| Background and objectiveHead and neck cancer(HNC)is the sixth common malignant cancer and the eighth leading cause of cancer-related death worldwide.More than 90% of HNCs are classified as squamous cell carcinoma(SCC),harboring high malignancy,and can invade the distant organ at the early stage.Currently,the therapies of the patients with HNCs were determined based on primary location and grading of invasion and metastasis,and the therapeutic strategies involve conventional surgery,chemotherapy and radiotherapy.Despite a large amount of advances in surgery,chemotherapy and radiotherapy,the survival and quality of life of HNC patients have not been significantly improved in clinic.Therefore,it is imperative to develop the novel therapeutic strategy for HNCs.Recently,biotherapy is the fourth most effective tumor treatment strategy after surgery,radiotherapy and chemotherapy.Replication-selective oncolytic adenoviruse(RSOA)has been recognized as a promising new therapeutic approach for cancer treatment.Virus immune gene therapy,based on RSOA as vectors,can selectively replicate in tumor tissues and lyse tumor cells,and the immune gene inserted by genetic engineering technology could express proteins in the local tumor tissue,which may enhance the antitumor efficacy of RSOA.The genetically-modified adenovirus H101(Sunway,Shanghai,China)is the first oncolytic virus to be accepted,receiving Chinese FDA approval in 2005 for the treatment of head and neck cance.We believe that encouraging adenoviral agents must be tested in well designed clinical trials as soon as possible.Interleukin-12(IL-12)has emerged as one of the most potent cytokines in mediating antitumor activity in a variety of preclinical models.IL-12 plays pleiotropic effects on different immune cells in the tumor microenvirnment,and IL-12 establishes a link between innate and adaptive immunity.The robust antitumor response exerted by IL-12 was associated with toxic side effects of hematology,and it is urgent to reduce systemic level of IL-12.Based on the preliminary studies,we constructed a new adenovirus armed ns IL-12: Bio TTT001(Ad-TD-ns IL-12),using Ad-TD(Patent no.ZL200910066130.4)as the backbone virus to enhance the anti-tumor efficacy markedly.Bio TTT001 has been verified to be excellent antitumor effect with low toxic in pancreatic cancer model peviously.Human Ad5 replication is generally species specific,replicating poorly in cells from most other species.In contrast to most other species examined,the Syrian hamster is permissive for human Ad5 replication.So the Syrian hamster has been an immunocompetent,replication-permissive animal model for the evaluation of oncolytic Ad5 vectors.The purpose of the current study is to evaluate the antitumor activity of the novel adenovirus Bio TTT001 and preliminarily investigate the mechanism of antitumor.These results will lay the foundation for clinical application of Bio TTT001.Chapter 1 Antitumor efficacy of the novel oncolytic adenovirus Bio TTT001 in vitroMethods1.Bacteria and mycoplasma contamination were detected by antibiotic-free LB medium and polymerase chain reaction(PCR)with mycoplasma specific primers,respectively.2.Cytotoxicity of the novel oncolytic adenovirus Bio TTT001 in tumor cell lines was detected by crystal violet staining and MTS.Apoptosis was detected by flow cytometry with Annexin V-FITC/PI staining in tumor cells infected with Bio TTT001.3.The replication and h IL-12 expression of Bio TTT001 were detected by TCID50 and enzyme linked immunosorbent assay(ELISA),respectively.4.Statistical treatment: All data were investigated by Graphpad Prism 5.0 and the comparisons of multiple samples were done by One-way ANOVA and Tukey’s multiple comparison test.P < 0.05 was considered as statistical significance.Results1.The viruses(Bio TTT001,Ad-TD-Luc and dl1520)applied in this study were both bacteria-free and mycoplasma-free.2.Adenoviruses lyse HCPC I cells(Syrian hamster cheek pouch carcinoma cell)and C666-1 cells(human nasopharynx cancer cell)more efficiently than the SCC7 cells(mouse