act at any stage of the enzyme gene, such as the transcription level, post-transcriptional regulation level, protein translation level, or post-translational modification level. Therefore, substances that inhibit the expression of B3GNT2 or B4GALT1 include, for example, substances that inhibit the transcription of the enzyme gene (e.g., antigene), substances that inhibit the processing of initial transcription products into mRNA, substances that inhibit the transport of mRNA into the cytoplasm, substances that inhibit the translation of mRNA into protein (e.g., antisense nucleic acid, miRNA) or that degrade mRNA (e.g., siRNA, ribozyme, miRNA), and substances that inhibit the post-translational modification of initial translation products. While substances that act at any stage can be used, substances that bind complementarily to mRNA to inhibit translation into protein or degrade mRNA are preferred.

[0053] Preferred examples of substances that specifically inhibit the translation of an enzyme gene from mRNA to protein (or degrade mRNA) include any of the following (i) to (iii): (i) a nucleic acid or a precursor thereof that has RNAi activity against the mRNA of an enzyme gene (ii) Antisense nucleic acid against the mRNA of the enzyme gene (iii) Ribozyme nucleic acid for the mRNA of the enzyme gene In a preferred embodiment, the shRNA can be expressed in a T cell-specific manner by using an expression vector in which DNA encoding the shRNA against the mRNA of the enzyme gene is functionally linked downstream of a T cell-specific promoter.

[0054] Examples of agents that reduce the production of UDP-GlcNAc, a substrate for LacNAc biosynthesis, include, but are not limited to, 4-F-GlcNAC and 3-F-GlcNAC, in which the 3-position is substituted with fluorine instead of the 4-position (see Patent Document 3 above). 4-F-GlcNAC is preferred. The structural formula of this compound is shown below.

[0055] [ka]

[0056] (b) A substance that binds to LacNAc and inhibits the binding of LacNAc to Gal-3 The substance (b) may be, for example, an antagonist that binds to LacNAc competitively with its ligand, Gal-3. Examples of such antagonists include lectins that specifically bind to LacNAc, or fragments containing all or part of their glycan-recognition domains. Examples of lectins that specifically bind to LacNAc include Urtica dioica agglutinin (UDA), potato lectin (Solanum tuberosum lectin (STL)), and tomato lectin (Lycopersicon esculentum lectin (LEL)), which belong to the chitin-binding lectin hevein family. However, LEL may be advantageous in terms of antigenicity. Alternatively, examples of LacNAc antagonists include free Gal-3 or fragments containing all or part of its glycan-recognition domain. The glycan-recognition domain of Gal-3 is approximately 130 amino acids from the C-terminus. Information on human Gal-3 protein can be obtained, for example, from the NCBI or UniProtKB database (UniProtKB accession no. P17931). Nucleic acid aptamers, small molecules, and other agents that bind to LacNAc are included as LacNAc antagonists. Antagonists that bind to LacNAc can also be obtained, for example, by constructing a competitive assay system using LacNAc and Gal-3 and screening a compound library.

[0057] In another embodiment, the substance (b) may be an antibody against LacNAc. The antibody may be either a polyclonal or monoclonal antibody. These antibodies can be produced according to publicly known methods for producing antibodies or antisera. The antibody isotype is not particularly limited, but is preferably IgG, IgM, or IgA, with IgG being particularly preferred. The antibody is not particularly limited as long as it has at least a complementarity-determining region (CDR) sufficient to specifically recognize and bind to LacNAc. It may be a complete antibody molecule, or a fragment thereof such as Fab, Fab', or F(ab')2; a genetically engineered conjugate molecule such as scFv, scFv-Fc, a minibody, or a diabody; or a derivative thereof modified with a molecule having a protein-stabilizing effect, such as polyethylene glycol (PEG).

[0058] (c) A substance that binds to Gal-3 and inhibits the binding of LacNAc to Gal-3 Examples of the substance (c) include antagonists that bind to Gal-3 competitively with the receptor LacNAc. Examples of such antagonists include Gal-3 inhibitors that specifically bind to Gal-3 and inhibit its interaction with LacNAc. Examples of Gal-3 inhibitors include GB1107, TD139 (Patent Documents 4 and 5), GB1211 (Zetterberg FR. J Med Chem 65: 12626-38 (2022)), GCS-100 (Streetly MJ, Blood 115(19): 3939-48 (2010)), and GR-MD-02 (Girard and Magnani, Trends in Glycoscience and Glycotechnology, 30(172): SE211-SE220 (2018)). The structural formulas of GB1107, TD139, GB1211 and GR-MD-02 are shown below.

