mRNA Cancer Vaccines

Katja Fiedler, Sandra Lazzaro, Johannes Lutz, Susanne Rauch

and Regina Heidenreich

Abstract
mRNA cancer vaccines are a relatively new class of vaccines, which combine the
potential of mRNA to encode for almost any protein with an excellent safety profile
and a flexible production process. The most straightforward use of mRNA vaccines
in oncologic settings is the immunization of patients with mRNA vaccines encoding
tumor-associated antigens (TAAs). This is exemplified by the RNActive®
technology, which induces balanced humoral and cellular immune responses in
animal models and is currently evaluated in several clinical trials for oncologic
indications. A second application of mRNA vaccines is the production of
personalized vaccines. This is possible because mRNA vaccines are produced by
a generic process, which can be used to quickly produce mRNA vaccines targeting
patient-specific neoantigens that are identified by analyzing the tumor exome. Apart
from being used directly to vaccinate patients, mRNAs can also be used in cellular
therapies to transfect patient-derived cells in vitro and infuse the manipulated cells
back into the patient. One such application is the transfection of patient-derived
dendritic cells (DCs) with mRNAs encoding TAAs, which leads to the presentation
of TAA-derived peptides on the DCs and an activation of antigen-specific T cells
in vivo. A second application is the transfection of patient-derived T cells with
mRNAs encoding chimeric antigen receptors, which allows the T cells to directly
recognize a specific antigen expressed on the tumor. In this chapter, we will review
preclinical and clinical data for the different approaches.

Keywords



RNActive® vaccines Personalized mRNA vaccines mRNA-pulsed dendritic


cells Chimeric antigen receptor (CAR) T cell immunotherapy

K. Fiedler (&)
S. Lazzaro
J. Lutz
S. Rauch
R. Heidenreich

CureVac AG, Paul-Ehrlich-Str. 15, 72076 Tübingen, Germany
e-mail: katja.fiedler@curevac.com

© Springer International Publishing Switzerland 2016 61

W. Walther (ed.), Current Strategies in Cancer Gene Therapy,
Recent Results in Cancer Research 209, DOI 10.1007/978-3-319-42934-2_5
62 K. Fiedler et al.

Abbreviations
A Adenine
APC Antigen-presenting cell
CAR Chimeric antigen receptor
CTL Cytotoxic T lymphocyte
CTLA4 Cytotoxic T lymphocyte antigen 4
DC Dendritic cell
DNA Deoxyribonucleic acid
EGFR Endothelial growth factor receptor
ELISA Enzyme-linked immunosorbent assay
GC Guanine and cytosine
GITR Glucocorticoid-induced tumor necrosis factor receptor
HLA Human leukocyte antigen
HMGB1 High-mobility group box 1
IDH1 Isocitrate dehydrogenase 1
IFN Interferon
Ig Immunoglobulin
LLC Lewis lung cancer
MAGEC1 Melanoma antigen family C1
MAGEC2 Melanoma antigen family C2
MHC Major histocompatibility complex
mRNA Messenger ribonucleic acid
MUC1 Mucin 1
NK Natural killer
NSCLC Non-small-cell lung cancer
NY-ESO-1 New York esophageal squamous cell carcinoma-1
ORF Open reading frame
OVA Ovalbumin
PAP Prostatic acid phosphatase
PD1 Programmed cell death 1
PDL1 Programmed cell death 1 ligand 1
PDL2 Programmed cell death 1 ligand 2
PFS Progression-free survival
PSA Prostate-specific antigen
PSCA Prostate stem cell antigen
PSMA Prostate-specific membrane antigen
RNA Ribonucleic acid
RT-PCR Reverse transcriptase polymerase chain reaction
STEAP1 Six-transmembrane epithelial antigen of the prostate 1
TAA Tumor-associated antigen
TERT Telomerase reverse transcriptase
Th T helper
TLR Toll-like receptor
mRNA Cancer Vaccines 63

Tmeso CAR T cells specifically targeting mesothelin

TPBG Trophoblast glycoprotein
UTR Untranslated region

1 mRNA Cancer Vaccines

1.1 Introduction

The concept of mRNA vaccines is founded on the observation that injection of

messenger RNA (mRNA) leads to local protein expression (Wolff et al. 1990) and
immune responses against the encoded antigen (Martinon et al. 1993). This was
rather unexpected given the abundant presence of RNases in body fluids and on the
skin, which leads to the rapid degradation of RNA, for example, in
serum-containing media (Rammensee 2006), and most work on nucleic acid-based
vaccine development continued to focus on DNA rather than on RNA vaccines.
This view has changed with the advent of several technological platforms, which
have demonstrated the potential of mRNA vaccines to express antigen and to
induce humoral and cellular immune responses.
While both nucleic acid-based platforms share many characteristics, mRNA
vaccines have several advantages over DNA-based platforms (Table 1). Firstly,
RNA only needs to pass the plasma membrane in order to induce protein synthesis,
facilitating vaccine delivery in comparison with DNA that needs to additionally
cross the nuclear membrane. Secondly, mRNA is unable to integrate in the genome
and therefore has no oncogenic potential. Finally, expression of mRNA-encoded
proteins is intrinsically transient, defined by the short half-life of mRNA. Hence,
mRNA vaccines feature a significantly increased safety profile over DNA.
In addition, mRNA vaccines provide important advantages over the more widely
used protein-based vaccine platforms: Endogenous production of proteins in the
cells of the vaccinee upon injection of mRNA supports correct protein modifica-
tions, such as glycosylation patterns, and abolishes the need for elaborate protein or
particle purification steps. From a technical perspective, mRNA, unlike
protein-based vaccines, can be produced in a fully synthetic production process and
allows the production of mRNA encoding any protein or combination of proteins of
choice using the same biologic compounds and production steps, greatly facilitating
vaccine manufacturing processes. Furthermore, the ability to support quick
sequence adjustments makes RNA vaccines highly versatile, which is of particular
importance in pandemic settings or for individualized therapies (see below).
64 K. Fiedler et al.

1.2 RNActive® Vaccines

The first section focuses on the RNActive® technology (CureVac AG), as an

example for an mRNA vaccine platform currently evaluated in several clinical trials
for oncologic indications.
RNActive® vaccines consist of formulated mRNA which encodes for the anti-
genic protein of choice and features modifications to enhance translation efficiency,
delay mRNA decay, and improve immune stimulation.
In 2000, Hoerr et al. described that the in vivo application of mRNA encoding
the model antigen β-galactosidase led to the induction of specific cytotoxic T
lymphocytes (CTLs) and antibodies (Hoerr et al. 2000). The RNA used for these
experiments had the basic design of a mRNA and consisted of a cap, an open
reading frame (ORF) encoding for β-galactosidase that was flanked 5′ and 3′ by the
untranslated regions (UTRs) of β-globin and a poly(A) tail. For the current
RNActive® technology, several aspects of the mRNAs have been modified to
increase the extent and duration of the antigen expression. The β-globin UTRs have
been replaced by UTRs selected for higher translation efficiency and stability of the
mRNAs, and the ORF was optimized for enhanced protein expression by enriching
the guanine and cytosine (GC) content according to a proprietary algorithm. These
changes, as well as the use of a template-encoded poly(A) tail of defined length,
optimized buffers, and purification led to an increase in protein expression by four
to five orders of magnitude in various test systems (Kallen and Theß 2014).
Importantly, this technology exclusively employs unmodified nucleotides. Studies
by Karikó (Karikó et al. 2008) and Anderson (Anderson et al. 2010) have
demonstrated that modified nucleotides can lead to increased protein expression.
However, they also reduced immunogenicity, making the use of modified
nucleotides unfavorable in the context of vaccines.
In addition to antigen availability, adjuvanticity is essential for inducing strong
immune responses. In RNActive® vaccines, immunostimulatory capacity is
achieved via suitable formulation, e.g., by employing protamine, a cationic peptide
that forms stable complexes with nucleic acids. Protamine binds to the mRNA and
leads to the formation of larger particles that activate the immune system in a
process involving the endosome-resident TLR7 (Toll-like receptor 7)
(Fotin-Mleczek et al. 2011; Kallen et al. 2013; Scheel et al. 2005; Kowalczyk et al.
2016) (Table 1).
Hence, RNActive® vaccines are containing two components that serve com-
plementary functions: “naked” mRNA, which serves as a translation template
responsible for strong expression of the encoded antigen, and mRNA–protamine
complexes, which enhance the immune stimulatory capacity of the vaccine. Indeed,
vaccination with RNActive® vaccines has been shown to induce strong and bal-
anced immune responses, i.e., Th1 and Th2, humoral and cellular, and effector and
memory responses. Encouraging results have been gained in various animal model
systems, among others mice, ferrets, and pigs, both in the field of cancer
mRNA Cancer Vaccines 65

Table 1 Advantages of RNA over DNA vaccines

DNA RNA
Delivery DNA needs to cross both cell and RNA only needs to gain entry into the
nuclear membranes and be first cytoplasm, where translation and thus
transcribed in the nucleus before protein protein expression directly occur
expression occurs
Integration DNA vaccines are able to integrate into RNA cannot integrate into the genome
the host genome, which might result in and therefore has no oncogenic potential
insertional mutagenesis and
chromosomal instability
Expression Long-term expression possible (months Transient expression
to years), depending on vector

immunotherapy (Fotin-Mleczek et al. 2011, 2012) and prophylactic vaccines

(Petsch et al. 2012; Kowalczyk et al. 2016; Schnee et al. 2016).

