4-Peptide Vaccine of The Future

REVIEW Special Issue: Immunopeptidomics
The Peptide Vaccine of the Future

Authors
Annika Nelde, Hans-Georg Rammensee, and Juliane S. Walz
Correspondence Graphical Abstract

juliane.walz@med.uni-tuebingen.
de
In Brief
Therapeutic peptide-based
vaccination approaches for the
treatment of cancer patients
have shown first glimmers of
success. However, to achieve
broad clinical efficacy and
implement peptide vaccinations
in the standard treatment of
cancer patients future peptide
vaccines need further
optimization in terms of target
antigen selection, adjuvant
choice, vaccination schedules,
delivery routes, biomarkers, and
combinatorial drugs.
Highlights
• Several prerequisites are mandatory for clinical effective peptide-based vaccines
• Overview of current approaches regarding antigen selection and adjuvants
• Suitable combinations will enhance the efficacy of peptide vaccines in the future
• Biomarkers could be useful to preselect eligible patients for vaccination
2021, Mol Cell Proteomics 20, 100022

© 2020 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and
Molecular Biology.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
https://doi.org/10.1074/mcp.R120.002309
REVIEW Special Issue: Immunopeptidomics
The Peptide Vaccine of the Future

Annika Nelde1,2,3 , Hans-Georg Rammensee2,3,4 , and Juliane S. Walz1,3,*
The approach of peptide-based anticancer vaccination the surface of cells on human leukocyte antigen (HLA) mole-
has proven the ability to induce cancer-specific immune cules and are recognized by the T cell receptor of CD4+ and
responses in multiple studies for various cancer entities. CD8+ T cells.
However, clinical responses remain so far limited to single
patients and broad clinical applicability was not achieved.
Therefore, further efforts are required to improve peptide ANTIGEN SELECTION—THE DEVIL IS IN THE DETAILS
vaccination in order to integrate this low-side-effect The most essential aspect for a clinically effective peptide
therapy into the clinical routine of cancer therapy. To vaccination is indeed the selection of optimal antigen targets,
design clinically effective peptide vaccines in the future,
which should exhibit a highly frequent and tumor-exclusive
different issues have to be addressed and optimized
presentation on the surface of tumor cells and must be
comprising antigen target selection as well as choice of
optimal adjuvants and vaccination schedules. Further- capable of recognition by the patients’ T cells. In recent years
more, the combination of peptide-based vaccines with it has become apparent that neoepitopes derived from tumor-
other immuno- and molecular targeted therapies as well specific nonsynonymous somatic mutations are the central
as the development of predictive biomarkers could further specifications of immune checkpoint inhibitor-induced T cell
improve efficacy. In this review, current approaches in the responses (8). This is in line with the correlation of response to
development of peptide-based vaccines and critical im- checkpoint inhibitor therapy with tumor mutational burden
plications for optimal vaccine design are discussed. (9, 10). Owing to their true tumor specificity, neoepitopes are
