Re-designing Interleukin-12 to enhance its safety and potential as an anti-tumor immunotherapeutic agent

Abstract

Interleukin-12 (IL-12) has emerged as one of the most potent agents for anti-tumor immunotherapy. However, potentially lethal toxicity associated with systemic administration of IL-12 precludes its clinical application. Here we redesign the molecule in such a way that its anti-tumor efficacy is not compromised, but toxic effects are eliminated. Deletion of the N-terminal signal peptide of IL-12 can effect such a change by preventing IL-12 secretion from cells. We use a newly designed tumor-targeted oncolytic adenovirus (Ad-TD) to deliver non-secreting (ns) IL-12 to tumor cells and examine the therapeutic and toxic effects in Syrian hamster models of pancreatic cancer (PaCa). Strikingly, intraperitoneal delivery of Ad-TD-nsIL-12 significantly enhanced survival of animals with orthotopic PaCa and cured peritoneally disseminated PaCa with no toxic side effects, in contrast to the treatment with Ad-TD expressing unmodified IL-12. These findings offer renewed hope for development of IL-12-based treatments for cancer.

Conflict of interest statement

Y.W., P.W., D.G., and N.R.L. are inventors of a filed patent application relevant to this study. The remaining authors declare no competing financial interests.

Figures

Fig. 1

A novel oncolytic adenovirus Ad-TD-LUC is tumor-selective in vitro and in vivo. a The basic structures of the Ad5 genome and derived mutants are shown. The arrows indicate the deleted regions in the genome of adenovirus. IL-12 comprises p40 subunit and p35 subunit linked by an elastic peptide (VPGVGVPGVG), with a signal peptide linked to the p40 subunit. Non-secreting (ns) IL-12 lacks this signal peptide. b−k Tumor selectivity was confirmed by assessing replication of Ad-TD-LUC and wild type AdV in normal cells and in a panel of human cancer cells. Cell lines examined were normal human bronchial epithelial cells (NHBE); primary culture hamster hepatocytes; pancreatic cancer cell lines (PaTu8988T; SUIT2; MiaPaCa2); lung cancer (A549); prostate cancer (PC3); colorectal cancer (HCT116); ovarian cancer (SKOV3) and breast cancer (MCF-7). NHBE Cells were infected at an MOI of 100 particles/cell, the rest were infected at an MOI of 5 PFU/cell. Replication assays were carried out over a period of 72 h. Infectious virus production was assessed by titration on JH293 cells and the titer as PFU/cell calculated and shown as mean and standard error of the mean (SEM). Statistical analysis was performed using a two-way ANOVA test with Bonferroni post-test; *p < 0.05, **p < 0.01; ***p < 0.001. Experiments were performed in triplicate. l Orthotopic PaCa tumors were established in Syrian hamsters using 3 × 106 Hap-T1 cells. Six days later Ad5 or Ad-TD-LUC were injected i.p. (n = 9/group) on day 0, 2, 4, 6, 8, and 10 at a dose of 2.5 × 109 PFU/injection. Animals were killed on day 3, 7, and 14 after the last viral treatments. Tumors, lung, and livers were analyzed by qPCR for copy numbers of the viral E1A gene. The sensitivity of the assay is illustrated by the dotted line. Mean and SEM are shown for each group and compared using an independent t-test. ***p < 0.001

Fig. 2

Ad-TD-LUC and IL-12-armed viruses replicate in and are cytotoxic cancer cell lines. a, b To assess virus cytotoxicity, cell proliferation assays (Promega) were carried out in all human a and Syrian hamster b tumor cell lines. Cells were infected with Ad5, Ad-TD-LUC or Ad-TD-IL-12 or Ad-TD-nsIL-12 and incubated for 6 days at 37 oC. The amount of virus particles required to kill 50% of the cells is shown as EC50 (PFU/cell) and shown as mean and SEM compared using an independent t-test. c−g Replication assays were carried out in hamster pancreatic cancer cell lines, HPD1NR, Hap-T1, SHPC6 and IPAN, and hamster kidney cancer cells (HaK) at an MOI of 5 PFU/cell. Mean and SEM are shown for each group and compared using an independent t-test. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

