Review

mRNA-Based Cancer Vaccines: A Therapeutic Strategy for the

Treatment of Melanoma Patients
Maryam Bidram 1 , Yue Zhao 2,† , Natalia G. Shebardina 3,† , Alexey V. Baldin 4,5 , Alexandr V. Bazhin 2,6 ,
Mohamad Reza Ganjalikhany 1 , Andrey A. Zamyatnin, Jr. 3,4,7,8, * and Mazdak Ganjalikhani-hakemi 9, *

1 Department of Cell and Molecular Biology, Faculty of Biological Science and Technology,
University of Isfahan, Isfahan 8174673441, Iran; [email protected] (M.B.);
[email protected] (M.R.G.)
2 Department of General, Visceral and Transplant Surgery, Ludwig-Maximilians University of Munich,
81377 Munich, Germany; [email protected] (Y.Z.);
[email protected] (A.V.B.)
3 Institute of Molecular Medicine, Sechenov First Moscow State Medical University, 119991 Moscow, Russia;
[email protected]
4 Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University,
119992 Moscow, Russia; [email protected]
5 V.I. Kulakov National Medical Research Center of Obstetrics, Gynecology and Perinatology,
117997 Moscow, Russia
6 German Cancer Consortium (DKTK), Partner Site Munich, 81377 Munich, Germany
7 Department of Biotechnology, Sirius University of Science and Technology, 1 Olympic Ave,



354340 Sochi, Russia

 8 Faculty of Health and Medical Sciences, University of Surrey, Guildford GU2 7X, UK
9 Department of Immunology, Faculty of Medicine, Isfahan University of Medical Sciences,
Citation: Bidram, M.; Zhao, Y.;
Isfahan 8174673441, Iran
Shebardina, N.G.; Baldin, A.V.; * Correspondence: [email protected] (A.A.Z.J.); [email protected] (M.G.-h.)
Bazhin, A.V.; Ganjalikhany, M.R.; † Authors have equal contribution.
Zamyatnin, A.A., Jr.;
Ganjalikhani-hakemi, M.
Abstract: Malignant melanoma is one of the most aggressive forms of cancer and the leading cause
mRNA-Based Cancer Vaccines: A
of death from skin tumors. Given the increased incidence of melanoma diagnoses in recent years,
Therapeutic Strategy for the
it is essential to develop effective treatments to control this disease. In this regard, the use of
Treatment of Melanoma Patients.
cancer vaccines to enhance cell-mediated immunity is considered to be one of the most modern
Vaccines 2021, 9, 1060. https://
doi.org/10.3390/vaccines9101060
immunotherapy options for cancer treatment. The most recent cancer vaccine options are mRNA
vaccines, with a focus on their usage as modern treatments. Advantages of mRNA cancer vaccines
Academic Editor: Fabio Grizzi include their rapid production and low manufacturing costs. mRNA-based vaccines are also able
to induce both humoral and cellular immune responses. In addition to the many advantages of
Received: 10 August 2021 mRNA vaccines for the treatment of cancer, their use is associated with a number of challenges.
Accepted: 17 September 2021 For this reason, before mRNA vaccines can be used for the treatment of cancer, comprehensive
Published: 23 September 2021 information about them is required and a large number of trials need to be conducted. Here, we
reviewed the general features of mRNA vaccines, including their basis, stabilization, and delivery
Publisher’s Note: MDPI stays neutral methods. We also covered clinical trials involving the use of mRNA vaccines in melanoma cancer and
with regard to jurisdictional claims in
the challenges involved with this type of treatment. This review also emphasized the combination of
published maps and institutional affil-
treatment with mRNA vaccines with the use of immune-checkpoint blockers to enhance cell-mediated
iations.
immunity.

Keywords: melanoma cancer; mRNA vaccine; therapeutic; delivery systems; immune checkpoint

Copyright: © 2021 by the authors.

Licensee MDPI, Basel, Switzerland.
This article is an open access article
1. Introduction
distributed under the terms and
conditions of the Creative Commons
Melanoma is a malignant tumor that originates from melanocytes. It is one of the most
Attribution (CC BY) license (https:// aggressive forms of skin cancer and has a low survival rate. In the United States, in 2021,
creativecommons.org/licenses/by/ the estimated number of new cases has already reached 106,000, with the number of deaths
4.0/). being about 7000, which are constantly growing [1]. Although advances in melanoma

Vaccines 2021, 9, 1060. https://doi.org/10.3390/vaccines9101060 https://www.mdpi.com/journal/vaccines

Vaccines 2021, 9, 1060 2 of 33

diagnosis have improved the chance of early detection, the prognosis for patients with
advanced or metastatic melanoma remains poor. One of the most threatening properties
of malignant melanoma is its ability to metastasize rapidly. To predict the likelihood of
metastasis, the following factors are assessed: the depth of invasion of the primary tumor,
the presence of an ulcer, micrometastases in regional lymph nodes, and the number of
mitoses in thin tumors [2]. The stage of malignancy in melanoma patients determines the
type of treatment required [3]. Nowadays, there are several therapies available, including
chemotherapy, radiation therapy, immunotherapy, and surgery. Of these, immunotherapy
is the most modern method and is continuing to evolve [4]. Ipilimumab (anti-CTLA-4
monoclonal antibody), nivolumab, and pembrolizumab (anti-PD-1 monoclonal antibodies)
were the first immunotherapies approved by the US Food and Drug Administration
(FDA) for the treatment of metastatic cutaneous melanomas [5,6]. These immunotherapies
represent checkpoint inhibitors to enhance the immune system. Combination therapy with
ipilimumab and nivolumab was also approved in 2015 for the treatment of stage III and
IV melanoma patients [7]. Immunotherapy is considered a standard treatment option for
advanced melanoma.
Conventional treatments such as chemotherapy do not specifically target tumor cells,
so they also damage the normal dividing cells. Additionally, the use of suboptimal doses of
cancer chemotherapeutic agents to reduce side effects can lead to treatment failure. It has
been found that oncological diseases are capable of progressing only when the adequate
functioning of the immune system is disrupted, as this ensures the control over oncogenic
viruses and abnormal cells. Accordingly, active studies on cancer immunotherapies that
suppress tumor development continue. Immunotherapies can be differentiated in terms of
their activity and specificity. As an active therapy type, cytokines or synthetic molecules
are usually used. To activate a specific immune response, vaccines based on tumor or viral
antigens are used. The main type of passive, nonspecific therapy is adoptive cell therapy, in
which effector cells are activated outside the body and then injected back into the patient.
With the help of tumor-specific monoclonal antibodies, a passive-specific immune response
can be achieved [4,8,9].
Unlike vaccines against infectious diseases, cancer vaccines are focused on the treat-
ment of the disease rather than on its prevention. The aim of therapeutic cancer vaccines
is to induce specific stimulation of the patient’s immune system using tumor antigens to
ultimately trigger an antitumor response, leading to tumor removal. There are several types
of cancer vaccines: (1) whole-cell vaccines, which are subdivided into autologous, modified
and unmodified, and allogeneic; (2) vaccines based on heat shock proteins, gangliosides, or
peptides; (3) vaccines based on dendritic cells; (4) vaccines based on recombinant viruses;
and (5) vaccines based on DNA or mRNA [10]. A potential target for cancer vaccines is
somatic point mutations in tumors that trigger or control the neoplastic process. Melanoma,
thus, represents an excellent target for cancer vaccine treatment since it is a malignant
tumor with one of the highest mutation prevalence, namely a high tumor mutation burden
(TMB) [11]. Such a feature of melanoma makes it highly immunogenic, providing a lot
of antigens to choose from for vaccine formulation. Moreover, tumors with high TMB
are characterized by tumor microenvironment highly infiltrated by lymphocytes. Such
tumors, including melanoma, in particular, have the highest response rates to checkpoint
inhibitors [12,13].
The most recently developed cancer vaccine options are mRNA vaccines. Initially, for
cancer immunotherapy, mRNA was used only as a template encoding tumor-associated
antigens, but due to its versatility and design variability, the therapeutic potential of
mRNA is now considered limitless. The simplicity of mRNA vaccines greatly reduces
complications generally associated with the production of biological vaccines, such as the
handling of infectious agents and genetic variability or environmental risks. mRNA-based
vaccines can be produced easily and rapidly. A major reason for the use of mRNA vaccines
is their superior safety profile compared with those of pDNA and viral vectors. mRNA
represents the minimal genetic vector and contains only the elements directly required
Vaccines 2021, 9, 1060 3 of 33

for the expression of the encoded protein [14]. mRNA can now be used for the following
purposes: (1) to deliver tumor-specific monoclonal antibodies, monoclonal antibodies
that block immune checkpoints, and their fragments to create bispecific antibodies or
chimeric antigen receptors; (2) to deliver toxic proteins that induce the death of cancer cells;
(3) to modulate tumor-associated dendritic cells; (4) to modulate the suppressive tumor
microenvironment; (5) to modulate the cytokines in the tumor microenvironment; (6) and
to generate cancer T cells. These mRNA applications were reviewed in detail by Hoecke
et al. [15].

2. Melanoma Antigens
As melanoma cells progress, they express intracellular or cell-surface molecules, which
can be both tumor-specific and ectopic normal proteins. An ideal immunotherapy target
would be an antigen that is immunogenic and specific to cancer cells. The identification
and characterization of such antigens can contribute to gaining a better understanding of
tumor progression and differentiation, and they could also be regarded as targets for im-
munotherapies. Different melanoma-associated antigens are characterized by their unique
compositions, cellular locations, and the stages at which they begin to be expressed [16].
Depending on their characteristics, the following melanoma antigens can be distinguished:
antigens associated with melanocyte differentiation, oncofetal or cancer-germline antigens,
and cell membrane proteins [17].
Melanocyte differentiation antigens include tyrosinase, tyrosinase-related proteins
(TRP-1 and TRP-2), melanocyte antigen (MELAN-A/MART-1), glycoprotein 75 (gp75), and
glycoprotein 100 (gp100) [16,18,19]. Melanocyte differentiation antigens are not mutant;
however, they are only expressed in melanoma cells and melanocytes at different stages
of differentiation [20]. These antigens are localized in compartments where melanin is
synthesized, that is, in the corresponding organelles and melanosomes, and they act as
highly specific markers of melanocyte differentiation [21,22]. Thanks to tumor-infiltrating
lymphocytes, which can detect antigens associated with melanocyte differentiation, it has
become possible to use such antigens as targets for immunotherapy [23].
Cancer-germline antigens include more than 80 proteins, which are combined into
families. These are the melanoma-associated antigen family (MAGE-family), the BM anti-
gen family (BAGE-family), the G-antigen family (GAGE-family), the synovial sarcoma
family of the X chromosome breakpoint (SSX-family), and NY-ESO-1, which has no ho-
mologs. Normally, cancer-germline antigens are expressed in the placenta, trophoblasts,
medullary epithelial cells of the thymus, and germ cells of the testes at various stages of
spermatogenesis [24,25]. The expression of some types of antigens has been observed in
somatic tissues, mainly at the developmental stage [24,25]. The expression of this group of
antigens is generally limited to germ cells, so their appearance in adults can be associated
with the occurrence of various types of cancer, including melanoma.
Membrane-associated proteins are considered targets for immunotherapies, since their
expression is increased in melanoma cancer cells [26]. At this stage, the most commonly
studied melanoma receptors include integrins, melanoma chondroitin sulfate proteoglycan
(MCSP), immunoglobulin superfamily molecules, melanotransferrin (MTf), and S100. In a
review by Pitcovski et al., different types of membrane-associated antigens are considered
in more detail [17].
Melanoma-associated antigens are considered targets for CAR therapy. For example,
CAR-lymphocytes that recognize NY-ESO have been associated with a strong immune
response [27]. CAR-lymphocytes, which recognize melanoma antigens such as MART-
1 and gp100, have been shown to cause an autoimmune reaction that affects healthy
melanocytes of the skin, eyes, and other tissues, resulting in side effects in the form of
vitiligo and the impairment of vision and hearing [28]. Peptide vaccines against melanoma
are most often based on gp100, MAGE, Melan A (MART-1), and NY-ESO. For example,
after the introduction of a vaccine based on gp100 HLA-A2, positive patients with stage I–c
Vaccines 2021, 9, 1060 4 of 33

