Atabani, Suha
(2023)
Pan-cancer vaccines: breaking tolerance by targeting stress-induced post-translational modifications in tumours.
PhD thesis, University of Nottingham.
Abstract
Cancer immunotherapy aims to induce potent pro-inflammatory immune responses that can lead to elimination of tumours. The challenge of this approach is to induce cancer-specific immune responses that do not cross react with healthy tissues and are not subject to the anti-inflammatory suppressive tumour environment. One approach is to use a vaccine therapy that targets cancer-associated antigens.
The objective of this study was to evaluate the immunogenicity of citrullinated and homocitrullinated epitopes in cancer patients. Here it is shown that cancer patients have a repertoire to these stress induced post-translational modification (siPTM) peptides which can be measured when their PBMCs (peripheral blood mononuclear cells) are cultured and stimulated with the modified peptides. The number of lung cancer patients with a CD4 proliferative response to citrullinated enolase 241-260 (E241) (p=0.0021) and vimentin 28-49 (V28) (p=0.0230) peptides or ovarian cancer patients with a CD4 proliferative response to citrullinated E241 (p=0.0078) and citrullinated binding immunoglobulin protein 189-208 (BIP) (p=0.0286) peptides was significantly reduced compared to healthy donors. In addition, the proliferating CD4 T-cells in cancer patients produced less of the cytokine IFNγ (Interferon-γ) compared to healthy donors (p<0.0001). A population of activated antigen specific cells that do not proliferate was identified in cancer patients. This population was defined as CD4+ T-cells expressing the transferrin receptor CD71 which is expressed on CD4+ T-cells following activation by an antigen. In lung cancer patients, this population (CD4+CD71+) was higher in the expression of Latency Associated Peptide (LAP, a peptide that associates with the inhibitory transforming growth factor-β (TGF-β)) than in healthy donors (p=0.0424) and was inversely correlated with proliferation (p=0.0003). In a suppression assay, CD4+T-cells expressing LAP were isolated from PBMCs of lung cancer patients and found to suppress proliferation (p=0.0156). These results suggest that upon stimulation with a modified antigen, cells either become proliferative Th1 (T-helper 1) cells producing IFNγ or convert into a suppressive Th3 (T-helper 3) cells expressing LAP.
In a lung tumour microarray (TMA) the expression of vimentin (a cytoskeletal protein expressed during epithelial mesenchymal transition), citrullinated vimentin, peptidyl arginine deiminase enzymes (PADI2 and PADI4 involved in the process of citrullination), CD4 T-cell infiltration, MHC-II, programmed cell death ligand-1 (PDL-1), autophagy markers and LAP was measured. Vimentin expression was significantly (p=0.025) associated with a bad prognosis, whereas citrullinated vimentin (p=0.016), PADI2 (p=0.048) and Ambra1 (p=0.009) were associated with a good prognosis. There was a positive correlation with expression of citrullinated vimentin and patients’ age (p=0.003) but not with gender, TMN stage or tumour size. There was also a positive correlation between expression of PADI2 (p<0.0001) and PADI4 (p=0.001) enzymes, MHC-II expression (p=0.008), p62 (p=0.001) and PD-L1 expression (p=0.012) with citrullinated vimentin expression. There was a negative correlation between the expression of LAP on tumour cells and citrullination of vimentin (p=0.018) suggesting LAP maybe inhibiting the expression of citrullinated epitopes. The co-expression of LAP in tumour infiltrating lymphocytes (TILs) and citrullinated vimentin was a prognostic indicator (p=0.018) with patients whose tumours expressed citrullinated vimentin and had low LAP in TILs surviving 78 months while in contrast those whose tumours did not express citrullinated vimentin and had high LAP in TILs survived 33 months.
In murine experiments with C57Bl/6 mice bearing Py8119 breast cancer, immunisation with citrullinated peptides resulted in 50% improved survival (p=0.0270) but no synergy was seen with an anti-LAP antibody. Upon culling the mice when their tumours reached a size of ≥10mm, flow cytometry was used to examine the expression of LAP and LAG3 (lymphocyte activation gene 3) on CD4+ T-cells isolated from the murine tumours. Th3 cells were described as CD4 highly suppressive cells expressing LAP and LAG3. In the group vaccinated with citrullinated peptides, the levels of LAP+LAG3+ on CD4+ TILs were lower compared to the controls (p=0.0400) suggesting that the vaccination was able to polarize responses from Th3 to Th1. Vaccination of the same model with citrullinated peptides in combination with anti-LAG3 resulted in improved survival (p=0.0056) and lower levels of Th3 cells identified by expression of CD4, LAP and LAG3 (p=0.0435). The Py8119 tumour model was identified as a model that expressed high levels of both LAP+ and LAG3+ CD4+TILs. However, in a model that expressed lower levels of LAP+ CD4 TILs, the B16F1 melanoma model, vaccination with citrullinated peptides lowered tumour volume on day 15 and 18 (p=0.0057 and 0.0269 respectively) but following this time point tumours exponentially outgrew the immune control and overall survival was not improved although was statistically close to significance (p=0.0637). In the latter model, vaccination with citrullinated peptides lowered the levels of CD4+LAP+ TILs (p=0.0087) suggesting that this tumour model escapes immune control through regulatory mechanisms other than Th3 cells.
These results suggest that modified antigens may be a good target for anti-tumour immunity but that some cancer patients may have developed an antigen specific LAP+ Th3 regulatory population which would either need to be polarized to Th1 with vaccination or inhibited with an antibody targeting either TGF-β/LAP or LAG3. The ModiFy trial, is the first in human trial which will reveal whether vaccination with citrullinated peptides alone or in combination with a checkpoint inhibitor such as agents targeting Programmed cell death receptor-1 (PD-1) can control tumour growth and polarize responses to Th1.
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