METHODS FOR ISOLATING, EXPANDING AND ADMINISTERING CANCER SPECIFIC CD8+ T CELLS

Cross-Reference to Related Applications

This application claims the benefit of the filing date of U.S. application

Serial No. 62/479,057, filed on March 30, 2017, the disclosure of which is incorporated by reference herein.

S tatement of Government Rights

This invention was made with government sup port under grant number R35CA196878 awarded by the National Institutes of Health. The government has certain rights in the invention.

Background

Treatment with autologous CD 8 positive patient-specific T cells has produced long term remissions in some patients with melanoma and other cancers (Rosenberg and Restifo, 2015). However, it has been very difficult to identify, and expand such cells. Usually, the so-called Tumor Infiltrating Lymphocytes (TILs) must be isolated from surgical or biopsy specimens, and expanded for weeks in tissue culture until a sufficiently large number of cells can be recovered for re-infusion (Rosenberg et al, 2008) (Figure 1). For these reasons, autologous T cell therapy of cancer has not been widely adopted.

Recently, the Rosenberg group at the NCI was successful in identifying neo-antigen specific T cells in the peripheral blood of some melanoma patients, using a combination of surface markers and functional assessment of reactivity to tumor antigens (Gros et al., 2016). However, the number of patients in which such cells can be recovered and expanded is meager. Accordingly, there is an unmet and critical need for simple and practical methods to increase the numbers of TILs in the blood, to demonstrate their tumor reactivity, and to expand them for re-administration.

S ummary

A major goal of cancer immunotherapy is the expansion and/or reactivation of cytotoxic CD8 ⁺ T cell responses against malignant cells. Poorly immunogenic tumor cells evade host immunity and grow even in the presence of an intact immune sy stem, but the complex mechanisms regulating tumor immunogenicity have not been elucidated. Here, in three different murine syngeneic tumor models (B16, SCC7, and 4T 1), it was demonstrated that loss of the Hippo pathway kinases LATS1/2 (large tumor suppressor 1 and 2) in tumor cells inhibits tumor growth. Tumor regression by LATS1/2 deletion requires adaptive immune responses, and LATS1/2 deficiency enhances tumor vaccine efficacy . Mechanistically, LATSl/2-null tumor cells secrete nucleic acid-rich extracellular vesicles (EVs), which induce a type I interferon response via the Toll-like receptors-M YD 88/T RTF pathway. LATS1/2 deletion in tumors thus improves tumor immunogeni city, leading to tumor destruction by enhancing anti-tumor immune responses. These observations uncover a role of the Hippo pathway in modulating tumor immunogeni city and demonstrate a proof of concept for targeting LATS1/2 in cancer immunotherapy .

In one embodiment, a protocol that allows maximizing the therapeutic effect of anti-PD l therapy for the clonal expansion of tumor specific CD8+ T cells in TILs and spleen by local administration of an immune stimulating agent (e.g., a TLR7 or TLR9 agonist), is provided. This clonal expansion is a great predictor of efficacy of anti-PDl therapy and hence a guiding indicator patients should be administered/treated with this therapy .

In one embodiment, a patient with cancer is administered specific drugs that promote the release of immunogenic tiny EVs from cancer cells, in the absence of cytotoxicity, which in turn induces clonal expansion of tumor specific CD8+ T cells, without knowing the exact antigen specific for the tumor cells. In one embodiment, after drug therapy , tumor specific CD8+ T cells are expanded in tissue culture, which can be made more efficient by purification of activated CD 8+ T cells and by co-culture of the T cells with the immunogenic EVs derived from the blood of the same patient in the presence or absence of feeder cells. In one embodiment, the treatment of cancer patients with anti-PD 1 and other checkpoint inhibitors should be undertaken specifically in those subjects who have circulating clonal T cells after drug treatment, and at the time when both EVs and TIL concentrations in the blood reach maximal levels. In one embodiment, antigen presenting cells, like dendritic cells and macrophages, release exosomes (EVs) containing antigen and immune stimulating DNA/RNA which may function as "artificial antigen presenting cells" that travel through the lymphatics to distant sites, in order to initiate a good immune response. In one embodiment, a method to isolate and expand cancer-specific CD8+ T cells is provided. In one embodiment, the method includes

administering to a mammal having a tumor an effective amount of a composition comprising an agent that promotes the release of extracellular vesicles from tumor cells; and collecting from the mammal extracellular vesicles and immune cells including tumor specific CD8+ T cells. In one embodiment, the

composition comprises a TLR7 or TLR9 agonist. In one embodiment, the extracellular vesicles and the immune cells are collected from blood of the mammal. In one embodiment, the extracellular vesicles and the immune cells are collected contemporaneously . In one embodiment, the agent inhibits or inactivates LATS1 and/or LATS2. In one embodiment, the method further includes culturing the immune cells to expand and/or activate cancer-specific CD8+ T cells. In one embodiment, the collected extracellular vesicles are cultured with the immune cells. In one embodiment, the collected extracellular vesicles are less than about 0.5 microns in diameter. In one embodiment, the collected extracellular vesicles are isolated. In one embodiment, in the isolated extracellular vesicles are less than about 0.5 microns in diameter. In one embodiment, the cultured and expanded cancer-specific CD8+ T cells are isolated from non-cancer specific CD8+ T cells. In one embodiment, the method further includes culturing the immune cells in the presence of feeder cells. In one embodiment, the method further includes collecting the expanded, or activated and expanded, cancer-specific CD8+ T cells. In one embodiment, the composition is orally or parenterally administered or by intrapulmonary routes. In one embodiment, the composition is administered to the tumor by direct injection. In one embodiment, the composition is administered sy stemically using liposomes, antibodies or other targeting mechanisms. In one embodiment, the mammal is subjected to chemotherapy before the immune cells are collected. In one embodiment, the mammal is subjected to chemotherapy after the immune cells are collected. In one embodiment, the method further includes isolating the cultured cancer-specific CD8+ T cells. In one

embodiment, the cancer-specific CD8+ T cells are administered to the mammal. In one embodiment, the mammal is subjected to chemotherapy after the cancer- specific CD8+ T cells are administered. In one embodiment, wherein thetherapy is anti-PD therapy . In one embodiment, thetherapy is a checkpoint inhibitor therapy . In one embodiment, the inhibitor comprises pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, or ipilimumab.

In one embodiment, a method to isolate and expand cancer-specific CD8+ T cells is provided. In one embodiment, the method includes collecting from a mammal having a tumor extracellular vesicles and immune cells including tumor specific CD8+ T cells; culturing the immune cells and enriching for cancer-specific CD8+ T cells; and culturing the enriched cancer-specific CD8+ T cells and the extracellular vesicles. In one embodiment, the mammal is subjected to chemotherapy before the immune cells are collected. In one embodiment, the mammal is subjected to chemotherapy after the immune cells are collected. In one embodiment, the method further includes isolating the cultured cancer-specific CD8+ T cells. In one embodiment, the cancer-specific CD8+ T cells are administered to themammal. In one embodiment, the mammal is subjected to chemotherapy after the cancer-specific CD8+ T cells are administered. In one embodiment, wherein the therapy is anti-PD therapy . In one embodiment, the therapy is a checkpoint inhibitor therapy . In one embodiment, the inhibitor comprises pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, or ipilimumab.

In one embodiment, a method to enhance the immunogenicity of tumor cells is provided. In one embodiment, the method includes modifying ex vivo tumor cells of a mammal to provide for tumor cells that do not express or have reduced expression of LAT l and/or LAT2; and administering to themammal an amount of the modified cells effective to enhance the immune response to the tumor in the mammal.

Although direct injection of toll-like receptor 7 (TLR7) agonists into primary tumors can induce tumor-specific oligoclonal T cell responses whose magnitude correlates with therapeutic efficacy, tumors are not always accessible to local therapy . Herein below, it is demonstrated that a single systemic administration of a phospholipid conjugated TLR7 agonist can also expand tumor-specific cytotoxic T cells that are shared by different animals. The expansion can be achieved without causing apparent toxicity . Similar technology combining immune repertoire analysis and immunomodulatory drugs can help to guide the development of optimal immunotherapeutic regimens in cancer patients. Brief Description of the Figures

Figure 1. Current protocol for TIL isolation and expansion. The procedure in the current TIL therapy requires collection of surgical specimens. Circulating tumor specific T cells can be collected from peripheral blood, however, the frequency of such cells is very low, Because pan T cell stimulators, e.g IL-2 and OKT3, are used to expand T cells, proliferation of non-specific T cells may overcome expansion of tumor specific clones.

Figures 2A-2F. Combination therapy with the TLR7 agonist 1V270 and systemic anti-PD-1 agent increases activated CD8 ⁺ population in TILs and spleens. (A-F) 1V270 increased CD8 ⁺ population in TILs. C3H mice (n=5- 8/group) were implanted with SCC7 and carcinoma cells 1A. Tumors and spleens were harvested on day 21 and T cells in TILs or spleens were analyzed by flow cytometry . (A and B) Tumor infiltrating CD8 ⁺ T cells were gated on CD45 ⁺ CD3 ⁺ CD8 ⁺ populations. Number of CD8 ⁺ T cells (A) and IFNy ⁺ CD8 ⁺ cells (B) per tumor volume (mm ³ ) were calculated and plotted. (C)

Representativeimmunohistochemistry images of the tumors (day 21) stained for CD 8 (red) and D API (blue). Scale bar: 20 urn. (D) Number of IFNy ⁺ CD8 ⁺ T cells in spleens. Bars indicate mean ± SEM . Each dot represents an individual animal and bars indicate mean ± SEM in A, B and D. *<0.05, **P<0.01 (Kruskal-Wallis test with Dunn's post hoc test), n=5-8/group . (E) Tumor volumes at the injected sites were plotted against the log of the number of IFNy ⁺ CD8 ⁺ T cells in the tumor microenvironment. Significant negative correlation was demonstrated by Spearman correlation test. (F) The tumor volumes (day 21, injected side) were plotted against the log of the numbers of IFNy ⁺ CD8 ⁺ T cells in the spleen.

Figures 3A-3E. Systemic anti-PD-1 agent treatment or combination treatment increased TCR clonality of CD8 ⁺ T cells. (A-D) SCC7-bearing mice (n=4/group) were treated as described in Figure 2. Tumors and spleens were harvested on day 21 and CD8 ⁺ T cells were isolated using M ACSMicroBead. RNA was isolated and next-generation sequencing was performed. (A)

Representative data of TCR rep ertoire clonality of CD8 ⁺ T cells. TheX and Y- axes show the combination of V and J genes (TRA V and TRAJ families), and the Z-axis shows their frequency of usage. (B and C) Clonality indices (1- normalized Shannon index) in injected and distant uninjected tumors (B) and spleens (C). Higher value of clonality index reflects TCR clonal expansions. Closed and open symbols indicate injected and uninjected tumors, respectively .

(D) Percentage of common TCR clones in injected, uninjected tumors and spleens. (E) The tumor volumes on day 21 were plotted against the log of % common TCR clones. n=16.

Figures 4A-4G. Loss of LATSl/2 in tumors inhibits tumor growth in vivo (A) Equal numbers of WT or LATSl/2 dKO B16-OVA cells were transplanted into C57BL/6 mice and tumor growth was monitored after the indicated times. Data are represented as mean ± SEM; n = 8 tumors for each group . ***p < 0.001, two-way ANOVA test. (B) C57BL/6 mice were injected with WT or

LATSl/2 dKO B16-OVA melanoma and tumor weight was determined 20 days after transplantation. Data are represented as mean ± SEM; n = 6 tumors for WT, n = 8 tumors for LATSl/2 dKO (note that two of the tumors were completely rejected). **p < 0.01, Mann-Whitney test.

(C) Kaplan-Meier tumor-free survival curves for mice injected with WT or

LATSl/2 dKO B16-OVA cells are shown (n = 14 mice for each group). ***p < 0.001, log-rank test. (D) WT or two independent clones of LATSl/2 dKO SCC7 cells were transplanted into C3H/HeOu mice and tumor growth was monitored after the indicated times. Data are represented as mean ± SEM; n = 8 tumors for each group . p values were determined using two-way ANOVA test, comparing each group toWT group . ***p < 0.001.

(E) Kaplan-Meier tumor-free survival curves for mice injected with WT or LATSl/2 dKO SCC7 cells are shown (n = 4 mice for each group), p values were determined using log-rank test, comparing each group toWT group . **p < 0.01. (F) BALB/c mice were injected with WT or LATSl/2 dKO 4T 1 cells and primary tumor weight was determined 28 days after transplantation. (G) WT or LATSl/2 dKO 4T 1 cells were transplanted into the mammary fat pad of BALB/c mice and lung metastasis of the primary tumor was determined 28 days after transplantation. Normal lung tissue was stained with black India ink, whereas tumor nodules remain white. The gross appearance of the lungs (left panel) and tumor nodules on lungs (right panel) were examined. Data are represented as mean ± SEM; n = 16 tumors (F) and n = 8 mice (G) for each group . ***p < 0.001, Mann-Whitney test. Figures 5A-5G. LATS1/2 deletion in tumors stimulates host adaptive immunity and enhances tumor vaccine efficacy (A) WT or L ATS 1/2 dKO B 16- OVA melanoma cells were injected into C57BL/6 mice and tumor growth was monitored after the indicated times (left panel). For co-injection experiments, WT and LATS1/2 dKO cells were injected into opposite flanks in the same mouse (right panel). "WT [withLATSl/2 dKO(#l)]" (blue line) indicates WT tumor growth, and "LATS1/2 dKO(#l) [with WT]" (yellow line) indicates LATS1/2 dKO tumor growth, in the co-injected mice. Data are represented as mean ± SEM; n = 8 tumors for WT or LATS1/2 dKO group, n = 6 tumors for each co-injectioned group . p value was determined using two-way ANOVA test, comparing WT [withLATSl/2 dKO(#l)] group toWT group . ***p < 0.001. (B) C57BL/6 mice were immunized intradermally at the base of the tail with equal numbers of irradiated WT or LATSl/2 dKO B16-OVA cells (or PBS control). 12 days after immunization, mice were challenged with WT B16-OVA melanoma and tumor growth was monitored after the indicated times. Data are represented as mean ± SEM; n = 8 tumors for each group . PBS, phosphate buffered saline.

Figures 6 A-6F. Extracellular vesicles released from LATS 1/2 null cells are immunostimulatory . (A) Bone marrow-derived dendritic cells (BMDCs) were p retreated with conditioned medium from WT or LAT Sl/2 dKO B 16-0 VA melanoma cells (or control medium) and pulsed with OVA protein. BMDCs were then subjected to an in vitro cross-presentation assay using CFSE-labeled CD8 ⁺ T cells isolated from OVA-specific T cell receptor transgenic OT-I mice. OT-I CD8 ⁺ T cells proliferate when they were stimulated with OVA antigen via cross-presentation by BMDCs, resultingin dilution of CFSE content.

Representative histograms of the gated CD8 ⁺ T cells are shown in the left panel. The division index was calculated and data are presented as means ± SEM of 3 independent experiments in the right panel. ***p < 0.001, one-way ANOVA test followed by Tukey 's multiple comp arison test. (B) BMDCs were stimulated with conditioned medium or EVs from WT or LAT Sl/2 dKO B 16-0 VA cell culture supernatants and then IL-12 levels in the culture supernatants were determined by ELISA. Data are represented as mean ± SEM; n = 3 independent experiments for conditioned medium stimulation, n = 4 independent experiments for EVs stimulation. *p < 0.05; ***p < 0.001, one-way ANOVA test followed by Tukey's multiple comparison test. (C) C57BL/6 mice were inoculated with irradiated WT B16-OVA cells at the base of the tail and EVs freshly isolated from culture sup ernat ants of equal numbers of WT or LATSl/2 dKO B16-OVA cells were injected every 3 days (day 0, 3, 6, and 9). At day 12, mice were challenged with WT B 16-0 VA melanoma and tumor growth was monitored. Data are represented as mean ± SEM; n = 8 tumors for each group . The tumor growth curves shown in Figure 4B are presented in a lighter color for reference. p value was determined using two-way ANOVA test, comparing WT EV- immunized group [WT + WT EVs→ WT] toLATSl/2 dKO EV-immunized group [WT + LATS1/2 dKO EVs→WT]. ***p < 0.001. (D) EVs isolated from culture sup ernat ants of equal numbers of WT or LATS1/2 dKO B l 6-0 VA cells were subjected to nanop article tracking analysis (NanoSight) to quantify the number and size distribution. The numbers of particles are presented as means ± SEM of 3 independent experiments. **p < 0.01, unpaired t-test. (E) Protein concentrations of EVs isolated as in (D) were determined. Data are means ±

SEM of 6 independent experiments. ***p < 0.001, unpaired t-test. (F) EVs were isolated from culture supernatants of equal numbers of WT, LATS1/2 dKO, or YAP(5SA)-overexpressing B16-OVA cells and RNA concentrations were determined by Agilent TapeStation. Data are means ± SEM of 3 independent experiments. ***p < 0.001, one-way ANOVAtest followed by Tukey's multiple comparison test.

Figure 7. Exemplary protocol for collection and expansion of autologous T cell therapy .

Figures 8A-8E. LATS1/2 Deletion Enhances Anchorage-Independent Tumor Cell Growth/^ Vitro (A) Wild-type (WT) and two independent clones of LATS1/2 double knockout (dKO) B16-OVA melanoma cells were serum starved or treated with Latrunculin B (LatB) and then subjected to immunoblot (IB) analysis with antibodies to the indicated proteins. (B) LatB-treated or non- treated (control) B16-OVA cells were subjected to immunostaining analysis. YAP/TAZ subcellular localization was determined by immunofluorescence staining for endogenous YAP/TAZ (green) along with DAPI for DNA (blue). Representative images are presented in the left panel, (right panel) Cells in five randomly selected views (about 100 cells) were selected for the quantification of YAP/TAZ localization. N, nuclear; C, cytoplasmic. (C) B16-OVA cells were subjected to soft-agar colony -formation assay, and the colonies were stained with cry stal violet for quantification. (D) Soft-agar colony -formation assay of SCC7 squamous cell carcinoma cells. E) Soft-agar colony -formation assay of 4T 1 breast cancer cells. Data are presented as means ± SD from three independent experiments (C-E). Thep values were determined using a one-way ANOVA test followed by Tukey 's multiple comparison test (C and D) or an unpaired t test (E). **p < 0.01; ***p < 0.001. See also Figure SI .

Figures 9A-9G. Loss of LATSl/2 in Tumors Inhibits Tumor Growth/^ Vivo (A) Equal numbers of WT or LATSl/2 dKO B 16-OVA cells were transplanted into C57BL/6 mice, and tumor growth was monitored after the indicated times. Data are presented as means ± SEM; n = 8 tumors for each group . ***p < 0.001, two-way ANOVA test. (B) C57BL/6 mice were injected with WT or LATSl/2 dKO B 16-OVA melanoma, and tumor weight was determined 20 days after transplantation. Data are presented as means ± SEM; n = 6 tumors for WT, and n = 8 tumors for LATSl/2 dKO (note that two of the tumors were completely rejected). **p < 0.01, Mann-Whitney test. (C) Kaplan- Meier tumor-free survival curves for mice injected with WT or LATSl/2 dKO B 16-OVA cells are shown (n = 14 mice for each group). ***p < 0.001, logrank test. (D) WT or two independent clones of LATSl/2 dKO SCC7 cells were transplanted into C3H/HeOu mice, and tumor growth was monitored after the indicated times. Data are presented as means ± SEM; n = 8 tumors for each group . The p values were determined using a two-way ANOVA test, comparing each group to the WT group . ***p < 0.001. (E) Kaplan-Meier tumor-free survival curves for mice injected with WT or LATSl/2 dKO SCC7 cells are shown (n = 4 mice for each group). The p values were determined using a log- rank test, comparing each group to the WT group . **p < 0.01. F and G) In (F), BALB/c mice were injected with WT or LATSl/2 dKO 4T 1 cells, and primary tumor weight was determined 28 day s after transplantation. (G)WT or LATSl/2 dK04T l cells were transplanted into the mammary fat pad of BALB/c mice, and lung metastasis of the primary tumor was determined 28 days after transplantation. Normal lung tissue was stained with black India ink, whereas tumor nodules remained white. The gross appearance of the lungs (left panel) and the tumor nodules on the lungs (right panel) were examined. Data are presented as means ± SEM; n = 16 tumors in (F), and n = 8 mice in (G) for each group . ***p < 0.001, Mann-Whitney test. See also Figure 16.

Figures 10A-10I. LATS1/2 Deficiency in Tumor Cells Induces Host Anti-tumor Immunity (A) WT or LATS1/2 dKO B 16-OVA melanoma cells were injected into C57BL/6 mice. Tumors were paraffin embedded and stained withH&E 12 days after transplantation. Arrowheads indicate infiltration of inflammatory cells. (B) Frozen sections from WT or LATS1/2 dKO B16-OVA melanomas were subjected to immunostaining analysis of CD45 (red) along with D API for DNA (blue). (C) WT and two independent clones of LATS1/2 dKO B 16-OVA melanoma cells were subjected to immunoblot (IB) analysis with antibodies to the indicated proteins. (D-F) In (D), C57BL/6 mice were injected (or not injected) with WT or LATS1/2 dKO B16-OVA melanoma cells, and serum anti-OVA IgG concentrations were determined by ELISA 12 day s after transplantation. (E) Splenocy tes from C57BL/6 mice injected as in (D) were re- stimulated ex vivo with SIINFEKL p eptide and then subjected to flow- cytometric analy sis. SIINFEKL is an OVA-derived peptidebeing presented through the major histocompatibility complex class I (MHC class I) molecule H- 2Kb. Frequency of CD8 ⁺ T cells expressing activation markers, GranzymeB or interferon g (IFNg), was determined. (F) Splenocytes from C57BL/6 mice injected as in (D) were subjected to flow-cytometric analysis. OVA-specific CD8 ⁺ T cells were quantified using Kb-SIINFEKL tetramers and plotted as a percentage of total CD8 ⁺ T cells. Dataare presented as means ± SEM; n = 4mice for the uninjected group, n = 10 mice for the WT -injected group, and n = 10 mice for the LATSl/2 dKO-injected group . ***p < 0.001, one-way ANOVA test followed by Tukey 's multiple comparison test. (G) C57BL/6 mice were injected as in (D), and the inguinal lymph nodes were cultured ex vivo with OVA protein. IFNg levels in the culture supernatants were determined by ELISA. Data are presented as means ± SEM of triplicate cultures of pooled lymph node cells from 4 mice per group . ***p < 0.001, one-way ANOVA test followed by Tukey 's multiple comparison test. (H) C57BL/6 mice were injected as in (D) and CD8 ⁺ T cells were isolated from splenocytes. T cell cytotoxicity assay was performed with CFSE (carboxy fluorescein succinimidyl ester)-labeled EL4 cells ex vivo and the percentage of specific killing was plotted. Dataare presented as means ± SEM of five independent experiments with pooled CD8 ⁺ T cells from 3-4 mice per group . ***p < 0.001, one-way ANOVA test followed by Tukey 's multiple comparison test. (I) WT or LATS1/2 dKO B 16-OVA melanoma cells were injected into C57BL/6 mice, and tumors were subjected to flow-cytometric analy sis 12 day s after transplantation. Data are presented as means ± SEM of the percentage of CD8 ⁺ T cells infiltrating into tumors among total CD45 ⁺ cells; n = 4 tumors for each group . ***p < 0.001, unpaired t test. See also Figure 17.