not affected by central or peripheral tolerance, which renders
them ideal targets. The encouraging potential of neoantigen-
PEPTIDE-BASED VACCINATION
based vaccination approaches was already investigated in
The idea of activating the immune system to combat cancer preclinical and early-phase clinical trials assessing
cells was already documented in ancient Egypt (1) and was neoantigen-based peptide vaccines in several different cancer
then again pursued in the late 19th century by William Coley, entities (11–14). Yet, substantial challenges remain in the
who had been exploiting streptococcus-mediated tumor effective identification of immunogenic and naturally pre-
rejection (2, 3), probably after hearing from similar experiments sented neoepitopes derived from point mutations, insertion–
by Wilhelm Busch (4). Such antigen-independent strategies deletions or frame-shift mutations. Therefore, in recent
build the cornerstone for a broad and promising research area years, several different bioinformatic tools have been devel-
aiming to actively induce antitumor T cell responses using oped to improve the selection and identification of candidate
various different strategies. Today, multiple antitumor immu- neoepitopes for vaccine design. Based on whole-exome
notherapy approaches (5), including immune checkpoint sequencing data algorithms such as NeoPredPipe, MuPeXI,
blockade, CAR T cells, and antibody-based therapies, have pVAC-Seq, and CloudNeo identify patient-individual muta-
found their way into the clinic and even established in the tions, predict the affinity of neoantigen-derived peptides to
routine of anticancer treatment. Therapeutic cancer vaccines, HLA molecules regarding to the individual patient’s HLA al-
such as protein-, peptide-, DNA-, RNA-, and dendritic cell– or lotypes, integrate tumor mutation and expression data, predict
tumor cell–based vaccines (6, 7), are designed to generate the immunogenicity of these peptides, and evaluate their po-
new or amplify preexisting antigen-specific T cell responses tential as T cell epitopes (15–18). Still, this selection and pre-
against malignant cells. Peptide-based vaccines represent a diction process remains challenging especially for HLA class
low-side-effect vaccination approach using synthetic tumor- II–presented neoepitopes recognized by CD4+ T cells (19). In
associated or -specific peptides or peptide combinations addition, the use of mutation-derived vaccine peptides har-
that are designed to induce and activate peptide-specific tu- bors two main issues: the majority of tumor-specific mutations
mor-reactive T cells in vivo. These peptides are presented on are patient-specific and therefore not suitable for broadly
From the 1Clinical Collaboration Unit Translational Immunology, German Cancer Consortium (DKTK), University Hospital Tübingen, Tübingen,
Germany; 2Department of Immunology, Institute for Cell Biology, 3Cluster of Excellence iFIT (EXC2180) “Image-Guided and Functionally
Instructed Tumor Therapies”, University of Tübingen, Tübingen, Germany; 4German Cancer Consortium (DKTK) and German Cancer Research
Center (DKFZ), Partner Site Tübingen, Tübingen, Germany
* For correspondence: Juliane S. Walz, juliane.walz@med.uni-tuebingen.de.
Mol Cell Proteomics (2021) 20 100022 1

