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Peptide‐based therapeutic cancer vaccine: Current trends in clinical application

Abstract The peptide‐based therapeutic cancer vaccines have attracted enormous attention in recent years as one of the effective treatments of tumour immunotherapy. Most of peptide‐based vaccines are based on epitope peptides stimulating CD8+ T cells or CD4+ T helper cells to target tumour‐associated antigens (TAAs) or tumour‐specific antigens (TSAs). Some adjuvants and nanomaterials have been exploited to optimize the efficiency of immune response of the epitope peptide to improve its clinical application. At present, numerous peptide‐based therapeutic cancer vaccines have been developed and achieved significant clinical benefits. Similarly, the combination of peptide‐based vaccines and other therapies has demonstrated a superior efficacy in improving anti‐cancer activity. We delve deeper into the choices of targets, design and screening of epitope peptides, clinical efficacy and adverse events of peptide‐based vaccines, and strategies combination of peptide‐based therapeutic cancer vaccines and other therapies. The review will provide a detailed overview and basis for future clinical application of peptide‐based therapeutic cancer vaccines.


| INTRODUC TI ON
Immunotherapeutic strategies have dramatically revolutionized cancer treatments, including dendritic cell (DC)-based cancer vaccines, immune checkpoint inhibitors and chimeric antigen receptor T-cell immunotherapies (CAR-T). For example, checkpoint inhibitor-based immunotherapies that could activate T cells result in an improvement in clinical success, but the tumour targeting was deficient. Despite specific tumour targeting, CAR-T therapy showed risks of cytokine release syndrome and neurotoxicity, and it could not gain clinical benefits on solid tumours, which caused the limitation of clinical application. Therefore, developing the safe and effective treatments to enhance the specific anti-tumour activity has become a hot topic in the current field of tumour immunotherapy.
The peptide-based therapeutic cancer vaccines could offer many advantages with regard to convenient production, cost-effective manufacture, low carcinogenic potential, insusceptible pathogen contamination and high chemical stability. This type of vaccine contains the

| TARG E T CHOI CE S OF PEP TIDE-BA S ED THER APEUTI C C AN CER VACCINE S
The CD8 + T cells are capable of recognizing the peptide-HLA complex to produce a persistent memory CTL response against target cells expressing the antigen. Therefore, the critical factor is the selection of proper TA for therapeutic cancer vaccines to exert specific cytotoxicity against tumour cells.
TAs can be classified into tumour-associated antigens (TAA) and tumour-specific antigens (TSA). Despite TAAs can express in both normal cells and tumour cells, they overexpress in tumour cells but at a low level in normal cells. Therefore, TAAs are attractive targets for developing immunotherapeutic cancer vaccines. Some studies reported that characteristics of proper TAA should be the following: i) differential expression between normal cells and tumour cells; ii) involvement in cell cycle; and iii) association with cell survival. 1 Normally, most of TAAs with low self-tolerance and strong immunogenicity were used as targets in preclinical studies and clinical trials to evaluate safety and efficacy of peptide-based therapeutic cancer vaccines. 2 On the other hand, TSA only expressed in tumour cells rather than in normal cells, including mutations of normal proteins, 3 cancer testis antigen, 4,5 neoantigens 6 and virus-related antigens. 7,8 Boon et al reported melanoma antigen-A1 (MAGE-A1) as the first TSA in humans at 1991. 9 Human leucocyte antigen (HLA) / TSA-derived peptide complex, could exert higher avidity specific T cells to lead to effective and safe immune response of cancer vaccines against tumour. 10,11 TSAs as targets of cancer vaccines demonstrated similar results in both animal models and clinical trials due to the loss of TSA expression in normal tissues, which means non-immunologically tolerant to TSA and non-immunity targeting normal tissues. 12 TSAs are attractive for personalized cancer immunotherapy, but it is not cost-effective. 13 Besides, some studies emerge for the selection of specific epitopes, such as T-cell epitopes associated with impaired peptide processing (TEIPP), 14 which only express on transporter associated with antigen processing (TAP)-deficient tumour cell surface. The preprocalcitonin (ppCT) [16][17][18][19][20][21][22][23][24][25] antigenic peptide, derived from the calcitonin hormone precursor, as the first human TEIPP Ag, provides a new strategy to counteract immune evasion by antigenic processing machinery defects. 15 Currently, many TAAs and TSAs have been identified as targets for peptide-based therapeutic cancer vaccines ( Figure 1, Table 1), in which most focus on targeting melanoma, 16 lung cancer, 17 breast cancer 18 and leukaemia, 19,20 whereas most of them are in phase I and phase II. Recent clinical trials in phase III are only including HER2 (human epidermal growth factor receptor 2)/neu targeting breast cancer 21 ; tyrosinase, gp100 antigen, and MART-1 (melanoma antigen recognized by T cells 1) antigen targeting melanoma; PR3 (proteinase-3) targeting leukaemia. 22 TAs, such as Survvin, VEGFR (vascular endothelial growth factor receptor), MUC1 (mucin 1) and TTK (TTK protein kinase), were used most extensively as targets for developing peptide-based therapeutic cancer vaccines, targeting lung cancer, gastrointestinal cancer and melanoma ( Figure 2).