melanoma displayed a specific cytotoxic immune response involving the formation of a

pool of memory T cells [29].
In addition to the tumor-associated antigens (TAAs) mentioned above, tumor-specific
antigens (TSAs) are also considered an important target for immunotherapies. Tumor-
specific antigens, known as neoantigens, are new proteins caused by mutations that occur in
tumor DNA. In fact, these mutational antigens are specifically expressed in tumor cells and
are not present in normal cells. Neoepitopes can be divided into two classes: neoantigens
that are only found in a particular type of cancer are called shared, and those that are
specific to a patient are called personalized [30]. BRAF and NRAS mutations, for example,
are shared neoepitopes that are observed in approximately 50% and 15–25% of melanomas,
respectively [31,32]. These characteristics suggest that neoantigen cancer vaccines could
be a promising prospect for cancer immunotherapy, as studies show that these vaccines
may elicit stronger immune responses than TAA-loaded DC vaccines [33]. For vaccination
involving neoantigens, the whole genome is sequenced after tumor biopsy and compared
with healthy tissues to identify existing mutations. Each mutation is then evaluated using
bioinformatics algorithms to determine its affinity to bind to the MHC molecule and elicit
an immune response [34,35].
The analysis of whole-genome sequences from major melanoma subtypes, including
cutaneous, acral (hands and feet), and mucosal subtypes, has demonstrated a different
genomic landscape. A lower TMB has been observed for acral and mucosal melanomas than
for cutaneous melanoma. The heavily mutated landscape of cutaneous melanoma, which
results from coding and non-coding mutations, is attributed to its underlying mechanism of
ultraviolet radiation-induced DNA damage. In contrast, mucosal and acral melanomas are
dominated by structural variants that are not attributed to ultraviolet radiation. Therefore,
these subtypes differ in terms of their pathogenesis and therapeutic targets (detailed by
Hayward, N.K. et al.) [36].
The combination of melanoma antigens can be used for diagnosis and determination
of the disease stage and prognosis, which is certainly important for determining treatment
tactics and developing a timely response to pathology. Knowledge about the composition,
cellular location, and stage at which antigens begin to be expressed may assist with the
development of melanoma immunotherapies based on antigen-specific immunization.
At the same time, it is necessary to take into account the limitations associated with the
characteristic properties of each antigen group. One of the progressive directions in the
melanoma immunotherapies is the development of personalized vaccines.

3. mRNA Vaccines: General Features

3.1. The Basis of mRNA Vaccines
Currently, mRNA-based vaccines are attracting more and more interest in the scientific
and medical communities. Against the background of the global COVID-19 pandemic,
the issue of developing vaccines for disease prevention has become the focus. One of
the most progressive and effective options is the development of mRNA-based vaccines.
One of the advantages of mRNA-based vaccines is their ability to induce both humoral
and cellular immunity, in particular, through the induction of the CD8+ T cell response,
which is of great importance in the fight against tumors [37]. At the same time, mRNA
vaccines, unlike DNA vaccines, do not have serious side effects such as integration into the
patient’s genome. Such integration side effects might include gene disruption, insertional
mutagenesis, cell death, and even tumorogenesis [38]. In addition, mRNA functions in
the cytoplasm and does not penetrate into the nucleus of the target cell that facilitates
delivery. Finally, a significant advantage of mRNA vaccines is their rapid and inexpensive
production, allowing high yields of the desired product to be produced under in vitro
conditions.
RNA vaccines can be divided into two types: (1) vaccines based on conventional
non-replicating mRNA that only encode the antigen of interest, and (2) vaccines based on
self-amplifying mRNA (saRNA), which is produced from single-stranded RNA viruses
x FOR PEER REVIEW 5 of 33

amplifying mRNA (saRNA), which is produced from single-stranded RNA viruses and
Vaccines 2021, 9, 1060 5 of 33
encodes the viral replication apparatus (Figure 1A). The mechanisms of action of saRNA
vaccines have been discussed in detail in previous reviews [39]. Conventional non-repli-
and encodes
cating mRNA consists the structural
of five viral replication apparatus
elements: (Figure
(1) cap 1A). The mechanisms
structures; of action of
(2) a 5′-untranslated
region (5′-UTR);saRNA
(3) anvaccines have been discussed in detail in previous reviews [39]. Conventional
open reading frame (ORF) encoding antigens of interest; (4) a 3′-
non-replicating mRNA consists of five structural elements: (1) cap structures; (2) a 50 -
UTR; and (5) untranslated
an adenineregion repeating nucleotide
(50 -UTR); (3) an opensequence that(ORF)
reading frame forms a polyadenine
encoding antigens of
(poly(A)) tail. Unlike
interest;saRNA, 0 ordinary
(4) a 3 -UTR; and (5) mRNA is small
an adenine due nucleotide
repeating to its simpler structure
sequence anda
that forms
the presence of only one ORF.
polyadenine (poly(A)) tail. Unlike saRNA, ordinary mRNA is small due to its simpler
structure and the presence of only one ORF.

Figure 1. Non-replicating and self-amplifying mRNA (A) and administration routes of mRNA vaccines (B).
Figure 1. Non-replicating and self-amplifying mRNA (A) and administration routes of mRNA vac-
cines (B). saRNA encodes not only an antigen-encoding gene, but also a gene responsible for
viral RNA replication [40]. saRNA, due to amplification of the RNA template in the target
cells, expresses
saRNA encodes not only high
anlevels of the gene of interest.
antigen-encoding gene,Depending
but also aongene
the preparation
responsible method,
for
saRNA can be divided into (1) plasmid-based DNA saRNA, (2) virus-like particle delivery
viral RNA replication
saRNA,[40]. saRNA,
and (3) in vitrodue to amplification
transcribed saRNA [41].ofBased the RNA template
on these in the
three types of target
saRNA,
cells, expresses Beissert
high levels of the gene
et al. developed of interest. Depending
trans-amplifying RNA (taRNA)on to the preparation
activate the immunemethod,
response,
saRNA can be divided
a processinto
that (1) plasmid-based
is safe, processable, and DNAeasysaRNA,
to optimize (2)[42].
virus-like particle delivery
mRNA vaccines are associated
saRNA, and (3) in vitro transcribed saRNA [41]. Based on these with a set of problems thatthree
need totypes
be considered.
of saRNA, First,
these vaccines are highly sensitive to nuclease degradation and immunogenicity. Over the
Beissert et al. developed trans-amplifying RNA (taRNA) to activate the immune response,
past decade, a wide variety of options have been explored to overcome these obstacles
a process that is(Figure
safe, processable,
2). The aim was andto easy
obtaintoanoptimize [42]. quiet mRNA product, since the
immunologically
mRNA vaccines
decisiveare associated
factor withthis
associated with a set of problems
drawback is the rapidthat need toofbe
recognition mRNAconsidered.
molecules
by innate immunity sensors. The uridine-rich sequence
First, these vaccines are highly sensitive to nuclease degradation and immunogenicity. is believed to be a key factor in
the activation of Toll-like receptors [43]. Nelson et al. considered and demonstrated a
Over the past decade, a wide variety of options have been explored to overcome these
variant containing N1-methyl-pseudouridine (1mΨ)-modified mRNA with the removal
obstacles (Figure 2). The aim was
of double-stranded RNA to(dsRNA)
obtain an immunologically
impurities quietTheir
[44] (Figure 2A,B). mRNAstudy product,
confirmed
since the decisivethat replacing uridine with 1mΨ in mRNA changes its interaction with patternof
factor associated with this drawback is the rapid recognition mRNA
recognition
molecules by innate immunity
receptors sensors.the
(PRR), suppresses The uridine-rich
stimulation of innatesequence
immunity, is and
believed
increasestothe
bestability
a key
of the molecule. In addition, the combination of this technique
factor in the activation of Toll-like receptors [43]. Nelson et al. considered and demon- with purification of the
product to reduce dsRNA impurities, which are formed during transcription and also affect
strated a variantinnate
containing N1-methyl-pseudouridine (1mΨ)-modified mRNA with the
immune activity, contributes to the sequence of interest having the most favorable
removal of double-stranded RNAThis
level of expression. (dsRNA)
is due toimpurities [44]
the fact that (Figure 2A,B).
mammalian Their
Toll-like study
receptor con-
3 (TLR3)
firmed that replacing uridine with 1mΨ in mRNA changes its interaction with pattern
recognition receptors (PRR), suppresses the stimulation of innate immunity, and increases
the stability of the molecule. In addition, the combination of this technique with purifica-
tion of the product to reduce dsRNA impurities, which are formed during transcription
and also affect innate immune activity, contributes to the sequence of interest having the
 5-methylcytidine (m5C); the replacement of uridine with 5-methyluridine (m5U), 2-thiur-
idine (s2U), 5-methoxyuridine (5moU), or pseudouridine (ψ); and the replacement of
adenosine with N1-methyladenosine (m1A) or N6-methyladenosine (m6A) have been in-
vestigated [46]. Another way to avoid an innate immune response and to ensure sufficient
Vaccines 2021, 9, 1060 6 of 33
expression of the required protein might be optimization of the mRNA sequence in com-
bination with purification from dsRNA, as was demonstrated by Thess et al. [47]. In their
recognizes
study, for each amino dsRNA
acid and induces in
contained activation of NF-κB
the protein [45]. Accordingly,
of interest, only thetherichest
purification
guanine
of the product from dsRNA contributes to a decrease in immunogenicity. Other types of
and cytosine codons were used. Although the selection of GC-rich mRNA is described as
chemical sequence modifications, such as the replacement of cytidine with 5-methylcytidine
a method to(m5C);
improve mRNA half-life,
the replacement it is with
of uridine controversial. Indeed,
5-methyluridine AU-rich
(m5U), elements
2-thiuridine (s2U), located
5-
in the 3′-UTR can act to destabilize
methoxyuridine mRNA [48–50].
(5moU), or pseudouridine It has
(ψ); and been notedofthat
the replacement mRNAs
adenosine withthat
N1-methyladenosine
harbor coding region instability (m1A) or N6-methyladenosine
elements happen to be(m6A) GC-poorhave (i.e.,
been investigated
factor VIII,[46].IL2, c-
Another way to avoid an innate immune response and to ensure sufficient expression
myc, c-fos, HPV, HIV-1 mRNAs). However, it is not known whether a general correlation
of the required protein might be optimization of the mRNA sequence in combination
exists between
with cellular mRNA
purification lifetimeasand
from dsRNA, wasGC content. Moreover,
demonstrated it has
by Thess et al. [47].been shown
In their study,that
GC contentfor oneach
mRNA aminodoes
acidnot significantly
contained affectofthe
in the protein cellular
interest, onlymRNA lifetime,
the richest guanine while
and the
effect of increased expression
cytosine codons level Although
were used. of GC-rich the genes
selectionwas due to mRNA
of GC-rich their several-fold
is described as higher
a
method
transcription efficacyto improve
[51]. mRNA half-life, it is controversial. Indeed, AU-rich elements located
in the 30 -UTR can act to destabilize mRNA [48–50]. It has been noted that mRNAs that
Additional methods for stabilizing the mRNA molecule include the use of synthetic
harbor coding region instability elements happen to be GC-poor (i.e., factor VIII, IL2,
analogs of the cap
c-myc, andHPV,
c-fos, capHIV-1
enzymes,
mRNAs). regulatory
However, itelements
is not known in the 5′-UTR
whether andcorrelation
a general 3′-UTR, and
the addition of poly(A) tails that screen mRNA. These methods significantly increase
exists between cellular mRNA lifetime and GC content. Moreover, it has been shown thatpro-
GC content
tein translation on mRNA
[52–55]. does not
Strategies forsignificantly
improvingaffect the the cellular mRNA
translation lifetime,
efficiency arewhile the
discussed
effect of increased expression level of GC-rich genes was due to their several-fold higher
in detail in a review by Miao et al. [56].
transcription efficacy [51].