[0059] [ka]

[0060] [ka]

[0061] [ka]

[0062] [ka]

[0063] Gal-3 antagonists include nucleic acid aptamers, small molecules, and other active substances that bind to Gal-3.Gal-3 antagonists can also be obtained by, for example, constructing a competitive assay system using Gal-3 and LacNAc, and screening a compound library.

[0064] In another embodiment, the substance (c) may be an antibody against Gal-3. The antibody may be either a polyclonal or monoclonal antibody. These antibodies can be produced according to publicly known methods for producing antibodies or antisera. The antibody isotype is not particularly limited, but is preferably IgG, IgM, or IgA, with IgG being particularly preferred. The antibody is not particularly limited as long as it has at least a complementarity-determining region (CDR) sufficient to specifically recognize and bind to Gal-3. It may be a complete antibody molecule, or a fragment thereof such as Fab, Fab', or F(ab')2; a genetically engineered conjugate molecule such as scFv, scFv-Fc, a minibody, or a diabody; or a derivative thereof modified with a molecule having a protein-stabilizing effect, such as polyethylene glycol (PEG).

[0065] When an antibody is used as one of the substances (a) to (c), the antibody is used as a pharmaceutical intended for administration to humans. Therefore, the antibody (preferably a monoclonal antibody) is an antibody with a reduced risk of exhibiting antigenicity when administered to humans, specifically, a fully human antibody, a humanized antibody, a mouse-human chimeric antibody, or the like, with fully human antibodies being particularly preferred. Humanized antibodies and chimeric antibodies can be produced by genetic engineering using standard methods. Furthermore, fully human antibodies can also be produced from human-human (or mouse) hybridomas. However, to provide large amounts of antibodies stably and at low cost, it is desirable to produce them using human antibody-producing mice or phage display methods.

[0066] (d) Substances that inhibit Gal-3 expression The substance (d) may act at any stage of the Gal-3 gene, such as the transcription level, post-transcriptional regulation level, protein translation level, or post-translational modification level. Therefore, substances that inhibit Gal-3 expression include, for example, substances that inhibit the transcription of the gene (e.g., antigene), substances that inhibit the processing of initial transcription products into mRNA, substances that inhibit the transport of mRNA into the cytoplasm, substances that inhibit the translation of mRNA into protein (e.g., antisense nucleic acid, miRNA) or that degrade mRNA (e.g., siRNA, ribozyme, miRNA), and substances that inhibit the post-translational modification of initial translation products. While substances that act at any stage can be used, substances that bind complementarily to mRNA and inhibit translation into protein or degrade mRNA are preferred.

[0067] Preferred examples of substances that specifically inhibit translation of the Gal-3 gene from mRNA to protein (or degrade mRNA) include any of the following (i) to (iii): (i) a nucleic acid or a precursor thereof having RNAi activity against the mRNA of the Gal-3 gene (ii) Antisense nucleic acid against the mRNA of the Gal-3 gene (iii) a ribozyme nucleic acid for the mRNA of the Gal-3 gene In a preferred embodiment, the shRNA can be expressed in a T cell-specific manner by using an expression vector in which DNA encoding the shRNA against the mRNA of the Gal-3 gene is functionally linked downstream of a T cell-specific promoter.

[0068] Since Gal-3 contains the NWGR motif common to the bcl-2 survival protein family in its carbohydrate-recognition domain, the substances (c) and (d) above are expected to not only act on HTLV-1-uninfected T cells to inhibit their interaction with LacNAc and thus inhibit cell-to-cell contact infection, but also induce cell death in HTLV-1-infected cells. In fact, as shown in the Examples below, GB1107 exhibits a more pronounced inhibitory effect on cell infection than 4-F-GlcNAC at the same concentration, suggesting that it also exerts an inhibitory effect on infected cell division, another mechanism by which HTLV-1 infection spreads.

