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Supplementary Figure S1. Dasatinib-induced rapid lymphocyte mobilization and tumor growth rate correlation Supplementary Figure S2. Immunohistochemical staining of CD8+ T cells in the tumor tissues of A) vehicle treated control group and B) dasatinib treated group Supplementary Figure 3. Confirmation of CD4+, CD8+ T cells and NK cell depletion by FACS Supplementary Figure 4. Immunophenotypic analysis of B16.OVA tumor infiltrating leukocytes Supplementary Figure 5. Immunophenotypic analysis of 1956 tumor infiltrating leukocytes Supplementary Figure 6. Immunophenotypic analysis of MC38 tumor infiltrating leukocytes Supplementary Figure 7. Immunophenotypic analysis of 4T1 tumor infiltrating leukocytes Supplementary Table S1. A summary of anti-mouse antibodies used for CyTOF mass cytometry analysis Supplementary Table S2. Frequencies of intratumoral T cell subsets analysed by CyTOF.

Supplementary Materials and Methods

Supplementary Figure Legends

Supplementary Figure S1: Expression levels of intratumoral chemokines following adenovirus injections.

Breast cancer cell lines BT-474, SKBR3, JIMT-1, and MDA-MB-436 were infected with indicated viruses at increasing plaque-forming units per cell (pfu/cell). The pfu-titers were first determined for each virus in a standard tissue culture infectious dose 50 (TCID50) assay. Ad5/3-Î"24-tras mediated significantly higher cancer cell killing as compared to its oncolytic control virus Ad5/3-Î"24 (P

Supplementary Figure S5: Expression of immune checkpoint molecules negatively regulating T-cell function in tumors post-treatment.

To confirm that observed ADCC mediated by virus-produced trastuzumab (Fig. 3) requires immune cells, supernatants from mock or Ad5/3-Î"24-tras infected 293 cells were purified and concentrated (virus-free, antibody-containing), and then added on N87 HER2+ gastric cancer cells without human peripheral blood mononuclear cells, followed by measuring lactate dehydrogenase (LDH) release after 6 hours as in Figure 3. As expected when lacking the effect of immune cells, neither virus-produced trastuzumab, mock-treated supernatant, nor irrelevant IgG control antibody showed cell killing (LDH release) at the 6-hour timepoint. Of note, similarly treated HER2-positive N87 cells showed dose-dependent susceptibility to commercial and virus-produced trastuzumab after a longer, 6-day incubation (Supplementary Fig. S1). Maximum LDH Release reagent was used as a positive control.

Supplementary Figure S2: Treatment schedule and phenotypic characterization of OT-I splenocytes before and after CD8a+ enrichment.

3-4 week-old female nude/NMRI mice (Harlan Laboratories) were xenografted in both flanks with MDA-MB-436 triple-negative breast cancer cells (6Ã-106 cells/tumor). When tumors reached 5 mm in diameter, mice were randomized and treated intratumorally with 2Ã-109 VP of Ad5/3-Î"24 or Ad5/3-Î"24-tras, or mock-treated (PBS only) on indicated days, and tumor growth was monitored. Trastuzumab-coding Ad5/3-â^†24-tras virus treatment resulted in significant tumor-growth inhibition as compared mock (P

Dasatinib, a broad-range tyrosine kinase inhibitor, induces rapid mobilization of lymphocytes and clonal expansion of cytotoxic cells in leukemia patients. Here, we investigated whether dasatinib could induce beneficial immunomodulatory effects in solid tumor models. The effects on tumor growth and on the immune system were studied in four different syngeneic mouse models (B16.OVA melanoma, 1956 sarcoma, MC38 colon, and 4T1 breast carcinoma). Both peripheral blood (PB) and tumor samples were immunophenotyped during treatment. Although in vitro dasatinib displayed no direct cytotoxicity to B16 melanoma cells, a significant decrease in tumor growth was observed in dasatinib-treated mice compared with vehicle-treated group. Further, dasatinib-treated melanoma-bearing mice had an increased proportion of CD8+ T cells in PB, together with a higher amount of tumor-infiltrating CD8+ T cells. Dasatinib-mediated antitumor efficacy was abolished when CD4+ and CD8+ T cells were depleted with antibodies. Results were confirmed in sarcoma, colon, and breast cancer models, and in all cases mice treated daily with dasatinib had a significant decrease in tumor growth. Detailed immunophenotyping of tumor tissues with CyTOF indicated that dasatinib had reduced the number of intratumoral regulatory T cells in all tumor types. To conclude, dasatinib is able to slow down the tumor growth of various solid tumor models, which is associated with the favorable blood/tumor T-cell immunomodulation. The assessment of synergistic combinatorial therapies with other immunomodulatory drugs or targeted small-molecule oncokinase inhibitors is warranted in future clinical trials. Cancer Immunol Res; 5(2); 157–69. ©2017 AACR.