Figure 2. Methods
Figureof mRNA nuclease
2. Methods of degradation reduction.
mRNA nuclease The chemicalreduction.
degradation modification of uridine
The to pseudouridine
chemical modification (A),
of uridine
purification of the product from double-stranded RNA using high-performance liquid chromatography (HPLC) (B), and
to pseudouridine (A), purification of the product from double-stranded RNA using high-perfor-
choice of codons containing more G:C pairs (C) are performed to reduce the sensitivity of mRNA to digestion by nucleases.
mance liquid chromatography (HPLC) (B), and choice of codons containing more G:C pairs (C) are
performed to reduce the sensitivity
Additional methods of
formRNA to digestion
stabilizing the mRNA bymolecule
nucleases.
include the use of synthetic
analogs of the cap and cap enzymes, regulatory elements in the 50 -UTR and 30 -UTR, and the
Anotheraddition of poly(A)
problem tails that
associated screen
with mRNA. These vaccines
mRNA-based methods significantly increase
is the delivery protein
of molecules
into the cytoplasm. mRNA is a large, hydrophilic, negatively charged molecule thatincan-
translation [52–55]. Strategies for improving the translation efficiency are discussed
detail in a review by Miao et al. [56].
not freely enterAnother
target cells through the lipid bilayer membrane. In recent years, methods
problem associated with mRNA-based vaccines is the delivery of molecules
of delivering
intomRNA to target
the cytoplasm. mRNAcellsis have
a large,been actively
hydrophilic, studiedcharged
negatively [57] (Figure
molecule 3).that
However,
cannot it
is known that,
freelyunder certain
enter target conditions,
cells through the naked mRNA
lipid bilayer can enter
membrane. cells years,
In recent and induce
methodsan of im-
mune response against the encoded antigen.
 Vaccines 2021, 9, 1060 7 of 33

x FOR PEER REVIEW 7 of it33

delivering mRNA to target cells have been actively studied [57] (Figure 3). However, is
known that, under certain conditions, naked mRNA can enter cells and induce an immune
response against the encoded antigen.

Figure 3. Various carriers for mRNA vaccine delivery.

Figure 3. Various carriers for mRNA vaccine delivery.
The choice of mRNA-based vaccine administration method plays an equally important
role in determining the quality and intensity of the immune response. Intradermal, subcu-
The choicetaneous,
of mRNA-based vaccine administration method plays an equally im-
and intramuscular drug administration methods are usually used to inoculate
portant role in cancer
determining the quality
mRNA vaccines (Figureand
1B).intensity
In a study ofby the
Pardiimmune response.
et al., mRNA-lipid Intrader-
nanoparticles
mal, subcutaneous, and intramuscular
(mRNA-LNP) drugthe
were used to assess administration methods
effects of different are usually used
vaccine administration routes to
on
antigen expression [58]. With intramuscular and subcutaneous
inoculate cancer mRNA vaccines (Figure 1B). In a study by Pardi et al., mRNA-lipid na- injections, expression of the
mRNA-LNP protein was higher than with intradermal injection. Intranodal immunization
noparticles (mRNA-LNP) were used to assess the effects of different vaccine administra-
under ultrasound control is also an attractive option due to the high concentration of
tion routes on antigen
dendriticexpression [58]. With
cells in the draining lymph intramuscular and subcutaneous
nodes [59]. Additionally, intratumoral injections,
delivery is
expression of the mRNA-LNP
a potential method, protein
since it was higher
provokes thanresponse
a quick with intradermal
by local immune injection.
cells. InIntran-
a study
by Jeught
odal immunization underet al., intratumoral
ultrasound delivery
control of fusion
is also mRNA wasoption
an attractive shown due
to have
to significant
the high
therapeutic potential, which was enhanced by immune checkpoint
concentration of dendritic cells in the draining lymph nodes [59]. Additionally, intra- inhibitors [60].

tumoral delivery3.2.isVaccine
a potential method,
Optimization since mRNA
by Improving it provokes a quick
Translation response by local im-
and Stability
mune cells. In a study by Jeught
Because mRNA et al., intratumoral
is sensitive delivery
and vulnerable of fusionenzymes,
to environmental mRNAits was shown
stabilization
to have significant
shouldtherapeutic
guarantee potential, which was
protein expression. enhanced
Making various by immune checkpoint
modifications in-
to the structural
hibitors [60]. elements of in vitro transcribed (IVT) mRNA significantly increases the duration, and
hence amount, of encoded protein production. Modifiable elements include the 5 cap, 0

50 - and 30 -UTRs, the coding region, and the poly(A) tail [61]. During the transcription
3.2. Vaccine Optimization by Improving mRNA
process, a 7-methylguanosine (m7G)Translation
cap is linkedand Stability
to mRNA by a 50 -50 -triphosphate (ppp)
bridge is
Because mRNA (m7GpppNpNp
sensitive and . . .vulnerable
), which is attached to eukaryotic translation
to environmental enzymes, initiation factor
its stabiliza-
4E (EIF4E) in the early stages of translation [62]. Therefore,
tion should guarantee protein expression. Making various modifications to the structural the mRNA capping process
is vital for the stability and maturity of mRNA. The use of recombinant vaccinia virus
elements of in capping
vitro transcribed (IVT) mRNA significantly increases the duration, and
enzymes and synthetic cap analogs are two important approaches that can be used
hence amount, of encoded
to create protein
different production.
versions of the 50 capModifiable
for use in IVTelements include
mRNA [53,63]. Duringthe this
5′ cap, 5′-
process,
and 3′-UTRs, thethecoding
cap may region,
also beand the poly(A)
inserted tail direction,
in the reverse [61]. During the that
an issue transcription
can be overcome process,
with
the use of anti-reverse cap analogs to increase translation
a 7-methylguanosine (m7G) cap is linked to mRNA by a 5′-5′-triphosphate (ppp) bridge efficiency [54].
(m7GpppNpNp…), which is attached to eukaryotic translation initiation factor 4E (EIF4E)
in the early stages of translation [62]. Therefore, the mRNA capping process is vital for the
stability and maturity of mRNA. The use of recombinant vaccinia virus capping enzymes
and synthetic cap analogs are two important approaches that can be used to create differ-
ent versions of the 5′ cap for use in IVT mRNA [53,63]. During this process, the cap may
Vaccines 2021, 9, 1060 8 of 33

Modification of the 50 - and 30 -UTRs is another way to improve the translation and
increase the half-life of IVT mRNA. The 50 - and 30 -UTRs contain regulatory sequence ele-
ments that are located upstream and downstream of the initiation and termination codons,
respectively [64]. Studies have shown that, in orthopoxviruses, mRNA 50 -UTR is involved
in the inhibition of both decapping and 30 -50 exonucleolytic degradation activities [65]. The
30 -UTR of α-globin and β-globin mRNAs has been widely targeted in clinical trials [66].
The 30 -UTR plays a crucial role in gene expression due to containing sequences such as AU-
rich elements (AREs) and the poly(A) tail. In some therapeutic applications, limited-length
proteins have required the presence of an AU-rich area [67].
The addition of a suitable length poly(A) tail at the 30 end of mRNA also plays an
important role in its successful translation and stability. The poly(A) tail can be added to
IVT mRNA either through a template vector or by recombinant poly(A) polymerase after
the transcription process has occurred [52,61].
Coding region modification is another way to strengthen mRNA stability and trans-
lation. Codon optimization is performed to avoid the occurrence of rare codons, which
are replaced with repeatedly used synonymous codons. These codons do not alter the
amino acid sequence of the protein, as they have the same cognate and tRNA, but they
accelerate the translation process and increase the protein yield [68,69]. However, some
proteins require slower translation rates for reasons such as ensuring correct folding, as is
the case when rare codons are present [70]. Usually, to evaluate codon optimization, the
protein resulting from DNA plasmids that carries the wild-type sequence is compared with
the protein with the modified and optimized sequence [71]. Codon-optimization of IVT
mRNA has been successfully conducted in studies focused on producing vaccines for viral
and non-viral infections [72,73].

