Randomized dose-finding clinical trial of oncolytic immunotherapeutic vaccinia JX-594 in liver cancer
Jeong Heo1,17, Tony Reid2,17, Leyo Ruo3, Caroline J Breitbach4, Steven Rose2, Mark Bloomston5, Mong Cho1, Ho Yeong Lim6, Hyun Cheol Chung7, Chang Won Kim1, James Burke4, Riccardo Lencioni8, Theresa Hickman4, Anne Moon4, Yeon Sook Lee9, Mi Kyeong Kim9, Manijeh Daneshmand10, Kara Dubois4, Lara Longpre4, Minhtran Ngo11,12, Cliona Rooney11–13, John C Bell4,10, Byung-Geon Rhee14, Richard Patt15, Tae-Ho Hwang9,16,18 & David H Kirn4,18
© 2013 Nature America, Inc. All rights reserved. Oncolytic viruses and active immunotherapeutics have complementary mechanisms of action (MOA) that are both self amplifying in tumors, yet the impact of dose on subject outcome is unclear. JX-594 (Pexa-Vec) is an oncolytic and immunotherapeutic vaccinia virus. To determine the optimal JX-594 dose in subjects with advanced hepatocellular carcinoma (HCC), we conducted a randomized phase 2 dose-finding trial (n = 30). Radiologists infused low- or high-dose JX-594 into liver tumors (days 1, 15 and 29); infusions resulted in acute detectable intravascular JX-594 genomes. Objective intrahepatic Modified Response Evaluation Criteria in Solid Tumors (mRECIST) (15%) and Choi (62%) response rates and intrahepatic disease control (50%) were equivalent in injected and distant noninjected tumors at both doses. JX-594 replication and granulocyte-macrophage colony-stimulating factor (GM-CSF) expression preceded the induction of anticancer immunity. In contrast to tumor response rate and immune endpoints, subject survival duration was significantly related to dose (median survival of 14.1 months compared to 6.7 months on the high and low dose, respectively; hazard ratio 0.39; P = 0.020). JX-594 demonstrated oncolytic and immunotherapy MOA, tumor responses and dose-related survival in individuals with HCC. Despite advances in cancer treatment over the past 30 years with chemotherapy and biologics, the majority of solid tumors remain incurable once they are metastatic. Truly new agents with multiple complementary MOA are required to move beyond the modest benefits achieved so far. Extensive study in the field of active immunotherapy has recently culminated in regulatory approvals of sipuleucel-t (Provenge; Dendreon) and ipilimumab (Yervoy; BristolMyers Squibb). Although these agents comprise the first approvals for a new therapeutic class, their limited long-term benefit warrants development of more potent active immunotherapies. The oncolytic and immunotherapeutic herpes simplex virus T-Vec (Amgen), which expresses GM-CSF after local intratumoral injection, recently demonstrated anticancer immune induction and durable objective responses in an intratumoral phase 2 melanoma study1. Oncolytic immunotherapies are designed to selectively replicate within, and subsequently lyse, cancer cells2–5 while inducing tumorspecific immunity. JX-594 (also called Pexa-Vec; Jennerex Inc.) is a vaccinia virus (Wyeth vaccine strain) with disruption of the viral
1Department 2Moores

thymidine kinase gene (TK) for cancer selectivity and insertion of human granulocyte-macrophage colony-stimulating factor (hGMCSF) and β-galactosidase transgenes for immune stimulation and replication assessment, respectively6–8. JX-594 is designed to induce both virus replication–dependent oncolysis and tumor-specific immunity6,9,10. The advantages of a vaccinia virus include intravenous (i.v.) stability and delivery11, enhanced potency12, extensive safety experience as a live vaccine, demonstrated ability to induce efficient immune responses and a large transgene-encoding capacity6. The onco­lytic immunotherapies JX-594 (Pexa-Vec), T-VEC (Amgen)13 and an adenoviral construct expressing GM-CSF (Ad-GM-CSF)3 comprise a new platform that has advantages over the currently approved immunotherapeutics given their direct tumor lysis, induction of subject tumor-specific immunity and use as off-the-shelf products. In contrast to other agents in this class, JX-594 showed both complete responses of bulky tumors and systemic efficacy in phase 1 studies14,15. In a phase 1 clinical trial of intratumoral JX-594 (ref. 14), as can be expected with a self-amplifying agent, injection-site

of Internal Medicine, Pusan National University and Medical Research Institute, Pusan National University Hospital, Seo-Gu, Busan, South Korea. Cancer Center, University of California San Diego (UCSD), La Jolla, California, USA. 3Department of Surgery, McMaster University Medical Centre, Hamilton, Ontario, Canada. 4Jennerex Inc., San Francisco, California, USA. 5Department of Surgery, The Ohio State University Comprehensive Cancer Center, Columbus, Ohio, USA. 6Department of Medicine, Division of Hematology-Oncology, Samsung Medical Center, Ilwon-Dong, Gang Nam-Gu, Seoul, South Korea. 7Yonsei Cancer Center, Yonsei University College of Medicine, Seodaemun-gu, Yongdong Severance Hospital, Seoul, South Korea. 8Division of Diagnostic Imaging and Intervention, Pisa University School of Medicine, Lungarno Pacinotti, Pisa, Italy. 9SillaJen Inc., GeumJung-Gu, Busan, South Korea. 10Centre for Innovative Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada. 11Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas, USA. 12Baylor College of Medicine, Houston, Texas, USA. 13Texas Children’s Hospital, Houston, Texas, USA. 14Green Cross Corporation, Giheung-gu, Yongin, South Korea. 15RadMD, Doylestown, Pennsylvania, USA. 16Department of Pharmacology, Pusan National University, Busan, South Korea. 17These authors contributed equally to this work. 18These authors jointly directed this work. Correspondence should be addressed to D.H.K. ( or T.-H.H. ( Received 28 September 2012; accepted 14 January 2013; published online 10 February 2013; doi:10.1038/nm.3089

nature medicine   advance online publication 

induction of immunity to both cancer cells and vaccinia. Baseline subject characteristics and prognostic factors were well balanced between the two groups (Table 1).d.7 13 (81) 3 (19) 9 (56) 7 (44) 9 (56) 3 (19) 6 (37) 0 (0) 7 (44) 16 (100) responses were seen at all doses. All rights reserved. All subjects were evaluable for safety (as they all received at least one dose) and all but one (because of a major undocumented protocol deviation.d. bPatients may have received more than one type of therapy. including fever. None of the differences between the two study groups was significant (P ≥ 0. Treatment-related adverse events are summarized by grade and treatment arm in Supplementary Table 1.6 ± 3. this included two subjects who had clinical disease progression and died without a scan at week 8 (and were considered to have had progression).2–1. RESULTS Subject enrollment and baseline and treatment characteristics Between December 2008 and May 2011. phase 1 trial15.6 ± 3.     Median     Range   Baseline aPTT (s)     Mean ± s.8 ± 0. proof of active immunotherapy induction in subjects with these agents was lacking. In addition.7 10.8 13. Barcelona Clinic Liver Cancer staging system. including previous sorafenib treatment (all four subjects with previous sorafenib treatment in the high-dose group had tumor progression while on this therapy).2 32. aPTT. The frequency and grade (according to the National Cancer Institute Common Terminology Criteria for Adverse Events) of the adverse events were similar between the two dose groups. intrahepatic tumor response.6 0. Safety and toxicity JX-594 was generally well tolerated at both doses.1 0. Twenty-eight subjects were considered evaluable for radiographic endpoints. Inc.     Median     Range   Baseline bilirubin (mg/dl)     Mean ± s. we screened 49 subjects for enrollment. The objectives of this randomized trial were to compare outcomes with low-dose (108 PFU) and high-dose (109 PFU) JX-594 in a uniform advanced solid tumor population (HCC). P = 0. however.1–14.  advance online publication  nature medicine . High-dose JX-594 was also required for i.7 0.4 12 (86) 2 (14) 3.4 ± 3. the high-dose subjects were more likely to have failed previous systemic therapy.8 0. however.9 ± 12.3 4.05). four highdose and four low-dose).7 12. Ten non–treatment-related serious adverse events were reported (in eight subjects. activated partial thromboplastin time.6 ± 5.4–3. Fisher’s exact test).7–4. 1).9 28.9–4. as reported by the principal investigators.8 10.9 ± 2.2 ± 0. Thus far. Therapies given after JX-594 treatment and off protocol. One treatment-related serious adverse event was reported in the high-dose group (nausea and vomiting requiring prolonged hospitalization).d. Twenty-nine subjects received all three doses. BCLC.8 37. We enrolled 30 subjects and stratified (viral or nonviral tumor etiology) and randomized them (16 high-dose arm and 14 low-dose arm. and overall survival.9 31.3 ± 1.d.8 2.v.3 1 (7) 0 (0) 16 (100) 0 (0) 4. 