viruses
Review
Virotherapy in Germany—Recent Activities in Virus
Engineering, Preclinical Development, and Clinical Studies
Dirk M. Nettelbeck 1,*,†, Mathias F. Leber 1,2,† , Jennifer Altomonte 3, Assia Angelova 1, Julia Beil 4,5 ,
Susanne Berchtold 4, Maike Delic 6, Jürgen Eberle 7, Anja Ehrhardt 8, Christine E. Engeland 1,2,8 ,
Henry Fechner 9, Karsten Geletneky 10, Katrin Goepfert 6, Per Sonne Holm 11,‡, Stefan Kochanek 12,
Florian Kreppel 13, Lea Krutzke 12, Florian Kühnel 14, Karl Sebastian Lang 15, Antonio Marchini 16,17 ,
Markus Moehler 6, Michael D. Mühlebach 18, Ulrike Naumann 19 , Roman Nawroth 11, Jürg Nüesch 20 ,
Jean Rommelaere 1, Ulrich M. Lauer 4,5 and Guy Ungerechts 1,2,21,*
Citation: Nettelbeck, D.M.; Leber,
M.F.; Altomonte, J.; Angelova, A.;
Beil, J.; Berchtold, S.; Delic, M.; Eberle,
J.; Ehrhardt, A.; Engeland, C.E.; et al.
Virotherapy in Germany—Recent
Activities in Virus Engineering,
Preclinical Development, and Clinical
Studies. Viruses 2021,13, 1420.
https://doi.org/10.3390/v13081420
Academic Editors: Manfred Schmidt
and Irene Gil-Farina
Received: 2 June 2021
Accepted: 16 July 2021
Published: 21 July 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1Clinical Cooperation Unit Virotherapy, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280,
2Department of Medical Oncology, National Center for Tumor Diseases and Heidelberg University Hospital,
Im Neuenheimer Feld 460, 69120 Heidelberg, Germany
3
Department of Internal Medicine II, Klinikum rechts der Isar, Technical University of Munich, Ismaningerstr.
4
Virotherapy Center Tübingen (VCT), Department of Medical Oncology and Pneumology, Medical University
5German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Partner Site Tübingen,
Otfried-Müller-Str. 10, 72076 Tübingen, Germany
6Department of Internal Medicine, University Medical Center, Johannes Gutenberg University Mainz,
7Department of Dermatology, Venereology and Allergology, Skin Cancer Centre Charité,
Charité—Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany; [email protected]
8Virology and Microbiology, Center for Biomedical Research and Education (ZBAF), Faculty of Health,
9Department of Applied Biochemistry, Institute of Biotechnology, Technical University of Berlin,
10 Department of Neurosurgery, Klinikum Darmstadt, Grafenstraße 9, 64283 Darmstadt, Germany;
11 Department of Urology, Rechts der Isar Medical Center, Technical University of Munich, Ismaninger Str. 22,
12 Department of Gene Therapy, Ulm University, Helmholtzstraße 8/1, 89081 Ulm, Germany;
13 Chair of Biochemistry and Molecular Medicine, Center for Biomedical Research and Education (ZBAF),
Faculty of Health, University Witten/Herdecke (UW/H), Stockumer Str 10, 58453 Witten, Germany;
14 Department of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School (MHH),
Carl-Neuberg-Str.1, 30625 Hannover, Germany; Kuehnel.Florian@mh-hannover.de
15 Institute of Immunology, Medical Faculty, University of Duisburg-Essen, Hufelandstrasse 55,
16
Laboratory of Oncolytic Virus Immuno-Therapeutics (LOVIT), German Cancer Research Center (DKFZ), Im
17
Laboratory of Oncolytic Virus Immuno-Therapeutics (LOVIT), Luxembourg Institute of Health, 84 Val Fleuri,
L-1526 Luxembourg, Luxembourg
18 Division of Veterinary Medicine, Paul-Ehrlich-Institut, Paul-Ehrlich-Str. 51-59, 63225 Langen, Germany;
19 Hertie Institute for Clinical Brain Research and Center Neurology, Molecular Neurooncology, University of
20 Division of Virus-Associated Carcinogenesis, German Cancer Research Center, Im Neuenheimer Feld 242,
21 Cancer Therapeutics Program, Ottawa Hospital Research Institute, 501 Smyth Road,
Ottawa, ON K1H 8L6, Canada
*Correspondence: [email protected] (D.M.N.); guy.ungerechts@nct-heidelberg.de (G.U.)
Viruses 2021,13, 1420. https://doi.org/10.3390/v13081420 https://www.mdpi.com/journal/viruses
Viruses 2021,13, 1420 2 of 29
† These authors contributed equally to this work.
‡ Presently at the Department of Oral and Maxillofacial Surgery, Medical University Innsbruck,
6020 Innsbruck, Austria.
Abstract:
Virotherapy research involves the development, exploration, and application of oncolytic
viruses that combine direct killing of cancer cells by viral infection, replication, and spread (oncolysis)
with indirect killing by induction of anti-tumor immune responses. Oncolytic viruses can also be
engineered to genetically deliver therapeutic proteins for direct or indirect cancer cell killing. In this
review—as part of the special edition on “State-of-the-Art Viral Vector Gene Therapy in Germany”—
the German community of virotherapists provides an overview of their recent research activities that
cover endeavors from screening and engineering viruses as oncolytic cancer therapeutics to their
clinical translation in investigator-initiated and sponsored multi-center trials. Preclinical research
explores multiple viral platforms, including new isolates, serotypes, or fitness mutants, and pursues
unique approaches to engineer them towards increased safety, shielded or targeted delivery, selective
or enhanced replication, improved immune activation, delivery of therapeutic proteins or RNA,
and redirecting antiviral immunity for cancer cell killing. Moreover, several oncolytic virus-based
combination therapies are under investigation. Clinical trials in Germany explore the safety and
potency of virotherapeutics based on parvo-, vaccinia, herpes, measles, reo-, adeno-, vesicular
stomatitis, and coxsackie viruses, including viruses encoding therapeutic proteins or combinations
with immune checkpoint inhibitors. These research advances represent exciting vantage points for
future endeavors of the German virotherapy community collectively aimed at the implementation of
effective virotherapeutics in clinical oncology.
Keywords:
oncolytic virus; virotherapy; research in Germany; virus engineering; virus targeting;
therapeutic transgene; immunotherapy; combination therapy; clinical trials
1. Introduction
Virotherapy research covers a continuum of activities, ranging from endeavors for
screening and engineering viruses as oncolytic cancer therapeutics, using cutting edge tech-
nology to concerted programs for their clinical translation and implementation. Oncolytic
viruses (OVs) feature at least two modes of action: (i) direct tumor cell killing by productive
infection and viral spread, and (ii) induction of local and systemic anti-tumor immunity
by release of tumor antigens in the context of danger- and pathogen-associated molecular
patterns, triggering an immunogenic tumor microenvironment [
1
–
10
]. With the rise of
immunotherapy in clinical oncology, induction of anti-tumor immunity by viral oncolysis
has received increasing attention, with OVs being explored as components of combination
regimens that induce primary anti-tumor responses and/or trigger immune cell infiltration
of tumors. Virotherapy exploits the diversity of viruses as pharmacophores featuring a
wide range of particle and genome sizes, structures and replication modalities [
2
,
9
,
11
].
The defining feature of OVs is a tumor-selective infection or replication. This oncotropism
can be achieved by, e.g., the exploitation of defective anti-viral host responses in cancer
cells, allowing for the application of interferon-sensitive viruses or virus vaccine strains [
6
].
Furthermore, OVs have been engineered to target cancer cells, either at the cell entry level,
e.g., by fusion of cancer cell-binding ligands to viral surface proteins, or during post-entry
replication steps [
2
,
11
–
13
]. The latter has been achieved by deletion of viral genes or gene
functions required for replication in normal, but not in cancer cells, as well as expression
of essential viral genes from tumor-selective promoters, respectively. Many viral pharma-
cophores were further optimized in terms of therapeutic potency by genome modifications
aimed at enhanced cancer cell lysis or anti-tumor immune activation [
2
,
5
,
9
]. A prominent
approach is to insert therapeutic transgenes into viral genomes, deriving so-called “armed”
OVs [
2
,
14
]. Beyond and in addition to that, combination regimens with immuno-, chemo-,
radio- and targeted therapies, as well as surgery, are being explored [
15
–
19
]. Proof-of-
Viruses 2021,13, 1420 3 of 29
principle has been obtained for both, successful OV development and clinical translation.
As such, a plethora of effective OVs have been characterized in preclinical models, and
hundreds of clinical studies have been conducted, both exploring the treatment of a wide
spectrum of cancer entities and different application routes [
20
,
21
]. The demonstration of
favorable safety profiles in patients represents a key milestone for the clinical implementa-
tion of virotherapy. Furthermore, while most clinical studies have explored intratumoral
virus application, OVs have been shown to reach tumors after intravenous application and
a durable response of a patient with advanced metastatic disease after a single systemic
virus application has been reported [
22
,
23
]. The first marketing approval of an OV, T-VEC
(Imlygic
®
), was obtained in the US and EU in 2015 [
24
], establishing virotherapy in routine
clinical oncology.
However, formidable challenges remain to be addressed in order to implement OVs as
an effective cancer treatment modality for wider use in routine clinical oncology. Foremost,
more potent OVs are needed in order to facilitate improved therapeutic outcomes in
an increasing range of tumor entities. Limitations that need to be overcome include
virus-neutralizing blood factors and virus-sequestering phagocytes [
25
–
27
], blocks to viral
replication and spread in cancer cells and in the tumor microenvironment (TME) [
2
,
6
,
9
],
and sub-optimal immune activation or an immunosuppressive TME [
1
,
3
,
5
,
7
,
8
,
10
]. These
roadblocks must be addressed to facilitate effective systemic virus application, more potent
direct tumor cell killing, and/or immune cell-mediated systemic cancer cell eradication
(even after local OV application), respectively. OV combination regimens provide further
opportunities, as has been explored so far primarily in prime-boost regimens for OV-
mediated tumor vaccination [
17
]. At the same time, adverse host defenses and side effects
need to be kept to a minimum. With the development of more powerful OVs and the
manufacturing capabilities to produce and, thus, administer them at higher doses, targeting
strategies will be of increasing importance. Of note, emerging virus pharmacophores, a
panel of engineering technologies, as well as opportunities for novel combination regimens
are available to address these challenges. Finally, more OVs need to progress towards
clinical exploration and ultimately marketing approval, a complex process involving
various scientific, medical, infrastructural, and regulatory challenges, many of them specific
to the OV of choice. While the first approved virus is a herpes virus, it is expected that
several virus platforms will provide successful virotherapeutics in the future, depending
on the targeted tumor entity, route of application, therapeutic modality (e.g., “arming”),
and engineering opportunities.
With this review being part of the special edition on “State-of-the-Art Viral Vector Gene
Therapy in Germany”, the German virotherapy community, represented by the authors
of this review, provides an overview of German virotherapy research activities. While
conceptually different, there is major overlap with gene therapy, which frequently exploits
virus-based, replication-deficient gene transfer vectors. Examples include virus engineering
approaches towards target cell specificity, understanding and manipulating anti-viral host
responses, or process development for virus manufacturing. In fact, some efforts for
OV development have evolved from gene therapy research, such as the development
of oncolytic adenoviruses, which exploited established approaches for engineering of
adenoviral gene transfer vectors. Furthermore, OVs “armed” with therapeutic genes
actually represent—from the gene therapists’ perspective—replication-competent gene
transfer vectors delivering a therapeutic gene that mediates direct or indirect tumor cell
killing. Virotherapy and gene therapy research have always been strongly integrated
in Germany and have been active within the German Gene Therapy Society (DG-GT).
While the German virotherapy landscape was introduced in a review in 2017 [
28
], here we
present an update with a focus on recent and on-going research activities in pre-clinical
and translational virotherapy research.
Viruses 2021,13, 1420 4 of 29
2. Recent Preclinical Virotherapy Research Activities in Germany
Exploring novel OVs, advanced OV engineering, and establishing innovative virother-
apeutic modalities have been a key stronghold of virotherapy research in Germany [
28
].
This chapter highlights recent research activities ordered according to the virus platform,
i.e., adenovirus, arenavirus, coxsackievirus, herpes simplex virus, measles virus, par-
vovirus, vaccinia virus, and vesicular stomatitis virus. A comprehensive overview of
scientific strategies pursued is provided in Table 1and Figure 1.
Figure 1. Overview of recent pre-clinical and clinical virotherapy research activities in Germany. Created with BioRender.com.
Viruses 2021,13, 1420 5 of 29
2.1. Adenovirus Platform
Oncolytic adenovirus (oAd) engineering and development has been a pillar of German
preclinical virotherapy research over the last decades with several groups exploring a
spectrum of strategies for developing novel oAds or optimizing them: (i) shielding of oAd
particles; (ii) targeting, controlling, or enhancing oAd replication; (iii) “arming” of oAds
with therapeutic or imaging genes; (iv) oAd-based immunotherapeutic approaches; and
(v) oAd combination treatment (see [
28
]). In the following, we discuss recent activities in
preclinical oAd research in Germany.
The group of Anja Ehrhardt at Witten/Herdecke University focuses on exploring the
natural diversity of human adenoviruses (Ads) for the development of oAds. More than
100 distinct human Ads have been identified, but only a limited number of these Ads have
been converted into oAds. In a recent study, the group established a genome engineering
system enabling cloning of complete Ad DNA genomes from different sources, such as
purified virions or infected cells [
29
]. The technology is based on advanced linear-linear
homologous recombination (LLHR) and linear-circular homologous recombination (LCHR).
In this initial study, 34 Ad genomes were cloned and tagged with reporter genes. Screening
of this reporter-tagged Ad library revealed an Ad-type dependent uptake and oncolysis
efficiency in osteosarcoma-derived [
29
] and breast cancer cell lines [
30
]. Meanwhile, this
cloning system was applied to obtain genetic access to other emerging human Ads [
31
]. The
group is currently adopting interesting candidates into oAds by adding, for instance, tumor-
specific promoters driving expression of early virus transcription units and developing
novel serotype-derived oAds for targeting of GI cancers (collaboration with the group of
Dirk Nettelbeck within the Clinical Cooperation Unit Virotherapy in Heidelberg headed
by Guy Ungerechts). In the future, this platform, based on a broad spectrum of different
human Ads, may provide the potential to customize OVs to target specific cancer types.
Florian Kreppel’s group at the University Witten/Herdecke continued its work on
the modification of Ad vector capsids by covalent chemical, non-covalent chemical, and
genetic means (see [
28
]). The group’s main focus is to improve systemic vector delivery
through the blood stream and to understand molecular mechanisms underlying the toxic
effects of mistargeted/sequestered Ad vector particles in mice. One central tool of the
group is combined genetic and chemical capsid modification that allows for site-specific
attachment of ligand or shielding moieties to the virus capsids [
32
]. This enables studies
on the relevance of different capsid positions for shielding by polymer molecules and
for targeting by ligands. The group has set up a model to study the
in vivo
fate of Ad
immunocomplexes by chemical decoration of the vector capsid with carbohydrates [
33
].