3.3. Various Carriers for mRNA Vaccine Delivery

Another challenge for mRNA vaccines is their passage through the membrane and
delivery to the cells, because mRNA is an unstable, large, negatively charged molecule that
has difficulty crossing the membrane structure. Because mRNA cannot pass through the
membrane by passive diffusion, RNA-based drugs are usually taken up by endocytosis.
Although naked mRNA vaccines can be injected directly into cells, such as dendritic cells,
delivery carriers are required for significant rates of expression and inhibition to occur [74].
Therefore, various strategies have been developed to introduce mRNA vaccines to cells,
including viral-vector-based delivery, lipid-based delivery, polymer-based delivery, hybrid-
carrier-based delivery, and peptide-based delivery (Figure 3). These delivery carriers
prevent the degradation of mRNA by RNase enzymes and facilitate its entry into cells and
subsequent escape from the endosome, allowing it to reach lymphoid organs and cellular
targets to eventually produce the desired antigen and immune response [75].

3.3.1. Naked mRNA Vaccines

Unlike carrier-based mRNA vaccines, naked mRNA delivery is usually realized by
direct injection of the mRNA solution. naked mRNA is commonly dissolved in Ringer’s
solution or lactated Ringer’s solution for vaccination [76,77]. Ca2+ is used as a component of
these two solutions, since it is known to improve the uptake of mRNA in an ion-dependent
manner [78]. naked mRNA dissolved in Ringer’s solution has already been tested in
anticancer clinical trials. For instance, in a study by Sahin et al., naked mRNA was diluted
in Ringer’s solution at a concentration of 1.0 mg mL−1 and injected into separate inguinal
lymph nodes in thirteen melanoma patients [79]. Patients showed good tolerance, and
promising results were achieved in this clinical trial.
The cellular uptake mechanism of naked mRNA remains elusive. It is known that
naked mRNA molecules cannot penetrate the cell membrane freely. Several scientists
hypothesized that the uptake of naked mRNA mainly occurs due to a variety of DC-
mediated endocytic pathways [74,80–82]. This process ensures the translation of antigen-
encoding mRNA and promotes the activation of DC and T cells, leading to the formation
Vaccines 2021, 9, 1060 9 of 33

of antitumor adaptive immunity. However, to determine the mechanism underlying this

process, further investigation is required.
Unlike DNA vaccines, naked mRNA shows superiority in terms of its low level of
toxicity due to the bacteria-free manufacturing procedures used for its production [83]. Fur-
thermore, patients who receive naked mRNA injections are free from the threat of genome
integration, which occurs in DNA vaccination. Naked mRNA also has the advantage of
quicker protein translation than DNA molecules. Instead of complex antigen generation,
beginning from DNA transcription to mRNA, followed by translation processes, once
naked mRNA molecules reach the cytosol, ribosomes combine with mRNA and launch the
translation procedure promptly, resulting in a rapid immune response following naked
mRNA administration. Thus, mRNA vaccines usually achieve the targeted immunity of
hosts more quickly than DNA vaccination methods. However, the limitations of naked
mRNA molecules as vaccines are obvious. The instability of mRNA molecules frequently
leads to their degradation by host RNases. Moreover, their low translation efficiency
reduces their ability to generate sufficient antigens to trigger an immune response when
used as vaccines. Further, unwanted innate immunity against naked mRNA may be stimu-
lated. To overcome these disadvantages, several mRNA structure modifications have been
applied, for example, sequence optimization, modification of nucleosides, and purification
of mRNA, as mentioned above. Furthermore, following alteration of the administration
pathway to avoid having RNase in the bloodstream, for example, by using intranodal,
intradermal, and intramuscular administration methods, naked mRNA vaccines have
still shown promising results. Notably, Sahin and colleagues administered naked, un-
modified mRNA encoding neoantigens in an intranodal manner, and this was shown to
bolster robust specific T cell immune responses in melanoma patients, demonstrating that
the challenges associated with the use of naked mRNA vaccines in clinical practice are
surmountable [79].

3.3.2. Viral Vectors

Delivery systems can be based on viral or non-viral vectors. In vaccines based on viral
vectors, modified viruses are used to deliver the genetic code of the antigen to the target
cells. These vaccines work similarly to natural infections caused by viruses, which, after
infecting cells, produce large concentrations of antigens that eventually trigger an immune
response [84]. Due to the high transduction efficiency of adenoviruses, there is great interest
in engineering them as vectors for carrying genes/mRNA, although this would depend on
the presence of specific receptors for internalization [85]. The genes coding for these viruses
are partially or completely replaced by the desired antigen genes, and these modified
viruses act as vectors for the delivery of these nucleic acid cargoes. It is noteworthy
that positive-strand RNA viruses, such as alphaviruses [86], picornaviruses [87], and
flaviviruses [88] have been used to deliver mRNA. The replicative features of positive-
strand RNA viruses cause these saRNA vector systems to act as adjuvants, leading to high
and transient expression of exogenous proteins [14]. However, the use of viral vectors poses
a number of challenges, including genome integration, toxicity, and immunogenicity [89].
For this reason, the use of non-viral vectors based on polymers, lipids, etc. as mRNA
delivery systems has advantages [90].

3.3.3. Lipid-Based Carriers

Different synthetic and natural lipids can be used to deliver nucleic acids [91]. The
lipids used for the delivery of mRNA vaccines are in the forms of liposomes or lipid
nanoparticles (LNPs). Liposomes have long been used as carriers for drugs due to their
desirable properties, such as their easy preparation and low toxicity [92]. A variety of
liposomes have been designed and studied for the delivery of mRNA vaccines and have
shown promise for use in the treatment of diseases such as cancer [93]. The term lipoplexe
refers to a nucleic acid and liposome complex. During a self-assembly process, cationic
liposomes form complexes with RNA through electrostatic interactions, leading to the for-
Vaccines 2021, 9, 1060 10 of 33

mation of lipoplexes [91]. 1,2-di-O-octadecenyl-3-trimethylammonium-propane (DOTMA)

and 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) are examples of cationic lipids
that are used to deliver mRNA vaccines [93]. In addition, neutral lipids are used in cationic
liposomes to achieve high transfection and low toxicity [94]. Therefore, the activity and
functional properties of lipoplexes are related to various factors, including the overall
particle charge, lipid content, and the cationic lipid to mRNA ratio [95]. The results of
previous studies show that lipoplexes encoding viral antigens or neoantigens elicit memory
T cell responses and prevent tumor progression. The two challenges for cationic lipids are
their rapid clearance and high toxicity. These disadvantages can be overcome by replacing
them with ionizable lipids [96]. At acidic and physiological pHs, ionizable lipids have
positive and neutral charges, respectively. This can help them to maintain their transfec-
tion efficiency and reduce their toxicity, but these lipids are not widely used in lipoplex
formulations and should be used in LNPs [97].
LNPs are one the most broadly used tools for in vivo mRNA delivery [98]. They usu-
ally consist of four components: an ionizable or cationic lipid, cholesterol, phospholipids,
and lipid-linked polyethylene glycol (PEG). As previously mentioned, ionizable lipids
are positively charged under acidic conditions, which leads to the encapsulation of RNA,
and at physiological pH, they have a neutral or partial cationic charge. This property
is essential, as it allows mRNA to escape from the endosome and be released into the
cytoplasm [99]. Cholesterol, as a stabilizing agent, and phospholipid, as a supporting agent
for the formation of the lipid bilayer structure, are components of LNPs. Polyethylene
glycol increases the LNP circulation time because it prevents the binding of mRNA to
proteins in plasma [100]. LNPs have been used to deliver mRNA vaccines that act against
viral infections, such as the Zika virus [101], and for cancer immunotherapies, such as that
used to treat B16F10 melanoma [102]. The use of LNPs to deliver mRNA encoding antigens
that act against B16F10 melanoma tumors has been associated with a reduction in tumor
size and an increase in survival following the induced immune response.

3.3.4. Polymer-Based Carriers

Polymer-based carriers have been used less frequently in clinical trials than lipids;
however, they have significant potential for use in nucleic acid delivery. Polymers include
cationic and anionic structures; however, the use of cationic polymers as nucleic acid
carriers is more applicable. Cationic polymers form complexes with anionic mRNA via
electrostatic interactions [57]. Polyplex nanoparticles and micelleplex nanoparticles result
from such interactions and have differences from and similarities with each other [103].
Polyethylenimine (PEI) is the most widely used cationic polymer and the most commonly
used transfection agent for nucleic acids [104,105]. Polyethylenimine has a branched struc-
ture with a high cationic charge density [104]. The presence of several amine groups in the
polyethylenimine backbone as well as other cationic polymers means that it can bind easily
to nucleic acid phosphate groups and form Polyplex nanoparticles, which increases the
transfection efficiency and mRNA protection [106]. Cationic polymers establish electrostatic
interactions with the negatively charged endosome membrane through these amine groups
which contribute to endosomal escape and mRNA release into the cytosol. In addition, the
buffering capacity of amine groups on cationic polymers causes a proton-sponge effect that
finally induces osmotic swelling and destruction of the endosome membrane [104,107].
In a study of mRNA vaccines, a polyethylenimine-polyplex nanoparticle contain-
ing mRNA encoding the influenza virus hemagglutinin and nucleocapsid was used. In
this study, mRNA was successfully delivered to dendritic cells, transferred to the cy-
tosol, and translated into proteins, leading to both humoral and cellular immune reac-
tions [108]. However, highly positively charged polyethylene-based formulations have
increased toxicity, as they bind to negatively charged serum proteins; therefore, other
cationic polymers have been developed, including poly(2-dimethylaminoethyl methacry-
late) (PDMAEMA) [109], polyamidoamine (PAMAM) dendrimer [110], biodegradable
poly(β-amino ester) (PBAE) [111], poly(amino-co-ester) (PACE) [112], and polysaccha-
Vaccines 2021, 9, 1060 11 of 33

ride [113] polymers. Although cationic polymers have more applications than anionic ones,
anionic polymers such as poly D,L-lactide-co-glycolide may sometimes be used. However,
cationic lipids need to be added to establish a stable complex with negatively charged
mRNA [114].
Micelleplexes are another polymer-based nucleic acid delivery system form. They
consist of a hydrophobic inner core and a hydrophilic outer shell. In fact, micelleplexes
are produced by amphiphilic copolymers consisting of one or more cationic blocks, and
due to having a positive charge, they interact with negatively charged nucleic acids and
form stable complexes. Micelleplexes have a remarkable ability to co-deliver drugs (such
as chemotherapeutic agents) and nucleic acids [115,116]. The first micelleplex-based de-
livery system for mRNA vaccines was developed using branched PEI2k and stearic acid
conjugates (PSA). These PSA/mRNA micelles can be effectively transmitted into cells and
escape endosomally. In addition, they are better at inducing dendritic cell maturation and
immune profiles than PEI/mRNA complexes, indicating that these nanomicelles have the
potential for vaccine delivery [117].
Research related to polymer-based delivery systems is in the early preclinical trial
stage. Thus far, these polymer-based carriers have been shown to offer a promising platform
for the effective delivery of mRNA.