Ad-TD-nsIL-12 produced IL-12 is functionally active in Syrian hamster cells. a−h To detect IL-12 expression from Ad-TD-IL-12/nsIL-12 in vitro, tumor cells were infected with Ad-TD- IL-12/nsIL-12 or Ad-TD-LUC at an MOI of 5 PFU/cell. Human cancer cells a or Syrian hamster tumor cell supernatants b−f or disrupted cell lysates g were collected every 24 h for 96 h and assayed for IL-12 by ELISA h Intracellular levels of IL-12 were detected in HapT1 cells infected with Ad-TD-IL-12 or Ad-TD-nsIL-12 at an MOI of 5 PFU/cell i mRNA levels of IL-12 were detected in HapT1 cells infected with Ad-TD-IL-12 or Ad-TD-nsIL-12 at an MOI of 5 PFU/cell. mRNA levels relative to β-actin are shown. j, k Recombinant human IL-12 protein (rhIL-12) induced TNF-α j and IFN-γ k expression in hamster splenocytes. mRNA levels relative to β-actin are shown. l Human, hamster, and mouse PBMCs were incubated in the presence of (rhIL-12) or lysate from Hap-T1 cells infected with Ad-TD-IL-12 or Ad-TD-nsIL-12, each at 2 ng/ml, or Ad-TD-LUC infected lysate as a control. Cell proliferation was measured using MTS assay 48 h post-treatment. All experiments were performed in triplicate and the mean and standard error of the mean are shown for each group and compared using an independent t-test. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

Ad-TD-nsIL-12 is an effective treatment for subcutaneous hamster PaCa. 2 × 106 HPD1NR cells were seeded into the right flank of Syrian hamsters. When tumor volumes reached 300 mm3, seven hamsters per group were each injected i.t. with 100 µl PBS, 1 × 109 PFU Ad-TD-LUC or Ad-TD-nsIL-12/IL-12 on days 0, 2, 4, 6, 8, and 10. a Mean tumor sizes and SEM are shown for each group and compared using a one-way ANOVA with post hoc Tukey’s Multiple Comparison Test. *p < 0.05, ***p < 0.001. b Kaplan–Meier survival curves were generated and a log-rank (Mantel–Cox) test used to analyze significance. **p < 0.01, ***p < 0.001. Treatment with both Ad-TD-IL-12 and Ad-TD-nsIL-12 resulted in 100% survival in this model. c Hamsters that had cleared tumors after i.t treatment with Ad-TD-nsIL-12 during efficacy experiments were re-challenged 4 weeks later in the opposite flank with 4 × 106 HPD1NR or 5 × 106 HaK cells, or were injected i.p with anti-CD3 mAb (500 μg/injection) on the day before re-challenge with HPD1NR cells. In parallel, naive hamsters were injected with 5 × 106 HaK cells at the same sites and tumor growth measured and analyzed using a one-way ANOVA with post hoc Tukey’s Multiple Comparison Test. ***p < 0.001

Fig. 5

Ad-TD-nsIL-12 is a safe and effective treatment for peritoneally disseminated PaCa. 1 × 107 SHPC6 cells were seeded into the peritoneal cavity of Syrian hamsters. Four days later, 10 hamsters per group were each injected i.p with PBS or viruses. a PBS or 1 × 109 PFU each virus was injected i.p on days 0, 2, and 4 and survival monitored. b PBS or varying doses of Ad-TD-IL-12 (2.5 × 108 to 1 × 109) were injected i.p. on days 1, 2, and 4 and survival monitored. c, d Using the same tumor model, nine hamsters per group were each injected i.p with 500 µl PBS, 1 × 109 PFU Ad-TD-LUC, 5 × 108 PFU Ad-TD-IL-12 or 1 × 109 PFU Ad-TD-nsIL-12 on day 0. Serum was collected on days 1, 3, and 5 for detection of the levels of ALT, AST, and ASP, respectively, c or IL-12 d detected by ELISA. Mean and SEM are shown. Statistical analysis was carried out using a one-way ANOVA with post hoc Tukey’s Multiple Comparison Test or independent t-test. *p < 0.05, **p < 0.01, ***p < 0.001. e 3 × 109 PFU Ad-TD-nsIL-12 or PBS was administered i.p on day 0 and survival monitored (n = 10). f PBS or 1 × 109 PFU Ad-TD-nsIL-12 was injected i.p on days 0, 2 and 4 (n = 10). Forty days later, surviving hamsters previously treated with Ad-TD-nsIL-12 were re-challenged with 1 × 107 SHPC6 cells. Age-matched naive hamsters also were peritoneally injected with SHPC6 cells as control. Survival was monitored. In each case, Kaplan–Meier survival curves were generated and significance assessed using the log-rank (Mantel–Cox) test. **p < 0.01, ***p < 0.001