Despite the rapid progress in the development of novel adoptive T-cell therapies, the clinical benefits in treatment of established tumors have remained modest. Several immune evasion mechanisms hinder T-cell entry into tumors and their activity within the tumor. Of note, oncolytic adenoviruses are intrinsically immunogenic due to inherent pathogen-associated molecular patterns. Here, we studied the capacity of adenovirus to overcome resistance of chicken ovalbumin-expressing B16.OVA murine melanoma tumors to adoptive ovalbumin-specific CD8+ T-cell (OT-I) therapy. Following intraperitoneal transfer of polyclonally activated OT-I lymphocytes, control of tumor growth was superior in mice given intratumoral adenovirus compared with control mice, even in the absence of oncolytic virus replication. Preexisting antiviral immunity against serotype 5 did not hinder the therapeutic efficacy of the combination treatment. Intratumoral adenovirus injection was associated with an increase in proinflammatory cytokines, CD45+ leukocytes, CD8+ lymphocytes, and F4/80+ macrophages, suggesting enhanced tumor immunogenicity. The proinflammatory effects of adenovirus on the tumor microenvironment led to expression of costimulatory signals on CD11c+ antigen-presenting cells and subsequent activation of T cells, thus breaking the tumor-induced peripheral tolerance. An increased number of CD8+ T cells specific for endogenous tumor antigens TRP-2 and gp100 was detected in combination-treated mice, indicating epitope spreading. Moreover, the majority of virus/T-cell–treated mice rejected the challenge of parental B16.F10 tumors, suggesting that systemic antitumor immunity was induced. In summary, we provide proof-of-mechanism data on combining adoptive T-cell therapy and adenovirotherapy for the treatment of cancer. Cancer Immunol Res; 3(8); 915–25. ©2015 AACR.

Supplementary Figure S3: SPECT/CT imaging experiment to study biodistribution and trafficking of radiolabeled OT-I cells in B16.OVA bearing mice.

A) Breast cancer BT-474, SKBR3, JIMT-1, and MDA-MB-436 cells, B) ovarian cancer OVCAR3, SKOV3, CaOV3, and HEY cells, and C) gastric N87, MKN-28, MKN-45 and esophageal cancer OE19 cells were treated with commercial or virus-produced trastuzumab (N87 cell line) in escalating doses (left panels), or infected with indicated viruses at increasing virus particle per cell (VP/cell) titers (right panels). Three HER2-positive (HER2+) cancer cell lines, BT-474, N87 and OE19, showed dose-dependent susceptibility to anti-HER2 antibody treatment with IC50-value at 80.3 ng/mL, 196.3 ng/mL, and 239.1 ng/mL on day 6 post-treatment, respectively. Equivalent doses of virus-produced trastuzumab purified from supernatant of Ad5/3-Î"24-tras infected 293 cells resulted in comparable cytotoxicity of HER2-positive N87 gastric cancer (C, top left, dashed line: data from identical separate experiment). Trastuzumab-coding virus Ad5/3-Î"24-tras and the oncolytic control virus Ad5/3-Î"24 showed significant cytotoxicity in all tested cancer cell lines (right panels, A-C), while non-replicating control viruses lacked efficacy. Potency of Ad5/3-Î"24-tras virus in vitro was pronounced in cell lines expressing HER2 (comparable to oncolytic control virus in BT-474, SKBR3, OVCAR3 and N87), while HER2-negative MDA-MB-436 cells were also found equally susceptible to oncolytic viruses. Importantly, both oncolytic viruses were able to mediate total cytotoxicity at the relevant 100 VP/cell titer. Of note, because Ad5/3-Î"24 lacks a transgene and its genome is therefore smaller, the virus packages more efficiently leading to a lower VP/pfu ratio (24 versus 203 in Ad5/3-Î"24-tras). Therefore cytotoxicity was also tested according to functional titers, which rendered higher potency for the recombinant Ad5/3-Î"24-tras virus (see Supplementary Fig. S2). IC50, half-maximal inhibitory concentration.