9 (56) 7 (44) 6 (43) 8 (57) 2 (12) 14 (88) 6 (37) 10 (63) 10 (63) 9. systemic tumor responses and delivery through the blood to distant tumors required a high dose.1–59.8 are shown as the mean ± s. biopsy-confirmed cholangiocarcinoma) were evaluable for survival. However.0 37. these included sorafenib treatment (full-dose sorafenib for ≥8 weeks in two high-dose subjects and one low-dose subject) and local-regional palliative therapies (two high-dose subjects and one low-dose subject). Flu-like symptoms (grade 1–2) occurred in all subjects over the first 12–24 h after treatment.6 ± 1.5 10 (71) 4 (29) 8 (57) 6 (43) 9 (64) 3 (21) 6 (43) 2 (14) 3 (21) 14 (100) 62.8 26.8 ± 0.     Median     Range aData Low dose (n = 14) 67.v. were similar in the two groups.9 ± 9. the trial profile is shown in Supplementary Fig. nausea or vomiting.1 3 (21) 3 (19) 13 (81) 11 (69) 3.d.6 1.Articles Table 1  Demographic and baseline characteristics of the subjects according to treatment group (safety population) High dose (n = 16) Demographics   Age (years)a   Sex (n (%))     Male     Female   Region (n (%))     North America     Asia   Cause of disease (n (%))     Virally associated     HCV     HBV     Alcohol only     Other   Karnofsky performance status (KPS) of 70–100 (n (%))   Baseline neutralizing antibodies to vaccinia (n (%))     Positive     Negative Tumor burden   BCLC stage (n (%))     B (intermediate)     C (advanced)   Baseline tumors (n (%))     Single     Multiple     At least four   Baseline tumors (longest diameter (sum))a   Baseline tumors (hepatic or portal vein invasion) (n (%))   Extrahepatic spread (n (%))     Noted     Unknown   Previous therapy (n patients (%))b     Locoregional therapy     Number of previous locoregional interventions (per patient)a     Progressed on previous systemic therapyc     Progressed on previous sorafenib therapyc Baseline liver function   Child-Pugh classification (n (%))     A (5–6)     B (7–9)   Baseline albumin (gm dl−1)     Mean ± s. Subjects treated at the high dose showed a larger temperature increase after the first © 2013 Nature America. We noted no statistically significant differences for prognostic factors between groups. cPatients receiving systemic therapies had tumor progression while on therapy (refractory disease).6 3. Study enrollment was halted early by an independent data safety monitoring board because of a significant survival benefit favoring the high-dose group.3 ± 2. delivery and efficacy in a dose-escalation i. one subject received only two doses because of an unrelated adverse event.2 12.     Median     Range   Baseline prothrombin time (s)     Mean ± s.3 0.5–16.2 6 (38) 4 (25) 1 (7) 13 (93) 11 (79) 2. including safety.9 3 (19) 1 (7) 13 (93) 4 (29) 10 (71) 6 (43) 10. no rando­m­ ized dose-finding trial has been reported with these self-amplifying active immunotherapies.09.4 12. which is a negative prognostic factor (with six high-dose subjects compared to one low-dose subject failing previous systemic therapy. rigors.1 ± 11.0–39. No treatmentrelated deaths were reported.2 3.

with response rates of 57% and 67% for the high-dose and low-dose groups. The mean change in longest tumor diameter (mRECIST) may have reflected dose-related edema formation (changes of 23. we performed tumor contrast enhancement measurements according to the modified Choi criteria18 to assess effects on perfusion and the development of tumor necrosis. Pharmacokinetics. n = 22 patients who had measurable and evaluable tumors at baseline and at least one follow-up time point. 2). Baseline Week 8 Month 21 Figure 1  Radiographic antitumor activity after JX-594 therapy determined by dynamic MRI with a central radiographic reader blinded to treatment group. The mean changes in the Choi parameter were −35. A tumor with an increase >100% is indicated with a + above the bar. as JX-594 has been shown to disrupt tumor blood flow and induce tumor necrosis17. Intrahepatic disease control and mRECIST and Choi responses We performed serial dynamic magnetic resonance imaging (MRI) scans of the liver and abdomen. including small masses (<1 cm) that were poorly visualized at baseline.00 mm/div 10. high dose). respectively. nature medicine   advance online publication  . A single high-dose subject developed eight to ten grade 1 skin pustules measuring <1 cm diameter each on the extremities. (d) Greatest decrease in tumor contrast enhancement or longest diameter from baseline (Choi criteria) in noninjected 10. respectively). We found objective mRECIST responses and decreased tumor perfusion and contrast enhancement in both injected and noninjected tumors within both dose groups (Fig. (a) Greatest decrease in the sum of the longest diameter (LD) of 10. and these were subsequently read by expert independent central readers who were blinded to treatment arm. The intrahepatic mRECIST disease control rate at week 8 was 46% overall (28 evaluable ­ ubjects. thus resulting in systemic distribution. replication and transgene expression JX-594 injections into tumors resulted in acute diffusion of JX-594 genomes into the blood. Increases in serum transaminase concentrations were reported in six subjects (four lowdose and two high-dose).8% and −8. Inc. t test) and had a higher incidence of anorexia (31% compared to 0%. high dose). As demonstrated in a previous study14. n = 8 evaluable. 1a–d). A tumor with an increase >100% is indicated with a + above the bar.00 mm/div (mRECIST criteria) in livers of Baseline Week 8 Week 14 individual patients after JX-594 treatment.00 mm/div liver tumors of individual patients after JX-594 treatment. 1a–d and Supplementary Fig. Four objective mRECIST responses (one complete and three partial.11). whichever was better).00 mm/div 10. with control rates of 47% and 46% for the high-dose and s low-dose groups. we did not consistently perform subsequent scans and as a result did not assess time to tumor progression. (g) Example of the effects of JX-594 on contrast enhancement and perfusion in a noninjected (distant) tumor (JX7-1403. In some cases.00 mm/div 10. Fig.0023.73).04). One possibly related grade 4 event of lymphopenia (2 week duration) was reported in a high-dose subject. n = 8 evaluable. n = 24 patients who had measurable and evaluable tumors at baseline and at least one follow-up time point.6% and −28. As the primary radiographic endpoint was at week 8. tumors with decreased contrast enhancement showed swelling and edema (Fig.00 mm/div 10. The lesions developed approximately 4 d after treatment and resolved completely without scar formation within approximately 6 weeks. All rights reserved. 1f) and ten cases of stable disease were reported. dose did not correlate with intrahepatic response (Fig. The modified Choi response rate was 62% overall (26 evaluable subjects. e Baseline Week 8 f g JX-594 treatment compared with low-dose subjects ( P = 0. The subject received two subsequent doses of JX-594 without delays. We applied the mRECIST response criteria developed for individuals with HCC16 to assess the effects of JX-594 treatment in the liver.7% in the high-dose and low-dose groups.00 mm/div target tumors from baseline 10. ( c) Greatest decrease in the sum of the longest tumor diameter from baseline (RECIST criteria) in noninjected liver tumors of individual patients after JX-594 treatment. (e) Example of the effects of JX-594 on contrast enhancement and perfusion in an injected tumor (JX7-1401. 1e). The red circles (e–g) indicate the same (responding) tumors over time. (f) Example of the effects of JX-594 on the longest diameter of an injected tumor (complete mRECIST response) (JX7-1715.8% in the highdose and low-dose groups. 1g).articles a Maximum LD change from baseline (%) + High dose Low dose Maximum LD change from baseline (%) 100 80 60 40 20 0 –20 –40 –60 –80 –100 b Maximum enhancement or LD change from baseline (%) 100 + 80 60 40 20 0 –20 –40 –60 –80 –100 High dose Low dose c 80 60 40 20 0 –20 –40 –60 High dose Low dose d 60 Maximum enhancement or LD change from baseline (%) 40 20 0 –20 –40 –60 –80 High dose Low dose © 2013 Nature America. In addition. P = 0. (b) Greatest decrease in tumor contrast enhancement or longest diameter from baseline (Choi criteria) in target tumors in livers of individual patients after JX-594 treatment. respectively (P = 0. forehead and trunk. We found similar effects on tumor vascularity in noninjected tumors after high-dose treatment (Fig.00 mm/div 10. low dose). respectively) and was 50% at any time point (meaning at week 8 or any other time. P = 0. both doses were associated with intrahepatic antitumor activity.

