Antitumour Efficacy Of A Novel Oncolytic Adenovirus Armed With Non-secreting IL-12 In The Immunocompetent Syrian Hamster Pancreatic Cancer Models

Background and aimPancreatic cancer(PaCa)is currently the most aggressive human cancers,the fourth leading type of cancer death in USA and the fifth leading cause of cancer death in Europe.Despite decades of effort,the five-year survival rate remains at only 5%,was the lowest one of all kinds of cancers.Because most patients have no recognizable symptoms or signs at the early stage,are not diagnosed until in well-advanced stages of local invasion and metastasis in their disease.Moreover,only 15%of patients have a potential to resect the neoplasm of pancreas when diagnosed,and even in operable cases,16%of patients have macroscopically incomplete resections.New therapeutic strategy is clearly imperative.Viral immunotherapy is a promising method to treat cancers.Replication-selective oncolytic viruses are attractive therapeutic candidates for cancer treatment due to their inherent ability to specifically target and lyse tumour cells and induce antitumor effects,good vectors to deliver therapeutic genes.The first generation replication-selective oncolytic human adenovirus 5 ONYX-015(dl1520)was tested for human pancreatic cancer treatment.The treatments were well tolerated,but no objective responses with virus therapy alone were seen in any of the patients,and only 10%(2/21)patients showed objective response when Gemcitabine was combined.Interleukin-12(IL-12)is one of the most potent cytokines in antitumor activity,has pleiotropic effects on different immune cells that form the tumor microenvironment,has not yet been successfully translated into the clinics due to inducing severe side-effects.So,it is important to development a strategy to deliver IL-12 into the tumor microenvironment,achieve durable,local,non-toxic antitumor responses.Replication-deficient and replication-selective adenoviruses expressing IL-12 have shown promising results in different tumor models.Other kinds of oncolytic viruses expressing IL-12 also showed antitumor efficacy.The bad news was most of the oncolytic viruses released high level of IL-12 to the blood or induced a lot of systemic IFN-y,no matter how to deliver the viruses,locally or systemically.The antitumor efficacy of oncolytic viruses is dependent on the interaction of virus,tumor cells,and the host immune response to the virus as well as to tumor cells.Most of the oncolytic adenoviruses had defects in viral structures with E1B 55K and E3 region genes deletion.Based on the knowledge of E1A CR2,E1B 19K and E3 gp19K genes of adenovirus,we constructed a new adenovirus armed non-secreting IL-12(nsIL-12)with these three genes deletion(triple deletion,TD).Adenovirus 5 replicates well in Syrian hamster tissues,permissive cancer cell lines are available to be used for antitumor studies,and Syrian hamster and human have similar symptoms of pancreatic cancer.To investigate whether Ad-TD-nsIL-12 has superior targeting and antitumor potency and safety,the tests were performed in immunocompetent Syrian hamster.Part ? Construction and identification of the novel oncolyticadenoviruses1 ObjectiveTo construct and identify the novel tumor targeting oncolytic adenovirus expressing immune therapeutic gene.2 Methods2.1 Construction of shuttle verctors for homologous recombinationCloning the genes using high fidelity enzyme,inserting the gene fregements into shuttle vectors with left and right homologous arms to construct the homologous shuttle vectors.2.2 Homologus recombination of adenovirus by electroporationLinerizing the homologous shuttle vector by Pme I,transfering the shuttle vector and adenovirus vector into BJ5183 to do homologous recombination by electroporation.2.3 Production and identification of the novel oncolytic adenovirusLinerizing the adenovirus