[0069] When administered to asymptomatic HTLV-1 carriers, the inhibitors of the present invention can suppress the number of infected cells due to cell-to-cell contact infection, i.e., the increase in HTLV-1 proviral load, thereby preventing or delaying the onset of HTLV-1 infections such as HAM, ATL, and HTLV-1 uveitis (HU). Furthermore, when administered to patients infected with HTLV-1, the inhibitors can treat or suppress the progression of the disease. In particular, since it is known that the severity of motor disorder in HAM patients gradually worsens over the natural course and that the severity of motor disorder correlates with the HTLV-1 proviral load, the inhibitors of the present invention can also be used to suppress the progression and ameliorate the severity of HAM motor disorder by reducing the HTLV-1 proviral load. Therefore, the present invention provides a therapeutic agent for HAM, as well as a therapeutic agent for preventing the onset of HTLV-1 infection in asymptomatic carriers and for treating the disease in HTLV-1 infected patients.

[0070] When the inhibitor of the present invention is used as a pharmaceutical, it can be administered orally or parenterally to a person in need thereof (i.e., asymptomatic HTLV-1 carriers, HAM patients, etc.) as a liquid preparation or as a pharmaceutical composition in an appropriate dosage form.

[0071] When the active ingredient is a proteinaceous molecule such as the above-mentioned antibody or a low molecular weight compound, it may be administered as such or as an appropriate pharmaceutical composition. The pharmaceutical composition used for administration may contain the active ingredient and a pharmacologically acceptable carrier, diluent, or excipient. Such a pharmaceutical composition is provided in a dosage form suitable for oral or parenteral administration.

[0072] Compositions for parenteral administration include, for example, injections, which may take the form of intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, or drip infusions. Such injections can be prepared according to known methods. For example, injections can be prepared by dissolving, suspending, or emulsifying the antibody or low-molecular-weight compound of the present invention or a salt thereof in a sterile aqueous or oily liquid typically used for injections. Examples of aqueous solutions for injection include physiological saline, isotonic solutions containing glucose or other adjuvants, and the like, which may be used in combination with appropriate solubilizers such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)). Examples of oily liquids include sesame oil and soybean oil, and solubilizers such as benzyl benzoate and benzyl alcohol may be used in combination. The prepared injection solution is preferably filled into an appropriate ampule.

[0073] Compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups, emulsions, suspensions, etc. Such compositions are produced by known methods and may contain carriers, diluents, or excipients commonly used in the pharmaceutical field. Examples of carriers and excipients for tablets include lactose, starch, sucrose, and magnesium stearate.

[0074] The parenteral or oral pharmaceutical compositions are conveniently prepared in dosage unit forms that correspond to the dosage of the active ingredient. Examples of such dosage unit forms include tablets, pills, capsules, and injections (ampoules). Protein molecules such as antibodies and low-molecular-weight compounds are typically contained in an amount of 0.1 to 500 mg per dosage unit, preferably 5 to 100 mg for injections and 10 to 250 mg for other dosage forms.

[0075] The dosage of the above-mentioned pharmaceutical containing the above-mentioned antibody or other proteinaceous molecule, or a low molecular weight compound, or a salt thereof varies depending on the subject, symptoms, and route of administration. For example, a single dose of an antibody or low molecular weight compound is typically about 0.0001 to 20 mg / kg body weight, and low molecular weight compounds are administered orally or parenterally about 1 to 5 times daily, while proteinaceous molecules such as antibodies are administered by intravenous injection once a day to once every several months. Similar amounts can also be administered for other parenteral and oral administrations. When symptoms are particularly severe, the dosage may be increased depending on the symptoms.

[0076] When the active ingredient of the inhibitor of the present invention is a nucleic acid molecule, it can be administered orally or parenterally (e.g., intravascular administration, subcutaneous administration, topical application, etc.) as a liquid preparation or as a pharmaceutical composition in an appropriate dosage form to humans or non-human warm-blooded animals (e.g., rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, chickens, etc.; including HTLV-1-infected model animals such as humanized mice), preferably humans.

[0077] Pharmaceuticals containing the nucleic acid molecule as an active ingredient can be formulated and administered according to methods known per se. That is, the nucleic acid molecule may be used alone, or it may be functionally inserted into an expression vector for suitable mammalian cells, such as a retroviral vector, adenoviral vector, or adenovirus-associated viral vector. The nucleic acid may be administered directly or together with an adjuvant to promote uptake, using a gene gun or a catheter such as a hydrogel catheter. Alternatively, it may be aerosolized and administered locally into the trachea as an inhalant. Furthermore, for the purposes of improving pharmacokinetics, prolonging half-life, and improving cellular uptake efficiency, the nucleic acid may be formulated alone or together with a carrier such as liposomes into a pharmaceutical preparation (injectable) and administered intravenously, subcutaneously, or the like.