2a). 3a. high-dose JX-594. All rights reserved.e. CDC was evident even after serum dilution down to 5%. Radiographic evidence included progressive near-complete tumor hypovascularity and necrosis associated with a markedly enhancing rim in noninjected tumors developing over 3–4 months in four subjects (Fig. 3).) for all cycles was 273.650 genomes ml−1 for the high-dose group and 31. transgene expression and GM-CSF protein function. 15. As can be expected because of replication of JX-594 at both doses. Neutralizing antibody titers to vaccinia (and.v. phase 1 trial). We first assessed antibody development to the β-galactosidase (β-gal) protein (the lacZ transgene product).000 400. The detection of JX-594 genomes in blood at any of these time points after JX-594 injection would suggest intratumoral replication and subsequent leakage into the systemic circulation. intratumoral. as β-gal protein expression is associated with virus replication) (Fisher’s exact test). Third.v.090 genomes ml−1 and 2. t test). t test).b). 3). all baseline samples were negative for hGM-CSF) (Fig. We monitored the concentrations of JX-594 in the blood over time at 15 min. Neutrophils and eosinophils are GM-CSF–responsive subsets of white blood cells. P = 0. JX-594 expresses the hGM-CSF transgene under control of the synthetic early-late promoter such that high-level expression would occur during replication of the product in cancer cells 6–8. β-gal–specific antibodies developed in most of the subjects during the treatment phase: 75% of high-dose and 62% of low-dose subjects developed these antibodies (Fig. We were able to detect JX-594 genomes in the blood of three subjects (two high dose and one low dose) at the late time points (days 15–36).098 ± 488 genomes ml−1 for the high-dose and low-dose groups.5 pg ml−1 for the low-dose subjects.m. white bars. We assessed JX-594 replication and dual transgene expression using three methods. these findings are considered highly unusual for  advance online publication  nature medicine . Whereas i.m.15. we assessed antibodymediated complement-dependent cytotoxicity (CDC) in subjects’ serum over time after treatment.000 0 Tr ea t Tr me ea nt tm 1 Tr e ea nt 2 t Tr me ea nt 3 t Tr me ea nt 1 t Tr me ea nt Al tm 2 e l tre high nt 3 at -d Al me os e l tre low nts at -d m os en e ts b P = 0.e. low-dose JX-594. the peak concentrations in the high-dose group in this trial were above the threshold for i. (b) The percentage of patients with evidence of β-gal transgene expression (+95% confidence interval (CI)) after JX-594 treatment (generation of antibodies (Ab) to the β-gal transgene product within 29 d of treatment is indicative of JX-594 replication.000 200. Acute blood concentrations at 15 min after injection were consistent with a substantial fraction of the input dose acutely entering the blood during or immediately after the injection procedure (half-life of ~60 min). 5–7 d and 14 d after completion of each injection procedure. P < 0. respectively. whereas the concentrations in the low-dose group were not. six subjects with CDC induction had hepatitis B–associated HCC. delivery to tumors (as defined in the i. mean concentrations of 10. 3 h. hGM-CSF protein was quantifiable in the plasma on day 5 in 69% of the high-dose and 46% of the low-dose subjects (P = 0. 36. ( d) Maximum induction of neutrophil concentration in blood after treatment cycle 1 by dose group (using blood obtained on days 5 and 15 after treatment). 22. which is dependent on viral replication and associated protein expression. JX-594 can acutely stimulate production of multiple cytokines. As a measure of induction of antitumoral immunity.6 pg ml−1 compared to 1.000 100. Black bars.27 High dose Low dose d Absolute neutrophil increase from baseline (103 cells µl–1) 12 10 8 6 4 2 0 –2 –4 hGM-CSF induction (day 5) Figure 2  Laboratory evidence for JX-594 replication. respectively. we evaluated the delayed re-emergence of JX-594 genomes in the blood on days 5. t test). © 2013 Nature America. The pharmacokinetic profiles were similar for all three injections per subject (Fig. genomes were detectable in blood at this time point in all subjects on both arms. Second. JX-594) were detectable at baseline (from childhood vaccination) in 50% of subjects.v. the mean concentration (± s. The frequency and concentrations of detectable JX-594 genomes in the blood were also significantly greater in the high-dose subjects at 3 h after injection (78% of subjects had detectable JX-594 compared to 45% for the high-dose and low-dose groups. hence.317 genomes ml−1 for the low-dose group (P = 0. The peak concentrations of JX-594 were significantly greater for subjects in the high-dose arm (Fig. 2b). ( a) The mean (± s. Induction of humoral and cellular anticancer immunity We analyzed subject blood. 43 and 57. for all treatment cycles. ( c) The percentage of patients with evidence of hGM-CSF transgene expression (+95% CI) on day 5 after JX-594 treatment (Fisher’s exact test). for all treatment cycles.0006. Concentrations were highest at 15 min after injection.700 ± 55.Articles a Genomes per ml blood 15 min after IT treatment 500. The absolute neutrophil and eosinophil concentrations increased substantially between 5–14 d after treatment in 65% of subjects with quantifiable hGM-CSF in the blood (Fig. two had hepatitis C–associated HCC and three had non-viral HCC.69 c 100 Percentage of patients with hGM-CSF induction 80 60 40 20 0 P = 0.450 ± 2. All HCC cell lines tested contained hepatitis B virus DNA except HepG2 (which is not virally associated). 29. Fisher’s exact test. infusion at similar doses in a phase 1 trial15.27. Eleven of 16 (69%) subjects evaluated developed CDC against at least one of four HCC cell lines (six high-dose and five low-dose subjects) (Fig. IT. The peak concentrations in these subjects were similar to peak concentrations in subjects after i.000 300. Exploratory data from a subset of subjects treated with the same manufacturing lot of clinical trial material showed that hGM-CSF concentration (day 5) was higher in the high-dose subjects than in the low-dose subjects (median for the high-dose subjects was 47.v.) peak concentration of JX-594 (genomes measured by quantitative PCR (qPCR)) in blood after each treatment cycle (using blood obtained 15 min after the completion of treatment) by dose group (t test). tumor tissue and radiographic imaging to assess the induction of anticancer immunity (Fig.0002 100 Percentage of patients with Ab induction 80 60 40 20 0 β-gal Ab induction (day 29) P = 0. 2d).650 ± 5. Inc.001. delayed hGM-CSF expression 5 or more days after infusion (when other cytokine concentrations have returned to baseline levels) was indicative of hGM-CSF expression from JX-594 in the context of replication14. all subjects developed detectable titers by day 29. 2c). 2a).0002.