© 2020 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1074/mcp.R120.002309
Peptide Vaccine of the Future
applicable immunotherapy, but rather can be used for reviewed journal. Furthermore, comparative transcriptome
personalized approaches. Furthermore, in cancer entities with and proteome expression profiling between tumor and normal
low mutational burden, the role and impact of neoepitope- tissues as provided, for example, by the Genotype-Tissue
directed T cell responses appear to be marginal, as several Expression Consortium (48) and the Human Protein Atlas (49),
studies have demonstrated that the frequency of naturally can help to reconsider vaccine candidates and filter out genes
presented HLA-presented neoepitopes is less than 1% of the and proteins highly expressed in normal tissues. However, the
number of nonsynonymous mutations based on mass above-described distorted correlation of gene expression,
spectrometric analysis (11, 20–22). Mass spectrometry also proteome, and immunopeptidome (21, 32–35) clearly limited
revealed that hotspot sequence regions within individual the informative value of these analysis. Furthermore, it is
proteins generate the majority of HLA-presented peptides in important to note that tumor antigens are not always deter-
contrast to other protein domains that are not efficiently pro- mined at the protein level. The individual peptides can be
cessed for HLA presentation (21, 23, 24), which highlight the clearly tumor-associated, even if the source protein itself is
need for immunopeptidomics to select truly processed and not. This can occur through differential processing, post-
naturally presented peptides and neoepitopes. Therefore, translational modifications, or antigen processing in the tumor
future large-scale immunopeptidomics studies may reveal cell and highlights the need for immunopeptidomic databases
such hotspots in the human proteome leading then to of benign tissues and cells.
improved bioinformatics tools that prioritize and select neo- In addition to the selection of optimal antigens, additional
epitopes based on their location within such hotspot regions. questions rise about the ideal peptide length, the choice of
Besides mutation-derived neoepitopes unmutated tumor- CD4+ versus CD8+ T cell epitopes, as well as the number of
associated self-peptides have gained renewed attention. different peptides within the vaccine cocktail. In recent years it
These unmutated peptides can arise from aberrantly has become evident that the selection of HLA class
expressed or processed antigens in the tumor cells owing to II–restricted antigen targets recognized by CD4+ T cells in
differential tumor-specific gene expression, posttranslational addition to CD8+ T cell–recognized HLA class I–presented
modification, antigen processing, or other tumor-specific peptides is of paramount importance for clinical outcome.
cellular processes (25, 26). For the selection of suitable CD4+ T cells harbor a crucial role for effective antitumor im-
tumor-associated unmutated peptides different strategies munity (50) and could further improve and sustain T cell
have been applied. For several years, the identification of reactivity (51) and tumor clearance (52). CD4+ T helper cells
tumor-associated antigens was based solely on gene induce and intensify more permanent tumor immune control
expression analyses identifying overexpressed proteins, fol- (53), epitope spreading (54), CD8+ T cell expansion and sur-
lowed by in silico predictions of HLA-presented peptides vival (55), as well as tumor immune cell infiltration (56). In
(27–29) with algorithms such as SYFPEITHI (30) and addition, CD4+ T cells also exhibit direct antitumor effector
NetMHCpan (31). Subsequently, the tremendous number of function (57). Therefore, the artificial elongation of tumor-
predicted candidate peptides had to be screened for immu- associated CD8+ Tcell epitopes or the application of multi-
nogenicity by extensive in vitro assays. A major drawback of valent long peptides comprising at the same time CD4+ and
such prediction-based approaches lies in the distorted cor- CD8+ T cell epitopes displays promising attempts to improve
relation of gene expression with HLA-restricted antigen pre- the efficacy and clinical outcome of peptide vaccination
sentation, since the immunopeptidome is an independent (58–63). The enhanced induction of T cell responses with long
complex layer formed by the antigen presentation machinery vaccine peptides could also be explained by the fact that
and therefore does not necessarily mirror the transcriptome or exact epitopes bind directly to HLA class I molecules of any
the proteome (21, 32–35). In contrast to epitope prediction somatic cell and thus also to nonprofessional antigen-
algorithms, the large-scale and in-depth direct identification of presenting cells (APCs) without further processing, which
naturally HLA-presented peptides, known as HLA ligandome could then lead to suboptimal T cell priming and induce T cell
or immunopeptidome, from primary patient samples by mass tolerance (51, 64, 65). Long peptides, on the other hand,
spectrometry (36–44) is the only unbiased approach to require processing and are therefore only presented by pro-
comprehensively identify tumor rejection antigen candidates fessional APCs (66, 67). We therefore believe that HLA class
and validate the natural processing and presentation of these II–restricted target antigens also in terms of elongated CD8+ T
antigens (45). For the identification of tumor-associated cell epitopes are an indispensable component for clinically
unmutated self-peptides not presented on benign cells and effective peptide vaccines. This is further supported by the
tissues, the extensive screening of immunopeptidomes higher promiscuity of HLA class II molecules leading to a not
derived from benign tissues is of paramount importance. The so tight HLA restriction of the presented peptides (68) in
HLA ligand atlas (www.hla-ligand-atlas.org/) is an open- contrast to CD8+ T cell epitopes. This enables the presenta-
source tool providing a comprehensive collection of mass tion of the same CD4+ T cell epitope on different HLA class II
spectrometry–identified HLA-presented peptides on benign molecules and allows the vaccination of all patients with the
tissues (46, 47), which has currently been submitted to a peer- same cocktail, regardless of their HLA allotype.
2 Mol Cell Proteomics (2021) 20 100022