Supplementary Figure S4: Immune cell subsets in the tumors following combination treatment.

3-4 week-old male nude/NMRI mice (Taconic) bearing HER2-expressing N87 gastric cancer xenografts were treated in a mechanism of action experiment as in figure 4, but with adjusted doses: 3Ã-108 VP/tumor or saline on days 0, 4, 8, and 15, while one saline-treated group also received 5.0 µg/g of systemic commercial trastuzumab intraperitoneally (tras). On day 20, tumors, draining lymph nodes (DLNs) and spleens were collected, homogenized and analyzed by flow cytometry with CD49b NK-cell marker. Overall absolute levels of NK-cells did not differ between groups in any compartment. However, when distribution of NK cells into draining lymph nodes (left panel) and tumors (right panel) over the spleens of the animals was assessed, the oncolytic trastuzumab-coding virus (OV-tras) showed the highest ratio of NK cells in both target tissues; In particular, NK-cells showed homing towards the draining lymph nodes where they have been reported to e.g. mediate DC-editing and polarize activated CD4+ T-cells into T-helper 1 phenotype (36). Ratio of NK cells between compartments was calculated by dividing the average NK-cell frequency in tumors/lymph nodes by the average of NK-cells in spleens, each per 5Ã-104 cells. n=3 mice per group. Mock, animals treated with intratumoral saline injections; DLN, draining lymph node; OV-tras, oncolytic adenovirus coding for trastuzumab; OV, oncolytic control virus; tras, systemic trastuzumab therapy.

PDF file - 100K, PBMCs were isolated from patients before and circa one month after treatment and pulsed with an adenovirus-derived peptide pool and analyzed with IFN-gamma ELISPOT.

Purpose: Multiple injections of oncolytic adenovirus could enhance immunologic response. In the first part of this article, the focus was on immunologic aspects. Sixty patients previously naïve to oncolytic virus and who had white blood cells available were treated. Thirty-nine of 60 were assessed after a single virus administration, whereas 21 of 60 received a “serial treatment” consisting of three injections within 10 weeks. In the second part, we focused on 115 patients treated with a granulocyte macrophage colony-stimulating factor (GM–CSF)–coding capsid chimeric adenovirus, CGTG-102.Results: Following serial treatment, both increase and decrease in antitumor T cells in blood were seen more frequently, findings which are compatible with induction of T-cell immunity and trafficking of T cells to tumors, respectively. Safety was good in both groups. In 115 patients treated with CGTG-102 (Ad5/3-D24-GMCSF), median overall survival was 111 days following single and 277 days after serial treatment in nonrandomized comparison. Switching the virus capsid for avoiding neutralizing antibodies in a serial treatment featuring three different viruses did not impact safety or efficacy. A correlation between antiviral and antitumor T cells was seen (P = 0.001), suggesting that viral oncolysis can result in epitope spreading and breaking of tumor-associated immunologic tolerance. Alternatively, some patients may be more susceptible to induction of T-cell immunity and/or trafficking.Conclusions: These results provide the first human data linking antiviral immunity with antitumor immunity, implying that oncolytic viruses could have an important role in cancer immunotherapy. Clin Cancer Res; 19(10); 2734–44. ©2013 AACR.

PDF file - 62K, Adverse reactions of all CGTG-102 patients.

PDF file - 67K, Adverse reactions in patients na�ve to oncolytic adenovirus.