squamous cell carcinoma cell).The EC50 values of Bio TTT001 in a panel human cancer cell lines were below 0.5.Apoptosis occurred in HCPC I and C666-1 cell lines at 48 h after the infection with viruses,but not in SCC7 cells.3.The replication ability of Bio TTT001 in HCPC I and C666-1 cells was similar to dl1520.The replication ability of viruses in C666-1 cells reached peak at 48 h,whereas the replication level in HCPC I cells was low,with time progression,the replication ability gradually increased.The expression of h IL-12 in HCPC I and C666-1 celllines displayed in time-dependent manner.In addition,the h IL-12 level reached 1000pg/m L in various human tumor cell lines after infected with Bio TTT001.Chapter 2 Antitumor efficacy of the novel oncolytic adenovirus Bio TTT001 in vivoMethods1.5.0×106and 1.0×107HCPC I cells were subcutaneously injected into Syrian hamsters,respectively,and tumor forming time and size were observed.Tumor histomorphology was verified by Hematoxylin-eosin(HE)staining.2.1.0×107HCPC I cells were subcutaneously injected in the right flanks of Syrian hamsters,when tumors volume reached 350mm3,7 hamsters per group were injected intratumorally(IT)with 2.0×108PFU dl1520,Ad-TD-Luc,Bio TTT001 or100μl PBS on days 1,2,3,4,5 and 6 or days 1,3,5,7,9 and 11;another treatment schedule was on days 1,3,5,7,9 and 11 with 5.0×107PFU virus.Tumor volume was measured twice weekly,and the tumor growth curve was made using Graphpad Prism 5.0 software.3.1.0×107 HCPC I cells were subcutaneously injected in the right flanks of Syrian hamsters,when tumors volume reached 350mm3,7 hamsters per group were injected introperitoneally at doses of 500μg/injection with PBS,GK1.5(anti-mouse CD4 m Ab),4F11(anti-hamster CD3 m Ab),or Ig G control.24 h later,5.0×107PFU of dl1520,Ad-TD-Luc,Bio TTT001 or 100μl PBS was injected IT on days 1,3,5,7,9 and 11.The antibodies were injected twice a week untill the ending of experiment.4.The tumor-free hamsters under the treatment with Bio TTT001 were rechallenged 4weeks later in the opposite flanks with 2.0×107HCPC I cells,and partial of hamsters injected intraperitoneally with 4F11.5.Statistical treatment: All data were investigated by Graphpad Prism 5.0 and the comparisons of multiple samples were done by One-way ANOVA and Tukey’s multiple comparison test.P < 0.05 was considered as statistical significance.Results1.Subcutaneous inoculation with 1.0×107HCPC I cells was superiority over low dose 5.0×106 in tumor formation.After HE staining,tumor tissues appeared obvious tumor features.2.In the HCPC I subcutaneous model,interval viral treatment with intratumor injection showed a better antitumor efficacy compared to consecutive viral treatment.When the viral treatment dose reduced to 5.0×107PFU,Bio TTT001 still has a strong antitumoral effect,compared to backbone virus and control virus,and the tumor free rate was 85.7%(6/7).3.CD3+and CD4+T lymphocyte were depleted from hamsters before treatment with Bio TTT001.Depletion of CD3+ T lymphocyte had a significantly detrimental effect on the viral treatment efficacy,but not CD4+ T lymphocyte.4.Tumor free hamsters after intratumoral treatment with Bio TTT001 were rechallenged with HCPC I cells,did not form tumor;while tumors kept growing when hamsters were injected with 4F11 antibody intraperitoneally.Conclusions1.The cancer cell lines drived from Syrian hamster and human are more sensitive to oncolytic adenovirus than mice.Bio TTT001 harbores strong killing efficacy on a panel of human tumor cells,and thus may be a promising tumor agent for a large number of tumors in clinic.2.The antitumor efficacy of interval treatment with Bio TTT001 virus is superiority over that of consecutive viral treatment.Bio TTT001 can effectively suppress tumor growth in HNC subcutaneous model,and its antitumor efficacy is stronger than that of the first generation virus dl1520.3.Bio TTT001 can induce long-term immune protection.Most notably,CD3+T lymphocytes play an essential role in antitumour efficacy mediated by Bio TTT001. |
PDF Full Text Request