1.2.1 Induction of Antitumor Responses with RNActive®

Vaccines
The antitumor efficacy of RNActive® vaccines was initially studied in the E.
G7-OVA tumor model. This system uses C57BL/6 mice, which are inoculated
subcutaneously with the syngeneic E.G7-OVA cell line, a clone of the mouse
thymoma EL4 cell line that has been stably transfected to express ovalbumin
(Moore et al. 1988). The vaccine was first studied in a prophylactic setting. Mice
were immunized twice intradermally with the mRNA vaccine encoding ovalbumin
and challenged one week later with E.G7-OVA cells. Compared to control mice
that had received an mRNA vaccine encoding for an irrelevant antigen, the
OVA-RNActive®-treated group displayed a significant delay in tumor growth
(Fotin-Mleczek et al. 2011). Additionally, vaccination with OVA-RNActive®
induced superior tumor protection in comparison with vaccination with ovalbumin
protein or OVA-peptide SIINFEKL (Fotin-Mleczek et al. 2012).
A detailed analysis revealed that the antigen-specific vaccination had induced a
balanced humoral and cellular immune responses with high titers of
ovalbumin-specific IgG1 and IgG2a antibodies and cytotoxic T cells, which
secreted IFN-γ upon stimulation with an OVA-derived peptide in vitro and killed
OVA-peptide-loaded cells in vivo. Further experiments employing the antigen
PSMA showed that the cytotoxic response was boostable and led to the formation
of T memory cells. Increasing the number of biweekly vaccinations with the
PSMA-RNActive® vaccine from 2 to 4 or 6 vaccinations induced a significant
increase in both number of IFN-γ-secreting CD8+ T cells and in vivo cytotoxicity
during the acute phase. When analyzing these mice in the memory phase 8 weeks
after the last immunization, a similar increase in the number of IFN-γ-secreting
CD8+ T cells and in vivo cytotoxicity was observed by increasing the number of
vaccinations. Importantly, these IFN-γ-secreting CD8+ T cells displayed the
memory T cell marker CD44 and predominantly exhibited an effector memory
66 K. Fiedler et al.

phenotype (CD44+, CCR7−, CD62L−). Moreover, repeated vaccinations did not

induce detectable frequencies of regulatory T cells in mice in comparison with
controls (Fotin-Mleczek et al. 2011).
To test the efficacy of this technology in a therapeutic setting, the E.G7-OVA
model was again employed. Mice were challenged subcutaneously with the tumor
cells and received the OVA-RNActive® vaccine twice a week once the tumor was
palpable. The treatment led to a significant delay in tumor growth but could not
eradicate the tumor. A subsequent quantitative RT-PCR analysis showed that the
ovalbumin expression in the outgrown tumors of all mice treated with the
ovalbumin-encoding mRNA vaccine was reduced or even absent, while this was
only the case in 1/5 of the untreated mice. This indicates that the tumors in
RNActive® vaccine-treated mice had escaped immunotherapy due to downregu-
lation of ovalbumin (Fotin-Mleczek et al. 2011).
Analysis of the cellular composition of tumors at several time points after vac-
cination demonstrated an increased and sustained influx of activated CD8+ T cells
into the tumors in vaccinated mice. Depletion of CD8+ T cells, but not of CD4+ T
cells, during the effector phase completely abolished the antitumor effect of the
vaccine. In contrast, CD4+ T cells are required during the priming phase of the
immune response as their depletion during vaccination significantly reduced vaccine
efficacy (Fotin-Mleczek et al. 2011).

1.2.2 Clinical Studies with RNActive® Vaccines

Based on the encouraging preclinical results, the decision was taken to advance this
technology to clinical testing in cancer patients (Table 2). The first-in-class
first-in-man clinical trial using mRNA as a therapeutic approach was CV9103 for
patients with prostate cancer. CV9103 is an RNActive® vaccine targeting the fol-
lowing 4 tumor-associated antigens: prostate-specific antigen (PSA),
prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), and
six-transmembrane epithelial antigen of the prostate 1 (STEAP1). The vaccine was
tested in a phase I/IIa study that enrolled 44 eligible patients with castrate-resistant
prostate cancer with rising levels of PSA (all patients) and metastatic disease in 84 %
of patients and comprised 12 men in the phase I and 32 in the phase IIa study (Kübler
et al. 2015). The study was conducted as an open, uncontrolled, multicenter,
international, and prospective trial with safety and tolerability defined as the primary
endpoints and the induction of antigen-specific humoral and cellular immune
responses as secondary endpoints. The recommended dose was established via a
dose escalation in the phase I trial (259, 640, and 1280 µg total RNA tested), while
the phase IIa study was designed to confirm safety and address the induction of
antigen-specific cellular and humoral immune responses following injection with the
highest dose. This study demonstrated that CV9103 exhibits a favorable safety
profile with most frequent adverse events being injection site reactions, fatigue,
pyrexia, chills, and flulike symptoms. The majority of related adverse events was of
mild-to-moderate intensity and resolved upon therapy.
Importantly, immune responses against all mRNA-encoded antigens were
detectable independent of the cellular localization of the antigen and the HLA
Table 2 Overview of clinical trials using RNActive® vaccines
Biologic Indication Phase Antigens n Outcome Reference
CV9103 Hormone refractory I/IIa PSA 44 – Favorable safety profile Kübler et al. (2015)
prostate cancer PSMA – Induction of multiple
PSCA antigen-specific immune
STEAP1 responses
– Induction of immune responses
mRNA Cancer Vaccines

against all encoded antigens

CV9104 Metastatic I/IIb MUC1 Ph IIb Double-blind placebo-controlled
castrate-refractory PAP 197 study
prostate cancer PSA Recruitment completed
Asymptomatic, minimally PSMA
symptomatic, chemonaive PSCA
STEAP1
CV9104 Intermediate- or high-risk II MUC1 48 Open-label randomized trial of
prostate cancer prior to PAP presurgical CV9104 vs no
radical prostatectomy PSA treatment
PSMA Recruitment completed
PSCA
STEAP1
CV9201 Advanced-stage I/IIa MAGEC1 46 – Favorable safety profile Sebastian et al.,
non-small-cell lung cancer MAGEC2 – Induction of multiple manuscript in
NY-ESO-1 antigen-specific immune preparation
Survivin responses
5T4 – Induction of immune responses
against all encoded antigens
CV9202+ local radiation Advanced-stage Ib MAGEC1 26 Study ongoing
non-small-cell lung cancer MAGEC2
MUC1
NY-ESO-1
Survivin
67

5T4
68 K. Fiedler et al.

subtype of the patient. More specifically, antigen-specific cellular immune

responses were detected in 76 % of all patients treated with the highest dose. Of
note, 58 % of responding patients and 45 % of all evaluable patients at the highest
dose level showed responses to multiple antigens. The assessment of humoral
immune responses was restricted to PSA and PSCA, since no proteins suitable for
ELISA were available for the other vaccination antigens at that time. An increase of
PSA-specific antibodies could be detected in 12 % of patients, but no increase of
anti-PSCA antibodies was observed.
Clinical efficacy of CV9103 was assessed mainly by progression of PSA serum
levels, since radiographic progression-free survival (PFS) was not assessed in this
clinical trial. Similar to other cancer vaccine trials, a median time to PSA-related
PFS is 1.8 months and an objective PSA response in only one patient was observed.
Additionally, the study estimated a median survival of 31.4 months for a sub-
group of 36 patients with metastatic castrate-resistant prostate cancer. In this group,
a non-significant correlation between survival time and multivalent immune
response was observed. Interestingly, outcome further improved in patients with
responses to multiple vaccination antigens. Yet a correlation between immune
responses against more than one antigen and improved survival time does not
necessarily imply a therapeutic effect of vaccination. The ability to mount an
immune response after vaccination might be instead a surrogate of an improved
prognosis (Kübler et al. 2015).
Nevertheless, these encouraging results justified the investigation of RNActive®
vaccines in a controlled clinical trial. Hence, 197 patients with castrate-resistant
asymptomatic to minimally symptomatic castration-resistant prostate cancer were
enrolled and randomized in an ongoing placebo-controlled phase IIb study with
CV9104. This updated vaccine is based on CV9103 and encodes two additional
tumor-associated antigens, namely prostatic acid phosphatase (PAP) and mucin 1
(MUC1), a glycoprotein that is overexpressed and aberrantly glycosylated in var-
ious cancers including prostate cancer. The clinical efficacy and safety of PAP as
vaccine antigen have previously been demonstrated in the clinical trial of
Sipuleucel-T (Kantoff et al. 2010), while MUC1 has mainly been used in
immunotherapy approaches against non-small-cell lung cancer with a favorable
safety profile (Palmer et al. 2001). Primary endpoint of the study is overall survival
from time of randomization (up to 3.5–4 years). Secondary endpoints include PFS
from date of randomization and from start of first subsequent systemic therapy,
respectively, immune response against the CV9104 antigens, time to symptom
progression, and change in quality of life.
In a second study, CV9104 is tested in patients with high- and intermediate-risk
non-metastatic prostate cancer. For these patients, radical prostatectomy is a stan-
dard treatment option. After surgery, the estimated risk of relapse is considerable,
even more in case of positive surgical margins. Hence, new adjuvant or neoadjuvant
treatments that can prevent relapses after primary therapy are highly needed.
The study is aimed to assess the immune responses in the peripheral blood as
well as immunological parameters in the tumor tissue such as cellular immune
infiltrates, cytokines, and gene expression upon application of CV9104.
mRNA Cancer Vaccines 69

In addition to its application in the treatment of prostate cancer, RNActive®

vaccines were also tested as an immunotherapeutic against non-small-
cell lung carcinoma (NSCLC) in a clinical phase I/IIa trial (Sebastian et al.,
manuscript in preparation).
In this study, the immunotherapeutic CV9201 was intradermally administered at
different dose levels to evaluate the safety and tolerability in patients with advanced
NSCLC. CV9201 encodes five NSCLC-associated cancer antigens selected for their
role in NSCLC oncogenesis, differential expression between malignant and normal
tissue and to induce cellular or humoral immune responses: New York esophageal
squamous cell carcinoma-1 (NY-ESO-1), melanoma antigen family C1
(MAGEC1), melanoma antigen family C2 (MAGEC2), survivin, and trophoblast
glycoprotein (TPBG; synonym 5T4). Eligible patients had stage IIIB/IV NSCLC
and at least stable disease after first-line treatment. During the open, uncontrolled,
multicenter, and prospective trial, 9 patients received CV9201 in the phase I and 37
patients in the phase IIa part of the study. Overall, 45/46 patients received at least
two treatments and 33/46 patients received all five planned doses of CV9201. This
study demonstrated that similar to CV9103, also CV9201 exhibits a favorable
safety profile with most frequent adverse events being injection site reactions,
including injection site erythema, pruritus, discoloration, and pain. Other treatment-
related adverse events were pyrexia and fatigue, chills, and flulike symptoms.
Furthermore, immune responses against all encoded antigens were detectable. In
detail, 65 % of all patients exhibited antigen-specific cellular or humoral immune
responses and 65 % of responders reacted against multiple antigens. The strength of
the induced T cell response varied, but most patients displayed a more-than-twofold
increase in frequency and number of antigen-specific T cells detected ex vivo, with
some patients showing very strong responses. Moreover, 48 % of the patients
treated in phase IIa had antigen-specific humoral responses, with more than twice as
many patients exhibiting IgM than IgG responses.
Tumor response was evaluable in 29 patients of which nine patients had stable
disease and twenty patients experienced progressive disease as best overall
response. The median PFS in the total study population was 2.7 months. Impor-
tantly, the study revealed an encouraging clinical course in five patients with stage
IV NSCLC being without further systemic cancer therapy after 1 year.
Overall, these results demonstrate that RNActive® vaccines represent a highly
promising new vaccine platform that is safe, highly specific, versatile, and able to
induce a balance immune response in the context of clinical studies.