Each graph shows the mean (–) 300 300 percentage cell viability (+s.5 3/13 (23%) 8/13 (62%) 8/12 (67%) 2.articles a P = 0.00 mm/div 10. 3f). radiographic and biopsy evidence for JX-594– P = 0. (a) Antibody-mediated P = 0. 3e).31 0.75 1.0 × 103 ± 0.650 13.e. Table 2  Correlations by dose High dose Mean peak acute pharmacokinetics (genomes ml−1 blood)a Median overall survival in patients with multiple tumors (months) Median overall survival (months) GM-CSF induction (yes/no) Peak GM-CSF concentration (day 5) (pg ml−1)a Best mRECIST response (yes/no) β-gal–specific antibody induction (day 29) (yes/no) Best Choi response (yes/no) Peak neutrophil induction (day 5–15) (µl−1)a mRECIST disease control (week 8) (yes/no) CDC induction versus SNU739 cell line (≤50% cell viability (yes/no)) CDC induction versus HepG2 cell line (≤50% cell viability (yes/no)) CDC induction versus SNU475 cell line (≤50% cell viability (yes/no)) aData We also assessed cellular immunity.d. JX7-0311. The data shown are from serum of patients that induced ≤50% cell viability (>50% cell killing) on at least one follow-up time point. 50 µm (high-magnification inset).0001 complement-dependent cytotoxicity induction after JX-594 therapy in P = 0. We biopsied one of these masses 1.d. 3d). nature medicine   advance online publication  .650 ± 5. ( f) ELISPOT analysis detecting T cells producing interferon.317 4.018 0. (b) Antibody-mediated CDC induction against HCC cell 1 29 57 1 29 57 lines in individual patients over time after JX-594 therapy (serum diluted Time after JX-594 treatment (d) Time after JX-594 treatment (d) to 5%. β-gal (–) 400 400 n = 3.0 1.γ in response to stimulation with β-gal peptides at baseline and after JX-594 treatment.561 associated induction of anticancer immunity.00 mm/div 10.7 6/13 (46%) 38.e) indicate the same (responding) tumors over time. Figure 3  Laboratory.0 Statistical test t test Gehan-Breslow Gehan-Breslow Fisher’s exact t test Fisher’s exact Fisher’s exact Fisher’s exact t test Fisher’s exact Fisher’s exact Fisher’s exact Fisher’s exact 273.0002 0.00 mm/div f tumor progression in HCC. Inc. (−).00 mm/div 10.d. two high0 0 dose patients.5 years after the last JX-594 treatment (Fig. Cytotoxic T cells were induced to vaccinia peptides (data not shown) and the JX-594 transgene product β-gal (enzyme-linked immunosorbent spot (ELISPOT) analysis) (Fig.76 × 103 7/15 (47%) 6/8 (75%) 3/8 (38%) 5/8 (63%) are shown as the mean ± s.8 ± 38.) after incubation with each individual 200 200 patient’s serum (diluted to 5%) collected on day 43 after the initiation 100 100 of treatment compared to baseline. n = 4) cell lines. but we could not completely rule out progression.002 1712 1713 1601 1401 1402 1702 SNU739 1703 1704 1705 301 302 1712 1713 1401 1402 1702 1703 SNU475 1704 1705 301 140 120 100 80 60 40 20 0 1702 1703 1704 HepG2 1705 301 302 0 10 20 30 40 50 Time after JX-594 treatment (d) Week 20 60 0 10 20 30 40 50 60 Time after JX-594 treatment (d) Week 50 c d e Baseline Week 8 Week 38 Baseline Week 8 Week 14 10. low dose).0 1.m.7 × 103 ± 0. data are expressed as the mean number of spot-forming cells (SFC) per 10 5 cells (+s.02 0.00 mm/div 10. β-gal cytotoxic T cell activity was present in a SFC per 105 cells (JX7-0310) Low dose 31.69 0. high dose).5 years after the initiation of JX-594 treatment.002 b 120 100 80 60 40 20 0 P = 0.00 mm/div © 2013 Nature America.) (JX7-0310. 10. HUVEC and MRC-5) and non-HCC (RCC.3 6.70 0.0 1.009 SN U Percentage cell viability (JX7-1702) SN -44 U 9( -4 H H 75 CC e ) ( SN pG HC U 2 (H C) -3 M 98 CC R ) H C5 (HC U VE (no C) C rm R (n al M en orm ) el an al ( al) om n = 3 a (n ) = 4) 120 100 P = 0.1 1/15 (7%) 12/16 (75%) 8/14 (57%) 3. (e) Radiographic evidence of progressive necrosis and peripheral enhancement over time in a noninjected tumor (JX7-0301.002 β-gal 500 500 HCC (n = 4). Scale bars.). low dose). melanoma.9 ± 18.33 0. the biopsy showed diffuse lymphocytic infiltration (Fig.27 0.102 P < 0.00 mm/div 10. Red circles (c. All rights reserved. ( c) Radiographic evidence of progressive necrosis and peripheral enhancement over time in noninjected tumors (JX7-0307.87 × 103 6/13 (46%) 5/8 (63%) 3/8 (38%) 4/8 (50%) SFC per 105 cells (JX7-0311) P 0. mean ± s. The P values in a and f were calculated by t test.700 ± 55.1 11/16 (69%) 83. 100 µm (low magnification).6 14.00 mm/div 10. JX7-1704 and JX7-1702. low dose. normal (n = 2. ( d) H&E staining of a biopsy sample from a tumor collected from patient JX7-0301 (low dose) 1.003 SN Percentage cell viability U (JX7-1704) SN -47 5 U (H SN -44 CC U 9 (H ) -3 9 C H 8 ( C) ep H M G2 CC R ) C ( H -5 HC U VE (no C) C rm al ( ) R n M en orm el an al ( al) om n = a 3 (n ) = 4) Percentage cell viability Percentage cell viability 100 90 80 70 60 50 40 30 20 10 0 80 60 40 20 0 0 10 20 30 40 50 60 70 Time after JX-594 treatment (d) 160 140 120 100 80 60 40 20 0 Percentage cell viability P = 0. negative control peptide.