This model can serve to analyze the effects of neutralizing and non-neutralizing pre-existing
antibodies on the
in vivo
biodistribution and toxicity. Since Ad vectors based on different
Ad serotypes and species are currently used as vectored vaccines against the COVID-19
pandemic (and will be used during the next years), it can be expected that a very large
fraction of the global human population will develop neutralizing antibodies and T cells
against Ad species and types that were so far considered to be rare. Therefore, it is even
more important than before to understand the effects of (cross-) neutralizing anti-Ad
antibodies and cross-reactive anti-Ad T cells in order to safely and efficiently use Ads as
virotherapeutic agents (for an innovative approach to exploit Ad-neutralizing antibodies
for cancer therapy, see next paragraph). In addition, it can be foreseen that effective
shielding will become an important tool for oAds. The Kreppel group has contributed to
the analysis of novel amphiphilic dendrimers designed to non-covalently interact with the
vector surface. Upon systemic delivery through the bloodstream, vectors coated with such
amphiphilic dendrimers showed significantly altered vector biodistribution [
34
]. Overall,
these results demonstrate the potency of non-covalent capsid modifications.
Viruses 2021,13, 1420 6 of 29
The group of Florian Kühnel at MHH in Hannover has previously explored various
aspects of oAd engineering and molecular retargeting of oAds using bispecific adapter
proteins (see [
28
]). In contrast to adapter proteins, genetic modification of the viral receptor-
binding fiber protein allows for the cross-generational maintenance of the altered target cell
tropism during infection. However, options of fiber engineering without compromising the
structural integrity of the virus are limited. In collaboration with the group of Rita Gerardy-
Schahn (Department Clinical Biochemistry, MHH), a chimeric fiber was designed wherein
the knob domain is genetically replaced by the bacteriophage-derived endosialidase En-
doNF. This protein combines retargeting properties with essential structural features for
a genetic fusion with the adenoviral fiber shaft. The resulting chimeric fiber facilitates a
stable molecular retargeting of oAds to infect polysialic acid expressing tumors, such as
glioblastoma or small cell lung cancer [
35
]. In a recent work, the Hannover groups devel-
oped an innovative approach to re-direct Ad-neutralizing antibodies to mediate tumor
cell killing. Neutralizing antibodies against OVs, either treatment-induced or pre-existing
(after childhood infection or vaccination with Ad-vectored vaccines), are a limiting factor
of virotherapy because they inhibit effective virus spread. Nevertheless, these neutralizing
antibodies represent a potent, yet unexploited immunological resource: Niemann et al.
have developed a strategy to convert this undesired immune response against OVs into a
tumor-directed immune attack [
36
]. They generated a tumor-directed adapter molecule
which harbors a dominant immunogen of Ad serotype 5, being a frequently used OV.
In Ad-immunized mice, intravenous injection of these adapters delayed the growth of
syngeneic tumors. The antitumor effect was attributable to CD8
+
T cells but also required
the activity of natural killer (NK) cells. Interestingly, retargeting of preexisting antiviral an-
tibodies also enabled a significant tumor response to PD-1 inhibition. Antibody retargeting
after intratumoral virotherapy was highly effective in a murine MC-38 colon cancer and
additional application of a PD-1 checkpoint inhibitor resulted in long-term survival of the
majority of treated mice. These results confirm that virotherapy and antibody retargeting
may promise to activate immunologically ‘cold’ tumors for checkpoint inhibitors.
The group of Stefan Kochanek and Lea Krutzke, with Astrid Kritzinger, Robin Nilson,
and Frederik Wienen pursue different projects in the areas of oncolytic virotherapy, genetic
vaccination and use of mesenchymal stroma cells (MSCs) as carriers. Therapeutic target
with replicating Ads is the head and neck squamous cell carcinoma (HNSCC). In their
research, they pay special attention on targeting not only the actual cancer cells but also
cells of the tumor microenvironment (TME). In the last years, they have developed a
capsid-modified oAd, which shows not only substantially enhanced and Coxsackievirus
and adenovirus receptor (CAR)-independent infection of cancer cells, but also significant
infection of cells of the TME. The virus is currently being investigated in preclinical tumor
animal studies. Prospective studies will analyze if the introduced mutation can also be
transferred to other Ad types. Important aspects are also production- and quality-related
issues that are currently being addressed. The Kochanek group further investigates the use
of MSCs as carrier cells to enable systemic administration of oAds. Allogenic or autogenic
MSCs naturally migrate towards tumor sites; hence, represent promising shuttle cells to
transport viruses to their site of action while protecting them from cellular or non-cellular
sequestration mechanisms en route. The group developed a readily applicable method to
substantially enhance the ex vivo virus transduction of infection-refractory MSCs, which,
to date represents a major hurdle for the use of MSC as carrier cells in the context of oAds
(Nilson et al., submitted). Moreover, they recently established the chicken chorioallantoic
membrane (CAM) assay as a quick and low-cost high-throughput tumor model system
for the
in vivo
analysis of systemically or locally injected OVs [
37
]. The method was
already successfully used to investigate tumor targeting capabilities of modified Ads and
AAVs [38]; however, it is currently also being evaluated for its applicability to other OVs.
Viruses 2021,13, 1420 7 of 29
The groups of Henry Fechner at Technical University of Berlin and Jürgen Eberle at
Charité-Universitätsmedizin Berlin explore the “arming” of oAds for targeting malignant
melanoma. In previous studies, they had constructed AdV-TRAIL, a melanoma-specific
oAd with inducible expression of TNF-related apoptosis-inducing ligand (TRAIL) and
reported superior therapeutic activity in melanoma cells by TRAIL-induced apoptosis
(see [
28
]). However, melanoma cells characteristically develop resistance to TRAIL [
39
]. In
a recent project, the groups showed that TRAIL resistance can be efficiently overcome in
melanoma cells by inhibition of the antiapoptotic Bcl-2 protein Mcl-1. Using siRNAs for
transcriptional silencing of five different antiapoptotic Bcl-2 proteins, Mcl-1 was highlighted
as the most efficient target to overcome TRAIL resistance [
40
]. In a follow-up study
in cooperation with Florian Kreppel (University Witten/Herdecke) the Berlin groups
significantly enhanced AdV-TRAIL cytotoxicity in melanoma cells by Mcl-1 silencing. The
effects are the result of enhanced apoptosis and necrosis seen both in TRAIL-resistant
and in TRAIL-sensitive melanoma cell lines [
41
]. The Berlin groups now aim to “arm”
AdV-TRAIL with (artificial) microRNAs for Mcl-1 downregulation. In addition, computer-
designed Mcl-1 inhibitors [42] shall be used to establish strategies for Mcl-1 silencing and
enhancement of the oncolytic activity of TRAIL-“armed” oAds in melanoma.
The groups of Per Sonne Holm and Roman Nawroth at the Technical University of
Munich (PSH is now at Medical University Innsbruck), in collaboration with the group of
Ulrike Naumann at the Hertie Institute for Clinical Brain Research in Tübingen and the
group of Uwe Thiel and Sebastian Schober at Children’s Hospital Schwabing are focus-
ing on further developing their previously established YB-1-dependent oAd XVir-N-31
(see [
28
]) for treatment of glioblastoma, bladder cancer, and sarcoma. Explored treatment
modalities include the combination with targeted therapy approaches, radiation or immune
checkpoint inhibition. In this regard, the investigators have recently demonstrated that tu-
mor irradiation, temozolomide or STAT 3/5 inhibitors further strengthened the therapeutic
impact of XVir-N-31 in glioma-bearing mice, as well as in bladder cancer [
43
–
45
], indicat-
ing the benefit of combining oAds with established therapies. Currently, the therapeutic
effects of XVir-N-31 are evaluated in combination with cyclin-dependent kinase (CDK) 4/6
and bromodomain inhibitors (BETi), both known to affect the cell cycle upon targeting
the RB/E2F pathway. Results from the past years indicate strong synergistic effects, and
ongoing research is focusing on the molecular basis for the observed strong increase in cell
killing. Importantly, the increase in cell killing is accompanied with a further stimulation of
the immune response. Furthermore, immune-stimulatory effects of a derivate of XVir-N-31
that expresses a humanized antibody against PD-L1 are being evaluated in experimental
glioma using immuno-humanized mice. Initial results support previous findings from
other groups, indicating that the immune response against tumor antigens plays a central
role in the therapeutic effect of oAd. Based on the results from the past years, and together
with the spin-off company XVir Therapeutics GmbH and supported by the German Min-
istry of Education and Research (BMBF) and German Cancer Aid, clinical phase I/II trials
with XVir-N-31 are in preparation for the treatment of glioblastoma and sarcoma.
Viruses 2021,13, 1420 8 of 29
Table 1. Recent preclinical virotherapy research activities in Germany according to scientific strategies.
Scientific Strategy Description of Research
Approach Virus Refs
Identifying new viruses as OVs
Screening and/or cloning of virus
strains, serotypes, or mutants
adenovirus
coxsackievirus
parvovirus
[29–31]
[46]
[47]
Shielding virus particles from
blood factors or from cellular
sequestration
Combining genetic and chemical
capsid engineering or exploiting
adapter molecules for shielding
and targeting of virus particles
and for exploration of host
interactions
adenovirus [33,34]
Exploring carrier cells to enable
systemic administration of OVs adenovirus on-going work
Exploring/Targeting/Enhancing
efficiency of OV cell binding and
entry
Unraveling the virus cell entry
pathway parvovirus [48,49]
Genetically replacing the
cell-binding domain of a viral
capsid protein with a
tumor-specific ligand
adenovirus [35]
Genetic engineering of virus
capsid for enhanced entry into
tumor cells, cells of the TME, and
carrier cells
adenovirus Nilson et al., submitted,
on-going work
Replacing OV glycoproteins by
those of other viruses VSV [50]
Genetic engineering of viral
glycoproteins using highly stable
and affine targeting domains and
selected protease recognition
motifs for combined receptor and
protease targeting
measles virus [51]
Combining genetic and chemical
capsid engineering or exploiting
adapter molecules for shielding
and targeting of virus particles
and exploration of host
interactions
adenovirus [33,34]
Post-entry targeting of OV
replication
Expression of essential viral genes
from tumor-selective promoters adenovirus on-going work
Insertion of microRNA target sites
into viral genes for mRNA
destruction and/or translational
inhibition in healthy tissues
coxsackievirus
measles virus [52–54]
[55,56]
Enhancing oncolytic activity or
tumor-specificity of OVs
Enhancing oncolytic activity of
OVs: production of fitness
mutants with enhanced
oncosuppressive capacity
coxsackievirus
parvovirus on-going work
[47]
Enhancing the tumor-specificity
of OVs by selecting mutated
viruses in a fast evolution
platform
arenavirus on-going work
Viruses 2021,13, 1420 9 of 29
Table 1. Cont.
Scientific Strategy Description of Research
Approach Virus Refs
Immune effects of OVs and
enhancing their
immuno-stimulatory potency
OV-induced activation of innate
and (anti-tumor) adaptive
immunity
arenavirus
measles virus
parvovirus
vaccinia virus
[57,58]
[59]
[60]
[61,62]
Enabling OV-induced syncytia
formation as immunogenic cell
death by replacing viral
glycoproteins with heterologous
fusogenic envelope proteins
VSV [50]
Expression of immunomodulators
(ICIs, bispecifics, cytokines)
adenovirus
coxsackievirus
measles virus
on-going work
on-going work
[63–65]
Expression (and presentation) of
tumor antigens for genetic
vaccination measles virus [66,67]
OV stability Analysis of genomic stability of
OVs measles virus [68]
Expression of therapeutic
proteins or shRNAs by OVs
Induction of apoptosis by
expression of death ligands or
RNAi-mediated inhibition of
anti-apoptotic proteins of intrinsic
apoptosis pathways
adenovirus [41] and on-going work
Insertion of suicide genes into the
virus genome for genetic prodrug
activation
measles virus
vaccinia virus [51,56,69]
[61,70]
Expression of immunomodulators
(ICIs, bispecifics, cytokines)
adenovirus
coxsackievirus
measles virus
on-going work
on-going work
[63–65]
Expression (and presentation) of
tumor antigens for genetic
vaccination measles virus [66,67]
Combination therapy with
OVs
Combination therapy with
radiotherapy adenovirus
measles virus [43]
on-going work
Combination therapy with
chemotherapy measles virus
vaccinia virus [71]
[61]
Combination therapy with
apoptosis induction adenovirus
parvovirus [41] and on-going work
[72]
Combination with targeted
therapy
adenovirus
measles virus
vaccinia virus
[44] and on-going work
on-going work
[73]
Combination with epigenetic
therapy adenovirus on-going work
Combination therapy with
starvation measles virus [74]
Combination therapy with ICI adenovirus
parvovirus [36]
[60,62]
Combination therapy with
adoptive T cell or NK cell transfer
measles virus
VSV [75]
[76]
Combination therapy with
anti-viral antibody-retargeting via
recombinant adapters adenovirus [36]
Control of OV replication
(safety measure) OV inhibition by virostatic drugs herpes virus [77]
Viruses 2021,13, 1420 10 of 29
2.2. Arenavirus Platform
Karl Sebastian Lang’s group at the University Hospital Essen has previously de-
veloped a new concept for using arenaviruses in immunovirotherapy (see [
28
]). Work
included the lymphocytic choriomeningitis virus (LCMV), which is presently being devel-
oped within Abalos Therapeutics GmbH. The overall aim is to maximize the inflammatory
signals within the tumor tissue and thereby activate several anti-tumoral immune effector
mechanisms within the tumor. One ideal condition for the success of this therapeutic
approach is a pre-existing anti-tumoral immunity. To achieve a strong and locally restricted
(re-)activation of the immune system the LCMV is used as viral backbone. LCMV is almost
non-cytopathic and can persist for several days to months in cell culture or mice [
78
].
This is one feature, which characterizes LCMV as a strong immune activator [
79
]. LCMV
is hardly neutralized by antibodies and initial control will be achieved by CD8
+
T cell
infiltration in infected organs. The Lang group reported that intravenous application of
LCMV into tumor-bearing mice can lead to specific replication of the virus in primary
tumors and metastasis for several days [
58
]. This prolonged locally restricted replication of
the virus was correlated with a relatively weak responsiveness to type I interferon (IFN-I).
Replication within the tumor led to recruitment and activation of anti-tumoral monocytes,
tumor-specific CD8
+
T cells and NK cells [
57
,
58
], thereby resulting in tumor shrinkage.
These findings are in line with earlier studies in humans using an LCMV-related virus in
patients with different tumors [
80
]. Six of these patients had a beneficial effect (i.e., an
altered course of disease) with a distinct destructive effect on the malignant tissues. This
hints at a so far completely ignored opportunity to treat cancer patients with an arenavirus-
based therapy. In current studies of the Lang group, an adaptation of the virus to the tumor
tissue is achieved by specific selection of tumor-prone virus mutations. This so-called Fast
Evolution Platform aims to enhance the efficiency, e.g., via accelerated viral replication,
and specificity of the virus for specific tumor types, the latter in order to limit potential
side effects. Whether such newly tumor-tropic viruses will enhance the already known
anti-tumoral effects of LCMV will have to be explored in more detail in the near future.