| S TR ATEG IE S FOR SCREENING EPITOPE PEP TIDE S
The anti-tumour effects of DC-mediated T-cell activation are through the stimulation of peptides, terms epitopes, instead of the entire antigen molecule. Normally, the epitope for developing peptide-based therapeutic cancer vaccines is a short amino sequence derived from TA with immunogenicity and HLA allele compatibility. It has been reported many screening strategies for immunodominant epitopes, such as bioinformatic analysis and HLA ligandome. The affinity of HLA-I allele and epitopes can be measured and predicted by many methods (Table 2), including the method based on structural analysis, the position-specific scoring matrix (PSSM), artificial neural network (ANN) method and machine learning. 23 Structural analysis identifies neoepitopes by calculating the minimal free energy of epitope-HLA complex. 24,25 PSSM is produced by measuring the interaction between peptides and specific MHC molecule. 26 The correlation of different positions in sequence was considered into ANN analysis to predict affinity between peptides and MHC molecule. Machine learning could predict affinity of peptides and MHC molecule by learning the affinity of known functional regions with peptides. The immune epitope database (IEDB) predicts the optimal amino binding positions of MHC-I molecule through a large variety of HLA allele algorithms, thereby being broadly applicated for identifying the epitope peptides. 27 Additionally, HLA ligandome approach could identify naturally HLA-presented peptides existed in tumour cells by mass spectrometry analysis. 28 It could also be used to identify specifically overexpressed protein-derived peptides, signal peptide-derived peptides and antigenic mutation-derived peptides. 29 This approach could combine with computational biology and bioinformatics, such as functional annotation and gene expression analysis, to identify potential TSA (including neoantigens) and TAA. Based on ligandome analysis, we can observe a few peptides of 11 amino acids, 12 amino acids and 13 amino acids, as their length is outside the consensus of the computer programs for motif prediction of class I peptides. tor T-cell activation, which contributes to the persistence and survival of effector cells in vivo. 42 Therefore, these peptide-based therapeutic cancer vaccines have been reported to be well tolerated and have shown clinical benefits against tumours. In the following paragraphs, we focused on introductions of targets, sequences and research progress of epitope peptides in recent 5 years (Table 3).

| Study design and treatment
Peptide-based therapeutic cancer vaccines are usually administered in a 7-to 15-day interval with subcutaneous axillary and/or inguinal injection of 1-3 mg/dose per peptide per person. Patients usually complete a course of at least 2 months to a maximum of 12 months unless patients experience disease progression or unacceptable toxicity. The primary end points are safety, tolerability, immunogenicity and operational feasibility of the peptide-based vaccines.
The secondary end points are evaluations of anti-tumour effects, overall survivals (OS) and disease-free survivals (RFS).