020.6 months.4 Baseline 0.3 months in the low-dose group (hazard ratio 0.39. respectively. Inc. T cells collected from healthy donors not exposed to JX-594 did not have reactivity to β-gal (Supplementary Fig. several key were 35% and 11%.1 months for the high. Third. functional the time of study enrollment. neutrophil induc. high-dose JX-594 minant of overall survival duration in subjects with advanced was associated with a significant survival benefit. respectively. a Probability of survival by dose Probability of survival in patients with multiple tumors by dose 1. basis of data from randomized phase 3 trials with sorafenib 19. JX-594 to evaluate dose and peak genome less important than it is with other therapies in cancer.0 0. the low this trial (8.  advance online publication  nature medicine .0 0. Gehan-Breslow-Wilcoxon test) (Fig. respectively). Compared to T cells derived ratio of 0. (c) Overall survival in the entire evaluable study population by the presence or absence of baseline neutralizing antibody status. the median over.4 0. In subjects with multiple tumors at base.6 months. to our knowledge. Amgen).6 0. four in this group antitumoral immunity.6 0. the relative High-dose subjects in this systemic therapy failure subgroup had importance of each MOA remains to be determined.8 months compared to 16. we demonstrated the negative subjects.4 0. Breslow-Wilcoxon test. phenotypic generating analysis. hazard ferences from peripheral blood T cells. In a melanoma clinical trial with 24 subjects were evaluable for all variables. 3). associated antigen recognized by T cells (MART-1)-specific T cells Six high-dose subjects had previously failed systemic therapy at in tumors undergoing regression after vaccination. we demonstrated that JX-594 dose was an important deterline (ten high-dose and nine low-dose subjects). Survival did not correlate with tumor findings for JX-594 and the field of oncolytic immunotherapies. Although data on diverse MOA were obtained. P = 0.018. and at 18 months We report here for the first time.2 0.68 in favor of baseline antibody-positive compared to antibody. 4c). 4d).­ carcinoma. dose group compared to 6.19. one-sided test for superiority of the high dose) to our knowledge. Kaplan-Meier survival estimates for the high-dose and low-dose DISCUSSION groups at 1 year were 66% and 23%. Gehan-Breslow-Wilcoxon test. The dose in this trial had clear anticancer efficacy. All rights reserved.Articles Figure 4  Kaplan-Meier analysis of overall survival. one-sided test for superiority of the a median survival of 13. 4b).6 0. 0 Number at risk High dose 16 Overall 29 Low dose 13 0 Number at risk High dose 10 Low dose 9 c d 1. this is the first randomized clinical trial showing (Fig.comparison to placebo might have been.2 0 5 10 15 20 25 Time since randomization (months) 8 5 3 2 2 2 0 30 Low dose High dose 1.First. other variables did not add additional overall survival from nontreated control patients. there was an increase in melanomapredictive value after dose. including JX-594 replication and immune stimulation. (a) Overall survival in the entire evaluable study population (dashed line) and by dose group (n = 29 total).8 0.0 0. and two such subjects were high dose). had failed previous treatment with sorafenib. as only ing in antibody-dependent CDC. Gehan. but others for this subject population was estimated to be ~2–4 months on the remain unanswered. The only ­variable © 2013 Nature America. at risk at risk Positive 14 8 5 3 2 2 1 High dose 6 4 3 2 2 2 Overall survival was significantly longer Negative 15 10 6 4 2 2 in the high-dose arm compared to in the low-dose arm (hazard ratio 0. active induction of functional antitumor immunity with an oncolytic We performed an exploratory multivariate stepwise regression analysis virus in multiple subjects within a homogenous population.358).4 for the entire study population. (d) Overall survival in high-dose subjects having previously failed systemic therapy (n = 6). this was only a hypothesis.8 0.6 months in the high-dose group compared to has been predicted that maximizing the dose to the subject would 4. In contrast.24. We assessed antibody 0.8 Probability of survival in high-dose patients with prior systemic therapy Probability of survival by presence of baseline antibody to vaccina high-dose subject 1.6 The median overall survival was 9. P = 0. Of note. however. 3). The median survival These trial results address a number of key questions. showpresence or absence of detectable neutralizing antibodies to vaccinia ing a significant survival impact for the high-dose group compared at baseline did not correlate with survival duration (hazard ratio of to low-dose active controls was presumably a higher hurdle than a 0. it all survival was 13. and therefore. P = 0. Only dose 0 0 cohort and peak JX-594 blood concentration 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 Time since randomization (months) Time since randomization (months) Number Number correlated significantly with overall survival.7 months for the low-dose group (Fig. etiology (viral or nonviral). was not assessed1. Subjects with multiple tumors (n = 19) had a median sur.that an oncolytic virus or gene therapy agent was associated with vival that was half that of subjects with single tumors (n = 10) in significantly improved overall survival duration.2 the correlation between various baseline variNo baseline antibody ables and overall survival (Table 2).0 months 0. The median overall survival was 14. 4a).20.0 0.2 0 5 10 15 20 25 30 Time since randomization (months) 13 9 5 4 4 1 18 11 7 4 4 1 5 2 2 35 High dose Low dose Overall b 1. given that oncolytic viruses replicate in tumor cells.8 Overall survival 0. (b) Overall survival in subjects with multiple tumors at baseline (ten high-dose and nine low-dose subjects).treatment induced a polyclonal humoral immune response resulttion and antibody induction to the JX-594 β-gal transgene.herpes simplex virus (HSV) hGM-CSF (T-Vec.5 years after treatment initiation (Supplementary Fig.still alive after more than 2 years (Fig.0085. Second. This multivariate analysis confirmed that dose analysis of T cells derived from tumor samples suggested distinct difgroup was the best predictor of overall survival (χ2 = 2. as described above.

Targeted and armed oncolytic poxviruses: a novel multimechanistic therapeutic class for cancer.C. these include cytokines.B. Kirn.. as demonstrated by tumor-lysing antibody generation and lymphocyte infiltration into tumors. Kaufman.R..3089. R.L. D. In summary. administration. K. 749–758 (2012). a targeted oncolytic poxvirus.H. GM-CSF or interleukin-2 (IL-2)). single-chain antibodies and tumor antigens (reviewed in ref. designed the study. Transgene SA (Illkirch. Heo. Clin.H. this durable efficacy could reflect chronic viral oncolysis or immune-mediated effects.L. R. 105.B.J.B. M.R. C.N. and Mol.H. Local and distant immunity induced by intralesional vaccination with an oncolytic herpes virus encoding GM-CSF in patients with stage IIIc and IV melanoma. Nevertheless. S. nature medicine   advance online publication  . 7.C. Galanis.v.L. followed by sorafenib in hepatocellular carcinoma: preclinical and clinical demonstration of combination efficacy. of note. Cancer 2. Trials in other solid tumor populations are underway.C. analyzed the data and wrote the manuscript.. All rights reserved. risk management and future directions.P. more data are needed to know how to interpret these mRECIST and Choi responses. The application of mRECIST and Choi criteria in HCC trials is increasing.H..B. 19. E. Sequential therapy with JX-594. Invest.D. the survival benefit of high-dose JX-594 was most pronounced in patients with the most extensive tumor burdens. blood concentrations in the high-dose group were above the threshold concentration required for i.C. Clin.N. M. the oncolytic and immunotherapeutic vaccinia virus JX-594 (Pexa-Vec) holds promise for the treatment of advanced solid tumors. 101–117 (2007). C. a statistically significant survival benefit was demonstrated because of the large effect size. Large randomized trials adequately powered for overall survival effects are underway or have been planned with JX-594 in patients with advanced HCC. Ministry for Health. 