Abalos Therapeutics develops such an optimized virus candidate for clinical testing.
2.3. Coxsackievirus Platform
Coxsackieviruses are a more recent addition to the German OV research portfolio. The
group of Henry Fechner at the Technical University of Berlin, in addition to his work on
oAds (see above), has investigated the oncolytic potential of coxsackievirus B3 (CVB3), a
single stranded RNA virus of the picornavirus family, for treatment of colorectal carcinomas.
The group showed that the CVB3 strain PD, which has a unique receptor tropism to N- and
6-O-sulfated heparan sulfate, was more cytotoxic in colorectal cancer cells than other CVB3
viruses, which use the CAR for cell entry. In a xenograft mouse model of colorectal cancer,
PD and two other CVB3 strains significantly inhibited tumor growth, but only PD showed a
sufficient safety profile [
46
]. Pancreatitis and myocarditis may represent serious side effects
induced by CVB3s. Thus, Fechner’s group applied a microRNA-dependent de-targeting
strategy to prevent virus replication in both organs. After evaluation of potential insertion
sites within the viral genome [
54
], target sites of microRNAs specifically expressed in
pancreas and heart were inserted into the 3
0
UTR of the highly pancreato- and cardiotropic
CVB3 strain H3. Long-term
in vivo
investigations after intratumoral virus injection into
subcutaneously established colorectal tumors in nude mice confirmed that H3 replication
was completely prevented in pancreas and heart. Importantly, virus replication in tumors
remained unaffected, and tumor growth was significantly inhibited [
52
,
53
]. Currently, the
group is also investigating anti-tumoral immune mechanisms induced by PD, and they
aim to increase the oncolytic activity of PD further by tumor cell-specific adaptation of the
virus and by insertion of immunomodulatory transgenes into the viral genome [81].
Viruses 2021,13, 1420 11 of 29
2.4. Herpes Simplex Virus Platform
Herpes simplex virus type-1 is a double-stranded DNA virus and is frequently used in
oncolytic virotherapy. An important representative of this virus family is T-VEC (Imlygic
®
),
the only virus construct approved in the western hemisphere for virotherapy to date, which
is characterized by the additional integration of the gene encoding human granulocyte-
macrophage colony-stimulating factor (hGM-CSF), intended to trigger an enhancement
of the virus-mediated anti-tumor immune response. The group of Ulrich Lauer at the
University Hospital Tübingen performed a preclinical assessment of this state-of-the-art
OV, using a panel of human neuroendocrine tumor (NET) cell lines. NETs represent a
rare and heterogeneous group of tumors originating from the neuroendocrine system
and occurring at various anatomic sites, such as the pancreas, lung, and intestine, and
their therapy remains a challenge in oncology. It was demonstrated that T-VEC is able to
infect human NET cells, already at very low virus concentrations, with a high oncolytic
efficiency, to replicate and to subsequently lyse the cells. Moreover, the virostatic drug
ganciclovir (GCV) was found to lower viral titers in all cell lines tested and efficiently
limit T-VEC-mediated cytotoxicity, representing an important safety feature for future
treatments of NET patients [77].
The group of Markus Moehler at the Johannes Gutenberg University Mainz explored
the immunostimulatory effects of the T-VEC virus by comparing it with JS-1, which is
identical to it except for the hGM-CSF transgene. Moreover, they analyzed the putative
synergistic biological and immunological effects of T-VEC with cytotoxic agents in human
tumor-immune cell co-culture experiments (Delic and Moehler, manuscript in preparation).
They documented increased activation of human CTLs after infection by both HSV-1 strains,
as previously reported for H-1PV as well [
82
]. After human melanoma cell infection with
T-VEC or JS-1, human DC maturation was not substantially increased, similar to a previous
report for oAd-infected melanoma cells [83].
2.5. Measles Vaccine Virus Platform
The exploration of Measles vaccine viruses (MeV) as OVs is another stronghold of
German virotherapy research. In previous preclinical work, several teams in Germany
addressed various aspects of oncolytic MeV (oMeV) development towards maximum
tumor specificity and therapeutic efficacy, i.e., shielding, entry and post-entry targeting,
replication control, enhancing oncolytic potency, “arming” with therapeutic genes, potenti-
ating antitumor immunity and combination regimens (see [
28
]). Here, we report on recent
preclinical activities.
The group of Michael Mühlebach at the Paul-Ehrlich-Institut, Langen, has further elab-
orated on the advantages of using designed ankyrin repeat proteins (DARPins) as targeting
domains to direct cell entry of oMeV to tumor-specific surface antigens. Employing these
highly affine and stable domains to redirect the tropism of MeV hemagglutinin to EGFR
yielded viruses with the same oncolytic potential on receptor-positive tumor cells than
non-targeted MeV. Thereby, it became feasible for the group to generate dual-targeted MeV,
the entry of which also becomes restricted to a protease-rich environment as found e.g., in
glioblastoma multiforme [
51
]. This approach should be useful especially when applying
MeV with higher cytotoxic potential due to arming with a suicide gene, e.g., super cytosine
deaminase (SCD see below) [
51
], since for only EGFR-targeted agents, on-target side-effects
have been described, previously. On the other hand, the inherent potential of vaccine-strain
derived oMeV to stimulate innate immune pathways and modulate the immunopeptidome
as shown in a collaboration spearheaded by the group of Ghazaleh Tabatabai, University
Hospital Tübingen [
59
] has re-enforced the research of the Mühlebach group on analyzing
replicating MeV as a tumor vaccine platform. It is a well-established concept that the
immunogenic properties of vaccine strain MeV can be readily used to induce immunity
against other pathogens by encoding critical antigens of those [
84
] as the Mühlebach lab
recently demonstrated also for SARS-CoV-2 [
85
]. The group could demonstrate that this
vaccine platform technology even breaks tolerance to homologous tumor-associated au-
Viruses 2021,13, 1420 12 of 29
toantigens when encoding the unmodified autoantigen or presenting the autoantigen on
retroviral virus-like particles (VLPs) [
67
]. These VLPs are highly immunogenic, per se, but
their immunogenicity can be further strengthened by co-display of GM-CSF [
86
] pointing
at further potential for optimization.
Mathias Leber’s group within Guy Ungerechts’ Clinical Cooperation Unit Virotherapy
in Heidelberg focuses on genetic engineering strategies to improve the therapeutic index
of OVs with a focus on oMeV (as reviewed in [
87
]). Recently, the team has explored
strategies to enhance oMeV safety and efficacy and analyzed oMeV genomic stability. In
this context, the Leber group has previously developed a microRNA-based, post-entry
restriction system for oMeVs [
88
,
89
]. This system is based on the incorporation of target
sequences for differentially expressed microRNAs into the MeV genome. In a recent study,
this system was systematically analyzed and further optimized [
55
]. Viruses harboring
microRNA target sites in various positions within the MeV genome were generated and
the critical importance of the insertion position on the overall efficacy of virus regulation
was reported. Furthermore, it was shown that the mechanism of microRNA-mediated
virus control is dependent on the actual microRNA sequence, and likely encompasses both,
direct cleavage of target sites and translational inhibition. In a second line of research, the
Leber team has systematically analyzed the genomic stability of oMeV during continuous
serial passaging in tumor and producer cells [
68
]. This approach was chosen to mimic
extended periods of virus replication in a clinical virotherapy setting. Since RNA viruses
can quickly adapt to changing environmental pressures by selecting quasispecies with
superior fitness based on beneficial genetic alterations, these changes could potentially
weaken their safety profile. The distribution of consensus mutations detected after a full
year of serial passaging was non-random, indicating different levels of genetic constraints
in different regions of the genome. Altogether, the number of consensus mutations detected
in the genomes of serially passaged viruses was remarkably small, further underlining
the genomic stability and excellent safety profile of oMeV. In a third line of research, the
Leber group aimed at combining the priorities of safety and efficacy in a single engineered
virus for chemovirotherapy of pancreatic cancer [
56
]. Here, target sequences for miR-148a,
which is downregulated in pancreatic ductal adenocarcinoma (PDAC) but expressed in
multiple healthy tissues of the gastrointestinal tract, were inserted along with the prodrug-
converting enzyme cytosine deaminase-uracil phosphoribosyl transferase (CD-UPRT here
E.coli-derived) into the genome of an oMeV. The microRNA target sites restricted replication
and spread of the virus in miR-148a-expressing cells, while allowing for unaltered oncolytic
efficacy in PDAC cell lines. The prodrug convertase CD-UPRT converts systemically
administered, non-toxic 5-FC (5-fluorocytosin) into the chemotherapeutic drug 5-FU (5-
fluorouracil), thus allowing for a localized chemovirotherapy. The group could demonstrate
superior anti-tumor efficacy of the MeV-CD-UPRT virus in combination with 5-FC both,
in vitro
and
in vivo
. Taken together, this approach demonstrated the feasibility to generate
dually modified oMeVs for enhanced safety and efficacy. Currently, the Leber team is
working on novel small RNA-based engineering technologies as well as on combination
therapy approaches including radio-, immuno- and pharmacovirotherapy.
The group of Christine Engeland within the Clinical Cooperation Unit Virotherapy in
Heidelberg headed by Guy Ungerechts (C.E.E. is now at Witten/Herdecke University) has
a strong focus on MeV for targeted immunomodulation [
90
–
92
]. This strategy employs the
viral vector for delivery of immunomodulators to the tumor site, thereby increasing the
therapeutic window. Moreover, immunomodulators may complement anti-tumor immune
effects of oncolysis, leading to synergistic effects. Following this approach, bispecific T
cell engagers (BiTEs) were introduced into the MeV platform [
64
]. BiTEs consist of two
antibody single-chain variable fragments (scFv) binding a tumor surface antigen and CD3
on T cells, thereby mediating tumor-directed T cell cytotoxicity. BiTEs have demonstrated
clinical efficacy against hematological malignancies. However, difficulties with delivery
and toxicities have so far hampered broader application, also against solid tumors. The
Heidelberg team showed that MeV-encoded BiTEs are functional and recruit endogenous
Viruses 2021,13, 1420 13 of 29
T cells
in vivo
. MeV BiTE prolonged survival compared to MeV encoding a non-binding
BiTE, parental MeV, and BiTE only. Gene expression profiling revealed signatures linked to
T cell activation, but also exhaustion, indicating potential for combination with immune
checkpoint inhibition. Mice achieving complete tumor remission subsequently rejected
tumor re-engraftment, demonstrating induction of durable anti-tumor immunity. More-
over, in patient-derived xenograft models, the combination of MeV BiTE and adoptive
immune cell transfer significantly prolonged survival compared to monotherapies. This
was the first study to demonstrate efficacy of an OV encoding a tumor-targeting BiTE in
both, syngeneic and patient-derived xenograft models, highlighting the potential of this
combination [
93
]. To further improve effector T cell function, Engeland and colleagues had
previously demonstrated strong anti-tumor efficacy of an oMeV encoding IL-12, achieving
90% complete tumor remissions in the MC38cea model, a colorectal cancer model in fully
immunocompetent C57BL/6 mice [
65
]. However, analysis of tumor-infiltrating immune
cells had indicated induction of activation-induced cell death (AICD) by MeV-encoded
IL-12. To prevent AICD, the group designed MeV encoding an IL-15 superagonist [
63
].
Despite intratumoral T cell and NK cell activation, MeV IL-15 was less effective in both
the B16-CD46 and MC38cea tumor models compared to MeV IL-12. This was associated
with stronger viral gene expression and immune activation by MeV IL-12, emphasizing the
superior efficacy of this MeV construct. Based on these results, clinical translation of MeV
IL-12 is now being actively pursued. While BiTEs and cytokines non-specifically activate all
lymphocytes, priming and activation of tumor-antigen specific T cells is a key goal in cancer
immunotherapy. To this end, MeV harboring epitope cassettes derived from the model anti-
gen ovalbumin and the melanoma antigen trp-2 were generated [
66
]. In ex vivo co-culture
models, these vectors were shown to mediate efficient antigen presentation, priming of
naïve and activation of effector CD8
+
T cells. Vectors encoding secreted epitope variants
or epitope strings targeted to the proteasome mediated the strongest IFN-
γ
responses.
This concept can be adapted to diverse heterologous antigens, both cancer-derived (for
immunovirotherapy) and pathogen-derived (for vaccination against infectious diseases).
Further, MeV vectors can be combined with vectors derived from other virus platforms in
prime-boost regimens to enhance antigen-specific immune responses.
Ulrich Lauer’s group at the University Hospital Tübingen not only works on herpes
viruses (above) and vaccinia viruses (below), but also has a special focus on MeV. In this re-
gard, the group investigated a combinatorial approach employing oncolytic MeV together
with activated human NK cells (or PBMCs) in human sarcoma cell lines. In an earlier
preclinical study, the Lauer group had demonstrated that MeV exhibits potent oncolytic
activity in pediatric sarcomas [
94
]. However, since there were also sarcoma cell lines that
showed primary resistance to MeV-mediated oncolysis, thoughts turned towards combina-
tion therapies. It was shown that a combination of oncolytic MeV-GFP virotherapy and
activated NK cells resulted in enhanced oncolysis of human sarcoma cell lines compared
with the respective monotherapies. In addition, NK cells were activated upon coculture
with MeV-infected A673 sarcoma cells [
75
]. These results not only support the initiation of
clinical trials combining oncolytic virotherapy with NK cell-based immunotherapies, but
also provide the rationale for potential triple combinatorial approaches, for instance with
immune checkpoint inhibitors. The same viral construct was used by the Lauer team in a
study investigating the influence of starvation on the oncolytic efficacy in human colorectal
carcinoma (CRC) cells. Since it is known that starvation sensitizes tumor cells to chemother-
apy while protecting healthy cells in a process called differential stress resistance, the group
of Ulrich Lauer examined whether this phenomenon also applies to OVs. It was shown
that long-term low-serum, standard-glucose starvation significantly enhanced the efficacy
of oMeV-mediated cell killing of CRC cells, whereas it was diminished in normal colon
cells [
74
]. With regard to the treatment of patients, a personalized starvation-enhanced
virotherapy could provide benefits for distinct CRC cancer patients; however, possible
contraindications such as cachexia, sarcopenia and malnutrition as well as the individual
perseverance must be considered in the decision for this particular therapy. In further
Viruses 2021,13, 1420 14 of 29
work of the Lauer group, a MeV was combined with gemcitabine to achieve an enhanced
chemovirotherapy for pancreatic cancer. Gemcitabine is a first-line chemotherapeutic agent
widely used as a palliative treatment option for pancreatic cancer patients. Moreover,
gemcitabine, just like many other cytostatic drugs, is able to induce senescence in tumor
cells, resulting in permanent cell cycle arrest and consequently in maintaining cells in a
less malignant/less proliferative state. In a previous study, the group showed that MeV
can infect senescent cells, including pancreatic cancer cells, replicate in them, and even
lyse them more efficiently than non-senescent cells [
95
]. The Lauer laboratory therefore
investigated whether gemcitabine-induced senescent tumor cells can be oncolyzed more
efficiently during chemovirotherapeutic combination therapy. It was shown that different
pancreatic cancer cell lines treated with both gemcitabine and MeV were lysed with higher
efficacy than those treated with the respective monotherapy. Furthermore, gemcitabine-
induced tumor cell senescence was not impaired by MeV [
71
]. These findings pave the
way for a new therapeutic option for patients with advanced pancreatic cancer. Moreover,
the group of Ulrich Lauer pursues strategies to integrate suicide genes into the genome of
OVs which has been reported to increase oncolytic efficiency through bystander killing.
