Developing a novel therapeutic vaccine combining IL-10 inhibitor for late stage cancers with targeted Clostridial spore oncolysis of the tumour microenvironment

Abstract

Background: Cancer is a class of diseases characterised by uncontrolled proliferation of cells. Cancer represents as a major public health problem accounting for approximately 8.8 million deaths each year worldwide. Current treatments include surgical removal of tumour mass, chemotherapy, radiotherapy, or a combination of these therapies. Often these options result in side effects that can be quite detrimental to the patients. Therefore, new treatments are urgently needed. Cancer therapeutic vaccines are aiming at killing tumour cells without affecting normal cells. Vaccine induced cytotoxic T lymphocyte (CTL) responses are pivotal for the killing tumour cells. Interleukin 10 (IL-10) is a cytokine with multiple biological functions. Blocking IL-10 signalling with an anti-IL-10 receptor antibody at the time of immunisation drastically increases vaccine induced immune responses and prevents tumour growth in animal models. However, it is not clear whether there are unwanted side effects with such a blockade, and no clinical grade IL-10 inhibitor for human is available. Advanced cancers show a significant increase in angiogenesis resulting in the formation of abnormal blood vessels, which restrict blood supply and result in reduced oxygen levels within the cancer. Therefore, some areas of the late stage tumour are hypoxic and necrotic. Also, the solid tumour microenvironment (TME) is immunosuppressive. Our laboratory has developed an anaerobic Clostridium ghonii strain. When these clostridial spores are administered and regerminated in hypoxic and necrotic cancer central region, they cause significant oncolysis, leading to tumour regression in animal models. The oncolytic activity may also destroy the TME. Hypothesis and Aims: The hypothesis of the project is (1) Blocking IL-10 at the time of immunisation should increase vaccine induced T cell responses without eliciting unwanted side effects; (2) Humanised and active IL-10 inhibitors could be designed by using computerised programs; (3) Clostridial spores induce oncolysis of cancer should also improve immune cell penetrations to cancer in situ, thus achieving a robust anti-cancer immune response. To test these hypothesis, the thesis is specifically aimed: 1) To investigate whether an antibody-mediated blocking of IL-10 signalling at the time of an immunisation will generate any unwanted side effects; 2) To establish whether new anti-IL-10 peptides designed through computer modelling are bioactive in both vitro and in vivo; 3) To determine whether oncolytic events in anaerobic Clostridium ghonii spore treatment of modelled cancers will improve the immune suppressive TME. Results and discussion: (1) In a mouse TC-1 tumour model, blocking of IL-10 with an antibody at the time of immunisation increases the numbers of IL-10 producing CD4+ T cells in the spleen and draining lymph nodes, and does not result in blood cell infiltration to the intestines and does not cause intestinal pathological changes. These results indicate that immunisation with an IL-10 inhibitor may facilitate a generation of safe and effective therapeutic vaccines against chronic viral infection and cancer. (2) Computer designed Peptides P1, FFKKF FKKFF KKFFK K-OH and P2, FFRRF FRRFF RRFFR R-OH are based on the hydrophobic and hydrophilic pattern of the IL10R-binding helix with IL-10. It could be bound with either IL- 10R1 or IL-10, and inhibit inflammatory signals for a long duration and result in negligible cytotoxicity in vitro. Furthermore, P2 can enhance the vaccine induced antigen specific CD8+ T cell’s responses in mice. As evidenced by both experimental and molecular dynamic simulation, the N-terminal hydrophobic peptide constructed with repeating hydrophobic and hydrophilic pattern of residues is more likely to inhibit IL-10. In addition, the sequence length and the ability of protonation are also important for inhibition activity. (3) Intravenous administration of a derivative of Clostridium ghonii (DCG) spore leads to pro-inflammatory responses characterised by increased levels of IFN-γ or/and Interleukin-9 (IL-9), depending on the dosage of DCG administration. Antigen specific CD8+ T cell responses are elicited after DCG spore administration, and IFN-γ secreting T cells were attracted to the tumour site. Conclusion: Taken together, blocking IL-10 signalling at the time of immunisation does not increase unwanted side effects in mice; a peptide P2 designed through computer modelling is able to inhibit IL-10-mediated signalling both in vitro and in vivo; the length of the peptide and the ability of protonation are critical for the peptide’s function. Finally, systematic administration of DCG spore attracts more IFN-γ secreting T cells to the tumour site. Thus, it is believed that if administration of DCG spore is combined with a therapeutic vaccine, more antigen specific T cells may be attracted to the tumour site, therefore a better outcome for cancer treatment could be achieved. In this project, it is the first time to demonstrate that blocking IL-10 at the time of immunisation increases immune response without significant side effects; Also, the newly developed peptide based IL-10 signalling inhibitors are active, paving the way for the testing of a humanised IL-10 inhibitor; the project also showed a Clostridium ghonii spore-mediated oncolysis could disrupt the TME, may improve vaccine mediated anti-cancer immune responses. If such approaches are combined, an exciting new way of cancer therapy may be developed for advanced cancers.