by Derek Hoi-Hang Ho, and Roger Hoi-Fung Wong TNP-470 skews DC differentiation to Th1-stimulatory phenotypes and can serve as a novel adjuvant in a cancer vaccine by Derek Hoi-Hang Ho, and Roger Hoi-Fung Wong BloodAdv Volume 2(14):1664-1679 July 24, 2018 © 2018 by The American Society of Hematology

Derek Hoi-Hang Ho, and Roger Hoi-Fung Wong Blood Adv 2018;2:1664-1679 © 2018 by The American Society of Hematology

TNP-470 potentiates DC immunogenicity upon LPS stimulation in vitro. TNP-470 potentiates DC immunogenicity upon LPS stimulation in vitro. (A) In vitro inhibitory effects of TNP-470 on T lymphocytes. Lymphocytes were and CD28 with different concentrations of TNP-470 for 72 hours. 3H-Thymidine was added 16 hours prior to sample harvest. Proliferation of T lymphocytes was evaluated by the level of incorporated 3H-thymidine. Representative data from 3 independent studies are shown (left). Data presented as mean ± standard error of the mean (SEM; n = 3). IL-2 secretion from lymphocytes upon CD3/CD28 stimulation (right). Lymphocytes were stimulated with anti-mouse CD3e and CD28 in the presence of vehicle/TNP-470 (0.5 and 1 µM) for 24 hours. Culture supernatant was harvested and detected for IL-2 secretion. Data presented as mean ± SEM (n = 3) from 2 independent studies. ***P < .001 compared with vehicle. ###P < .001 TNP-470 (0.5 µM) vs TNP-470 (1 µM). BMDCs upon vehicle and TNP-470 treatment (5 nM) were stimulated with LPS (200 ng/mL) for 4 hours to evaluate cytokine mRNA expression and for 16 hours to evaluate cytokine secretion. (B) Cytokine secretion profile of TNP-470/vehicle-treated DCs upon LPS stimulation. **P < .01 and ***P < .001 TNP-470–treated DCs (TNP-DC) vs vehicle-treated DCs (CTR-DC). Data presented as mean ± SEM from 2 independent experiment. (C) Fold-of-change in expression of IL-12 and IL-10 from BMDCs, which were normalized with GAPDH. Data presented as mean ± SEM from 3 independent experiment. ***P < .001 TNP-470 DC vs CTR-DC. (D) Cytokine IL-12 and IL-10 secretion from culture supernatant. Data presented as mean ± SEM from 5 independent experiment. ***P < .001 TNP-DC vs CTR-DC; ###P < .001 imDC vs CTR-DC. (E) BMDCs was stimulated with LPS for 4 hours to evaluate gene expression levels (left) and 8 hours to examine protein levels (right) of SOCS1 and SOCS3. ***P < .001 TNP-DC vs CTR-DC. Data presented as mean ± SEM from 2 independent experiment. Derek Hoi-Hang Ho, and Roger Hoi-Fung Wong Blood Adv 2018;2:1664-1679 © 2018 by The American Society of Hematology

TNP-470–treated DCs induce T-cell activation in vitro and in vivo. TNP-470–treated DCs induce T-cell activation in vitro and in vivo. (A) BMDCs were cultured with syngeneic (left) and allogenic (middle) lymphocytes at different ratios (1:5, 1:10, 1:20) for 72 hours (syngeneic culture) and 48 hours (allogenic culture). 3H-labeled thymidine (0.5 μCi/well) was added in the last 16 hours of culture and cell proliferation was measured by incorporated 3H-labeled thymidine. Data presented as mean ± SEM from 3 independent experiment. ***P < .001 TNP-DC coculture vs CTR-DC coculture. Culture supernatant from 72-hour syngeneic culture and 48-hour allogenic culture (DC:T-cell ratio = 1:5) were harvested and detected for IL-2 secretion (right). ***P < .001 TNP-DC coculture vs CTR-DC coculture. Data presented as mean ± SEM from 3 independent experiment. (B) Activation of in vitro OVA-specific CD4 T cells. CD4 cells from OVA-immunized mice were collected and cultured with TNP-470/vehicle- treated and OVA-pulsed DCs for 24 hours. Culture supernatant was detected for IL-2 secretion (left). ***P < .001 OVA-pulsed CTR-DC coculture vs OVA-pulsed TNP-DC coculture. Data presented as mean ± SEM from 3 independent experiment. Activation of OVA-specific lymphocytes in vivo. C57BL mice were immunized with OVA-pulsed DCs (TNP-470/vehicle-treated). Lymphocytes from mice in different groups were harvested and stimulated with OVA ex vivo. Culture supernatant from 24-, 48-, and 72-hour culture was detected for IL-2 secretion (right). *P < .05 lymphocytes from mice immunized with OVA-pulsed TNP-DC vs OVA-pulsed CTR-DC counterpart. Data presented as mean ± standard deviation (SD) from 3 independent experiment. Derek Hoi-Hang Ho, and Roger Hoi-Fung Wong Blood Adv 2018;2:1664-1679 © 2018 by The American Society of Hematology