In contrast to other antigen-specific treatments, e.g., CAR T induction of peptide-specific T cell responses was demon-
cells, a remarkable advantage of peptide-based vaccines is strated against mutated and self-peptides equally (71) as well
the possibility of a simple and cost-effective combination of as short (77) and long peptides (11). However, patient and
several different tumor-associated antigens in a single injec- antigen selection, dosing, vaccine schedule, as well as usage
tion. Multipeptide vaccines could therefore overcome the of adjuvants and combinatorial treatments differ in all of these
issue of antigen loss and reduce the risk of immune escape, studies, which hampers a comprehensive comparison and
which often occurs under therapeutic pressure (69). The conclusion for the development of improved peptide vaccines.
approach of multipeptide vaccination is further supported by The chemical or sequence modification of the individual
the observation that patients with immune responses to mul- vaccine peptides represents an additional option to improve
tiple tumor-associated antigens showed higher levels of dis- the efficiency of the vaccine-induced antitumor responses
ease control and harbor a survival benefit (38, 70). (reviewed in 81, 82). Amino acid substitutions of self-antigens
Furthermore, validation of target antigen expression on could circumvent tolerance against unmutated self-peptides.
patients’ tumor cells prior to the administration of a peptide Altering the amino acid sequence could not only increase
vaccine to a patient could considerably improve the propor- the peptide binding affinity to its respective HLA molecule, but
tion of patients who can benefit from a specific vaccine. also improve its immunogenicity (83–85). Besides the impact
Validation of both unmutated and mutated target antigen on the strength of a T cell response, altered peptide se-
expression can be carried out at different levels such as quences could also modulate the character and polarization of
transcriptome, proteome, or immunopeptidome. For mutated the peptide-specific immune responses (86, 87). Especially in
antigens, the proof of the presence of the somatic mutation by chronic diseases such as cancer or viral infections heteroclitic
whole-exome sequencing or RNA-Seq is required, but the peptides can help to restore T cell function and proliferation
additional evidence of the natural presentation within the (88) and repeal frequently occurring T cell exhaustion caused
immunopeptidome is usually rather difficult for these antigens by antigen persistence (89). However, such changes also
(71). However, for unmutated target antigens the validation of harbor the potential risk of weakening the T cell response or
antigen expression using immunopeptidomic analysis is highly potentially inducing the expansion of T cells with irrelevant or
recommended, especially in contrast to transcriptomic or pro- even autoreactive specificities. Therefore, the careful evalua-
teomic detection of antigen expression, which often shows a tion of altered peptide ligands is of paramount importance.
distorted correlation to HLA antigen presentation (21, 32–35). In addition to the modified amino acid composition, direct
Besides the presentation of the target antigens on the pa- conjugation of ligands of pattern recognition receptors such
tients’ tumor cells, the immunogenicity of the individual vac- as CpG or Pam3CSK4 for Toll-like receptors (TLRs) (90, 91) or
cine peptides is of paramount importance and has to be C-type lectin receptor–specific mannosylation (92) on long
proven in T cell–based assays prior to inclusion of the specific vaccine peptides could enhance antigen uptake and guide
peptide in a vaccine cocktail (72, 73). Generally, two different intracellular trafficking, thereby favoring antigen presentation.
approaches can be utilized to assess T cell responses to a
ADJUVANTS AND DELIVERY MODE—IT IS ALL ABOUT ACTIVATION
specific antigen: 1) in vitro priming of naive T cells or 2) ex vivo
or in vitro stimulation of preexisting memory T cells. For the For the induction of effective T cell responses through
detection of preexisting memory T cell responses patient- peptide-based vaccines the choice of optimal, strong adju-
derived peripheral blood mononuclear cells or tumor- vants or immunostimulators and ideal delivery routes is of
infiltrating lymphocytes are needed since tumor antigen- paramount importance to ensure that the vaccine peptides are
specific T cells are normally not abundantly present in appropriately sensed by and activate the immune system. The
healthy individuals. Depending on the abundance of preex- main focus of peptide vaccination is here to induce a type
isting memory T cells, their detection can be performed 1–polarized, cell-mediated immunity rather than a type
directly ex vivo or after a short in vitro peptide-specific 2–polarized and humoral response (93). For achieving an
amplification with cytokines such as interleukin 2 (IL-2). As optimal T cell–mediated antitumor effect, the activation and
readout different methods can be applied such as enzyme- expansion of antigen-specific T cells is mandatory, which
linked immunospot assay (74), intracellular cytokine staining necessarily requires the three well-known signals of T cell
(75), or multimer staining (76). Such immunogenicity tests receptor stimulation, appropriate costimulation, and specific
should also be performed during a clinical trial to assess the cytokines (94, 95). Therefore, adjuvants need to deliver the
induction of vaccine-induced T cell responses in vaccinated peptide to dendritic cells and activate and mature these APCs
patients. to accomplish a solid T cell response. The purpose of such
In recent years, multiple peptide-based vaccination trials adjuvants thus comprises the protection of the peptide and
have tested different combinations of short (77) versus elon- prevention of immediate degradation, the efficient uptake by
gated peptides (11), of single peptide–based (78) versus APCs, as well as the appropriate and full activation of APCs.
multipeptide vaccines (79), or of mutated (11) versus unmu- Delivery vehicles, which ideally should have a depot effect,
tated (80) peptides in a variety of different cancer entities. The can be composed, for example, of oil depots such as