1.2.3 Combination of RNActive® Vaccines with Chemotherapy

or Radiation Therapy
From a clinical perspective, it is important to assess whether RNActive® vaccines
can be combined with the current standard of care. Therefore, the combination of
these vaccines with chemotherapy or radiation therapy was investigated.
The combination with chemotherapy was tested in the E.G7-OVA tumor model
using docetaxel or cisplatin and the mRNA vaccine against ovalbumin.
70 K. Fiedler et al.

Having received a subcutaneous tumor challenge followed by RNActive® vac-

cinations, mice were subsequently treated with an intraperitoneal application of
docetaxel followed by further vaccinations. This treatment schedule led to a sig-
nificant delay in tumor growth compared to docetaxel or RNActive® vaccine alone.
Similar results were observed when using the same treatment schedule but cisplatin
as a chemotherapeutic drug. In agreement with published results using viral vector
vaccines (Garnett et al. 2008), a delay of tumor growth was not detectable when
chemotherapy preceded vaccination, indicating a negative effect of docetaxel during
the induction phase of the immune response (Fotin-Mleczek et al. 2012).
An interesting option is the combination of vaccination with radiation therapy
because of the complementary features of the two approaches, which can synergize
to restrict tumor growth. Radiation is a well-established therapeutic method,
especially for the treatment of single solid tumors. It acts on the tumor tissue locally
and, for a very short period of time, is effective on tumors with a local immuno-
suppressive environment due to increased release of proinflammatory cytokines
(Formenti and Demaria 2013) and induces local MHC expression (Reits et al.
2006). Moreover, radiotherapy-induced cell death seems to represent a form of
immunogenic cell death characterized by cell surface translocation of calreticulin
and extracellular release of ATP and the high-mobility group box 1 (HMGB1)
protein (Golden et al. 2014). These factors are able to activate dendritic cells
(DCs) via pattern recognition receptors and promote the cross-presentation of tumor
antigens between DCs and T cells (Demaria and Formenti 2012).
Vaccination, on the other hand, is able to induce systemic immune responses
against non-symptomatic metastatic disease that are long-lasting and boostable.
Hence, the combination of radiotherapy and RNActive® vaccines was tested in
an E.G7-OVA tumor model. After the establishment of large subcutaneous tumors,
the mice received three local radiations on consecutive days. Concomitantly, sev-
eral vaccinations with OVA-RNActive® vaccine were administered. These exper-
iments demonstrated a pronounced tumor regression in the group treated with
combination therapy and complete eradication of large established E.G7-OVA
tumors in 3 out of 7 mice, while single therapies remained significantly less
effective (Fotin-Mleczek et al. 2014). Additional experiments were performed in the
Lewis lung cancer (LLC) model as a second tumor system, which features low
immunogenicity and is resistant to different kinds of therapeutic regimens (Savai
et al. 2007; Shojaei et al. 2007; Knapp et al. 2003). After subcutaneous tumor
challenge, the mice were treated with three local radiations on consecutive days in
combination with several vaccinations with RNActive® vaccines encoding the
described tumor-associated antigens EGFR and connexin (Mandelboim et al. 1995).
The combined treatment showed a synergistic antitumor effect demonstrating the
efficacy of radioimmunotherapy even in a low immunogenic carcinoma model
system (Fotin-Mleczek et al. 2014).
mRNA Cancer Vaccines 71

1.2.4 Clinical Study with RNActive® Vaccines in Combination

with Radiotherapy
Based on the promising preclinical results, a phase Ib study is ongoing, evaluating
the clinical potential of the RNActive® vaccine CV9202 in combination with local
radiation of individual metastases as consolidation and maintenance treatment for
patients with stage IV non-small-cell lung cancer. CV9202 is an updated version of
CV9201 that encodes in addition to NY-ESO-1, MAGEC1, MAGEC2, survivin,
and 5T4, the antigen MUC1. As described earlier, the glycoprotein MUC1—
overexpressed and aberrantly glycosylated in various cancers—has previously been
used in immunotherapy approaches against non-small-cell lung cancer (Palmer
et al. 2001). The exploratory, open-label multicenter trial assesses the safety and
tolerability of CV9202 vaccination combined with local radiation in patients with
stage IV NSCLC of different subtypes (squamous and non-squamous cell with or
without EGFR mutations) who achieved a response or stable disease after first-line
therapy (chemotherapy or EGFR tyrosine kinase inhibitors). All patients receive
two initial vaccinations with CV9202 prior to local radiotherapy followed by fur-
ther vaccinations until disease progression. The primary endpoint of the study is the
number of patients experiencing treatment-related adverse events above grade 3.
Secondary endpoints include evaluation of cellular and humoral immune responses
to antigens encoded by CV9202 and antigens not covered by the vaccine allowing
the investigation of potential antigen spreading in response to the treatment. In
addition, assessment of PFS, time to start of second-line cancer treatment, response
to second-line treatment, and overall survival are secondary efficacy endpoints of
the study (Sebastian et al. 2014).

1.2.5 Combination of RNActive® Vaccines with Immune

Checkpoint Inhibitors
The immune response to cancer evolves over many years and ultimately fails due to
immunoediting (downregulation of MHC I and immunogenic antigens) and
immunoevasion (immunosuppressive microenvironment) (Vesely and Schreiber
2013). Moreover, the antitumor immune response has many characteristics of a
chronic immune response, with T cell exhaustion mediated by immune checkpoint
receptors such as programmed cell death 1 (PD1). PD1 is also expressed by B cells,
NK cells, and regulatory T cells in addition to activated T cells and is engaged by
two different ligands PDL1 (B7-H1) and PDL2 (B7-DC). In contrast, the immune
checkpoint receptor cytotoxic T lymphocyte antigen 4 (CTLA4) is only expressed
by activated T cells or regulatory T cells, and binding of its ligands CD80 (B7-1) or
CD86 (B7-2) leads to inhibition of T cells by antagonizing the costimulatory signals
delivered by CD28 during T cell activation (Melero et al. 2015).
Various antibodies targeting these immune checkpoints are currently in clinical
testing and show promising results for some of the patients. But there are still a
substantial proportion of patients not reacting to immune checkpoint blockade.
Newly published studies imply a strong correlation between the occurrence of
antigen-specific T cells and response to immune checkpoint blockade (reviewed in
Schumacher and Schreiber 2015). Hence, the induction of antigen-specific cellular
72 K. Fiedler et al.

response against tumor-associated antigens by vaccination would be beneficial

during immune checkpoint blockade therapy. To test whether checkpoint inhibitors
can also be combined with mRNA vaccines, an RNActive® vaccine was combined
with an anti-CTLA-4 antibody. Mice were challenged with E.G7-OVA tumors and
treated alternatingly with an ovalbumin-encoding mRNA vaccine and an
anti-CTLA-4 antibody. The combination led to significantly reduced tumor growth,
while anti-CTLA-4 treatment alone did not delay tumor growth. In addition, mice in
which treatment had led to complete rejection were rechallenged with the parental
tumor cell line, which lacks OVA expression. These experiments showed that the
mice were nonetheless completely protected, strongly indicative of the induction of
epitope spreading (Fotin-Mleczek et al. 2012).
In summary, the superior antitumor response of RNActive® vaccine treatment in
combination with immune checkpoint blockade could be demonstrated in a pre-
clinical animal model.