In a study with acute myeloid leukemia (AML) cell lines and primary AML cells the on-
colytic efficacy of a MeV construct armed with super cytosine deaminase (MeV-SCD), a
yeast-derived CD-UPRT, which catalyzes the conversion of the inactive prodrug 5-FC into
the therapeutically active and clinically approved compound 5-FU, was investigated. It
was demonstrated that MeV-SCD infected the leukemic blasts and significantly reduced
the number and viability of leukemic cells by induction of apoptosis. The conversion of
5-FC to 5-FU was found to further potentiate- this effect [69].
2.6. Parvovirus Platform
The development of oncolytic parvoviruses (PVs), the smallest viruses clinically
explored as OVs, has been pioneered by the group of Jean Rommelaere at the German
Cancer Research Center in Heidelberg. The group has explored rodent PVs, in particular H-
1PV, for therapeutic applications with the initial report published in 1982 [
96
], and the first-
in-human clinical virotherapy study initiated in Germany in 2011 at Heidelberg University
Hospital (EudraCT 2011-000572-33, [
97
]). In parallel, previous preclinical research at the
German Cancer Research Center focused on enhancing delivery, oncolytic potency and
immunostimulation of oncolytic PVs (see [28,98]).
Recent work by the group of Markus Moehler at the Johannes Gutenberg Univer-
sity Mainz, in collaboration with Jean Rommelaere’s group, explored H1-PV to further
improve the therapeutic success of immune checkpoint inhibitors. The H-1PV-induced
immunogenic cell death was accompanied by increased expression of the immune check-
point proteins CTLA-4, PD-1, and PD-L1 [
60
,
62
]. Nevertheless, H-1PV-infected human
melanoma and colorectal cancer cells triggered maturation of human antigen-presenting
cells such as dendritic cells (DC). Combining H-1PV with the immune checkpoint in-
hibitors ipilimumab, tremelimumab or nivolumab strengthened cytokine release during
DC maturation [60,62,99].
The Laboratory of Oncolytic Virus Therapeutics (LOVIT) at the German Cancer Re-
search Center in Heidelberg and at the Luxembourg Institute of Health, headed by Antonio
Marchini, pursues three main areas of research to improve the anticancer efficacy of on-
colytic PVs: (i) the development of novel combinatorial treatments, which combine PVs
with other anticancer agents (recently reviewed in [
100
]); (ii) the generation of novel engi-
neered PVs with improved oncolytic and immunomodulatory activities (recently reviewed
in [
101
]); and (iii) the characterization of H-1PV life cycle in order to identify cellular factors
that could serve as biomarkers to predict the response of PV-based treatments or guide
the identification of new drugs synergizing with PVs in killing cancer cells [
102
]. The
LOVIT laboratory recently found that sublethal doses of BH3 mimetics, namely ABT-737
and ABT-199, potentiate the anticancer activity of H-1PV by cooperating with H-1PV in
inducing immunogenic cell death. The co-treatment triggers major disturbances at the
Viruses 2021,13, 1420 15 of 29
level of mitochondria, lysosomes and the endoplasmic reticulum, and it is associated
with oxidative stress and calcium release [
72
,
102
]. On a more fundamental level, the
Marchini group characterized the entry pathway of H-1PV in cancer cells. First, it was
shown that laminins, in particular those containing the laminin
γ
1 chain, modulate H-1PV
cell attachment and entry. Silencing of LAMC1, the gene encoding the laminin
γ
1 chain,
strongly decreased H-1PV cell transduction by impairing H-1PV attachment at the cell
membrane. In particular, H-1PV binds to sialic acid moieties present in laminins. A direct
correlation between H-1PV oncolytic activity and LAMC1 mRNA levels was found in 59
cancer cell lines from different tumor entities, suggesting that tumors with elevated levels
of
γ
1-containing laminins are more susceptible to H-1PV-based therapies [
49
]. Second,
Marchini’s laboratory found that H-1PV cell internalization occurs via clathrin-mediated
endocytosis, a process that is dependent on dynamin. H-1PV traffics from early to late
endosomes, with acidic pH being necessary for a productive infection [
48
]. This study also
revealed that siRNA-mediated silencing of caveolin-1 increased H-1PV transduction of
cancer cells, suggesting that caveolin-1 is a negative modulator of the H-1PV life cycle.
Further studies are required in order to translate this new knowledge into more effective
H-1PV-based therapies.
Jürg Nüesch’s group, also at the German Cancer Research Center in Heidelberg,
explores PV fitness mutants for improved oncolytic potency. Although H-1PV proved
to efficiently infect and kill a variety of tumor cell lines, success of virotherapy may be
hampered in certain cancer entities and/or distinct patients. Such a limitation could be due
to the restricted tissue tropism of H-1PV and/or its inability to produce progeny viruses
and spread through the patient’s neoplastic tissue. To generate propagation-competent
H-1PV variants endowed with increased therapeutic impact on brain cancers, the Nüesch
group performed serial adaptation of H-1PV in patient-derived human glioblastoma cell
lines. This led to the isolation of H-1PV variants characterized by an in-frame deletion in the
NS region and 1–3 amino acid substitutions in the capsid surface [
47
]. To further enlarge the
spectrum of oncolytic PVs, ongoing work of the group determines the genome sequences
of PV strains derived from different species and originally isolated as contaminants of
various human cancer cell lines ([
103
]). Obtained sequences are currently used to produce
replication-competent molecular clones. In addition, diagnostic tools (e.g., mAbs) are
prepared to enable assessment of the oncolytic potential of H-1PV and other PV strains in
various cancer entities.
The group of Assia Angelova together with Jean Rommelaere, within Guy Ungerechts’
Clinical Cooperation Unit Virotherapy in Heidelberg, currently develops a heterotypic
pancreatic ductal adenocarcinoma (PDAC) spheroid system. Spheroids are generated with
PDAC cells, fibroblasts and endothelial cells and allow further coculture with immune cells.
They offer a relevant preclinical tumor model for analysis of the tumor-suppressive and
immunostimulating capacity of oncolytic PVs and other OVs presently in development.
2.7. Vaccinia Virus Platform
The group of Ulrich Lauer at the University Hospital Tübingen not only investigates a
suicide gene-armed MeV construct (MeV-SCD, see above and clinical research chapter),
but also a vaccinia virus (VV) Lister derivative (GLV-1h94) encoding the same prodrug-
converting enzyme, which locally converts the prodrug 5-FC into the chemotherapeutic
compound 5-FU. In a systematic evaluation of the NCI-60 tumor cell panel using GLV-1h94
as monotherapy, different levels of cellular resistance were observed within the cell lines,
which, however, could be completely overcome by activation of the prodrug system. A
more detailed study of the prodrug system revealed that the cytotoxic effect of converted
5-FU is directed either against the cells or against the viral particles, and this process
apparently relies on the balance between cell line-specific susceptibility to GLV-1h94-
induced oncolysis and 5-FU sensitivity [
70
]. In further work by the Lauer lab, the oncolytic
VV derivative GLV-1h68, which the group previously explored in a clinical study for
treatment of peritoneal carcinomatosis (see [
28
]), showed great promise in neuroendocrine
Viruses 2021,13, 1420 16 of 29
tumors (NETs). The Lauer group demonstrated that GLV-1h68, which includes three
expression cassettes encoding
β
-glucuronidase,
β
-galactosidase and green fluorescent
protein (GFP), exhibits stable cytotoxicity in human NET cells of various anatomical origins
and also a highly efficient production of progeny virus particles. Moreover, additional
combination with the mTOR inhibitor everolimus, which is approved for treatment of
metastatic NETs, did not impair replication of GLV-1h68 suggesting that combinatorial
treatment is not an obstacle for further development of the approach [73].
Markus Moehler at Johannes Gutenberg University Mainz analyzed the two oncolytic
VVs, JX-594-GFP
+
/hGM-CSF (JX-GFP), which is derived from JX-594 [
22
] and TG6002 [
104
]
which are genetically modified by secreting hGM-CSF or encoding CD-UPRT for converting
5-FC into 5-FU, respectively [
61
]. In their human melanoma model, they compared the
properties to kill human tumor cells and again induce immunogenic cell death (ICD).
Combined treatment of JX-GFP or TG6002 with 5-FU resulted in strongly reduced tumor cell
viability. TG6002 in combination with 5-FC did not significantly strengthen the reduction
of cell viability in this setting. After viral infection, the ICD markers calreticulin and high
mobility group 1 protein (HMGB1) and strong DC maturation were detected. Thus, JX-GFP
and TG6002 lyse human melanoma cells and induce immunostimulatory effects to promote
human antitumor immune responses [61].
2.8. Vesicular Stomatitis Virus Platform
Vesicular stomatitis virus (VSV), and its application in virotherapy, has been the focus of
research activities of the group of Jennifer Altomonte at the Klinikum rechts der Isar, Technical
University of Munich. VSV is a promising candidate for oncolytic virotherapy, due to its
broad host cell tropism and rapid and robust replication and tumor cell lysis; however, the
clinical translation of VSV has been substantially hindered by concerns surrounding the known
neurotoxic side effects associated with this virus [
105
]. Newcastle disease virus (NDV) offers
the aspect of viral spread via syncytia and has also demonstrated a promising safety profile
in humans [
106
]; however, as it is a deadly pathogen in its avian hosts, the use of oncolytic
strains of NDV has been severely restricted due to the severe environmental risk it poses. The
Altomonte group has recently reported a strategy to exploit the beneficial aspects of these
viruses, while eliminating the safety concerns of each [
50
]. The novel chimeric virus, VSV-NDV,
utilizes the rapidly replicating VSV backbone, wherein the targeting glycoprotein of VSV was
replaced with the fusogenic envelope proteins of NDV. By further engineering the fusion (F)
protein in the recombinant vector, the group was able to achieve extensive tumor-specific
syncytia formation, which is known to be a beneficial mechanism of direct oncolysis, as well as
a potent inducer of ICD [
107
,
108
].
In vivo
, intravenous administration of VSV-NDV led to a
nearly twofold increase in survival time in mice bearing multifocal, orthotopic HCC, as well as
a 1000-fold elevation in the maximum tolerated dose, compared with VSV [
50
]. Based on these
and additional unpublished data, the group is now working towards clinical translation of
VSV-NDV in thecontext of aplannedspin-out, Fusix Biotech. In ordertofurther exploretheVSV-
NDV platform as a potential treatment partner with established cancer immunotherapeutics,
Altomonte and colleagues have recently reported the enhancement of adoptive T cell therapy
through combination with fusogenic VSV-NDV in an immunocompetent murine model of
melanoma [
76
]. In this study, it was shown that pre-treatment with VSV-NDV allowed for
upregulation of MHC-I on tumor cells and enhanced recruitment of adoptively transferred
cytotoxic T cells, resulting in synergistic treatment responses. Strikingly, therapeutic responses
were not limited to tumors directly injected with VSV-NDV, but abscopal effects in contralateral
tumors were evident as well, which resulted in a significant survival prolongation. The group
now focuses on establishing an expanded proof-of-concept in more challenging preclinical
tumor models, as well as the development of enhanced immunostimulatory VSV-NDV vectors
through arming and optimized combination approaches with other cancer immunotherapeutics.
Viruses 2021,13, 1420 17 of 29
3. Recent Clinical Virotherapy Research Activities in Germany
As with most novel therapeutics, their translation from the laboratory into clinical trials
and, ultimately, clinical routine, represents a tremendous undertaking often spanning many
years, if not decades. This is especially true for completely new classes of drugs (such as
OVs) with a potential for previously unknown adverse side effects. As reflected in the study
protocols of completed and ongoing early virotherapy trials, considerable emphasis is put on
safety aspects including biodistribution and shedding of virotherapeutics. Along the way from
bench to bedside, the vast majority of therapeutic candidates drop out and the many reasons
for this include lack of efficacy, severe adverse events, regulatory hurdles, manufacturing
issues and financial bottlenecks. While most OVs are still being developed pre-clinically or
clinically, the first OV therapeutic (Talimogene laherparepvec, T-VEC, trade name: Imlygic
®
)
has received approval for late-stage melanoma therapy by the FDA and EMA in 2015 [
21
]. This
is widely regarded as a breakthrough for the whole virotherapy field, opening up the potential
for routine use of virotherapeutics in the clinic. While T-VEC has demonstrated safety and
efficacy in the respective phase III trial (OPTIM [
109
]) against malignant melanoma, we strongly
believe that clinical testing of additional and potentially improved oncolytics will add to our
armamentarium in the fight against cancer.
The Paul Ehrlich Institut (PEI), which is the Federal Higher Authority being responsible
for all clinical virotherapy activities in Germany, actively supports the transfer of virotherapy
research results to clinical virotherapy trials in cancer patients. Interaction with the PEI works
mainly via the institutionalized platform of the German Cancer Consortium, DKTK [
110
].
Researchers planning a clinical virotherapy trial are supported at the DKTK platform by
counseling sessions in partnership with PEI, which are offered already at an early stage in the
development of new active substances and therapeutic methods. As part of their partnership,
PEI and DKTK have established special consulting formats to promote the initiation of clinical
trials in the academic environment. Research-based physicians and scientists at DKTK who
are planning a clinical virotherapy trial are supported by free kick-off meetings to answer
general questions and by scientific advisory meetings on product-specific issues. An overview
of all ongoing virotherapy studies is provided by the DKTK Study Register of the Clinical
Communication Platform
(dktk.org/ccpstudienregister).
Taken together, the close interaction
with PEI has worked out to be very helpful and instrumental, especially in the setup and
configurationofnovelphaseIprotocol types. Thus, virotherapyinGermany receives continuous
and sustained support.
In the following, we will summarize recent clinical trials that were initiated by or
involved investigators in Germany (for an overview see Table 2).
Viruses 2021,13, 1420 18 of 29
Table 2.
Recent virotherapy trials initiated by or involving the authors of this article. Pexa-Vec = Pexastimogene devacirepvec;
T-Vec = Talimogene laherparepvec; SCD = super cytosine deaminase; GALV-GP-R
-
= gibbon ape leukemia virus glycoprotein;
ICI = immune checkpoint inhibition; CPA = cyclophosphamide; GBM = glioblastoma multiforme; PDAC = pancreatic ductal
adenocarcinoma; GI = gastrointestinal; NSCLC = non-small cell lung cancer; HCC = hepatocellular carcinoma; TNBC = triple-
negative breast cancer; CRC = colorectal carcinoma; CSCC = Cutaneous Squamous Cell Carcinoma; NET = neuroendocrine
tumors; C = completed; P = planned; T = terminated; O = ongoing.