| Clinical efficacy and immune response
Analysis on patients treated with peptide-based vaccines showed that the production of epitope-specific CTLs could be induced in most patients, and even tumour infiltrating lymphocyte (TIL) activation could be induced in individual patients. 43 The CD8 + T cells in lymph nodes and the infiltration of CD8 + T cells in the tumour microenvironment increased in about 30%-60% of patients, and the secretion of granzyme B and interferonγ (IFNγ) also increased. Patients who showed a strong epitope-specific CTL response had longer OS than those with non-or low immune response, demonstrating that peptide-based vaccines could be effective in patients who showed a peptide-specific immune response. Compared with the placebo group, patients receiving the peptide-based vaccine showed a tendency of improved OS and RFS, and their condition was more stable. The peptide-based vaccine therapy usually shows delayed immune response and tumour growth inhibition, but does not show significant tumour shrinkage. 44,45 Additionally, the epitope peptide could induce anti-tumour response over a long period of time. 46 Kjeldsen et al reported that 13.3% of patients showed anamnestic immune response 6 years after primary immunization. 47 In another case of oesophageal cancer, the patient received 8 vaccinations every 6 months, a total of 38 vaccinations, and finally obtained a complete response (CR) lasting for 5 years. 48 Although peptide-specific responses also were elicited in high-risk patients, previous studies showed that patients in the early stage of tumour progression or with a low disease burden could obtain better clinical benefits. [49][50][51]

| Adverse events
The AEs, which ensured the long-term safety of peptide-based vaccines. 47 Sawada et al found the TA-specific CD8 + T cells showed exhausted phenotypes in individual patients, which may be due to over-activation of CD8 + T cells in patients with high tumour mutation burden or overfrequent vaccinations. 66 In summary, patients could gain clinical benefits from peptide-based therapeutic cancer vaccines with distinct advantages of safety, good tolerance and effective immunization.

| COMMON PHARMACEUTIC AL FORMUL ATIONS OF PEP TIDE-BA S ED THER APEUTI C C AN CER VACCINE S
The peptide-based therapeutic cancer vaccines can improve the prognosis of cancer patients, while a more effective vaccine is needed to improve PFS and OS of patients. One of the strategies is developing

| Immune stimulation adjuvants
Immune stimulation adjuvants could enhance humoral immune and has been considered to be toxic. Besides, CD8 + T-cell immune response induced by poly-ICLC may be marginally more responsive than LPS.

| Vaccine design and delivery system
Optimized delivery systems have been developed to design rational vaccines, which usually consist of comparable size, such as liposomes, microemulsions, immune-stimulating complexes, and other nanometre or microparticle systems. The delivery system being especially suitable for the development of vaccines could improve clinical benefits of vaccines.
In recent years, more and more attention has been paid to the design of peptide-based nanoparticle vaccines for tumour immunotherapy ( Figure 3C). The optimized liposome-based vaccines could co-deliver peptides and adjuvants to promote their delivery to lymphoid organs and to draining lymph nodes (dLNs), which shows the acceptable clinical potential of liposome as delivery system. 73 The bioconjugation strategy links the target to the particle

| COMB INATI ON OF PEP TIDE-BA S ED THER APEUTI C C AN CER VACCINE S AND OTHER THER APIE S
Although many studies have demonstrated the effectiveness of peptide-based therapeutic cancer vaccines, no vaccine has shown significant OS benefits in randomized phase III clinical trials.
However, combination of therapies aimed at controlling immune tolerance might improve outcomes, such as chemotherapy, radiotherapy (RT), biological agents and immune checkpoint inhibitors (Table 4). In addition to TA-derived peptide vaccination, the personalized peptide vaccination (PPV), a novel immunotherapeutic approach based on a specific pool of peptides, is usually used on the combination strategy with other therapies in clinical trials. The peptide pool of PPV includes all information on the HLA-A type, and the peptide candidate library includes mutated peptides and highly expressed peptides. Considering the heterogeneous antigen expressions of different patients before vaccination, four specific epitopes aiming to the individual patient were selected from the candidate peptides into combination application strategy of peptide-based therapeutic cancer vaccines.