Figures 11 A-l 1G. LATS1/2 Deletion in Tumors Stimulates Host Adaptive Immunity and Enhances Tumor Vaccine Efficacy (A) WT or LATSl/2 dKO B 16-OVA melanoma cells were injected into C57BL/6 mice, and tumor growth was monitored after the indicated times (left panel). For coinjection experiments, WT and LATS1/2 dKO cells were injected into opposite flanks in the same mouse (right panel). "WT [withLATSl/2 dKO(#l)]" (blue line) indicates WT tumor growth, and "LATS1/2 dKO(#l) [with WT]" (yellow line) indicates LATS1/2 dKO tumor growth, in the co-injected mice. Data are presented as means ± SEM; n = 8 tumors for WT or LATS1/2 dKO group, n = 6 tumors for each co-injected group . Thep value was determined using two-way ANOVA test, comparing the "WT [withLATSl/2 dKO(#l)]" group to the WT group . ***p < 0.001. (B and C) In (B), C57BL/6 mice were immunized intradermally at the base of the tail with equal numbers of irradiated WT or LATS1/2 dKO B16-OVA cells (or PBS control). 12 day s after immunization, mice were challenged with WT B16-OVA melanoma, and tumor growth was monitored after the indicated times. Data are presented as means ± SEM; n = 8 tumors for each group . (C) Kaplan-Meier tumor-free survival curves for mice immunized and challenged as in (B) are shown (n = 12 mice for each group). The survival curve of C57BL/6 mice challenged with WT B16-OVA melanoma without vaccination in Figure 16C is also shown in light gray for reference. A schematic representation of vaccination experiment with irradiated-tumor cells is shown in the lower panel. Thep value was determined using a two-way

ANOVA test (B) or a log-rank test (C), comparing the WT -immunized group [WT / WT] to the LATS1/2 dKO-immunized group [LATS1/2 dKO(#l)/WT]. ***p < 0.001. (D) C3H/HeOu mice were first injected with non-irradiated LATS1/2 dKO SCC7 cells. 60 day s after the initial injection, mice designated tumor-free were rechallenged withWT SCC7 cells, and tumor growth was monitored [LATS1/2 dKO(#l)/WT]. The tumor growth curve of WT SCC7 injected into naive C3H/HeOu mice in Figure 2D is also shown in light gray for reference (WT). Data are presented as means ± SEM; n = 8 tumors for each group . ***p < 0.001, two-way ANOVA test. A schematic representation of the rechallenge experiment is shown in the lower panel. (E) WT or LATS1/2 dKO B 16-0 VA cells were transp lanted into Rag- 1 knockout (KO) mice that lack mature B and T lymphocytes. Tumor growth was monitored after the indicated times. Data are presented as means ± SEM; n = 8 tumors for each group . ns, not significant (p > 0.05, two-way ANOVA test). (F) Kaplan-Meier tumor-free survival curves for mice transplanted as in (E) are shown (n = 10 mice for each group), ns, not significant (p > 0.05, log-rank test). (G) WT and LATS1/2 dKO B 16-OVA melanoma cells were injected into opposite flanks in the same Rag-1 KO mouse. Data are presented as means ± SEM; n = 6 tumors for each group . The tumor growth curves shown in (E) are shown in a lighter color for reference. The p value was determined using a two-way ANOVA test, comparing the "WT [with LATSl/2 dKO(#l)]" group to the WT group , ns, not significant (p > 0.05).

Figures 12 A- 12F. EVs Released from LATSl/2-Null Tumor Cells Stimulate Host Immune Responses (A) Bone marrow-derived dendritic cells (BMDCs) were pretreated with conditioned medium from WT or LATSl/2 dKO B 16-OVA melanoma cells (or control medium) and pulsed with OVA protein. BMDCs were then subjected to an in vitro crosspresentation assay using CFSE- labeled CD8 ⁺ T cells isolated from OVA-specific T-cell-receptor transgenic OT- I mice. OT-I CD8 ⁺ T cells proliferate when stimulated with OVA antigen via crosspresentation by BMDCs, resultingin dilution of CFSE content.

Representative histograms of the gated CD8 ⁺ T cells are shown in the left panel. The division index was calculated, and data are presented as means ± SEM of three independent experiments in the right panel. ***p < 0.001, one-way ANOVA test followed by Tukey 's multiple comparison test. (B) BMDCs were stimulated with conditioned medium or EVs from WT or LATSl/2 dKO B16- OVA cell culture supernatants, and then IL-12 levels in the culture supernatants were determined by ELISA. Data are presented as means ± SEM; n = 3 independent experiments for conditioned medium stimulation, n = 4 independent experiments for EVs stimulation. *p < 0.05; ***p < 0.001, one-way ANOVA test followed by Tukey 's multiple comparison test. (C) C57BL/6 mice were inoculated with irradiated WT B16-OVA cells at the base of the tail, and EVs freshly isolated from culture supernatants of equal numbers of WT or LATSl/2 dKO B 16-OVA cells were injected every 3 days (days 0, 3, 6, and 9). At day 12, mice were challenged with WT B16-OVA melanoma and tumor growth was monitored. Data are presented as means ± SEM; n = 8 tumors for each group . The tumor growth curves shown in Figure 1 IB are presented in a lighter color for reference. Thep value was determined using a twoway ANOVA test, comparing WT EV-immunized group (WT + WT EVs / WT) to LATSl/2 dKO Evimmunized group (WT + LATSl/2 dKO EVs/WT). ***p < 0.001. (D) EVs isolated from culture supernatants of equal numbers of WT or LATSl/2 dKO B 16-OVA cells were subjected to nanop article tracking analysis (NanoSight) to quantify the number and size distribution. The numbers of particles are presented as means ± SEM of three independent experiments. **p < 0.01, unpaired t test. (E) Protein concentrations of EVs isolated as in (D) were determined. Data are presented as means ± SEM of six independent experiments. ***p < 0.001, unpaired t test. (F) EVs were isolated from culture supernatants of equal numbers of WT, LATSl/2 dKO, or YAP(5SA)-overexpressing B16-OVA cells, and RNA concentrations were determined by Agilent TapeStation. Data are presented as means ± SEM of three independent experiments. ***p < 0.001, one-way ANOVA test followed by Tukey 's multiple comparison test. See also Figures 18-20.

Figure 13A-13I. LATSl/2-Deleted Tumor EVs Stimulate Anti-tumor Immunity via the TLRs-Type I IFN Pathway (A-G) WT or LATSl/2 dKO B 16- OVA cells were transplanted into mice deficient in My d88 (A; n = 12 mice for the WT group, and n = 13 mice for the LATSl/2 dKO group), Ticaml (also known as TRIF) (B; n = 6 mice for each group), Tmeml73 (also known as STING) (C; n = 4 mice for each group), Casp 1 (also known as caspase-1) (D; n = 4 mice for the WT group, and n = 5 mice for the LATSl/2 dKO group), Tlr4 (E; n = 9 mice for each group), Tlr7 (F; n = 8 mice for each group), or Tlr9 (G; n = 7mice for each group), and Kaplan-Meier tumor-free survival curves are shown. The survival curves of WT C57BL/6 mice injected withWT or LATSl/2 dKO B 16-0 VA in Figure 9C are shown in a lighter color for reference. The p value was determined using a log-rank test, comparing KO mice injected with LATSl/2 dKO B16-OVA cells (orange) to corresponding wild-type C57BL/6 mice injected with LATSl/2 dKO B16-OVA cells (light red), ns, not significant (p > 0.05); *p < 0.05; **p < 0.01; ***p < 0.001. (H) WT or LATSl/2 dKO B 16- OVA cells were injected into Ifnarl KO mice that lack functional typel lFN receptor, and tumor growth was monitored after the indicated times. The tumor growth curves of WT or LATSl/2 dKO B 16-0 VA cells injected into wild-ty p e C57BL/6 mice in Figure 2A are shown in a lighter color for reference. Data are presented as means ± SEM; n = 8 tumors for each group . (I) Kaplan-Meier tumor-free survival curves for mice injected as in (H) are shown (n = 8 mice for each group). The survival curves of WT C57BL/6 mice injected with WT or LATSl/2 dKO B 16-0 VA in Figure 2C are shown in a lighter color for reference. Thep value was determined using a log-rank test, comparing Ifnarl KO mice injected withLATSl/2 dKO B 16-OVA cells (orange) to corresponding WT C57BL/6 mice injected with LATSl/2 dKO B 16-OVA cells (light red). ***p < 0.001. See also Figure 21.

Figure 14. Proposed Model for the Regulation of Anti-tumor Immunity by the Hippo Pathway in Tumors Poorly immunogenic tumor cells evade host immune defenses, despite expressing antigenic neoep it opes. LATSl/2 deletion in tumor cells stimulates nucleicacid-rich EV secretion, which induces a type I IFN response via the TLRs-MYD88/TRIF pathway. Typel lFN stimulates multiple components of host immune responses, including cross-presentation of tumor-derived antigens by antigen-presenting cells and T cell activation.

Activated T cells, in turn, facilitate tumor-specific responses of cytotoxic T cells and antibody production by B cells, promoting tumor destruction. Thus, loss of LATSl/2 in tumors leads to the rejection of both LATSl/2-deficient and

LATSl/2-adequate tumor cells by enhancing host anti-tumor immune responses.

Figures 15 A- 15G. LATSl/2 Deletion Enhances Anchorage-Independent Tumor Cell Growth Jw Vitro, Related toFigure 8. (A) LATSl/2 dKO B16-OVA cells grow similarly to WT on regular cell culture plates. Wild-type (WT) and two independent clones of LATSl/2 double knockout (dKO) B16-OVA melanoma cells (1 x 10 ⁵ ) were plated in 6-well culture dishes and cell number was determined with a hemocytometer after the indicated times. Data are means ± SD of triplicate cultures from a representative experiment, ns, not significant (p > 0.05, two-way ANOVA test). (B) Deletion of LATSl/2 in SCC7 cells abolishes YAP phosphorylation in response to actin polymerization inhibitor Latrunculin B (LatB) treatment and glycolysis inhibitor 2-deoxy-D-glucose (2- DG) treatment. WT and two independent clones of LATSl/2 dKO SCC7 squamous cell carcinoma cells were subjected to immunoblot (IB) analysis with antibodies to the indicated proteins. The mouse YAP SI 12 is equivalent to human YAP S127, which is themajor regulatory site responsible for YAP cytoplasmic localization. Where indicated, gels containing phos-tagwere employ ed for assessment of YAP phosphorylation status. YAP proteins canbe separated into multiple bands in the presence of phos-tag depending on differential phosphorylation levels, with phosphorylated proteins migrating more slowly . (C) Loss of LATS1/2 promotes YAP/T AZ nuclear localization. LatB- treated or non-treated (control) SCC7 cells were subjected to immunostaining analy sis. YAP/TAZ subcellular localization was determined by

immunofluorescence staining for endogenous YAP/TAZ (green) along with DAPI for DNA (blue). Representative images are presented in the left panel. (Right panel) Cells in five randomly selected views (about 100 cells) were selected for the quantification of YAP/TAZ localization. N, nuclear; C, cytoplasmic. (D) LATS1/2 deletion promotes anchorage-independent growth of SCC7 cells in vitro. Representative images of the soft-agar colony -formation assay in Figure 8D are shown. (E) Deletion of LATS1/2 in 4T 1 cells abolishes YAP phosphorylation in response to serum starvation, LatB treatment, and 2-DG treatment. WT and LATS1/2 dKO 4T 1 breast cancer cells were subjected to immunoblot and phos-tag analysis. (F) Loss of LATS1/2 promotes YAP/TAZ nuclear localization. LatB-treated or non-treated (control) 4T 1 cells were subjected to immunostaining analysis. YAP/TAZ subcellular localization was determined by immunofluorescence staining for endogenous YAP/TAZ (green) along with DAPI for DNA (blue). Representative images are presented in the left panel. (Right panel) Cells in five randomly selected views (about 100 cells) were selected for the quantification of YAP/TAZ localization. N, nuclear; C, cytoplasmic. (G) LATS1/2 deletion promotes anchorage-independent growth of 4T 1 cells in vitro. Representative images of the soft-agar colony -formation assay in Figure IE are shown.

Figures 16A-16C. Loss of LATS1/2 in Tumor Cells Inhibits Tumor Growth In Vivo, Related to Figure 2 (A) Deletion of LATS1/2 in B 16-OVA melanoma inhibits tumor growth in vivo. Wild-type (WT) or clone #2 of LATSl/2 double knockout (dKO) B16-OVA cells were injected into C57BL/6 mice and tumor weight was determined 16 days after transplantation. Data are represented as mean ± SEM; n = 6 tumors for each genotype. **p < 0.01, Mann- Whitney test. (B) Deletion of LATSl/2 in SCC7 squamous cell carcinoma inhibits tumor growth in vivo. The gross appearance of C3H/HeOu mice injected withWT or two independent clones of LATSl/2 dKO SCC7 cells was examined 18 day s after transplantation. (C) Deletion of LATSl/2 in 4T 1 breast cancer inhibits tumor growth in vivo. The gross appearance of the primary tumors of WT or LATSl/2 dK0 4T l cells injected into BALB/c mice was examined 28 day s after transp lantation.

Figures 17A-17F. LATSl/2 Deficiency in Tumor Cells Stimulates Host Anti-tumor Immunity, Related to Figure 10. (A) LATSl/2 deletion induces immune responses. Wild-type (WT) or LATSl/2 double knockout (dKO) 4T l breast cancer cells were injected into BALB/c mice. Tumors were paraffin- embedded and stained with hematoxylin and eosin (H&E) 28 day s after transplantation. Arrowheads indicate infiltration of inflammatory cells. (B) CD45+ leukocytes infiltrate into LATSl/2 null tumors. Frozen sections from WT or LATSl/2 dK0 4T l breast cancers were subjected to immunostaining analy sis of CD45 (red) along with D API for DNA (blue). (C) LATSl/2 null tumors activate CD8+ T cells. C57BL/6 mice were injected (or not) with WT or LATSl/2 dKO B16-OVA melanoma cells. 12 days after transplantation, splenocytes were collected and re-stimulated ex vivo with SIINFEKL peptide and then subjected to flow cytometric analysis. SIINFEKL is an OVA-derived peptidebeing presented through the class I major histocompatibility complex (MHC class I) molecule, H-2Kb. Representative scatterplots of the gated CD8+ T cells in Figure 10E are shown. Gating of CD8+ T cells was performed after background assessment. Numbers indicate the percentage of GranzymeB or interferon γ (IFNy) positive cells in the gated CD8+ population. FSC, forward scatter. (D) LATSl/2 deletion in tumors increases OVA-specific CD8+ T cells. Splenocytes from C57BL/6 mice injected as in (C) were subjected to flow cytometric analy sis. OVA-specific CD8+ T cells were quantified using K ^b - SIINFEKL-tetramers. Representative scatterplots of the gated CD 8+ T cells in Figure 10F are shown. Gating of CD8+ T cells was performed after background assessment. Numbers indicate the percentage of tetramer positive cells in the gated CD 8+ population. (E) CD8+ T cells from LATSl/2 dKO tumor- challenged mice show increased OVA-specific cytotoxicity . C57BL/6 mice were injected (or not) with WT or LATSl/2 dKO B 16-0 VA melanoma cells and CD8+ T cells were isolated from splenocytes. T cell cytotoxicity assay was performed with CFSE-labeled EL4 cells ex vivo. The frequency of CSFE^ ¹¹ (irrelevant peptide control) and CFSE ^low (SIINFEKL loaded target) EL4 cells was determined by flow cytometric analysis 18 hours after incubation.

Representative histograms of the gated CSFE ⁺ EL4 cells in Figure 10H are shown in the up per panel. Gating of CSFE ⁺ EL4 cells was performed after background assessment. Numbers indicate the percentage of the gated cells in CSFE ⁺ population. Schematic representation of ex vivo cytotoxicity assay using CFSE-labeled EL4 cells is shown in thelower panel. CFSE, carboxy fluorescein succinimidyl ester. (F) CD8+ T cells infiltrate into LATSl/2 dKO tumors. WT or LATSl/2 dKO B16-OVA melanoma cells were injected into C57BL/6 mice and tumors were subjected to flow cytometric analy sis 12 day s after

transplantation. Representative scatterplots of the gated CD45+ cells in Figure 31 are shown. Gating of CD45+ T cells was performed after background

assessment. Numbers indicate the percentage of CD8 positive cells in the gated CD45+ p opulation.

Figures 18A-18H. Overexpression of YAP or TAZ in Tumor Cells

Partially Suppresses Tumor Growth/^ Vivo, Related to Figure 11. (A)

YAP/TAZ activation in LATSl/2 null tumors. Wild-type (WT) or LATSl/2 double knockout (dKO) B16-OVA melanoma cells were injected into C57BL/6 mice. 20 day s after transplantation, tumor samples were harvested and then subjected to immunoblot (IB) analy sis with antibodies to the indicated proteins. YAP dephosphorylation and TAZ accumulation were evident in tumors deficient for LATSl/2. n = 3 tumors for each group . (B) Increased YAP/TAZ

transcriptional activity in LATSl/2 null tumors. The tumor samples, same as in (A), were subjected to RT and real-time PCR analy sis of the indicated

YAP/TAZ target gene mRNA. Normalized data are expressed relative to the corresponding value for WT tumors and are mean ± SEM; n = 3 tumors for each group . *p < 0.05, unpaired t test. (C) Expression levels of YAP(5SA) or

TAZ(4SA) in B 16-0 VA melanoma cells. YAP (5 S A) and TAZ (4 SA) are active mutants of YAP/TAZ with all five/four LATSl/2 phosphorylation sites mutated to alanine, thereby unresponsive to inhibition by the LATSl/2 kinase. B 16-OVA cells stably expressing YAP(5SA), TAZ(4SA), or control vector were subjected to immunoblot analysis with antibodies to theindicated proteins. (D) YAP(5SA) or TAZ(4SA) overexpression promotes anchorage-independent growth of B 16- OVA cells in vitro. Soft-agar colony -formation assay was performed and the colonies were stained with cry stal violet for quantification. Data are means ± SD from 3 independent experiments. **p < 0.01; ***p < 0.001, one-way ANOVA test followed by Tukey 's multiple comparison test. (E) YAP(5SA) or TAZ(4SA) overexpression in B16-OVA melanoma inhibits tumor growth in vivo. B16-OVA cells stably expressing YAP(5SA), TAZ(4SA), or control vector were injected into C57BL/6 mice and tumor growth was monitored after theindicated times. Data are represented as mean ± SEM; n = 8 tumors for each group , p values were determined using two-way ANOVA test, comparing each group to control group . ***p < 0.001. (F) Expression levels of YAP(5SA) or YAP(5SA/S94A) in B 16-0 VA melanoma cells. TEAD1-4 are the major YAP-associated

transcription factors, and their binding to YAP requires the Ser94 residue in YAP. YAP(5SA/S94A) is unable tobind TEAD l-4 and thus fails to promote TEAD-dependent transcription. (G) YAP(5SA), but not YAP(5SA/S94A), promotes downstream target gene transcription in B 16-0 VA cells. Total RNA extracted from B 16-0 VA cells stably expressing the indicated constructs was subjected to RT and real-time PCR analysis of the indicated YAP/TAZ target genes. Data are means ± SD of triplicates from a representative experiment. (H) Tumor growth suppression by YAP in v/vo requires TEAD-binding of YAP. B 16-OVA cells stably expressing YAP(5SA/S94A) were injected into C57BL/6 mice and tumor growth was monitored after the indicated times. The tumor growth curves shown in (E) are presented in a lighter color for reference. Data are represented as mean ± SEM; n = 8 tumors for each group , p value was determined using two-way ANOVA test, comparing YAP(5SA) group to YAP(5SA/S94A) group . ***p < 0.001.

Figures 19A-19B. EVs Released from LATSl/2-Null Tumor Cells

Stimulate Host Immune Responses, Related to Figure 12. (A) Enrichment of the EV protein markers, such as CD81, ALIX, and FLOT 1 in theEV preparation. Whole cell lysate (WCL) of wild-type (WT) or LATS1/2 double knockout (dKO) B16-OVA melanoma cells as well as EVs isolated from their culture supernatants were subjected to immunoblot (IB) analy sis with antibodies to the indicated proteins. Equal amounts of protein samples (2.5 mg) were resolved in SDS-PAGE in non-reducing conditions. (B) Detergent treatment abolishes the activity of EV preparations in stimulating bone marrow-derived dendritic cells (BMDCs). Culture supernatants ofWT or LATS1/2 dKO B 16-OVA melanoma cells were either treated or untreated with detergent (1% Triton X-100) and then ultracentrifuged to isolate EVs. The resulting EV pellets were re-suspended in PBS and used to stimulate BMDCs. 18 hours after incubation, IL-12 levels in the culture supernatants ofBMDC were determined by ELISA. Data are represented as mean ± SEM of 3 independent experiments, p value was determined using one-way ANOVA test followed by Tukey 's multiple comparison test. ***p < 0.001; ns, not significant (p > 0.05).

Figures 20A-20F. EVs Released from LATSl/2-Null Tumors Contain More Nucleic- Acid-Binding Proteins in Comparison to WT EVs, Related to Figure 12. (A) LATS1/2 null tumor cells secrete more EVs. EVs isolated from culture supernatants ofwild-type (WT) or LATS1/2 double knockout (dKO) B 16-OVA melanoma cells were subjected to nanop article tracking analysis (NanoSight) to quantify the number and size distribution. Representative histograms in Figure 12D are shown. (B) Proteomic profiling of total proteins identified in EVs show enrichment of previously reported exosomal and microvesicle cargo proteins. EVs were isolated from culture supernatants of WT or LATS1/2 dKO B16-OVA cells and subjected to mass spectrometry analysis. Enrichment analysis of the Gene Ontology (GO) cellular component of total EV proteins identified (1,772 proteins) was done using the PANTHER program. The enrichment p value of each term was transformed to a -logio(p value). The top 3 most significantly enriched cellular components are indicated. (C) Heatmap of the total EV proteins identified. Absolute protein abundances were estimated using the iBAQ algorithm. The iBAQ-scaled protein expression (Ex) was transformed to a log2(Ex) and the scale indicates relative expression. Data rep resent two independent biological replicates. See Table SI for protein contents. (D) RNA binding and nucleic-acid-binding proteins are enriched in EVs from LATS1/2 null tumor cells. Enrichment analysis of the GO molecular function of the top 100 most significantly increased proteins in LATS1/2 dKO EVs is shown. The enrichment p value of each term was transformed to a - logio(p value). (E) EVs from LATSl/2-deficient tumor cells or YAP(5SA)- overexp res sing tumor cells contain higher amounts of RNA than EVs from WT tumor cells. EVs were isolated from culture supematants of equal numbers of WT, LATSl/2 dKO, or YAP(5SA)-overexpressing B 16-OVA cells and RNA concentrations were determined by Agilent TapeStation. Representative histograms in Figure 5F are shown. (F) EVs from B16-OVA cells contain single stranded RNA. EVs were isolated from culture supematants of equal numbers of WT or LATSl/2 dKO B 16-OVA cells. RNA was then purified from EV samples and either treated or untreated with single-strand-specific ribonuclease (RNase A, the reaction was performed under high salt concentrations to achieve single- strand specificity), followed by agarose gel electrophoresis in non-denaturing conditions.

Figures 21A-21D. EVs from LATSl/2-Deleted Tumor Cells Stimulate Anti-tumor Immunity via the TLRs-Type-I-IFN Pathway, Related to Figure 13. (A) Schematic representation of the endogenous nucleic-acid-sensing pathways and TLR signaling in mice (Junt and Barchet, 2015). TLR, toll-like receptor; LPS, lip op oly saccharide; dsRNA, double-stranded RNA; ssRNA, single- stranded RNA; rRNA, ribosomal RNA; TRIF, TIR-domain-containing adaptor- inducing interferon-b; MYD88, myeloid differentiation primary response 88; STING, stimulator of interferon genes; IL, interleukin; IFN, interferon. (B) Wild-type (WT) or LATSl/2 double knockout (dKO) B 16-OVA cells were injected into Myd88 knockout (KO) mice and tumor growth was monitored after the indicated times. Data are represented as mean ± SEM; n = 8 tumors for each group . (C) WT or LATSl/2 dKO B16-OVA cells were injected into Ticaml (also known as TRIF) KO mice and tumor growth was monitored after the indicated times. Data are represented as mean ± SEM; n = 10 tumors for each group . (D) LATSl/2 deletion has little effect on the mRNA abundance of typel IFN in tumor cells. WT or LATSl/2 dKO B16-OVA cells were stimulated (or not) with 2 mg/ml poly(LC) complexed with 4 mg/ml Poly Jet for24 hours. Total RNA was extracted from the cells and then subjected to RT and real-time PCR analysis of the indicated mRNA. Data are means ± SD of triplicates from a rep resentative exp eriment .

Figures 22A-22C. Systemic administration of 1V270 inhibits lung metastasis in the4T lmurine syngeneic breast cancer model in a CD8 ⁺ T cell dependent manner. (A) Protocol of spontaneous metastesis model. 4T 1 cells (5 x 10 ⁵ ) were inoculated in both 4th mammary pads of Balb/c mice (n = 13/group) 1V270 (20, 80, 04 200 was administered on days 7, 10, 14, 17, 21 and 24. (B) The numbers of lung nodules were counted by staining with India ink after harvesting lungs on day 27. (C) The mice (n = 6-15/group) were orthotopically implanted with 4T 1 cells and i.p . treated with 1 V270 (20 μ^ί^εΛίοη) as shown in Figure 1A. CD8 ⁺ cells were depleted by administration of anti-CD8 or isotypemAbs on days 5, 8, 11, 16, 19, and 23. The numbers of lung nodules were counted on day 27. Each dot indicates an individual mouse and horizontal and vertical bars indicate means ± SEM . Data shown are pooled from 2 independent experiments. *P<0.05, **P<0.01 by Kruskal-Wallis test with Dunn's post hoc test comparing treatment groups against vehicle group , n.s; statistically not significant.