[0078] The nucleic acid molecule may be administered by itself or as an appropriate pharmaceutical composition. The pharmaceutical composition used for administration may contain the nucleic acid of the present invention and a pharmacologically acceptable carrier, diluent, or excipient. Such a pharmaceutical composition is provided in a dosage form suitable for oral or parenteral administration.

[0079] The oral or parenteral pharmaceutical compositions are conveniently prepared in dosage unit forms that correspond to the dosage of the active ingredient. Examples of dosage unit forms include tablets, pills, capsules, and injections (ampoules). The nucleic acid of the present invention is preferably contained in an amount of, for example, about 0.01 to 500 mg per dosage unit.

[0080] The dosage of the above-mentioned pharmaceuticals containing nucleic acid molecules as active ingredients varies depending on the subject, symptoms, and route of administration. For example, a single dose of nucleic acid molecule is typically about 0.0001 to 20 mg / kg body weight, administered by intravenous injection approximately once a day to once every six months. Similar amounts can also be administered for other parenteral and oral administrations. When symptoms are particularly severe, the dosage may be increased depending on the symptoms. When using Gal-3 inhibitors under clinical development, such as GB1107, TD139, GB1211, GCS-100, and GR-MD-02 (belapectin), as the active ingredient, the dosage and administration methods used in each clinical trial can be used as a reference.

[0081] By combining the inhibitors of the present invention with drugs capable of specifically damaging HTLV-1-infected CD4-positive T cells, it is possible to address both of the two modes of HTLV-1-infected cell count increase, and this is expected to result in a stronger anti-HTLV-1 effect and preventive and therapeutic effects against HTLV-1 infection. Examples of such concomitant drugs include ABL tyrosine kinase inhibitors (e.g., imatinib, nilotinib, dasatinib, etc.) and anti-CCR4 monoclonal antibodies (e.g., mogalizumab). Combination with an ABL tyrosine kinase inhibitor, which is expected to be effective regardless of clonal antibody phenomenon, is particularly desirable. The dosage of these concomitant drugs may be the same as when they are administered alone.

[0082] The above-mentioned concomitant drug can be formulated separately from the inhibitor of the present invention, and if possible, can also be prepared as a single pharmaceutical composition (combined drug). When formulated separately, the inhibitor of the present invention and the concomitant drug can be administered simultaneously or at intervals via the same or different administration routes.

[0083] The present invention will be explained in more detail below by way of examples, but these are merely illustrative and do not limit the scope of the present invention in any way. [Example]

[0084] Clinical trials of oral administration of peracetylated 4-fluoroglucosamine 4-F-GlcNAc, a metabolic inhibitor of LacNAc biosynthesis, to humans have not yet been conducted. Although there has been little research on the inhibition of LacNAc synthesis in N-glycans, there has been a relatively large amount of research on the inhibition of LacNAc synthesis in O-glycans. Therefore, the administration methods used in animal studies of LacNAc in O-glycans can be used as a reference for considering actual administration methods for humans.

[0085] Examples of in vivo administration of 4-F-GlcNAc in mice include the following: Sixty mice received intraperitoneal injections of 4-F-GlcNAc at 100 mg / kg / day for nine consecutive days, with no acute side effects (Cedeno-Laurent F. J Invest Dermatol 132(2):410-20.(2012)). Similarly, 6- to 8-week-old mice received intraperitoneal injections of 4-F-GlcNAc at 50-250 mg / kg / day for six days, demonstrating anti-inflammatory effects, with no toxicity other than limited toxicity at 250 mg / kg / day (Dimitroff CJ. J. Clin Invest 112:1008-18.(2003)). The anti-inflammatory effect at 100 mg / kg / day was observed as inhibition of PSGL-1 (a selectin ligand) with a polylactomine backbone and reduced migration of inflammatory cell infiltration. A dose of 100 mg / kg / day for mice is equivalent to a dose of 5,000 mg (5 g) / day for a human with a body weight of 50 kg. Four mice were also administered a 0.25 mg / ml solution of 4-F-GlcNAc for seven days, with an average daily intake of 4.5–5.0 ml / day, resulting in an oral intake of 2.25–2.5 mg / day. Serum MS-TOF analysis revealed a mean serum concentration of 0.66 ± 0.20 mM (660 ± 200 μM). In experimental autoimmune encephalomyelitis (EAE) groups, the untreated group had a 90% incidence of disease and a 40% mortality rate, while the treated group had an 82% incidence and 0% mortality rate. The Th1 / Th17 response was suppressed in the treated group. In the above example, assuming a mouse weighing 20 g, an intake of 2.0 mg corresponds to approximately 1 / 10,000 of the body weight, which corresponds to a 5 g / day dose for a 50 kg human. Roughly speaking, a 760 mg / day dose for a 50 kg body weight, or approximately 1,500 mg / day for a 75 kg body weight, would result in a blood concentration of 100 μM. The IC50 of 4-F-GlcNAc for leukemia cell death is 34 μM (Sharma M. Carbohydr. Res. 198, 205-221.(1990)). In this paper, a dose of 200 mg / kg / day to mice was toxic. This is equivalent to an intake of 10 g / day for a 50 kg human. If this amount is considered the minimum intake required for side effects to appear, then the above dose would be considered to have a safety factor of about 10.

