Peptide-based vaccines for cancer therapy

Abstract

Interest for cancer vaccination started more than 30 years ago after the demonstration that both in animal models and later on in patients it is possible to generate anti-tumor immune responses. The clinical application of this knowledge, however, was disappointing.

In this review we summarize results on peptides epitopes recognized by T cells that have been studied thanks to their easy synthesis and the lack of significant side effects when administered in-vivo. To improve the clinical efficacy, peptides were modified in their aminoacid sequence to augment their immunogenicity. Peptides vaccines were recently shown to induce a high frequency of immune response in patients that were accompanied by clinical efficacy. These data are discussed at the light of recent progression of immunotherapy caused by the addition of check-point antibodies thus providing a general picture of the potential therapeutic efficacy of the peptide-based vaccines and their combination with other biological agents.

Keywords:

• cancer
• vaccination
• antigens
• epitopes
• immune responses

Background and Introduction

Immunotherapy of cancer has represented an active area of anti-cancer therapeutic investigation during the last 2–3 decades world-wide. This interest was generated early in the fifties through the sixties of the last century by the fathers of tumor immunology (i.e., Richmond Prehn, Philadelphia; George Klein, Stockholm; Lloyd J. Old/Edward Boyse, New York; Robert Baldwin, Nottingham). These investigators demonstrated in different animal models of tumors induced by a variety of carcinogens (e.g., chemicals, UV, viruses) that both unique tumor-associated antigens (TAAs) and shared self TAAs can coexist in the same tumor. Particularly the unique, mutation-derived TAAs were found to express a strong immunogenicity that can generate transplantation immunity leading to rejection of a challenge of the same tumor cells that will otherwise take and kill 100% mice.^1,2

These studies unequivocally demonstrated that experimental tumors can be recognized and rejected by the immune system though mechanisms explaining such features were not worked out at that time. In the subsequent 30 y or so numerous attempts were done to translate in the clinical arena such a concept without much success until recently (see below).

The Issue of TAAs to Be Used in Clinical Trials (Shared vs. Mutated)

The main explanation for this limited clinical outcome, in our opinion, lies in the type of TAAs that were targeted in these early clinical trials. In fact, the immunization of cancer patients with the aim of generating tumor cytotoxic immune responses started with the use of whole irradiated autologous tumor cells, then transduced with different cytokine genes (reviewed in³ with the hope that such cells could express immunogenic TAAs recognized by the immune system as determined by in vitro assays with autologous T cells.

The self TAAs

The clinical outcome of these trials, however, while proving the concept that under certain conditions tumor-derived peptides can elicit a tumor-restricted T cell response⁴ remained limited (10–20% response rate).

A clear progress in the field was the finding that TAAs of melanoma patients could be molecularly identified as normal protein involved in melanin production and melanocyte differentiation.^5,6 Many similar self TAAs were described in different human tumors⁷ and several of them were shown to be immunogenic thanks to the lack of complete tolerance of the body to these normal proteins. The next step in the study of human TAAs was the identification of the aminoacid sequences (9–13 aa long peptides) that were recognized by T cell receptor in the context of MHC molecules (epitope).

This evidence was also obtained by biochemical techniques that allowed the separation of the peptide/epitope from the MHC/peptide complex directly eluting them from tumor cells.^8,9 Thus these peptides were studied as potential immunogens in animal models and in cancer patients, particularly in those bearing metastatic melanoma.

Based on the identification of these shared peptides recognized by T cells in the context of HLA class I- and/or HLA-Class II several clinical studies were performed showing that tumor-specific T cell response can be activated even in the majority of patients without, however, a parallel induction of clinical response.^10-12

However, antigen peptides remained a focus of many investigations aimed at improving their immunogenicity and clinical efficacy. This occurred even because, peptide-based vaccines have distinct practical advantages and known manageable disadvantages in comparison with other vaccine formulation (e.g., tumor cells, viral vectors, DNA/RNA), and that are summarized as follows.

Advantages: (1) Known aminoacid sequence and length that allows the prediction of molecular interactions with the given MHC allele; (2) unlimited availability owing to their easy synthesis; (3) possibility to assess the T cell response ex-vivo against a known, target peptide/antigen.