Figures 23A-23G. Systemic administration of 1V170 induces tumor- specific CD8 ⁺ T cells. (A) Protocol of IV metastasis model. (B) BALB/c mice (n=B-15/group) were i.v. injected with 4T l cells (2 x 10 ⁴ ) on day 0. 1V270 (2, 20, or 200 μ^ή^εΛίοη) was i.p . administered on day s -1, 7, 10, and 14. The numbers of lung nodules were counted on day 21. (C) 200 μ^ί^εΛίοη 1V270 was i.p . administered on days -1 or 0 followed by days 7, 10, and 14 where indicated. The numbers of lung nodules were counted on day 21. Each dot indicates an individual mouse and horizontal and vertical bars indicate means± SEM . *P<0.05, **P<0.01 Kruskal-Wallis test with Dunn's post hoc test comp aring treatment group s against vehicle group . (D-G) BALB/c mice

(n=10/group) were treated with 1V270 (200 pgfinjection) on day-1 and 4T 1 cells were inoculated on day 0. (D, E) Three weeks later, mLNs cells were stained for CD3 and CD8. Intracellular granzyme B (D) and IFNy (E) were analyzed by flow cytometry . Data are representative of 2 independent experiments showing similar results. *P<0.05, by the Mann-Whitney U test comparing the 1V270 treatment group s against the vehicle treated group . (F) Histological analysis of lungs on day 21. Representative images of H&E staining and

immunohistochemical staining for CD3 and CD45. Scale bar: 100 um. Original magnification x 200. (G) Tumor-specific cytotoxicity was examined using 4T 1 cells as target cells and BALB/3T3 cells as irrelevant target cells. The splenocy tes incubated with 4T 1 cell ly sate and IL-2 were cocultured with 4T 1 and BALB/3T3 cells at 16: 1 and 2:1 effector to target cell ratios (E:T), respectively, for 16 hours. The percent sp ecific killing was calculated. Data were analyzed by two-way ANOVA using a Bonferroni post hoc test comparing treatment group s against vehicle group . *P<0.05. Data are representative of 3 independent experiments showing similar results.

Figures 24A-24G. Tumor infiltrating T cells in 1V270 treated mice show high clonalities and increased frequency of intra- and inter-individual common clones (A) Experimental protocols of the secondary challenge studies. Two groups of BALB/c mice (n=5/group) were i.p, treated with 1V270. One cohort of mice was i.v. injected with 4T 1-GLF cells (2 x 10 ⁴ ) on day 0 and tumor growth in the lungs was monitored by IVIS on day 20. Another cohort did not receive i.v. tumor injection (no tumor-exposed mice). Native BALB/c mice served as controls. 4T 1 cells were orthotopically inoculated on day 21. (B) Tumor growth was measured with a caliper and calculated using the formula: volume

(mm3)=(width)2 x length/2. (C) TILs s were isolated from the secondarily challenged tumor on day 39 and stained for CD8 ⁺ T cells (CD3 ⁺ CD8 ⁺ ) andPD- 1. The numbers of tumor-infiltrating cos* T cells were expressed per tumor volume (mm ³ ). Data are representative of 2 independent experiments showing similar results. Results were analyzed by the Mann-Whitney U test comparing the 1V270 treatment group s against the vehicle treated group . **P<0.01. (D G) TCRrepertoire of CD8 ⁺ T cells from TILs s and splenocytes were examined. (D) The clonality index (1-normalized Shannon index) of CD8 ⁺ T cells infiltrating to the left side of the secondarily challenged tumor was plotted against tumor volume in the tumor exposed mice. Correlation between the clonality index and the tumorvolume was evaluated by Pearson's method (R ² = 0.97, p<0.05). (El BUB overlap index of CD8 ⁺ T cells TCRa (green) or TCR (blue) between Tls and splenocytes in each individual. Higher BUB index shows higher similarity of TCRrepertoire between TILs and splenocytes. Each point shows the BUB index of individual mouse. (F) Heat map of BUB overlap index of TCRa or TCR in the same group . The color scale bar on the left shows that white is zero and red is 1. (G) BUB overlap index of TCRa (green) or TCR (blue) between individual mice was plotted. Statistical analysis was performed by the Mann- Whitney U test for comparing two groups. *P<0.05. Each point represents the BUB overlap index of TCRa or TCRJ3 between pairs of individual mice in the same groups.

Figures 25A-25F. Innate immune cells, NK cells and dendritic cells are activated following 1 V270 therapy and contribute to anti-metastatic effects (A- C) Systemic administration of 1V270 activates dendritic cells and increases CD8 ⁺ cell recruitment in the lung draini lymph node. (A) BALB/c mice

(n=S/group) were treated with 1 V270 on day -1 and then tumor cells were i.v. administered on day 0. Seven day s later, mediastinal lymph node (mLN) cells were stained for dendritic cells (DC; CD45 ⁺ CD1 lc ⁺ MHC classl ). (B) DCs were further stained for CD80 and CD86. (C) ThemLN cells were also stained for central memory CD8 ⁺ T cells (CD3+CD8 ⁺ CD44 ⁺ CD62L ⁺ ), effector memory CD8 ⁺ T cells (CD3+CD8 ⁺ CD44 ⁺ CD62L ^" ), and naive CD8 ⁺ T cells

(CD3+CD8 ⁺ CD44 ^" CD62L ⁺ ). Each dot indicates an individual mouse and horizontal and vertical bars indicate means ± SEM . Data are representative of 3 independent experiments showing similar results. *P<0.05, **P<0.01 by Mann- Whitney U test comparing the individual groups. (D-F)In vivo imaging analyses using GLF expressing 4T 1 cells (4T 1-GLF). (D) BALB/c mice (n=14-15/group) were i.p, administered with 200 μg 1V270 or vehicle. Next day, 2 x 10 ⁴ 4T 1- GLF cells were i.v. injected through thetail vein. Tumor signals were quantified by IVIS. Data (mean ± SEM) were pooled from 3 independent experiments showing similarresults. *P<0.05, **P<0.01 by two-way ANOVAusing a Bonferroni post hoc test comparing treatment group s against the vehicle group . The data are displayed as radiance on a color bar with a range of 1 x 10 ⁵ to 1 x 10 ⁶ . (E,F) NK cells recruited by 1V270 therapy prevent colonization of 4T 1 cells. (E) BALB/c mice (n=G-7/group) were treated with 1 V270 (200 μ^ί^εΛίοη) on day -1 and then tumor cells were i.v. administered on day 0. On the next day, lung cells were stained for NK markers (CD45 ⁺ CD3 ^"

NKp46 ⁺ CD49 ⁺ ) and analyzed by flow cytometry . Mann-Whitney U test was used to compare treatment group s against thevehicle group . **P<0.01. (F) 1V270 treatedBALB/c mice (n=10 /group) received 4T 1 cells i.v. on day 0. NK cells were depleted by administration of anti-asialo-GM 1 antibody

(80μ^ίη]εϋΐίοη) on day -4, and -1. Tumor signals were analyzed as described above. Data(mean± SEM) were pooled from 2 independent experiments showing similar results. *P<0.05 by two-way ANOVA using Bonferroni post hoc test was used to compare treatment group s against the vehicle in the first 24 hours, n.s.: not significant

Figures 26A-26D. lntranasally administered 1 V270 inhibits pulmonary colonization in experimental metastasis models (A) Protocol for testing the therapeutic efficacy of i.n. treatment with 1V270 in IV metastasis model.

BALB/c mice (n=6-8/group) were i.n. administered with 1 V270 (20 or 200 μg) or vehicle on days -3, -1, 3, 7, and 10. 4T 1 cells were v. injected on day 0. (B) The numbers of lung nodules were counted on Day 21. *P<0.05 calculated by Kruskal-Wallis test with Dunn's post hoc test. (C) Intranasal 1V270 therapy attenuated the growth of secondarily challenged tumors. BALB/c mice (n=S) were i.n. treated with 200 μg 1V270 on days -3, -1, 3, 7 and 10. 4T 1 cells were injected on day 0. On day 21, the surviving mice were orthotopically inoculated with 4T l cells and tumor growth was monitored. The mice treated with 1V270 without i.v. tumor cell injection served as controls. (D) TILs were isolated from the secondarily challenged tumor, and the numbers of CD8 ⁺ T cells and PD-1 expressing CD8 ⁺ T cells were analyzed by flow cytometric assay . Data were analyzed by the M ann-Whitney U test comparing two groups. *P<0.05. Data are representative of 2 independent experiments showing similar results.

Figures 27A-27D. Systemic 1V270 therapy effectively inhibits lung colonization in melanoma and Lewis lung cell carcinoma (LLC) models.

Therapeutic effects of 1V270 were evaluated in IV metastatic models of 816 melanoma (A, B) and LLC (C, D). BG-albino mice (n=B-10) (A, C), C57BL6 wild typemice (n= 15-20) (B and D), were i.p . administered with 200 μg 1V270 or vehicle!iNext day, 5 x 10 ⁵ B16-GLF cells (A) or 1 x 10 ⁵ LLC- GLF cells (c) were i.v. administered (A,C). The tumor signals were quantified by IVIS at day 14. The data are displayed as a radiance on a color bar with a range of 1 x 10 ⁶ to 1 x 10 ⁷ to 1 x 10 ⁶ (C). (B,D) The mice were monitored daily and were euthanized upon reaching the criteria according to UCSD IACUC guidelines. The survival data were analyzed by Log-Rank test. Datawere pooled from 2 independent experiments showing similar results and were analyzed by Living Image ^® software. Each dot indicates an individual mouse and horizontal bars represent means. Statistical differences were analyzed by the Mann-Whitney U test comparing 1V270 treatment group s against thevehicle. **P<0.05. Figures 28A-28C. Systemic administration of 1V270 inhibits lung metastasis, but not the primary tumor growth(A) Growth curves of the orthotopically implanted primary tumor in the spontaneous metastasis model. 4T1 cells (5 x 10 ⁵ ) were inoculated to both 4th mammary pads of BALB/c mice (n=I 3/group). 1V270 (20, 80, or 200 μ^ί^εΛίοη) was i. p . administered on days 7, 1 0, 14, 17, 21, and 24 as shown in Figure 1A. Tumor growth was measured with a caliper and calculated using the formula: volume (mm ³ )=(width) ² x length/2. (B) Experimental protocol of T cell depletion. BALB/c mice (n=6- 15/group) were i. p . treated with anti-CDS- mAb. (C) Growth curves of the orthotopically implanted primary tumor in the CD ⁸⁺ T cells depleted mice. Data shown are mean ± SEM and representative of two independent experiments showing similar data.

Figures 29A-29B. Representative gating process of CD8 ⁺ T cells in TILs by flow cytometry (A) Representative gating process of CD8 ⁺ T cells

(CD45+CD3 ⁺ CD8 ⁺ ). (B) Representative flow cytometric plots of CD8 ⁺ T cells on day 26 following CD8 ⁺ T cell depletion. Over 80 % CD8 ⁺ T cell population was depleted both in vehicle and 1V270 treated groups.

Figures 30A-30C. 1 V270 therapy increases frequency of commonly shared clones between individuals (A) The clonality index (I-normalized

Shannon index) of CD8 ⁺ T cells infiltrating to left side of the secondarily challenged tumor were plotted against both sides of tumor volume in the notumor exposed mice. The correlation was evaluated by a Pearson's correlation test (R ² = 0.15, p = 0.61). (B) The Venn diagram of "shared clones" among the individual mice in the same group . Based on the sequence of CDR3 region, number ofTCR clones shared between individual mice were counted. The shared clone was defined as the TCR clone consisting of identical V and D genes and amino acid sequence of CDR3 shared among 3 or more mice in the groups. (C) The percentage frequency of shared clones in the total reads in TILs. The frequency was calculated by dividing the sum of number of sequence reads from shared clones by the total number of reads. Each dot represents the frequency of the shared clones of either TCR (green) or TCRP (blue) in the individual mice.

Figures 31A-31C. Systemic administration of lV270 activates local dendritic cells in the lungs (A) Representative gating process of CD1 lc+MHC classll+ dendritic cells in the lungs. (B) BALB/c mice (n=S/group) were treated with 1 V270 (200 p^injection) on day -1 and then tumor cells were i.v.

administered on day 0. Seven day s later, single cell suspensions derived from the lungs were stained for CD1 k'MHC class Ir' dendritic cells (left panel). The cells were further assayed for costimulatory molecules (CD80, and CD86) expression (right panel). Each dot indicates an individual mouse and horizontal and vertical bars indicate means ± SEM . *P<0.05, **P<0.0 I by Kruskal-Wallis test with Dunn's post hoc test comparing treatment group s against vehicle.

Figures 32A-32B. In vivo imaging in the IV metastasis model (A) In vivo imaging protocol in IV metastasis model. (B) Representative of lung tumor signals by IVIS in Figure4D.

Figures 33A-33C. 1V270 therapy recruits innate immune cells to the lungs. (A-C) Representative flow cytometric plots and gating process of immune cells in the lungs. BALB/c mice (n=6- 7 /group) were treated with 1 V270 (200 Pi^injection) on day -1 and then tumor cells were i.v. administered on day 0. Next day, single cell suspensions derived from the lungs were stained for (A) NK cells (CD45 ⁺ CD3 ^_ NKp46 ⁺ CD49 ⁺ ), (B) M-MDSCs (CD45 ⁺ CD 1 lb ⁺ Ly6G ^_ Lye^) and (C) PMN-MDSCs (CD45 ⁺ CD 1 lb ⁺ Ly6G ⁺ ) were analyzed by flow cytometric assay . (C) Frequencies of M-MDSCs and PMN-DSCs were compared between 1V270 treated and vehicle-treated mice. Each dot indicates an individual mouse and horizontal and vertical bars indicate means ± SEM . Data are representative of 2 independent experiments showing similar results. M ann-Whitney U test was used to comp are treatment group s against the vehicle group . *P<0.05, **P<0.01.

Figures 34A-34C. Antibody-mediated depletion of NK cells reverses inhibition of tumor cell colonization by 1V270 therapy in the early phase. (A) Experimental protocol ofNK cell depletion. Anti-asialo GM I rabbit polyclonal Ab (aGMl) or rabbit IgG polyclonal Ab was injected on day s -4, -1, 3, and 10. 1V270 (200 p^injection) was administered on day -1 and 2 x 10 ⁴ 4T 1 cells were i.v. injected on day 0. (B) Representative flow cytometric plots of NK cell frequency in the lung on day 14. (C) Representative of lung tumor signals of NK cell depleted mice by IVIS in Figure 4F.

Figure 35. Toll-like receptor 7 expression in 4T1, B16, and LLC cells. Expression of TLR7 in the cell lines used in this study was assessed by quantitative RT-PCR. Figure 36. Systemic administration of 1V270 induces significantly lower levels of cytokine induction by systemically administrated 1V270 and 1V136. BALB/c mice (n=5) were i.p . administered IV270 (200 μg = 185 nmol/injection) or 2V136 (58 μg = 185 nmol/injection) and sera were collected 2, 4, 6, and 24 hours following the administration. The levels of TNFa, IL-12, and IP -10 were measured by Luminex assay . *P<0.05, **P<0.01 by twoway ANOVA using a Bonferroni post hoc test comparing treatment group s.

Figures 37 A-37B. The correlation between the tumor signal in the lung at day 10, and the number of lung nodules or overall survival. (A) BALB/c mice (n=S) were injected with 2 x 10 ⁴ 4T 1 cells on day 0. Tumor cell signals were monitored using IVIS. Tumor signals on day 10 were plotted with the number of lung tumor nodules examined on day 21. (B) BALB/c mice (n=8 /group) were injected with 1V270 on day -1 and with 2 x 10 ⁴ 4T1 cells on day 0. Tumor signals were monitored using IVIS. Tumor signals on day 10 were plotted with survival (day s) of individual mice. The representative tumor signal images by IVIS for (A) and (B) are shown at right. The correlation was evaluated by a Pearson's correlation test.

DetailedDescription

Definitions

A "phospholipid" as the term is used herein refers to a glycerol mono- or diester bearing a p hosp hate group bonded to a gly cerol hy droxy 1 group with an alkanolamine group being bonded as an ester to the phosphate group, of the general formula

wherein R ¹¹ and R ¹² are each independently hydrogen or an acyl group, and R ¹³ is a negative charge or a hydrogen, depending upon pH. When R13 is a negative charge, a suitable counterion, such as a sodium ion, can be present. For example, the alkanolamine moiety can be an ethanolamine moiety, such that m = 1. It is also understood that the NH group can be protonated and p ositively charged, or unprotonated and neutral, depending upon pH. For example, the phospholipid can exist as a zwitterion with a negatively charged phosphate oxy anion and a positively charged p rot onated nitrogen atom. The carbon atom bearing OR ¹² is a chiral carbon atom, so the molecule can exist as an R isomer, an S isomer, or any mixture thereof. When there are equal amounts of R and S isomers in a sample of the compound of formula (II), the sample is referred to as a "racemate." For example, in the commercially available product 1,2-dioleoy 1- sn-glycero-3-phosphoethanolamine, as used in Example I below, the R ³ group is of the chiral structure

, which is of the R absolute configuration.

A phospholipid can be either a free molecule, or covalently linked to another group for example as shown

wherein a wavy line indicates a point of bonding.

Accordingly, when a substituent group, such as R ³ of the compound of formula (I) herein, is stated to be a phospholipid what is meant that a

phospholipid group is bonded as specified by the structure to another group, such as to an N-benzyl heterocyclic ring system as disclosed herein. The point of attachment of the phospholipid group can be at any chemically feasible position unless specified otherwise, such as by a structural depiction. For example, in the phospholipid structure shown above, the point of attachment to another chemical moiety can be via the ethanolamine nitrogen atom, for example as an amide group by bonding to a carbonyl group of the other chemical moiety, for example

wherein R represents the other chemical moiety to which the phospholipid is bonded. In this bonded, amide derivative, the R ¹³ group can be a proton or can be a negative charge associated with a counterion, such as a sodium ion. The acylated nitrogen atom of the alkanolamine group is no longer a basic amine, but a neutral amide, and as such is not protonated at physiological pH.

An "acyl" group as the term is used herein refers to an organic structure bearing a carbonyl group through which the structureis bonded, e.g., to glycerol hydroxy 1 groups of a phospholipid, forming a "carboxylic ester" group .

Examples of acyl groups include fatty acid groups such as oleoyl groups, that thus form fatty (e.g, oleoyl) esters with the glycerol hydroxyl group s.

Accordingly, when R ¹¹ or R ¹² , but not both, are acyl groups, the phospholipid shown above is a mono-carboxylic ester, and when both R ¹¹ and R ¹² are acyl groups, the phospholipid shown above is a di-carboxylic ester.

Within the present disclosure it is to be understood that a compound of the formula (I) or a salt thereof may exhibit the phenomenon of tautomerism whereby two chemical compounds that are capable of facile interconversion by exchanging a hydrogen atom between two atoms, to either of which it forms a covalent bond. Since the tautomeric compounds exist in mobile equilibrium with each other they may be regarded as different isomeric forms of the same comp ound. It is to be understood that the formulae drawings within this specification can represent only one of the possible tautomeric forms. However, it is also to be understood that the disclosure encompasses any tautomeric form, and is not to be limited merely to any one tautomeric form utilized within the formulae drawings. The formulae drawings within this specification can represent only one of the possible tautomeric forms and it is to be understood that the specification encompasses all possible tautomeric forms of the compounds drawn not just those forms which it has been convenient to show graphically herein. For example, tautomerism may be exhibited by a pyrazolyl group bonded as indicated by the wavy line. While both substituents would be termed a 4-py razolyl group, it is evident that a different nitrogen atom bears the hydrogen atom in each structure.

Such tautomerism can also occur with substituted pyrazoles such as 3- methyl, 5 -methyl, or 3,5-dimethylpyrazoles, and the like. Another example of tautomensm is amido-imido (lactam-lactim when cyclic) tautomerism, such as is seen in heterocyclic compounds bearing a ring oxygen atom adjacent to a ring nitrogen atom. For example, the equilibrium:

is an example of tautomerism.

Accordingly, a structure depicted herein as one tautomer is intended to also include the other tautomer.

Optical Isomerism

It will be understood that when compounds of the present disclosure contain one or more chiral centers, the compounds may exist in, and may be isolated as pure enantiomeric or diastereomeric forms or as racemic mixtures. The present disclosure therefore includes any possible enantiomers,

diastereomers, racemates or mixtures thereof of the compounds of the disclosure.

The isomers resulting from the presence of a chiral center comprise a pair of non-sup erimposable isomers that are called "enantiomers." Single

enantiomers of a pure compound are optically active, i.e., they are capable of rotating the plane of plane polarized light. Single enantiomers are designated according to the Cahn-Ingold-Prelog system. The priority of substituents is ranked based on atomic weights, a higher atomic weight, as determined by the systematic procedure, having a higher priority ranking Once the priority ranking of the four groups is determined, the molecule is oriented so that the lowest ranking group is pointed away from the viewer. Then, if the descending rank order of the other groups proceeds clockwise, the molecule is designated (R) and if the descending rank of the other groups proceeds counterclockwise, the molecule is designated (S). In the example in Scheme 14, the

Cahn-Ingold-Prelog ranking is A > B > C > D. The lowest ranking atom, D is oriented away from the viewer.

(R) configuration (S) configuration The present disclosure is meant to encompass diastereomers as well as their racemic and resolved, diastereomerically and enantiomerically pure forms and salts thereof. Diastereomeric pairs may be resolved by known separation techniques including normal and reverse phase chromatography, and

crystallization.

"Isolated optical isomer" means a compound which has been

substantially purified from the corresponding optical isomer(s) of the same formula. In one embodiment, the isolated isomer is at least about 80%, e.g., at least 90%, 98% or 99% pure, by weight.

Isolated optical isomers may be purified from racemic mixtures by well- known chiral separation techniques. According to one such method, a racemic mixture of a compound of the disclosure, or a chiral intermediate thereof, is separated into 99% wt.% pure optical isomers by HPLC using a suitable chiral column, such as a member of the series of DAICEL ^® CHIRALPAK ^® family of columns (Daicel Chemical Industries, Ltd., Tokyo, Japan). The column is operated according to the manufacturer's instructions.

As used herein, "pharmaceutically acceptable salts" refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, gly colic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxy maleic, phenylacetic, glutamic, benzoic, behenic, salicylic, sulfanilic, 2- acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like.

The pharmaceutically acceptable salts of the compounds useful in the present disclosure can be synthesized from theparent compound, which contains a basic or acidic moiety, by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile may be employed. Lists of suitable salts are found in Remington's Pharmaceutical Sciences. 17th ed., Mack Publishing Company, East on, PA, p . 1418 (1985), the disclosure of which is hereby incorporated by reference.

The compounds of the formulas described herein can be solvates, and in some embodiments, hydrates. Theterm "solvate" refers to a solid compound that has one or more solvent molecules associated with its solid structure. Solvates can form when a compound is cry stallized from a solvent. A solvate forms when one or more solvent molecules become an integral part of the solid cry stalline matrix upon solidification. The compounds of the formulas described herein can be solvates, for example, ethanol solvates. Another type of a solvate is a hydrate. A "hydrate" likewise refers to a solid compound that has one or more water molecules intimately associated with its solid or crystalline structure at the molecular level. Hydrates can form when a compound is solidified or

cry stallized in water, where one or more water molecules become an integral part of the solid crystalline matrix

The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio.

The following definitions are used, unless otherwise described: halo or halogen is fluoro, chloro, bromo, or iodo. Alkyl, alkoxy, alkenyl, alkynyl, etc. denote both straight and branched groups; but reference to an individual radical such as "propyl" embraces only the straight chain radical, a branched chain isomer such as "isopropyl" being specifically referred to. Aryl denotes a phenyl radical or an ortho-fusedbicyclic carbocyclic radical having about nine to ten ring atoms in which at least one ring is aromatic. Het can be heteroary 1, which encompasses a radical attached via a ring carbon of a monocyclic aromatic ring containing five or six ring atoms consisting of carbon and one to four heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(X) wherein X is absent or is H, O, (Ci-C4)alkyl, phenyl or benzyl, as well as a radical of an ortho-fusedbicyclic heterocycle of about eight to ten ring atoms derived therefrom, particularly a benz-derivative or one derived by fusing a propylene, trimethylene, or tetramethylene diradical thereto.

It will be appreciated by those skilled in the art that compounds of the disclosure having a chiral center may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism. It is to be understood that the present disclosure encompasses any racemic, optically- active, polymorphic, or stereoisomenc form, or mixtures thereof, of a compound of the disclosure, which possess the useful properties described herein, it being well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recry stallization techniques, by synthesis from optically -active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase) and how to determine agonist activity using the standard tests described herein, or using other similar tests which are well known in the art. It is also understood by those of skill in the art that the compounds described herein include their various tautomers, which can exist in various states of equilibrium with each other.