361–370 (2006).R. et al.R. However. T. D. Hwang. Limacher. L.W. Liu. Lusky (all from Transgene SA) gave insightful comments on the manuscript.C.R. 9. The survival analysis from this stratified and randomized clinical trial demonstrated a significant dose-dependent survival prolongation. Further study will be needed to assess the relative importance of each MOA in specific patient populations. enrolled and managed the patients.J. J. 17. 64–71 (2009).L. reprints/index.B. 6).H. 7. Nat. Ther. could be expressed from the same virus backbone. et al. Furthermore. made the final decision to submit for publication. as well as complementary cytokines (for example.P.articles that correlated with overall survival duration. Methods Methods and any associated references are available in the online version of the paper..K.. H.L. 847–851 (2000). & Kirn. Note: Supplementary information is available in the online version of the paper.. M.H. et al. In particular. Republic of Korea (A091047). C.22. 10.K. Oncol. D. D. these findings should be extended in larger randomized trials with a placebo control arm included. 2. Replication-selective virotherapy for cancer: biological principles.-G. whereas low-dose concentrations were not.H.. Kim. Systemic armed oncolytic and immunologic therapy for cancer with JX-594. Pract.v. 15). systemically effective oncolytic poxvirus. including colorectal carcinomas with a mutant K-RAS genotype. France) and the Green Cross Corporation. a decade of progress. and J. 19. Ministry for Health. Inc. & Thorne. Nat. delivery in phase 1 (ref.H. Rational strain selection and engineering creates a broadspectrum. Acknowledgments J. C..H.. et al. Ther. checkpoint inhibitors. patients are being randomized to either JX-594 plus the best supportive (palliative) care or to the best supportive care alone (ClinicalTrials.H. number NCT00554372. Hence. 6. Ann.nature. Nat. besides dose group. Ther.. J. larger trials will allow the exploration of correlations between immune and virus-replication endpoints and patient survival. Parato. T. et al.A.K. Mol. The oncolytic poxvirus JX-594 selectively replicates in and destroys cancer cells driven by genetic pathways commonly activated in cancers. in patients with metastatic melanoma. 1. 3.C. and preliminary data suggest that these criteria may be better predictors of overall survival in HCC than the standard RECIST criteria21. although helper T cell engagement was demonstrated by IgG class switching and the induction of T cells specific for the β-gal transgene. In a randomized trial involving patients having failed treatment with sorafenib. Mol.M.B. and D. Heise.R.S. tumor antigens. Nat. B. B. J. & Zwiebel. performed bioanalytical analyses. tumor oncolysis and necrosis and antitumor immunity. J. Surg. Prognostic factors were well-balanced between the two arms. were supported by a grant of the Korea Healthcare technology Research and Development Project. This trial was registered with ClinicalTrials. 1170–1179 (2011). T..H. Kirn. L. Rev. J.K.. Reprints and permissions information is available online at http://www. 4. Oncolytic viruses. In addition.. with the exception that more high-dose patients had the poor prognostic factor of having failed previous systemic therapy. H. 4. including sorafenib treatment. 5. Despite the relatively small sample size and an active control-group treatment.D.H.K. 938–950 (2002). Replication-selective adenoviruses as oncolytic agents. and M. et al.A. M. grants to T. had access to all the data in the trial.M... H. Chiocca. As predicted on the basis of this hypothesis. C. Invest. was supported by a pilot grant from the Dan Duncan Cancer Center. 8. to J. Clinical trial results with oncolytic virotherapy: a century of promise. the survival duration in this subgroup on high-dose therapy was similar to that of systemic treatment–naive patients. prodrug-activating enzymes. S.. J. Although not proven. the survival benefit of high-dose JX-594 cannot be explained by imbalances in known prognostic factors. 3350–3358 (2007). A. A mechanistic proof-of-concept clinical trial with JX-594. Choi response criteria include changes in tumor density and maximum diameter...Y. We selected the sample size of 30 patients to give sufficient power for toxicity analyses in both arms.K. Funding was provided by Jennerex. Y. AUThOR CONTRIBUTIONS D. K. M. Clin. Bastien and M. J. Homerin. from Korea Healthcare technology R&D Project. is supported by the Ontario Institute for Cancer Research and the Terry Fox Foundation.K. Oncol.1038/nm. T. Published online at http://www. and a pilot grant to C. Mol. 14. 20.C.. from the Dan Duncan Cancer Center. 11.K. JX-963.H... NCT01387555). D. a targeted poxvirus expressing GM-CSF. and M. 781–787 (2001).R. T. was acute peak JX-594 concentration in the blood. Ther. Cancer 9. 718–730 (2010). M. S.L.html. Welfare and Family Affairs. Republic of Korea. targeted oncolytic vaccinia viruses such as JX-594 can be engineered to express diverse biologics with varied and potentially synergistic MOA.B. J. multiple tumor antigens.nature. was supported by the Robert and Janice McNair Foundation and Baylor Research Advocates for Student Scientists Fund. R. from the Terry Fox Foundation and the Canadian Institute for Health Research (CIHR). and further clinical trials are warranted. COMPETING FINANCIAL INTERESTS The authors declare competing financial interests: details are available in the online version of the paper. Replication and hGM-CSF transgene expression were associated with three MOA: acute vascular disruption in tumors. Likewise.-M. 1913–1922 (2011).H.H.-H. further data on tumor-specific T lymphocyte induction will need to be obtained in future trials. Therefore. these data suggest that systemic tumor control and improved survival may be achieved with JX-594 through high-dose i. The long-term survival of highdose patients (~35% at 2 years) after dosing over only 4 weeks suggests a potential durable systemic benefit. Welfare and Family Affairs. a targeted multi-mechanistic oncolytic poxvirus.-H. Martuza. Given the large transgene-encoding capacity of vaccinia & Kirn.K. E. Med. Rev. exploration of tumor antigen expression is warranted given our demonstration in this trial that a T cell response was induced to the JX-594 transgene β-gal. © 2013 Nature America. 117. and C. T.

and anti-HBV activities in patients with hepatocellular carcinoma. B. Use of a targeted oncolytic poxvirus. 22. Modified RECIST (mRECIST) assessment for hepatocellular carcinoma. 4504–4512 (2011). Comparison of tumor response by Response Evaluation Criteria in Solid Tumors (RECIST) and modified RECIST in patients treated with sorafenib for hepatocellular carcinoma. J. 378–390 (2008). T. placebo-controlled trial. Clin. JX-594. Liver Dis. Engl. 359. Lencioni. Park. J.L. H. Lancet Oncol. 99–102 (2011). Bell. All rights reserved.H. second-generation oncolytic herpesvirus in patients with unresectable metastatic melanoma. R. 1317–1324 (2003). N. Validation and analysis of a mathematical model of a replication-competent oncolytic virus for cancer treatment: implications for virus design and delivery. 30. Oncol. & Llovet. 147–156 (2012). antivascular. Park. Clin. J. C. et al. et al. N.C. in patients with refractory primary or metastatic liver cancer: a phase I trial. Cancer Res.M. 20. 63. 25. Llovet. Correlation of computed tomography and positron emission tomography in patients with metastatic gastrointestinal stromal tumor treated at a single institution with imatinib mesylate: proposal of new computed tomography response criteria. Faivre. 21. Wu. J. Mol.H. & Kirn. Edeline.N. et al. 13.H. J. 1753–1759 (2007). Senzer.M. 27. 15. 18.H. Sorafenib in advanced hepatocellular carcinoma. T.J. & Kirn.. et al. S. Liu.  advance online publication  nature medicine . D. 52–60 (2010). Wein.. Intravenous delivery of a multi-mechanistic cancer-targeted oncolytic poxvirus in humans. L. Cancer 118. 1637–1642 (2008). Lancet Oncol. 5763–5771 (2009). J. 17. Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma: a phase III randomised. 19. A. Nature 477. Phase II clinical trial of a granulocyte-macrophage colonystimulating factor–encoding. 17. 16.M. D. Semin.Articles 12. 16. doubleblind. 533–542 (2008). et al. B. The targeted oncolytic poxvirus JX-594 demonstrates antitumoral. et al. 25–34 (2009). Breitbach. Choi.. Hwang. et al. Oncol. 14. Cheng. © 2013 Nature America. J. Inc. et al.. 9. Med. Ther. Changes in tumor density in patients with advanced hepatocellular carcinoma treated with sunitinib. Clin.T. J. Cancer Res. 10.