vector by Pac I,transfecting the 293 cells,producing the infectious adenovirus;producing and the purifying the adenovirus;sequencing the genome of the addenocirus;detecting the expression of IL-12 by ELISA.2.4 The effects of human IL-12 on immune cells of Syrian hamsterPBMCs were separated and incubated with PHA for three days.Then,PBMCs were seeded in a 96-well plate and incubated with human IL-12 for 48 hours.PBMCs proliferation was determined by MTS assay.Splenocytes were separated and incubated with PHA for three days,incubated with human IL-12 for 24 and 48 hours.qPCR detects the expressions of IFN-? and TNF-?.3.Results3.1 Construction of shuttle verctors for homologous recombinationSequencing results showed the pssIL-12,pssnsIL-12 and pssLUC vectors were successfully constructed.3.2 Homologus recombination of adenovirus by electroporationSequencing results showed the novel oncolytic adenovirus vectors Ad-TD-IL-12?Ad-TD-nsIL-12 and Ad-TD-LUC were successfully constructed.3.3 Production and identification of the novel oncolytic adenovirusHigh titer adenoviruses were produced.Genomic sequencing of the adenoviruses showed the nucleotides were correct.The expression of IL-12 was detected in Ad-TD-IL-12 and Ad-TD-nsIL-12-infected tumor cells.3.4 The effects of IL-12 on immune cells of Syrian hamsterHuman IL-12 espressed by Ad-TD-IL-12 and Ad-TD-nsIL-12 has effects on the proliferation of huam and Syrian hamster PBMC,but not mouse.Human IL-12 induced IFN-? and TNF-? expression in Syrian hamster splenocytes.4 ConclusionsThe novel tumor targeting oncolytic adenovirus expressing immune therapeutic gene was successfully constructed and identified.Human IL-12 has effects on immune calls of Syrian hamster.Part ? The antitumor effects of the novel oncolytic adenoviruses onadevanced pancreatic cancer in immunocompetent hamster 1 ObjectiveTo investigate the antitumor potency of the novel oncolytic adenovirus on pancreatic cancer in immunocompetent Syrian hamster.2 Methods2.1 The cytotoxicity of the novel oncolytic adenovirus in tumor cellsInvestigating the cytotoxicity of dl1520 and the novel oncolytic adenoviruses in pancreatic cancer cells and renal tumor cells of Syrian hamster by MTS assay.2.2 The viral replication of the novel oncolytic adenovirus in normal and tumor cellsComparing the viral replication of dl1520 and the novel oncolytic adenovirus in normal and tumor cells.2.3 The antitumor potency of the novel oncolytic adenovirus in subcutaneous pancreatic cancer model in Syrian hamsterEstabishing the Syrian hamster pancreatic cancer HPD1NR subcutaneous tumormodel,when the tumor volume reaches to 80 mm3 or 160 mr m monitor the tumor growth after dl1520 and Ad-TD-LUC treatments.when the tumor volume reaches to 310 mm3,monitor the tumor growth after Ad-TD-IL-12,Ad-TD-nsIL-12 and Ad-TD-LUC treatments.2.4 The antitumor potency of the novel oncolytic adenovirus in orthotopic pancreatic cancer model in Syrian hamsterEstabishing the orthotopic pancreatic cancer Hap-T1 model in Syrian hamster,monitor the survival time of hamster after Ad-TD-IL-12,Ad-TD-nsIL-12 and Ad-TD-LUC treatments.2.5 The antitumor potency of the novel oncolytic adenovirus in peritoneally disseminated pancreatic cancer model in Syrian hamsterEstabishing the peritoneally disseminated pancreatic cancer model in Syrian hamster,monitor the survival time of hamster after Ad-TD-IL-12,Ad-TD-nsIL-12 and Ad-TD-LUC treatments.3 Results3.1 The cytotoxicity of the novel oncolytic adenovirus in tumor cellsThe novel oncolytic adenoviruses had more oncolytic effects compared with dll 520 in pancreatic cancer cells and renal tumor cells of Syrian hamster.3.2 The replication of the novel oncolytic adenovirus in normal and tumor cellsReplication of the novel oncolytic adenovirus was greatly attenuated compared with that of dl1520 after infection in normal cells.In