Montanide ISA 51 (incomplete Freund’s adjuvant analog) TREATMENT CONCEPTS—BROADLY APPLICABLE OR

(96, 97). Montanide is therefore mixed with the peptides prior PERSONALIZED?
to vaccination to generate a water-in-oil emulsion. For efficient For the clinical application of immunotherapeutic ap-
uptake, the peptides can also be encapsulated in structures proaches three different study concepts and strategies
such as liposomes (98) and nanoparticles (99) or can be comprising distinct levels of personalization of drug products
covalently conjugated to adjuvants (90, 92). In multipeptide and anticancer therapies have been proposed: 1) stratifica-
vaccination approaches and especially in patient- tion, 2) warehouse-based personalization, and 3) individual-
individualized approaches the covalent linkage of adjuvants ization (114, 115). Each of these three concepts harbors
to each individual and single peptide is cumbersome for several advantages as well as disadvantages that need to be
clinical translation. considered. Stratification-based treatment decisions as the
So far the cytokine granulocyte-macrophage colony basic level of personalization are already standard in clinical
stimulating factor (GM-CSF) that initiates the recruitment, routine, for example in the biomarker-based application of
maturation, and activation of dendritic cells (100) has been targeted therapies for specific mutations (116) or antibody-
one of the most common adjuvants applied in anticancer based immunotherapies (117). Stratification selects suitable
peptide vaccination trials but its adjuvant effect remains patients who will benefit from a specific therapy based on the
weak (71, 101, 102). For activation and maturation of APCs, availability of predefined tumor-associated criteria and will
signaling through TLRs and their ligands is known to induce then all be treated with one invariant drug product. Only
optimal and strong activation. Therefore, potent adjuvants patients harboring the respective tumor feature will receive
often mimic such TLR ligands. Especially TLR4 ligands are the therapy. Thereby, stratification represents an approach
known to enable a potent activation of APCs (103). However, with an enhancement of treatment efficacy and in the same
the prototype among TLR4 ligands—lipopolysaccharide time minimization of side effects. However, for peptide-
(LPS)—is not sustainable for clinical application owing to based vaccination approaches stratification alone seems
substantial toxicity (104, 105). Its chemically detoxified form not suitable owing to the high intraindividual differences
MPL (3-O-desacyl-4’-monophosphoryl lipid A) (106) is an already starting with patient–individual HLA allotypes.
approved adjuvant, for example, in human papillomavirus Therefore, stratification-based peptide vaccine approaches
vaccines and furthermore investigated in different vaccina- focus on very common HLA allotypes such as HLA-A*02 or
tion approaches (107, 108). The most commonly used TLR HLA-A*24 thereby excluding a substantial proportion of pa-
agonist (71) poly-ICLC (Hiltonol) is a polyinosinic- tients (70, 71). Consequently, current vaccine designs are
polycytidylic acid (poly-IC) stabilized by lysine and more and more focusing on warehouse approaches (77, 80,
carboxymethylcellulose (109), which enhances vaccine- 118), patient-individualized concepts (119), or also a com-
induced T cell responses (110) by TLR3 signaling. bination of both (71). The so-called warehouse concept en-
Recently, the novel, water-soluble adjuvant XS15, a synthetic ables the composition of patient-specific drug products
TLR1/2-binding Pam3-Cys-derivate covalently linked to a assembled from a collection of predefined and pre-
single synthetic—nonvaccine—peptide (GDPKHPKSF), was manufactures high-frequent tumor-associated peptides.
described as an effective vaccine adjuvant inducing unpre- Therefore, each peptide within the warehouse is separately
ceded strong and long-lasting CD8+ and CD4+ T cell re- manufactured and depending on the patient’s individual
sponses in first-in-man proof-of-concept experiments (111). characteristics such as HLA allotypes or tumor-presented
The first clinical trials using XS15, including antitumor peptides the peptide vaccine cocktail is then individually
(EudraCT 2020-002367-65) as well as highly relevant anti- assembled using these off-the-shelf peptides. Thus, the
SARS-CoV-2 peptide vaccinations (EudraCT 2020-002502- warehouse concept enables the individualization of vaccine
75; EudraCT 2020-002519-23), will start recruitment within cocktails in a time- and cost-saving manner. For example, for
2020. Since the induction of the antitumor T cell responses a hypothetical warehouse peptide vaccination study covering
and the clinical outcome has so far been unconvincing, it is eight different HLA allotypes with five peptides each and
mandatory to investigate novel adjuvants like XS15 in clinical including 25 patients, who should be vaccinated with a
trials to further increase the strength and unlock the power of multipeptide cocktail comprising, for example, as commonly
peptide-based vaccines. practiced and approved by regulatory authorities (71, 80), 10
Further improvement of imaging technologies to track either peptides, only 40 different peptides have to be produced.
the peptide vaccine at the molecular and (sub)cellular level The number of 40 peptides is calculated by multiplying the
(112) or the peptide-specific T cells in the organism (113) will number of unique peptides per allotype by the number of
further expand our knowledge of antigen delivery, uptake, and different HLA allotypes covered within the respective ware-
processing as well as of antigen-specific T cell routes. This house. A warehouse covering, for example, eight of the
knowledge can then be utilized to further improve peptide- world’s most common HLA class I allotypes could be used to
based vaccination regarding delivery vehicles, adjuvants, treat approximately 90% of a patient cohort, which could be
and administration route.