TNP-470–treated DCs promote in vitro and in vivo Th1 polarization and induce strong CTL responses. TNP-470–treated DCs promote in vitro and in vivo Th1 polarization and induce strong CTL responses. Naive CD4 cells were primed with TNP-470/vehicle-treated DCs for 7 days. Primed CD4 cells were further stimulated with PMA (50 ng/mL) for 4 hours as mitogenic stimulation and anti-CD3e for 6 hours as TCR-mediated stimulation. Representative data of IFN-γ–producing CD4 cells and T-bet expressing CD4 cells by intracellular staining were showed (A, left) and percentages of corresponding cell population were summarized (A, right). ***P < .001 CD4 cells primed with TNP-DC vs CTR-DC. Data presented as mean ± SEM from 3 independent experiment. (B) In vitro OVA polarization assay. TNP-470/vehicle- treated and OVA-pulsed DCs were cultured with OVA-specific CD4 cells for 24 hours. Culture supernatant were detected for IL-12 (left) and IFN-γ (right) secretion. ***P < .001 Coculture of TNP-DCs and OVA-specific CD4 cells vs that of CTR-DCs and OVA-specific CD4 cells. Data presented as mean ± SEM from 3 independent experiment. (C) In vivo OVA polarization assay. C57BL mice were immunized with OVA-pulsed DCs (TNP-470/vehicle-treated) on day 0 and day 7. Lymphocytes from mice in different groups were harvested on day 14 and stimulated with OVA ex vivo. Culture supernatant from 24-, 48-, and 72-hour culture was detected for IFN-γ secretion (left). Data presented as mean ± SD from 3 independent experiment. Percentages of OVA-specific CD4 (middle) and CD8 cells (right) were quantified by flow cytometric analysis of IFN-γ+CD4+ and CD8+ cells upon 72 hours of ex vivo stimulation of OVA. Data presented as mean ± SD from 3 independent experiment. (D) OVA-specific CTL response. C57BL mice were immunized with OVA or OVA peptide (SIIFEKL)–pulsed DCs (TNP-470/vehicle- treated) on day 0, 7, and 14. Lymphocytes harvested on day 21 were stimulated with OVA peptide ex vivo and culture supernatant from 24-, 48-, and 72-hour culture was collected to detect IFN-γ secretion. **P < .01 and ***P < .001 TNP-DC vs CTR-DC (OVA /SIIFEKL-pulsed). ##P < .01 OVA-pulsed TNP-DC vs SIIFEKL-pulsed TNP-DC. Data presented as mean ± SD (n = 3). Derek Hoi-Hang Ho, and Roger Hoi-Fung Wong Blood Adv 2018;2:1664-1679 © 2018 by The American Society of Hematology