anticancer therapy within 4 weeks before the first treatment. McMaster University Medical Center. Clinical trial material (CTM). adefovir.0 times the upper normal limit.15. 29. Patients. We administered JX-594 by imaging-guided IT injection using a multi-pronged Quadrafuse needle (Rex Medical Inc) to ensure even distribution of the virus throughout the tumor when possible (if this was not technically feasible. The manuscript was written and edited by Jennerex Inc. Exclusion criteria included liver tumors in a location that could result in clinical adverse effects as a result of tumor swelling after treatment (for example. masking and treatment. tumors impinging on the biliary tract). We read the absorbance at 405 nm and subtracted the absorbance at 630 nm. After enrollment and stratification for HCC etiology (hepatitis B or C virus infection–associated or not virally associated). The tissue was formalin fixed and paraffin embedded.). hence limit of detection of the assay was 10.ONLINE METHODS © 2013 Nature America. An independent data-safety monitoring board reviewed major safety assessments and periodically assessed safety and efficacy. 43 and 57. b-gal–specific antibody ELISA. arbitrarily assigned a titer of 8. use of certain antiviral agents with activity against vaccinia (for example. We measured JX-594–specific neutralizing antibody titers at baseline and on days 5. We evaluated hGM-CSF protein concentrations in plasma at baseline and 5 d after the first treatment by solid-phase sandwich ELISA using the Quantikine hGM-CSF Kit (R&D Systems) as directed by the manufacturer. 15.5T or 3. The primary endpoint was assessed by independent expert radiologists who were blinded to treatment group. Randomization. We assessed antibody induction to β-gal at baseline and day 29. 15. we elected to assess tumor vascularity (contrast enhancement) at baseline and at each time point using both the mRECIST for HCC criteria16 (performed by R. symptomatic ascites. 29 and 57 by cytopathic effect inhibition assay as previously described14.L.P. Patients with household contacts at known risk for vaccinia vaccine complications (for example. Portal vein thrombosis or invasion and extrahepatic disease were allowed. Three patients in this trial were included in a publication on the use of sorafenib after JX-594 treatment10. 36.P. as well as the Institutional Review and Infection Control Committees at the following centers: Moores University of California San Diego (UCSD) Cancer Center. Human IgG antibodies to β-gal were measured by ELISA. severe immune deficiency or eczema) who could not relocate were excluded. Cells were harvested after approximately 48 h and lysed. adventitious viruses (absent). The academic investigators vouch for the validity of the results.250 cells mm−3.500 cells mm−3 and <50. severe or unstable cardiac disease. 15 and 29. We measured β-gal–specific antibody titers at baseline and on day 29 by ELISA as previously described15. Ohio State University. the lowest dilution tested was 10%. We assessed white blood cell counts at baseline and on days 5.23. Briefly. The study protocol and consent forms were approved by the United States Food and Drug Administration. We collected samples before each injection and 15 min. serum chemistry and urinalysis. scans were optional every 6 weeks thereafter. in collaboration with the principal investigators. We performed multiphase dynamic contrast-enhanced MRI of the abdomen on either a 1. white blood cell count >2. The total dose per patient per treatment session was fixed at 109 or 108 PFU. We performed white blood cell counts by routine laboratory testing as defined in the protocol. aspartate aminotransferase (AST) and alanine aminotransferase (ALT) <5. potency measured in PFU ml−1 and hGM-CSF production. JX-594 was diluted in bicarbonate-buffered saline. multinational randomized and stratified.1038/nm. All rights reserved. qPCR. and the cellular debris was removed by a standard filtration step.3089 nature medicine . we added the colorimetric substrate alkaline phosphatase yellow (pNPP. ELISA grade (Sigma)). This trial was a multicenter. Histopathology analysis. Titers are expressed as the reciprocal of the highest dilution (least concentrated) of heat-inactivated serum that resulted in ≥50% cell viability. coagulation studies. CTM was generated after infection of human carcinoma cells. Physical and laboratory assessments.).0) and standard laboratory toxicity grading for hematology. platelet count ≥50. and the principal investigators together in collaboration with Green Cross Corporation after a joint decision to publish. necrosis and inflammation. Study design. We added diluted serum (1:50.5 times the upper normal limit and Child-Pugh score of A or B) and a KPS of 70 or greater. Immediately before the intratumoral (IT) injection. All patients gave written informed consent according to the principles of Good Clinical Practice. Given the mechanism of tumor destruction on the basis of vascular disruption previously described for JX-594 (ref. Data collection and monitoring were performed by Jennerex Inc. Patients were stratified (viral or nonviral) and randomized one-to-one to either the high-dose or low-dose cohort. Sigma) and added NaOH 10 min later to stop color development. increased risk of vaccination complications (exfoliative skin conditions such as eczema or ectopic dermatitis). The volume of JX-594 solution to be injected was proportional to the volume of the tumor to be injected (25% of the tumor volume). Severance Hospital Yonsei University Health System and Pusan National University Hospital. Radiology acquisition and analysis.000. and R. Samsung Medical Center. the Korea Food and Drug Administration and Health Canada. We evaluated the concentration of JX-594 genomes in blood over time using qPCR as previously described14. evaluation and analysis were performed by independent expert contractors. 1:100 or 1:200 in PBS with 0. Inc. CTM was manufactured according to Good Manufacturing Practice guidelines. we incubated plates (NUNC MaxiSorp.5 times the upper normal limit) and organ function (including serum creatinine <2 mg dl−1. Scans were evaluated by two independent radiologists with expertise in liver cancer assessment (R.L. 3 h and 5 d or 7 d after each treatment and on days 43 and 57 (2 and 4 weeks after the final treatment). 14). a straight needle was used). liver and renal function. Data management. Choi response criteria doi:10. Detection of neutralizing antibodies. We stained sections with H&E to assess tissue histology. adequate hematological function (absolute neutrophil count >1. hemoglobin ≥9 g dl−1.0T MR system using extracellular gadolinium chelate contrast agent. We performed physical assessments and interval medical histories at each visit over the first 8 weeks of study.000 cells mm−3 and International Normalized Ratio (INR) ≤1. Thermo Fisher Scientific) with wells containing β-gal (Sigma) or bicarbonate or carbonate buffer overnight at 4 °C and washed the plates with PBS-Tween before incubation with blocking buffer (PBS with 1% BSA. After washing. total bilirubin ≤2. hGM-CSF ELISA. Final-product qualitycontrol tests included assays for sterility and endotoxin (absent).000. We performed scans at baseline (days −14 to 0) and at week 8. ribavirin. immunodeficiency caused by underlying illness. principal investigators were not blinded to dose group. We obtained core-needle biopsies of liver tumors in a subset of patients after treatment. 22. known central nervous system malignancy. We subtracted control-well values to account for nonspecific binding and calculated titer values by comparison to a standard curve of positive sera. The date of data cutoff for the survival analysis was the survival followup data collected when the last patient reached the week 8 primary endpoint assessment visit. cidofovir and PEG-IFN) and pregnancy or nursing. statistics. the readers were blinded to treatment group. parallel-group dose-finding study in patients with advanced HCC. and Green Cross Corporation. Inclusion criteria included unresectable and injectable (defined tumor margins) HCC (histologically confirmed or clinical and laboratory diagnosis defined according to the American Association for the Study of Liver Diseases guidelines).05% Tween and 1% BSA) to β-gal–coated and control wells in duplicate and incubated them at 23 °C. We washed the plates and incubated them with alkaline-phosphatase–labeled goat human-specific IgG (Abcam) diluted 1:2.) and a modification of Choi response assessments24 (performed by R. The remaining supernatant was purified by tangential flow filtration. and radiographic image management. DNA and protein content. This study was designed by Jennerex Inc. Safety monitoring included adverse event monitoring according to the National Cancer Institute Common Toxicity Criteria (version 3. we randomized patients centrally using permuted blocks to receive a dose of 109 or 108 PFU distributed among up to five intrahepatic tumors on days 1. safety reporting management.000 cells mm−3.

