| Global cancer incidence and mortality are rapidly growing in recent years.The reasons are complex but reflect both aging and rapid growth of the population,as well as the changes of social living environment.According to the report of global cancer statistics 2018 published in CA:A Cancer Journal for Clinicians,there will be an estimated 18.1 million new cancer cases and 9.6 million cancer deaths globally in 2018,indicating that the global cancer burden is further aggravated.Therefore,approaches to reduce the global cancer burden,including early screening and early detection and early treatment of cancer and active comprehensive intervention are the most important tasks to prevent and control cancer disease.Conventional treatments for cancer including chemotherapy,radiotherapy and so on,have some major problems,such as serious adverse effects(SAE),failure to control the progress of advanced malignant cancers and improve the quality of life of patients with refractory and extensive metastatic cancer,and thus cannot effectively control cancer.In recent years,targeted cancer therapy has made some breakthroughs in some types of cancer such as lung cancer,leukemia and melanoma.However,targeted cancer therapy works by targeting the cancer’s specific genes and proteins that contribute to cancer cell growth,which is only beneficial to a small subset of patients with these specific genes and proteins.Another major problem is that the continuous evolution of cancer cells leads to the appearance of new mutations while on treatment or after treatment,and thus leads to drug resistance or recurrence.All factors stated above limit the clinical application of cancer-targeted drugs.Cancer immunotherapy,such as anti-PD-1/PD-L1 therapies,is regarded as an immense breakthrough in cancer therapeutics due to their good safety profile and remarkable efficacy.The most prominent advantage of cancer immunotherapy compared to traditional cancer treatments is that patients can acquire durable clinical benefits even after immunotherapy is discontinued.Although anti-PD-1/PD-L1 therapy have durable effects in some cancer types,multiple factors restrict the therapeutic efficacy(overall objective response rate was only about 20～30%),including PD-L1 expression level on cancer cells,tumor mutation burdens,immune cell infiltration,microsatellite instability and mismatch repair system in the tumor microenvironment.Still a large number of the cancer patients cannot benefit from immunotherapy.Therefore,there is still an urgent need to develop other novel and effective treatments so that more cancer patients will benefit from these cancer treatments.Most recently,there are mounting evidences that oncolytic virotherapy is effective in treating cancer in both in pre-clinical animal models and clinical trials with human cancer patients.Oncolytic virotherapy is a new approach for cancer treatment that utilizes natural viruses that have a preferential tropism for tumors or genetically engineered viruses that selectively replicate in cancer cells and lyse these infected cancer cells,and then releases tumor-associated antigens and damage-related molecular patterns and induces immunogenic cell death,and finally initiates host anti-tumor immunity.Compared with other cancer treatments,oncolytic virotherapy has some outstanding advantages,including selective replication and killing of cancer cells,good safety record both in preclinical models and in clinical trials,targeting a broad-spectrum of cancer types and having the capability of transforming "cold" tumors into "hot"tumors and thus synergistically improving the clinical benefits of PD-1/PD-L1 blockade.In 2015,the U.S.FDA approved the world’s first oncolytic virus T-VEC for the treatment of melanoma.In 2018,The New England Journal of Medicine published the results of a Phase Ⅰ trial of PVSRIPO in recurrent glioblastoma,showing the 3-year survival rate of patients with advanced glioblastoma treated with oncolytic poliovirus PVSRIPO was more than five times compared to the historical controls(21%vs 4%).In this thesis,we developed a rational engineered hTERT promoter-regulated oncolytic HSV-1 virus(OVH)and comprehensively explored its safety,anti-tumor activity and mechanism of actions in multiple tumor models.The goal of the first part in this thesis was to rationally design and screen tumortargeting oncolytic virus(OVH),and comprehensively evaluate its tumor selectivity,neurotoxicity and acute toxicity.OVH was modified from wild-type HSV-1 virus(KOS strain),in which the major virulence gene y34.5(encodes ICP34.5 protein)and RL2 gene(encodes ICP0 protein,which was mainly involved in activating viral β and γ gene expression and balancing the latent infection and lyticity,and also involved in viral immune escape)were deleted,and the essential gene ICP27 is under the regulation of the tumor-specific hTERT promoter.In vitro and in vivo results showed that OVH has extremely good tumor selectivity and low neurotoxicity compared with wild-type KOS strains,ICP0-null viruses(dICP0),ICP34.5 and ICP0-null viruses(OVN).Moreover,OVH showed good safety profile in acute challenge model.These results lays the foundation for further exploring the application of