TNP-470–treated DCs trigger NF-κB transactivation of IL-12 and potentiate p38/JNK activation. TNP-470–treated DCs trigger NF-κB transactivation of IL-12 and potentiate p38/JNK activation. (A) TNP-470/vehicle-treated DCs were stimulated with different TLR agonists (5 nM, 16hr). **P < .01 TNP-DC vs CTR-DC upon agonist stimulation. Data presented as mean ± SEM from 3 independent experiment. (B) TNP-470/vehicle-treated DCs were stimulated with LPS (1 µg/mL) for 0, 30, and 60 min. Total protein lysate was blotted with antibodies against p38, phosphor-p38, JNK, phosphor-JNK and GAPDH. Representative images from 2 independent experiment were showed. (C) TNP-470/vehicle-treated DCs were stimulated with LPS for 24 hours in the presence of 2 µM and 10 µM p38 inhibitor SB203580 and MAPK inhibitor PD98059. IL-12 secretion was detected by cytokine ELISA. ***P < .001 compared with TNP-470–treated DCs without inhibitor. Data presented as mean ± SEM from 2 independent experiment. (D) TNP-470/vehicle-treated DCs were stimulated with LPS (200 ng/mL) for 24 hours in the presence of 2 µM and 10 µM NF-κB inhibitor CAPE. IL-12 secretion was detected by cytokine ELISA. ***P < .001 compared with TNP-470–treated DCs without inhibitor. Data presented as mean ± SEM from 2 independent experiment. (E) Fold-of-change in expression of different NF-κB subunits, p52, p65, c-Rel and relB, which were normalized with GAPDH, in TNP-470/vehicle-treated DCs (200 ng/mL LPS for 4 hours). *P < .05, **P < .01 and ***P < .001 TNP-DC vs CTR-DCs. Data presented as mean ± SEM from 2 independent experiment. (F) TNP-470/vehicle-treated DCs was stimulated with LPS for 30 minutes. Expression of cRel, p65 and histone H3 (loading control) from nuclear fractions were detected. Representative images from 2 independent experiment were showed. (G) NF-κB p65 translocation assay. TNP-470/vehicle-treated DCs were stimulated with LPS for 30 minutes. The subcellular localization of p65 was visualized by immunofluorescence using anti-p65 antibody (red) and nuclei were stained by DAPI (blue). Representative images from 3 independent experiment were showed. Original magnification ×40. (H) Recruitment of NF-κB subunit c-Rel to IL-12 promoter. TNP-470/vehicle-treated DCs were stimulated with LPS for 0 and 2 hours. Cells were then cross-linked and lysed. DNA was immunoprecipitated with anti-cRel and control IgG. Degree of c-Rel recruitment to IL12 promoter was quantified by real time PCR. *** P < .001 TNP-DC vs CTR-DC. Representative data presented as mean ± SEM from 2 independent experiment. Derek Hoi-Hang Ho, and Roger Hoi-Fung Wong Blood Adv 2018;2:1664-1679 © 2018 by The American Society of Hematology

Enhanced tumor-specific immunogenicity and protective effect(s) of TNP-470–treated DC vaccine in prophylactic B16 melanoma vaccination model. Enhanced tumor-specific immunogenicity and protective effect(s) of TNP-470–treated DC vaccine in prophylactic B16 melanoma vaccination model. (A) Schematic diagram of prophylactic setting of vaccination model. Mice were immunized with PBS (solvent control), DC without tumor lysate pulsing (DC control), tumor lysate-pulsed TNP-470/vehicle-treated DC vaccine. (B) Lymphocytes from immunized mice in different groups were collected and stimulated with tumor lysate (50 µg/mL) for 24, 48 and 72 hours ex vivo. Culture supernatant was detected for secretion of IL-2 (left) and IFN-γ (right). **P < .01 and *** P < .001 TNP-DC vaccine vs vehicle DC vaccine. (C) To quantify amount of tumor-specific CTLs from mice in different groups, lymphocytes were stimulated with tumor lysate (50 µg/mL) for 72 hours ex vivo, followed by re-stimulation of PMA/Ionomycin for 6 hours. Cells were then stained with CD3, CD8 and IFN-γ. Tumor-specific CTLs were determined by flow cytometric analysis of CD3+CD8+IFN-γ+ cells. *P < .05 vehicle DC vaccine vs PBS solvent control. **P < .01 TNP-DC vaccine vs DC control. ***P < .001 TNP-DC vaccine vs PBS solvent control. ##P < .01 TNP-DC vaccine vs vehicle DC vaccine. Data presented as mean ± SD (n = 6) from 2 independent studies. (D) Tumor-specific cytolytic activity. Lymphocytes from mice in different groups were collected and stimulated with tumor lysate (50 µg/mL) for 72 hours ex vivo, then harvested as effector cells (E), and B16-F10 cells were used as target cells (T). Specific lysis was detected by LDH assay at E:T ratio = 100:1. **P < .01 TNP-DC vaccine vs vehicle DC vaccine; ***P < .001 TNP-DC vaccine vs PBS solvent control and DC control. Data presented as mean ± SD (n = 6) from 2 independent studies. (E) Percentages of tumor-free mice in different vaccination groups (n = 6). *P < .05 TNP-DC vaccine vs PBS solvent control. #P < .05 TNP-DC vaccine vs DC control. (n = 6, Modified Kaplan-Meier Survival Analysis). (F) Weight of solid tumor developed on mice on day 26 (end point). *P < .05 TNP-DC vaccine vs PBS solvent control. **P < .01 TNP-DC vaccine vs DC control. Data presented as mean ± SD (n = 6) from 2 independent studies. Derek Hoi-Hang Ho, and Roger Hoi-Fung Wong Blood Adv 2018;2:1664-1679 © 2018 by The American Society of Hematology