Methods to Enhance Cancer Specific CD8+ Cells

Cellular transformation, tumor growth, and metastasis constitute a multistep process that requires the continuous rewiring of signaling pathways and alterations of the reciprocal interaction between cancer cells and the tumor microenvironment, thereby allowing cells to acquire features to become fully neoplastic and eventually malignant (Hanahan and Weinberg, 2011). The Hippo pathway has gained great interest in recent years as being strongly involved in several of these key hallmarks of cancer progression (Harvey et al., 2013;

Moroishi et al, 2015a) and, in general, serves important regulatory functions in organ development, regeneration, and stem cell biology (Johnson and Haider, 2014; Yu et al, 2015). The heart of the mammalian Hippo pathway is akinase cascade involving mammalian STE20-like protein kinase 1 (M ST 1; also known as STK4) and M ST2 (also known as STK3) (homologs of Drosophila Hippo), as well as two groups of MAP4Ks (mitogen-activated protein kinase kinase kinase kinases)— MAP4K 1/2/3/5 (homologs of Drosophila Happyhour) and MAP4K4/6/7 (homologs of Drosophila Misshapen)— and the large tumor suppressor 1 (LATSl) and LATS2 (homologs of Drosophila Warts) (Meng et al., 2016). When the Hippo pathway is activated, M ST 1/2 or MAP4Ks phosphorylate and activate the LATS1/2 kinases, which, in turn, directly phosphorylate and inactivate Yes -associated protein (YAP) and transcriptional coactivator with PDZ -binding motif (TAZ; also known as WWTRl), the two major downstream effectors that mediate transcriptional output of the Hippo pathway (Hansen et al., 2015). Activation of LATS1/2 kinases (and inactivation of YAP/T AZ) rep resents the major functional output of the Hipp o p athway .

Previous studies have convincingly established the Hipp o p athway as a suppressor signal for cellular transformation and tumorigenesis, though other studies revealed its oncogenic functions in certain contexts (Moroishi et al, 2015a; Wang et al., 2014). Deletion of M ST l/2 in mouse liver results in tissue overgrowth and tumor development, demonstrating the tumor suppressor function of these kinases (Zhou et al, 2009). Complementarity, overexpression of YAP in mouse liver also promotes tissue overgrowth and tumorigenesis (Camargo et al., 2007; Dong et al., 2007). These studies have demonstrated an inhibitory role of the Hipp o p athway in tumor initiation. However, effects of the Hippo pathway in tumor growth, especially in the context of reciprocal interactions between tumor cells and host anti-tumor immune responses, remain largely unknown.

Recent advances in cancer immunotherapy have improved patient survival. However, only a minority patients with pulmonary metastatic disease resp onds to treatment with checkpoint inhibitors. As an alternate approach, we have tested the ability of systemically administered 1V270, a toll-like receptor 7 (TLR7) agonist conjugated to a phospholipid, to inhibit lung metastases in two variant murine 4T 1 breast cancer models, as well as in B 16 melanoma, and Lewis lung c models. In the4T l breast cancer models, 1V270 therapy inhibited lung metastases if given up to a week after primary tumor initiation. The treatment protocol was facilitated by the minimal toxic effects exerted by the phospholipid TLR7 agonist, compared- to the unconjugated agonist. The lV270 therapy inhibited colonization by tumor cells in the lungs in a NK cell dependent manner. Additional experiments revealed that single administration of 1V270 led to tumor-sp ecific CD8 ⁺ cell-dependent adaptive immune responses that suppressed late stage metastatic tumor growth in the lungs. T cell receptor (TCR) repertoire analy ses showed that 1V270 therapy induced oligoclonal tumor-sp ecific T cells in the lungs and regional lymph nodes. Different animals displayed commonly shared TCR clones following 1V270 therapy . Intranasal administration of 1V270 also suppressed lung metastasis and induced tumor- specific adaptive immune resp onses. These results indicate that sy stemic 1 V270 therapy can induce tumor-specific cytotoxic T cell responses to pulmonary metastatic cancers, and that TCR rep ertoire analy ses can be used to monitor, and to predict, the responseto therapy .

Exemplary TLR7 1igands are shown below.

In one embodiment, the TLR7 ligand has formula (I):

wherein X ¹ is -0-, -S-, or -NR ^C -;

R ¹ is hy drogen, (Ci-Cio)alkyl, substituted (Ci-Cio)alkyl, C6-ioaryl, or substituted C6-ioaryl, Cs-siheterocyclic, substituted Cs-siheterocy clic;

R ^c is hy drogen, Ci-ioalkyl, or substituted Ci-ioalkyl; or R ^c and R ¹ taken together with the nitrogen to which they are attached form a heterocy clic ring or a substituted heterocyclic ring;

each R ² is independently -OH, (Ci-C ₆ )alkyl, substituted (Ci-C ₆ )alkyl, (Ci-C ₆ )alkoxy, substituted (Ci-C ₆ )alkoxy, -C(0)-(Ci-C ₆ )alkyl (alkanoyl), substituted -C(0)-(Ci-C ₆ )alkyl, -C(0)-(C ₆ -Cio)ary 1 (aroyl), substituted -C(O)- (C ₆ -Cio)aryl, -C(0)OH (carboxyl), -C(0)0(Ci-C ₆ )alkyl (alkoxycarbonyl), substituted -C(0)0(Ci-C ₆ )alkyl, -NR ^a R ^b , -C(0)NR ^a R ^b (carbamoyl), halo, nitro, or cyano, or R ² is absent;

each R ^a and R ^b is independently hydrogen, (Ci-C ₆ )alkyl, substituted (Ci-C ₆ )alkyl, (C ₃ -C ₈ )cycloalkyl, substituted (C ₃ -C ₈ )cycloalkyl, (Ci-C6)alkoxy, substituted (Ci-C6)alkoxy, (Ci-C ₆ )alkanoyl, substituted (Ci-C ₆ )alkanoyl, aryl, aryl(Ci-C6)alkyl, Het, Het (Ci-C ₆ )alkyl, or (Ci-C6)alkoxycarbonyl; wherein the substituents on any alkyl, aryl or heterocyclic group s are hydroxy, Ci-6alkyl, hydroxyCi-6alkylene, Ci-6alkoxy, C3-6cycloalkyl, Ci-

6alkoxyCi-6alkylene, amino, cyano, halo, or aryl;

n is 0, 1, 2, 3 or 4;

X ² is a bond or a linking group; and

R is a phospholipid comprising one or two carboxylic esters;

or a tautomer thereof;

or a pharmaceutically acceptable salt or solvate thereof.

In one embodiment, the composition of the disclosure comprises nanoparticles comprising a compound of formula (I). As used herein, a nanop article has a diameter of about 30 nm to about 600 nm, or a range with any integer between 30 and 600, e.g, about 40 nm to about 250 nm, including about 40 to about 80 or about 100 nm to about 150 nm in diameter. The nanop articles may be formed by mixing a compound of formula (I), which may spontaneously form nanoparticles, or by mixing a compound of formula (I) with a preparation of lipids, such as phospholipids including but not limited to phosphatidylcholine, phosphatidylserine or cholesterol, thereby forming a nanoliposome. Optionally, a compound of formula (I), a lipid preparation and a glycol such as propylene glycol are combined.

In one embodiment, a composition comprises an amount of a compound of Formula (I):

wherein X ¹ is -0-, -S-, or -NR ^C -;

R ¹ is hydrogen, (Ci-Cio)alkyl, substituted (Ci-Cio)alkyl, C6-ioaryl, or substituted C6-ioaryl, Cs-siheterocyclic, substituted Cs-siheterocy clic;

R ^c is hydrogen, Ci-ioalkyl, or substituted Ci-ioalkyl; or R ^c and R ¹ taken together with the nitrogen to which they are attached form a heterocyclic ring or a substituted heterocyclic ring; each R ² is independently -OH, (Ci-C ₆ )alkyl, substituted (Ci-C ₆ )alkyl, (Ci-Ce)alkoxy, substituted (Ci-Ce)alkoxy, -C(0)-(Ci-C ₆ )alkyl (alkanoyl), substituted -C(0)-(Ci-C ₆ )alkyl, -C(0)-(C ₆ -Cio)ary 1 (aroyl), substituted -C(O)- (C ₆ -Cio)aryl, -C(0)OH (carboxyl), -C(0)0(Ci-C ₆ )alkyl (alkoxycarbonyl), substituted -C(0)0(Ci-C ₆ )alkyl, -NR ^a R ^b , -C(0)NR ^a R ^b (carbamoyl), halo, nitro, or cyano, or R ² is absent;

each R ^a and R ^b is independently hydrogen, (Ci-C ₆ )alkyl, substituted (Ci-C ₆ )alkyl, (C3-C ₈ )cycloalkyl, substituted (C3-C ₈ )cycloalkyl, (Ci-C ₆ )alkoxy, substituted (Ci-C ₆ )alkoxy, (Ci-C ₆ )alkanoyl, substituted (Ci-C ₆ )alkanoyl, aryl, aryl(Ci-C ₆ )alkyl, Het, Het (Ci-C ₆ )alkyl, or (Ci-C ₆ )alkoxycarbonyl;

wherein the substituents on any alkyl, aryl or heterocyclic group s are hydroxy, Ci-6alkyl, hydroxyCi-6alkylene, Ci-6alkoxy, C3-6cycloalkyl, Ci- 6alkoxyCi-6alkylene, amino, cyano, halo, or aryl;

n is 0, 1, 2, 3 or 4;

X ² is a bond or a linking group ; and

R ³ is a phospholipid comprising one or two carboxylic esters;

or a tautomer thereof;

or a pharmaceutically acceptable salt or solvate thereof. Optionally , the comp osition further comprises an antigen. In one embodiment, the comp osition having an antigen is administered concurrently, priorto or subsequent to administration of the comp osition having a compound of formula (I).

3 can comprise a group of formula

wherein R ¹¹ and R ¹² are each independently a hydrogen or an acyl group, R ¹³ is a negative charge or a hydrogen, and m is 1 to 8, wherein a wavy line indicates a position of bonding, wherein an absolute configuration at the carbon atom bearing OR ¹² is R, S, or any mixture thereof.

For example, m can be 1, providing a glycerophosphatidylethanolamine. More specifically, R ¹¹ and R ¹² can each be oleoyl groups. In various embodiments, the phospholipid of R can comprise two carboxylic esters and each carboxylic ester includes one, two, three or four sites of unsaturation, ep oxidation, hydroxy lation, or a combination thereof.

In various embodiments, the phospholipid of R ³ can comprise two carboxylic esters and the carboxylic esters of are the same or different. More specifically, each carboxylic ester of the phospholipid can be a C17 carboxylic ester with a site of unsaturation at C8-C9. Alternatively, each carboxylic ester of the phospholipid can be a CI 8 carboxylic ester with a site of unsaturation at C9- C10.

In various embodiments, X ² can be a bond or a chain having one to about 10 atoms in a chain wherein the atoms of the chain are selected from the group consisting of carbon, nitrogen, sulfur, and oxygen, wherein any carbon atom can be substituted with oxo, and wherein any sulfur atom can be substituted with one or two oxo groups. The chain can be interspersed with one or more cycloalkyl, aryl, heterocyclyl, or heteroaryl rings.

In various embodiments X ² can be C(O), or can be any of

In various embodiments, R can be dioleoylphosphatidyl ethanolamine (DOPE).

In various embodiments, R ³ can be l,2-dioleoyl-sn-glycero-3-phospho ethanolamine and X ² can be C(O).

In various embodiments, X ¹ can be oxygen.

In various embodiments, X ¹ can be sulfur, or can be -NR ^C - where R ^c is hydrogen, Ci-6 alkyl or substituted Ci-6 alkyl, where the alky 1 substituents are hydroxy, C3-6cycloalkyl, Ci-6alkoxy, amino, cyano, or aryl. More specifically, X ¹ can be -NH-.

In various embodiments, R ¹ and R ^c taken together can form a

heterocyclic ring or a substituted heterocyclic ring. More specifically, R ¹ and R ^c taken together can form a substituted or unsubstituted morpholino, piperidino, pyrrolidino, or piperazino ring.

In various embodiments R ¹ can be a C1-C10 alkyl substituted with CI -6 alkoxy .

In various embodiments, R ¹ can be hydrogen, Ci-4alkyl, or substituted Ci- ₄ alkyl. More specifically, R ¹ can be hydrogen, methyl, ethyl, propyl, butyl, hydroxy Ci-4alkylene, or Ci-4alkoxyCi-4alkylene. Even more specifically, R ¹ can be hydrogen, methyl, ethyl, methoxy ethyl, or ethoxy ethyl.

In various embodiments, R ² can be absent, or R ² can be halogen or Ci- 4alkyl. More specifically, R ² can be chloro, bromo, methyl, or ethyl.

In various embodiments, X ¹ can be O, R ¹ can be Ci-4alkoxy -ethyl, n can be 1, R ² can be hydrogen, X ² can be carbonyl, and R ³ can be 1,2- dioleoylphosphatidyl ethanolamine (DOPE).

In various embodiments, the compound of Formula (I) can be:

In various embodiments, the compound of formula (I) can be the R- enantiomer of the above structure:

In one embodiment, the TLR& ligand is:

a compound of formula (II) a compound of formula (III)

Thiazolopyrimidines Purines

X ^{1 =} -0- -S-, or -NR ^c - wherein R ^c hydrogen, Ci-ioalkyl, or Ci-ioalkyl substituted by C3-6 cycloalkyl, or R ^c and R ¹ taken together with the nitrogen atom can form a heterocyclic ring or a substituted heterocyclic ring, wherein the substituents are hydroxy, Ci-6 alkyl, hydroxy Ci-6 alkylene, Ci-6 alkoxy, Ci-6 alkoxy Ci-6 alkylene, or cyano;

wherein R ¹ is (Ci-Cio)alkyl, substituted (Ci-Cio)alkyl, C6- ₁₀ aryl, or substituted C ₆ -io ary 1, C5-9 heterocyclic, substituted C5-9 heterocyclic; wherein the substituents on the alkyl, aryl or heterocyclic groups are hydroxy, Ci-6 alkyl, hydroxy Ci-6 alkylene, Ci-6 alkoxy, Ci-6 alkoxy Ci-6 alkylene, amino, cyano, halogen, or aryl;

each R ² is independently -OH, (Ci-C ₆ )alkyl, substituted (Ci-C ₆ )alkyl, (Ci-C ₆ )alkoxy , substituted (Ci-C ₆ )alkoxy , -C(0)-(Ci-C ₆ )alkyl (alkanoyl), substituted -C(0)-(Ci-C ₆ )alkyl, -C(0)-(C ₆ -Cio)ary 1 (aroyl), substituted -C(O)- (C ₆ -Cio)aryl, -C(0)OH (carboxyl), -C(0)0(Ci-C ₆ )alkyl (alkoxycarbonyl), substituted -C(0)0(Ci-C ₆ )alkyl, -NR ^a R ^b , -C(0)NR ^a R ^b (carbamoyl), -O- C(0)NR ^a R ^b , -(Ci-C ₆ )alkylene-NR ^a R ^b , -(Ci-C ₆ )alkylene-C(0)NR ^a R ^b , halo, nitro, or cyano;

wherein each R ^a and R ^b is independently hydrogen, (Ci-6)alkyl, (C3- C ₈ )cycloalky, (Ci-66)alkoxy, halo(Ci-6)alkyl, (C3-C ₈ )cycloalkyl(Ci-6)alkyl, (Ci- ₆ )alkanoyl, hydroxy(Ci-6)alkyl, aryl, ary l(Ci-6)alkyl, aryl, aryl(Ci-6)alkyl, Het, Het (Ci-6)alkyl, or (Ci-6)alkoxycarbony 1; wherein X ² is a bond or a linking group; wherein R ³ is a phospholipid comprising one or two carboxylic esters wherein n is 0, 1, 2, 3, or 4; or a tautomer thereof; or a pharmaceutically acceptable salt thereof. In cases where compounds are sufficiently basic or acidic to form acid or base salts, use of the compounds as salts may be appropriate. Examples of acceptable salts are organic acid addition salts formed with acids which form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, a-ketoglutarate, and a-glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts.

Acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.

Alkyl includes straight or branched Ci-io alkyl groups, e.g., methyl, ethyl, propyl, butyl, pentyl, isopropyl, isobutyl, 1-methylpropyl, 3-methylbutyl, hexyl, and the like.

Lower alkyl includes straight or branched Ci-6 alkyl groups, e.g., methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 2-methylpropyl, 1,1- dimethylethyl, pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1,1- dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, and the like.

The term "alkylene" refers to a divalent straight or branched hydrocarbon chain (e.g., ethylene: -CH2-CH2-).

C3-7 Cycloalkyl includes groups such as, cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like, and alkyl-substituted C _3- 7 cycloalkyl group, preferably straight or branched Ci-6 alkyl group such as methyl, ethyl, propyl, butyl or pentyl, and C5-7 cycloalkyl group such as, cyclopentyl or cyclohexyl, and the like.

Lower alkoxy includes Ci-6 alkoxy groups, such as methoxy, ethoxy or propoxy, and the like.

Lower alkanoyl includes Ci-6 alkanoyl groups, such as formyl, acetyl, propanoyl, butanoyl, pentanoyl or hexanoyl, and the like.

C7-11 aroyl, includes groups such as benzoyl or naphthoyl;

Lower alkoxy carbonyl includes C2-7 alkoxy carbonyl groups, such as methoxy carbonyl, ethoxy carbonyl or propoxy carbonyl, and the like. Lower alky lamino group means amino group substituted by Ci-6 alkyl group, such as, methy lamino, ethy lamino, p ropy lamino, buty lamino, and the like.

Di(lower alkyl)amino group means amino group substituted by the same or different and Ci-6 alkyl group (e.g., dimethy lamino, diethylamino,

ethy lmethy lamino) .

Lower alkylcarbamoyl group means carbamoyl group substituted by Ci-6 alkyl group (e.g., methy lcarbamoyl, ethylcarbamoyl, propylcarbamoyl, butylcarbamoyl).

Di(lower alkyl)carbamoyl group means carbamoyl group substituted by the same or different and Ci-6 alkyl group (e.g., dimethy lcarbamoyl,

diethy lcarbamoy 1, ethy lmethy lcarbamoy 1).

Halogen atom means halogen atom such as fluorine atom, chlorine atom, bromine atom or iodine atom.

Aryl refers to a C ₆ -io monocyclic or fused cyclic aryl group, such as phenyl, indenyl, or naphthyl, and the like.

Heterocyclic or heterocycle refers to monocyclic saturated heterocyclic groups, or unsaturated monocyclic or fused heterocyclic group containing at least one heteroatom, e.g., 0-3 nitrogen atoms NR ^C , 0-1 oxygen atom (-0-), and 0-1 sulfur atom (-S-). Non-limiting examples of saturated monocyclic heterocyclic group includes 5 or 6 membered saturated heterocyclic group, such as tetrahydrofuranyl, pyrrolidinyl, morpholinyl, piperidyl, piperazinyl or pyrazolidinyl. Non-limiting examples of unsaturated monocyclic heterocyclic group includes 5 or 6 membered unsaturated heterocyclic group, such as furyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, thienyl, pyridyl or pyrimidinyl. Non- limiting examples of unsaturated fused heterocyclic group s includes unsaturated bicyclic heterocyclic group, such as indolyl, isoindolyl, quinolyl, benzothizolyl, chromanyl, benzofuranyl, and the like. A Het group can be a saturated heterocyclic group or an unsaturated heterocyclic group, such as a heteroaryl group .

R ^c and R ¹ taken together with the nitrogen atom to which they are attached can form a heterocyclic ring. Non-limiting examples of heterocyclic rings include 5 or 6 membered saturated heterocyclic rings, such as 1- pyrrolidinyl, 4-morpholinyl, 1-piperidyl, 1-piperazinyl or 1-pyrazolidinyl, 5 or 6 membered unsaturated heterocyclic rings such as 1-imidazolyl , and the like.

The alkyl, aryl, heterocyclic groups of R ¹ can be optionally substituted with one or more substituents, wherein the substituents are the same or different, and include lower alkyl; cycloalkyl, hydroxyl; hydroxy Ci-6 alkylene , such as hy droxy methy 1, 2-hy droxy ethy 1 or 3 -hy droxy p ropyl; lower alkoxy ; C i- ₆ alkoxy

Ci-6 alkyl , such as 2-methoxy ethyl, 2-ethoxy ethyl or 3 -methoxy propyl; amino; alkylamino; dialkyl amino; cyano; nitro; acyl; carboxyl; lower alkoxy carbonyl; halogen; mercapto; Ci-6 alkylthio, such as, methy lthio, ethylthio, propylthio or buty lthio; substituted Ci-6 alkylthio, such as methoxy ethylthio,

methy lthioethy lthio, hydroxy ethylthio or chloroethy lthio; aryl; substituted C ₆ -io monocyclic or fused-cyclic aryl, such as 4-hy droxy phenyl, 4-methoxy phenyl, 4- fluorophenyl, 4-chlorophenyl or 3,4-dichlorophenyl; 5-6 membered unsaturated heterocyclic, such as furyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, thienyl, pyridyl orpyrimidinyl; and bicyclic unsaturated heterocyclic, such as indolyl, isoindolyl, quinolyl, benzothiazolyl, chromanyl, benzofuranyl or phthalimino.

In certain embodiments, one or more of the above groups can be expressly excluded as a substituent of various other groups of the formulas.

The alkyl, aryl, heterocyclic groups of R ² can be optionally substituted with one or more substituents, wherein the substituents are the same or different, and include hydroxyl; Ci-6 alkoxy , such as methoxy, ethoxy or propoxy;

carboxyl; C2-7 alkoxy carbonyl, such as methoxy carbonyl, ethoxy carbonyl or p rop oxy carbony 1) and halogen.

The alkyl, aryl, heterocyclic groups of R ^c can be optionally substituted with one or more substituents, wherein the substituents are the same or different, and include C3-6 cycloalkyl; hydroxyl; Ci-6 alkoxy; amino; cyano; aryl;

substituted aryl, such as 4-hy droxy phenyl, 4-methoxy phenyl, 4-chlorophenyl or

3,4-dichlorophenyl; nitro and halogen.

The heterocyclic ring formed together withR ^c and R ¹ and the nitrogen atom to which they are attached can be optionally substituted with one or more substituents, wherein the substituents are the same or different, and include Ci-6 alkyl; hydroxy Ci-6 alkylene; Ci-6 alkoxy Ci-6 alkylene; hydroxyl; Ci-6 alkoxy; and cyano. A specific value for X ¹ is a sulfur atom, an oxygen atom or -NR ^C -. In other embodiments, the TLR7 ligand has formula (I) wherein R is hydrogen, (Ci-Cio)alkyl, substituted (Ci-Cio)alkyl, C6-ioaryl, or substituted C ₆ - loaryl, Cs-siheterocyclic, substituted Cs-siheterocyclic.

In other embodiments, the TLR7 ligand has formula (I), wherein R ³ is independently -OH, (Ci-C ₆ )alkyl, substituted (Ci-C ₆ )alkyl, (Ci-C6)alkoxy, substituted (Ci-Ce)alkoxy , -C(0)-(Ci-C ₆ )alkyl (alkanoy 1), substituted -C(O)- (Ci-C ₆ )alkyl, -C(0)-(C ₆ -Cio)aryl (aroyl), substituted -C(O)- (C ₆ -Cio)aryl, -C(0)OH (carboxyl), -C(0)0(Ci-C ₆ )alkyl (alkoxycarbonyl), substituted -C(0)0(Ci-C ₆ )alkyl, -NR ^a R ^b , -C(0)NR ^a R ^b (carbamoyl), halo, nitro, or cyano, or R ² is absent; each R ^a and R ^b is independently hydrogen, (Ci- C ₆ )alkyl, substituted (Ci-C ₆ )alkyl, (C ₃ -C ₈ )cycloalkyl, substituted (C ₃ - C ₈ )cycloalkyl, (Ci-C6)alkoxy, substituted (Ci-C6)alkoxy, (Ci-C ₆ )alkanoyl, substituted (Ci-C ₆ )alkanoyl, aryl, aryl(Ci-C6)alkyl, Het, Het (Ci-C6)alkyl, or (Ci-C6)alkoxycarbonyl; wherein the substituents on any alkyl, aryl or heterocy clic group s are hy droxy , Chalky 1, hy droxy Ci-6alky lene, Ci-6alkoxy , C _{3- 6} cycloalkyl, Ci-6alkoxy Ci-6alkylene, amino, cyano, halo, or aryl.

The invention will be further described by the following non-limiting examples.

Example 1

The present disclosure provides several discoveries. First, the local administration of an immune stimulating agent (specifically a TLR7 or TLR9 agonist) to a syngeneic cancer in a mouse can cause the clonal expansion of tumor specific CD8 ⁺ T cells in TILs and spleen, as detected by RNA sequencing of T cell receptor variable region genes (Figure 2). Second, the population of common T cell clones correlates with the clinical efficacy of the immune stimulating TLR therapy, and with the efficacy of checkpoint inhibitor therapy using an anti-PD-1 monoclonal antibody (Figure 3). These results strongly suggest that clonal expansion of the tumor specific T cell is a biomarker for an immune reaction against the cancer (common clones between tumors and spleens in Figure 3). The data also leads to the proposal that the tumor specific T cell receptor variable region gene products induced by a drug or othertherapy can identify clonal TIL cells in the blood, permitting their more efficient isolation and expansion. Thus, drug treatment may cause the release of tumor specific T cell clones in TILs from the tumor, and their subsequent expansion in the blood and lymphoid tissues, outside of the suppressive tumor micro- environment.