1.3 Personalized mRNA Vaccines

In the past, development of cancer vaccines was mainly dedicated toward the
so-called tumor-associated proteins as antigenic source. Mostly, these
tumor-associated antigens can be divided into two classes: Antigens derived from
fetal genes overexpressed in cancer cells (van der Bruggen et al. 1991; Van den
Eynde et al. 1991) or antigens derived from tissue-specific proteins also expressed
in cancer cells (Boon et al. 1994). To offer broadly applicable immune therapies
against cancer, the latest developmental activity in the field of cancer vaccines was
focused on shared tumor-associated antigens that are expressed not only in abun-
dance in different cancer types but also in a certain amount of cancer patients.
But the use of such shared tumor-associated antigens is accompanied with certain
drawbacks: Firstly, T cells easily recognize foreign antigens but in general are
unable to recognize self-antigens due to the negative selection process in the thymus
preventing induction of autoimmunity. Unfortunately, most tumor-associated anti-
gens fall into the group of self-antigens hampering the initiation of a proper immune
response toward these antigens. Secondly, tumor-associated antigens are not soli-
tarily expressed in tumor tissue leading to possible on-target effects in healthy tissue
through the induced immune response. Finally, the expression of tumor-associated
antigens in different tumor tissues or different cancer patients can be highly variable
due to normal biologic heterogeneity. Recent research has shown that ninety-five
percent of the mutations in a given patient seem to be unique to that tumor (Stratton
2011) and that even on a subclonal level a high variability can be found in a certain
tumor (Gerlinger et al. 2015; Martincorena et al. 2015). Therefore, it can be assumed
that expression of shared tumor-associated antigens is subjected to a similar vari-
ability. Moreover, tumor tissues often exploit different escape mechanisms to evade
the antitumoral immune response like downregulation of tumor-associated antigens
or preferential outgrowth of non-expressing clones (Matsushita et al. 2012).
mRNA Cancer Vaccines 73

The use of really tumor-specific antigens or neoantigens instead of tumor-

associated antigens would be advantageous for the development of cancer vaccines
for obvious reasons: Owed to the tumor-specific mutations of these neoantigens, the
specific T cell repertoire is not affected by negative selection in the thymus. In
addition, neoantigen-specific T cells would not cause on-target effects on healthy
tissue as mutated antigens are only expressed in cancer cells. Already in the 1970s
and 1980s, the tumor-rejecting abilities of neoantigen-induced immune responses
were described in transplantable tumor models (Boon and Kellermann 1977; Lur-
quin et al. 1989). Moreover, different studies could show that single mutations in
defined genes are able to induce antigen-specific cytolytic T cell responses (Wölfel
et al. 1995; Coulie et al. 1995; Echchakir et al. 2001).
The advent of next-generation sequencing technologies over the last years allows
nowadays the definition of such tumor-specific antigens or neoantigens by com-
paring genome, exome, or transcriptome data of tumor tissue to healthy tissue for an
individual patient. This approach allows the description of non-synonymous
mutations solely expressed in tumor cells. Moreover, exome or transcriptome
analysis permits the definition of protein abundance for defined non-synonymous
mutations in the tumor tissue. This conglomerate of tumor-specific mutations, also
named “mutanome,” displayed by an individual tumor is a valuable source for
highly tumor-specific antigens.
Two studies in mice provided the first direct evidence that neoantigens identified
by mutanome analysis can be recognized by T cells (Castle et al. 2012; Matsushita
et al. 2012). Briefly, potential MHC-binding peptides were predicted for all
tumor-specific mutations that result in the formation of novel protein sequences and
the most promising mutated peptides were used to query T cell reactivity in vivo.
Castle and colleagues could show that 16 out of 50 mutation-coding peptides elicit
a measurable immune response in immunized mice. Moreover, the induced immune
response conferred a significant antitumor effect in a B16F10 tumor model. In the
study of Matsushita et al., it was demonstrated that one in silico-predicted highly
immunogenic neoantigen derived from a mutant spectrin-β2 is sufficient to induce
tumor cell elimination in an unedited tumor.
Subsequent studies in a clinical setting have added further evidences for the
ability of neoantigens to induce significant antitumoral immune responses and the
predictability of such neoantigens by mutanome mining. Robbins and coworkers
could identify neoantigens recognized by adoptively transferred tumor-reactive T
cells mining exome sequencing data (Robbins et al. 2013), whereas van Rooji and
colleagues discovered neoantigen-specific T cell reactivity in an
ipilimumab-responsive melanoma by tumor exome analysis (van Rooij et al. 2013).
Particularly, the correlation between successful immune checkpoint blockade and
the occurrence of tumor-specific neoantigens has been demonstrated in several
publications over the last years. Gubin and colleagues identified neoantigens fol-
lowing anti-PD1 or anti-CTLA-4 therapy of mice bearing progressively growing
sarcoma. Additionally, they could show that therapeutic synthetic long-peptide
vaccines incorporating these mutant epitopes are able to induce tumor rejection
comparably to immune checkpoint blockade (Gubin et al. 2014). Even more
74 K. Fiedler et al.

interesting are the results of Snyder et al. and Rizvi et al. demonstrating a close
correlation between the abundance of clonal neoantigens and sensitivity to immune
checkpoint blockade by anti-PD1 or anti-CTLA-4 treatment in patients suffering
from non-small-cell lung cancer or melanoma. Moreover, the predicted neoantigens
elicited specific CD8+ T cell responses in both studies (Snyder et al. 2014; Rizvi
et al. 2015). Also for therapeutic approaches with adoptive tumor-infiltrating
lymphocyte transfer in melanoma patients, a similar correlation between neoantigen
load and therapy efficacy was revealed (Lu et al. 2014; Linnemann et al. 2015).
Additionally, Tran and colleagues could provide evidence that immunotherapy
based on mutation-specific CD4+ T cells in a patient with epithelial cancer is
possible (Tran et al. 2014).
The above-mentioned studies and several further publications demonstrated the
feasibility of tumor exome mining plus immunogenicity prediction based on HLA
allotypes and peptide-binding probability to identify patient-specific highly
immunogenic neoantigens in different types of cancer with a variety of approaches
(Rajasagi et al. 2014; Duan et al. 2014; Yadav et al. 2014; Tran et al. 2015).
Moreover, the potential value of such neoantigens for personalized immunotherapy
approaches has been frequently implied (Gubin et al. 2014; Tran et al. 2014). One
possibility to target specifically neoantigens in cancer patients is to engineer
neoantigen-specific T cell receptors and adoptively transfer these T cells (Leisegang
et al. 2016) using methods also described here. A further option would be the
immunization with vaccines incorporating the mutated sequences of neoantigens.
The efficacy of such an immunization approach using peptides is currently under
evaluation in a phase I clinical trial in IDH1R123H-mutated grade III–IV gliomas
(NCT02454634). This clinical study is based on the results published by Schu-
macher and colleagues showing the antitumor potential of mutant IDH1 peptide
vaccination in tumor-bearing MHC-humanized mice (Schumacher et al. 2014).
However, mutant IDH1 seems to be a unique exception, since the mutation can be
found frequently in diffuse grade II and grade III gliomas.
This high penetrance of a neoantigen appears to be in contrast to other tumor
entities. In most human tumors, a large fraction of the mutations is not shared
between patients at a meaningful frequency. Moreover, only a small portion of
mutations within expressed genes have antigenic potential leading to T cell reac-
tivity (Lu et al. 2014; Linnemann et al. 2015). Due to these limitations, broadly
applicable neoantigens that can be used in huge patient cohorts for vaccinations are
highly unlikely. Hence, mRNA vaccines represent a superior approach to satisfy the
specific demands of a personalized immunotherapy based on neoantigens. The
obvious advantages of mRNA vaccines in comparison with peptide vaccines were
already mentioned above. Particularly, the production under well-defined and
controlled conditions by in vitro transcription and the possibility to produce vac-
cines against different neoantigens by a common process and in a relatively short
period of time fulfill the requirements for a personalized immunotherapy approach.
In a recent study of Kreiter and colleagues, the tumor-rejecting properties of
neoantigen-based RNA vaccines were demonstrated in murine tumor models. In
principle, mutanome analysis and MHC-binding prediction lead to selection of
mRNA Cancer Vaccines 75

neoepitopes with proven in vivo immunogenicity for several of the selected

neoepitopes. Additionally, the immunization of tumor-bearing mice with an mRNA
vaccine coding for one B16-specific neoantigen resulted in a significantly increased
survival of immunized mice in comparison with untreated mice. For the clinical
setting, a combination of several neoantigens in one vaccine would be preferable to
address tumor heterogeneity and immune editing which could mediate clinical
failure of vaccines in humans. It was recently demonstrated that immunotherapy of
CT26 lung metastasis after i.v. injection of tumor cells employing mRNA-encoded
pentatope resulted in a drastic reduction of the tumor burden in the lung of vac-
cinated mice (Kreiter et al. 2015).
Based on these convincing results, a first phase I clinical trial was started in 2014
with melanoma patients, where a poly-neoepitope coding RNA vaccine is admin-
istered that targets the unique mutation signature of an individual patient
(NCT02035956). In addition, a second clinical trial was set up in 2015 using a
slightly different approach for the treatment of triple-negative breast cancer
(NCT02316457).
In summary, induction of neoantigen-specific T cell reactivity via mRNA vac-
cines seems to be a promising strategy for successful cancer immunotherapies.
Because of the tumor-restricted expression of the antigens that are being targeted,
these personalized antitumor therapies offer the promise of high specificity and
safety. Additionally, T cell reactivity that can be achieved with such personalized
immunotherapies will further increase the spectrum of human malignancies that
respond to cancer immunotherapy. Particularly, the correlation between immune
checkpoint blockade and neoantigen burden implies synergistic effects by combi-
nation of personalized cancer immunotherapy with checkpoint blockade.