3.3.5. Hybrid Carriers

Carrier systems for delivering mRNA vaccines may involve a combination of sev-
eral different substances. These hybrid carriers include lipopolyplexes and cationic na-
noemulsions. Lipopolyplexes consist of an inner core, a complex containing a nucleic acid
and polycationic component (cationic polymer or cationic peptide), and an outer lipid
shell [118–120]. These hybrid carriers are actually a combination of a lipoplex and poly-
plex and offer more advantages than non-hybrid systems [121]. Poly(lactide-co-glycolide)
(PLGA), polycaprolactone, and polylactic acid are examples of polymers, and DOTAP,
1,2-dilauroyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine,
lecithin, DSPE, and PEG are examples of lipids that are mostly used in the formulation of
hybrid nanoparticles [122]. One study showed that the use of histidylated lipopolyplexes
of mRNA encoding MART1 in tumor models significantly prevents the growth and pro-
gression of B16F10 melanoma tumors and induces a cellular immune response [123]. These
results indicate that the use of this type of mRNA formulation as a vaccine is more efficient
than using a lipoplex and polyplex alone.
Cationic nanoemulsions (CNE) are another form of hybrid vector with other structural
compounds in addition to lipids and polymers. The Novartis Institute developed an MF59-
based cationic nanoemulsion method to self-amplify mRNA vaccine delivery [124]. MF59
is a proprietary immunologic adjuvant based on squalene. The cationic nanoemulsion
has an oil phase consisting of squalene, DOTAP, and sorbitan trioleate. The addition of
DOTAP allows electrostatical binding to mRNA. This oil phase combines with an aqueous
phase, which consists of compounds such as Tween 80 [125]. The use of these cationic
nanoemulsions as carriers of saRNA vaccines has been associated with an increase in
immunogenicity. The advantage of cationic nanoemulsions is the use of oils and surfactants
that have already been used successfully in clinical trials and whose safety has been
proven [126].

3.3.6. Peptide-Based Carriers

Peptides can be used as non-viral mRNA delivery systems because of their relative
stability, low immunogenicity, and low toxicity [127]. Cationic peptides easily interact
with negatively charged mRNA due to their positive charge resulting from the amino
groups contained in their amino acids [128]. Protamines and cell-penetrating peptides
(CPPs) are two important cationic peptides that can be used to deliver mRNA to cells.
A desirable feature of protamine is the protection of mRNA from degradation by serum
nucleases [129]. However, due to the extremely tight connection of protamine with mRNA,
Vaccines 2021, 9, 1060 12 of 33

the use of protamine–mRNA complexes alone prevents the translation process and reduces
the effectiveness of a vaccine. This can be compensated for by using RNActive technology
in which the mRNA–protamine complex acts as a stimulant of the immune system and
does not play a role in the expression system [130,131]. In fact, with this platform, the
vaccine consists of two compounds: naked mRNA and mRNA complexed with protamine
in which naked mRNA is expressed to the desired antigen. The mRNA–protamine complex
acts as an adjuvant that induces an adaptive immune response through TLR7-mediated
signaling [130,132].
Another study used the fusion of protamines and a class of CCP for mRNA trans-
fection to encode reporter genes into human cells [133]. Cell-penetrating peptides are
small peptides, 8 to 30 amino acids in length, that have the ability to penetrate biological
membranes and transfer cargo to cellular targets [134]. These peptides are divided into
three categories: natural protein-derived peptides, chimeric peptides, and synthetic pep-
tides. Cell-penetrating peptides have been shown to be highly effective for transfection.
Some can escape from the endosome through the proton-sponge effect, which leads to
osmotic swelling and rupture of the endosome, and some, through interaction with the
membrane, can cause its destruction and pore formation [135,136]. Fusogenic lipids, such
as dioleoylphosphatidylethanolamine (DOPE), are commonly added to CPPs to enhance
endosomal escape.
Anionic peptides can also be used in delivery systems, but because the negative
peptide charge and negative mRNA charge repel each other, a cationic copolymer is needed
to encapsulate the mRNA. For example, the pHDPA copolymer is used to encapsulate
OVA-mRNA, which is then conjugated to an anionic peptide, GALA. Anionic peptides
aid in the process of cell uptake via sialic acid on dendritic cells. This vector has been
associated with an increase in transfection and the induction of an immune response [137].

3.3.7. Dendritic-Cell-Based mRNA Vaccines

As the most potent antigen-presenting cells in the immune system, dendritic cells
(DCs) can internalize, process, and present antigens to CD8+ or CD4+ T cells on major
histocompatibility complexes (MHCs), including MHC class I and MHC class II. Due to
the prominent biological features of DCs that are associated with initiation of the adaptive
immune response, DCs are no doubt an ideal vaccine target to prevent cancers. Moreover,
DCs secrete various kinds of cytokines and chemokines, which are indispensable for T cell
proliferation, activation, and recruitment [138,139]. Thus, numerous preclinical and clinical
trials have been initiated in previous decades to assess DC-based mRNA vaccines. There
are two approaches for the delivery of DC-based mRNA vaccines, loading DCs ex vivo
and targeting DCs in vivo, as we described previously [140]. The advantages of ex vivo
DC loading possesses are precise antigen stimulation, superior cellular condition control,
and high transfection efficiency. However, the major obstacles to the development of such
vaccination methods are the labor-intensive procedures and high costs involved. Although
in vivo targeting methods are superior in terms of rapid manufacture and low costs, specific
and efficient cell-type delivery is still hard to reach. These two delivery approaches have
been investigated, and some promising results have been achieved recently.
Myeloid DCs and plasmacytoid DCs represent the two major types of DCs found in
the peripheral blood and the most potent antigen-presenting cells in the immune system.
Nevertheless, these two kinds of DC constitute only 0.1% of peripheral blood mononuclear
cells (PBMCs), and it is difficult and expensive to obtain a sufficient concentration of DCs
for use in vaccination against cancers. The most commonly used approach to generate
large numbers of DCs for vaccination is the establishment of monocyte-derived DCs
(MDDCs). In this process, immature DCs are differentiated from monocytes in the presence
of GM-CSF and IL-4 for five days and then stimulated to mature through the addition
of maturation stimuli [141]. Although DC can directly uptake antigen-encoding mRNA
molecules via endocytosis, various methodologies have been developed to enhance the
internalization efficiency ex vivo, including electroporation, lipofection, nucleofection, and
Vaccines 2021, 9, 1060 13 of 33

sonoporation [142–144]. Among these methods, electroporation is utilized for ex vivo

transfection the most frequently. Under a high-voltage pulse, cell membrane pores are
formed and mRNA molecules can pass through effortlessly, leading to antigen translation
in the cytoplasm [142]. In 2012, Aarntzen et al. electroporated MDDCs with mRNA
encoding gp100 and tyrosinase and administered them intranodally to forty-five stage III
and IV melanoma patients [145]. Robust tumor antigen-specific CD4+ and CD8+ T-cell
responses were evident, and clinical benefits were observed in this trial, favoring the use
of mRNA-electroporated dendritic cell vaccines against melanoma. Lipofection is also an
attractive approach for ex vivo DC mRNA vaccine generation. Markov and colleagues
reported the application of cationic liposomes for the transfection of mRNA in DCs [146].
The injection of DCs pulsed with cationic liposomes resulted in an obvious suppression
of metastasis in a model of murine B16 melanoma in vivo. However, as an adoptive
cell transfer technique, ex vivo DC-based mRNA vaccines still face challenges, as their
production is time and labor intensive.
In vivo DC-targeting mRNA vaccines are administered intranodally. Previous work
showed that intranodally delivered mRNA is almost exclusively taken up and translated by
lymph-node-resident DCs [147]. Furthermore, intranodal administration achieves the same
level of immunogenicity as other administration routes with lower vaccine doses [59,148].
A completed clinical trial has already revealed the possibility of using in vivo mRNA
delivery to DCs through intranodal injection of naked neoepitope-encoding mRNA in
advanced melanoma patients [79]. To enhance the stimulation efficiency of in vivo DC
targeting mRNA delivery, the use of TriMix, which contains three different mRNA en-
coding immune-stimulatory proteins—CD40 ligand (CD40L), CD70, and constitutively
active Toll-like receptor 4 (TLR4)—as an adjuvant combined with tumor antigen-encoding
mRNA was investigated [149]. This combination demonstrated superiority in the stim-
ulation of DCs when administered intranodally [147]. Recently, a preclinical evaluation
of mRNA trimannosylated lipopolyplexes revealed that in vivo targeting DCs can be re-
alized through intradermal administration [150]. This delivery system is equipped with
mannose-containing glycolipids, which specifically target endocytosis receptors presented
on the membranes of DCs, highlighting the in vivo targeting DC potency of this mRNA
formulation.

4. mRNA Vaccines in Combination with Checkpoint Blockade for the Treatment of

Melanoma Cancer
The expression of immune-checkpoint molecules is a mechanism that regulates the
functioning of the immune system. Such molecules include cytotoxic T-lymphocyte-
associated antigen-4 (CTLA-4) and programmed death-1 receptor (PD-1). Both immune
checkpoints are expressed on T cells. CTLA-4 interacts with CD80 and CD86 with a higher
affinity than the co-stimulant CD28, which leads to the limited activation of T lympho-
cytes [151]. PD-1 interacts with programmed death ligands (PD-L1 and PD-L2), which
are expressed by a wide range of cells (tumor cells, myeloid dendritic cells derived from
monocytes, epithelial cells, T and B lymphocytes) and sends a negative signal to T cells,
which leads to the depletion of the latter [152]. Thus, these molecules, along with Treg cells,
can suppress the anti-tumor T-cell response.
Immune checkpoint inhibitors have already been licensed as a therapeutic in the
form of monoclonal antibodies against CTLA-4, PD-1, and PD-L1. These are aimed, in
particular, at combating melanoma [153]. Initially, efforts were directed toward the study of
CTLA-4. Preclinical trials have shown that blocking CTLA-4 with monoclonal antibodies
leads to a significant delay in tumor growth in murine models of melanoma and many
other cancers [154], which is associated with increased T cell infiltration [155]. Based on
these results, many clinical trials involving the use of monoclonal antibodies for CTLA-4
inhibition have been performed. One of the first drugs approved for the treatment of
patients with metastatic melanoma was Ipilimumab, which increased median overall
survival (OS) by 10 months [156].
Vaccines 2021, 9, 1060 14 of 33