Virus
Platform
Virus
Name Transgene Combined
With Name Identifier Entity Phase Status
PV ParvOryx/
H1-PV ParvOryx01 Eudra-CT
2011-000572-33 GBM I/IIa C
ParvOryx/
H1-PV ParvOryx02 Eudra-CT
2015-001119-11 Metastatic
PDAC II C
MeV MeV-IL12 IL-12 CanVirex01 GI basket
trial I/II P
MeV-SCD SCD 5-FC + ICI NSCLC P
MeV-SCD SCD 5-FC + ICI GI basket
trial P
VV Pexa-Vec/
JX-594 GM-CSF TRAVERSE NCT01387555 HCC IIb C
Pexa-Vec/
JX-594 GM-CSF sorafenib PHOCUS NCT02562755 HCC III T
HSV
T-Vec/
Imlygic GM-CSF ICI MASTERKEY-
265 NCT02263508 Melanoma Ib/III T
T-Vec/
Imlygic GM-CSF ICI Eudra-CT
2015-005480-16
TNBC
and CRC
with liver
metas-
tases
Ib O
T-Vec/
Imlygic GM-CSF ICI Eudra-CT
2019-001906-61 Melanoma II O
T-Vec/
Imlygic GM-CSF ICI Eudra-CT
2014-005386-67
HCC &
non-HCC
liver
metas-
tases
Ib/II O
RP-1 GM-CSF,
GALV-GP-
R- ICI CERPASS Eudra-CT
2018-003964-30 CSCC II O
AdV AdVince CPA RADNET Eudra-CT
2014-000614-64
NET with
liver
metas-
tases
I/IIa O
PeptiCRAd-
1CD40L,
OX40L ICI START Eudra-CT
2021-000642-18 Basket
trial I P
CoxV V937/CVA21 ICI NCT04521621 Basket
trial Ib/II O
ReoV Pelareorep ICI GOBLET Eudra-CT
2020-003996-16 GI basket
trial I/II P
VSV VSV-GP GP of
LCMV ICI Basket
trial I P
3.1. H-1 Parvovirus (H-1PV)
The first PV clinical trial, ParvOryx01 (EudraCT 2011-000572-33), performed at Hei-
delberg University Hospital, demonstrated the excellent safety profile of H-1PV upon both
local and systemic administration in glioblastoma patients. In addition, trial-accompanying
research provided a first hint of PV treatment-induced enhanced inflammation (“warming
up”) in the tumor microenvironment [
97
]. Further detailed analysis of resected glioblastoma
tissues revealed the formation of large intratumoral immune infiltrates composed of acti-
vated (CD25
+
, granzyme B- and perforin-expressing) cytotoxic T lymphocytes (CTLs) [
111
].
Importantly, tumor-infiltrating CTLs were PD-1-negative and only scarcely scattered Treg
Viruses 2021,13, 1420 19 of 29
cells were detected within the infiltrates. Glioblastoma-associated microglia/macrophages
similarly displayed an activated phenotype characterized by increased CD68, cathepsin B
and iNOS expression. Production of proinflammatory cytokines, in particular interferon-
gamma (IFN-
γ
) and interleukin-2 (IL-2), was also observed in a subset of ParvOryx01
patients’ tumor samples. The above findings provided valuable first-in-man experience and
laid the ground for future parvoviro-immunotherapy clinical developments. Among these,
one approach, namely combining H-1PV with bevacizumab and checkpoint inhibitors,
deserves special consideration based on encouraging data from recent compassionate
use programs in recurrent glioblastoma [
112
]. Partial to complete tumor remission was
documented in patients who received the PV in combination with bevacizumab and PD-1
blockade. The response rate was significantly higher than reported in the literature for
bevacizumab and nivolumab applied as monotherapy.
Clinical evidence that immune mechanisms underlie PV-mediated tumor suppression
also came from the second H-1PV single center trial in Heidelberg, ParvOryx02 (EudraCT
2015-001119-11) in patients with metastatic pancreatic cancer. In this study, virus adminis-
tration was found to be associated with a favorable clinical outcome in two out of seven
patients, with radiologically proven partial response and remarkably long survival. More-
over, the findings of accompanying research confirmed immunological effects of H-1PV
on the tumor microenvironment associated with a favorable clinical outcome (manuscript
under review). Therapy was very well tolerated without any clinically detectable adverse
events, except elevation of C-reactive protein (CRP) in four out of seven patients. No
dose-limiting toxicities occurred, accordingly. Viral shedding data attest an excellent safety
profile of H-1PV with consistent formation of anti-drug antibodies after virus administra-
tion and no subsequent detection of infectious viral particles in body fluids on day 18 or
thereafter. Viral tumor homing after intravenous administration could be determined in
patients of all dose levels. Altogether, the clinical experience gathered so far provides a
strong impetus for further H-1PV-based cancer immunotherapy development (recently
reviewed in [98,100]).
3.2. Measles Viruses
The Heidelberg Team headed by Guy Ungerechts has engineered multiple transgene-
encoding oMeVs for increased therapeutic efficacy (see preclinical research highlights
and [
28
]). One of the lead candidates, an oMeV encoding a secreted form of interleukin
12 [
65
] (MeV-IL12), is currently being moved into clinical testing. A phase I/II investigator-
initiated trial in Heidelberg is scheduled to be launched in 2022. This trial is sponsored
by the Heidelberg University Hospital spin-off company CanVirex AG and will assess
the immunovirotherapeutic efficacy of MeV-IL12 against multiple refractory solid tumors
(basket trial). With multiple patent families, the Heidelberg team along with CanVirex
AG is aiming to launch a series of such immunovirotherapy trials over the next years.
Importantly, these trials will be accompanied by a comprehensive translational research
program to unravel immune signatures associated with response to immunomodulating
oMeV using state-of-the-art techniques, including laser capture microdissection and auto-
mated microscopy after immunohistochemistry to quantify tumor-infiltrating lymphocyte
subpopulations, cytokine and chemokine profiling by multiplex arrays, TCR repertoire
sequencing, analysis of humoral and cellular anti-tumor immune responses as well as
tumor expression profiling with neoepitope discovery.
As described previously ([
28
]), the group of Ulrich Lauer is investigating an oMeV
armed with the prodrug-converting enzyme SCD (MeV-SCD), which locally converts the
prodrug 5-FC into the chemotherapeutic compound 5-FU. A monocenter investigator-
initiated trial (IIT) sponsored by University Hospital Tübingen is scheduled by the Lauer
team in Tübingen, in which safety and potential efficacy of MeV-SCD plus prodrug 5-FC
combined with pembrolizumab is evaluated in patients with stage III/IV non-small cell
lung cancer (NSCLC). The current standard of care for NSCLC is the anti-PD-1 immune
checkpoint inhibitor (ICI) pembrolizumab, although in some cases with a low objective
Viruses 2021,13, 1420 20 of 29
response rate. Accordingly, there is an urgent need for novel combination treatments
that further enhance the antitumoral efficacy of pembrolizumab. This study aims to
additionally administer MeV-SCD IT into NSCLC tumor lesions of patients who are under
pembrolizumab monotherapy, however with limited response. The analyses accompanying
the study will include the characterization of the tumor-specific immune response in blood
samples as well as in tumor biopsies, the investigation of viral parameters such as infection,
replication and marker gene expression of MeV-SCD, as well as the determination of
the conversion rates of 5-FC to cytotoxic 5-FU derivatives. In addition, the antibody-
/nanobody-based immuno-imaging (immunoPET) established at the University Hospital
Tübingen will be applied in this study in order to guide and predict the efficacy of this
combined immunovirotherapeutic approach. Another projected monocenter IIT (sponsor:
University Hospital Tübingen) will investigate MeV-SCD in patients with gastrointestinal
(GI) tumors. In this study, patients with GI tumors will be treated IT by endoscopic
guidance with MeV-SCD alone or in combination with the prodrug 5-FC or the anti-PD-
1 checkpoint inhibitor pembrolizumab. Primary objectives are to determine the safety,
tolerability and immunogenicity of each treatment regimen.
3.3. Vaccinia Virus
Pexastimogene devacirepvec (Pexa-Vec) is a VV-based oncolytic immunotherapy
designed to preferentially replicate in and destroy tumor cells while stimulating anti-tumor
immunity by expressing GM-CSF. Markus Moehler (Johannes Gutenberg University Mainz)
with investigators from multiple German sites (including Hamburg, Heidelberg, and
Munich) promoted a randomized, open-label, international phase IIb trial that investigated
whether Pexa-Vec improved overall survival (OS, primary endpoint) over Best Supportive
Care (BSC) alone in HCC patients who failed sorafenib (TRAVERSE study) [
113
]. 129
patients were randomized 2:1. Pexa-Vec was given as a single intravenous (IV) infusion
followed by up to 5 IT injections. Unfortunately, a high drop-out rate in the control
arm (63%) confounded the response-based endpoints. Median OS for the generally well-
tolerated Pexa-Vec plus BSC vs. BSC alone was 4.2 and 4.4 months, respectively. However,
induction of immune responses to VV antigens and HCC associated antigens were clearly
observed in patients by ELISPOT analyses of immune response to VV,
β
-galactosidase and
tumor antigens before (pre-dose) and 6 weeks after treatment (post-dose). Detection of
T-cells specific for tumor-associated antigen peptides with detectable increased responses
against MAGE-A1 and MAGE-A3, as well as HCV peptides in HCV-positive patients
were also documented. Despite a tolerable safety profile and induction of T cell responses,
Pexa-Vec did not improve OS as second-line therapy. The true potential of OVs may thus lie
in the treatment of patients with earlier disease stages or minimal residual disease, which
should be addressed in future studies.
Since 2019, the PHOCUS multi-center phase III clinical trial has been completed
(NCT02562755). Multiple German sites (incl. Aachen, Bonn, Dresden, Frankfurt am Main,
Hamburg, Hannover, Heidelberg, Mainz, München, Tübingen, Ulm) participated. In this
trial, IT-administered Pexa-Vec followed by sorafenib was compared to sorafenib treatment
alone in the first-line treatment of patients with advanced hepatocellular carcinoma. The
study enrolled a total of 459 patients of which 234 received Pexa-Vec followed by sorafenib
and 225 received sorafenib alone. The trial was terminated early after an interim analysis
came to the conclusion that the trial was unlikely to meet its primary objective at the
initially planned study end. In July 2020, data collection for primary outcome measure was
completed, and we are currently awaiting publication of the final trial results.
3.4. Herpes Virus
The MASTERKEY-265/KEYNOTE-034 trial (NCT02263508), sponsored by Amgen
was a phase Ib/III trial in unresectable late stage IIIB to IVM1c melanoma with talimogene
laherparepvec (T-VEC, Imlygic
®
) in combination with pembrolizumab and was launched
back in 2014. However, the phase Ib part was conducted in overseas only. Results were
Viruses 2021,13, 1420 21 of 29
extremely promising with no dose-limiting toxicities, a confirmed objective response rate
of 62% and a complete response rate of 33%. Patients who responded to combination
therapy had increased CD8
+
T cells, elevated PD-L1 protein expression, as well as IFN-
γ
gene expression on several cell subsets in tumors after T-VEC treatment [
114
]. German
trial centers (Berlin, Dresden, Erlangen, Essen, Hannover, Heidelberg, Kiel, Leipzig, Mainz,
Mannheim, München, Regensburg, Tübingen, Würzburg) participated later in the phase
III part of the trial which was stopped for futility after an interim analysis by the Data
Monitoring Committee. No new safety signals were observed and results are anticipated
to be presented at ESMO 2021.
Besides, T-VEC was evaluated in several other multi-center clinical trials involving
German study sites. This list includes early (phase Ib and/or II) trials for treatment of
triple-negative breast cancer and colorectal carcinoma with liver metastases (Eudra-CT No:
2015-005480-16; German study sites: Berlin, Bonn, Tübingen), melanoma (Eudra-CT No:
2019-001906-61; Germany study sites: Berlin, Dresden, Hannover, Heidelberg, Regensburg,
Tübingen), or non-resectable liver tumors (Eudra-CT No: 2014-005386-67; German study
sites: Berlin, Bonn, Reutlingen, Tübingen).
CERPASS (Eudra-CT No: 2018-003964-30; sponsor Replimune): besides T-VEC, an-
other herpes simplex virus type 1, named RP-1, is investigated in a phase II trial in combina-
tion with cemiplimab (anti-PD-1 mAb) in patients with advanced cutaneous squamous cell
carcinoma (CSCC). RP-1 is a selectively replication competent HSV-1 virus which contains
two additional sequences, one for hGM-CSF and one for gibbon ape leukemia virus fuso-
genic glycoprotein (GALV-GP-R
-
). The expression of GALV-GP-R- causes enhancement
of viral spreading through the tumor, triggered by the induction of syncytia formation
in infected tumor cells. The immunogenic cell death evoked by this pathway together
with the local expression of hGM-CSF and the additional combination with the checkpoint
inhibitor cemiplimab is expected to result in a synergistically enhanced anti-tumor immune
response, which is intended to lead to an improvement of clinical benefit. The primary
objective of this study is to assess the clinical benefit of cemiplimab applied intravenously
as monotherapy compared to cemiplimab in combination with intratumorally adminis-
tered oncolytic RP-1 in patients with advanced CSCC. The study will now be expanded to
include study centers in Germany (e.g., Tübingen and other skin cancer centers).
3.5. Adenovirus
RADNET (Eudra-CT no: 2014-000614-64): this single-center phase I/IIa clinical study
of oAd AdVince was initially launched in 2016 in Sweden, evaluating the safety of repeated
infusions of AdVince into the hepatic artery of patients with metastatic neuroendocrine
tumors (NETs). By inserting the human chromogranin A (CgA) promoter and the mouse
H19 insulator as well as microRNA target sequences in the 3
0
UTR of E1A, AdVince is
designed to replicate specifically in neuroendocrine tumor cells but not to damage any
normal hepatocytes. Furthermore, patients receive cyclophosphamide, if tolerated, as a
concomitant therapy to transiently suppress antiviral immunity and potentially increase
the therapeutic effect of AdVince. The primary aim of this study is to evaluate the safety
and the maximum tolerated dose (MTD) of AdVince for patients suffering from advanced
NETs with multiple liver metastases refractory to surgical resection. Secondary objectives
include the anti-tumor efficacy and the replication profile of AdVince as well as the humoral
and cytokine-mediated immune response triggered by this virotherapy. The study is now
scheduled to be expanded with an additional study center in Tübingen, Germany, in order
to increase the number of participating patients.
START (Eudra-CT No: 2021-000642-18): the START (
S
afety and anti-
T
umor
A
ctivity
of PeptiC
R
Ad-1 in
T
reatment of Cancer) study is an open-label, non-randomized, first-
in-human phase I trial of PeptiCRAd-1 against multiple solid tumors (melanoma, triple-
negative breast cancer and NSCLC). The adenovirus-based oncolytic encodes two addi-
tional therapeutic transgenes (CD40L, OXO40L), which will be expressed in tumor cells
upon infection to further stimulate the innate and adaptive immune response. Moreover,
Viruses 2021,13, 1420 22 of 29
the virus is coated with NY-ESO-1 and MAGE-A3 peptides to direct the immune system
against NY-ESO-1 or MAGE-A3-positive tumor cells. Patients will be pre-treated with
intravenous cyclophosphamide to enhance the therapeutic efficacy of PeptiCRAd-1, which
will be administered intratumorally in six individual doses. As a common theme with other
OV trials, PeptiCRAd-1 is combined with immune checkpoint inhibition (pembrolizumab).