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Purpose: Oncolytic adenoviruses are promising tools for cancer therapy. Although several clinical reports have indicated both safety and promising antitumor capabilities for these viruses, there are only a few examples of complete tumor eradication. Thus, the antitumor efficacy of oncolytic adenoviruses needs to be improved. One potentially useful approach is combination with radiotherapy.Experimental Design: To target systemically administered radioiodide to tumors, we created Ad5/3-Δ24-human sodium iodide symporter (hNIS), a Rb-p16 pathway selective infectivity enhanced oncolytic adenovirus encoding hNIS.Results: Ad5/3-Δ24-hNIS replication effectively killed prostate cancer cells in vitro and in vivo. Also, the virus-mediated radioiodide uptake into prostate cancer cells in vitro and into tumors in vivo. Furthermore, Ad5/3-Δ24-hNIS with radioiodide was significantly more effective than virus alone in mice with prostate cancer xenografts.Conclusions: These results suggest that oncolytic adenovirus-mediated targeted radiotherapy might be a potentially useful option for enhancing the efficacy or adenoviral virotherapy. (Clin Cancer Res 2009;15(17):5396–403)

Purpose: Twenty-one patients with cancer were treated with a single round of oncolytic adenovirus ICOVIR-7.Experimental Design: ICOVIR-7 features an RGD-4C modification of the fiber HI-loop of serotype 5 adenovirus for enhanced entry into tumor cells. Tumor selectivity is mediated by an insulator, a modified E2F promoter, and a Rb-binding site deletion of E1A, whereas replication is optimized with E2F binding hairpins and a Kozak sequence. ICOVIR-7 doses ranged from 2 × 1010 to 1 × 1012 viral particles. All patients had advanced and metastatic solid tumors refractory to standard therapies.Results: ICOVIR-7 treatment was well tolerated with mild to moderate fever, fatigue, elevated liver transaminases, chills, and hyponatremia. One patient had grade 3 anemia but no other serious side effects were seen. At baseline, 9 of 21 of patients had neutralizing antibody titers against the ICOVIR-7 capsid. Treatment resulted in neutralizing antibody titer induction within 4 weeks in 16 of 18 patients. No elevations of serum proinflammatory cytokine levels were detected. Viral genomes were detected in the circulation in 18 of 21 of patients after injection and 7 of 15 of the samples were positive 2 to 4 weeks later suggesting viral replication.Conclusions: Overall, objective evidence of antitumor activity was seen in 9 of 17 evaluable patients. In radiological analyses, 5 of 12 evaluable patients had stabilization or reduction in tumor size. These consisted of one partial response, two minor responses and two cases of stable disease, all occurring in patients who had progressive disease before treatment. In summary, ICOVIR-7 treatment is apparently safe, resulting in anticancer activity, and is therefore promising for further clinical testing. Clin Cancer Res; 16(11); 3035–43. ©2010 AACR.

PDF file - 889K, Individual anti-tumor (survivin) and anti-viral (Ad5) ELISPOT results in single (A) and serial (B) treated patients.

Supplementary Data from Targeted Radiotherapy for Prostate Cancer with an Oncolytic Adenovirus Coding for Human Sodium Iodide Symporter

PDF file - 71K, Characteristics of patients na�ve to oncolytic adenovirus.

We have recently reported that low tumor levels of SMAD4, a key mediator of transforming growth factor-β superfamily signaling, can predict the probability of recurrence in patients with Dukes C colorectal cancer who had surgery as the only form of treatment. However, standard treatment for Dukes C colorectal cancer patients currently involves the administration of 5-fluorouracil (5-FU)–based adjuvant chemotherapy after surgery. Approximately 30% to 40% of these patients present with recurrence and die within 5 years, and there is great need for markers capable of predicting poor prognosis after the combined surgery/adjuvant treatment. In this study, we evaluate the prognostic value of SMAD4 in patients treated with surgery and 5-FU-based adjuvant therapy. We used immunohistochemistry and quantitative real-time reverse transcription-PCR to measure the levels of SMAD4 protein and mRNA expression in the primary tumors and a number of lymph node metastases from a series of 75 Dukes C colorectal cancer patients with at least 6 years of follow-up. Patients with tumors expressing low levels of SMAD4 protein or mRNA showed significantly shorted disease-free and overall survival than patients with high tumor levels of SMAD4. The median survival of patients with low SMAD4 protein or mRNA tumor levels was 1.4 and 1.2 years, respectively, whereas patients with high SMAD4 tumor level had a median survival of >9.3 years. In addition, the protein and mRNA levels of SMAD4 in lymph node metastases was significantly lower than in primary tumors (P = 0.006). In contrast, allelic imbalance in chromosome 18q21 was of no prognostic significance in these patients. In conclusion, low SMAD4 tumor levels identified a subset of patients with poor prognosis following surgery and 5-FU-based adjuvant therapy; therefore, these patients could be good candidates to receive combined treatment with additional chemotherapeutic agents such as CPT-11 and/or oxaliplatin.