In a separate series of experiments, it was found that inactivation in cancer cells of two enzymes, called LATSl and LATS2, markedly increases the response of the immune system to the cancers, leading to tumor eradication in several examples (Figures 4 and 5). Without causing any detectable cytotoxic effects, the reduction in enzyme activity induces the release from the malignant cells of small extracellular vesicles (EVs) (e.g., less than about 0.2 microns in diameter). After isolation by filtration and ultracentrifugation, the small EVs initiate and activate tumor specific T cells in vivo (Figure 6). When re- administered to mice, the isolated tumor EVs trigger a therapeutic immune resp onse against the cancer, without detectable cytotoxicity to normal cells. Previous investigations have shown that various drugs, radiation, and heat shock can induce therelease of EVs from tumor cells (Andaloussi et al, 2013; Vader et al., 2014). However, in almost every instance, the EV release was associated with cytotoxicity, exactly oppositeto what is described herein. Moreover, most previous studies have shown that EVs released from cancers are immune suppressive and actually promote metastasis, rather than causing cancer specific immune stimulation, as described herein (Moroishi et al., 2016). Based upon these unexpected revelations, specific drugs that promote the release of immunogenic EVs from cancer cells, in the absence of cytotoxicity, are predicted to induce clonal expansion of tumor specific CD8+ T cells, and can be identified by the same protocols used in experiments with TLR agonists, without knowing the exact antigen specific for the tumor cells. The drugs need not be locally administered to a cancer. In one embodiment, the agents could be administered orally, parenterally, or by inhalation. The latter route may be particularly useful in patients with lung cancer or pulmonary metastases.

After drug therapy, the isolation and expansion of the tumor specific CD8 ⁺ T cells in tissue culture can be made much more efficient by purification of activated CD8 ⁺ T cells, and by co-culture of the T cells with the

immunogenic tiny EVs derived from the blood of the same patient in the presence or absence of feeder cells. Moreover, the treatment of cancer patients with anti-PDl and other checkpoint inhibitors should be undertaken specifically in those subjects who have circulating clonal T cells after drug treatment, and at the time when both EVs and TIL concentrations in the blood reach maximal levels.

To identify drugs that can induce immunogenic EV release with discernible toxic effects, in one embodiment, a mouse melanoma cell line is contacted with the drugs, a maximal non-toxic concentration is identified, released EVs with less than 0.2 micron diameter into the medium are measured, and the ability of the released EVs to stimulate tumor reactive T cells and to cause clonal expansion is assessed.

In contrast to current methods for the isolation of tumor specific autologous CD8 ⁺ T cells, which typically requires the processing of surgical specimens or biopsies to release the Tumor Infiltrating Lymphocytes (TILs) (Figure 1), the method described herein does not require tissue processing. In addition, although previously in a few patients, tumor specific T cells have been identified in the blood, by their expression of PD-1, of activation antigens, and by reactivity with synthetic pep tides derived from mutated oncogene products e.g., Ras (Arafeh et al, 2015), the presently disclosed method does not require any specific knowledge of the tumor antigens

Further, the current method for the expansion of TILs in tissue culture before re-infusion often leads to the co-expansion of non-specific T cells that can outgrow the effector T cells against the cancer. This problem can lead to therapeutic failure. In contrast, the presently disclosed methods involve a combination of drug treatments that enhances tumor specific T cells in the peripheral circulation before isolation and expansion of T cells in tissue culture, combined with stimulation with autologous immune EVs that contain tumor antigens and immunostimulatory molecules. This approach renders TIL therapy much more efficient and less expensive.

In one embodiment, the following protocol may be employed:

1. Administration of a non-toxic drug that induces EVs release and

expansion of tumor specific T cells in TILs to a patient, e.g., via the oral, parenteral, or intrapulmonary routes. The drugs may include TLR agonists, enzyme inhibitors, antibiotics, hormones, and the like.

2. Before, and each day after drug administration, up to aboutl4 days, a heparinized blood sample (10 mL) is withdrawn. PD-1 positive CD8 ⁺ T cells are isolated, for example, using magnetic beads, and the TCR alpha and beta mRNAs are reverse transcribed, and subjected toNexgen RNA sequencing. The clonal expansion of tumor sp ecific T cells is

demonstrated by the increasing clonal dominance of one or more TCR alpha and TCR beta sequence mRNAs, compared to the p re-treatment specimen.

3. Small EVs in the p re-treatment and p ost-treatment blood sp ecimens are isolated by filtration of plasma through an about 0.2 up to about 0.4 micron sterile filter, followed by high speed centrifugation. RNA and protein content are assessed.

4. At the time of substantial EV release, and TCR clonal dominance, anti- PD-1 antibody treatment is initiated.

5. The activated clonal CD8 ⁺ T cells are isolated, e.g., using beads coated with anti-TCR antibodies or by limiting dilution and tissue culture. T cells are expanded as previously described (Dudley et al., 2003). Briefly, T cells are expanded with a rapid expansion protocol using, for instance, OKT3 (anti-CD3) antibody and IL2, e.g., about 30 ng/mL OKT3 (anti- CD3) antibody and about 6,000 IU/mL IL2, in the presence of irradiated allogeneic feeder cells. The tissue cultures are supplemented once at initiation with EVs from thetreated patients.

6. T cell cultures are expanded using cytokines, to yield 100 million to one billion cells; their TCR clonality may be confirmed.

7. In p atients who did not resp onds fully to checkp oint inhibitor therapy as described above, the expanded TILs can be reinfused.

The disclosure will significantly improve isolation and expansion of autologous tumor sp ecific T cells for cancer immunotherapy . Biopsies or surgical resection will no longer be required. Patients who are likely to respond to the T cell therapy will be identified early, before expansion of autologous T cells. Then all patients with metastatic cancer will be potential candidates for treatment.

Example 2

In the present study, therole of theLATSl/2 kinases in the growth of established tumors in the context of anti-tumor immunity was investigated.

Surprisingly, inactivation of the "tumor suppressor" LATS1/2 in tumor cells strongly suppresses tumor growth in immune-competent, but not immune- compromised, mice due to the induction of host anti-tumor immune responses. The data indicate a new paradigm for how tumor immunogenicity is regulated through the Hippo signaling pathway in tumor cells and also have implications for targeting LATS1/2 in cancer immunotherapy .

Experimental Model and Subject Details

Animals

C57BL/6, C3H/HeOu, or BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). M d88, Tlr4, Tlr7, and Tlr9 KO mice were kind gifts from Dr. Shizuo Akira (Osaka University, Osaka, Japan). Ticaml (also known as TRIF) KO mice were kindly provided by Dr. Bruce Beutler (University of Texas Southwestern Medical Center, Dallas, TX, USA). Caspl (also known as Caspase-l)KO mice were kindly provided by Dr. Richard A. Flavell (Yale University School of Medicine, New Haven, CT, USA). Ragl KO mice, (also known as STING) KO mice, and OT -I transgenic mice were purchased from The Jackson Laboratory . Ifiiarl KO mice were purchased from B&K Universal (East Yorkshire, United Kingdom). These mouse strains were backcrossed for 10 generations onto the C57BL/6 background at the University of California, San Diego. Mutant mice were bled by the University of California, San Diego Animal Care Program. 7-12 weeks old mice were used and all animal experiments were approved by the University of California, San Diego,

Institutional Animal Care and Use Committee.

Method Details

Cell culture and gene deletion by CRISP R/Cas9 system

All cell lines were cultured under an atmosphere of 5% C0 ₂ at 37°C. B 16-OVA cells (B16F 10 cells expressing ovalbumin) were cultured in DMEM (GIBCO) supplemented with 10% fetal bovine serum (FBS, GIBCO), penicillin (lOO U/ml), and streptomycin (100 mg ml). SCC7, 4T 1, EL4, bone marrow- derived dendritic cells (BMDCs), mouseprimary lymph node cells and CD8 ⁺ T cells were cultured in RPMI 1640 (GIBCO) supplemented with 10% FBS (GIBCO), penicillin (100 U/ml), and streptomycin (100 mg/ml).

LATSl/2-deficient cells were created through the CRISPR (clustered regularly interspaced short palindromic rep eats)/Cas9 system (Ran et al, 2013). We use a transient CRISPR strategy for the deletion of LATS1/2 to avoid any potential unspecific effects mediated by stable Cas9/sgRNA genome integration. Cells were transiently transfected with a Cas9 and single-guide RNA (sgRNA) expression plasmid encoding puromycin resistance (PX459; Addgene plasmid #48139). The CRISPR-transfected cells will thus acquire transient resistance to puromycin. The guide sequences were designed using the Optimized CRISPR Design at http ://crispr.mit.edu. The guide sequences used are 5'- AGACGTTCTGCTCCGAAATC-3' (SEQ ID NO: l) or 5'- ACGTTTCCATTGGCGAATGA-3' (SEQ ID NO :2) for mouse Latsl; 5'- GAGTGT CC AGCTTAC AAGCG-3 ' (SEQ ID NO:3) or 5'- GCTGGGTGGTGCAAACTACG-3' (SEQ ID NO:4) for mouse Lats2.

Following transfection and transient selection with puromy cin for 3 day s, cells were single-cell sorted by fluorescence-activated cell sorting (F ACS) into 96- well plate without puromycin. Knockout clones were selected by immunoblot analysis for the lack of LATS1/2 proteins and YAP phosphorylation. LATS1/2 dKO cells were sensitive to puromycin after expansion, indicating a transient expression of CRISPR/Cas9 systemin those cells. Two independent clones were analyzed as indicated and the parental LATSl/2 WT cells (not transfected with PX459) were used as control.

Retroviral Infection

B16-OVA cells stably expressing empty vector, YAP(5SA),

YAP(5SA/S94A), or TAZ(4SA) were generated by retroviral infection. 293

Phoenix retrovirus packaging cells were transfected with pB ABE empty vector, pBABE YAP(5SA), p B ABE YAP (5 SA/S94 A), or pBABETAZ(4SA) constructs. Forty -eight hours after transfection, retroviral supernatant was supplemented with 5 mg/mL polybrene, filtered through a 0.45 mm filter, and used for infection. Forty -eight hours after infection, cells were selected with 4 mg/mL puromy cin in culture medium.

Immunoblot Analysis

Equal amount of protein samples were resolved in SDS-PAGE in reducing conditions unless otherwise mentioned in the Figure Legends.

Antibodies to YAP (#14074), pYAP (S127 in humans and SI 12 in mice; #4911), YAP/TAZ (#8418), LATS1 (#3477), CD81 (#10037), and ALIX (#2171) were obtained from Cell Signaling; those to actin (#ab3280) and ovalbumin (OVA, #abl221) were from Abeam; those to LATS2 (# A300-479 A, also weakly recognize LATS1) were from Bethyl Laboratories; those toFLOT l (#610821) and HSP90 (#610418) were from BD Biosciences. The phos-tag electrophoresis was performed as described previously (Moroishi et al, 2015b). YAP proteins can be separated into multiple bands in the presence of phos-tag depending on differential phosphorylation levels, with phosphorylated proteins migrating more slowly . Where indicated, cells were treated with serum starvation (DMEM or RPMI 1640 without other supplements), 1 mg/ml Latrunculin B (LatB), or 25 mM 2-deoxy-D-glucose (2-DG) for 1 hour before harvest.

Immunostaining of Cells

Cells were treated with or without 1 mg ml Latrunculin B (LatB) for 1 hour before harvest. Cells were then fixed for 10 minutes at room temperature with 4% paraformaldehyde in phosphate-buffered saline (PBS) and were permeabilized with 0.1% Triton X-100 in PBS for 10 minutes at room temperature. Cells were then incubated consecutively with primary antibodies to YAP/TAZ (Santa Cruz, #sc-101199) (overnight at 4°C) and Alexa Fluor 488- labeled goat secondary antibodies (for 90 minutes at room temperature) in PBS containing 1% bovine serum albumin (BSA). Cells were covered with a drop of ProLong Gold antifade reagent with D API (Invitrogen) for observation. Cells in five randomly selected views (about 100 cells) were selected for the

quantification of YAP/TAZ localization.

Reverse Transcription (RT) and Real-Time PCR Analysis

Total RNA (500 ng) isolated from cells with the use of RNeasy Plus Mini Kit (QIAGEN) was reverse-transcribed to complementary DNA using iScript cDNA Synthesis Kit (Bio-Rad). Complementary DNA was then diluted and used for quantification by real-time PCR, which was performed using KAPA SYBR FAST qPCR Kit (Kapa Biosystems) and the 7300 real-time PCR system (AppliedBiosy stems). The sequences of thePCR primers (forward and reverse, respectively) are 5'-GCCTGGAGAAACCTGCCAAGTATG-3' (SEQ ID NO:5) and 5'-GAGTGGGAGTTGCTGTTGAAGTCG-3' (SEQ ID NO:6) for mouse Gapdh; 5'-AGCTGACCTGGAGGAAAACA-3' (SEQ ID NO:7) and 5'- G AC AGGCT T GGCG ATTT TAG-3 ' (SEQ ID NO:8) for mouse Ctgf, 5'- GCTCAGTCAGAAGGCAGACC-3' (SEQ ID NO:9) and 5'- GTTCTTGGGGACACAGAGGA-3' (SEQ ID NO:10) for mouse Cyr61 5'- AGGAGAAGAGTTGCCCACCTATGAG-3' (SEQ ID NO:l 1) and 5'- T CGA AG AGCT TC ATCCTGTCGC-3 ' (SEQ ID NO: 12) for mouse Amotl2; 5'- CCTGAGAAAGAAGAAACACAGCCTC-3' (SEQ ID NO:13) and 5'- GC A AGT T GGTTG AGGA AGAGAGGG-3 ' (SEQ ID NO: 14) for mouse Ifha4 5'-GAAGAGTTACACTGCCTTTGCCATC-3' (SEQ ID NO: 15) and 5'- A A AC ACT GT CTGCTGGTGGAGTTC-3 ' (SEQ ID NO:16) for mouse Ifiibl. Reactions for Gapdh mRNA were performed concurrently on the same plate as those for the test mRNAs, and results were normalized by the corresponding amount of Gapdh mRNA.

Soft Agar Colony Formation Assay

Each 6-well plate was coated with 1.5 mL of bottom agar (DMEM or RPMI 1640 containing 10% FBS and 0.5% Difco agar noble). Cells (5 x 10 ³ cells for B16-OVA and SCC7, 2.5 x 10 ³ cells for 4T 1) were suspended in 1.5 mL of top agar (DMEM or RPMI 1640 containing 10% FBS and 0.35% Difco agar noble) into each well. Cells were cultured for approximately two weeks and replaced with fresh medium every three days. Colonies were stained using 0.005%) crystal violet in 5%> methanol and quantified using ImageJ software. Tumor Transplantation and Immunization

B 16-0 VA cells (2 x 10 ⁵ ) were subcutaneously transplanted into both back flanks of C57BL/6 mice. Tumor height and width were measured with a caliper every 2-3 days to calculate tumor volume ( = width ² x height x 0.523). Mice were sacrificed when tumors reached maximum allowed size (15 mm in diameter) or when signs of ulceration were evident. Likewise, 1 x 10 ⁵ of SCC7 cells were subcutaneously transplanted into both back flanks of C3H/HeOu mice and 2.5 x 10 ⁵ of 4T l cells were transplanted into both mammary fat pads of BALB/c mice. For 4T 1 lung metastasis assay, lungs were tracheally injected with India ink 28 day s after transplantation, and then destained in Fekete' s solution to count tumor nodules.

For tumor vaccination experiments, C57BL/6 mice were immunized intradermally at the base of the tail with irradiated B16-OVA cells (100 Gy, 1 x 10 ⁶ ) 12 days priorto challenge withB 16-OVA cells (one time vaccination, without any adjuvant). For immunization with EVs, C57BL/6 mice were inoculated with irradiated B16-OVA cells (100 Gy, 1 x 10 ⁶ ) at the base of the tail and EVs freshly isolated from culture supernatants ofB 16-OVA cells (6 x 10 ⁶ ) were injected every 3 day s (days 0, 3, 6, and 9) into the same place until challenged withB 16-OVA cells at day 12. Histopathology and Immunostaining of Tumors

Tumors were fixed with 4% paraformaldehyde in PBS, embedded in paraffin, sectioned with a microtome, and stained with hematoxylin-eosin according to standard procedures. Immunostaining of tumors was performed with frozen cry ostat sections with PE-conjugated antibodies to CD45

(eBioscience, #12-0451-82).

Measurement of OVA Specific Antibodies

Serum anti-OVA IgG concentrations were measured by enzyme-linked immunosorbent assay (ELISA). Briefly, half area 96-well plates (Corning) were coated with 5 mg ml OVA protein (Worthingt on Biochemical, #LS003056) in

PBS overnight at 4°C. Plates were washed and then blocked for 3 hours at room temperature with blocking buffer [1% BSA (bovine serum albumin) in PBS], followed by wash and incubation with serum samples tested at a 1 : 100 to 1 : 125,600 dilutions in blocking buffer overnight at 4°C. Plates were then washed and incubated with HRP -conjugated detecting antibody in blocking buffer at room temperature for 2 hours. Plates were washed and incubated with TMB substrate (KPL, #95059-286), and then read at 450 nm and 650 nm after stoppingthe development with 1 M phosphoric acid. Each ELISA plate contained a titration of a previously quantified serum to generate a standard curve. Anti-OVA IgG concentrations were determined from the lowest dilution of serum samples within a standard curve and reported as U/ml.

Flow Cytometry

Flow cytometry was performed using a BD LSREortessa and results were analyzed using FlowJo software (Treestar). Cell suspensions were incubated in mouse Fc block (anti CD16/CD32; BD Biosciences, #553142) priorto staining. Fluorochrome conjugated anti-mouse CD45 (clone 30F-11), CD3e (clone 145- 2C11), CD8a (clone 53-6.7), GranzymeB (clone GB11), and IFNy (clone XMG1.2) antibodies were used following the manufacturers protocol. K ^b - SIINFEKL-tetramer was used for identifying OVA-specific CD8 ⁺ T cells.

Propidium iodide (PI) was used to stain dead cells.

To analyze intracellular cytokine expression, cells were re-stimulated ex v/vowith 10 mg/ml SIINFEKL pep tide (AnaSpec, #AS-60193-1) for 5 hours in the presence of protein transport inhibitor (BD biosciences, #555029) for the last 4 hours. Intracellular cytokine staining was then performed using

Fixation/Permeabilization Solution Kit (BD Biosciences, #554714).

Ex Vivo Cytotoxicity Assay

EL4 cells were pulsed with 8 mg/ml SIINFEKL peptide or irrelevant peptides for 2 hours at 37°C, and then labeled with 0.25 mM or 2.5 mM CFSE (carboxy fluorescein succinimidyl ester; Thermo Fisher Scientific, #C34554) for 10 minutes at 37°C, respectively . CFSE ^low (SIINFEKL loaded target) and CSFE^ ¹¹ (irrelevant peptide control) EL4 cells were mixed at 1 :1 ratio, and then co-cultured with CD8 ⁺ T cells isolated from splenocytes of C57BL/6 mice challenged (or not) with WT or LATS1/2 dKO B 16-0 VA cells at 8 : 1 effector to target cell ratio (E:T). CD8 ⁺ T cells were isolated using Easy Sep Mouse CD8a Positive Selection Kit (STEM CELL, #18753) from pooled splenocytes of 3-4 mice per group for each experiment. The frequencies of CFSE ^low and CSFE^ ¹¹ EL4 cells in CFSE positive fraction were determined by flow cytometric analysis 18 hours after incubation and the percent of specific killing was calculated. Specific killing (%) = [1-" Sample ratio'V'Negative control ratio"] x 100; "Sample ratio" = [CFSE ^low (target)/CSFE ^M irrelevant)] value of each samples co-cultured with CD8 ⁺ T cells; "Negative control ratio" =

[CFSE ^low (target)/CSFE ^Mg irrelevant)] value of EL4 cells not cultured with CD 8 ⁺ T cells.

In Vitro Cross-Presentation Assay

For conditioned medium preparation, B 16-0 VA cells were seeded on culture plates and incubated in DMEM supplemented with 10% FBS for 24 hours at 37°C to allow cell attachment. The cells were then washed with PBS, and culture medium was switched to DMEM without serum. After incubation for 48 hours, conditioned medium was collected and centrifuged at 2,000 g for 10 minutes at 4°C to remove cell debris. The resulting supernatant was used for the experiment.

Naive CD8 ⁺ T cells were isolated from OVA-specific T cell receptor transgenic OT-I mice using Easy Sep Mouse CD8a Positive Selection Kit

(STEM CELL) and labeled with 2 mM CFSE. Bone marrow derived dendritic cells (BMDCs) were generated by 6 days of GM-CSF (20 ng/ml; eBioscience, #14-8331-80) differentiation, and then incubated (or not) for 18 hours with conditioned medium (10% of the total volume) from WT or LATSl/2 dKO B16- OVA melanoma cells and pulsed with OVA protein (10 mg/ml) for the last 4 hours. The cells were washed and cultured with CFSE-labeled OT-I CD8 ⁺ T cells at 1 : 1 ratio for 3 days. OT-1 T cell proliferation was monitored by CFSE dilution using a flow cytometer and a division index was determined using FlowJo software (Treestar).

Cytokine ELISA

IFNy or IL-12 levels in culture sup ernat ants were determined by ELISA. For ex vivo IFNy production from lymph node cells, draining lymph nodes (inguinal lymph nodes) were isolated from C57BL/6 mice challenged (or not) with B l 6-0 VA cells and cultured with OVA protein (100 mg ml) for 3 day s. For IL-12 production from BMDCs, BMDCs were generated by 6 days of GM-CSF (20 ng/ml) differentiation and stimulated (or not) for 18 hours with conditioned medium (10% of the total volume) or EVs isolated from culture supernatants of equal numbers of WT or LATSl/2 dKO B16-OVA cells (EVs from 2 x 10 ⁵ cells were used to stimulate 1 x 10 ⁶ BMDCs). Both cultures were done in RPMI 1640 supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 mg/ml) under an atmosphere of 5% C0 ₂ at 37°C, and then aliquots of cell culture supernatants were used for cytokine ELISA. For cell conditioned medium preparation, B 16-0 VA cells were seeded on culture plates and incubated in DM EM supplemented with 10% FBS for 24 hours at 37°C to allow cell attachment. The cells were then washed with PBS, and culture medium was switched to DMEM without serum. After incubation for 48 hours, conditioned medium was collected and centrifuged at 2,000 g for 10 minutes at 4°C to remove cell debris. Theresulting supernatant was used for EV isolation, which is described in the "EV isolation and analysis" section.

IFNy concentrations were determined using Mouse IFN-gamma DuoSet ELISA (R&D Systems, #DY485-05) according to a manufacturer' s protocol. For IL-12 ELISA, half area 96-well plates were coated with capture antibody (Purified Rat Anti-Mouse IL-12 p40/p70; BD Biosciences, #551219) in PBS overnight at 4°C. Plates were washed and then blocked for 3 hours at room temperature with blocking buffer (1% BSA in PBS), followed by wash and incubation with culture supernatants overnight at 4°C. Plates were then washed and incubated with biotinylated detection antibody (Biotin Rat Anti-Mouse IL- 12 (p40/p70; BD Biosciences, #554476) in blocking buffer at room temperature for 1 hour, followed by wash and incubation with streptavidin-HRP conjugate for 20 minutes. Plates were washed and incubated with TMB substrate (KPL, #95059-286), and then read at 450 nm and 650 nm after stoppingthe

development with 1 M phosphoric acid. Concentrations were determined by comparison to a standard curve.

EV Isolation and Analysis

B16-OVA cells were seeded in 150 mm culture plate and incubated in DMEM supplemented with 10% FBS for 24 hours at 37°C to allow cell attachment. The cells were then washed with PBS twice, and culture medium was switched to 35 mL of DMEM without serum. After incubation for 48 hours, conditioned medium was collected and centrifuged at 2,000 g for 10 minutes at 4°C to thoroughly remove cell debris. The resulting supernatant was then filtered through a 0.22 mm PVDF filter (Millipore, #SLGV033RB) to remove cell debris and microvesicles (for the detergent treatment experiment, the resulting flow- through was treated with 1% Triton X-100 for 10 minutes at 4°C prior to the ultracentrifugation). The flow -through was transferred into ultracentrifuge tubes (BECKMAN COULTER, #344058) and then ultracentrifuged in a Beckman SW32Ti rotor at 30,000 rpmfor 90 minutes at 4°C. The resulting pellets were washed with 35 mL of PBS and then ultracentrifuged again at 30,000 rpm for 90 minutes at 4°C. The resulting EV pellets were re-suspended in PBS for experimental use. Protein concentrations of EVs were determined using Micro BCA Protein Assay Kit (Thermo, #23235). RNA in EVs was isolated using TRIzol reagents (Thermo, #15596026) according to the manufacturer's protocol and concentrations were determined using Agilent 2200 TapeStation (Agilent Technologies). Forribonuclease treatment, total RNA isolated from EVs was digested for 30 minutes at 37°C with 100 mg/ml RNase A (Thermo, #EN0531) in a buffer comprising 10 mM Tris-HCl (pH 7.5), 5 mM EDT A, 300 mM NaCl. RNA was then resolved in agarose gels in non-denaturing conditions.