Fig. 6

Ad-TD-nsIL-12 is an effective and safe treatment for orthotopic PaCa in Syrian hamsters. Six days after Hap-T1 cells were seeded into the tail of the pancreas, hamsters were injected i.p. with 500 µl PBS, 1 × 109 PFU Ad-TD-LUC, Ad-TD-IL-12 or Ad-TDnsIL-12 (a) or 2.5 × 109 PFU Ad-TD-LUC, 2.5 × 109 PFU Ad-TD-nsIL-12 or 5 × 108 PFU Ad-TD-IL-12 (b) (n = 7/group) on days 0, 2, 4, 6, 8, and 10. Kaplan–Meier survival curves were generated. Significance was assessed using the log-rank (Mantel–Cox) test. *p < 0.05, **p < 0.01. c−h Animals were treated as b (n = 3/time point/group) and killed on day 3, 7, and 14 after last viral treatments. c Representative images of immunohistochemical staining for Hexon at day 7. Hexon-positive cells were counted in five high-power fields (HPF) from each tumor section (×200). ND: not detected. d Infectious virion recovery from tumor tissue was determined by TICD50 using JH293 cells. e Tumors, lung, and livers were analyzed by qPCR for the copy numbers of the viral E1A gene after treatment with Ad-TD-nsIL-12 at 2.5 × 109 PFU/injection on day 0, 2, and 4. The sensitivity of the assay is illustrated by the dotted line. Mean and SEM are shown for each group and compared using an independent t-test. *p < 0.05, **p < 0.01. f Mean tumor volumes and SEM are shown for each group. Statistical analysis was carried out using a one-way ANOVA with post hoc Tukey’s Multiple Comparison Test. *p < 0.05. g Representative images of immunohistochemical staining for CD3 at day 7. Quantitative scores of lymphocyte infiltration within tumors are shown (right panel) from five HPF from each tumor section (×200). The scoring was conducted within the tumor and stroma and necrotic areas were avoided. The extent of positive cells was categorized into the following four grades: 1, <15 cells/HPF; 2, 16–30 cells/HPF; 3, 31–45 cells/HPF; 4, >45 cells/HPF. ND: not detected. h IL-12 expression in sera was detected by ELISA. i Representative histopathology of HE staining of livers after three i.p injections of virus into hamsters bearing orthotopic PaCa tumors (n = 3/group) on day 0, 2, and 4, using the same dose as in b. Livers were collected one day following the last injection and analyzed using HE staining (×200)

Fig. 7

Ad-TD-nsIL-12 efficacy is dependent on hamster CD3+/CD4− immune cell subsets. a, b Syrian hamsters were inoculated subcutaneously with 2 × 106 HPD1NR cells. The established tumors were injected i.t. with 1 × 109 PFU Ad-TD-nsIL-12 or PBS (n = 7/group) on day 0, 2, 4, 6, 8, and 10. Control IgG and either mouse anti-hamster CD3 mAb (4F11) a or CD4 mAb b were injected i.p. at doses of 500 μg/injection every fourth day from the day before the viral therapy to the end of the experiment and FACS analysis used to confirm the depletion. Mean tumor volumes and SEM are shown for each group. Statistical analysis was carried out using a one-way ANOVA with post hoc Tukey’s Multiple Comparison Test. *p < 0.05, ***p < 0.001. c–g 2 × 106 HPD1NR cells were seeded into the right flank of Syrian hamsters. When tumor volumes reached 300 mm3, nine hamsters per group were each injected i.t with PBS, 1 × 109 PFU Ad-TD-LUC or Ad-TD- IL-12/nsIL-12 on day 0. On days 3, 7, and 21 tumors were collected and processed for IHC. c Representative images of immunohistochemical staining for CD3 and CD4 at day 7 (×200). d−f Quantitative scores of lymphocyte infiltration within tumors. Lymphocytes were counted in 5 HPFs randomly selected from each tumor section (×200). The scoring was conducted within the tumor and stroma; necrotic areas were avoided. The extent of lymphocyte infiltration was categorized into the following four grades: 1, <25 cells/HPF; 2, 25–49 cells/HPF; 3, 50–75 cells/HPF; 4, >75 cells/HPF. Statistical analysis was carried out using a one-way ANOVA with post hoc Tukey’s Multiple Comparison Test. *p < 0.05, **p < 0.01, ***p < 0.001. g Spleens and lymph nodes were collected and analyzed by FACS for CD3 and CD4 expression at the time points shown. Mean expression and SEM is plotted (n = 3/group). Statistical analysis was carried out using a one-way ANOVA with post hoc Tukey’s Multiple Comparison Test. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 8

Ad-TD-nsIL-12 efficacy is mediated by production of inflammatory mediators. 2 × 106 HPD1NR cells were seeded into the right flank of Syrian hamsters. When tumor volumes reached 300 mm3, nine hamsters per group were each injected i.t with 100 µl PBS, 1 × 109 PFU Ad-TD-LUC or Ad-TD- IL-12/nsIL-12 on day 0. Spleens, draining lymph nodes (DLN) and tumors were collected and analyzed by qPCR to analyze expression levels of CD83, IFN-γ, IP10, IL-1β, IL-2, IL-6, IL-10, IL-12, and IL-21 at the indicated time points. All experiments were performed in triplicate and the mean and SEM are shown for each group compared using an independent t-test. *p < 0.05, **p < 0.01, ***p < 0.001