tumour cells,replication of the novel oncolytic adenoviruses were promoted.3.3 The antitumor potency of the novel oncolytic adenovirus in subcutaneous pancreatic cancer model in Syrian hamsterThe Syrian hamster pancreatic cancer HPD1NR subcutaneous tumor model was estabished.When the tumor volume reaches to 80 mm3,Both Ad-TD-LUC and dl1520 demonstrated anti-tumour efficacy.However,when the tumour volume reaches to 160 mm3,dl1520 had no distinct antitumor efficacy,Ad-TD-LUC still resulted in a superior antitumor efficacy compared with PBS-treated animals.When the tumor volume reaches to 310 mm3,treatments with Ad-TD-IL-12 and Ad-TD-nsIL-12 resulted in superior antitumor efficacy with 100%of hamsters showing tumor clearance compared with PBS-treated animals.Ad-TD-LUC treatment showed no distinct tumor regression.3.4 The antitumor potency of the novel oncolytic adenovirus in orthotopic pancreatic cancer model in Syrian hamsterThe orthotopic pancreatic cancer Hap-T1 model was estabished in Serian hamster.Treatment with low dose Ad-TD-nsIL-12 resulted in a significant difference in survival rate compared with PBS treatment.Ad-TD-LUC treatment showed no distinct promotion of survival time.All the hamsters treated with Ad-TD-IL-12 died earlier than PBS group's.High dose Ad-TD-nsIL-12 treatment resulted in a significant difference in survival rate compared with PBS treatment.Ad-TD-LUC and low dose Ad-TD-IL-12 treatments showed no promotion of survival rate.3.5 The antitumor potency of the novel oncolytic adenovirus in peritoneally disseminated pancreatic cancer model in Syrian hamsterThe peritoneally disseminated pancreatic cancer model was estabished in Syrian hamster.Ad-TD-LUC treatment increased the survival rate compared with PBS-treated animals.Ad-TD-nsIL-12 treatment cured the animal with 100%survival.There was no significant difference in survival rates between Ad-TD-IL-12-treated and PBS-treated groups.One injection of high dose of Ad-TD-nsIL-12 also resulted in 100%survival.4 ConclusionsAd-TD-nsIL-12 has superior antitumor potency and safety in different tumor models of Syrian hamster pancreatic cancer.Part III The functional mechanisms of antitumor potency and saftyof the novel oncolytic adenovirus1 ObjectiveTo investigate the mechanism of antitumor potency and safty of Ad-TD-nsIL-12.2 Methods2.1 The effect of Ad-TD-nsIL-12 on liver function.Detecting the liver functional enzymes and IL-12 expression in the sera of Syrian hamster peritoneally disseminated pancreatic cancer model after oncolytic adenovirus treatment.2.2 The study of the specific antitumor immunity and long-term immune protection induced by the novel oncolytic adenovirus.Syrian hamsters that had cleared tumors after intratumoral treatment with Ad-TD-IL-12 or Ad-TD-nsIL-12 during efficacy experiments were rechallenged 4 weeks later in the opposite flank with HPD1NR or HaK cells or were intraperitoneal injected with anti-CD3 mAb at the day before the rechallenge with HPD1NR cells.Forty days later,Syrian hamsters bearing peritoneally disseminated pancreatic cancer were rechallenge with SHPC6 after viral treatments.2.3 The analysis of T cell populations mediating the antitumor potency of Ad-TD-nsIL-12Estabishing the Syrian hamster pancreatic cancer HPD1NR subcutaneous tumor model,when the tumor volume reaches to 300 mm3,CD3+ and CD4+ immune cells were depleted from hamsters before treatment with Ad-TD-nsIL-12,monitored the tumor growth.2.4 The effects of Ad-TD-nsIL-12 on tumor microenvironment and host immunity.Estabishing the Syrian hamster pancreatic cancer HPD1NR subcutaneous tumor model,when the tumor volume reaches to 300 mm3,one injectin of oncolytic adenoviruses was delivered into tumors,spleens,draining lymph nodes and tumors were collected.FACS,qPCR and IHC were performed to investigate the alterations of immune cells and cytokines in