Abstract
The immune responses generated against tumour cells are not always capable of controlling tumour growth. Immunotherapeutic vaccines utilise the immune system’s ability to detect and destroy tumour cells and are a promising strategy for effective cancer treatment. There is however a need for the development of improved immunotherapy strategies as clinically tested immunotherapies have not yet provided the hoped for effective anti-tumour responses. Co-delivery of tumour antigen and vaccine adjuvant into the same antigen-presenting cell (APC) at the same time has been shown to be a more effective way at inducing anti-tumour immune responses compared to mixtures of antigen and adjuvant. The co-delivery of antigen and adjuvant is believed to generate this enhanced efficacy by mimicking the immunological processing of complex pathogens to induce cytotoxic T lymphocyte (CTL) responses, as the complex pathogens naturally comprise both antigen and adjuvant. Direct chemical conjugation of the antigen to adjuvant may however limit their processing by the APC for immune stimulation. The aim of this research was to test this theory and improve the effectiveness of soluble vaccine conjugates. Antigen-adjuvant conjugates were designed to be cleaved by an intracellular trigger to release antigen and adjuvant from each other following internalisation by APC. The intracellular triggers used for targeted release of antigen from adjuvant were redox and enzyme triggers. For reduction triggered release a disulphide linker was used, we generated two disulphide conjugates with different chemistries. For enzyme triggered release a peptide sequence was used which was specific for cleavage by Cathepsin S.
These different linking systems were used to conjugate ovalbumin (OVA) as a model tumour antigen and cytosine-phosphate guanosine (CpG) as a vaccine adjuvant. The activity of these cleavable conjugates was compared to non-cleavable conjugates and mixtures of antigen and adjuvant. Two stable conjugates were synthesised, one with a zero length spacer (C-4FB-O) and another that had a longer polyethylene glycol spacer (C-PEG-O) to determine whether spatial distance between antigen and adjuvant has an impact on the generated immune response. The reversibility of these linkers was tested in an in vitro cleavage assay using size-exclusion chromatography. The conjugates were incubated with the reductant glutathione (GSH) at extracellular and intracellular concentrations at pH 7.4 at 37 °C for 2 h, one disulphide conjugate (C-SPDP-O) was cleaved under extracellular reducing conditions while the second disulphide conjugate (C-4FBSS-O) was only cleaved at intracellular reducing conditions. The stable and peptide linked conjugates were not cleaved in either reducing condition. The peptide conjugate designed to be cleaved by Cathepsin S was not cleaved when exposed to the pure enzyme at pH 7.4 at 37 °C overnight. In vitro studies showed that the highest T-cell response was generated by the stable C-4FB-O and the intracellular cleavable C-4FBSS-O conjugates. The stable conjugate C-PEG-O, the extracellular cleaved conjugate C-SPDP-O and the C-peptide-O conjugate induced a lower T-cell response similar to the mixture of CpG and OVA. The conjugates were tested in vivo in a therapeutic tumour trial with B16-OVA cells injected subcutaneously into the left flank of C57BL/6 mice. After 80 days the surviving mice were re-challenged with B16-OVA tumour cells into the opposite flank. At day 160, a survival rate of 9/10 for mice vaccinated with the intracellular cleavable C-4FBSS-O conjugate was observed while 6/10 mice vaccinated with the stable C-4FB-O conjugate survived. All other conjugates and the mixture of CpG and OVA induced a survival rate of ≤ 3/10.
In summary this study supported our hypotheses and demonstrated that intracellular triggered antigen-adjuvant conjugates that release the antigen from the adjuvant induce an effective anti-tumour immunity. This intracellular cleavable conjugation strategy represents a promising approach to improve cancer immunotherapy.