Disadvantages: (1) high cost, due to the requirement of synthesizing the peptides under GMP conditions; (2) peptide stability may last for only 2–3 y and this may requires a re-synthesis and/or re-testing of peptides; (3) immunogenicity is variable according to each peptides since some of them may even induce immunosuppression.

Unique TAA-derived peptides

Recent advances in proteomic/genomic technologies (DNA sequencing) is allowing the identification of peptides deriving from non-synonymous somatic mutations that represent the truly tumor specific TAAs (previously defined as unique TAAs) absent in normal tissues and endowed with a higher immunogenicity as compared with self peptides.^9,13 Random mutagenesis throughout the genome is the hallmark of neoplastic transformation and occurs by nucleotide substitutions, deletions, insertions or gross chromosomal events (amplifications, deletions, inversions, translocations).^14,15 Mutations that confer a growth advantage are retained in the tumor genome by expansion of the clone bearing the mutation at the expense of other clones.¹⁴ This process generates many tumor-specific proteins bearing aminoacid substitutions, which frequently differ from tumor to tumor, therefore forming potential neo-antigens for the host's immune system as exemplified in melanoma and GBM patients.^16,17 There is evidence both in mice and humans that TAAs that result from mutations in cancer cell genes induce strong and specific anti-tumor immune responses though this may not occur for some mutations owing to the rapid elimination of the subpopulation of tumor cells by the immune system.

Peptide-Based Clinical Trials

It was soon realized, however, that ex-vivo immune response and clinical response of cancer patients to immunizing self peptides was limited in frequency, strength and/or duration thus making these vaccine of limited value in the clinics.^11,18,19

Polyspecific multipeptide approach

Since many different peptide/proteins TAAs may be express by cancer cells, a possible increase of anti-tumor response could be obtained using a vaccine containing several peptides and targeting them on cancer cells thus increasing the likelihood of inducing a T cell response able to destroy tumor cells.¹², ^{18, 20–23} Some of these trials were conducted as phase II randomized studies resulting also in a trend for clinical response.²⁴

The modified peptides

In addition, to overcome such weak tumor immunogenicity, altered peptide ligands containing single aminoacid substitution were constructed and used since they can improve immunogenicity by a higher affinity binding to HLA molecules. Such a strategy was shown to significantly increase T cell responses but without resulting in a parallel clinical benefit.²⁵ This clinical inefficiency was attributed to the low frequency of effective cross-recognition of melanoma cells TAAs. The use of a modified peptide from CEA interestingly showed a dose-dependent induction of anti-CEA (CAP-1) response with the high dose of 1mg causing T cell response in 100% of pancreatic cancer patients.²⁶

The long peptides

An additional modification of peptides was proposed by the group of Melief (Leiden) after an analysis of the structural modification and in vitro testing suggesting that such long (>13 aa) peptides appear to have e better immunogenicity and better efficacy most likely due to a higher affinity with the MHC molecules. This was proved in vivo in patients vaccinated against HPV peptides.²⁷

The Issue of Adjuvants

Immunological adjuvants are an heterogeneous group of compounds (e.g.Montanide, GM-CSF, AS15/MAGE-A3) that may increase the immune response of the immunizing antigen by different mechanism sharing, however, the capacity to recruit pro-inflammatory factors at the vaccination site and that have been used for a long time to improve the effect of vaccines in infectious diseases and, more recently in cancer. However, there is a limited evidence from phase III studies that one adjuvant may be superior to another in the clinical response to cancer vaccines. Adjuvants may include non-specific local stimulatory molecule like Montanide (an incomplete Freunds adjuvant like molecule),or chemokines that can recruit pro-inflammatory cells that may help the patient immune system to mount a systemic tumor cytotoxic response.^28-30

However, in a recent work Kruit and coworkers²⁹ have shown the superiority in overall survival of AS15 as compared with AS02_B in a randomized multicenter MAGE-A3-based vaccine in NSCLC patients.

We learned also how tumor cells defend themselves from the attack by the immune response (immune escape)³¹ an issue that needs to be addressed in each type of human tumors to be treated by immunobiotherapy.