calculated for individual allotype combinations using the comparison of different vaccination regimens and each clinical
population coverage tool of IEDB (http://tools.iedb.org/ trial uses different vaccination schedules, thus making evalu-
population/) (120, 121). ation extremely difficult.
In contrast, completely individualized peptide-vaccine
concepts are based on the selection and on-demand de COMBINATIONS—IT IS EASIER TOGETHER
novo production of single patient-specific drug products. The
Important considerations for clinically effective peptide
advantage of such approaches is the patient-specific design
vaccination encompass not only the actual design and
of such vaccines individually tailored on the patient’s HLA
formulation of the peptide vaccine itself, but also furthermore
allotypes, single rare mutations, and peptides presented on
require the awareness of effective combinatorial treatment
the individual tumor reaching the maximal drug benefit for
options. Cancer vaccines alone may be effective in cases of
each cancer patient. Personalized peptide vaccines can
early cancer diagnosis or in the setting of minimal residual
therefore comprise patient-tailored neoepitopes based on
disease to prevent relapse or recurrence. However, combi-
individual sequencing data and/or nonmutated tumor-
natorial treatments might enable to successfully treat even
associated peptides based on the patient-individual mass
established cancers as well as to overcome tumor-mediated
spectrometric analysis of tumor-presented peptides. There-
immunosuppression, immune escape mechanisms, impaired
fore, sequencing or mass spectrometry-based identification of
T cell infiltration to the tumor side, or profound immune de-
patient-individual neoepitopes and nonmutated tumor-
fects (126–129). The range of possible combinations is
associated peptides becomes indispensable. The translation
endless, e.g., combinatorial approaches of cancer vaccines
of sequencing data, mass spectrometry–based immuno-
with immune checkpoint inhibitors, chemotherapies, radio-
peptidomics and their combination is already investigated in
therapy, neutralizing antibodies to inhibitory cytokines, small
different clinical trials (71, 80, 122). However, the low-
molecule inhibition of regulatory T cells, or immunomodulatory
throughput sample capacity is still a major issue. Further
drugs have already been investigated in several studies (80,
limitations of completely individualized approaches are the
130–133) with the main goal to optimize the T cell compart-
huge peptide production costs and the limited drug produc-
ment. However, combinatorial cancer therapies might also
tion capacity. The enormous differences in the estimated
negatively impact T cell responses, as shown in a phase III
costs between peptide warehouse approaches and patient-
peptide vaccination study combined with the tyrosine kinase
individualized vaccines arise mainly from the increased
inhibitor sunitinib (134), which failed to confirm the vaccine-
number of different peptides required for patient-individual
induced immune responses and clinical outcome of the cor-
approaches. Compared with the warehouse approach, for
responding phase II trial testing the peptide vaccine without
the hypothetical study with 25 patients and 10 peptides per
sunitinib (70). This calls for the extensive preclinical and clin-
cocktail a fully individual approach would require the pro-
ical analysis of the effect of combinatorial drugs on T cell
duction of in total 250 peptides resulting in more than 5-fold
response.
higher peptide production costs.
In addition, anticancer drugs can also have marked effects
on the immunopeptidome of tumor cells, including HLA sur-
VACCINATION SCHEDULE—THE RIGHT TIME POINT ALSO MATTERS face expression (135), HLA allotype distribution (136), the
presentation of vaccine target peptides, as well as the in-
Besides the selection of the best fitting treatment concept
duction of novel, cryptic, treatment-associated ligands
also the schedule and timing of vaccination represent an
(136, 137). Therefore, it is of paramount importance to char-
essential pillar for clinically effective vaccination. The selection
acterize also the effects of combinatorial drugs not only on the
of the optimal time point for vaccination in the course of
effector cells but also on the antigenic landscape of the target
cancer treatment is extremely important. Peptide-based
cells (138). Furthermore, the induction of novel, treatment-
vaccination approaches should ideally be administered in
associated HLA ligands on cancer cells under specific treat-
the setting of an intact T cell compartment with an optimal
ments could be utilized for the identification of novel suitable
effector to target cell ratio (123). This could be optimally
antigen targets that could be eligible as novel treatment-
achieved in an adjuvant setting after surgery or in first-line
associated targets for combinatorial peptide vaccinations
remission, e.g., minimal residual disease induced by stan-
(137–139).
dard chemotherapy or radiation (124, 125). Furthermore, for
selection of the optimal vaccination time point, potential
concomitant therapies must be taken into account as these BIOMARKERS—IMPORTANT TO KNOW
drugs can influence the outcome and efficacy of peptide The identification of prognostic factors and predictive bio-
vaccination, as discussed below. Furthermore, the vaccination markers is very important to assist the selection of those
schedule, including primary and boost vaccinations, may have patients who are expected to respond well to peptide
an impact on the effectiveness and duration of the antitumor T vaccination, in the best case even before the start of therapy
cell response. However, there is hardly any systematic or at least early after treatment initiation. However, the criteria