The bicentric trial will be performed at study centers in Frankfurt and Heidelberg (sponsor:
VALO Therapeutics).
3.6. Coxsackievirus A21
V937 (NCT04521621): a phase Ib/II clinical study of intratumoral administration of
V937 (Coxsackievirus A21) in combination with pembrolizumab (MK-3475) in patients
with advanced/metastatic solid tumors. Safety and dose finding of the above-mentioned
combination is a primary objective, and again, synergistic effects of checkpoint inhibition
and oncolytic agent are anticipated (sponsor: MSD; participating trial centers in Germany
are Tübingen and Heidelberg).
3.7. Reovirus
GOBLET (Eudra-CT No: 2020-003996-16): a phase I/II multiple-indication biomarker,
safety, and efficacy study in advanced or metastatic Gastrointestinal cancers explOring
treatment comBinations with peLarEorep and aTezolizumab. In this study, the hypothesis
is that treatment with pelareorep will prime the TME for checkpoint blockade therapy,
thereby increasing PD-L1 expression and the number of new T cell clones within the tumor,
both of which are associated with increased response to checkpoint blockade [
115
]. In this
trial, the virus will be administered intravenously. The trial is sponsored by Oncolytics
Biotech Inc. and organized by the AIO-Studien-gGmbH (multiple trial centers in Germany).
3.8. Vesicular Stomatitis Virus
A phase I open-label, dose escalation trial is planned to investigate a novel replication-
competent vesicular stomatitis virus pseudotyped with the glycoprotein of the lymphocytic
choriomeningitis virus (VSV-GP) as monotherapy and in combination with an anti-PD-1
mAb in patients with advanced, metastatic or relapsed/recurrent malignant solid tumors.
This trial will evaluate the safety and tolerability of VSV-GP when given via intravenous
and/or intratumoral routes. Furthermore, early efficacy signals and the MTD or recom-
mended phase II dose for VSV-GP both as monotherapy and in combination with an
anti-PD-1 mAb are major trial objectives. This study is sponsored by Boehringer Ingelheim
Ltd. with Ulrich Lauer from University Hospital Tübingen as the European coordinat-
ing investigator.
4. Perspectives
Four years ago, we—the German virotherapy community—concluded in our state-of-
the art review in 2017 ([
28
]) that “successful translation of German preclinical activities has
the potential to inspire a boom in early clinical trials in the near future”. In this context,
connecting (i) academic research, (ii) technology transfer, and (iii) regulatory processes
was identified to be most critical. We do believe that significant advances within all three
sectors have been made.
The recent progress in preclinical, translational and clinical virotherapy research in
Germany reported in this state-of-the-art review establishes a vantage point for future
endeavors that aim at defining new and clinically effective virotherapeutics based on estab-
lished technology and advancing clinical development of established OVs. As such, it will
be of interest to further engineer and develop the emerging new virus strains, serotypes,
fitness mutants and chimeras for virotherapeutic applications. Furthermore, recent scien-
tific insights on virus entry, host-virus interactions after systemic virus application and
virus biodistribution should be exploited to further improve OVs or optimize application
modalities. We are curious to see whether the reported virus targeting approaches will
Viruses 2021,13, 1420 23 of 29
come to wider application when developing OVs with improved efficiency towards clinical
application. In this regard, various efficacy-enhanced OVs have become available resulting
from enhanced host cell lysis, improved immune activation, and/or encoded therapeutic
proteins or RNAs. In the context of immuno-oncology, it will be of interest to investigate
how the discussed new virus platforms (e.g., arenaviruses), genetically delivered therapeu-
tic molecules or tumor antigens, combination regimens, or innovative approaches, such as
the redirection of antiviral antibodies for cancer cell killing unfold therapeutic potential as
combination immuno-(viro-)therapeutics. Increasingly, the conduction of clinical trials will
give us the opportunity for systematic reverse-translational activities, which means new
assignments for our preclinical research programs.
Regarding the current limitations of and future challenges for clinical trials in the
virotherapy field, from the German perspective we believe that safety and feasibility has
been demonstrated in the last decade, and thus the focus should be shifted to oncolytic
potency. Virotherapists could learn from the brave investigators translating cellular thera-
peutics, such as CAR T cells, who have been managing severe side effects for years within
a specific clinical trial framework. Besides the virus engineering approaches discussed
above, future virotherapy trial protocols should include escalated virus doses and multi-
modal treatment regimens balancing the thus-far promising safety data with the need for
enhanced therapeutic efficacy. To ensure maximum patient safety, these trials should be
conducted in specialized centers only, which can provide the necessary infrastructure and
trained personnel at all times. Further, we believe that concerted efforts need to be under-
taken to select the patients most likely benefitting from virotherapy. This should include
the exploration of omics-data to identify and validate robust prognostic (bio-)markers of
virotherapy response.
In Germany, a decent number of spin-off biotech companies dedicated to OV devel-
opment launched within the last couple of years including Abalos Therapeutics GmbH
(LCMV), CanVirex AG (MeV-IL12), Oryx (H-1PV), and XVir (Ad XVir-N-31). Interna-
tionally, in a growing immuno-oncology market big pharma/biotech acquired several
virotherapy spin-offs with upfront payments of USD 300 million and more. If the focus
shifted to the above-mentioned German spin-offs, a booster of translational activities can
be anticipated. Accordingly, if big pharma companies keep consistent engagement, we can
expect a growing number of pivotal multicenter trials.
In terms of harmonization of regulatory processes, we recognized over the last few
years that there is an enhanced interaction and fruitful early dialog between the authorities
and the research community. Obviously, speed and shape of regulation is triggered by
the medical need, which is impressively demonstrated by EMA’s fast-track approvals of
(vector-based) vaccines against COVID-19. In oncology, this could be a future blueprint for
accelerated evaluation and assessment for certain cancer entities and disease stages.
Altogether, the German OV field has clearly advanced on all relevant levels, including
pre-clinical vector development and translational efforts. However, the ultimate benchmark
for our success needs to be the clinical benefit of virotherapy for cancer patients. This
can only be achieved on a regular basis and in a sustainable manner if we accomplish
marketing approval of several OVs for multiple tumor entities in the future.
Author Contributions:
Conceptualization, D.M.N., M.F.L. and G.U.; writing—original draft prepara-
tion, all authors; writing—review and editing, all authors; final review and editing, D.M.N., M.F.L.
and G.U.; visualization, M.F.L. All authors have read and agreed to the published version of the
manuscript.
Funding:
Research activities in the authors’ labs were funded by the Wilhelm Sander-Stiftung, grant
2020.069.1 (D.M.N.); by the German Research Association (DFG) Sonderforschungsbereich (SFB)
824 subproject C7, the German Cancer Aid, grant 70113272, and the European Research Council
(ERC) under the European Union’s Horizon 2020 research and innovation program, grant agreement
No. 853433 (J.A.); by the German Research Association (DFG), grant EH 192/5-1 (A.E.); by the
German Cancer Aid (Deutsche Krebshilfe), grant number: 70112382 (J.E.); by the German Research
Association (DFG), grant EN 1119/2-2, the Wilhelm Sander-Stiftung, grant 2018.058.1 and the Else
Viruses 2021,13, 1420 24 of 29
Kröner-Fresenius-Stiftung (2019_EKMS.02) (C.E.E.); by the German Cancer Aid (Deutsche Krebshilfe),
grant 70112382, the Wilhelm Sander-Stiftung, grant 2017.101.1, the Technische Universität Berlin
internal research tool ProTUTec, grant 17011/TUB and the excellence strategy of the Federation
and Federal States by the Berlin University Alliance (H.F.); by the German Federal Ministry of
Education and Research (BMBF) and the Federal States of Germany grant “Innovative Hochschule”
FKZ 3IHS024D (S.K.); by the Center for Biomedical Education and Research (ZBAF) at University
Witten/Herdecke (F.Kr.); by the German Cancer Aid (Deutsche Krebshilfe), grant number: 70113873
(F.Kü.); by the Luxembourg Cancer Foundation, Télévie and the Cooperational Research Program of
the German Cancer Research Center (DKFZ), Heidelberg with the Ministry of Science and Technology,
Israel and a generous donation from AndréWelter (A.M.); by the German Cancer Aid (Deutsche
Krebshilfe), grant number: 109614, the CI3 Cutting Edge Cluster Funding of the Federal German
Ministry of Education and Research of Germany (BMBF) grant 031A010B (M.D.M.); by Oryx GmbH
& Co. KG, Oncolytic Virus R&D program (J.R., A.M., A.A., and K.G.); by the German Cancer
Consortium (DKTK) of the German Cancer Research Center (DKFZ) (U.M.L.); by the Alois Hirdt-
Erben- und Wieland Stiftung grant 230/13/BA/00 (G.U.)
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest:
J.A. is co-inventor on a patent of the VSV-NDV technology. C.E.E. is listed as co-
inventor on patent (applications) related to oncolytic measles viruses as (cancer) immunotherapeutics.
H.F. has patented the CVB3 variant PD for use in cancer therapy and have a patent pending for
treatment of cancer using miR-TS in oncolytic CVB3. P.S.H. is cofounder of XVir Therapeutics
GmbH. K.S.L. is involved in the development of LCMV for clinical application in oncology in
cooperation with and as co-founder of Abalos Therapeutics GmbH. The clinical studies of ParvOryx
were sponsored by Oryx GmbH & Co. K.G., J.R., A.A., and A.M. received research grants from
Oryx GmbH. J.R., A.M., A.A., and K.G. are co-inventors of various patents (applications) relating to
the content of this review. M.M. received research grants from Amgen and Transgene SA, Illkirch-
Graffenstaden, Alsace, France (JX-GFP and TG6002 research). The MASTERKEY-265/KEYNOTE-034
trial (NCT02263508) was sponsored by Amgen Inc., Thousand Oaks, CA, USA. G.U. is founder,
and CMO/CSO of CanVirex AG, a company developing oncolytic measles viruses as (cancer)
immunotherapeutics. The other authors declare no conflict of interest. The funders had no role in
the design of the study; in the collection, analyses, or interpretation of data; in the writing of the
manuscript, or in the decision to publish the results.
References
1.
Breitbach, C.J.; Lichty, B.D.; Bell, J.C. Oncolytic Viruses: Therapeutics with an Identity Crisis. EBioMedicine
2016
,9, 31–36.
[CrossRef] [PubMed]
2.
Cattaneo, R.; Miest, T.; Shashkova, E.V.; Barry, M.A. Reprogrammed viruses as cancer therapeutics: Targeted, armed and shielded.
Nat. Rev. Microbiol. 2008,6, 529–540. [CrossRef] [PubMed]
3.
Fisher, K.; Hazini, A.; Seymour, L.W. Tackling HLA Deficiencies Head on with Oncolytic Viruses. Cancers
2021
,13, 719. [CrossRef]
[PubMed]
4.
Harrington, K.; Freeman, D.J.; Kelly, B.; Harper, J.; Soria, J.C. Optimizing oncolytic virotherapy in cancer treatment. Nat. Rev.
Drug Discov. 2019,18, 689–706. [CrossRef] [PubMed]
5.
Kaufman, H.L.; Kohlhapp, F.J.; Zloza, A. Oncolytic viruses: A new class of immunotherapy drugs. Nat. Rev. Drug Discov.
2015
,
14, 642–662. [CrossRef]
6.
Pikor, L.A.; Bell, J.C.; Diallo, J.S. Oncolytic Viruses: Exploiting Cancer’s Deal with the Devil. Trends Cancer
2015
,1, 266–277.
[CrossRef] [PubMed]
7.
Russell, L.; Peng, K.W.; Russell, S.J.; Diaz, R.M. Oncolytic Viruses: Priming Time for Cancer Immunotherapy. Bio. Drugs Clin.
Immunother. Biopharm. Gene. Ther. 2019,33, 485–501. [CrossRef]
8. Russell, S.J.; Barber, G.N. Oncolytic Viruses as Antigen-Agnostic Cancer Vaccines. Cancer Cell 2018,33, 599–605. [CrossRef]
9. Russell, S.J.; Peng, K.W.; Bell, J.C. Oncolytic virotherapy. Nat. Biotechnol. 2012,30, 658–670. [CrossRef]
10.
Woller, N.; Gürlevik, E.; Ureche, C.I.; Schumacher, A.; Kühnel, F. Oncolytic viruses as anticancer vaccines. Front. Oncol.
2014
,4,
188. [CrossRef]
11. Miest, T.S.; Cattaneo, R. New viruses for cancer therapy: Meeting clinical needs. Nat. Rev. Microbiol. 2014,12, 23–34. [CrossRef]
12.
Dorer, D.E.; Nettelbeck, D.M. Targeting cancer by transcriptional control in cancer gene therapy and viral oncolysis. Adv. Drug
Deliv. Rev. 2009,61, 554–571. [CrossRef] [PubMed]
Viruses 2021,13, 1420 25 of 29
13. Ruiz, A.J.; Russell, S.J. MicroRNAs and oncolytic viruses. Curr. Opin. Virol. 2015,13, 40–48. [CrossRef] [PubMed]
14.
Pearl, T.M.; Markert, J.M.; Cassady, K.A.; Ghonime, M.G. Oncolytic Virus-Based Cytokine Expression to Improve Immune
Activity in Brain and Solid Tumors. Mol. Ther. Oncolytics 2019,13, 14–21. [CrossRef]
15.
Binz, E.; Lauer, U.M. Chemovirotherapy: Combining chemotherapeutic treatment with oncolytic virotherapy. Oncolytic Virotherapy
2015,4, 39–48. [CrossRef] [PubMed]
16.
Bommareddy, P.K.; Shettigar, M.; Kaufman, H.L. Integrating oncolytic viruses in combination cancer immunotherapy. Nat. Rev.
Immunol. 2018,18, 498–513. [CrossRef]
17.
Martin, N.T.; Bell, J.C. Oncolytic Virus Combination Therapy: Killing One Bird with Two Stones. Mol. Ther. J. Am. Soc. Gene Ther.
2018,26, 1414–1422. [CrossRef]
18.
Ottolino-Perry, K.; Diallo, J.S.; Lichty, B.D.; Bell, J.C.; McCart, J.A. Intelligent design: Combination therapy with oncolytic viruses.
Mol. Ther. J. Am. Soc. Gene Ther. 2010,18, 251–263. [CrossRef]
19.
Twumasi-Boateng, K.; Pettigrew, J.L.; Kwok, Y.Y.E.; Bell, J.C.; Nelson, B.H. Oncolytic viruses as engineering platforms for
combination immunotherapy. Nat. Rev. Cancer 2018,18, 419–432. [CrossRef]
20.
Macedo, N.; Miller, D.M.; Haq, R.; Kaufman, H.L. Clinical landscape of oncolytic virus research in 2020. J. Immunother. Cancer
2020,8. [CrossRef]
21.
Pol, G.J.; Levesque, S.; Workenhe, S.T.; Gujar, S.; Le Boeuf, F.; Clements, D.R.; Fahrner, J.E.; Fend, L.; Bell, J.C.; Mossman, K.L.; et al.
