Taking into account that the immunopeptidome represents the whole spectrum of peptides presented in a cell, there is a need to identify the most promising cancer targets. Thus, there is a need to determine the structure of an MHC-bound peptide and its level of expression for vaccine development. There are numerous ongoing clinical trials utilizing different antigens and adjuvants as therapeutic cancer vaccines. Focusing on peptides with PTMs as vaccines, promising findings have resulted from the use of citrullinated peptides (Table 1). A citrullinated VIM peptide has been utilized as an antigen in combination with an adjuvant induced IFN-γ and granzyme B-secreting CD4+ T cells. Citrullinated VIM-specific Th1 cells induced by the vaccine had a potent antitumor response against established skin and lung tumors, as well as a long-term memory response [30]. Similarly, a citrullinated ENO1 peptide-based vaccine elicited a potent citrulline-specific Th1 cell response in pancreatic, skin, and lung cancers [29]. Additionally, ENO1 is commonly overexpressed in different tumor types, including melanoma, pancreatic, breast, and lung cancer, thus citrullinated ENO1 peptides are plausible antigens for a wide cancer spectrum [18,29,55]. Furthermore, the combination of citrullinated VIM and ENO1 peptides in a vaccine, designated Modi-1, induced a significant antitumoral response in a mouse model of ovarian cancer. Importantly, a substantial citrulline-specific T cell response was observed in more than half of ovarian cancer patients [103]. Moreover, analysis of the melanoma immunopeptidome led to the identification of MHC-II-bound citrullinated peptides are derived from MMP21, Cp450, and GRI proteins [33]. A combination of these citrullinated peptides did not induce a greater antitumoral response than citrullinated MMP21 and GRI peptides individually, pointing to the potential of a reduced response with multiple peptides with different MHC-II binding specificities [33]. Another source of citrullinated peptides is the NPM protein. Vaccination with a PADI2-mediated-citrullinated NPM peptide induced an antitumoral response which was therapeutic, increasing survival and resulting in protection against a second tumor challenge in melanoma and lung cancer mouse models. Interestingly, PADI4-mediated-citrullination of NPM peptide did not elicit a citrulline-specific Th1 response, in contrast to PADI2-mediated-citrullination [53]. The CD4 responses observed may result from binding of citrullinated peptides primarily by MHC-II [18] in HLA-DP4 and HLA-DR4 transgenic mice. Nevertheless, the vaccine-induced CD4 response was sufficient to inhibit tumor progression, indicating the effectiveness of responses that do not involve CD8+ T cells [10].

There is a more limited number of studies utilizing phosphorylation as a PTM for peptide vaccines, although immunopeptidome analysis has pointed to a substantial number of phosphorylated peptides. Immunopeptidome analysis of melanoma, ovarian carcinoma, B lymphoblastoid, and leukemia resulted in the identification of a large number of phosphopeptides that were cancer specific with CD8 T cell antigen specificity in patients [58,63]. Some of the identified phosphopeptides were derived from ISR2, BCAR, TNS2, SELH, CDC25b, and beta-catenin [58,62,63], concordant with the identification of phosphorylated ISR2, TNS2, and SELH peptides in the colorectal cancer immunopeptidome [67]. 

As for glycopeptide-based cancer vaccines, an initial source was the glycosylated MUC protein displaying the Tn antigen. Immunization of mice with a desialylated ovine MUC with substantial representation of the Tn antigen elicited primarily a CD4+ T cell response specific to the Tn antigen. Conversely, immunization with a deglycosylated MUC did not induce an immune response [73]. The induction of an immune response specifically against the PTM protein suggests that glycosylation may be a useful source of cancer-specific antigens given the findings of aberrant glycosylation in many cancers, notably breast cancer [78,106]. A case in point is a fully synthetic cancer vaccine, a dendrimeric multiple antigenic glycopeptide displaying a trimer of Tn antigens (MAG-Tn3) associated with a promiscuous CD4 epitope, the tetanus toxoid-derived P2 peptide, that has been shown to induce an antitumoral Tn-specific T cell response in monkeys [78]. This MAG-Tn3 vaccine has been used in a phase I clinical trial for high-risk relapsed breast cancer patients (NCT02364492). Another cancer vaccine in clinical trial is based on MUC1 bearing Tn antigens (Tn-MUC1) pulsed with autologous DCs [81]. This phase I/II clinical trial follows the same strategy used in rhesus macaques, which resulted in five out of seven castrate-resistant prostate cancer patients having a CD4 and/or CD8 response (NCT00852007).

 Given the complexity of glycan modifications, there has been a surge of various approaches to identify and develop glycopeptides as vaccines, as reviewed above, including the use of glyco-antigen microarrays to investigate immune responses to cancer vaccines [79,91]. Another development is the use of an antigen delivery system based on gold nanoparticles with Dectin-1 to target DC, conjugated with MHC-II glycopeptides. This gold nanoparticle glycopeptide vaccine elicited a strong humoral and cellular immune response in mice [84]. In all, much progress has been made in the identification of glycopeptides and their structural and other properties to enhance their effectiveness as cancer vaccines [92,93,95,98].