for patients selected for peptide vaccination and the selection Thus, multiple epitope vaccine approaches are already
of individual vaccine peptides vary greatly between different applied in the majority of clinical trials. Furthermore, the HLA
clinical trials, including preexisting T cell responses (77), allotype restriction of HLA class I–presented peptides might
expression of target antigens on the patient’s tumor (71, 80) or exclude patients with unmatching HLA class I allotypes, which
on soluble HLA molecules in the plasma (140–142), peptide- could be overcome by using HLA class II–presented promis-
specific preexisting immunoglobulin G responses (77), or in- cuous CD4+ T cell epitopes embedding multiple HLA class I
dividual transcriptome analysis (71). Several studies have re- peptides in the vaccine.
ported that the immunoglobulin G response against the Despite these rather peptide-specific limitations, there is in
vaccine peptides is a potential biomarker for the prediction of our view a major obstacle for the clinical success of all T cell–
overall survival (143, 144). However, the biological role of based immunotherapies including vaccines, which is the
peptide-specific antibodies remains still unknown. Increased limited accessibility of the tumor for cancer-specific T cells.
peptide-specific immunoglobulin G levels might reflect the Especially in solid tumors, T cell recruitment is often impaired
activation of CD4+ T cells (144). Further plasma biomarkers, by an immunosuppressive tumor environment (149). To
which were found to be useful predictive markers for clinical overcome the limited T cell recruitment combinatorial ap-
outcome after peptide vaccination, are the level and integrity proaches with direct or indirect microenvironment modifiers
of circulating cell-free DNA (145), or specific microRNAs (146). such as BRAF, MEK, or PARP inhibitors or VEGF-targeting
Recently, a study also demonstrated that the prevaccination, antibody- or inhibitor-based therapies have already been
but not postvaccination, blood cell composition (high evaluated (150).
lymphocyte cell counts and percentage) and the inflammatory Peptide-based cancer vaccines are in generally well toler-
signature (low levels of CRP, IL-6) are associated with overall ated with only a few side effects including local reaction on the
survival after personalized peptide vaccination (147). The vaccination site (70, 134). In a meta-analysis including 500
expression level of the immune checkpoints PD-1 and Tim-3 patients, only 1.2% of the vaccinated patients suffer from
on T cells has been demonstrated to correlate with overall vaccine-related serious adverse events (151). Augmented
survival in a peptide vaccination trial (148) further highlighting immune responses seem to be involved as both cellular and
the importance of combinatorial approaches with immune humoral responses to the vaccine peptides are boosted in
checkpoint inhibitors. affected patients. Beside allergic reaction against the vaccine,
The identification of biomarkers that can be utilized for the a major potential risk that can be anticipated for patients
selection of patients and predict the response to peptide- treated with peptide-based vaccines is autoimmunity induced
based immunotherapy is both important and attractive. by immune reactions against normal tissues expressing the
Since multiple factors may influence the induction of clinically vaccinated targets at considerable levels. Therefore, a precise
effective anticancer T cell responses after peptide vaccination, characterization of the target expression on normal tissues is
their predictive and prognostic capabilities have to be vali- of paramount importance. However, such autoimmunity
dated by further large-scale, prospective, and randomized events are very rare. In the phase I to III clinical trials of the off-
clinical trials. the-shelf multipeptide vaccine IMA901 for the treatment of
renal cell carcinoma with a total of 302 vaccinated patients
LIMITATIONS AND SIDE EFFECTS OF PEPTIDE-BASED VACCINATIONS (70, 134) as well as in the phase I/II trial of an antiprostate
AND THE WAY OUT—LITTLE BY LITTLE ONE GOES FAR
cancer vaccine treating 19 patients (152) no evidence of
Although the expectations placed in peptide-based vacci- autoimmunity was observed.
nation approaches have not been fulfilled so far and clinical To provide a maximum of safety for vaccinated patients and
success is still limited, more and more ideas and ways to to ensure early notice of side effects, especially allergic re-
overcome these disadvantages are emerging. In this section actions or autoimmunity, risk mitigation measures must be
we will discuss the drawbacks of peptide-based vaccine ap- implemented in each clinical trial. First, patients should be
proaches, take a look at the issue of adverse effects, and point monitored and kept under medical supervision for at least 2 h
out solutions to overcome these disadvantages, which have following each vaccination, including close monitoring of vital
already been partly applied and evaluated in different clinical parameters (pulse, blood pressure, temperature, oxygen
trials. A first limitation is the low immunogenicity of vaccine saturation) and subjective well-being, as allergic reactions
peptides reported in some clinical trials (11), which can be were reported to occur mostly in the first hour after vaccina-
overcome in different ways: optimized preclinical selection of tion (71, 134). In case of severe anaphylactic reactions, stan-
suitable antigens and the characterization of their immuno- dardized medical antiallergic treatment such as antihistamine
genicity, the modification and affinity maturation (81, 82), as should be applied. Furthermore, special emphasis should be
well as the use of improved strong but nontoxic adjuvants as put on any sign of the development of autoimmune disease.
discussed above are the most important points to address this Any unclear inflammation should be followed up closely until
problem. A further disadvantage is a potential immune escape it resolves, and pausing or stopping of vaccinations and
if vaccination is performed only with single target peptides. immunosuppressive treatment should be considered.

