- Title
- Enhancement of oncolytic coxasckievirus A21 with conventional chemotherapies and immune checkpoint inhibitors for the treatment of melanoma
- Creator
- Quah, Min Yuan
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2016
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Malignant melanoma is one of the most aggressive and lethal cancers of the skin. Clinical reports have shown patients diagnosed with stage IV of the disease have no longer than five years to live. Depending on the sub-stage of the disease, median survival of patients is between 6 - 18 months. To date, current Food and Drug Administration (FDA) approved anticancer drugs for melanoma have demonstrated limited response rates, few complete remissions and no significant survival benefits. The development of new therapies to effectively eradicate malignant melanoma are desperately in need. Recent growth in the field of oncolytic virotherapy has led to an increased number of clinical trials and acceptance of tumour selective viruses as a promising anticancer strategy. Numerous viruses have emerged as potent oncolytic agents because of their capacity to preferentially infect and destroy cancer cells while leaving normal cells intact. Like most anticancer modalities, it is very likely that oncolytic virotherapy will be used in combination with other existing therapeutics. Numerous groups have begun to explore the possibility of combining oncolytic virotherapy with conventional cancer therapies. The combination of oncolytic virotherapy with other existing cancer therapies may be an effective strategy to overcome the barriers faced by either therapeutic agents when used as monotherapies. The Kuykendall strain of Coxsackievirus A21 (CVA21) is a naturally occurring common-cold virus that has the inherent capacity to preferentially infect and destroy malignant cells. The oncolytic properties of CVA21 as an anticancer agent have been demonstrated both in vitro and in vivo studies, against numerous types of cancer, including breast cancer, prostate cancer, glioma, multiple myeloma, non-small cell lung carcinoma, and melanoma. In the clinic, tumour regression was observed in both injectable and distant non-injected lesion without any significant adverse events. Despite CVA21’s impressive progress to date, it is becoming clear the oncolytic viruses (OVs) cannot be viewed as monotherapies. The current trend is for the combination of virotherapy with mainstream therapies. A fundamental requirement for the success of this strategy is that the combination does not interfere with the life cycle of the oncolytic virus from the point of viral entry, to the release of viral progeny. In Chapter 4, we demonstrated that not only does the co-administration of clinically relevant cytotoxic anticancer drugs such as dacarbazine, paclitaxel and carboplatin not interfere with replication cycle of CVA21, enhanced cell destruction of melanoma cells was achieved when both oncolytic agent and chemotherapy were combined. To determine whether actual synergism was present, we subjected our data to the Chou-Talalay median effect equation which provides a robust measurement of drug combination relationships based on combination index values. When the constant drug-ratio design was applied to our combination assays, we discovered that CVA21 does indeed act synergistically with conventional chemotherapy in all melanoma cell lines except SK-Mel-28. However, we anticipate that it is possible to achieve synergistic or additive effects in all cell lines by carefully adjusting the drug concentrations used. Indeed, the combination of CVA21 with either of the chemotherapeutic agents at clinically relevant drug concentrations, resulted in increased cell death in SK-Mel-28 cells. Next, we were interested in identifying the underlying mechanism by which CVA21 is able to synergise with dacarbazine, paclitaxel and carboplatin. No increase in viral replication was observed in any of the test cell lines following combination chemotherapy treatment vs virus alone. Cell cycle analysis revealed an increase in the sub-G1/G₀ cell population after treatment with the combination of CVA21 and paclitaxel + carboplatin, but was not associated with the activation of caspase-3/7. Taken together, the combination of CVA21 plus chemotherapy has significant activity in a panel of melanoma cells though the mechanism behind this effect remains to be elucidated. In Chapter 5, we sought to determine whether the synergistic cancer cell death was reproducible in vivo. Two melanoma animal models were designed; an immunodeficient melanoma xenograft model and a syngeneic immunocompetent mouse model. Intratumoural injection of infectious CVA21 particles in the presence or absence of cytotoxic chemotherapy mediated complete regression of all injected melanoma xenografts. However, there was no statistical difference between single agent CVA21 and CVA21 in combination with chemotherapy in our immunodeficient mouse model. Such a result was not unexpected, as the absence of neutralising antibodies in the immunodeficient model allowed the virus to replicate indefinitely until all tumour cells were destroyed. Having proven that cytotoxic chemotherapy failed to interfere with the replication cycle of CVA21 in vivo, we developed a syngeneic immunocompetent mouse model and treated the animals with the same combinations as per the immunodeficient study. Overall, we observed an excellent level of treatment tolerability and tumour clearance in both single agent/combination therapy treatment groups. Results from both animal models show that the presence of either chemotherapy does not harm the infectivity or integrity of CVA21 virions in vivo and provides a strong rationale for clinical translation. In Chapter 6, we explored the immunotherapeutic potential of oncolytic CVA21. In the first section of this chapter, we examined whether vaccination of immunocompetent animals with cell lysates induced by CVA21 oncolysis (an oncolysate) could initiate the activation of anti-tumour immunity. Following immunisation with the oncolysates, animals were challenged with B16 cells and the development of flank tumours was recorded. Although B16 challenged animals showed reduced tumour growth, it became apparent that viral-mediated lysis of tumour cells alone was insufficient for complete tumour eradication. Thus, based on these observations, the next logical step was to combine oncolytic CVA21 with other immunotherapeutic agents to enhance the activation of anti-tumour immune responses. In the next section, we investigated if the immunomodulatory effects of two T cell regulatory receptors, PD-1 and CTLA-4, together with CVA21 could be harnessed to improve therapeutic efficacy. Animals were treated with intratumoural injections of CVA21 and intraperitoneally with either anti-PD-1 or anti-CTLA-4 antibodies. Animals were rechallenged with B16 cells lacking the CVA21 targeting receptor human ICAM-1 and the development of the secondary flank tumour was used as a measure for the generation of a protective anti-tumour immune response. Remarkably, the combination group displayed a superior anti-tumour response when compared to control animals or either of the agents as monotherapies. We were particularly successful in demonstrating this effect with the CVA21-CTLA-4 combination as indicated by the the complete regression of the primary tumour in all treated animals, followed by durable resistance to the secondary tumour challenge in 40% of the animals on study. The experimental data presented herein highlights the potential of CVA21 as a useful platform for combination strategies. Using the newly established, fully immunocompetent CVA21-susceptible mouse model of malignant melanoma, we showed that CVA21 in combination with dacarbazine or paclitaxel and carboplatin had significant advantages over either agents as monotherapies. Furthermore, the model was extended to evaluate the protective anti-tumour response generated from CVA21 oncolysis by challenging animals with murine melanoma cells without ICAM-1 surface receptors after the initial treatment. Favourable immune responses and increased therapeutic benefits were observed when CVA21 was administered with the immune checkpoint inhibitors PD-1 and CTLA-4. This study is further proof-of-concept that immunovirotherapy of cancer is achievable with CVA21, leading the way for future clinical trials combining checkpoint modulation with CVA21.
- Subject
- chemotherapy; melanoma; oncolytic coxsackievirus A21; skin cancers; oncolytic virotherapy
- Identifier
- http://hdl.handle.net/1959.13/1312749
- Identifier
- uon:22457
- Rights
- Copyright 2016 Min Yuan Quah
- Language
- eng
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