spleens,draining lymph nodes and tumors.2.5 The antitumor potency and safetyof Ad-TD-nsIL-12 in orthotopic pancreatic cancer model in Syrian hamsterEstabishing the orthotopic pancreatic cancer Hap-T1 model in Syrian hamster,collect the tumors,sera and livers at different time points after Ad-TD-IL-12,Ad-TD-nsIL-12 and Ad-TD-LUC treatments.ELISA,real time PCR,HE staining and IHC were performed to detect IL-12 expression,viral distribution and TIL.2.6 The antitumor potency of nsIL-12 in vaccinia virus and Adenovirus11 vectorsNsIL-12 gene was inserted in vaccinia virus and Adenovirus11 vectors to construct VV-nsIL-12 and Adll-nsIL-12.Their antitumor potency were tested in Syrian hamster bearing peritoneally disseminated pancreatic cancer.3 Results3.1 The effect of Ad-TD-nsIL-12 on liver function.There was no obvious elevation of liver enzymes in Ad-TD-nsIL-12 and Ad-TD-LUC-treated groups.However,significant elevations of liver enzymes were found in the Ad-TD-IL-12-treated group at three time points.The IL-12 expression level of Ad-TD-IL-12-treated group was dramatically higher than that of Ad-TD-nsIL-12-treated group.The level of IL-12 decreased rapidly in the sera of Ad-TD-IL-12-treated group,but kept the similar level in the sera of Ad-TD-nsIL-12-treated group.3.2 The study of the specific antitumor immunity and long-term immune protection induced by the novel oncolytic adenovirus.Hamsters that had cleared tumors after intratumoral treatments with Ad-TD-IL-12 or Ad-TD-nsIL,12 during efficacy experiments were rechallenged with HPD1NR or HaK cells.HPD1NR cells did not form tumor,but Hak tumors kept growing.When the hamsters were intraperitoneal injected with anti-CD3 mAb at the day before the rechallenge with HPD1NR cells,the HPD1NR tumors grew.90%of hamsters bearing peritoneally disseminated pancreatic cancer did not recur.3.3 The analysis of T cell populations mediating the antitumor potency of Ad-TD-nsIL-12The Syrian hamster pancreatic cancer HPD1NR subcutaneous tumor model was estabished.CD3+ and CD4+ immune cells were depleted from hamsters before treatment with Ad-TD-nsIL-12.Depletion of CD3+ cells had a significantly detrimental effect on the efficacy of treatment,but not CD4+.3.4 The effects of Ad-TD-nsIL-12 on tumor microenvironment and host immunityThere was similar TIL in Ad-TD-nsIL-12 and Ad-TD-IL-12-treated tumors.Ad-TD-IL-12 treatment resulted in higher levels of cytokines associated with antitumor effect compared with other treatments.The frequency of total CD4+ and CD3+CD4-cells significantly increased after treatment with Ad-TD-nsIL-12 at day 7 in draining lymph nodes,there was no alteration in spleen.Ad-TD-IL-12 treatment also resulted in CD3+CD4-cells significantly increased in draining lymph nodes at day 7,but resulted in CD3+CD4-cells significantly increased in spleen at day 3,7 and 21.3.5 The antitumor potency and safetyof Ad-TD-nsIL-12 in orthotopic pancreatic cancer model in Syrian hamsterThe tumor growth curve showed Ad-TD-IL-12 and Ad-TD-nsIL-12 inpressed tumor growth.Oncolytic viruses targeted and replicated in tumor cells by qPCR snd IHC assays.TILs significantly incressed in Ad-TD-IL-12 and Ad-TD-nsIL-12 treatment groups.There was no obvious toxicity in the livers.3.6 The antitumor potency of nsIL-12 in vaccinia virus and Adenovirusll vectorsThe Syrian hamsters bearing peritoneally disseminated pancreatic cancer were treated with 'VV-nsIL-12 or Ad11-nsIL-12 or control viruses.The survival rates were significantly increased.4 Conclusions4.1 The safty of Ad-TD-nsIL-12 is due to Ad-TD-nsIL-12 can durably and locally express low level of IL-12.Ad-TD-nsIL-12 only affects the local immune organ,draining lymph node,and alters the tumor microenvironment.4.2 Ad-TD-nsIL-12 treatment can induce specific antitumor immunity and long-term protection.CD3+CD4-T cells play an important role in antitumor immunity.