Recent technological developments have enabled significant progress in the prediction and discovery of neoantigens, resulting in numerous studies on tumor vaccines achieving promising outcomes. Currently, there are various types of cancer vaccines used for treatment, including tumor cell vaccines, DC vaccines, T-cell vaccines, DNA vaccines, RNA vaccines, viral vector vaccines, and protein and peptide vaccines (1–4). Each of these approaches has advantages and disadvantages: tumor cell vaccines are significantly limited by patient materials, while DC vaccines, despite being low in toxicity and the only FDA-approved cancer treatment, are not very effective and require additional steps to load and present the antigen. Viral vector vaccines accommodate large gene insertions and readily produce antigens presented by MHC Class I and Class II molecules but carry a high risk of infection or pre-existing immunity. DNA vaccines can deliver a variety of antigens and genes to express both short and long peptides, but it has proven difficult to translate mouse studies into human trials. mRNA vaccines have similar advantages to DNA vaccines, with fewer side effects and less autoimmune issues, but they rapidly degrade and are cleared from the body, reducing their effectiveness. Peptides are easy to produce and cost-effective, allowing multiple peptide sequences to be combined into a single formulation, and the vaccine’s immune effect can be directly monitored by studying the vaccine components. However, whole exon sequencing yields numerous neoantigen mutations, making it challenging to screen for neoantigen polypeptides with high immunogenicity. Therefore, immunogenicity screening becomes the most critical step in the entire process of tumor immunotherapy. Tumor neoantigens have thus become a focal point in this field, offering potential solutions to various challenges encountered in immunotherapy. Studies of individualized tumor vaccines based on tumor neoantigens have also achieved significant success, such as in clinical trials against lymph node metastasis of melanomas (5).

Neoantigens are abnormal proteins arising from genetic mutations in tumor cells. These neoantigens are degraded into short peptides (epitopes) within tumor cells or antigen-presenting cells (APCs). These epitopes bind to major histocompatibility complex (MHC) Class I or Class II molecules on the surface of APCs, forming MHC-polypeptide complexes presented on the APC surface. These complexes are recognized by T-cell receptors (TCRs) on T-cell surfaces, triggering a T-cell immune response and activating effector T-cells to attack and eliminate tumor cells (6). Different tumor types present various quantities and types of neoantigens, but neoantigen types also vary at the individual tumor level; this variation is termed “individualization” (7). Currently, the development of individualized vaccines follows these steps: (1) High-throughput sequencing of the whole exon genome is performed on DNA obtained from tumor tissues. (2) These sequencing results are utilized to predict potential neoantigen peptides and determine the affinity between each peptide and human leukocyte antigen (HLA) Class I molecules. (3) Neoantigen peptides with potential immune effects are screened by their MHC molecular affinity for use in complex immunological experiments, and those with strong immune activity are selected for preparing tumor vaccines for clinical treatment. Individualized tumor vaccines represent a major breakthrough in cancer treatment. Currently, next-generation sequencing (NGS) technologies for neoantigen identification include whole genome sequencing (WGS), whole exome sequencing (WES), and complete transcriptomic sequencing (RNA-seq). Screening for tumor neoantigens is essential for individualized precision therapy. However, since fewer than 10% of tumor cells mutate to form tumor neoantigens, it follows that fewer than 10% of these have sufficient immunogenicity to induce effective specific T-cell immunity, the primary anti-cancer immunity (8).

At present, most experimental studies screen for neoantigen peptides based on their MHC molecular affinity, but not all high-immunogenicity neoantigenic peptides exhibit correspondingly high MHC molecular affinity. Srivastava et al. suggest that neoantigens in transgenic mouse tumor models exhibit very different properties compared to viral antigens, generally displaying very low affinity for MHC Class I antigens (9). An individualized vaccine studied by Patrick A. Ott et al. included only neoantigenic peptides with high MHC molecular affinity variation and achieved significant efficacy when applied to melanoma patients (5). However, screening for neoantigen peptides based on MHC affinity or its variation leads to a high rate of misselection and omission of suitable peptides. Using immunoassays to confirm the immune effect of each antigen peptide in each patient is costly, time-consuming, and labor-intensive, preventing widespread clinical application. Therefore, simple and accurate detection of immunoeffective neoantigen peptides is crucial for the clinical viability of individualized vaccines. In this study, whole exon sequencing was conducted on colon cancer cells obtained from patients. The process of antigen presentation was simulated in vitro to analyze and clarify the relationship between the immune activity of neoantigen polypeptides, their HLA Class I molecular affinity, and HLA allele variations. With the rapid commercialization of whole exon sequencing and bioinformatics analysis, new methods for the swift and simple discovery of effective neoantigens have become feasible.