Nanoparticle tracking analysis was performed using NanoSight NS300 system (Malvern Instruments, Ranch Cucamonga, CA, USA) on isolated EVs diluted 5,000-fold with PBS for analysis. LC-M S/M S

EV samples were resolved in SDS-PAGE and the gels were cut into three regions, and then digested with trypsin. Extracted peptides were analyzed using a C18 column and an EASY-nLC-1000 (Thermo Scientific) coupled to a hybrid quadrupole-orbitrap Q-Exactive mass spectrometer (Thermo Scientific). A data- dependent, top 50 method was utilized for analy sis. The resulting RAW files were analyzed withProteome Discoverer 1.4 and MASCOT. Results were filtered with 1% FDR at the protein level and exported to our in-house

FileMakerPro database iSPEC and analyzed with Align!, which calculated intensity based absolute quantification (iBAQ) values (Schwanha ^" usser et al, 2011) that were used for subsequent analysis. The ratio of the iBAQ values for WT and LATSl/2 dKO EVs (DKO/WT ratio) in the individual experiments was calculated and scored according to the following criteria: score 2, > 5-fold; score 1, 5- to 2-fold; score 0, 2- to 0.5-fold; corel, 0.5- to 0.2-fold; and score 2, < 0.2- fold. We then added each

scores from the individual experiments and set the threshold as a score of > 3 for the top 100 most significantly increased proteins in LATSl/2 dKO EVs. Gene Ontology (GO) analysis was done using the PANTHER program (Mi et al, 2013). Heatmap s were generated using Net Walker (Komurov et al., 2012). Quantification and Statistical Analv sis

Statistical Analy sis

Statistical analyses were performed using GraphP ad Prism 5 software (GraphPad Software, Inc, La Jolla, CA, USA). Statistical parameters and methods are reported in theFigures and theFigure Legends. A value of p < 0.05 was considered statistically significant. Epidemiological data are obtained using the PrognoScan database (Mizuno et al, 2009). Association of gene expression with the survival of patients was evaluated using log-rank test and a value of p < 0.05 was considered statistically significant.

Results

LATSl/2 Deletion Enhances Anchorage-Indep endent Growth In Vitro

To elucidate the role of the Hippo pathway in anti-tumor immunity, murine syngeneic tumor models of three different cancer types in three different host genetic backgrounds were used; B 16-0 VA melanoma (B 16F 10 melanoma expressing ovalbumin [OVA]) in C57BL/6 mice, SCC7 head and neck squamous cell carcinoma in C3H/HeOu mice, and 4T 1 breast cancer in B ALB/c mice. These syngeneic allograft models have been well characterized and extensively used to study reciprocal interactions between tumor cells and host anti-tumor immune responses (Dranoff, 2011; Lei et al., 2016). Deletion of LATS1/2 almost completely abolished YAP/TAZ regulation by the Hippo pathway, while deletion of other components had only a partial or minor effect on YAP/TAZ activity (Meng et al, 2015). Therefore, LATS1/2 was deleted in B 16-OVA melanoma cells using CRISPR (clustered regularly interspaced short palindromic rep eats)/Cas9 genome-editing technology (Ran et al., 2013).

Multipleindependent LATSl/2 double-knockout (dKO) clones were obtained as verified by the lack of protein expression of both LATS1 and LATS2 (Figure 8 A). Two different clones generated by two independent CRISPR guide sequences were used for this study . Because YAP is a direct substrate of LATS1/2, of which phosphorylation can be readily detected with a phospho- YAP antibody or by mobility shift on a phos-tag gel, we use YAP

phosphorylation status as an indicator of LATS1/2 activity .Wefound

thatYAPphosphorylation levels were regulated in resp onse to LAT Sl/2- activating signals in wild-type (WT) B16-OVA cells; however, loss of LATS1/2 abolished YAP phosphorylation (Figure 8A). Phosphorylation of YAP/TAZ by LATS1/2 is known to promote YAP/TAZ cytoplasmic localization and inactivation (Zhao et al., 2007). Indeed, YAP/TAZ localized in the cytoplasmin resp onse to filamentous actin disruption (which activates LATS1/2) in WT B16- OVA cells, yet YAP/TAZ remains localized in the nucleus in LATS1/2 dKO cells under the same condition (Figure 8B). LATS1/2 inactivation or YAP/TAZ hyperactivation is known to promote cell growth (Zhao et al, 2008). Although LATS1/2 dKO B16-OVA cells showed identical growth on regular cell culture plates compared with WT cells (Figure 15A), LATSl/2 dKO B l 6-0 VA cells showed a significant increase in anchorage-independent growth in comparison to WT cells, in terms of both colony number and colony size (Figure 8C). These observations indicate that the Hippo pathway is still operational in B16-OVA melanoma cells and, in addition, that LAT Sl/2 deficiency activates YAP/TAZ and can further potentiate anchorage-independent growth of B16-OVA cells in vitro. LATS1/2 was also deleted in SCC7 squamous cell carcinoma cells and found that LATS 1/2 deficiency almost completely blocked YAP

phosphorylation (Figure 15B) and YAP/TAZ cytoplasmic localization (Figure 15C) in responseto LATSl/2-activating signals. Notably, WT SCC7 cells showed high YAP phosphorylation and cytoplasmic localization of YAP/TAZ, even in the absence of LATSl/2-activating signals, suggesting high basal LATS1/2 activity in these cancer cells. Loss of LATS1/2 again increased anchorage-independent growth of SCC7 cells (Figures 8D and 15D). LATSl/2- dependent regulation of YAP phosphorylation (Figure 15E), YAP/TAZ subcellular localization (Figure S1F), and anchorage-independent cell growth (Figures IE and S1G) were similarly observed in 4T 1 breast cancer cells.

Together, our data demonstrate that deletion of LATS1/2 in tumor cells promotes anchorage-independent tumor cell growth in vitro.

Loss of LATS1/2 Inhibits Tumor Growth In Vivo

To investigate the role of the Hippo pathway in tumor growth in vivo, equal numbers of WT or LATS1/2 dKO B16-OVA cells were subcutaneously transplanted into the back flanks of C57BL/6 mice and monitored their growth. Unexpectedly, deletion of LATS1/2 in B 16-OVA cells strongly inhibited tumor growth in vivo (Figures 9A and 9B). All mice died before day 22 in the WT B 16-OVA-injected group, whereas injection with LATS1/2 dKO B 16-OVA cells resulted in tumor-free survival in more than half of the mice (Figure 9C). The growth- suppressive effect of LATS1/2 deletion was confirmed with an independent clone of LATS1/2 dKO B16-OVA cells (Figure 16A). Next, we examined tumor growth of LATSl/2 dKO and WT SCC7 squamous cell carcinoma cells in syngeneic C3H/HeOu mice. All mice injected with the parental SCC7 cells showed aggressive tumor growth (Figures 9D and 16B), and 100% died before day 21 (Figure 9E). In contrast, none of the mice injected with LATS1/2 dKO SCC7 cells developed tumors, and all survived tumor free. In a 4T 1 orthotopic allograft model, 4T 1 breast cancer cells grow into solid tumors and can readily metastasize to the lung, liver, and brain when transplanted into the mammary fat pads of syngeneic BALB/c mice. Consistently, the parental 4T 1 cells developed tumors and metastasized to the lung in BALB/c mice (Figures 9F, 9G, and 16C). On the other hand, LATS1/2 dKO 4T 1 cells developed little tumors and had no metastasis when allografted in BALB/c mice. Thus, collectively, these observations indicate that loss of LATS1/2 in tumor cells dramatically inhibits tumor rowth in vivo in multiple types of cancer in different host backgrounds. Based on the current dogma, these results are totally unexpected, as LATS1/2 kinases supposedly function as tumor suppressors. LATS 1/2 Deletion Enhances Immunogenicitv of Tumor Cells

Since LATS1/2 deletion exerts completely opposite effects on tumor cell growth in vitro and in vivo (Figures 8-9), it was hypothesized that host factors may contribute to the apparent discrepancy between in vitro and in vivo p henoty pes of LATSl/2 dKO tumor cells. Therefore, we examined the histopathology of tumors from allografted mice. Massive infiltration of inflammatory cells in LATS1/2 dKO B16-OVA melanomas (Figure 10A), as well as in LATS1/2 dKO 4T 1 breast cancers (Figure 17A), was found, which were confirmed by staining with the pan-leukocyte marker CD45 (Figures 10B and 17B). These observations prompted the hypothesis that immune cells infiltrate, and thereby eliminate, LATS1/2 dKO tumor cells. Both innate and adaptive immune responses work together to constitute host anti-tumor immunity, but the adaptive immune system plays a pivotal role in mediating robust and highly specific immune responses against tumors (Gajewski et al., 2013). Therefore, host adaptive immune responses against tumor cells was examined. A B16-OVA melanoma model was used because B16-OVA melanoma cells express a non-secreted form of chicken OVA as a surrogate tumor antigen that can be conveniently used to follow immune responses directed against the OVA antigen. In addition, B16-OVA has been extensively used to study cancer immunity, and many genetically altered syngeneic mouse C57BL/6 strains are available.

Although WT and LATS1/2 dKO B 16-0 VA cells showed identical expression of OVA (Figure IOC), we detected significantly higher levels of serum anti-OVA antibody in mice injected with LATSl/2 dKO B 16-OVA cells (Figure 10D), suggesting an enhanced tumor-specific humoral immune response in the LATSl/2 dKO B16-OVA-injected mice. Next, cellular immune responses were examined. CD8 ⁺ T cells isolated from the spleens of LATS1/2 dKO B16- OVA -injected mice produced multiple effector cytokines (Figures 10E and 17C), indicative of T cell activation. Significantly higher CD8 ⁺ T cell crossp riming was observed when mice were injected withLATSl/2 dKO B 16-OVA cells (Figures 10F and 17D), and in addition, lymph node cells isolated from draining lymph nodes of LATSl/2 dKO B 16-OVAinjected mice showed a remarkably higher OVA-specific T cell response than did lymph node cells isolated from the parental B16-OVA-injected mice, as measured by interferon (IFN)g production (Figure 10G). These observations suggest that tumor specific cellular immune responses, particularly CD8 ⁺ T cell responses, are induced in mice injected with LATSl/2 Dko B 16-0 VA cells. Indeed, CD8 ⁺ T cells in LATSl/2 dKO B 16- OVA-injected mice possessed OVA-specific cytotoxic activity ex vivo (Figures 3H and S3E) and infiltrated tumors in vivo (Figures 101 and 17F). Together, the aforementioned data demonstrate that LATSl/2 deletion in tumor cells stimulate tumor-specific humoral and cellular immune responses, leading to the establishment of robust anti-tumor immunity .

LATSl/2 Deficiency Enhances Tumor Vaccine Efficacy via Adaptive Immunity Given that LATSl/2 deletion in tumor cells enhances host antitumor immune responses, it was hypothesizedthat LATSl/2-null tumor cells, by stimulating anti-tumor immunity, may protect the host from challenge with the corresponding LATSl/2 WT tumor cells. To test this, we performed two sets of experiments: co-injection of LATSl/2 dKO and WT tumor cells into each side of the same mouse (Figure 11A) or immunization of mice with LATSl/2 dKO tumor cells priortoLATSl/2 WT tumor cell injection (Figures 11B-C).

Strikingly, co-injection of LATSl/2 dKO B 16-0 VA cells significantly suppressed tumor growth of the co-injected WT B16-OVA cells (Figure 11A). Moreover, immunization of mice with irradiated LATSl/2 dKO B 16-0 VA cells, which were viable but unable to proliferate, strongly inhibited the corresponding LATSl/2 WT tumor growth, whereas immunization with irradiated parental B 16-OVA cells produced a much weaker effect (Figure 1 IB). Notably, in this experimental setting a single dose of tumor vaccination with irradiated WT B 16-OVA cells was not sufficient to extend survival (Figure 11C). In contrast, a single dose of tumor vaccination with irradiated LATSl/2 Dko B16-OVA cells showed a significant delay in tumor growth and prolonged survival.

Approximately 25% of themice immunized with irradiated LATSl/2 dKO B16- OVA cells were tumor free when challenged with WT B16-OVA cells. The aforementioned observations suggest that LATSl/2 deficiency renders B16- OVA cells highly immunogenic and improves tumor vaccine efficacy . Enhanced anti-tumor immunity was confirmed by LATSl/2 deletion with a different syngeneic model. C3H/HeOu mice having rejected LATSl/2 dKO SCC7 cells were resistant to rechallenge with the parental SCC7 cells (Figure 11D), indicating that these animals have established immunological memory against the given tumor cells.

Next, it was tested whether adaptive immunity is required for tumor suppression by LATSl/2 deletion. WT or LATSl/2 dKO B 16-OVA cells were subcutaneously transplanted into RAG- 1 (recombination activating gene 1) knockout (KO) mice that are immune-compromised due to the lack of mature T and B cells. LATSl/2 dKO B 16-OVA tumor cells grew similarly to WT cells

(Figure 1 IE) and showed comparable mortality (Figure 1 IF) in the absence of an adaptive immune system. Consistently, co-injection of LATSl/2 dKO B16-OVA cells failed to inhibit the corresponding LATSl/2 WT tumor growth in RAG-1 KO mice (Figure 11G). Based on the aforementioned data, LATSl/2 deletion in tumor cells enhances immunogenicity and provokes an adaptive immune response to eliminate tumor cells.

YAP or TAZ Overexpression in Tumor Cells Suppresses Tumor Growth In Vivo

YAP and TAZ are the most characterized downstream effectors of the Hippo pathway . LATSl/2 cells directly phosphorylate YAP/TAZ on multiple serine residues, leading to cytoplasmic retention, degradation, and thereby inactivation of YAP/TAZ. Because high YAP/TAZ activation was observed in LATSl/2 dKO B16-OVA tumors in vivo (Figures 18A-B), it was examined whether YAP/TAZ hyperactivation phenocopies the effect of LATSl/2 deletion in tumor growth. To this end, B16-OVA cells stably overexp res sing YAP(5SA) or TAZ (4SA) were generated. YAP(5 SA) and TAZ (4SA) are active mutants of YAP/TAZ, with the LATSl/2 phosphorylation sites mutated to alanine, thereby unresponsive to inhibition by LATSl/2. Notably, a mutual inhibition between YAP and TAZ protein abundance in YAP(5SA)- or TAZ(4SA)-overexpressing B 16-OVA cells (Figure 18C) was observed, consistent with the previously described negative-feedback response (Moroishi et al., 2015b). YAP(5SA)- or TAZ(18A)-overexpressingBl 6-0 VA cells showed increased anchorage- independent growth potential in comparison to the control cells in vitro (Figure 18D), while their tumor growth in vivo was significantly delayed (Figure 18E). Next it was investigated whether the effect of YAP(5SA) requires its transcriptional activity . YAP mainly binds to the TEA domain (TEAD) family of transcription factors (TEAD 1-4) to induce gene expression, and er94 in YAP is required for TEAD binding (Zhao et al, 2008). As expected, mutating Ser94 abolished the ability of YAP(5SA) to induce target gene transcription (Figures 18F-G). Imp ortantly , the TEAD-binding-defective YAP(5 SA/S94A) was unable to suppress B16-OVA tumor growth (Figure 18H), suggesting that tumor suppression by YAP requires TEAD-dependent transcription. Together, these observations indicate that hyperactivation ofYAP and TAZ significantly, though may not entirely, contributes to the in v/ ^' votumor growth suppression by

LATSl/2 deletion through a mechanism requiring TEAD-mediated transcription. Extracellular Vesicles Released from LATSl/2-Null Tumor Cells Stimulate Immune Responses

Next it was explored how LATSl/2 deficiency in tumors stimulates host anti-tumor immune responses. Because we observed a preeminent CD8 ⁺ T cell cross-priming in mice injected with LATSl/2 dKO B 16-OVA cells (Figure

10F), it was hypothesized that LATSl/2 dKO B16-OVA cells stimulate cross- presentation by antigen-presenting cells. To test this, the effects of LATSl/2 dKO B 16-0 VA cells on MHC (major histocompatibility complex) class I- restricted cross-presentation was examined using bone marrow-derived dendritic cells (BMDCs) as antigen-presenting cells. It was found that pre-treatment of BMDCs with conditioned medium from LATSl/2 dKO B16-OVA cells significantly augmented antigen cross-presentation in comparison to WT conditioned medium (Figure 12 A), and consistent with this, LATSl/2 dKO conditioned medium enhanced BMDC activation, as assessed by interleukin (IL)-12 production (Figure 12B). Theseresults imply that factors released from LATSl/2 dKO B16-OVA cells activate BMDCs and thereby enhance antigen cross-presentation. Recent studies have revealed the emerging roles of extracellular vesicles (EVs) in immune regulation, both in an

immunosuppressive and in an immunostimulatory manner (Robbins and Morelli, 2014). Therefore, it was investigated whether Evs secreted from LATSl/2 dKO B 16-OVA cells are capable of stimulating immune responses. EVs were isolated from culture sup ernatants of WT or LATSl/2 dKO B l 6-0 VA melanoma cells by ultracentrifugation (Figure 19A) and it wasfound that EVs from LATSl/2 dKO B 16-OVA cells were more p otent than EVs from WT B16-OVA cells in activating BMDCs, as assessed by IL-12 production in vitro (Figure 12B). To discriminate EVs from extracellular non-membranous particles that may be enriched by ultracentrifugation, we treated the culture sup ernatants with detergent (Triton X- 100) prior to EV purification. The detergent -treated EV pellets failed to activate BMDCs (Figure 19B). More importantly, LATSl/2 dKO EVs improved the tumor vaccine efficacy of irradiated WT B l 6-0 VA cells and conferred a strong immunity against tumor challenge in vivo (Figure 5C). Thus, these results show that EVs released from LATSl/2-deficient tumor cells induce immune responses and are sufficient to render LATSl/2-adequate tumor cells highly immunogenic.

LATSl/2-Deficient Tumor Cells Secrete Nucleic-Acid-Rich EVs

To elucidate the mechanistic basis for immunostimulatory effects of LATSl/2 dKO EVs, the nature of EVs released from WT or LATSl/2 dKO B 16-OVA cells was characterized. It was found that LATSl/2 dKO B16-OVA cells produced slightly more EVs compared with WT cells, as assessed by nanop article tracking analysis (Figures 12D and 19 A) as well as by protein quantification (Figure 12E). The proteome of EVs was analyzed using quantitative mass spectrometry . A total of 1,772 proteins were identified in EVs, which showed enrichment of previously reported exosomal and microvesicle cargo proteins (Figure 20B), sup porting the quality of EV purification. Most of the protein expression was almost identical between WT and LATSl/2 dKO EVs, but a subset of proteins were highly elevated in LATSl/2 dKO EVs (Figure 20C). Among the top increased proteins in LATSl/2 dKO EVs were those involved in RNA and nucleic acid binding (Figure 20D). These observations promptedus to hypothesizethat LATSl/2-null tumor EVs contain higher amounts of nucleic acids, which are previously reported contents of EVs (Yanez- Mo et al., 2015) and are also wellknown immunostimulators (Junt and Barchet, 2015). Because RNA is the most abundant nucleic acid in EVs (Robbins and Morelli, 2014), we characterized total RNA isolated from EVs. RNA contents in LATSl/2 dKO or YAP(5SA)-overexpressing EVs were dramatically increased in comparison to WT EVs (Figures 12F and 20E). The RNAs in EVs were sensitive to single-strand-specific ribonuclease treatment (Figure 20F). Taken together, our observations suggest a model in which LAT SI /2-deficient tumor cells secrete higher amounts of nucleic-acid-rich EVs that may contribute to the potent immunostimulatory effects.

EVs from LATSl/2 dKO Tumor Cells Stimulate the Toll-like Receptors-Tvpe l Interferon Pathway

To test the hypothesis that nucleic-acid-rich EVs released from

LATSl/2-null tumors stimulate host anti-tumor immunity, it was examined whether alterations in the host nucleic-acid-sensing pathway s imp air the tumor- protective effects of LATSl/2 deletion in vivo. Both microbial (non-self) and self nucleic acids can be recognized by distinct families of pattern recognition receptors, including endosomal Toll-like receptors (TLRs) and cytosolicnon- TLR sensors (Figure 21A). Activation of these pathways results in the production of inflammatory cytokines as well as type I IFN, which stimulates innate and adaptive immunity (Junt and Barchet, 2015). WT or LATSl/2 dKO B 16-OVA cells were subcutaneously transplanted into C57BL/6 mice deficient in the following key molecules in the endogenous nucleic-acid-sensing pathway s: M YD 88 (myeloid differentiation primary response 88) and TRIF (TIR-domain-containing adaptor-inducing interferon-b, also known as

TICAM l), two adaptorproteins required for TLR signaling; STING (stimulator of interferon genes, also known as TMEM 173), an adaptor protein required for the cGAS (cyclic GMP-AMP synthase, also known as MB21D 1) cytoplasmic DNA-sensing pathway; and caspase-1 (also known as CASP 1), an effector protein involved in IL-lb maturation under the AEVI2 (absent in melanoma 2) cytoplasmic DNA-sensing pathway . Wefound that deletion of MYD88 largely (Figures 13 A and 2 IB), and TRIF deficiency considerably (Figures 13B and 21C), attenuated the tumor-suppressive effects of LATSl/2 deletion, as assessed by tumor mortality . In contrast, deletion of STING (Figure 13C) or caspase-1 (Figure 13D) in recipient mice had no effect on tumor protection by LATSl/2 deficiency, suggesting that the TLRs-MYD88/TRIF nucleic acid sensing pathway, not the cytop lasmic DNA-sensing pathway , is required for

immunostimulatory effects of LATSl/2 deletion.

Distinct types of TLRs utilize MYD88 or TRIF as adaptor proteins and specifically respond to a wide range of ligands on the cell surface, as well as in the endosome (Figure 21 A). The endosomal TLRs are intrinsically capable of detecting nucleic acids. It was further investigated which TLR is required for tumor suppression by LATSl/21oss. Whereas LATSl/2 deletion in tumors still protected mice from tumor challenge in TLR4 (which senses bacterial lip op oly saccharides) KO mice (Figure 13E), deletion of TLR7 (which senses single-stranded RNA) (Figure 13F) or TLR9 (which senses double-stranded DNA) (Figure 13G) in recipient mice partially, but significantly, impaired tumor protection by LATSl/2 loss. Thus, our data suggest that multiple TLRs, and probably not a single TLR, cooperatively sense the nucleic acid-rich EVs secreted from LATSl/2-null tumors and trigger immune responses through the MYD88/TRIF signaling pathway .

Activation of TLRs-M YD88/TRIF signaling results in pro-inflammatory cytokine production as well as typel lFN (in particular, IFNa and IFNb) production, which stimulates anti-tumor immune responses (Figure S7A).

Particularly, type I IFN plays a central role in anti-tumor immunity by promoting dendritic cell maturation, antigen cross-presentation, and CD8 ⁺ T cell clonal expansion (Fuertes et al, 2013). Therefore, it was examined whether host type I IFN signaling is required for establishing host anti-tumor immunity induced by LATSl/2 dKO tumor cells. To test this, we subcutaneously transplanted WT or LATSl/2 dKO B16-OVA cells into IFNARl (interferon a and b receptor subunit l) KO mice that are deficient in a functional typel lFN receptor. We found that loss of host ty p e I IFN signaling largely obliterated the p rotective role of LATSl/2 deletion in tumor growth (Figure 13H) as well as tumor mortality (Figure 131). Thus, collectively, the data provide in vivo evidence supportinga model in which EVs secreted from LATSl/2-deficient tumor cells stimulate the host TLRs-MYD88/TRIF nucleic-acid-sensing pathways to incite typel lFN signaling and establish robust anti-tumor immunity .