oncolytic virus OVH to treat cancers.The second part of this thesis is to evaluate the oncolytic efficacy and mechanism of actions of OVH in vitro and in vivo.We first examined the cancer cell-killing effects of OVH on 53 cultured human cancer cell lines,representing different cancer types.The in vitro results showed that OVH remarkably induced cancer cell death.Of the 53 cancer cell lines that we tested,at 72 h after OVH infection at a MOI=1,40 cancer cell lines showed more than 50%decrease in cell viability,and 8 cancer cell lines showed more than 20%decrease but less than 50%in cell viability.To test the hypothesis that the tumor cell-killing capability of OVH correlated with virus replication,we tested the replication titers of OVH in different cell line sets based on their sensitivity to OVH(high sensitivity(HS),sensitivity(S)and refractory to OVH(R)).The result showed that positive correlation between viral titers and cancer cell-killing capability.Subsequently,we isolated primary liver cancer cells,primary glioma cells and primary ovarian cancer cells from clinical specimens separately,and tested their sensitivity to OVH.In vitro cancer cell-killing results showed that OVH completely kill all the primary cultured cancer cells.All together,the results above showed that OVH can target a broad-spectrum of cancer cells in vitro.To further analyze OVH mediated cell death,we found that OVH infection can induce significant apoptosis and immunogenic cell death(increased extracellular release of ATP and HMGB1,calreticulin membrane translocation),which suggest that OVH may produce "in situ cancer vaccine" to initiate systemic anti-tumor immune response.To explore the oncolytic efficacy of OVH in vivo,we firstly established seven subcutaneous xenograft model implanted with different human cancer cell lines(H1299,Hep3B,CEN-2,MCF-7 and HCT-116)or primary isolated cancer cells(GBM,DHL0712).The results showed that OVH significantly inhibit the growth of xenografted tumors in immunodeficient mice.We next tested the oncolytic efficacy of OVH in three syngeneic bilateral tumor models,OVH treatment can significantly inhibit the growth of established tumors on the injected flank and distant flank tumors,and eradicate the majority of established tumors.These data indicate that intratumoral injection of OVH can induce systemic anti-tumor immune response to inhibit the growth of distant tumors.Additionally,relapse was not observed in OVH cured tumor-bearing mice,and those long-term survived cured mice were resistant to the rechallenge with the same cancer cells implanted before OVH treated,but not resistant to the rechallenge with the non-related cancer cells.Furthermore,we found that the sera from OVH cured mice can specifically react with the same cancer cells but not with the other cancer cells.Those results suggest that the observed anti-tumor effect in the distant tumors was dependent on the injected cancer type and not a result of nonspecific inflammation generated by OVH infection or others.To evaluate the functions of these endogenous antibodies,we developed an approach to deplete all the antibodies from the sera of OVH-cured mice by protein A/G beads,and then carried out the sera adoptive transfer experiments.We found that OVH-elicited specific antibodies has therapeutic potential,sera transferred from OVH-cured mice can reject the same tumor growth of naive recipients.Finally,we analyzed the immune cell subsets within the injected and distant tumor microenvironment.An increase in innate immune cells,including myeloid cells,NK cells,and NKT cells,was observed in tumors of both flanks.Increased infiltration of CD45+ leukocytes,CD3+ lymphocytes,CD4+T cells and CD8+T cells,but decreased Treg+ cells were observed in both flanks,suggesting that abundant immune cells were recruited into both injected and distant tumors.Interestingly,we observed that PD-1+T cells and PD-L1+cancer cells were also upregulated,suggests that concurrent induction of adaptive immune resistance after OVH therapy.Considering the induction of PD-1 on T lymphocytes and PD-L1 on cancer cells,we hypothesized that anti-PD-1 therapy may synergize with OVHmediated therapy.Indeed,combination therapy with anti-PD-1 antibodies and OVH had significantly improved therapeutic efficacy compared with either treatment alone.Depletion experiments revealed that B cells,CD8+ T cells,macrophages and natural killer cells were required for maximal antitumor efficacy of oncolytic immunotherapy.The therapeutic effects of combination therapy was largely dependently on CD8+ T cells.In summary,we have rationally designed a novel oncolytic virus(OVH)that both viral γ34.5 gene and RL2 gene were deleted,and the essential gene ICP27 was under the regulation of the tumor-specific hTERT promoter.OVH can target a broadspectrum of cancer types in vitro and in vivo.OVH infection can induce significantly cancer cell apoptosis and immunogenic cell death,and thus reprogram the tumor microenvironment by inducing robust innate and adaptive immune responses in a setting of large established tumors.Combined with anti-PD-1 antibody therapy can further improve the anti-tumor effect of oncolytic virotherapy. |
PDF Full Text Request