[0086] In addition, IC 100 was found in an in vitro growth inhibition test of human T cells. 10 The 10% inhibitory concentration (10%) was at least 200 μM or less than 150 μM (based on a 36-hour doubling time culture). Treatment with 50 μM 4-F-GlcNAc suppressed P-selectin ligand (PSGL-1) expression on T cells by 96% (Dimitroff C. Blood 101(2): 602-10. (2003)). Treatment with 10-100 μM suppressed E-selectin ligand expression by 50% (Descheny LJ Invest Dermatol 126(9): 2065-2073. (2006)). Thus, we observed that selectin ligands (modified with LacNAc on the O-glycan) on T cells could be inhibited at concentrations that did not affect T cell growth, which is consistent with the blood concentrations that can be achieved by an estimated oral dose in humans.

[0087] No human clinical trials have yet been reported for 4-F-GlcNAc, and only one has been reported for the similar compound, N-acetylglucosamine (GlcNAc) (Tomonaga A. Exp Ther Med. 12(3):1481-1489.(2016)). In this study, 75 healthy volunteers were divided into three groups and administered a placebo, 500, or 1000 mg (0.5 or 1.0 g) per day. No side effects were observed, and the cartilage protective effect was observed.

[0088] Oral administration of 4-F-GlcNAc to humans can be achieved using the dose for mice as a reference: 760 mg / day for a 50 kg human, or approximately 1,500 mg / day for a 75 kg human, resulting in a blood concentration of 100 μM. This amount generally has a safety margin of approximately 10.

[0089] Since we believe that the LacNAc N-glycan on T cells, which we target, can be suppressed at similar blood concentrations, oral administration of 4-F-GlcNAc at a dose of 760 mg / day in a 50 kg human body, or approximately 1,500 mg / day in a 75 kg human body, would result in a blood concentration of 100 μM. This dose would suppress the LacNAc N-glycan on T cells of HTLV-1-infected individuals and inhibit cell-to-cell contact infection within the body of HTLV-1-infected individuals, at a concentration that does not affect T cell growth. [Example]

[0090] As with 4-F-GlcNAc, the oral dosage for humans of the Gal-3 inhibitor GB1107 has not yet been tested in clinical trials, so the dosage for mice can be used as a reference for conversion.

[0091] Gal-3 is expressed in human T cells, macrophages, fibroblasts, epithelial cells, and cancer cells. The dosage for humans of the Gal-3 inhibitor GB1107 can be determined from data reported on the administration of GB1107 to a mouse tumor-bearing model (Vuong L. Cancer Res 79(7): 1480-92. (2019)). Mouse Lewis lung carcinoma (LLC1) cells: 2.5 × 10 5 gal3- / - C57B1 / 6 mice, 3 × 10 human lung cancer cell line A549 cells 6 When GB1107 was transplanted into CD-1 nude mice, LLC1 cells did not grow at all in the gal3- / - mice, and M2 macrophages were reduced in the mice. This demonstrates the necessity of Gal-3 for lung cancer. Furthermore, in mice implanted with the human lung cancer A545, Gal-3 levels and tumor weight were significantly reduced in the GB1107-treated group. With oral administration of GB1107 at 10 mg / kg once daily, lymphocyte counts such as CD3 and CD4 were unchanged compared to the control group, while CD8 counts were higher in the treated group. This translates to a daily oral administration of 500 mg of GB1107 for a 50 kg person, or 750 mg for a 75 kg person.