we seeded 2. & Meier. 42. Lonza) were cultured in the endothelial cell medium EBM-2 (Lonza. b-gal–specific T cell analysis. Ther. we developed the plates and sent them to Zellnet Consulting. We used a pepmix of a testis cancer antigen. ATCC) were cultured in MEM containing 10% FBS with penicillin and streptomycin. The sponsor of the study participated in study design. Smallpox and pan-orthopox virus detection by real-time 3′-minor groove binder TaqMan assays on the roche LightCycler and the Cepheid smart Cycler platforms. MRC-5 nontransformed cells (lung fibroblast. Secondary objectives included safety and antitumor activity (change in contrast enhancement. 23. 24. Twenty-eight patients were evaluable for overall mRECIST response: 2 patients died before the week 8 efficacy assessment and were categorized as progressive disease. E. MD. Clin. Choi. We seeded each cell line onto 96-well plates and incubated the cells overnight. and a linear regression was calculated after excluding plateau data points. AJR Am. VA)) supplemented with 1. data collection. SNU398. SNU449 and SNU739 (human hepatocellular carcinoma. SK-MEL-5 and WM-266-4 cells (human melanoma cells. The GehanBreslow-Wilcoxon test was selected because dosing occurred only over the first 4 weeks.) and incubated cells at 37 °C for 2 h. nature medicine doi:10. All patients who received at least one dose of JX-594 were evaluable for safety. HUVEC (endothelial cells.1 µg per peptide per well and PHA (phytohemagluttinin) at 2 µl (1 mg ml−1) as negative and positive controls. 53.5 × 104 to 105 T cells in triplicate with individual pepmixes spanning β-gal at 0. U. 4 patients were evaluable by mRECIST but did not have measurable tumors. Gerdemann. and comparisons were made using the Gehan-Breslow-Wilcoxon test and a one-sided α for the statistical significance of high-dose superiority. CT evaluation of the response of gastrointestinal stromal tumors after imatinib mesylate treatment: a quantitative analysis correlated with FDG PET findings. 26. All rights reserved. Kaplan. obtained from KCLB) were cultured in minimum essential medium (MEM) containing 10% FBS with penicillin and streptomycin.000 U ml−1 IL-4 (R&D Systems. patients with evaluable baseline and week 8 scans were evaluable for radiographic efficacy endpoints.1038/nm. and therefore a test that weighted the early part of the overall survival curves more heavily was appropriate and justifiable. 15 evaluable patients were to be enrolled at each dose to test the hypothesis that the true success rate was at most 33% (meaning little or no activity) versus the alternative hypothesis that the true success rate was 66% or greater independently in each treatment arm (78% power. We drew peripheral blood mononuclear cells (PBMCs) from two patients before and after treatment with JX-594 and stimulated them with autologous dendritic cells pulsed with an overlapping peptide library (20-mer amino acids overlapping by 15 amino acids) spanning the entire protein sequence of β-gal. Inc. immune parameters and overall survival were other endpoints. Assoc. Dichotomous variables were evaluated by Fisher’s exact test. USA) supplemented with 2% FBS with penicillin and streptomycin. obtained from ATCC) were cultured in DMEM medium containing 10% FBS with penicillin and streptomycin. Twenty-six patients were evaluable for overall Choi response: 2 patients died before the week 8 efficacy assessment and were categorized as nonresponders. Overall survival was assessed by Kaplan-Meier analysis26. © 2013 Nature America. Am. 183. Dojindo. SNU349. data analysis. 20. H. We then exposed cells to PBS and 10 µl Cell counting kit-8 (CCK-8) solution (CCK-8 kit. The primary objective was intrahepatic disease control rate (mRECIST complete or partial response or stable disease) at week 8. Rapidly generated multivirus-specific cytotoxic T lymphocytes for the prophylaxis and treatment of viral infections. HepG2 cells (human hepatocellular carcinoma. et al. A onesided P value was appropriate for this dose-finding study because the objective was to determine whether the high dose was more effective for tumor control and overall survival to justify its potentially higher risk of side effects.L. SNU482 and SNU267 (human renal cell carcinoma. and 22 patients had measurable and evaluable disease. Two patients who were evaluable for mRECIST longest diameter changes were not evaluable for contrast enhancement changes.include tumor decreases from baseline of >10% in longest diameter or >15% decrease in tumor density. Cell viability was measured by optical density at 450 nm. not virally associated. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. et al. NJ for quantification. mRECIST for HCC). We maintained T cells in T cell medium (TCM) (RPMI 1640 (Hyclone) supplemented with 45% Click’s medium (Irvine Scientific). data interpretation and writing of the report. 601–609 (2004). respectively. at 0. We used ELISPOT analysis as a semiquantitative measure of antigen-specific effector T cells as previously described25. We subsequently incubated cells with DMEM (no FBS) and the serum sample at 37 °C for 4 h.03). We also drew PBMCs 1. J. Kulesh. 471–481 (1958). Lox IMVI cells (human melanoma cells. SFC counts and input cell numbers were plotted. Minneapolis. We normalized cell viability in serum after JX-594 administration to the cell viability of patient serum at baseline (before JX-594 treatment). Stat. MN) and 10 ng ml−1 IL-7 (PeproTech). All patients who received at least one dose and had no major documented protocol deviations were evaluable for overall survival. J. obtained from KCLB) cells were cultured in RPMI 1640 (Gibco) supplemented with 10% FBS (Hyclone) with penicillin and streptomycin. pharmacokinetics. Complement-dependent cytotoxicity assay. By protocol. 2 mmol l−1 GlutaMAX TM-I (Invitrogen) and 5% Human AB Serum (Valley Biomedical. We assessed CDC activity by measuring cell viability after incubation with 5% serum in 96-well plates. Role of the funding source. Statistical analyses. et al. Roentgenol. Winchester. We expressed the frequency of T cells specific to each antigen as specific SFC per input cell numbers. whereas continuous variables were compared by t test using the GraphPad Prism 5. Inc. 1622–1632 (2012). We also isolated PBMCs from four healthy donor controls. P. Mol. hepatitis-B associated. 1619–1628 (2004). one-sided type I error rate of 0. these criteria were modified compared to the standard published Choi criteria because standard Choi uses computed tomography scans rather than MRI scans. Microbiol. After 18 h of incubation.5 years after the last JX-594 dose in an additional patient. The trial was designed to enroll 30 patients evaluable for safety and radiographic endpoints. Nonparametric estimation from incomplete observations.0 software (GraphPad Software). Briefly. SK-MEL-2.1 µg per peptide per well. 25.3089 . obtained from KCLB) were cultured in RPMI 1640 (Gibco) supplemented with 10% FBS (Hyclone) with penicillin and streptomycin. J. NY-ESO1. and 24 patients had measurable tumors. obtained from Korean Cell Line Bank (KCLB)) and SNU475. D. We harvested cells on day 9 and tested for β-gal specificity using ELISPOT assay.A.

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