In recent years, advancements in bioinformatics have catalyzed significant progress in personalized tumor vaccines. Examination of tumor mutations enables testing of immune responses to mutant proteins, or neoantigens, through computer simulations. This approach facilitates screening for immunogenic neoantigens and the development of polypeptide or mRNA tumor vaccines for clinical trials. Whole exome sequencing was performed on tumor tissue and matched healthy tissue (typically peripheral blood), followed by comparison to identify somatic gene mutations. This method typically identifies the majority of single nucleotide variants with sufficient coverage. In 2013, Rosenberg et al. pioneered whole exome sequencing to identify mutated proteins in patients. They utilized an MHC molecule-epitope affinity algorithm to predict and synthesize candidate epitopes for immune response validation. This approach enables rapid identification of mutated antigens expressed on tumor cells, facilitating their recognition by tumor-infiltrating lymphocytes (TIL). Subsequently, this method was clinically applied in 2014, again by Rosenberg’s team. High-depth exon sequencing technology and immune response verification enabled identification of a high-frequency mutant gene in a patient with metastatic cholangiocarcinoma, along with its corresponding TIL clone. Amplification of the TIL clone and retrotransfusion therapy effectively controlled the patient’s disease (10). Also in 2013, another team utilized exon sequencing technology alongside transcriptome sequencing and high-throughput proteomic analysis to predict MHC molecule-antigen epitope affinity, facilitating T cell recognition and efficient activation peptide vaccine production. This personalized tumor vaccine demonstrated efficacy comparable to both preventive and therapeutic vaccines (11). With the discovery of additional HLA alleles, software tools like NetChop, NetMHCpan, NetMHC, and SYFPEITHI have been developed to predict MHC molecular ligands of neoantigen peptides and their affinities. However, due to variations in neoantigens between patients and their limited immunogenicity, this discovery process is complex and time-consuming (12, 13). Certain immunoeffective neoantigens induce immune tolerance, impotence, and even immunosuppression. These factors hinder the clinical application of personalized vaccines, underscoring the need to rapidly identify effective neoantigen peptides and enhance the immune response of those with weak immunogenicity (5, 14).

Currently, many algorithms are capable of predicting the affinity between peptides and MHC molecules. However, the MHC affinity of peptides does not correlate with their immunogenicity; it is believed that immunogenicity is more closely associated with the stability of the peptide-MHC complex (15, 16). Kristensen et al. demonstrated that predicting the stability of binding between peptides and MHC molecules can increase the hit rate of candidate neoantigens (i.e., successful predictions of immunoeffective neoantigens) (17). Assarsson et al. conducted a systematic analysis of putative and actual vaccinia virus epitopes, determining that epitope dominance had little relationship with HLA peptide affinity, stability, TCR affinity, or the number of processed epitopes (18). Another study suggested that the NETMHC score was not a valid predictor of immunogenicity or tumor rejection. The authors developed the DAI algorithm, which subtracts the NETMHC scores of unmutated copies of predicted mutated epitopes from those of the corresponding mutated epitopes, accounting for the NETMHC scores of those copies. Although the DAI algorithm identified far more tumor-protective epitopes than those derived from the highest NETMHC or MHC-binding scores, most epitopes identified by DAI still fail to elicit tumor protection (9).