Trial Watch: Oncolytic viro-immunotherapy of hematologic and solid tumors. Oncoimmunology
2018
,7, e1503032. [CrossRef]
[PubMed]
22.
Breitbach, C.J.; Burke, J.; Jonker, D.; Stephenson, J.; Haas, A.R.; Chow, L.Q.; Nieva, J.; Hwang, T.H.; Moon, A.; Patt, R.; et al.
Intravenous delivery of a multi-mechanistic cancer-targeted oncolytic poxvirus in humans. Nature
2011
,477, 99–102. [CrossRef]
[PubMed]
23.
Russell, S.J.; Federspiel, M.J.; Peng, K.W.; Tong, C.; Dingli, D.; Morice, W.G.; Lowe, V.; O’Connor, M.K.; Kyle, R.A.; Leung, N.;
et al. Remission of disseminated cancer after systemic oncolytic virotherapy. Mayo Clin. Proc. 2014,89, 926–933. [CrossRef]
24.
Rehman, H.; Silk, A.W.; Kane, M.P.; Kaufman, H.L. Into the clinic: Talimogene laherparepvec (T-VEC), a first-in-class intratumoral
oncolytic viral therapy. J. Immunother. Cancer 2016,4, 53. [CrossRef]
25. Atasheva, S.; Shayakhmetov, D.M. Oncolytic Viruses for Systemic Administration: Engineering a Whole Different Animal. Mol.
Ther. J. Am. Soc. Gene Ther. 2021,29, 904–907. [CrossRef] [PubMed]
26.
Chakradhar, S. Viral vanguard: Designing cancer-killing viruses to chase metastatic tumors. Nat. Med.
2017
,23, 652–655.
[CrossRef]
27.
Marchini, A.; Scott, E.M.; Rommelaere, J. Overcoming Barriers in Oncolytic Virotherapy with HDAC Inhibitors and Immune
Checkpoint Blockade. Viruses 2016,8, 9. [CrossRef]
28.
Ungerechts, G.; Engeland, C.E.; Buchholz, C.J.; Eberle, J.; Fechner, H.; Geletneky, K.; Holm, P.S.; Kreppel, F.; Kühnel, F.;
Lang, K.S.; et al. Virotherapy Research in Germany: From Engineering to Translation. Hum. Gene Ther.
2017
,28, 800–819.
[CrossRef]
29.
Zhang, W.; Fu, J.; Liu, J.; Wang, H.; Schiwon, M.; Janz, S.; Schaffarczyk, L.; von der Goltz, L.; Ehrke-Schulz, E.; Dörner, J.; et al.
An Engineered Virus Library as a Resource for the Spectrum-wide Exploration of Virus and Vector Diversity. Cell Rep.
2017
,19,
1698–1709. [CrossRef]
30.
Mach, N.; Gao, J.; Schaffarczyk, L.; Janz, S.; Ehrke-Schulz, E.; Dittmar, T.; Ehrhardt, A.; Zhang, W. Spectrum-Wide Exploration of
Human Adenoviruses for Breast Cancer Therapy. Cancers 2020,12, 1403. [CrossRef]
31.
Zhang, W.; Mese, K.; Schellhorn, S.; Bahlmann, N.; Mach, N.; Bunz, O.; Dhingra, A.; Hage, E.; Lafon, M.E.; Wodrich, H.; et al.
High-Throughput Cloning and Characterization of Emerging Adenovirus Types 70, 73, 74, and 75. Int. J. Mol. Sci.
2020
,21, 6370.
[CrossRef] [PubMed]
32.
Kreppel, F.; Gackowski, J.; Schmidt, E.; Kochanek, S. Combined genetic and chemical capsid modifications enable flexible and
efficient de- and retargeting of adenovirus vectors. Mol. Ther. J. Am. Soc. Gene Ther. 2005,12, 107–117. [CrossRef] [PubMed]
33.
Kratzer, R.F.; Espenlaub, S.; Hoffmeister, A.; Kron, M.W.; Kreppel, F. Covalent decoration of adenovirus vector capsids with
the carbohydrate epitope
α
Gal does not improve vector immunogenicity, but allows to study the
in vivo
fate of adenovirus
immunocomplexes. PLoS ONE 2017,12, e0176852. [CrossRef]
34.
Wu, Y.; Li, L.; Frank, L.; Wagner, J.; Andreozzi, P.; Hammer, B.; D’Alicarnasso, M.; Pelliccia, M.; Liu, W.; Chakrabortty, S.; et al.
Patchy Amphiphilic Dendrimers Bind Adenovirus and Control Its Host Interactions and in Vivo Distribution. ACS Nano
2019
,13,
8749–8759. [CrossRef]
35.
Martin, N.T.; Wrede, C.; Niemann, J.; Brooks, J.; Schwarzer, D.; Kühnel, F.; Gerardy-Schahn, R. Targeting polysialic acid-abundant
cancers using oncolytic adenoviruses with fibers fused to active bacteriophage borne endosialidase. Biomaterials
2018
,158, 86–94.
[CrossRef] [PubMed]
36.
Niemann, J.; Woller, N.; Brooks, J.; Fleischmann-Mundt, B.; Martin, N.T.; Kloos, A.; Knocke, S.; Ernst, A.M.; Manns, M.P.; Kubicka,
S.; et al. Molecular retargeting of antibodies converts immune defense against oncolytic viruses into cancer immunotherapy. Nat.
Commun. 2019,10, 3236. [CrossRef] [PubMed]
37.
Krutzke, L.; Allmendinger, E.; Hirt, K.; Kochanek, S. Chorioallantoic Membrane Tumor Model for Evaluating Oncolytic Viruses.
Hum. Gene Ther. 2020,31, 1100–1113. [CrossRef] [PubMed]
Viruses 2021,13, 1420 26 of 29
38.
Feiner, R.C.; Kemker, I.; Krutzke, L.; Allmendinger, E.; Mandell, D.J.; Sewald, N.; Kochanek, S.; Müller, K.M. EGFR-Binding
Peptides: From Computational Design towards Tumor-Targeting of Adeno-Associated Virus Capsids. Int. J. Mol. Sci.
2020
,21,
9535. [CrossRef]
39. Eberle, J. Countering TRAIL Resistance in Melanoma. Cancers 2019,11, 656. [CrossRef] [PubMed]
40.
Sarif, Z.; Tolksdorf, B.; Fechner, H.; Eberle, J. Mcl-1 targeting strategies unlock the proapoptotic potential of TRAIL in melanoma
cells. Mol. Carcinog. 2020,59, 1256–1268. [CrossRef]
41.
Tolksdorf, B.; Zarif, S.; Eberle, J.; Hazini, A.; Dieringer, B.; Jönsson, F.; Kreppel, F.; Kurreck, J.; Fechner, H. Silencing of Mcl-1
overcomes resistance of melanoma cells against TRAIL-armed oncolytic adenovirus by enhancement of apoptosis. J. Mol. Med.
2021. [CrossRef] [PubMed]
42.
Berger, S.; Procko, E.; Margineantu, D.; Lee, E.F.; Shen, B.W.; Zelter, A.; Silva, D.A.; Chawla, K.; Herold, M.J.; Garnier, J.M.;
et al. Computationally designed high specificity inhibitors delineate the roles of BCL2 family proteins in cancer. eLife
2016
,5.
[CrossRef]
43.
Czolk, R.; Schwarz, N.; Koch, H.; Schötterl, S.; Wuttke, T.V.; Holm, P.S.; Huber, S.M.; Naumann, U. Irradiation enhances the
therapeutic effect of the oncolytic adenovirus XVir-N-31 in brain tumor initiating cells. Int. J. Mol. Med.
2019
,44, 1484–1494.
[CrossRef]
44.
Hindupur, S.V.; Schmid, S.C.; Koch, J.A.; Youssef, A.; Baur, E.M.; Wang, D.; Horn, T.; Slotta-Huspenina, J.; Gschwend, J.E.; Holm,
P.S.; et al. STAT3/5 Inhibitors Suppress Proliferation in Bladder Cancer and Enhance Oncolytic Adenovirus Therapy. Int. J. Mol.
Sci. 2020,21, 1106. [CrossRef] [PubMed]
45.
Lichtenegger, E.; Koll, F.; Haas, H.; Mantwill, K.; Janssen, K.P.; Laschinger, M.; Gschwend, J.; Steiger, K.; Black, P.C.; Moskalev, I.;
et al. The Oncolytic Adenovirus XVir-N-31 as a Novel Therapy in Muscle-Invasive Bladder Cancer. Hum. Gene Ther.
2019
,30,
44–56. [CrossRef] [PubMed]
46.
Hazini, A.; Pryshliak, M.; Brückner, V.; Klingel, K.; Sauter, M.; Pinkert, S.; Kurreck, J.; Fechner, H. Heparan Sulfate Binding
Coxsackievirus B3 Strain PD: A Novel Avirulent Oncolytic Agent Against Human Colorectal Carcinoma. Hum. Gene Ther.
2018
,
29, 1301–1314. [CrossRef] [PubMed]
47.
Nüesch, J.; Thomas, N.; Plotzky, C.; Rommelaere, J. Modified Rodent Parvovirus Capable of Propagating and Spreading through
Human Gliomas. U.S. Patent 9029117, 12 May 2015.
48.
Ferreira, T.; Kulkarni, A.; Bretscher, C.; Richter, K.; Ehrlich, M.; Marchini, A. Oncolytic H-1 Parvovirus Enters Cancer Cells
through Clathrin-Mediated Endocytosis. Viruses 2020,12, 1199. [CrossRef] [PubMed]
49.
Kulkarni, A.; Ferreira, T.; Bretscher, C.; Grewenig, A.; El-Andaloussi, N.; Bonifati, S.; Marttila, T.; Palissot, V.; Hossain, J.A.; Azuaje,
F.; et al. Oncolytic H-1 parvovirus binds to sialic acid on laminins for cell attachment and entry. Nat. Commun.
2021
,12, 3834.
[CrossRef]
50.
Abdullahi, S.; Jäkel, M.; Behrend, S.J.; Steiger, K.; Topping, G.; Krabbe, T.; Colombo, A.; Sandig, V.; Schiergens, T.S.; Thasler, W.E.;
et al. A Novel Chimeric Oncolytic Virus Vector for Improved Safety and Efficacy as a Platform for the Treatment of Hepatocellular
Carcinoma. J. Virol. 2018,92. [CrossRef]
51.
Hanauer, J.R.H.; Koch, V.; Lauer, U.M.; Mühlebach, M.D. High-Affinity DARPin Allows Targeting of MeV to Glioblastoma
Multiforme in Combination with Protease Targeting without Loss of Potency. Mol. Ther. Oncolytics
2019
,15, 186–200. [CrossRef]
52.
Hazini, A.; Dieringer, B.; Pryshliak, M.; Knoch, K.P.; Heimann, L.; Tolksdorf, B.; Pappritz, K.; El-Shafeey, M.; Solimena, M.; Beling,
A.; et al. miR-375- and miR-1-Regulated Coxsackievirus B3 Has No Pancreas and Heart Toxicity But Strong Antitumor Efficiency
in Colorectal Carcinomas. Hum. Gene Ther. 2021,32, 216–230. [CrossRef] [PubMed]
53.
Pinkert, S.; Pryshliak, M.; Pappritz, K.; Knoch, K.; Hazini, A.; Dieringer, B.; Schaar, K.; Dong, F.; Hinze, L.; Lin, J.; et al.
Development of a new mouse model for coxsackievirus-induced myocarditis by attenuating coxsackievirus B3 virulence in the
pancreas. Cardiovasc. Res. 2020,116, 1756–1766. [CrossRef] [PubMed]
54.
Pryshliak, M.; Hazini, A.; Knoch, K.; Dieringer, B.; Tolksdorf, B.; Solimena, M.; Kurreck, J.; Pinkert, S.; Fechner, H. MiR-375-
mediated suppression of engineered coxsackievirus B3 in pancreatic cells. FEBS Lett. 2020,594, 763–775. [CrossRef] [PubMed]
55.
Leber, M.F.; Baertsch, M.A.; Anker, S.C.; Henkel, L.; Singh, H.M.; Bossow, S.; Engeland, C.E.; Barkley, R.; Hoyler, B.; Albert, J.; et al.
Enhanced Control of Oncolytic Measles Virus Using MicroRNA Target Sites. Mol. Ther. Oncolytics 2018,9, 30–40. [CrossRef]
56.
Singh, H.M.; Leber, M.F.; Bossow, S.; Engeland, C.E.; Dessila, J.; Grossardt, C.; Zaoui, K.; Bell, J.C.; Jäger, D.; von Kalle, C.; et al.
MicroRNA-sensitive Oncolytic Measles Virus for Chemovirotherapy of Pancreatic Cancer. Mol. Ther. Oncolytics
2021
. [CrossRef]
[PubMed]
57.
Bhat, H.; Zaun, G.; Hamdan, T.A.; Lang, J.; Adomati, T.; Schmitz, R.; Friedrich, S.K.; Bergerhausen, M.; Cham, L.B.; Li, F.; et al.
Arenavirus Induced CCL5 Expression Causes NK Cell-Mediated Melanoma Regression. Front. Immunol.
2020
,11, 1849. [CrossRef]
[PubMed]
58.
Kalkavan, H.; Sharma, P.; Kasper, S.; Helfrich, I.; Pandyra, A.A.; Gassa, A.; Virchow, I.; Flatz, L.; Brandenburg, T.; Namineni, S.;
et al. Spatiotemporally restricted arenavirus replication induces immune surveillance and type I interferon-dependent tumour
regression. Nat. Commun. 2017,8, 14447. [CrossRef]
59.
Rajaraman, S.; Canjuga, D.; Ghosh, M.; Codrea, M.C.; Sieger, R.; Wedekink, F.; Tatagiba, M.; Koch, M.; Lauer, U.M.; Nahnsen, S.;
et al. Measles Virus-Based Treatments Trigger a Pro-inflammatory Cascade and a Distinctive Immunopeptidome in Glioblastoma.
Mol. Ther. Oncolytics 2019,12, 147–161. [CrossRef]
Viruses 2021,13, 1420 27 of 29
60.
Goepfert, K.; Dinsart, C.; Rommelaere, J.; Foerster, F.; Moehler, M. Rational Combination of Parvovirus H1 With CTLA-4 and
PD-1 Checkpoint Inhibitors Dampens the Tumor Induced Immune Silencing. Front. Oncol. 2019,9, 425. [CrossRef]
61.
Heinrich, B.; Klein, J.; Delic, M.; Goepfert, K.; Engel, V.; Geberzahn, L.; Lusky, M.; Erbs, P.; Preville, X.; Moehler, M. Immunogenicity
of oncolytic vaccinia viruses JX-GFP and TG6002 in a human melanoma
in vitro
model: Studying immunogenic cell death,
dendritic cell maturation and interaction with cytotoxic T lymphocytes. Onco Targets Ther. 2017,10, 2389–2401. [CrossRef]
62.