Discussion

In this study, it was demonstrate that LATSl/2 deletion unmasks a malignant cell's immunogenic potential and restrains tumor growth due to the induction of anti-tumor immune responses. The effects of LATSl/2 deletion on tumor growth are striking insofar as LATSl/2 dKO completely abolishes the tumor growth potential of SCC7 and dramatically reduces tumor growth and the metastasis of B 16 and 4T 1 cells. LATSl/2-null B 16 melanomas secrete nucleic- acid-rich EVs that stimulate the host TLRs-MYD88/TRIF-IFN pathway sto induce anti-tumor immunity and the eventual elimination of tumor cells (Figure 7). LATS 1/2 deletion similarly stimulates host immune responses in both SCC7 and 4T 1 syngeneic models (Figures 11D, 17A, and 17B), though the

involvement of EVs has only been examined in theB 16 model in this study . Dual Functions of LATS1/2 in Cancer

It is generally accepted that the Hippo pathway is a tumor suppressor that inhibits proliferation and survival of normal cells, preventing tumorigenesis (Harvey et al, 2013; Moroishi et al, 2015a; Wang et al, 2014), yet a few studies did suggest an oncogenic role of the Hippo pathway in certain contexts (Barry et al., 2013; Cottini et al., 2014). We have analyzed human epidemiological data using the PrognoScan database (Mizuno et al, 2009) to find any correlation between LATS1/2 mRNA expression levels and patient outcome in different types of human cancer. Among 107 epidemiological datasets available, 26 studies show significant (p < 0.05) correlation between LATS2 mRNA levels and patient outcome, which includes 17 studies showing better patient survival with low LATS2 expression. Moreover, 12 studies show significant correlation between LATS1 mRNA levels and patient outcome, which includes 5 studies showing betterpatient survival with low LATS1 expression. In addition, low YAP expression predicted worse patient survival in human colorectal cancer (Barry et al, 2013) and multiple myeloma (Cottini et al, 2014). Therefore, although YAP/TAZ hyperactivation is frequently observed in human cancers (Harvey et al, 2013; Moroishi et al, 2015a), theprecise role of the Hippo pathway in human cancer might be context dependent. In this study , we show that deletion of LATS1/2 in tumor cells strongly suppresses tumor growth in vivo. On the surface, the present data cannot be easily reconciled with the tumor sup pressor model of LATS1/2 in the Hippo field.

The following model is proposed : LATS1/2 suppress tumor initiation as well as inhibit immunogeni city . These two activities are important for the physiological role of LATS1/2 in maintaining tissue homeostasis. LATS1/2 normally provide growth inhibitory signals to the cells; therefore, they function cell autonomously to limit tissue overgrowth. It is also proposed that LATS1/2 suppresses immunogenicity, serving as a built-in mechanism to prevent overgrowth of undesirable cells at the wrong places in the organism. For example, inactivation of LATS1/2 is needed to promote cell proliferation during wound healing and tissue regeneration. However, cells with imp aired LATS1/2 activity may over-proliferate and migrate to the wrong place. Such undesirable cells should be eliminated to maintain tissue homeostasis and integrity . This can be achieved because inactivation of LATS1/2 in these cells can induce a strong immune response. Therefore, the immunosuppressive function of LATS1/2 is consistent with its physiological roles in tissue homeostasis.

In the established tumor cell lines of B16, SCC7, and 4T 1, YAP and T AZ are not constitutively active. In fact, YAP and T AZ are readily regulated (in B16 and 4T 1 cells) or even largely inactive (in SCC7 cells). Therefore, the tumorigenicity of these cancer cell lines is independent of the Hipp o pathway . Nevertheless, deletion of LATS1/2 causes a moderate increase of anchorage- independent growth of these tumor cells in vitro, consistent with the growth inhibitory effect of LATS1/2. However, the enhanced immunogenicity unmasked by theLATSl/2 deletion in these cells can induce strong immune responses and overwhelm any growth advantage that might be gained due to LATS1/2 deletion, leading to strong inhibition of tumor growth in the immune- competent mice. The dual functions of L ATS 1/2 in sup pressing cell growth and immunogenicity can explain previous observations along with the present data. Hippo Pathway in Inflammation and Tumor Immunogenicity

The present results indicate that inactivation of the Hippo pathway in tumor cells induces host inflammatory responses. Interestingly, recent studies revealed that the Hippo pathway can respond to (Nowell et al, 2016; Taniguchi et al., 2015) and mediate (Liu et al., 2016) inflammatory signals. This study, together with these recent findings, suggests a reciprocal interaction between the Hippo pathway and inflammatory responses. LATSl/2-deficient tumor-derived EVs contain higher amounts of nucleic acids, which stimulate the host TLRs- MYD88/TRIF nucleic-acid sensing pathways, provokinga typel lFN response to establish robust anti-tumor immunity . Recent studies indicate that tumor cells themselves can produce type I IFN in responseto chemotherapy, thus enhancing anti-tumor immune responses (Chiappinelli et al., 2015; Sistigu et al., 2014). Because WT and LATS1/2 dKO B16-OVA cells showed similar expression levels of type I IFN genes such as Ifna4 and Ifnbl (Figure S7D), it is less likely that typel IFN secreted from LATSl/2-null tumor cells is the main mechanism conferring the anti-tumor immunity evoked by LATS1/2 deletion. Given that nucleic-acid-rich EVs from LATSl/2-deficient tumors can stimulate dendritic cells in vitro (Figure 12B), and that the host TLRs-MYD88/TRIF nucleic acid sensing pathways are required for immunostimulatory effects of LATSl/2 deletion in vivo (Figure 13), host immune cells in the tumor microenvironment may be the major source of type l lFN (Fuertes et al, 2013).

A series of unbiased Hippo pathway interact ome studies have linked endosomal compartments to the Hippo pathway (Moya and Haider, 2014). It is possible that the Hippo pathway may regulate endocytic trafficking and, therefore, regulate EV biogenesis. Little is known about the signaling

mechanisms involved in EV biogenesis and incorp oration of proteins or nucleic acids intoEVs. Given the known effect of YAP on global microRNA (miRNA) biogenesis (Mori et al, 2014) and the functional importance of miRNA in EVs (Yanez-Mo et al, 2015), one may speculate that the effect of YAP/TAZ on miRNA biogenesis may increase immunogenicity of LATSl/2-null cells.

However, TEAD mediated transcription is required for tumor suppression by YAP (Figure 18H), whereas TEAD -dependent transcription is dispensable for YAP-influenced miRNA biogenesis (Mori et al., 2014). Moreover, LATS 1/2 dKO cells do not increase miRNA contents in EVs (Figure 20E). Therefore, YAP/TAZ hyperactivation suppresses tumor growth in vivo via a transcription- dependent, but miRNA -biogenesis-independent, mechanism. The tumor growth suppression by YAP/TAZ overexpression (Figure 20E) is not as strong as that of LATS1/2 deletion (Figure 9A), suggesting that LATSl/2 may have additional targets to suppress immune responses. Recent studies revealed new LATS1/2 substrates in spindle orientation (Dewey et al, 2015; Keder et al, 2015).

Because aneuploidy play s a role in tumor immunogenicity (Senovilla et al, 2012), these new LATS1/2 substrates in spindle regulation could contributeto immunosuppression.

Targeting the Hippo Pathway for Cancer Immunotherapy

Recent advances in cancer immunotherapy have provided new therapeutic approaches for cancer, and several immune checkpoint inhibitors indeed show impressive effects in the clinic (Sharma and Allison, 2015).

However, individual immune resp onse to cancer immunotherapy often relies on tumor immunogenicity that varies extensively between different cancer types and different individuals; therefore, immune checkpoint inhibitors may not work in cases where tumor immunogenicity is intrinsically limited (Pico de Coana et al., 2015). Thepresent study revealed that inactivation of LATS1/2 in tumor cells increases tumor immunogenicity and enhances tumor vaccine efficacy . Therefore, it is speculated that inhibiting LATS 1/2 may enhance anti-tumor immune response and, therefore, would be an attractive approach to treat cancer. Furthermore, LATS1/2 inhibition to imp rove immunogenicity of tumor cells may enhance immune checkpoint inhibitor efficacy . Thus, a combination of LATS1/2 inhibitors and immune checkpoint inhibitors would be a novel and exciting therapeutic approach for poorly immunogenic cancers, especially in cases where malignancy is driven by oncogenic alterations that leave the Hippo signaling pathway intact. It is noteworthy that germline or somatic mutations affecting the core components of the Hippo pathway are uncommon in human cancers (Harvey et al., 2013; Moroishi et al., 2015a). Therefore, inhibition of LATS 1/2 may enhance tumor immunity in most cancer types. However, the caveat remains that the immune system of mice is considerably different from that of humans, and whether our findings in mice can directly be translated to humans remains to be determined. Moreover, the effect of LATS1/2 inhibition as an intervention for established tumors needs to be explored. Nevertheless, future studies expanding the therapeutic potentials of the Hippo pathway will have important clinical implications.

Example 3

Recent advances in cancer treatment have improved the survival and quality of life for patients, especially those who are in the early clinical stages (Steeg, 2016). However, prolonged survival is still unachievable in most patients with advanced cancer that have distal metastases. In particular, metastases of primary tumors contribute to 90% of patient deaths (Lambert et al., 2017). The lungs are the second most frequent site of metastases from extra-thoracic malignancies (Mohammed et al, 2011). Cancer in the lungs can create an immunosuppressive and angiogenic microenvironment (Kitamura et al., 2015; O strand-Rosenberg and Fenselau, 2018). Recent studies suggest that

immunotherapy with checkpoint inhibitors can overcome the immunosuppressive networks to prevent, and in some cases, to eradicate lung metastases (Sharma and Allison, 2015, Wolchok et al, 2013). However, checkpoint inhibitors achieve a progression-free survival in only 10-30% of patients with metastasis (Wolchok et al., 2017). Hence, there are strong demands for new immunotherapeutic approaches to improve survival rates of metastatic cancer patients.

A synthetic TLR7 agonist, imiquimod, has already been approved for human use and shows favorable clinical efficacy in patients with dermatologjcal tumors, including basal cell carcinoma and actinic keratosis (Geisse et al., 2004). However, the drug's applications are limited to topical use because of

immunotoxicity induced by systemic administration (Savage et al., 1996; Engel et al., 2011). To improve the pharmacokinetics and reduce severe immune adverse effects, our laboratory developed 1V270, a small molecule TLR7- specific ligand (1V136, SM360320) conjugated to a phospholipid moiety (Chan et al., 3009; Wu et al., 2014). Herein below it is demonstrated that intratumoral administration of 1V270 induces tumor-specific adaptive immune responses and inhibits primary tumor growth in murine syngeneic models of head and neck cancer and melanoma (Hayashi et al., 2011; Sato-Kaneko et al, 2017). The local 1V270 treatment activates tumor-associated macrophages and converts an immune-sup p res sive tumor microenvironment to a tumoricidal environment without causing systemic cytokine induction (Sato-Kaneko et al, 2017).

Subsequently, 1V270 therapy induced tumor-specific adaptive immune resp onses that sup pressed tumor growth in uninjected tumors. Reports by others have demonstrated that the use of local or sy stemic TLR 7 agonists, alone or as vaccine adjuvants, can induce tumor-specific immune responses and reduce the growth of colon, renal and mammary carcinomas (Wange et al, 2010; Koga- Yamakawa et al., 2013).

In contrast to the therapeutic advantages of using TLR 7 agonists on the innate immune cells in the tumor microenvironment, some recent reports indicate that TLR7 signaling pathway may promote tumor growth in primary lung carcinoma (Cherfils-Vicini et al., 2010; Chatterjee et al, 2014). This phenomenon is attributable to increased recruitment of rnyeloid-derived suppressor cells (MDSCs) to the tumor following TLR7 therapy (Chatterjee et al., 2014; Ochi et al., 2012). Thus, TLR7 therapy can be a double-edged sword depending on the type of tumor, the levels of receptor expression, and infiltration of suppressor cells in the tumor microenvironment (Dajon et al, 2015).

In this study, the effects of 1V270 therapy on metastatic lung tumors was evaluated. Since metastatic lung tumors are not readily accessible to intratumoral drug delivery, parenteral drug administration was analyzed. Therapeutic effects of 1V270 were evaluated in three murine syngeneic tumor models, 4T1 breast cancer, B 16 melanoma, and Lewis lung carcinoma (LLC). The results showed that a single systemic dose of 1V270 reduced tumor lung colonization in all three models tested. Systemic 1V270 therapy activated local innate immune cells, including natural killer (NK) cells and antigen presenting dendritic cells. T cell receptor (TCR) repertoire analyses revealed that 1V270 therapy induced tumor-specific oligoclonal cytotoxic T cells, that were shared by different mice, and that suppressed the growth of metastatic tumors. Anti-metastatic effects of 1 V270 were also achieved by intranasal drug administration. These document that both local and sy stemictherapy with a phospholip id-conjugated TLR 7 agonist can safely induce tumor-specific cytotoxic T cell responses in pulmonary metastatic cancer, that peripheral T cell repertoire analysis may be used to monitor the effects of therapy .

Observations support the concept that an immunomodulatory drug and

TCRrepertoire analysis can be applied for monitoring prognosis of metastatic lung cancer.

Materials and Methods

Animals, and reagents

Wild-type female BALB/c mice, C57BL/6 and BG-albino mice were purchased from Jackson Laboratory (Bar Harbor, MA). The studies involving animals were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. A small molecule TLR71igand (1V136, SM360320) and phospholipid- conjugated TLR7 agonist, 1V270, were synthesized in our laboratory as described previously and was formulated in 20% hydroxypropylbeta- cyclodextrin (Chan et al, 2009). Endotoxin levels of these drugs and other reagents were determined by Endosafe® (Charles River Laboratory,

Wilmington, MA) and were less than 15 EU/mg

Lung metastasis models

The 4T 1 (mouse breast cancer cell line), B16 melanoma cell line, and LLC cell line and BALB/3T3 fibroblast (Clone A31, CLL163) were obtained from American Type Culture Collection (Rockville, MD, USA). The cells were tested for murine pathogens and were confirmed negative prior to inoculation in mice. A GLF -expressing 4T 1, B16 and LLC cells were prepared as described previously (Godebu et al., 2014). Two metastatic 4T 1 models, spontaneous and IV metastasis models, were used in this study .

In vivo imaging study of lung tumor growth

GFP and luciferase (GLF)-expressing tumor cells (2 x 10 ⁴ of 4T 1-GLF, 5 x 10 ⁵ of B16-GLF, and 1 x 10 ⁶ of LLC-GLF) were i.v. injected into BALB/c mice for 4T 1 models, BG-albino or wild-type C57BU6forB 16 melanoma or LLC models). To generate bioluminescence signals, D-luciferin (3 mg/100 uL /mouse) was injected i.p . 12-15 minutes prior to the image acquisition. Image data were acquired by 1 Ss exposure using the IVIS Spectrum and analyzed using the Living Image software, version 4.5.2 (Perkin Elmer, Waltham, MA). We confirmed that the tumor signals in the lungs at day 10 correlated with the number of lung metastasis determined on day 21, as well as the overall survival (Supplemental figures 10).

Flow cytometric analysis

The cells were labeled by incubating with cocktails of antibodies at 4°C for 30 minutes (Table 1) to identify various cell types.

Table 1 Antibodies used in flow cytometry analysis

Antibody Color Cat# Clone Host botype Vendor

CD3 PE/Cy7 552774 145-2cll Armenian BD

Hamster IgGl, K Biosciences

CD4 APC 17-0042 RM4-5 Rat IgG2a, K eBioscience

CD8a e450 48-0081 53-6.7 Rat IgG2a, K eBioscience

CD l ib FITC 11-0112 Ml/70 Rat IgG2b, K eBioscience

CD l ib e450 48-0112 M1/7C Rat IgG2b, K eBioscience

CDl lc APC/Cy7 117324 N418 Armenian Biolegend

Hamster IgG

CD44 APC/Cy7 103028 IM7 Rat IgG2b, K Biolegend

CD45 PE/Cy7 103114 30-F11 Rat IgG2b, K Biolegend

CD49b PE 553858 DX5 Rat Lewis IgM, K BD

Biosciences

CD62L FITC 11-0621 MEL-14 Rat IgG2a, K eBioscience

CD80 FITC 104706 16-10A1 ArmenianHamster Biolegend

IgG

CD86 PE 12-0862 GL1 Rat IgG2a, K eBioscience D279(PD-1) PE 135205 29F.1A12 Rat IgG2a, K Biolegend CD335( KP40) FITC 560756 29A1.4 Rat IgG2a, K BD

Biosciences

F4/80 FITC 11-4801 BM8 Rat IgG2a, K eBioscience LY6C Pacific 128014 HK1.4 Rat IgG2c,K Biolegend blue

LG6G APC 127614 1A8 Rat IgG2a, K Biolegend MHC Class2(l- APC 17-5323 AMS-32.1 Mouse IgG2b, K eBioscience ad )

Granzyme B FITC 515403 GB 11 Mouse IgGl, K BD

Biosciences

IFNr APC 17-7311 XMG1.2 Rat IgGl, K eBioscience

NK cells and CD 8 ⁺ cell depletion in vivo

Anti-asialo GM 1 rabbit poly clonal antibody (Waka, Richmond, VA) or rabbit lgG polyclonal antibody (Millipore, Temecula, CA) was used for NK cell depletion. Mouse anti-CD S (clone 2.43), and isotype control Ab (clone LFA-2) were used for CD8 ⁺ cell depletion. We confirmed over 90 % depletion of NK cells and CD8 ⁺ cells using flow cytometry (Figures 28 and 33).

Ex vivo tumor-specific cytotoxicity study

Tumor-specific cytotoxicity was examined using 4T 1 cells as target cells and BALB/3T3 cells as irrelevant cells. BALB/c mice were treated with 1 V270 (200 on day -1 and 4T 1 cells were inoculated on day 0. Three 4T 1 cell lysate and 10 units/mL of IL-2 for 3 days. 4T 1 and BALB/3T3 cells were labled with 2.5 uM and 0.25 uM CFSE, respectively, for 12 minutes at 37°C and were mixed at 1 :1 ratio. Splenocytes cultured for 3 days were then cocultured with 4T l and BALB/3T3 cells at 16:1 to 2: l effector to target cell ratio (E:T) for 16 hours. The frequencies of 4T 1 (CFSE high) and BALB/3T3 (CFSE low) cells were determined by flow cytometry, and the percent specific killing was calculated. Specific killing (%) = [l-"Sample ratio'V'Negative control ratio") x 100; "Sample ratio" = [4T 1 (target)/ BALB/3T3 (irrelevant)) value of each sample co-cultured with CD8 ⁺ T cells; "Negative control ratio" = 14T 1 (target)/ BALB/3T3 (irrelevant)) value cultured without CD8 ⁺ T cells.

TCR repertoire analysis

CD8 ⁺ T cells were isolated from single cell suspensions of tumors, or spleens using mouse CD8 ⁺ T cell isolation kit (Milteny i Biotec). Total RNA was extracted from CD8 ⁺ T cells withRNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Next-generation sequencing was performed with an unbiased TCR repertoire analysis technology (Repertoire Genesis Inc., Osaka, Japan) as described previously (Sato-Kaneko et al., 2017). Animal models and ethic statement

In the spontaneous metastasis model, othotopically implanted tumor cells spontaneously form lung metastases within three weeks after implantation. 5x10 ⁵ 4T1 cells were inoculated in both sides of the 4th mammary pads of female BALB/c mice. Tumor length and width were recorded, and tumor volumes were calculated using the formula: volume (mm ³ ) = (width) ² x length/2. On day 28, mice were euthanized and the lungs were stained with intratracheally injected India ink and destained in Fekete's solution to count tumor nodules. In the experimental metastasis model, 2 x 10 ⁴ 4T1 cells were i.v. injected. On day 21, mice were euthanized and tumor nodules in lungs were counted as described above. In the secondary challenge experiment, 1V270 treated- mice without tumor signals in the lungs on day 21 of the experimental metastasis protocol were included. 5x 10 ⁵ 4T1 cells were inoculated in both sides of the 4th mammary pads. Tumor length and width were recorded, and tumor volumes were calculated similar with

spontaneous metastasis model.

Histologic analysis

Lungs were fixed with 10% formalin for overnight, dehydrated and embedded in paraffin on the Excelsior ES tissue processor (Thermo Scientific) and sectioned at 5 um thickness on a rotary microtome. Antigen retrieval was performed in Citrate buffer, pH 6.0, heated to 98°C for 8 minutes and cooled at room temperature for 20 minutes. The sections were stained on a Lab Vision 360 automated immunostaining instrument (Thermo Scientific) using a 2 step immunoperoxidase protocol. Briefly, the slides were blocked for endogenous peroxidase activity, washed and incubated with protein blocking buffer, 5% normal donkey serum in TBS for 10 minutes. The primary antibodies, both rabbit polyclonal, CD45 (abl0558, AbCam, used at 1 ug/ml diluted in 5% NDS) and CD3 (ab 16669 from AbCam at a 1 : 100 dilution) were incubated for 1 hour at room temperature. After washing in TBS-tween, the slides were incubated with the secondary antibody - HRP -Donkey F(ab')2 anti rabbit (Jackson ImmunoResearch Laboratories diluted 1 :200) for 30 minutes at room temperature, washed and reacted with DAB as the brown color substrate, hematoxylin was used the counterstain. Antibody details are shown in Table 2. Images were acquired using Axio Imager Zeiss microscope (Zeiss, Thornwood, NY).

Table 2. Antibodies used for immunofluorescent staining

Primary Cat# Host botype Vendor antibody

CD45 abl0558 rabbit, polyclonal abeam

CD 3 ab 16669 rabbit, polyclonal abeam

S econdary Cat# Source Vendor antibody

HRP-F(ab')2 711-036-152 Donkey, Jackson anti rabbit polyclonal ImmunoRes earch

Laboratories

Cytokine analysis

The sera were collected from mice receiving 200 nmol of 1V136, 1V270 or vehicle 2, 4, 6, and 24 hours after i.p . administration (15). Cytokine levels in sera were determined by Luminex bead assay s (MILLIP LEX™ MAP kit,

Millip ore, Bill erica, MA).

Flow cytometric analysis

To make the single cell suspensions, tumors were dissociated using the mouse tumor dissociation kit (Miltenyi Biotec, San Diego, CA) and the gentleMACS Octo Dissociator according to the manufacture's protocol. Single cell suspensions of spleens, lungs and mLN were prepared in Hank's Balanced

Salt Solution (HBSS) supplemented with 20 μ^ιηΕ DNasel (Worthington,

Lakewood, NJ) and 0.6 mg/mL collagensse type I (Worthington). Total cell number was counted by the ViaCount assay (Millip oreSigma, Darmstadt,

Germany). Dead cells were excluded by propidium iodide staining.

NK cells or CD8 ⁺ cell depletion in vivo

For NK cell depletion, 50 [iL of anti-asialo GM1 rabbit polyclonal antibody (Wako, Richmond, VA) or rabbit IgG polyclonal antibody

(Millip ore, Temecula, CA) was injected on day s -1, 1, 5, 9, 13, and 17. Mouse anti-CDS (clone2.43) and isotyp e control Ab (clone LFA-2) were purchased from BioXcell (West Lebanon, NH). Anti-CDS and isotype control (200

were i.p administered on days 5, 8, 11, 14, 16,19, and 23 to mice. Statistical analysis

Means and standard errors of means (SEM) are shown in other analyses.

In dot plots, each dot represents a tumor, a spleen, or a lymph node from an individual mouse and the horizontal and vertical bars indicate mean and mean ± SEM . M ann-Whitney test was used to comp are two group s. Using tumor volumes collected over all time points, two-way repeated measures ANOVA was used to comp are different group s, with p air-wise contrasts made at the final time point using a Bonferroni post hoctest. To compare cross-sectional outcomes among more than two groups, Kruskal-Wallis tests with Dunn's post hoc test were applied. Correlations between tumor volumes and TCRreportire analy sis data were analyzed using a Pearson's correlation test, pooling data across the different treatment group s. Analysis of covariance (sometimes on the log scale) was used to test whether the correlation was mediated by differences among the treatment group s in both mean immune marker level and tumor volumes, p < 0.05 were considered statistically significant. Prism 6 (GraphPad Software, San Diego, C A) statistical software was used to carry out these analy ses.

Results

Systemic administration of IV270 inhibits spontaneous lung metastasis in a CD8 ⁺ dependent manner in a murine 4T1 orthotopic breast cancer model

1V270 induces tumor-specific adaptive immune resp onses when administered intratumorally (Hayashi et al., 2011; Sato-Kaneko et al., 2017). However, recent reports claim that TLR7 activation in the lung can promote primary tumor growth (Cherfils-Vicini et al., 2010; Chatterjee et al., 2014). Thus, it was examined whether sy stemic 1 V270 therapy would also promote tumor-specific adaptive T cell responses and if such responses could restrain pulmonary metastatic disease. For this purpose, the murine 4T 1 breast cancer model that exhibits characteristics similar to the human disease, in which orthotopically implanted tumor cells spontaneously metastasize to the lungs (Pulaski and Ostrand-Rosenburg, 2001), was employed. 4T 1 cells (5 x 10 ⁵ ) were inoculated into the 4th mammary pads on day 0, and 1V270 (20, 80, or 200 injection) was injected intraperitoneally (i.p .) twice a week for three weeks, starting on day 7 (Figure 22A). Both the primary tumor volumes and the numbers of lung metastases were evaluated on day 27. Systemic 1V270 therapy decreased the number of lung metastases (Figure 22B). However, the systemic administration did not retard tumor growth at the primary sites of implantation, suggesting that rapid tumor growth could overcome immune restraints (Figure 28).

To study the possible involvement of cytotoxic T cell immune responses in the anti-metastatic effects of 1V270, CD8 ⁺ cells were depleted with monoclonal antibody (mAbs) prior to treatment with the TLR agonist (Figures 22C and 28B). In both the 1 V270-treated and the vehicle-treated control mice, the numbers of lung nodules increased significantly (p<0.005) after CD8 ⁺ cell depletion did not alter the brisk tumor growth at the primary sites in the mammary glands (Figure 28C).