1.4 Cellular Vaccines: mRNA-Pulsed Dendritic Cells

mRNA-transfected dendritic cell (DC) vaccines represent a distinct type of vaccine

strategy involving RNA. Among different types of antigen-presenting cells (APCs),
DCs are considered to be the most potent ones because they can efficiently prime
naïve T cells, thereby stimulating an adaptive immune response. Because of their
ability to induce potent antitumor CTLs both in vitro and in vivo, DCs have been
employed as cancer vaccination platform. The basis of this therapeutic strategy is to
use cancer patient’s DCs expressing tumor-associated antigens (TAAs) in order to
activate antigen-specific T cells which after differentiation in cytotoxic effector T
cells will be able to eradicate tumor cells, irrespective of their location. Moreover,
these T cells can form an immunological memory providing in this way a defense
against recurring cancer cells.
For the delivery of tumor antigens to DCs, mRNA is considered as an attractive
vector as it overcomes some limitations associated with the use of antigen-derived
peptides, the most commonly used strategy of antigen loading (Cerundolo et al.
2004; Schuler et al. 2003; Jager et al. 2002). Synthetic peptides represent only
defined epitopes from known TAAs and are dependent on prior identification and
76 K. Fiedler et al.

on HLA restriction of the patients, meaning that the induced immune response by
the vaccine is limited to the peptides used and that only patients with specific HLA
allotypes can be treated (Van Nuffel et al. 2012). Instead, mRNA molecules can
encode the entire tumor antigen. Therefore, multiple immunogenic epitopes within
the same protein can be presented. In addition, since the protein is endogenously
expressed and presented after mRNA transfection of DCs, multiple peptide–MHC
complexes (pMHC) are generated; thereby, vaccine development is independent on
the patient’s genetic background.
Boczkowski and collaborators were the first ones to describe in the late 1990s
that DCs pulsed with mRNA encoding for tumor antigens are potent antigen-
presenting cells (Boczkowski et al. 1996). Only few years later, an early clinical
trial using DCs loaded with RNA encoding the PSA proved the feasibility and
safety of this approach. CTL responses against metastatic prostate tumors
expressing PSA were indeed induced in some patients (Heiser et al. 2002). This
approach was based on passive pulsing of DCs, which relies on the ability of these
cells to take up mRNA through micropinocytosis (Diken et al. 2011). But this
mechanism, involving transport into the endosomes, entails the risk that only a
fraction of the mRNA can reach the cytosol and can then be translated into the
protein. Therefore, several approaches have been established in order to deliver
mRNA directly to the cytoplasm such as electroporation, nucleofection, lipofection,
and more recently sonoporation (Benteyn et al. 2015). Above all, electroporation
has been shown to be a powerful technique to introduce tumor antigens into DCs
(Van Tendeloo et al. 2001).
mRNA encoding specific TAAs or total tumor RNA can be used to transfect
DCs. Vaccine strategies employing DCs transfected with defined TAA mRNA
avoid the need for growth of patient-specific tumor cells and reduce the risk of
autoimmunity, which can be induced in patients by the presence of normally
expressed self-proteins (Nair et al. 1999; Ponsaerts et al. 2003). However, there are
some limitations due to the fact that for many cancers, the TAAs are unknown. An
attractive alternative is to utilize DCs transfected with patient-derived total tumor
RNA. Through this approach, the entire spectrum of tumor-specific antigens is
displayed, thereby eliminating the need for identification of TAAs and allowing the
immune system to use the most effective antigens while reducing the risk of escape
mutants. Another advantage of using tumor-derived RNA as a source of
whole-tumor antigen is that it can be quickly and easily amplified by RT-PCR from
even a small amount of tumor (Heiser et al. 2002).
To date, several clinical trials have been reported using mRNA-transfected DCs.
The majority of these studies have shown that tumor-specific T cell responses can
be induced by mRNA-transfected DCs in several tumor entities such as brain
cancer, melanoma, lung adenocarcinoma, renal cell carcinoma, and ovarian cancer
(Bonehill et al. 2009; Caruso et al. 2004, 2005; Kyte et al. 2007; Nair et al. 2002;
Su et al. 2003; Morse et al. 2003; Dannull et al. 2005; Van Nuffel et al. 2012). In
particular, in the renal cell carcinoma study by Su et al., patients displayed no
evidence of dose-limiting toxicity or induction of autoimmunity (Su et al. 2003).
mRNA Cancer Vaccines 77

Similarly, brain tumor and neuroblastoma studies conducted in nine and seven
patients, respectively, showed a clinical response in a total of three of the patients.
Despite many progresses in mRNA-DC immunotherapies, clinical responses
remain modest and new strategies on how to enhance the efficacy of mRNA-DC
vaccines are being explored. Besides the delivery of tumor antigens, mRNA can be
used to deliver also proteins that can modulate the function of DCs. Cotransfection
of DCs with mRNA encoding TAAs and costimulatory molecules such as CD83
(Aerts-Toegaerr et al. 2007), OX40 (Dannull et al. 2005), and 4-1BBL (Grünebach
et al. 2005) has shown to improve mRNA-DC vaccine efficacy in preclinical
models. Moreover, DCs activated through electroporation with mRNAs encoding
four tumor antigens as well as mRNAs encoding CD40 ligand and constitutively
active TLR4 and CD70 (TriMix-DCs) resulted in a broad T cell response and
durable tumor response in chemorefractory advanced melanoma patients (Van
Nuffel et al. 2012). DCs have been also modified with mRNAs encoding
immunomodulating cytokines. For example, DCs transfected with mRNAs
encoding IL-12 and TAAs were shown to induce high-avidity cytotoxic T cells and
enhance their effector function. In addition, the migratory capacity of DCs has been
modulated using mRNA encoding a chimeric E/L-selectin, a strategy that led to
increased migration of the DCs to lymph nodes upon intravenous administration
(Dörrie et al. 2008).
Finally, further preclinical strategies have focused on increased DC function by
coadministration of DCs transfected with TAA-encoding mRNAs or mRNAs
encoding for antagonistic anti-CTLA-4 or agonistic anti-GITR antibodies (Pruitt
et al. 2011). Thus, the engineered DCs have the ability not only to present the tumor
antigen of interest but also to locally modulate immune checkpoints and the tumor
microenvironment. Because of promising results in preclinical studies, this
approach is currently under investigation in a phase I clinical trial for the treatment
of melanoma patients (NCT01216436).
Taken together, these strategies have the potential to improve cancer
immunotherapy.

1.5 CAR T Cells

Chimeric antigen receptor (CAR) T cell immunotherapy has recently emerged as a

promising strategy for treating tumors (Cheadle et al. 2012; Restifo et al. 2012;
Kershaw et al. 2013).
CAR T cells are a form of personalized cell therapy using patient-derived T
cells. After the collection of T cells, they are genetically engineered to express
receptors that allow them to recognize a specific antigen expressed by the patient’s
cancer cells and to attack the tumor, once the cells are infused back into the patient.
Chimeric receptors are usually composed of an extracellular TAA-specific
antibody-binding domain fused to intracellular T cell-signaling domains. CAR T
cells can be engineered to target virtually any tumor-associated antigen (Jensen and
Riddell 2015).
78 K. Fiedler et al.

Since CARs are based on TAA-specific antibody-binding domains, recognition

of the tumor antigen is HLA-independent, thereby extending its applicability to
many patients and overcoming some tumor escape mechanisms (Ramos and Dotti
2011).
The adoptive transfer of genetically modified T cells engineered to express a
CAR has shown early promising results in the treatment of hematologic malig-
nancies (Kochenderfer et al. 2012; Brentjens et al. 2013; Porter et al. 2011; Grupp
et al. 2013). However, by now, the use of CAR T cells was less successful for the
therapy of solid cancers, one of the major issues being toxicity. Indeed, clinical
trials have revealed the potential of engineered T cells to recognize and attack
normal cells that share the expression of the tumor CAR-specific antigen, causing
off-target toxicity (Lamers et al. 2006, 2013; Maus et al. 2013).
The main approach to modify the T cells uses viral vectors such as γ-retroviruses
and lentiviruses that are expensive to produce and involve safety concerns asso-
ciated with their integration into the genome (Kershaw et al. 2013).
Recently, mRNA electroporation has been used to engineer T cells with transient
CAR expression. Prof. June and his colleagues at the University of Pennsylvania’s
Perelman School of Medicine were the first to prove the feasibility of this approach
and the potential of mRNA-engineered T cells to induce robust antitumor effects in
preclinical tumor models (Zhao et al. 2010; Barett et al. 2011, 2013).
Consequently, they conducted the first clinical trial to evaluate the feasibility and
safety of repetitive infusions of mRNA CAR T cells in patients (Beatty et al. 2014).
Two patients, one with advanced mesothelioma and the other one having metastatic
pancreatic cancer, were recruited into a phase I clinical trial. Researchers used
mRNA electroporation to engineer patient-derived T cells with a CAR specifically
targeting mesothelin (Tmeso cells), a TAA overexpressed in several cancers. After
ensuring their viability and specificity, engineered T cells were repetitively infused
into the patients. Surprisingly, clinical and laboratory evidence of antitumor activity
without explicit evidence of off-tumor toxicity against normal tissues was shown in
both patients. In addition to the antitumor activity, CAR Tmeso cells were able to
trigger a broad antitumor immune response consistent with epitope spreading.
Moreover, mRNA CAR Tmeso cells were shown to persist transiently within the
peripheral blood after intravenous administration and migrate to tumor tissue.
More recently, mRNA has been used not only to engineer T cells to recognize
specific TAAs but also to transiently deliver a modified telomerase reverse tran-
scriptase (TERT) to CD19 CAR T cells. This study provided an effective and safe
method to extend the T cell replicative life span improving in this way the per-
sistence and antitumor effects of CAR T cell in mouse xenograft tumor models of B
cell malignancies compared with conventional CAR T cells (Bai et al. 2015).
All these findings support the development of mRNA-engineered T cells as a
novel approach for adoptive cell transfer, providing a flexible platform for the
treatment of cancer that may complement the use of retroviral and lentiviral engi-
neered T cells.
mRNA Cancer Vaccines 79

2 Conclusion

The versatility of mRNA in encoding TAAs, patient-derived tumor neoantigens, or

even chimeric antigen receptors makes mRNA vaccines a promising candidate for
use in cancer immunotherapy. The encouraging results of the first clinical studies
have prompted intensive research into strategies to increase the immunogenicity of
the vaccines, which include modifications to the mRNA and the application or the
combination with other treatments like checkpoint inhibitors or radiation therapy.
These efforts will hopefully result in novel treatment options in cancer therapy,
which will make full use of the unique properties of mRNA vaccines.

Acknowledgments We would like to thank Henoch Hong, Ulrike Gnad-Vogt, Ulrich Kruse, and
Mariola Fotin-Mleczek for discussions and suggestions.