Peptides can serve directly as T-cell epitopes due to low toxicity, precise targeting, cost-effectiveness, and ease of chemical synthesis, rendering them appealing for vaccine development. Peptides bound to MHC class I molecules are typically 8-11 amino acids long, reflecting their shorter gene sequence (19). Conversely, MHC class II molecules feature open grooves at both ends, accommodating longer peptides ranging from approximately 10 to 30 amino acids. Consequently, they possess more complex binding motifs and bind a broader range of exogenous antigenic peptides compared to MHC class I molecules. Moreover, neoantigens are endogenous and structurally straightforward, simplifying the study of peptides bound to MHC class I molecules compared to those acting as MHC class II antigens. The most prevalent peptide segment length is 9 amino acids, making it the most specific MHC binding ligand (20). In vitro synthesis of neoantigenic peptides can elicit antigen-specific T-cell responses (19). In this study, the 9 amino acid peptide mutant sequence was identified through whole exon sequencing, synthesized in vitro, and evaluated for antigen-specific T-cell responses. Dendritic cells, as antigen-presenting cells (APCs) with potent antigen-presenting capabilities, can present long peptides to immature T-cells for activation. Other APCs may present short peptides, but these can lead to anergy in stimulated T-cells due to the absence of a co-stimulatory molecule (21). Provenge (Sipuleucel-T), utilizing in vitro dendritic cells, received FDA approval in 2010 as the first therapeutic prostate cancer vaccine. Short peptides like neoantigen 9 amino acid peptides require effective adjuvants, such as TLR9 or TLR3 agonists, to induce an effective CD8+ T-cell response, mediated through DC activation and immune system stimulation (22). A study compared the effects of CpG ODN adjuvant and Freund’s adjuvant in combination with a tumor vaccine. CpG ODN demonstrated superior adjuvant effects over Freund’s adjuvant, with lower associated toxicity, highlighting its utility as a tumor vaccine adjuvant (23).

Granulocyte-macrophage colony-stimulating factor (GM-CSF) induces PBMC differentiation into macrophages and dendritic cells, while interleukin 4 (IL4) inhibits macrophage formation (24). Thus, dendritic cells can be prepared in vitro by isolating PBMCs from peripheral blood and culturing them in a medium supplemented with GM-CSF and IL4 cytokines. Dendritic cells prepared in this manner are referred to as immature dendritic cells (imDCs) (25, 26). In this study, DCs were loaded with neoantigen peptides to prepare DC vaccines by promoting DC maturation. The expression of marker molecules such as CD11c, CD80, CD86, and CD83, indicative of dendritic cell maturation, was assessed using flow cytometry. Detection results indicated that mature dendritic cell (mDC) levels in cell suspensions of DC vaccines exceeded 97%.

Peptides are easy to produce, allowing multiple sequences to be combined into a single formulation, thereby enabling direct monitoring of the vaccine’s immune effects through its individual components. Less than 10% of tumor cell gene mutations give rise to tumor antigens, and fewer than 10% of these neoantigens can successfully induce effective, specific T-cell immunity (the primary anticancer immunity) (8). Whole exon sequencing identifies a large number of potential neoantigen peptides, necessitating screening for those with high immunogenicity. Higher MHC molecule affinity increases the likelihood of becoming neoantigens; however, MHC affinity does not correlate with neoantigen immunogenicity. In a 2017 Nature study on neoantigen vaccines for melanomas (5), only neoantigen peptides with a substantial change in MHC affinity were included, showing a significant therapeutic effect. However, this does not constitute evidence of a broad correlation between immunogenicity and MHC affinity, and no relevant studies have confirmed such a relationship. This study confirmed that neoantigen immunogenicity did not correlate positively with changes in MHC class I molecular affinity. Only neoantigens with a significant change in MHC affinity were selected as polypeptide vaccines, leading to a high rate of misselection and omission.

This study utilized whole exon sequencing results from tumor tissues and predictions of MHC class I molecular affinity to screen for neoantigenic 9aa peptides. These peptides were combined with CPG adjuvant in vitro to simulate endogenous antigen presentation by APCs, specifically dendritic cells, to CD8+ T-cells, thereby activating their immunological response. Immunogenicity of each antigen was quantified using ELISpot, an enzyme-linked spot experiment. We observed that the immunogenicity of neoantigens did not show a positive correlation with the variation in affinity of MHC Class I molecules. The MHC affinity variation among neoantigens with the highest immunogenicity mostly ranged between 1 and 4, and the HLA-I genotype associated with the highest T-values predominantly exhibited binding to the greatest number of neoantigen peptides.

Neoantigenic 9aa peptides, as endogenous antigens, can specifically activate and proliferate CD8+ T-cells. However, several studies have demonstrated their capability to activate CD4+ T-cells as well (27–30). In this study, a DC vaccine was co-cultured with PBMCs. Flow cytometry analysis of T, B, and NK cells indicated that the neoantigen peptide not only activated CD8+ T-cells but also contributed to the proliferation of CD4+ T-cells and NK cells. This suggests that neoantigen peptides can activate both specific (CD8+ T-cell) and nonspecific (CD4+ T-cell and NK cell) immunity.

This study has helped elucidate the relationship between neoantigen immunogenicity and changes in MHC molecular affinity and HLA alleles, providing a novel method for rapid screening of strongly-immunoeffective neoantigens.