CONCLUSION 5. Taefehshokr, N., Baradaran, B., Baghbanzadeh, A., and Taefehshokr, S.

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4-Peptide Vaccine of The Future

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REVIEW Special Issue: Immunopeptidomics

The Peptide Vaccine of the Future

Correspondence Graphical Abstract

• Several prerequisites are mandatory for clinical effective peptide-based vaccines

• Overview of current approaches regarding antigen selection and adjuvants

• Biomarkers could be useful to preselect eligible patients for vaccination

2021, Mol Cell Proteomics 20, 100022

The Peptide Vaccine of the Future

Mol Cell Proteomics (2021) 20 100022 1

2 Mol Cell Proteomics (2021) 20 100022

Mol Cell Proteomics (2021) 20 100022 3

Montanide ISA 51 (incomplete Freund’s adjuvant analog) TREATMENT CONCEPTS—BROADLY APPLICABLE OR

4 Mol Cell Proteomics (2021) 20 100022

Mol Cell Proteomics (2021) 20 100022 5

6 Mol Cell Proteomics (2021) 20 100022

CONCLUSION 5. Taefehshokr, N., Baradaran, B., Baghbanzadeh, A., and Taefehshokr, S.

Mol Cell Proteomics (2021) 20 100022 7

8 Mol Cell Proteomics (2021) 20 100022

Mol Cell Proteomics (2021) 20 100022 9

10 Mol Cell Proteomics (2021) 20 100022

Mol Cell Proteomics (2021) 20 100022 11

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