Preclinical trials of PD-1 and PD-L1 inhibitors have shown favorable therapeutic re-
sponses in mice with melanoma [155,157]. The blockage of PD-1 has been shown to enhance
CD8+ T-cell infiltration by increasing the secretion of IFN-γ-inducible chemokines [158].
This led to clinical trials, including a significant study in 2012, which demonstrated positive
results in the treatment of advanced melanoma, non-small cell lung cancer, prostate cancer,
renal cell, and colorectal cancer [159]. Currently, Pembrolizumab and Nivolumab have
been approved and are now used to treat melanoma [153]. It is important to clarify that
the functioning of the considered immune checkpoint inhibitors depends on the stage of
T-cell activation; therefore, final effects may vary. Specifically, CTLA-4 limits the early stage
of T-cell activation; therefore, the inhibition of CTLA-4 leads to an increase in the effector
T-cell pool in the lymph nodes but not in the tumor microenvironment [160]. PD-1 exerts its
influence on T cells located in the periphery; therefore, T-lymphocyte infiltration increases
at the site of the tumor if PD-1 blockers are utilized [160]. Several studies have been based
on this difference. For example, a clinical study was conducted in patients with melanoma
to compare Ipilimumab (CTLA-4 blocker) and Pembrolizumab (PD-1 blocker) [161]. The
best response was given by patients receiving Pembrolizumab. Another study demon-
strated that the use of the combination of Ipilimumab and Nivolumab to treat patients with
melanoma yields a more significant response than the use of either separately [162].
Although showing partial effectiveness, immune checkpoint inhibitors affect late-
stage antitumor T-cell response. To achieve the best effect, cancer immunotherapies might
be combined, while other effects on tumor cells should be taken into account. A cocktail of
drugs that affect different signaling pathways is more likely to have a therapeutic effect
than the use of the same drugs individually. A study by Spranger et al. showed that
the blockade of immune checkpoints cannot restore tumor-specific T-cell responses to
completely unrecognized, non-infiltrated melanomas; that is, if they are inhibited, the T-cell
response will be nonspecific [163]. The network of suppressive myeloid cells may also
represent a barrier to immunotherapy development; therefore, it is likely that the inhibition
of colony-stimulating factor 1 receptor (CSF1R) in conjunction with blockade of immune
checkpoints may demonstrate a synergistic effect [164].
An example of rational immunotherapeutics combination is the simultaneous applica-
tion of immune checkpoint inhibitors and cancer vaccines. Recently, various combinations
of mRNA vaccines with immune checkpoint inhibitors have been actively explored. The
recruitment phase of a clinical trial is underway to analyze whether the combination of
mRNA-4157 (mRNA cancer vaccine targeting twenty tumor-associated antigens (TAAs))
with Pembrolizumab will improve relapse-free survival compared with the use of Pem-
brolizumab alone in patients with complete resection of skin melanoma and a high risk of
recurrence (NCT03897881). It has been shown that a potent immune response is generated
when Ipilimumab is combined with mRNA encoding TriMix (cluster of differentiation
70, ligand of cluster of differentiation 40, and constitutively active toll-like receptor (4)
and TAAs (TriMixDC-MEL) in two phase II clinical studies in patients with stage III/IV
melanoma (NCT01676779, NCT01302496)) [165,166]. More recently, BioNTech announced
a collaboration with Regeneron to begin a phase II clinical trial in patients with anti-PD1-
resistant/recurrent inoperable stage III or IV cutaneous melanoma to monitor the effects of
treatment with mRNA (BNT111) encoding four TAAs (NY-ESO-1, MAGE-A3, tyrosinase,
and TPTE) in combination with Celiplimab (PD-1 blocker) [167]. Another clinical trial that
is currently in the recruiting stage is aiming to compare the use of BMS-986016 (mono-
clonal antibody against lymphocyte activation gene (3) alone and in combination with
Nivolumab (PD-1-blocker) (NCT01968109)). There are now several clinical trials involving
mRNA encoding a neoantigen combined with PD-1 inhibitors that are in the recruiting
stage (NCT03289962, NCT03815058, NCT03897881). Sahin et al. presented data from a
preliminary interim analysis of their study in which patients with inoperable melanoma
who were already taking were given a vaccine containing RNA-LPX encoding four TAAs
(FixVac) either alone or in combination with PD1 blockade [168]. They observed that
Vaccines 2021, 9, 1060 15 of 33

although FixVac is active as a separate agent, it also acts synergistically with anti-PD1
therapy in patients who do not respond to checkpoint inhibitor monotherapy.
Another option is the combination of checkpoint blockers with mRNA encoding
cytokines to modulate the cytokine environment in the tumor microenvironment. In situ
delivery of mRNA encoding a fusokine called Fβ2, which consists of IFN-β fused to the
ectodomain of the transforming growth factor-β II receptor, has been performed. It was
shown that such constructions can delay tumor growth, and this can be further enhanced
by blocking the interaction of PD-1 with PD-L1 [60]. A trial of a combination therapy
consisting of mRNA encoding OX40L and Durvalumab is currently in the recruitment
stage (NCT03323398). Additionally, the combination of a mixture of IL-23/IL-36γ/OX40L
triplet mRNAs with checkpoint blockade has been shown to be more effective in models
that are otherwise resistant to the systemic inhibition of immune checkpoints [169].

5. Clinical Trials of In Vitro Transcription mRNA Vaccines in Melanoma

Although in vitro transcription (IVT) mRNA antitumor vaccines represent a minority
of melanoma immunotherapeutics in clinical trials in comparison with DC-based mRNA
vaccines, the promising results and progression of IVT mRNA vaccines collected from
preclinical studies indicates their great potential for use as immunotherapies for treating
melanoma. In 1990, the first report regarding the successful expression of IVT mRNA
transfected in mouse skeletal muscle cells revealed the possibility of converting IVT mRNA
into vaccines against infectious diseases or cancers [170]. However, limited advancement
in the development of mRNA cancer vaccines has been achieved since then mainly due
to concerns about mRNA instability, insufficient translation potency in vivo, and high
innate immunogenicity. Owing to the rapid development of molecular biotechnology, the
use of IVT mRNA for cancer treatment as a vaccine has become more feasible through
modulation of the mRNA structure, purification of mRNA by novel methods, and the
formulation of mRNA within delivery vehicles [14]. As pioneers of the pharmaceutical
industry, BioNTech, Moderna, and GenenTech have all announced clinical updates on
IVT mRNA vaccines against melanoma, as summarized in Table 1. Their representative
mRNA-based immunotherapies have been broadly evaluated in clinical trials, and some
encouraging results have been obtained, although some are still in the recruiting stage.
Another appealing alternative, saRNA, which originates from single-strand mRNA viruses,
has also gained significant attention due to its self-amplification property and persistent
antigen expression capability [56]. Currently, although saRNA can be synthesized after IVT
without viral particle packages, reducing safety concerns regarding the viral components,
no saRNA vaccines are being used in clinical trials of melanoma due to the debate over the
clinical benefits versus potential immunological damage to patients that such vaccines may
cause. Therefore, in this section, we only discuss mRNA vaccines delivered by non-viral
vectors, which have been extensively explored recently.
Vaccines 2021, 9, 1060 16 of 33

Table 1. Clinical trials of IVT mRNA-based vaccines for the treatment of melanoma.

Encoding Enrollment Target

Trial ID Start Date Phase Brand Formulation Route Combination Study Results Sponsor
Content Status Antigens
An increase of
vaccine-specific T cells
Melan-A,
was observed in two
MAGE-A1,
Protamine- of four University
NCT Jun MAGE-A3,
I/II Completed NA protected i.d. GM-CSF immunologically Hospital
00204607 2004 Survivin,
mRNA evaluable patients. Tuebingen
GP100,
One of seven patients
Tyrosinase
with measurable
diseases showed a CR.
Melan-A,
MAGE-A1,
University
NCT Apr MAGE-A3, Naked
I/II Completed NA i.d. GM-CSF Hospital
00204516 2007 Survivin, mRNA
Tuebingen
GP100,
Tyrosinase
MAAs NCT Jun NY-ESO-1, Naked
I Completed i.n. None BioNTech
01684241 2012 tyrosinase mRNA
More than 75%
showed immune
responses against at
least one MAA in 50
patients. In the FixVac
NY-ESO-1, monotherapy group
NCT Mar Active, not FixVac MAGE-A3, (n = 25), three patients
I RNA-LPX i.v. Anti-PD1 BioNTech
02410733 2015 recruiting (BNT111) TPTE, experienced a PR and
tyrosinase seven had SD, while
in the
FixVac/anti-PD1
combination group, 6
out of 17 patients
developed a PR.
Vaccines 2021, 9, 1060 17 of 33

Table 1. Cont.

Encoding Enrollment Target

Trial ID Start Date Phase Brand Formulation Route Combination Study Results Sponsor
Content Status Antigens
One-third of
pre-existing weak
responses against
neo-epitopes were
augmented while
two-thirds were de
novo responses. 13
mRNA patients in total. Eight
NCT Oct Naked encoding patients without
I Completed IVACMUTANOME Neopeptides i.n. BioNTech
02035956 2013 mRNA NY-ESO-1, measurable lesions at
tyrosinase the start of the trial
developed robust
immune responses
against neo-epitopes
and achieved
recurrence-free for the
whole follow-up
period.
NCT Dec BioNTech
Neoantigens I Recruiting RO7198457 Neopeptides LNP i.n. Atezolizumab
03289962 2017 GenenTech
NCT Jan BioNTech
II Recruiting RO7198458 Neopeptides LNP i.v. Pembrolizumab
03815058 2019 GenenTech
NCT Nov
I/II Terminated mRNA-4650 Neopeptides LNP i.m. None Moderna
03480152 2019
No ≥ grade III AEs
occurred. Of the 13
patients on
monotherapy, 12
patients remain
NCT Jul disease-free. In the Moderna
II Recruiting mRNA-4157 Neopeptides LNP i.v. Pembrolizumab
03897881 2019 combination group Merck
(n = 20), one CR, two
PR, and five SD were
observed for at least
five administration
cycles.
Vaccines 2021, 9, 1060 18 of 33

Table 1. Cont.