Supplementary Tables S1 & S2 and Figure S1 from SMAD4 Levels and Response to 5-Fluorouracil in Colorectal Cancer

PDF file - 94K, The phenotypic panel of circulating white blood cells.

PDF file - 95K, A panel of proinflammatory cytokines was analyzed to evaluate the safety of treatment.

Supplementary Data from Oncolytic Adenovirus ICOVIR-7 in Patients with Advanced and Refractory Solid Tumors

PDF file - 141K, Example of progressive inflammation in serial treatment.

Purpose: Transfer of prodrug activation systems into tumors by using replication-deficient viruses has been suggested to be an effective method for achieving high local and low systemic anticancer drug concentrations. However, most current suicide gene therapy strategies are still hindered by poor efficiency of in vivo gene transfer, inefficient tumor penetration, limited bystander cell killing effect, and need of large prodrug doses. We hypothesized that local amplification provided by a replication competent platform would help overcome these limitations.Experimental Design: We generated a transductionally and transcriptionally targeted oncolytic adenovirus Ad5/3-Δ24FCU1 expressing the fusion suicide gene FCU1. FCU1 encodes a bifunctional fusion protein that efficiently catalyzes the direct conversion of 5-FC, a relatively nontoxic antifungal agent, into the toxic metabolites 5-fluorouracil and 5-fluorouridine monophosphate, bypassing the natural resistance of certain human tumor cells to 5-fluorouracil.Results: We examined the efficacy of Ad5/3-Δ24FCU1 and the replication-defective control Ad5/3-FCU1 with and without 5-FC. FCU1 expression was confirmed by Western blot, whereas enzymatic conversion levels in vitro and in vivo were determined by high-performance liquid chromatography separation. Significant antitumor effect was observed in vitro and in vivo in a murine model of head and neck squamous cell carcinoma. Although we observed a decrease in viral DNA copy number in vitro and in tumors treated with Ad5/3-Δ24FCU1 and 5-FC, suggesting an effect on virus replication, the highest antitumor effect was observed for this combination.Conclusions: It seems feasible and efficacious to combine adenovirus replication to the FCU1 prodrug activation system. Clin Cancer Res; 16(9); 2540–9. ©2010 AACR.

Supplementary Data from Targeted Chemotherapy for Head and Neck Cancer with a Chimeric Oncolytic Adenovirus Coding for Bifunctional Suicide Protein FCU1

PDF file - 48K, Description of disease at the time of first treatment.

Supplementary Figures 1-6, Tables 1-3 from Oncolytic Adenovirus Coding for Granulocyte Macrophage Colony-Stimulating Factor Induces Antitumoral Immunity in Cancer Patients

PDF file - 1MB, PBMCs of a healthy donor was stimulated with supernatant collected from virus (CGTG-401 or control virus not coding CD40L) infected A549 lung cancer cells and IL-8 production was assessed 12, 24, 48, and 72 h later with FACSArray. The increase demonstrated at later time points indicates biological activity of the virus produced CD40L.