Enhanced tumor-specific immunogenicity and delayed tumor progression by TNP-470–treated DC vaccine in therapeutic B16 melanoma vaccination model. Enhanced tumor-specific immunogenicity and delayed tumor progression by TNP-470–treated DC vaccine in therapeutic B16 melanoma vaccination model. (A) Schematic diagram of therapeutic setting of vaccination model. Solid tumor was developed on mice by subcutaneous injection of B16 melanoma. When size of solid tumor reached around 150 mm3 (day 7), mice were immunized with PBS (solvent control), DC without tumor lysate pulsing (DC control), tumor lysate-pulsed TNP-470/vehicle-treated DC vaccine on day 7 and day 11, respectively. (B) Lymphocytes from immunized mice in different groups were collected and stimulated with tumor lysate (50 µg/mL) for 24, 48, and 72 hours ex vivo. Culture supernatant was detected for secretion of IL-2 (top) and IFN-γ (bottom). **P < .01 and *** P < .001 TNP-DC vaccine vs PBS solvent control; ##P < .01 TNP-DC vaccine vs vehicle DC vaccine. (C) To quantify amount of tumor-specific CTLs from mice in different groups, lymphocytes were stimulated with tumor lysate (50 µg/mL) for 72 hours ex vivo, followed by re-stimulation of PMA/Ionomycin for 6 hours. Cells were then stained with CD3, CD8 and IFN-γ. Tumor-specific CTLs were determined by flow cytometric analysis of CD3+CD8+IFN-γ+ cells. ***P < .05 TNP-/vehicle DC vaccine vs PBS solvent control; ##P < .01 TNP-DC vaccine vs DC control/ vehicle DC vaccine. Data presented as mean ± SD (n = 5). (D) Tumor-specific cytolytic activity. Lymphocytes from mice in different groups were collected and stimulated with tumor lysate (50µg/mL) for 72 hours ex vivo, then harvested as effector cells (E), and B16-F10 cells were used as target cells (T). Specific lysis was detected by LDH assay at E:T ratio = 100:1. **P < .01 vehicle DC vaccine vs PBS solvent control; ***P < .001 TNP-DC vaccine vs PBS solvent control. Data presented as mean ± SD (n = 5). (E) Solid tumor development in therapeutic setting of B16 melanoma vaccination model. Mice with solid tumor (around 150 mm3 on day 7) were vaccinated with vehicle PBS, DC without tumor lysate pulsing, tumor lysate-pulsed vehicle DC vaccine, TNP-470–treated DC vaccine. Tumor volume of solid B16 melanoma developed on mice from day 7 to day 15. Data are presented as mean ±SD (n = 5) *P < .05, TNP-DC vaccine vs vehicle DC vaccine; #P < .05, ##P < .01 TNP-DC vaccine vs PBS solvent control. Derek Hoi-Hang Ho, and Roger Hoi-Fung Wong Blood Adv 2018;2:1664-1679 © 2018 by The American Society of Hematology