[0092] GB1107 has been shown to have a relatively low clearance rate (T1 / 2 = 4.5 hours at 1.2 ml / min / kg intravenous infusion) and is highly bioavailable upon oral administration (F = 75%). Furthermore, GB1107 is reported to have a 38-fold lower affinity for mouse Gal-3 (see Vuong L, supra), but its equilibrium dissociation constant (Kd) for human Gal-3 is high at 37 nM (Zetterberg FR. Chem Med Chem 13(2):133-137 (2018)). Given these considerations, significantly lower doses than those calculated above may be sufficient. A more appropriate oral dose can be assessed by quantifying Gal-3 mRNA and protein in target human peripheral blood CD4+ T cells. [Example]

[0093] When using the LacNAc-specific chitin-binding lectin hevein family to inhibit the LacNAc-Gal-3 interaction, LEL is known to be non-toxic (Nachbar MS. J Biol Chem 255(5):2056-61. (1980)) and can be administered intravenously. Since there are no similar clinical trials in humans, administration examples in mice can be used as a reference for conversion.

[0094] Vascular endothelial cells, particularly those in the central nervous system, are known to express LacNAc and stain with LEL. Therefore, the use of labeled LEL in intravenous infusion in animals to visualize vascular networks and identify damaged areas, as well as for medical imaging, has been investigated. For example, it has been reported that intravenous injection of 100 μg / 100 μl of LEL into mice resulted in its disappearance from venous blood within two minutes, its staining of vascular endothelium between one minute and one hour later, and its detection in the same tissue after 12 hours (Robertson RT. Histochem Cell Biol. 143(2):225-34. (2015)).

[0095] This LEL amount corresponds to the concentration and amount of 250 mg / 250 ml administered intravenously in a 50 kg human. As with the other drugs in Examples 1 and 2, the appropriate dosage can be evaluated by quantifying Gal-3 mRNA and protein in target human peripheral blood CD4+ T cells. [Industrial Applicability]

[0096] The inhibitor of the present invention is the first drug consisting of a compound capable of inhibiting cell-to-cell contact infection of HTLV-1, and is extremely useful in that it can suppress the progression of and improve symptoms in HAM patients. Furthermore, when used in combination with a drug capable of specifically inducing cell death in HTLV-1-infected cells, it is extremely useful in that it can reduce or eliminate the HTLV-1 proviral load, and thus may serve as a radical treatment for HAM and HTLV-1 infection.

Claims

1. An inhibitor of human T-cell leukemia virus type 1 (HTLV-1) cell-to-cell contact infection, containing a substance that inhibits the receptor-ligand interaction between N-acetyllactosamine (LacNAc) and galectin-3 (Gal-3).

2. Substances that inhibit the receptor-ligand interaction between LacNAc and Gal-3 are (a) a substance that inhibits the expression of LacNAc on the membrane surface; (b) a substance that binds to LacNAc and inhibits the binding of LacNAc to Gal-3; (c) a substance that binds to Gal-3 and inhibits the binding of LacNAc to Gal-3, or (d) a substance that inhibits Gal-3 expression The inhibitor of claim 1, wherein

3. The substance (a) is contained, and the substance is (a1) an inhibitor of UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 2 (B3GNT2) or beta-1,4-galactosyltransferase 1 (B4GALT1), or (a2) metabolic LacNAc biosynthesis inhibitor The inhibitor of claim 2, wherein

4. The inhibitor according to claim 3, which contains the inhibitor (a2), and the inhibitor is peracetylated 4-fluoroglucosamine (4-F-GlcNAc).

5. The inhibitor according to claim 2, which contains the substance (b), and the substance is nettle lectin (UDA), potato lectin (STL), or tomato lectin (LEL).

6. The inhibitor according to claim 1, comprising the substance (c), wherein the substance is selected from the group consisting of GB1107, TD139, GB1211, GCS-100, and GR-MD-02 (belapectin).

7. The inhibitor according to any one of claims 1 to 6, which is for the treatment of HTLV-1 associated myelopathy (HAM) and HTLV-1 infection.

8. A method for inhibiting HTLV-1 cell-to-cell contact infection in an HTLV-1-infected individual, comprising administering to the infected individual an effective amount of a substance that inhibits the LacNAc-Gal-3 interaction.

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