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PDF file - 128K, T-cell analyses in C57Bl/6 mice bearing B16-Ova tumors C57Bl/6 mice bearing B16-Ova tumors were injected intratumorally. Lymph nodes, spleens and tumors were collected 8 days after first virus injection, homogenized and cultured for 24 hours. Analysis of Ova-specific CD8+CD19- was performed by flow cytometry gating the CD19- population and plotting SIINFEKEL marker of Ova with CD8. Results are represented as percentage of double positive CD8 and SIINFEKEL peptide and plotted as mean +SEM. *, P

PDF file - 1.1MB, (A) Patient serum samples were analyzed for Th1 induced cytokines interferon-� (IFN-�), tumor necrosis factor-� (TNF-�) and interleukin-2 (IL-2) and Th2 cytokines interleukin-4 (IL-4), interleukin-5 (IL-5) and Interleukin 10 (IL-10). The ratio between Th1 and Th2 type cytokines was assessed. Cytokines levels are reported relative to their baseline level, which was set as 1 and a ratio between Th1/Th2 was calculated for each time point. (B) Malignant ascites of patient R8 and pleural effusion of patient T181 were analyzed for Th1 induced cytokines IFN-� and IL-2, and for Th2 induced IL-10 before and after treatment.

PDF file - 1.2MB, Blood samples for all 9 patients were collected before (baseline) and at several time points after the treatment. Soluble CD40L (sCD40L) and RANTES concentrations in the serum were assessed. Data are presented as median � SD.

Granulocyte macrophage colony-stimulating factor (GMCSF) can mediate antitumor effects by recruiting natural killer cells and by induction of tumor-specific cytotoxic T-cells through antigen-presenting cells. Oncolytic tumor cell–killing can produce a potent costimulatory danger signal and release of tumor epitopes for antigen-presenting cell sampling. Therefore, an oncolytic adenovirus coding for GMCSF was engineered and shown to induce tumor-specific immunity in an immunocompetent syngeneic hamster model. Subsequently, 20 patients with advanced solid tumors refractory to standard therapies were treated with Ad5-D24-GMCSF. Of the 16 radiologically evaluable patients, 2 had complete responses, 1 had a minor response, and 5 had disease stabilization. Responses were frequently seen in injected and noninjected tumors. Treatment was well tolerated and resulted in the induction of both tumor-specific and virus-specific immunity as measured by ELISPOT and pentamer analysis. This is the first time that oncolytic virus–mediated antitumor immunity has been shown in humans. Ad5-D24-GMCSF is promising for further clinical testing. Cancer Res; 70(11); 4297–309. ©2010 AACR.

PDF file - 64K, Adverse reactions. 9 patients total, 6 received concomitant low-lose cyclophosphamide 50 mg/d, 3 did not. Numbers of patients experiencing the reaction at any time during their treatment (including 28d follow-up) is shown. The data is shown for the two subgroups separately in parentheses (cyclophosphamide/none).

Supplementary Figures 1-17, Tables 1-2, Methods and Materials from Epithelial Phenotype Confers Resistance of Ovarian Cancer Cells to Oncolytic Adenoviruses

PDF file - 91K, Nude mice bearing MB49 tumors were injected with Ad5/3-CMV-mCD40L, Ad5/3-Luc1 or PBS (Mock) on days 0,2 and 4. Eight days after first virus injection mice were killed and spleens and tumors were collected, smashed and cultured for 24 hours. Homogenates were analyzed for NK+ cells (left side) and CD19+ (right side). Results are presented as mean from 6 mice + SEM. *, P

PDF file - 2.2MB, (A) Cell monolayers were infected with 100 viral paticles /cell (VP/cell) and 24 hours later cells were analyzed by flow-cytometry measuring Annexin-V expression. Results are presented as percentage of positive cells relative to Mock uninfected cells. **, P

PDF file - 41K, Response evaluation of injected and non-injected lesions.

PDF file - 75K, (A) MB49 cells were infected with 100 viral particles/cell with Ad5/3-CMV-mCD40L and Ad5/3-Luc1 and Annexin-V was assessed 24 hours post-infection by flow cytometry. (B) Replication of Ad5/3-CMV-mCD40L and Ad5/3-Luc1 over time in MB49 cell line. Cell monolayers were infected at doses specified ranging from 101 to 108 viral particles and assays were stopped at different time points (days 2, 3, 4 and 8). Cell viability was measured and expressed overtime. (C) MB49 cells were infected with 100 viral particles and samples were collected at 0, 24, 48 and 72 hours post infection. Quantitative PCR was performed for E4 sequence and values were normalized to mouse beta-actin gene.