References
Aerts-Toegaert C, Heirman C, Tuyaerts S, Corthals J, Aerts JL, Bonehill A, Thielemans K,
Breckpot K (2007) CD83 expression on dendritic cells and T cells: correlation with effective
immune responses. Eur J Immunol 37(3):686–695
Anderson BR, Muramatsu H, Nallagatla SR, Bevilacqua PC, Sansing LH, Weissman D, Karikó K
(2010) Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR
activation. Nucleic Acids Res 38(17):5884–5892
Bai Y, Kan S, Zhou S, Wang Y, Xu J, Cooke JP, Wen J, Deng H (2015) Enhancement of the
in vivo persistence and antitumor efficacy of CD19 chimeric antigen receptor T cells through
the delivery of modified TERT mRNA. Cell Discov 1:Article number: 15040
Barrett DM, Zhao Y, Liu X, Jiang S, Carpenito C, Kalos M, Carroll RG, June CH, Grupp SA
(2011) Treatment of advanced leukemia in mice with mRNA engineered T cells. Hum Gene
Ther 22(12):1575–1586
Barrett DM, Liu X, Jiang S, June CH, Grupp SA, Zhao Y (2013) Regimen-specific effects of
RNA-modified chimeric antigen receptor T cells in mice with advanced leukemia. Hum Gene
Ther 24(8):717–727
Beatty GL, Haas AR, Maus MV, Torigian DA, Soulen MC, Plesa G, Chew A, Zhao Y, Levine BL,
Albelda SM, Kalos M, June CH (2014) Mesothelin-specific chimeric antigen receptor
mRNA-engineered T cells induce anti-tumor activity in solid malignancies. Cancer Immunol
Res 2(2):112–120
Benteyn D, Heirman C, Bonehill A, Thielemans K, Breckpot K (2015) mRNA-based dendritic cell
vaccines. Expert Rev Vaccines 14(2):161–176
Boczkowski D, Nair SK, Snyder D, Gilboa E (1996) Dendritic cells pulsed with RNA are potent
antigen-presenting cells in vitro and in vivo. J Exp Med 184(2):465–472
Bonehill A, Van Nuffel AM, Corthals J, Tuyaerts S, Heirman C, François V, Colau D, van der
Bruggen P, Neyns B, Thielemans K (2009) Single-step antigen loading and activation of
dendritic cells by mRNA electroporation for the purpose of therapeutic vaccination in
melanoma patients. Clin Cancer Res 15(10):3366–3375
Boon T, Kellermann O (1977) Rejection by syngeneic mice of cell variants obtained by
mutagenesis of a malignant teratocarcinoma cell line. Proc Natl Acad Sci USA 74(1):272–275
Boon T, Cerottini JC, Van den Eynde B, van der Bruggen P, Van Pel A (1994) Tumor antigens
recognized by T lymphocytes. Annu Rev Immunol 12:337–365
80 K. Fiedler et al.

Brentjens RJ, Davila ML, Riviere I, Park J, Wang X, Cowell LG, Bartido S, Stefanski J, Taylor C,
Olszewska M, Borquez-Ojeda O, Qu J, Wasielewska T, He Q, Bernal Y, Rijo IV, Hedvat C,
Kobos R, Curran K, Steinherz P, Jurcic J, Rosenblat T, Maslak P, Frattini M, Sadelain M
(2013) CD19-targeted T cells rapidly induce molecular remissions in adults with
chemotherapy-refractory acute lymphoblastic leukemia. Sci Transl Med 5(177):177ra38
Caruso DA, Orme LM, Neale AM, Radcliff FJ, Amor GM, Maixner W, Downie P, Hassall TE,
Tang ML, Ashley DM (2004) Results of a phase 1 study utilizing monocyte-derived dendritic
cells pulsed with tumor RNA in children and young adults with brain cancer. Neuro Oncol. 6
(3):236–246
Caruso DA, Orme LM, Amor GM, Neale AM, Radcliff FJ, Downie P, Tang ML, Ashley DM
(2005) Results of a Phase I study utilizing monocyte-derived dendritic cells pulsed with tumor
RNA in children with Stage 4 neuroblastoma. Cancer 103(6):1280–1291
Castle JC, Kreiter S, Diekmann J, Löwer M, van de Roemer N, de Graaf J, Selmi A, Diken M,
Boegel S, Paret C, Koslowski M, Kuhn AN, Britten CM, Huber C, Türeci O, Sahin U (2012)
Exploiting the mutanome for tumor vaccination. Cancer Res 72(5):1081–1091
Cerundolo V, Hermans IF, Salio M (2004) Dendritic cells: a journey from laboratory to clinic. Nat
Immunol 5(1):7–10
Cheadle EJ, Sheard V, Hombach AA, Chmielewski M, Riet T, Berrevoets C, Schooten E,
Lamers C, Abken H, Debets R, Gilham DE (2012) Chimeric antigen receptors for T-cell based
therapy. Methods Mol Biol 907:645–666
Coulie PG, Lehmann F, Lethé B, Herman J, Lurquin C, Andrawiss M, Boon T (1995) A mutated
intron sequence codes for an antigenic peptide recognized by cytolytic T lymphocytes on a
human melanoma. Proc Natl Acad Sci U S A. 92(17):7976–7980
Dannull J, Nair S, Su Z, Boczkowski D, DeBeck C, Yang B, Gilboa E, Vieweg J (2005)
Enhancing the immunostimulatory function of dendritic cells by transfection with mRNA
encoding OX40 ligand. Blood 105(8):3206–3213
Demaria S, Formenti SC (2012) Role of T lymphocytes in tumor response to radiotherapy. Front
Oncol 2:95
Diken M, Kreiter S, Selmi A, Britten CM, Huber C, Türeci Ö, Sahin U (2011) Selective uptake of
naked vaccine RNA by dendritic cells is driven by macropinocytosis and abrogated upon DC
maturation. Gene Ther 18(7):702–708
Dörrie J, Schaft N, Müller I, Wellner V, Schunder T, Hänig J, Oostingh GJ, Schön MP, Robert C,
Kämpgen E, Schuler G (2008) Introduction of functional chimeric E/L-selectin by RNA
electroporation to target dendritic cells from blood to lymph nodes. Cancer Immunol
Immunother 57(4):467–477
Duan F, Duitama J, Al Seesi S, Ayres CM, Corcelli SA, Pawashe AP, Blanchard T, McMahon D,
Sidney J, Sette A, Baker BM, Mandoiu II, Srivastava PK (2014) Genomic and bioinformatic
profiling of mutational neoepitopes reveals new rules to predict anticancer immunogenicity.
J Exp Med 211(11):2231–2248
Echchakir H, Mami-Chouaib F, Vergnon I, Baurain JF, Karanikas V, Chouaib S, Coulie PG (2001)
A point mutation in the alpha-actinin-4 gene generates an antigenic peptide recognized by
autologous cytolytic T lymphocytes on a human lung carcinoma. Cancer Res 61(10):4078–4083
Formenti SC, Demaria S (2013) Combining radiotherapy and cancer immunotherapy: a paradigm
shift. J Natl Cancer Inst 105(4):256–265
Fotin-Mleczek M, Duchardt KM, Lorenz C, Pfeiffer R, Ojkić-Zrna S, Probst J, Kallen KJ (2011)
Messenger RNA-based vaccines with dual activity induce balanced TLR-7 dependent adaptive
immune responses and provide antitumor activity. J Immunother 34(1):1–15
Fotin-Mleczek M, Zanzinger K, Heidenreich R, Lorenz C, Thess A, Duchardt KM, Kallen KJ
(2012) Highly potent mRNA based cancer vaccines represent an attractive platform for
combination therapies supporting an improved therapeutic effect. J Gene Med. 14(6):428–439
Fotin-Mleczek M, Zanzinger K, Heidenreich R, Lorenz C, Kowalczyk A, Kallen KJ, Huber SM
(2014) mRNA-based vaccines synergize with radiation therapy to eradicate established tumors.
Radiat Oncol 9:180
mRNA Cancer Vaccines 81

Garnett CT, Schlom J, Hodge JW (2008) Combination of docetaxel and recombinant vaccine
enhances T-cell responses and antitumor activity: effects of docetaxel on immune enhance-
ment. Clin Cancer Res 14(11):3536–3544
Gerlinger M, Catto JW, Orntoft TF, Real FX, Zwarthoff EC, Swanton C (2015) Intratumour
heterogeneity in urologic cancers: from molecular evidence to clinical implications. Eur Urol
67(4):729–737
Golden EB, Frances D, Pellicciotta I, Demaria S, Helen Barcellos-Hoff M, Formenti SC (2014)
Radiation fosters dose-dependent and chemotherapy-induced immunogenic cell death.
Oncoimmunology 3:e28518
Grünebach F, Kayser K, Weck MM, Müller MR, Appel S, Brossart P (2005) Cotransfection of
dendritic cells with RNA coding for HER-2/neu and 4-1BBL increases the induction of tumor
antigen specific cytotoxic T lymphocytes. Cancer Gene Ther 12(9):749–756
Grupp SA, Kalos M, Barrett D, Aplenc R, Porter DL, Rheingold SR, Teachey DT, Chew A,
Hauck B, Wright JF, Milone MC, Levine BL, June CH (2013) Chimeric antigen
receptor-modified T cells for acute lymphoid leukemia. N Engl J Med 368(16):1509–1518
Gubin MM, Zhang X, Schuster H, Caron E, Ward JP, Noguchi T, Ivanova Y, Hundal J,
Arthur CD, Krebber WJ, Mulder GE, Toebes M, Vesely MD, Lam SS, Korman AJ, Allison JP,
Freeman GJ, Sharpe AH, Pearce EL, Schumacher TN, Aebersold R, Rammensee HG,
Melief CJ, Mardis ER, Gillanders WE, Artyomov MN, Schreiber RD (2014) Checkpoint
blockade cancer immunotherapy targets tumour-specific mutant antigens. Nature 515
(7528):577–581
Heiser A, Coleman D, Dannull J, Yancey D, Maurice MA, Lallas CD, Dahm P, Niedzwiecki D,
Gilboa E, Vieweg J (2002) Autologous dendritic cells transfected with prostate-specific antigen
RNA stimulate CTL responses against metastatic prostate tumors. J Clin Invest. 109
(3):409–417
Hoerr I, Obst R, Rammensee HG, Jung G (2000) In vivo application of RNA leads to induction of
specific cytotoxic T lymphocytes and antibodies. Eur J Immunol 30(1):1–7
Jäger E, Jäger D, Knuth A (2002) Clinical cancer vaccine trials. Curr Opin Immunol 14
(2):178–182
Jensen MC, Riddell SR (2015) Designing chimeric antigen receptors to effectively and safely
target tumors. Curr Opin Immunol 33:9–15
Kallen KJ, Theß A (2014) A development that may evolve into a revolution in medicine: mRNA
as the basis for novel, nucleotide-based vaccines and drugs. Ther Adv Vaccines. 2(1):10–31
Kallen KJ, Heidenreich R, Schnee M, Petsch B, Schlake T, Thess A, Baumhof P, Scheel B,
Koch SD, Fotin-Mleczek M (2013) A novel, disruptive vaccination technology:
self-adjuvanted RNActive(®) vaccines. Hum Vaccin Immunother. 9(10):2263–2276
Kantoff PW, Higano CS, Shore ND, Berger ER, Small EJ, Penson DF, Redfern CH, Ferrari AC,
Dreicer R, Sims RB, Xu Y, Frohlich MW, Schellhammer PF; IMPACT Study Investigators
(2010) Sipuleucel-T immunotherapy for castration-resistant prostate cancer. N Engl J Med. 363
(5):411–22
Karikó K, Muramatsu H, Welsh FA, Ludwig J, Kato H, Akira S, Weissman D (2008)
Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with
increased translational capacity and biological stability. Mol Ther 16(11):1833–1840
Kershaw MH, Westwood JA, Darcy PK (2013) Gene-engineered T cells for cancer therapy. Nat
Rev Cancer 13(8):525–541
Knapp DC, Mata JE, Reddy MT, Devi GR, Iversen PL (2003) Resistance to chemotherapeutic
drugs overcome by c-Myc inhibition in a Lewis lung carcinoma murine model. Anticancer
Drugs 14:39–47
Kochenderfer JN, Dudley ME, Feldman SA, Wilson WH, Spaner DE, Maric I, Stetler-Stevenson
M, Phan GQ, Hughes MS, Sherry RM, Yang JC, Kammula US, Devillier L, Carpenter R,
Nathan DA, Morgan RA, Laurencot C, Rosenberg SA (2012) B-cell depletion and remissions
of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19
chimeric-antigen-receptor-transduced T cells. Blood 119(12):2709–2720
82 K. Fiedler et al.