Encoding Enrollment Target

Trial ID Start Date Phase Brand Formulation Route Combination Study Results Sponsor
Content Status Antigens
No AEs Grade 3 or
mRNA higher were reported.
encoding Vaccine-induced
CD70, tyrosinase, immune responses
NCT Jun Naked eTheRNA
Immunostimulants I Recruiting ECI-006 CD40L, i.n. gp100, were detected in 4/10
03394937 2017 mRNA immunotherapies
caTLR4 MAGE-A3, and 3/9 patients
MAGE-C2, treated with the low
PRAME and high dose,
respectively.
Abbreviation: MAAs: Melanoma-associated antigens; RNA-LPX: liposomal RNA; LNP: Lipid nanoparticle; AEs: Adverse events; CR: Complete response; PR: Partial response; SD: Stable disease; i.v.: Intravenous;
i.d.: Intradermal; i.n.: Intranodal; i.m.: intramuscular; NA: Not available.
Vaccines 2021, 9, 1060 19 of 33

The selection of antigens is a key issue in the establishment and development of cancer
vaccines. Various features of antigens, like their immunogenicity and avidity, impact the ef-
ficiency of cancer vaccines [171]. The choice of antigen is critical during the design of cancer
vaccines to better train and bolster the immune systems of hosts to fight against cancer and
eventually eliminate malignant cells. Tumor-associated antigens (TAAs), which have low
levels of expression in normal tissues while showing elevated expression in tumor cells,
have been well-studied and used in the development of cancer vaccines [172]. Nonetheless,
due to their “self-protein” characteristic, TAAs are not completely ideal targets for cancer
vaccines. Another emerging target, neoantigens, which originate from non-synonymous
mutations in tumor cells and are absent from normal cells, have been applied in cancer
vaccine manufacturing and have shown unique advantages in clinical trials, especially
for melanoma patients [173,174]. Currently, multiple mRNA-based cancer vaccines, either
encoding a cocktail of TAAs or personalized neoantigens, have been assessed in clinical
trials of melanoma (Table 1). Furthermore, the versatility of mRNA has paved a path
beyond its use as a source of tumor antigens. mRNA encoding immunostimulants, which
can induce the maturation and activation of antigen-presenting cells (APCs), promoting T
cell-mediated immune responses and modifying the suppressive tumor microenvironment,
have been applied as novel immunotherapies to combat cancer in combination with other
cancer vaccines or immunotherapeutic agents (e.g., immune checkpoint inhibitors) [15].
This mRNA-encoding immunostimulant strategy has evolved the landscape of mRNA-
based cancer vaccines, contributing to the eradication of tumor cells using a comprehensive
method.
The early-stage clinical trials of IVT mRNA cancer vaccines against melanoma targeted
melanoma-associated antigens (MAAs), which are preferentially expressed in melanoma
cells. In 2004, Weide et al. launched a clinical trial in which protamine-stabilized mRNAs
encoding Melan-A, Tyrosinase, gp100, Mage-A1, Mage-A3, and Survivin were used to
treat melanoma patients (NCT00204607) [175]. After receiving the vaccine intradermally,
enhancement of vaccine-specific T-cell immune response was observed in two out of four
immunologically evaluable patients. One out of seven stage IV patients with measurable
disease showed a complete response during a 36-month observation period. In 2015,
BioNTech initiated a multicenter, open-label, dose-escalation phase I trial (Lipo-MERIT,
NCT02410733) to assess the efficiency and safety of FixVac (BNT111), which is composed
of RNA-LPX encoding NY-ESO-1, MAGE-A3, TPTE, and tyrosinase, for the treatment
of advanced melanoma patients [168]. Patients expressing at least one of these MAAs
underwent eight vaccination cycles. This well-known nanoparticulate liposomal RNA
vaccine displayed its antitumor effects by bolstering the immune response against at least
one MAA in 39 out of 50 patients. In the FixVac monotherapy arm (n = 25), three patients
achieved a partial response, and seven attained a stable disease state. Seventeen patients
received the FixVac plus anti-PD1 antibody in the combination group, and six patients
developed a partial response in this arm. Results collected from this trial revealed that
the FixVac encoding four types of non-mutant MAAs has clinical benefits for melanoma
patients when combined with ICBs, especially for those with a lower tumor mutation
burden (TMB).
IVT mRNA vaccines encoding MAAs have already shown their feasibility for use in
the treatment of melanoma. However, central tolerance is still the major challenge that
threatens the efficiency of mRNA vaccines targeting MAAs. Furthermore, non-mutant
MAA mRNA vaccines could theoretically decrease in potency when applied to treat
melanoma, which is the TMB tumor type with the greatest magnitude [176]. In this new
era of using antigens for the cancer vaccine development, neoantigens have been shown
to have advantages in terms of inducing a highly specific, robust anti-tumor immune
response with an individual approach. This is in contrast to MAAs, especially for treating
high-TMB melanoma. Sahin et al. reported the first-in-human application of an RNA-
based polyneoepitope vaccine to treat melanoma (NCT02035956) [79]. Non-synonymous
mutations expressed by thirteen patients with melanoma were identified by exome and
Vaccines 2021, 9, 1060 20 of 33

RNA sequencing, and ten selected mutations per patient were constructed into two syn-
thetic RNAs. Each patient received at least eight doses of the neoepitope mRNA vaccine
intranodally. One-third of patients with pre-existing weak responses against neoepitopes
showed augmented responses, while two-thirds showed de novo responses. Eight patients
without radiologically detectable lesions at the start of the administration period generated
vigorous immune responses and experienced the absence of recurrence for a 12–23 month
postvaccination follow-up period, while the other five relapsed shortly after inclusion.
Interestingly, this trial revealed a stronger neoepitope-specific than TAA-specific response
in patients who received both vaccinations, indicating that a lack of central tolerance might
be the underlying mechanism.
With the rapid development of nanotechnology, LNP was designed as a powerful
delivery vehicle for mRNA vaccines with an effective internalization capability, endosomal
escape, and cell/organ-selective targeting. One well-known product named mRNA-4157
was launched by Moderna. This is a personalized mRNA vaccine encapsulated in LNP
that is used to treat patients with solid tumors including, but not limited to, melanoma
(NCT03897881) [177]. The formulation was found to be well-tolerated and no adverse
events were reported in this trial. Twelve out of the thirteen patients in the monother-
apy arm retained a disease-free status during an 8 month follow-up period, while in the
combination group (n = 20), there was one complete response, two partial responses,
and five patients with stable disease for at least five vaccination cycles. Furthermore,
BioNTech and GenenTech have also initiated multiple phase I and II trials to assess the
efficacy and safety of personalized LNP-encapsulated mRNA vaccines encoding neoanti-
gens (RO7198457, RO7198458) in combination with Atezolizumab or Pembrolizumab
(NCT03289962, NCT03815058).
Unlike antigen-targeting vaccines, immunostimulant-encoding vaccines fulfill their
function by priming APCs and T cell-mediated immunity and modifying the dysfunctional
tumor microenvironment [178,179]. Thus, the evaluation of mRNA encoding immunos-
timulants as monotherapies or for use in combination therapies with other immunological
agents has also been investigated in multiple clinical trials. One pioneer product invented
by eTheRNA known as the “TriMix” mRNA adjuvant vaccine, which encodes CD70 to
activate CD8+ T cells, CD40L to stimulate CD4+ T cells, and constitutively active TLR4
to promote antigen presentation in DCs [180], has been proven to be well tolerated and
immunogenic in clinical trials against melanoma [181–183]. However, TriMix is mainly
utilized in ex vivo DC transfection to induce the maturation and activation of DCs as one
major step in adoptive DC transfer therapy. Few clinical trials concerning direct TriMix
injection have been conducted. One trial aiming to assess the immunogenicity and safety
of the ECI006 vaccine (a combination of TriMix and MAA-encoding mRNA) via intranodal
administration was initiated in 2017 (NCT03394937) [184]. In this study, no serious adverse
events occurred in the postvaccination period, indicating the good tolerability and safety
of ECI006. Furthermore, a vaccine-induced immune response was demonstrated in four
out of ten and three out of nine patients treated with low and high doses, respectively.
More promising clinical results are expected in this ongoing trial. There are also other
adjuvant vaccine formulations, like mRNA-2752, a product developed by Moderna, which
is composed of OX40L, IL-23, and IL-36. This product has been shown to boost the anti-
cancer response by inducing the activation of T cells and modulating innate and adaptive
immunity (NCT03739931). Although the efficacy and tolerability of other adjuvant vaccines
against melanoma have not been tested in clinical trials yet, the guaranteed results achieved
from other types of cancer highlight its potential effects on melanoma.

6. DC mRNA Vaccines in Melanoma

Having an essential role in initiating the specific antitumor immune response, DCs can
be utilized to deliver mRNA-encoding tumor antigens in cancer immunotherapies based
on their biological features. The first demonstration of adoptive mRNA-pulsed DC transfer
was conducted by Boczkowski and colleagues in 1996 [185]. They found that DCs pulsed
Vaccines 2021, 9, 1060 21 of 33

with ovalbumin (OVA)-encoding mRNA were more effective than OVA peptide-pulsed
DCs for priming OVA-specific CTL responses in vitro. Furthermore, mice vaccinated
with DCs pulsed with OVA-encoding mRNA were protected from OVA-expressing tumor
cells. In their study, a dramatic reduction in lung metastases was observed in the poorly
immunogenic, highly metastatic B16/F10.9 tumor model after vaccination with DCs pulsed
with tumor-derived mRNA, revealing that DCs pulsed with mRNA represent an attractive
platform for cancer treatment and have the potential to be translated into clinical practice.
In addition to the ex vivo DC loading approach, in vivo DC targeting is an alternative for
the design of DC-based mRNA vaccines. However, this vaccine platform is usually realized
by intranodal administration, and the specific targeting of DCs is hard to guarantee due to
the diverse environment in lymph nodes. Thus, in this section, we only discuss the use of
ex vivo DC loading mRNA vaccines for the treatment of melanoma. Thus far, clinical trials
concerning DC-based mRNA vaccines have been initiated for patients with various types
of cancer, including colorectal cancer [186], glioblastoma [187], prostate cancer [188], and
acute myeloid leukemia [189].
Notably, DC-based mRNA vaccines have recently been widely applied to treat melanoma
patients, and promising results have been achieved from these clinical trials (Table 2).
Gaudernack and colleagues extracted autologous total mRNA from tumor biopsies of
melanoma patients and then electroporated it into DCs [190,191]. In vivo, vaccine-specific
immune responses elicited by DCs transfected with autologous tumor-derived mRNA
have been shown to be evident in melanoma patients. Although a broad spectrum of T cell
responses has been demonstrated after vaccination with tumor-derived mRNA transfected
DCs, tumor-derived mRNA generation requires a large number of tumor biopsies, which
means melanoma patients are usually in the late stage of disease, and limited effects
can be achieved by vaccine administration. Furthermore, tumor-derived mRNA also
encodes common host antigens; thus, the cytotoxicity against cancer prompted by mRNA
encoding tumor antigens is weakened by central tolerance. To achieve a more specific
immune response toward melanoma and avoid potential adverse events, TAA-encoding
mRNA, including MAGE-A3, MAGE-C2, tyrosinase, and gp100, is mostly chosen for
electroporation into DCs. Interestingly, unlike tyrosinase and gp100, which are widely
represented in melanoma but also expressed in normal tissues, MAGE-A3 and MAGE-C2,
as cancer-germline antigens, are exclusively expressed in germ cells as well as in various
types of cancers including, but not limited to, melanoma [192,193]. The existence of the
blood–testis barrier prevents the recognition of cancer-germline antigens by the immune
system. Once these antigens are move beyond the testis, a robust immune response against
them is aroused. This biological feature endows cancer-germline antigens great potential
for use in vaccine design [171,194]. In Wilgenhof’s trial, a specific MAGE-A3 and MAGE-C2
immune response was demonstrated in the postvaccination period in 7 and 10 out of 21
patients, respectively. This was superior to the responses of tyrosinase and gp100, revealing
the potency of cancer-germline antigens and their potential for use in mRNA vaccines
against melanoma [195]. In addition, as a pharmacological and immunological agent that
modifies the effect of the vaccine, immunological adjuvants function by stimulating and
amplifying immune responses to targeted antigens but do not confer immunity themselves.
The utilized adjuvant for DC-based mRNA vaccines against melanoma that has been used
most commonly in clinical trials is TriMix. This mRNA-based adjuvant contains three
naked mRNA molecules, which encode the activation stimulator CD40 ligand (CD40L),
which activates CD4+ T cells; the costimulatory molecule CD70, which activates CD8+
T cells; and constitutively active TLR4, which promotes DC antigen presentation [196].
The use of the TriMix adjuvant in combination with antigen-encoding mRNA has been
evaluated in multiple DC-based mRNA vaccine clinical trials and has been proven to be
well-tolerated and immunogenic [181,182,195,197,198].
Vaccines 2021, 9, 1060 22 of 33

Table 2. Completed clinical trials of DC-based mRNA vaccines against melanoma.