Combination of Peptide Vaccines with Other Biological Agents

A new and promising area of studies is that of the combination of peptide vaccines with other biotherapeutics like immunomodulating or anti-vascular antibodies,^32,33 and even radio/chemotherapay.³⁴ The best combination appears to be that involving immunomodulatory agents (e.g., Ipilimumab, anti-PD-1, etc.)³⁵ which can amplify T cell expansion over time and increase the duration of the effect of vaccination.³² In fact, the only peptide-based successful phase III trial included a combination of the gp100 peptide and high dose of IL-2 in metastatic melanoma patients³⁶ that was crucial for the in vivo maintenance and expansion of T cells induced by the peptide.

These immunotherapeutic combinations are being tested in several phase I-II trials by different groups of researchers worldwide in the hope of increasing the clinica efficacy of cancer vaccination.

Gene signatures for immunotherapy

The availability of gene signatures to identify cancer patients that can respond to vaccination has been the focus of many researches during the last few years. Recent work of Wang and Bertognetti however, has identified a gene signature that may predict a T cell response to the MAGE.A3 of immunotherapy-treated patients.³⁷

Conclusions

The peptide-based vaccines have been used in the past with a limited clinical success. However, during the last few years new knowledge has been provided on the biological characteristics of the peptides and their interaction with the immune system to be used in the clinic. New protocols have allowed to obtain significant immune and clinical responses in patients vaccinated with multiple class I and II peptides particularly by combining the peptides with a variety of other biological therapeutics in phase II and III trials (Table 1). This situation is now even more promising then before and we predict that such new peptide-based trials will provide other clinical success in a variety of human tumors.

Table 1. Phase II and III peptide-based vaccination trials with either clinical or immunological statistically significant response

Peptide	Tumor	N. patients	Clinical response	Immune response	Adjuvant	Year of publication
E75/HER2/Neu	Breast cancer	182	DFS 94vs86% (P < 0.04)	Yes, dose-dependent	GM-CSF	Mittendorf et al. Cancer 118: 2594, 2012
Telomerase GV1001	Pancreas, breast ca. NSCLC	1062	Not significant	ND	GM-CSF	Brunsvig et al. Clin Cancer Res 17: 6847, 2011
Idiotype peptide+KLH	NH Lymphoma	177	PFS 44 vs. 30mos P = 0.047	ND	GM-CSF	Schuster et al. JCO 29: 2787, 2011
GP100	MetastaticMelanoma	185	OS 17vs. Eleven mos P < 0.06	P = 0.01 pts with clinical response	IL2, Montanide	Schwartzentruber et al. NEJM 364: 2119, 2011
Survivin	Melanoma Stage IV	61	OS 9mos	32%	Cyclo+ Montanide	Becker et al. Cancer Immunol Immunother 61: 2001, 2012
MUC-1 Stimuvax(L-BLP25)	NSCLC Stage IIIB	34	Chemo+ vax vs vax P = 0.016	47%	Cyclo+MPL	Wu et al. BMC 11: 430, 2011
CIMAVAX (EGFR)	NSCLC Stage IIIB, IV	80	OS: NS	11 vs 5% P < 0.024	Montanide+ Cyclo	Neninger et al. JCO 26: 1452, 2008
GP2-Her2Neu	Breast cancer	172	NS	DTH 21vs 6mm P < 0.01	GM-CSF	Trappey et al., JCO; 31: Abstr 3005, 2013
IMA901	RCC	68	OS 23vs14 mos P = 0.023	74%	GM-CSF +Cyclo	Walter et al. Nature Med 18: 1254, 2012

Cyclo, Cyclophosphamide; DFS, disease-free survival; DTH, Delayed-type hypersensitivity; GM-CSF,Granulocyte Macrophage-Colony Stimulating Factor; KLH, Keyhole Limpet Hemocyanin; HER2, Human epidermal growth factor 2; MPL, Monophosphoryl Lipid A; ND, Not done; NS, Not Significant; NSCLC, Non small cell lung cancer; OS, Overall Survival; PFS, Progression-free Survival; Trials features as listed from clinicaltrials.gov of May 2014 and from Melero et al.¹²

Disclosure of Potential Conflicts of Interest

M.M. has received research funding by Bristol Myers Squibb (BMS) and participated in advisory Boards and communication activities from BMS and Roche. G.P. has participated in communication activities from BMS.

Acknowledgments

We thank Silvia Galvani for help in editing the manuscript.

Funding

Our work was supported by a Grant of AIRC (Italian Association for Cancer Research (Milan) to G.P.