Kowalczyk A, Doener F, Zanzinger K, Noth J, Baumhof P, Fotin-Mleczek M, Heidenreich R

(2016) Self-adjuvanted mRNA vaccines induce local innate immune responses that lead to a
potent and boostable adaptive immunity. Vaccine 34:3882–3893
Kreiter S, Vormehr M, van de Roemer N, Diken M, Löwer M, Diekmann J, Boegel S, Schrörs B,
Vascotto F, Castle JC, Tadmor AD, Schoenberger SP, Huber C, Türeci Ö, Sahin U (2015)
Mutant MHC class II epitopes drive therapeutic immune responses to cancer. Nature 520
(7549):692–696
Kübler H, Scheel B, Gnad-Vogt U, Miller K, Schultze-Seemann W, Vom Dorp F, Parmiani G,
Hampel C, Wedel S, Trojan L, Jocham D, Maurer T, Rippin G, Fotin-Mleczek M, von der
Mülbe F, Probst J, Hoerr I, Kallen KJ, Lander T, Stenzl A (2015) Self-adjuvanted mRNA
vaccination in advanced prostate cancer patients: a first-in-man phase I/IIa study. J Immunother
Cancer 3:26
Kyte JA, Kvalheim G, Lislerud K, thor Straten P, Dueland S, Aamdal S, Gaudernack G (2007) T
cell responses in melanoma patients after vaccination with tumor-mRNA transfected dendritic
cells. Cancer Immunol Immunother 56(5):659–675
Lamers CH, Sleijfer S, Vulto AG, Kruit WH, Kliffen M, Debets R, Gratama JW, Stoter G,
Oosterwijk E (2006) Treatment of metastatic renal cell carcinoma with autologous
T-lymphocytes genetically retargeted against carbonic anhydrase IX: first clinical experience.
J Clin Oncol 24(13):e20–e22
Lamers CH, Sleijfer S, van Steenbergen S, van Elzakker P, van Krimpen B, Groot C, Vulto A, den
Bakker M, Oosterwijk E, Debets R, Gratama JW (2013) Treatment of metastatic renal cell
carcinoma with CAIX CAR-engineered T cells: clinical evaluation and management of
on-target toxicity. Mol Ther 21(4):904–912
Leisegang M, Kammertoens T, Uckert W, Blankenstein T (2016) Targeting human melanoma
neoantigens by T cell receptor gene therapy. J Clin Invest 126(3):854–858
Linnemann C, van Buuren MM, Bies L, Verdegaal EM, Schotte R, Calis JJ, Behjati S, Velds A,
Hilkmann H, Atmioui DE, Visser M, Stratton MR, Haanen JB, Spits H, van der Burg SH,
Schumacher TN (2015) High-throughput epitope discovery reveals frequent recognition of
neo-antigens by CD4+ T cells in human melanoma. Nat Med 21(1):81–85
Lu YC, Yao X, Crystal JS, Li YF, El-Gamil M, Gross C, Davis L, Dudley ME, Yang JC,
Samuels Y, Rosenberg SA, Robbins PF (2014) Efficient identification of mutated cancer
antigens recognized by T cells associated with durable tumor regressions. Clin Cancer Res 20
(13):3401–3410
Lurquin C, Van Pel A, Mariamé B, De Plaen E, Szikora JP, Janssens C, Reddehase MJ, Lejeune J,
Boon T (1989) Structure of the gene of tum- transplantation antigen P91A: the mutated exon
encodes a peptide recognized with Ld by cytolytic T cells. Cell 58(2):293–303
Mandelboim O, Vadai E, Fridkin M, Katz-Hillel A, Feldman M, Berke G, Eisenbach L (1995)
Regression of established murine carcinoma metastases following vaccination with
tumour-associated antigen peptides. Nat Med 1(11):1179–1183
Martincorena I, Roshan A, Gerstung M, Ellis P, Van Loo P, McLaren S, Wedge DC, Fullam A,
Alexandrov LB, Tubio JM, Stebbings L, Menzies A, Widaa S, Stratton MR, Jones PH,
Campbell PJ (2015) High burden and pervasive positive selection of somatic mutations in
normal human skin. Science 348(6237):880–886 Tumor evolution
Martinon F, Krishnan S, Lenzen G, Magné R, Gomard E, Guillet JG, Lévy JP, Meulien P (1993)
Induction of virus-specific cytotoxic T lymphocytes in vivo by liposome-entrapped mRNA.
Eur J Immunol 23(7):1719–1722
Matsushita H, Vesely MD, Koboldt DC, Rickert CG, Uppaluri R, Magrini VJ, Arthur CD,
White JM, Chen YS, Shea LK, Hundal J, Wendl MC, Demeter R, Wylie T, Allison JP,
Smyth MJ, Old LJ, Mardis ER, Schreiber RD (2012) Cancer exome analysis reveals a
T-cell-dependent mechanism of cancer immunoediting. Nature 482(7385):400–404
Maus MV, Haas AR, Beatty GL, Albelda SM, Levine BL, Liu X, Zhao Y, Kalos M, June CH
(2013) T cells expressing chimeric antigen receptors can cause anaphylaxis in humans. Cancer
Immunol Res 1(1):26–31
mRNA Cancer Vaccines 83

