Identification of Four-Jointed Box 1 (FJX1)-Specific Peptides for Immunotherapy of Nasopharyngeal Carcinoma

Abstract

Nasopharyngeal carcinoma (NPC) is highly prevalent in South East Asia and China. The poor outcome is due to late presentation, recurrence, distant metastasis and limited therapeutic options. For improved treatment outcome, immunotherapeutic approaches focusing on dendritic and autologous cytotoxic T-cell based therapies have been developed, but cost and infrastructure remain barriers for implementing these in low-resource settings. As our prior observations had found that four-jointed box 1 (FJX1), a tumor antigen, is overexpressed in NPCs, we investigated if short 9-20 amino acid sequence specific peptides matching to FJX1 requiring only intramuscular immunization to train host immune systems would be a better treatment option for this disease. Thus, we designed 8 FJX1-specific peptides and implemented an assay system to first, assess the binding of these peptides to HLA-A2 molecules on T2 cells. After, ELISPOT assays were used to determine the peptides immunogenicity and ability to induce potential cytotoxicity activity towards cancer cells. Also, T-cell proliferation assay was used to evaluate the potential of MHC class II peptides to stimulate the expansion of isolated T-cells. Our results demonstrate that these peptides are immunogenic and peptide stimulated T-cells were able to induce peptide-specific cytolytic activity specifically against FJX1-expressing cancer cells. In addition, we demonstrated that the MHC class II peptides were capable of inducing T-cell proliferation. Our results suggest that these peptides are capable of inducing specific cytotoxic cytokines secretion against FJX1-expressing cancer cells and serve as a potential vaccine-based therapy for NPC patients.

Fig 1. Overexpression of FJX1 in biopsies and cell lines.

(a) Quantitative PCR on NPC cell lines, oral cancer cell line and biopsies from NPC patients showed increased levels of FJX1 mRNA compared to the normal nasopharynx tissue (TSE5 and NPC3). FJX1 is considered to be overexpressed when the relative expression ≥ 2 fold is observed when compared to normal. FJX1 overexpression in C666.1-A2 and corresponding control counterpart cell lines was confirmed at both (b) mRNA and (c) protein level.

Fig 2. HLA-A2 peptides demonstrated high binding affinity towards MHC class I molecule.

All 6 HLA-A2-restricted peptides demonstrate higher binding affinities towards HLA-A2 molecules suggesting stabilization of HLA-A2 molecule expression on T2 cell surface compared to the negative control (p < 0.001). Binding affinity was evaluated by comparing the mean fluorescence intensity (MFI) of HLA-A2 expression in the presence of peptides compared to the MFI in the absence of peptide. HLA-A2 restricted FluM-derived peptide was used as a positive control and samples with the absence of peptide were used as negative control.

Fig 3. FJX1 sequence specific peptides are immunogenic.

(a) Ex vivo ELISPOT demonstrated higher secretion of IFN-γ in healthy donors (n = 4) when compared to patients (n = 4; p = 0.002). (b) Ex vivo ELISPOT demonstrated higher secretion of granzyme B in donors (n = 4) when compared to patients (n = 4; p = 0.003).

Fig 4. FJX1 peptides stimulate higher cytokine secretion in NPC patients PBMCs when compared to donors equivalent after peptide pulsation.

(a) Augmentation of IFN-γ secreting CD8+ T-cells was significant higher in NPC patients (n = 4) when compared to healthy donors (n = 4) after peptide stimulation (p = 0.003). (b) Augmentation of granzyme B secreting CD8+ T cells was significant higher in NPC patients (n = 4) as compared to healthy donors (n = 4) after peptide stimulation (p < 0.001). Spot numbers was calculated by subtracting spots in the negative control wells with the absence of peptides.

Fig 5. FJX1-derived peptides are able to activate T-cells to recognize cancer cells using an optimal E:T (effector: target) ratio.

(a) Optimal cytotoxic potential of post-expansion T-cells was observed at E: T ratio of 20: 1, where most of the peptides were able to induce subjects’ T-cells secreting high level of IFNγ and granzyme B. Dotted lines represent granzyme B secretion and the solid lines represent IFNγ secretion. (b) Pep-01, Pep-03, Pep-05, Pep-06 and Pep-08 demonstrated a trend of specificity where the peptides were able to activate T-cells to recognize FJX1 overexpressing cancer cells (C666.1-A2/FJX1).

Fig 6. MHC class II peptides induced CD4 T-cell proliferation.

Pep-08 was able to induce T-cell proliferation in 6/9 of total samples while Pep-07 was only able to induce T cell growth in only 3/9 total samples tested.