Grade
Year Trial ID Phase Antigen Formulation Route Combination ≥3 Study Results Refs
Adverse Events
A vaccine-specific immune response
was demonstrated in 9/19 patients
evaluated by T-cell assays and in
Autologous 8/18 patients evaluated by DTH
2006 NA I/II Electroporation i.d. and i.n. None None [190]
tumor-mRNA reaction. The response rates do not
suggest an advantage in applying i.n.
vaccination compared with i.d.
vaccination.
The immunological data indicated
Autologous sustained T cell responses and
2007 NA I/II Electroporation i.d. and i.n. None None [191]
tumor-mRNA suggested an enhancing effect of
booster vaccinations.
Vaccinal antigen-specific DIL were
found in 0/6 patients tested at
MAGE-A3, vaccine initiation and in 12/21
Electroporation
MAGE-C2, (57.1%) assessed after the fourth
2011 NA NA with i.d. IFN-α-2b None [195]
tyrosinase, vaccine. During TriMixDC/IFN-a-2b
TriMix-mRNA
gp100 combination therapy, one PR and
five SD were observed in 17 patients
with evaluable disease at baseline.
MAGE-A3, Ex vivo-generated mRNA-modified
Electroporation
MAGE-C2, DCs can induce effector CD8+ and
2012 NA NA with i.v. and i.d. None NA [197]
tyrosinase, CD4+ T cells from the naive T-cell
TriMix-mRNA
gp100 repertoire of melanoma patients.
In a total of 15 patients, two patients
achieved a CR and two patients a PR.
MAGE-A3, All objective responders achieved a
Electroporation
NCT MAGE-C2, PFS. Antigen-specific SKILs were
2013 Ib with i.v. None None [181]
01066390 tyrosinase, documented in 6 of 12 patients, and
TriMix-mRNA
gp100 antigen-specific CD8+ T-cells were
detected in the blood of four of five
patients.
Vaccines 2021, 9, 1060 23 of 33

Table 2. Cont.

Grade
Year Trial ID Phase Antigen Formulation Route Combination ≥3 Study Results Refs
Adverse Events
MAGE-A1,
MAGE-A3,
The median relapse-free survival is
MAGE-C2, Electroporation
22 months (95 % CI 12–32 months),
2015 NA NA MelanA/MART- with i.d. IFN-α-2b None [182]
the 2-year and 4-year survival rates
1, TriMix-mRNA
are 93% and 70%, respectively.
tyrosinase,
gp100
The 6-month disease control rate
MAGE-A3, was 51% (95% CI, 36% to 67%), and
Electroporation
NCT MAGE-C2, the overall tumor response rate was
2016 II with i.v. and i.d. Ipilimumab 17 [183]
01302496 tyrosinase, 38%, seven CR and one PR are
TriMix-mRNA
gp100 ongoing after a median follow-up
time of 36 months
71% of patients in the study arm
MAGEA3, were free of disease compared with
Electroporation
NCT MAGE-C2, 35% in the control arm after one year.
2020 II with i.v. and i.d. None None [198]
01676779 tyrosinase, The median time to non-salvageable
TriMix-mRNA
gp100 recurrence was superior in the study
arm.
Antigen-specific CD8+ T cells were
found in 44% versus 67%, and
functional T cell responses in 28%
versus 19% in patients receiving DC
NCT tyrosinase, vaccination with and without
2020 II Electroporation i.v. and i.d. Cisplatin 1 [166]
02285413 gp100 cisplatin, respectively. A
significantly better OS is observed in
stage III patients treated with
combination therapy compared with
DC monotherapy.
Abbreviation: CR: Complete response; PR: Partial response; SD: Stable disease; PFS: Progression-free survival; i.v.: Intravenous; i.d.: Intradermal; i.n.: Intranodal; DTH: Delayed-type hypersensitivity; DIL:
DTH-infiltrating lymphocytes; MAGE: Melanoma-associated antigen; NA: No available.
Vaccines 2021, 9, 1060 24 of 33

All clinical trials indicated in Table 2 were performed using electroporation as the
mRNA transfection method for DCs. Electroporation is now a mature technique, and
its transfection potency has been demonstrated in various preclinical and clinical trials.
Nevertheless, other emerging transfection platforms, such as lipofection, nucleofection,
and sonoporation, are considered alternatives for electroporation, and further clinical
trial results are required to assess the transfection efficiency and biosafety of these meth-
ods [143,144]. For DC-based mRNA vaccines, intravenous and intradermal injections are
regular administration routes that have been widely applied in clinical trials. Several
studies have shown that only a small proportion of injected DCs reach the regional lymph
nodes after intradermal administration [199,200]. Therefore, intranodal injection seems
to be advantageous for DC-based vaccine administration compared with intradermal
injection.
Interestingly, Gaudernack et al. reported no difference between intranodal and in-
tradermal injection in relation to eliciting immune responses in the postvaccination pe-
riod [190]. This might be because (1) having a small percentage of successfully migrating
DCs is sufficient for T cell activation in the lymph nodes, (2) successfully migrating DCs
already receive further maturation signals during migration, and (3) damage to the lymph
node structure occurs during intranodal administration.
DC-based mRNA vaccines have shown promising antitumor effects against melanoma
in clinical trials. In 2011, Wilgenhof et al. reported that when TriMixDC/IFN-α-2b com-
bination therapy was applied to advanced melanoma patients, one partial response was
observed and 5 patients achieved a stable disease status out of 17 patients. The median OS
and progression-free survival (PFS) were 15.1 months and 3.1 months, respectively [195].
In 2013, Wilgenhof et al. reported on a phase Ib study in which intravenous TriMixDC-MEL
pulsed with MAAs was used to treat advanced melanoma patients. In this trial, two partial
responses were achieved and 2 patients attained a stable disease status out of 15 patients.
The median PFS and OS were 5 and 14 months, respectively [181]. In 2020, a randomized
controlled phase II clinical trial was conducted by Jansen and colleagues. In that study, the
one-year disease-free survival rate was 71% in the TriMixDC-MEL arm and 35% in the con-
trol arm. The time to non-salvageable recurrence or death was longer in the TriMixDC-MEL
arm than in the control arm [166]. These encouraging results from clinical trials provide
a solid foundation for the development of DC-based mRNA vaccines in clinical practice.
However, more firm clinical outcomes, especially from randomized two-arm studies, are
urgently needed. Moreover, to circumvent the limitations of monoimmunotherapies in can-
cer treatment, a rational DC-based mRNA vaccine combination approach requires further
investigation. Such an approach could impact the tumor and modulate the dysfunctional
tumor microenvironment in a comprehensive manner.

7. Therapeutic Considerations, Challenges, and Future Directions

Although mRNA vaccines have many advantages for the treatment of cancer, they are
still in the early stages of research. Currently, it is necessary to take all aspects related to
the safety of a product into account, including the above-mentioned problems associated
with the immunogenicity, stabilization, and efficacy of mRNA vaccines. The development
of an autoimmune response against the background of the immune-stimulating function of
mRNA vaccines is a significant clinical problem. The identification of highly immunogenic
tumor-specific antigens is challenging, since tumor antigens may differ greatly among
individuals. Finally, since antitumor vaccines are therapeutic rather than prophylactic
in most cases, and since cancer treatment requires long-term multiple administrations
of high-dose medicaments, the safety requirements for the creation and use of mRNA
vaccines must be significantly tightened. Nonetheless, mRNA vaccines have a promising
future and great potential to become one of the main strategies for cancer treatment. One
of the important advantages of mRNA cancer vaccines is their fast and scalable production,
allowing the product to be produced in a short time. This advantage is especially important
due to the frequent rapid disease progression in cancer patients.
Vaccines 2021, 9, 1060 25 of 33

The most important area of research in the cancer vaccine therapy field is the advance-
ment of the identification of individual cancer neoantigens derived from mutations or
other abnormal sequences that are technically challenging to identify. Therefore, further
studies to improve sequencing and bioinformatic approaches to analyze antigens and their
epitopes as well as predict their binding to MHC molecules are of considerable importance.
Further improvement to mRNA cancer vaccines could be obtained through combinations
with complementary immunotherapeutic approaches. Based on successful results obtained
from the combination of cancer vaccines with immune checkpoint inhibitors, combination
therapies should be considered as prominent approaches for cancer treatments, and further
experiments should be conducted in this area.
Overall, the number of ongoing studies in the field of mRNA cancer vaccines is
growing significantly (Tables 1 and 2). The experience gained through the development of
mRNA vaccines to prevent COVID-19 will undoubtedly play a role in the development
of mRNA cancer vaccines as well, especially in regard to production protocols, storage,
and distribution. Low costs, manufacturing benefits, and the ability to tailor personalized
preparations will pave the way for the production of mRNA vaccines.

Author Contributions: Conceptualization, M.B. and M.G.-h.; writing—original draft preparation,

M.B., Y.Z., N.G.S. and M.R.G.; visualization, Y.Z. and N.G.S.; A.V.B. (Alexey V. Baldin), A.V.B.
(Alexandr V. Bazhin) and A.A.Z.J. finalized the manuscript; funding acquisition, A.A.Z.J. All authors
contributed to the article writing. All authors have read and agreed to the published version of the
manuscript.
Funding: This research was funded by the Russian Science Foundation (grant # 21-75-30020).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: All figures in the present manuscript were prepared using Biorender instruments
(https://app.biorender.com, accessed on 20 June 2021).
Conflicts of Interest: The authors declare no conflict of interest.

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