Melero I, Berman DM, Aznar MA, Korman AJ, Pérez Gracia JL, Haanen J (2015) Evolving
synergistic combinations of targeted immunotherapies to combat cancer. Nat Rev Cancer 15
(8):457–472
Moore MW, Carbone FR, Bevan MJ (1988) Introduction of soluble protein into the class I
pathway of antigen processing and presentation. Cell 54(6):777–785
Morse MA, Nair SK, Mosca PJ, Hobeika AC, Clay TM, Deng Y, Boczkowski D, Proia A,
Neidzwiecki D, Clavien PA, Hurwitz HI, Schlom J, Gilboa E, Lyerly HK (2003)
Immunotherapy with autologous, human dendritic cells transfected with carcinoembryonic
antigen mRNA. Cancer Invest 21(3):341–349
Nair SK, Hull S, Coleman D, Gilboa E, Lyerly HK, Morse MA (1999) Induction of
carcinoembryonic antigen (CEA)-specific cytotoxic T-lymphocyte responses in vitro using
autologous dendritic cells loaded with CEA peptide or CEA RNA in patients with metastatic
malignancies expressing CEA. Int J Cancer 82(1):121–124
Nair SK, Morse M, Boczkowski D, Cumming RI, Vasovic L, Gilboa E, Lyerly HK (2002)
Induction of tumor-specific cytotoxic T lymphocytes in cancer patients by autologous tumor
RNA-transfected dendritic cells. Ann Surg 235(4):540–549
Palmer M, Parker J, Modi S, Butts C, Smylie M, Meikle A, Kehoe M, MacLean G,
Longenecker M (2001) Phase I study of the BLP25 (MUC1 peptide) liposomal vaccine for
active specific immunotherapy in stage IIIB/IV non-small-cell lung cancer. Clin Lung Cancer 3
(1):49–57 discussion 58
Petsch B, Schnee M, Vogel AB, Lange E, Hoffmann B, Voss D, Schlake T, Thess A, Kallen KJ,
Stitz L, Kramps T (2012) Protective efficacy of in vitro synthesized, specific mRNA vaccines
against influenza A virus infection. Nat Biotechnol 30(12):1210–1216
Ponsaerts P, Van Tendeloo VF, Berneman ZN (2003) Cancer immunotherapy using RNA-loaded
dendritic cells. Clin Exp Immunol 134(3):378–384
Porter DL, Levine BL, Kalos M, Bagg A, June CH (2011) Chimeric antigen receptor-modified T
cells in chronic lymphoid leukemia. N Engl J Med 365(8):725–733
Pruitt SK, Boczkowski D, de Rosa N, Haley NR, Morse MA, Tyler DS, Dannull J, Nair S (2011)
Enhancement of anti-tumor immunity through local modulation of CTLA-4 and GITR by
dendritic cells. Eur J Immunol 41(12):3553–3563
Rajasagi M, Shukla SA, Fritsch EF, Keskin DB, DeLuca D, Carmona E, Zhang W, Sougnez C,
Cibulskis K, Sidney J, Stevenson K, Ritz J, Neuberg D, Brusic V, Gabriel S, Lander ES,
Getz G, Hacohen N, Wu CJ (2014) Systematic identification of personal tumor-specific
neoantigens in chronic lymphocytic leukemia. Blood 124(3):453–462
Rammensee HG (2006) Some considerations on the use of peptides and mRNA for therapeutic
vaccination against cancer. Immunol Cell Biol 84(3):290–294
Ramos CA, Dotti G (2011) Chimeric antigen receptor (CAR)-engineered lymphocytes for cancer
therapy. Expert Opin Biol Ther 11(7):855–873
Reits EA, Hodge JW, Herberts CA, Groothuis TA, Chakraborty M, Wansley EK, Camphausen K,
Luiten RM, de Ru AH, Neijssen J, Griekspoor A, Mesman E, Verreck FA, Spits H, Schlom J, van
Veelen P, Neefjes JJ (2006) Radiation modulates the peptide repertoire, enhances MHC class I
expression, and induces successful antitumor immunotherapy. J Exp Med 203(5):1259–1271
Restifo NP, Dudley ME, Rosenberg SA (2012) Adoptive immunotherapy for cancer: harnessing
the T cell response. Nat Rev Immunol 12(4):269–281
Rizvi NA, Hellmann MD, Snyder A, Kvistborg P, Makarov V, Havel JJ, Lee W, Yuan J, Wong P,
Ho TS, Miller ML, Rekhtman N, Moreira AL, Ibrahim F, Bruggeman C, Gasmi B,
Zappasodi R, Maeda Y, Sander C, Garon EB, Merghoub T, Wolchok JD, Schumacher TN,
Chan TA (2015) Cancer immunology. Mutational landscape determines sensitivity to PD-1
blockade in non-small cell lung cancer. Science 348(6230):124–128
Robbins PF, Lu YC, El-Gamil M, Li YF, Gross C, Gartner J, Lin JC, Teer JK, Cliften P,
Tycksen E, Samuels Y, Rosenberg SA (2013) Mining exomic sequencing data to identify
mutated antigens recognized by adoptively transferred tumor-reactive T cells. Nat Med 19
(6):747–752
84 K. Fiedler et al.

Savai R, Schermuly RT, Pullamsetti SS, Schneider M, Greschus S, Ghofrani HA, Traupe H,
Grimminger F, Banat GA (2007) A combination hybrid-based vaccination/adoptive cellular
therapy to prevent tumor growth by involvement of T cells. Cancer Res 67(11):5443–5453
Scheel B, Teufel R, Probst J, Carralot JP, Geginat J, Radsak M, Jarrossay D, Wagner H, Jung G,
Rammensee HG, Hoerr I, Pascolo S (2005) Toll-like receptor-dependent activation of several
human blood cell types by protamine-condensed mRNA. Eur J Immunol 35(5):1557–1566
Schnee M, Vogel AB, Voss D, Petsch B, Baumhof P, Kramps T, Stitz L (2016) An mRNA vaccine
encoding rabies virus glycoprotein induces protection against lethal infection in mice and
correlates of protection in adult and newborn Pigs. PLoS Negl Trop Dis 10(6)
Schuler G, Schuler-Thurner B, Steinman RM (2003) The use of dendritic cells in cancer
immunotherapy. Curr Opin Immunol 15(2):138–147
Schumacher TN, Schreiber RD (2015) Neoantigens in cancer immunotherapy. Science 348
(6230):69–74
Schumacher T, Bunse L, Pusch S, Sahm F, Wiestler B, Quandt J, Menn O, Osswald M, Oezen I,
Ott M, Keil M, Balß J, Rauschenbach K, Grabowska AK, Vogler I, Diekmann J, Trautwein N,
Eichmüller SB, Okun J, Stevanović S, Riemer AB, Sahin U, Friese MA, Beckhove P, von
Deimling A, Wick W, Platten M (2014) A vaccine targeting mutant IDH1 induces antitumour
immunity. Nature 512(7514):324–327
Sebastian M, Papachristofilou A, Weiss C, Früh M, Cathomas R, Hilbe W, Wehler T, Rippin G,
Koch SD, Scheel B, Fotin-Mleczek M, Heidenreich R, Kallen KJ, Gnad-Vogt U, Zippelius A
(2014) Phase Ib study evaluating a self-adjuvanted mRNA cancer vaccine (RNActive®)
combined with local radiation as consolidation and maintenance treatment for patients with
stage IV non-small cell lung cancer. BMC Cancer 14:748
Shojaei F, Wu X, Malik AK, Zhong C, Baldwin ME, Schanz S, Fuh G, Gerber H-P, Ferrara N
(2007) Tumor refractoriness to anti-VEGF treatment is mediated by CD11b+ Gr1+ myeloid
cells. Nat Biotechnol 25:911–920
Snyder A, Makarov V, Merghoub T, Yuan J, Zaretsky JM, Desrichard A, Walsh LA, Postow MA,
Wong P, Ho TS, Hollmann TJ, Bruggeman C, Kannan K, Li Y, Elipenahli C, Liu C,
Harbison CT, Wang L, Ribas A, Wolchok JD, Chan TA (2014) Genetic basis for clinical
response to CTLA-4 blockade in melanoma. N Engl J Med 371(23):2189–2199
Stratton MR (2011) Exploring the genomes of cancer cells: progress and promise. Science 331
(6024):1553–1558
Su Z, Dannull J, Heiser A, Yancey D, Pruitt S, Madden J, Coleman D, Niedzwiecki D, Gilboa E,
Vieweg J (2003) Immunological and clinical responses in metastatic renal cancer patients
vaccinated with tumor RNA-transfected dendritic cells. Cancer Res 63(9):2127–2133
Tran E, Turcotte S, Gros A, Robbins PF, Lu YC, Dudley ME, Wunderlich JR, Somerville RP,
Hogan K, Hinrichs CS, Parkhurst MR, Yang JC, Rosenberg SA (2014) Cancer immunotherapy
based on mutation-specific CD4+ T cells in a patient with epithelial cancer. Science 344
(6184):641–645
Tran E, Ahmadzadeh M, Lu YC, Gros A, Turcotte S, Robbins PF, Gartner JJ, Zheng Z, Li YF,
Ray S, Wunderlich JR, Somerville RP, Rosenberg SA (2015) Immunogenicity of somatic
mutations in human gastrointestinal cancers. Science 350(6266):1387–1390
Van den Eynde B, Lethé B, Van Pel A, De Plaen E, Boon T (1991) The gene coding for a major
tumor rejection antigen of tumor P815 is identical to the normal gene of syngeneic DBA/2
mice. J Exp Med 173(6):1373–1384
van der Bruggen P, Traversari C, Chomez P, Lurquin C, De Plaen E, Van den Eynde B, Knuth A,
Boon T (1991) A gene encoding an antigen recognized by cytolytic T lymphocytes on a human
melanoma. Science 254(5038):1643–1647
Van Nuffel AM, Wilgenhof S, Thielemans K, Bonehill A (2012) Overcoming HLA restriction in
clinical trials: Immune monitoring of mRNA-loaded DC therapy. Oncoimmunology 1
(8):1392–1394
van Rooij N, van Buuren MM, Philips D, Velds A, Toebes M, Heemskerk B, van Dijk LJ,
Behjati S, Hilkmann H, El Atmioui D, Nieuwland M, Stratton MR, Kerkhoven RM, Kesmir C,
mRNA Cancer Vaccines 85

Haanen JB, Kvistborg P, Schumacher TN (2013) Tumor exome analysis reveals

neoantigen-specific T-cell reactivity in an ipilimumab-responsive melanoma. J Clin Oncol
31(32):e439–e442
Van Tendeloo VF, Ponsaerts P, Lardon F, Nijs G, Lenjou M, Van Broeckhoven C, Van
Bockstaele DR, Berneman ZN (2001) Highly efficient gene delivery by mRNA electroporation
in human hematopoietic cells: superiority to lipofection and passive pulsing of mRNA and to
electroporation of plasmid cDNA for tumor antigen loading of dendritic cells. Blood 98
(1):49–56
Vesely MD, Schreiber RD (2013) Cancer immunoediting: antigens, mechanisms, and implications
to cancer immunotherapy. Ann NY Acad Sci 1284:1–5
Wölfel T, Hauer M, Schneider J, Serrano M, Wölfel C, Klehmann-Hieb E, De Plaen E, Hankeln T,
Meyer zum Büschenfelde KH, Beach D (1995) A p16INK4a-insensitive CDK4 mutant
targeted by cytolytic T lymphocytes in a human melanoma. Science 269(5228):1281–1284
Wolff JA, Malone RW, Williams P, Chong W, Acsadi G, Jani A, Felgner PL (1990) Direct gene
transfer into mouse muscle in vivo. Science 247(4949 Pt 1):1465–1468
Yadav M, Jhunjhunwala S, Phung QT, Lupardus P, Tanguay J, Bumbaca S, Franci C, Cheung TK,
Fritsche J, Weinschenk T, Modrusan Z, Mellman I, Lill JR, Delamarre L (2014) Predicting
immunogenic tumour mutations by combining mass spectrometry and exome sequencing.
Nature 515(7528):572–576
Zhao Y, Moon E, Carpenito C, Paulos CM, Liu X, Brennan AL, Chew A, Carroll RG, Scholler J,
Levine BL, Albelda SM, June CH (2010) Multiple injections of electroporated autologous T
cells expressing a chimeric antigen receptor mediate regression of human disseminated tumor.
Cancer Res 70(22):9053–9061