Systemic administration of JV270 induces tumor-specific CD8 ⁺ T cells in an intravenous metastatic model of 4T1 breast cancer

Intravenous (IV) lung metastasis models have been used to evaluate in more detail the immune responses to circulating tumor cells induced by 1V270 therapy . Each animal received 2 x 10 ⁴ 4T 1 cells directly in the tail vein on day 0, and the numbers of lung nodules were counted on day 21 (Figure 23 A). Similar to the results after orthotopictumorinoculation, 1V270 inhibited lung metastasis in a dose-dependent manner, with the lowest effective dose being 20 μg (Figure 23B). A subsequent study indicated that a single administration of 1 V270 was sufficient to inhibit lung metastasis and that 1V270 had to be given 1 day prior to the tumor inoculation, which is important for maximal drug effects in this quickly developing lung metastasis model (Figure 23C).

To examine the role of CD8 ⁺ T cells after systemic 1 V270 treatment, mediastinal lymph node (mLN) cells, splenocytes, and lung tissues were analyzed in the IV metastasis model on day 21 (Figure 23D-2G). Activation of CD8 ⁺ T cells was assessed by intracellular staining for granzyme B and interferon γ (IFNy). Significantly higher percentages of granzyme Band IFNy positive CD8 ⁺ T cells were detected in the 1 V270-treated mice (p 0.05, Figure 2D and 2E). Histological analysis revealed a higher infiltration of CD45 ⁺ CD3 ⁺ cells in the pulmonary tumor microenvironmcnt of 1 V270-treated mice in comparison to those of vehicle-treated mice (Figure 23F). The tumor-specific effector function of the CD8 ⁺ cells was further evaluated by ex vivo cytotoxic T cell assays (Figure 23G). Briefly, splenic CD8 ⁺ T cells from lV270-treated or vehicle-treated mice were cultured with antigen (4T 1 tumor cell ly sates) and IL- 2, and then were incubated with carboxy fluorescein succinimidyl ester (CFSE)- labeled tumor cells. CD8 ⁺ T cells from 1 V270-treated mice showed significantly higher tumor-specific cytotoxicity at an effector to target cell ratio of 16:1 (p<0.05, Figure 23G). Theseresults demonstrated that a single administration of systemic 1V270 therapy can inhibit lung colonization by tumors in a CD8 ⁺ cell- dependent manner and that the administration of 1 V270 promotes adaptive CD8 ⁺ immune responses against tumor cells.

Tumor-infiltrating T cells in IV270 treated mice show high clonalities and intra- and inter-individual commonality by TCR repertoire analysis

Increased clonality of CD8 ⁺ T cells has been associated with hoth a positive clinical outcome and immune-related adverse events after immune checkpoint therapy (Ikedaet al., 2017; Dubudhi et al, 2016). Other studies have also indicated that clonal expansion of tumor-specific T cells is a biomarker for suppression of tumor growth (Straten et al., 1998; Kim et al., 2004). Intra- tumoral treatment with 1V270 induces local expansion an systemic dispersion of oligoclonal tumor-specific T cells by TCR repertoire analysis using next generation RNAseq methodology (Sato-Kaneko et al, 2017). Thus, it was important to determine whether systemic 1 V270 therapy also induced oligoclonal expansion of tumor-specific T cells.

To validate that 1V270 therapy induced tumor-specific adaptive immune responses, we monitored the growth of secondarily challenged tumors following prior 1V270 treatment. The mice treated with 1V270 using the IV metastasis protocol were orthot op ically re-challenged with 4T l cells on day 21 (Figure 24 A). We compared the growth of re-challenged tumors between 1 V270-treated mice that were not exposed to the tumor (no-tumor exposed), and 1 V270-treated and tumor-exposed mice (Figure 24A). Naive mice were also orthotop ically injected with tumor cells as a positive control. The tumor growth was significantly impaired in- the mice treated with 1V270 and exposed to the tumor cells compared to the naive mice or 1V270 treated no-tumor exposed mice (p<0.01, Figure 24B). As expected, there was no difference in the tumor growth between naive mice and 1V270 treated no-tumor exposure mice (Figure 24B). To examine whether tumor-specific T cells were recruited into the

microenvironment of the secondary challenged tumor, CD 8 ⁺ cells in the tumor infiltrating lymphocytes (TILs) were analyzed by flow cytometry (Figures 24C). Three-fold higher numbers of CD8 ⁺ T cells were detected in the TILs from the 1 V270-treated and tumor-exposed mice, compared to the 1 V270-treated and no- tumor exposed mice (p<0.05, Figure 24C). In this experiment, more than 80% of CD8 ⁺ T cells in TILs were positive for PD-1 (Figure 24C), indicating that they were exposed to antigen (tumor cells) and activated (Fernandez -Poma et al, 3027; Yoshida et al., 2000).

To examine clonal specificity of tumor-specific T cells, CD8 ⁺ cells were isolated from the spleens and the TILs of secondarily challenged tumors after initial 1V270 therapy . The TCR repertoires were assessed by next generation RNA sequencing of both TCRa and TCR β genes as previously described

(Yoshida et al, 2000). The clonality indices of CD8 ⁺ T cells in TILs, as assessed by I- Shannon index, were negatively correlated with the volumes of the secondarily challenged tumors only in the mice treated with 1V270 and exposed to tumor cells (Pearson's correlation coefficient, r ² = 0.97, P = 0.015, Figures 24D and 30A). When the frequencies of commonly shared TCR clones between TILs and splenocytes were evaluated using a binary similarity measure (Baroni- Urbani and Buser overlap index, BUB index) (Zhang et al, 2017), the 1V270- treated and tumor-exposed mice showed significantly higher BUB indices than the control non-tumor exposed mice (p<0.05, Figure 24E). The clonotypes in individual tumor-exposed mice showed a higher similarity in the 1V270 treated group in comparison to thenon-tumor exposed group (Figure 24F and 3G). Indeed, 31 TCRa and 11 TCR clonotypes were shared by 3 or more individuals in the tumor-exposed group, but only 5 TCRa clonotypes were shared in the non-tumor group (Figure 30B). The frequency of shared clones was significantly higher in tumor-exposed mice than in non-tumor exposed mice (Figure 30C). These experiments showed that 1 V270 therapy increased clonality of CD8 ⁺ T cells and the frequency of intra- and inter-individual shared clones, which correlated with the growth inhibition of secondarily challenged tumors. Dendritic cells in the lungs and draining lymph nodes are activated, and CDS+ T cells are recruited to the draining lymph nodes following 1V270 therapy

Previously it was demonstrated that 1V270 activates antigen presenting cells (APCs) and promotes cross-presentation of antigen to CD8 ⁺ T cells (Goff et al., 2015). Since the 1V270 therapy induced a tumor- specific CD8 ⁺ T cell response in the 4T 1 model, we evaluated whether the therapy activated APCs in the lungs, and/or in the draining lymph nodes. BALB/c mice were i. p .

administered with 1V270 on day -1, and 4T1 cells were injected the next day, and the dendritic cell populations in the draining mediastinal LNs (mLNs) and the lungs were examined on day 7 after the tumor injection. In the 1 V270-treated mice, a population of CD1 lc ⁺ dendritic cells was increased in both mLNs and the lungs (p <0.01, Figures 25A and 3 IB). Furthermore, the frequencies of CD11 ⁺ cells expressing co-stimulatory molecules (CD80 or CD86) were also significantly increased in mLN at both sites (p<0.01, Figures 25B and 31C). The activation of the dendritic cells was accompanied by the recruitment of CD8 ⁺ T cells with memory and naive phenotypes (Figure 25C); central memory CD8 ⁺ T cells (CD8 ⁺ CD44 ^" CD62L ⁺ ), effector memory CD8 ⁺ T cells (CD8 ⁺ CD44 ^" CD62L ⁺ ), and naive CD8 ⁺ T cells (CD8 ⁺ CD44-CD62L ⁺ ) (p<0.05 and p<0.01, Figure 25C). These findings demonstrated that 1 V270 activated local APCs and promoted their maturation, leading to expanded CD8 ⁺ T cell subsets.

1 V270 treatment activates innate immunity and inhibits lung colonization by tumor cells in a NKc-cell dependent manner

In the IV metastasis model, the administration of 1V270 one day before IV injection of tumor cells was required to restrain lung colonization. Since adaptive immune responses require several days to develop, this observation indicated that one or more innate immune cell types in the lung mediated the early therapeutic effect. To enable the monitoring of the detailed kinetics of the colonization process of 4T1 cells expressing both green fluorescent protein (GFP) and luciferase (4T1-GLF) were prepared using lentivirus vectors (Godebu et al., 2014). Subsequently, tumor implantation and growth were monitored using an IVIS Spectrum ^® in vivo imaging system. In both vehicle- and 1V270- treated mice, tumor cells accumulated in the lungs quickly after the injection (at Oh, Figures 25D and 32B). Thereafter, the signals progressed in two phases; they declined almost to baseline within the first 24 hours (the early phase) and then increased after day 7 (the late phase,). The 1 V270 treatment significantly suppressed the tumor signals both in the early (p<0.01 on 3 hours and 6 hours) and in the late phases (p<0.05 on days 7, 10, and 14).

To identify the types and functions of innate immune cells recruited into the lung following 1 V270 administration, the mice were injected with 1 V270 on day -1, 4T1 cells were i.v. administered on day 0, and the bronchoalveolar cells were isolated on day 7 after the tumor injection. A single cell suspension of lung cells was stained for natural killer (NK) cells and myeloid suppressor cells (MDSC) and analyzed by tlow cytometry (Figures 33 A-C and Table 1).

Significantly increased populations of NK cells, and monocytic-M DSC were recruited to the lungs following 1V270 treatment, in comparison to the vehicle treatment (p<0.01, Figures 25E and 33D). On the other hand, similar populations of granulocytic (PMN)MDSCs accumulated in the lungs in the lV270-treated mice compared to those of the vehicle-treated animals (Figure 33D).

NK cells can be activated directly by TLR7 agonists, and indirectly by typel lFN which is secreted from accessory dendritic cells (Liu et al., 2007; Hart et al., 2005). To examine the role of NK cells in the early therapeutic efficacy of 1 V270, this cell type was depleted by treatment with anti-asialo GM 1 polyclonal antibody (Kasai et al., 1980). Over 90 % of NK cells were depleted by antibody injection on days -4, -1, 3 and 10 (Figures 34A-B). IVIS analysis showed that lung colonization by tumor cells in 1V270 treated NK cell-depleted mice during the early phase developed similar to that in vehicle treated mice (Figures 25F and 34C). These data indicate that NK cells played a therapeutic role in the early phase.

Intranasal administration of 1 V270 is also effective in preventing metastasis and in inducing anti-tumor immunity

It was demonstrated that intranasal (i.n.) administration of 1V270 activates nasal and lung APCs, without causing sy stemic cytokine release (Wu et al., 2014). Therefore, we examined whetheri.n. 1V270 treatment could impair tumor growth in the IV metastasis model (Figure 26A). 1V270 (20 or 200 μg/50 uLdose) was i.n, delivered on days -3, -1, 3, 7, and 10 relative to the 4Tltumor cell injection (day 0) (Figure 26A). Intranasally administrated 1V270 inhibited the number of lung nodules in a dose-dependent manner (Figure 26B). It was examined whether i.n. administration of 1V270 could induce tumor-specific adaptive immune responses similar to the effects of systemic administration. Mice were treated with i.n. 1 V270 (200 or 500 μ§/50 uL) and then received 4T1 cells by the i.v. route. The surviving mice on day 21 were orthotopically re-challenged with tumor cells. There-challenged tumor growth was significantly inhibited in the mice which were i.n. treated with 1V270 (Figure 26C). The tumors grew similarly in narve mice when the mice received 1V270 treatment intranasally without tumor cell injection (Figure 26C). A higher number of CD8 ⁺ T cells and PD-1 ⁺ CD8 ⁺ T cells were detected in TILs from i.n. 1 V270-trcatcd mice exposed to the tumor cells, in comparison to 1V270 treated without tumor exposure or nafve mice (Figure 26D). These findings indicate that i.n. delivery of 1V270 effectively inhibited lung metastasis by inducing tumor-specific adaptive immune responses, similar to systemic 1V270 therapy .

Anti-metastatic effects of JV270 were observed in murine syngeneic melanoma and lung carcinoma models

To evaluate whether the 1V270 therapy can be effective in other cancer types, we employed two additional murine syngeneic metastasis models; B16 melanoma and Lewis lung cancer (LLC). Luciferase and GFP expressing cells (B16-GLF and LLC-GLF) were prepared using a lentivirus vector for in vivo imaging analy sis. Mice received sy stemic 1V270 treatment on day -1, and then B 16-GLF and LLC-GLF cells were i.v. administered on day 0. In both metastasis models, 1V270 inhibited lung metastasis by day 14 (Figures 27A and 27E) and prolonged mouse survival (Figures 27B and 27F).

Discussion

In patients with an advanced stage of cancer, the development of metastasis is almost inevitable since the metastatic niches are seeded with tumor cells long before clinical presentation (Valasty an and Weinberg, 2011). The ability of immune-checkp oint inhibitors to reactivate tumor-specific cytotoxic T cells provided evidence that immunotherapy can overcome these limitations, at least in some patients. Thus, there is an unmet medical need for additional agents that can increase the frequency of cytotoxic T cells at metastatic sites. Because each immunotherapy type exploits a distinct biological mechanism, biomarkers that predict efficacy and adverse effects are required (Topalian et al, 2000). Systemic 1V270 treatment systemically induced cytotoxic tumor-specific CD8 ⁺ T cells, as assessed by bot in vitro tumor-specific cytotoxicity assays, and tumor re-challenge experiments. TCRrepertoire analyses of TILs in the secondarily challenged tumors indicated that 1 V270 therapy strongly increased T cell clonality . The levels of clonality negatively correlated with the tumor volumes of secondarily challenged tumors. Of interest, the clonal similarity between tumor infiltrating and splenic T cells was increased in 1 V270 treated and tumor-exposed animals. A recent paper demonstrated that antitumor immune cells proliferate in the secondary lymphoid organs, including draining LNs and spleen, and can be detected in the peripheral blood during tumor rejection

(Spitzer et al, 2017). These findings suggested that immune monitoring should be possible by analyzing the TCRrepertoire of peripheral T cells.

Theoretically, the TCRrepertoire might be diverse among individual tumor-bearing mice, even though they share the same genetic background (Venturi et al., 2008). In a chronic virus infection, patients develop common clones which innteract with highly immunodommant antigens (Cerundolo et al., 2016; Miyamaet al., 2017). In thepresent study, an eight -fold higher number of shared clones in TILs was identified in the 1 V270 treated and tumor-exposed group, compared to the no-tumor exposed group . As increased frequency of shared clones suggested that the systemic 1V270 treatment may skew the TCR repertoire toward tumor-specific clones, that may recognize the same tumor antigens.

When administrated locally, synthetic TLR7 and TLR9 agonists are potent immune adjuvants, that can induce Thl and cytotoxic T cell responses over a period of week (Sato-Kaneko et al., 2017; Cho et al., 2002). When given systemically, however, some TLR agonists can cause a cytokine release syndrome that could potentially enhance metastatic growth by stimulating either angjogenesis or the development of M 2 macrophages (Hageman et al, 2005; Sanmarco et al., 2017). Therefore, effective sy stemic TLR7 therapy must clearly demonstrate that CD8 resp onses are induced without toxicity to the host or adverse changes in the tumor mi croenvironment. Thepresent data demonstrated that some monocyte linage, myeloid derived suppressor cells (MDSCs) were recruited to the lung after 1V270 administration. Immature MDSCs have the ability to suppress anti-tumor T cell responses (Quail and Joyce, 2013). Thus, we were concerned that systemic 1V270 treatment may promote tumor growth. However, other innate immune cells, including NK cells and dendritic cells, were also recruited to the lung after 1V270 administration, as reported previously in other models using TLR ligands (Smits et al, 2008). The recruitment and activation of the NK cells impeded tumor lung colonization, indicating that the NK cells could overcome the suppressive function of MDSC recruited by 1V270 administration. Another concern in immunotherapy using TLR7 ligands is that stimulation of a tumor TLR7 pathway could promote growth and chemo-resistance in some primary tumors expressing this receptor (Chrfils-Vincini etal, 2010; Chatterjee et al., 2014). In our study, 4T1, B16, and LLC cells did not express TLR7 by quantitative RT-PCR (Supplemental figures 8). We therefore conclude that systemic and i.n. TLR7 treatment is an effective therapy forTLR7 negative tumors.

1V270 inhibited the growth of small subcutaneous tumors when locally (intratumorally) injected (Hayashi et al, 2011; Sato-Kaneko et al, 2017). In the 4T1 metastatic model, orthotopically implanted primary tumors in the mammary gland were advanced at the time of initiation of 1V270 treatment. The ability of the TLR7 phospholipid agonist to prevent early lung metastasis may be attributable both to the lower tumor burden and to NK recruitment and activation. The in vivo imaging studies in NK cell depleted mice confirmed the critical role of this cell type in constraining tumor colonization, thus allowing for the development of a specific CD 8 T cell response in the later phases of metastasis.

Intratracheal administration of a low molecular weight TLR7 agonist (SM 276001) was reported to suppress metastatic lung tumors (Koga-Yamakawa et al., 2013). It was demonstrated that a low molecular weight TLR 7 agonist (SM360320) (1V136) induced high levels of systemic proinflammatory cytokines following parenteral and i.n. administration to mice and that conjugation of a TLR7 ligand to a phospholipid moiety could markedly reduce in vivo cytokine release (Figure 36) (Chan et al., 2009). Intranasally

administered 1V270 induced local (lung) immune cell activation without inducing sy stemic cytokine release (Wu et al., 2014). These results prompted us to assess whether the drug might have an immunotherapeutic effect in pulmonary metastatic disease. Our data demonstrated that i.n. administration of 1V270 suppressed lung metastasis similar to parenteral inoculation (Figure 26), suggesting that i.n. delivery might be clinically attractive for this drug.

In summary, single systemic administration of a phospholipid conjugated TLR7 agonist inhibited lung metastasis in three different murine syngeneic models of human malignancy, 4T 1 breast cancer, B 16 melanoma and Lewis lung carcinoma models. The drug quickly activated NK cells in the lung, and later induced a cytotoxic T cell response. These two different mechanisms, NK cell- mediated and tumor-specific adaptive T cell responses, were responsible for the early and late phases of tumor growth inhibition. The anti-tumor effects were achieved without significant systemic release of inflammatory cytokines following systemic administration. Furthermore, 1V270 therapy induced oligoclonal CD8 T cell responses as determined by TCRrepertoire analyses of both spleen and mediastinal lymph nodes. The emergence of shared T cell clones correlated with the development of adaptive immunity against tumor cells. These results suggest that TCR repertoire analyses may be used to guide clinical trials of TLR and other immunotherapies in patients with metastatic cancer.

References

Barry et al, Nature.493 : 106 (2013).

Brahmer et al., N. End. J. Med.. 373 :123 (2015).

Camargo et al, Curr. Biol.. 17:2054 (2007).

Cerundolo et al, J. Imnmnol. Ref. 175:6123 (2016).

Chan et al, Hioconiua Chem.. 20: 1194 (2009).

Chatterjee et al, Cancer Res.. 74:5008 (2014).

Cherfils-Vicini et al., J. Clin. Invest.. 120: 1285 (2010).

Chiappinelli et al., Cell, 162:974 (2015).

Cho et al, J. Immunol.. 168:4907 (2002).

Cottini et al., Nat. Med.. 20:599 (2014).

Dajon et al., Oncoimmunolog . 4:c991 (2015)

Dewey et al., Curr. Biol. 25:2751 (2015).

Dong et al, Cell, 130: 1120 (2007).

Dranoff, Nat. Rev. Immunol.. 12:61 (2011).

Engel et al., Expert Rev. Clin. Pharmacol.. 4:275 (2011).

Fernandez -Poma et al, Cancer Res.. 77:3672 (2017). Fuertes et al., Trends Immunol.. 34:67 (2013).

Gajewski et al., Nat. Immunol.. 14:1014 (2013).

Geissc et al., J. Am. Acad. Dermatol. 50:722 (2004).

Godebu et al., Transl. Med.. 12:27.1 (2014).

Goff et al., J. Virol. 89:3221 (2015).

Hagemann et al, J. Immunol. 175:1197 (2005).

Hanahan and Weinberg, Cell 144:646 (2011).

Hansen et al, Trends Cell Biol. 25:499 (2015).

Hart et al, J. Immunol. 175:1636 (2005).

Harvey et al, Nat. Rev. Cancer. 13 :246 (2013).

Hayashi et al, Melanoma Reseach.. 2J_:66 (2011).

Ikeda et al, Oncol. Rep .. 37:2603 (2017).

Johnson and Haider, Nat. Rev. Drug Discov.. 13 :63 (2014).

Junt and Barchet, Nat. Rev. Immunol. 15:529 (2015).

Kasai et al, Eur. J. Immunol. 10: 175 (1980).

Keder et al, Curr. Biol. 25:2739 (2015).

Kim et al, Surgery. 136:295 (2004).

Kitamura et al, Nat. Rev. Immunol. 15:73 (2015).

Koga-Yamakawa et al., Int. J. Cancer. 132:580 (2013).

Komurov et al, BMC Genomics. 13:282 (2012).

Lambert et al, Cell 168:670 (2017).

Lei et al, Onco Targets Ther.. 9:545 (2016).

Liu et al, Blood. 109:4336 (2007).

Liu et al, Cell 164:406 (2016).

Men et al. Genes Dev.. 30: 1 (2016).

Meng et al, Nat. Commun.. 6:8357 (2015).

Mi et al. Nat. Protoc. 8: 1551 (2013).

Miyamaet al, Sci. Rep .. 7:3663 (2017).

Mizuno et al, BMC Med. Genomics. 2: 18 (2009).

Mohammed et al, J. Thorac. Imaging. 1:26 (201 1).

Mori et al, Cell, 156:893 (2014).

Moroishi et al. Genes Dev.. 29: 1271 (2015b).

Moroishi et al, Nat. Rev. Cancer. 15:73 (2015a).

Moy a and Haider, Cell Res.. 24: 137 (2014). Nowell et al., Nat. Cell Biol. 18: 168 (2016).

Ochi and Graffeo, J. Clin. Invest.. 122:4118 (2012).

Ostraml-Rosenberg and Fenselau, J. Immunol.. 200:422 (2018).

Pico de Coana et al, Trends Mol. Med.. 21 :482 (2015).

Pulaski et al., Curr. Protoc. Immunol. 20:20.2 (2001).

Quail and Jovce. Oncologist. 13 :859 (2008).

Ran et al, Nat. Protoc. 8:2281 (2013).

Robbins and Morelli, Nat. Rev. Immunol.. 14:195 (2014).

Sanmarco et al, Biochim Biophys. Acta - Mn Basis Dis.. 1863 :R57

Sato-Kaneko et al, Insight. 2 (2017).

Savage et al., Br. T. Cancer. 74: 1482 (1996).

Schwanhausser et al, Nature. 473 :337 (2011).

Senovilla et al, Science. 337: 1678 (2012).

Sharma and Allison, Cell, 161 :205 (2015).

Sharma and Allison, CeU, 161 :205 (2015).

Sistigu et al, Nat. Med.. 20: 1301 (2014).

Spitzer et al., Cell, 168:487 (2017).

Steeg, Nat. Rev. Cancer. 16:201 (2016).

Straten et al., Proc. Natl. Acad. Sci. U.S.A.. 95:8785 (1998).

Subudhi et al., Proc. Natl. Acad. Sci.. 2:11919.

Taniguchi et al, Nature. 519:57 (2015).

Topalian et al, Nat. Rev. Cancer. 16:275 (2016).

Valastyan and Weinberg, CeU, 147:275 (2011).

Venturi et al., Nat. Rev. Immunol.. 8:231 (2008).

Wang et al., Mol. Cancer Ther.. 9:1788 (2010).

Wang et al., Cancer Metastasis Rev.. 33 : 173 (2014).

Wolchok et al, N. Engl. J. Med.. 369:122 (2013).

Wolchok et al, N. Engl. J. Med.. 377:1345 (2017).

Wu et al., Innate Immun.. 6:315 (2014).

Yanez-Mo et al, J. Extracell. Vesicles 4:27066 (2015).

Yoshida et al., Immunogenetics. 52:35 (2000).

Yu et al, CeU, 163 :811 (2015).

Zhang et al., BMC Bioinformatics.. 18: 129 (2017). Zhao et al., Genes Dev.. 21 :2747 (2007).

Zhao et al, Genes Dev.. 22:1962 (2008).

Zhou et al, Cancer Cell. 16:425 (2009).

All publications, patents and patent applications are incorporated herein by reference. While in the foregoing specification, this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details herein may be varied considerably without departing from the basic principles of the invention.