| The effect of combined chemotherapy and peptide-based vaccine
Causes of low immune responses may be associated with high Treg number. Since cyclophosphamide could selectively deplete Tregs 80 and regulate dendritic cell homoeostasis, the combination of low-dose cyclophosphamide and peptide-based therapeutic cancer vaccines may provide clinical benefits. 81,82 However, the peptide-based vaccines combined with low-dose IL-2 (interleukin-2) may exert negative effects on anti-cancer therapies due IL-2 may increase Tregs. 48 In addition, compared with Treg inhibitor gemcitabine alone, more than half of patients treated with peptide-based vaccine combined with gemcitabine showed long-lasting epitopespecific T-cell immune responses, reduced tumour burden, and long-term stable disease. 61 However, the peptide-based vaccine in combination with gemcitabine was not effective in patients with advanced metastatic disease, which was consistent with the opinion that the optimal condition for obtaining long-term clinical benefits was in the early stage of tumour or with a low disease burden described above. Besides, for prostate cancer patients treated with peptide-based vaccine and low-dose dexamethasone, OS was significantly prolonged compared with dexamethasone alone due to induction of the specific anti-tumour immunity. 83 In addition, OS also appeared to be improved when combined with peptidebased vaccines and platinum drugs. 84

| The effect of combined radiotherapy and peptide-based vaccine
The radiation may not reach all tumour focuses due to metastases or the large size of the tumour during radiotherapy. The combination of radiotherapy and peptide-based vaccines can effectively prevent tumours. 85 Release of danger-associated molecular patterns by RT-induced cell death, resulting in the facilitation of tumour antigen uptake by DCs and cross-presentation on MHC class I, is the molecular mechanism by which the combination strategy modifies the tumour microenvironment and enhances anti-tumour immune response. The other advantage is that the combination strategy is expected to reduce the dosage of chemotherapy drugs to avoid the side effects of chemotherapy, which has great potential clinical application values.

| The effect of combined other antineoplastic agents and peptide-based vaccine
The combination of anti-HER2 antibody trastuzumab with the HER2-targeting peptide-based vaccine in preclinical studies led to the proliferation of peptide-specific CTLs due to trastuzumabinduced improvement of cross-presentation of HER2 epitopepulsed DCs. 86,87 Clifton et al proved that the combination of HER2-targeting peptide vaccine nelipepimut-S and trastuzumab is well tolerated. Cardiac dysfunction of class III or IV was observed in the phase III trial of trastuzumab, and the combination of trastuzumab and HER2-derived peptide vaccine did not increase the cardiotoxicity. 88 Upregulation of immune checkpoint molecule expression on CD8 + T cells, such as PD-1 (programmed death 1), TIM-3 (T-cell immunoglobulin mucin 3) and TIGIT (T-cell immunoreceptor with Ig and ITIM domains), could inhibit immunopotentiation of the peptidebased vaccine. The peptide-based vaccine could also promote the infiltration of CD45RO + activation/memory T cells into the tumours, which in turn facilitate the increase of PD-1 + TILs. 89 These suggested that combination strategy of immune checkpoint inhibitors and peptide-based vaccines may be beneficial for tumour patients. 90,91 Indeed, the emergent of preclinical and clinical data demonstrated that the anti-tumour activity of immune checkpoint inhibitors can be enhanced by peptide vaccination.

| CON CLUS I ON AND PER S PEC TIVE
The peptide-based therapeutic cancer vaccines could be well WL wrote the manuscript. HT modified the review. WL, LL and XW collected and summarized the data. ZY and JL conceptualized the review. All authors read and approved the final manuscript.

CO N FLI C T S O F I NTE R E S T
The author declares that he/she has no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this published article.