Heinrich, B.; Goepfert, K.; Delic, M.; Galle, P.R.; Moehler, M. Influence of the oncolytic parvovirus H-1, CTLA-4 antibody
tremelimumab and cytostatic drugs on the human immune system in a human
in vitro
model of colorectal cancer cells. Onco
Targets Ther. 2013,6, 1119–1127. [CrossRef] [PubMed]
63.
Backhaus, P.S.; Veinalde, R.; Hartmann, L.; Dunder, J.E.; Jeworowski, L.M.; Albert, J.; Hoyler, B.; Poth, T.; Jäger, D.; Un-
gerechts, G.; et al. Immunological Effects and Viral Gene Expression Determine the Efficacy of Oncolytic Measles Vaccines
Encoding IL-12 or IL-15 Agonists. Viruses 2019,11, 914. [CrossRef]
64.
Speck, T.; Heidbuechel, J.P.W.; Veinalde, R.; Jaeger, D.; von Kalle, C.; Ball, C.R.; Ungerechts, G.; Engeland, C.E. Targeted BiTE
Expression by an Oncolytic Vector Augments Therapeutic Efficacy Against Solid Tumors. Clin. Cancer Res. Off. J. Am. Assoc.
Cancer Res. 2018,24, 2128–2137. [CrossRef] [PubMed]
65.
Veinalde, R.; Grossardt, C.; Hartmann, L.; Bourgeois-Daigneault, M.C.; Bell, J.C.; Jäger, D.; von Kalle, C.; Ungerechts, G.; Engeland,
C.E. Oncolytic measles virus encoding interleukin-12 mediates potent antitumor effects through T cell activation. Oncoimmunology
2017,6, e1285992. [CrossRef] [PubMed]
66.
Busch, E.; Kubon, K.D.; Mayer, J.K.M.; Pidelaserra-Martí, G.; Albert, J.; Hoyler, B.; Heidbuechel, J.P.W.; Stephenson, K.B.; Lichty,
B.D.; Osen, W.; et al. Measles Vaccines Designed for Enhanced CD8(+) T Cell Activation. Viruses 2020,12, 242. [CrossRef]
67.
Hutzler, S.; Erbar, S.; Jabulowsky, R.A.; Hanauer, J.R.H.; Schnotz, J.H.; Beissert, T.; Bodmer, B.S.; Eberle, R.; Boller, K.;
Klamp, T.; et al. Antigen-specific oncolytic MV-based tumor vaccines through presentation of selected tumor-associated antigens
on infected cells or virus-like particles. Sci. Rep. 2017,7, 16892. [CrossRef]
68.
Leber, M.F.; Hoyler, B.; Prien, S.; Neault, S.; Engeland, C.E.; Forster, J.M.; Bossow, S.; Springfeld, C.; von Kalle, C.; Jager, D.; et al.
Sequencing of serially passaged measles virus affirms its genomic stability and reveals a nonrandom distribution of consensus
mutations. J. Gen. Virol. 2020,101, 399–409. [CrossRef]
69.
Maurer, S.; Salih, H.R.; Smirnow, I.; Lauer, U.M.; Berchtold, S. Suicide gene-armed measles vaccine virus for the treatment of
AML. Int. J. Oncol. 2019,55, 347–358. [CrossRef]
70.
Berchtold, S.; Beil, J.; Raff, C.; Smirnow, I.; Schell, M.; D’Alvise, J.; Gross, S.; Lauer, U.M. Assessing and Overcoming Resistance
Phenomena against a Genetically Modified Vaccinia Virus in Selected Cancer Cell Lines. Int. J. Mol. Sci.
2020
,21, 7618. [CrossRef]
71.
May, V.; Berchtold, S.; Berger, A.; Venturelli, S.; Burkard, M.; Leischner, C.; Malek, N.P.; Lauer, U.M. Chemovirotherapy for
pancreatic cancer: Gemcitabine plus oncolytic measles vaccine virus. Oncol. Lett. 2019,18, 5534–5542. [CrossRef]
72.
Marchini, A.; Li, J.; Schroeder, L.; Geletneky, K.; Rommelaere, J. Cancer Therapy with a Parvovirus Combined with a bcl-2
Inhibitor. U.S. Patent 9889169, 13 February 2018.
73.
Kloker, L.D.; Berchtold, S.; Smirnow, I.; Beil, J.; Krieg, A.; Sipos, B.; Lauer, U.M. Oncolytic vaccinia virus GLV-1h68 exhibits
profound antitumoral activities in cell lines originating from neuroendocrine neoplasms. BMC Cancer 2020,20, 628. [CrossRef]
74.
Scheubeck, G.; Berchtold, S.; Smirnow, I.; Schenk, A.; Beil, J.; Lauer, U.M. Starvation-Induced Differential Virotherapy Using an
Oncolytic Measles Vaccine Virus. Viruses 2019,11, 614. [CrossRef] [PubMed]
75.
Klose, C.; Berchtold, S.; Schmidt, M.; Beil, J.; Smirnow, I.; Venturelli, S.; Burkard, M.; Handgretinger, R.; Lauer, U.M. Biological
treatment of pediatric sarcomas by combined virotherapy and NK cell therapy. BMC Cancer
2019
,19, 1172. [CrossRef] [PubMed]
76.
Krabbe, T.; Marek, J.; Groll, T.; Steiger, K.; Schmid, R.M.; Krackhardt, A.M.; Altomonte, J. Adoptive T Cell Therapy Is Comple-
mented by Oncolytic Virotherapy with Fusogenic VSV-NDV in Combination Treatment of Murine Melanoma. Cancers
2021
,13,
1044. [CrossRef]
77.
Kloker, L.D.; Berchtold, S.; Smirnow, I.; Schaller, M.; Fehrenbacher, B.; Krieg, A.; Sipos, B.; Lauer, U.M. The Oncolytic Herpes
Simplex Virus Talimogene Laherparepvec Shows Promising Efficacy in Neuroendocrine Cancer Cell Lines. Neuroendocrinology
2019,109, 346–361. [CrossRef] [PubMed]
78.
Recher, M.; Lang, K.S.; Navarini, A.; Hunziker, L.; Lang, P.A.; Fink, K.; Freigang, S.; Georgiev, P.; Hangartner, L.; Zellweger, R.; et al.
Extralymphatic virus sanctuaries as a consequence of potent T-cell activation. Nat. Med.
2007
,13, 1316–1323. [CrossRef] [PubMed]
79.
Zinkernagel, R.M. Lymphocytic choriomeningitis virus and immunology. Curr. Top. Microbiol. Immunol.
2002
,263, 1–5. [CrossRef]
80.
Webb, H.E.; Molomut, N.; Padnos, M.; Wetherley-Mein, G. The treatment of 18 cases of malignant disease with an arenavirus.
Clin. Oncol. 1975,1, 157–169.
81.
Geisler, A.; Hazini, A.; Heimann, L.; Kurreck, J.; Fechner, H. Coxsackievirus B3-Its Potential as an Oncolytic Virus. Viruses
2021
,
13, 718. [CrossRef]
82.
Moehler, M.H.; Zeidler, M.; Wilsberg, V.; Cornelis, J.J.; Woelfel, T.; Rommelaere, J.; Galle, P.R.; Heike, M. Parvovirus H-1-induced
tumor cell death enhances human immune response in vitro via increased phagocytosis, maturation, and cross-presentation by
dendritic cells. Hum. Gene Ther. 2005,16, 996–1005. [CrossRef]
83.
Schierer, S.; Hesse, A.; Knippertz, I.; Kaempgen, E.; Baur, A.S.; Schuler, G.; Steinkasserer, A.; Nettelbeck, D.M. Human dendritic
cells efficiently phagocytose adenoviral oncolysate but require additional stimulation to mature. Int. J. Cancer
2012
,130, 1682–1694.
[CrossRef]
84. Mühlebach, M.D. Vaccine platform recombinant measles virus. Virus Genes 2017,53, 733–740. [CrossRef]
Viruses 2021,13, 1420 28 of 29
85.
Hörner, C.; Schürmann, C.; Auste, A.; Ebenig, A.; Muraleedharan, S.; Dinnon, K.H., 3rd; Scholz, T.; Herrmann, M.; Schnierle, B.S.;
Baric, R.S.; et al. A highly immunogenic and effective measles virus-based Th1-biased COVID-19 vaccine. Proc. Natl. Acad. Sci.
USA 2020,117, 32657–32666. [CrossRef]
86.
Gogesch, P.; Schülke, S.; Scheurer, S.; Mühlebach, M.D.; Waibler, Z. Modular MLV-VLPs co-displaying ovalbumin peptides and
GM-CSF effectively induce expansion of CD11b(+) APC and antigen-specific T cell responses
in vitro
.Mol. Immunol.
2018
,101,
19–28. [CrossRef] [PubMed]
87.
Leber, M.F.; Neault, S.; Jirovec, E.; Barkley, R.; Said, A.; Bell, J.C.; Ungerechts, G. Engineering and combining oncolytic measles
virus for cancer therapy. Cytokine Growth Factor Rev. 2020,56, 39–48. [CrossRef]
88.
Baertsch, M.A.; Leber, M.F.; Bossow, S.; Singh, M.; Engeland, C.E.; Albert, J.; Grossardt, C.; Jager, D.; von Kalle, C.; Ungerechts, G.
MicroRNA-mediated multi-tissue detargeting of oncolytic measles virus. Cancer Gene 2014,21, 373–380. [CrossRef] [PubMed]
89.
Leber, M.F.; Bossow, S.; Leonard, V.H.; Zaoui, K.; Grossardt, C.; Frenzke, M.; Miest, T.; Sawall, S.; Cattaneo, R.; von Kalle, C.;
et al. MicroRNA-sensitive oncolytic measles viruses for cancer-specific vector tropism. Mol. Ther. J. Am. Soc. Gene Ther.
2011
,19,
1097–1106. [CrossRef] [PubMed]
90.
Heidbuechel, J.P.W.; Engeland, C.E. Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo. J.
Vis. Exp. JoVE 2019. [CrossRef] [PubMed]
91.
Pidelaserra-Martí, G.; Engeland, C.E. Mechanisms of measles virus oncolytic immunotherapy. Cytokine Growth Factor Rev.
2020
,
56, 28–38. [CrossRef] [PubMed]
92. Engeland, C.E.; Ungerechts, G. Measles Virus as an Oncolytic Immunotherapy. Cancers 2021,13, 544. [CrossRef]
93.
Heidbuechel, J.P.W.; Engeland, C.E. Oncolytic viruses encoding bispecific T cell engagers: A blueprint for emerging immunovi-
rotherapies. J. Hematol. Oncol. 2021,14, 63. [CrossRef] [PubMed]
94.
Berchtold, S.; Lampe, J.; Weiland, T.; Smirnow, I.; Schleicher, S.; Handgretinger, R.; Kopp, H.G.; Reiser, J.; Stubenrauch, F.; Mayer,
N.; et al. Innate immune defense defines susceptibility of sarcoma cells to measles vaccine virus-based oncolysis. J. Virol.
2013
,87,
3484–3501. [CrossRef] [PubMed]
95.
Weiland, T.; Lampe, J.; Essmann, F.; Venturelli, S.; Berger, A.; Bossow, S.; Berchtold, S.; Schulze-Osthoff, K.; Lauer, U.M.; Bitzer, M.
Enhanced killing of therapy-induced senescent tumor cells by oncolytic measles vaccine viruses. Int. J. Cancer
2014
,134, 235–243.
[CrossRef] [PubMed]
96.
Mousset, S.; Rommelaere, J. Minute virus of mice inhibits cell transformation by simian virus 40. Nature
1982
,300, 537–539.
[CrossRef] [PubMed]
97.
Geletneky, K.; Hajda, J.; Angelova, A.L.; Leuchs, B.; Capper, D.; Bartsch, A.J.; Neumann, J.O.; Schoning, T.; Husing, J.;
Beelte, B.; et al. Oncolytic H-1 Parvovirus Shows Safety and Signs of Immunogenic Activity in a First Phase I/IIa Glioblastoma
Trial. Mol. Ther. J. Am. Soc. Gene Ther. 2017,25, 2620–2634. [CrossRef] [PubMed]
98.
Marchini, A.; Daeffler, L.; Pozdeev, V.I.; Angelova, A.; Rommelaere, J. Immune Conversion of Tumor Microenvironment by
Oncolytic Viruses: The Protoparvovirus H-1PV Case Study. Front. Immunol. 2019,10, 1848. [CrossRef]
99.
Moehler, M.; Goepfert, K.; Heinrich, B.; Breitbach, C.J.; Delic, M.; Galle, P.R.; Rommelaere, J. Oncolytic virotherapy as emerging
immunotherapeutic modality: Potential of parvovirus h-1. Front. Oncol. 2014,4, 92. [CrossRef]
100.
Angelova, A.; Ferreira, T.; Bretscher, C.; Rommelaere, J.; Marchini, A. Parvovirus-Based Combinatorial Immunotherapy: A
Reinforced Therapeutic Strategy against Poor-Prognosis Solid Cancers. Cancers 2021,13, 342. [CrossRef] [PubMed]
101.
Bretscher, C.; Marchini, A. H-1 Parvovirus as a Cancer-Killing Agent: Past, Present, and Future. Viruses
2019
,11, 562. [CrossRef]
[PubMed]
102.
Hartley, A.; Kavishwar, G.; Salvato, I.; Marchini, A. A Roadmap for the Success of Oncolytic Parvovirus-Based Anticancer
Therapies. Annu. Rev. Virol. 2020,7, 537–557. [CrossRef]
103.
Hallauer, C.; Kronauer, G.; Siegl, G. Parvoiruses as contaminants of permanent human cell lines. I. Virus isolation from 1960-1970.
Arch. Gesamte Virusforsch 1971,35, 80–90. [CrossRef]
104.
Foloppe, J.; Kempf, J.; Futin, N.; Kintz, J.; Cordier, P.; Pichon, C.; Findeli, A.; Vorburger, F.; Quemeneur, E.; Erbs, P. The Enhanced
Tumor Specificity of TG6002, an Armed Oncolytic Vaccinia Virus Deleted in Two Genes Involved in Nucleotide Metabolism. Mol.
Ther. Oncolytics 2019,14, 1–14. [CrossRef]
105.
Johnson, J.E.; Nasar, F.; Coleman, J.W.; Price, R.E.; Javadian, A.; Draper, K.; Lee, M.; Reilly, P.A.; Clarke, D.K.; Hendry, R.M.; et al.
Neurovirulence properties of recombinant vesicular stomatitis virus vectors in non-human primates. Virology
2007
,360, 36–49.
[CrossRef] [PubMed]
106.
Freeman, A.I.; Zakay-Rones, Z.; Gomori, J.M.; Linetsky, E.; Rasooly, L.; Greenbaum, E.; Rozenman-Yair, S.; Panet, A.; Libson, E.;
Irving, C.S.; et al. Phase I/II trial of intravenous NDV-HUJ oncolytic virus in recurrent glioblastoma multiforme. Mol. Ther. J. Am.
Soc. Gene Ther. 2006,13, 221–228. [CrossRef]
107. Krabbe, T.; Altomonte, J. Fusogenic Viruses in Oncolytic Immunotherapy. Cancers 2018,10, 216. [CrossRef]
108.
Altomonte, J.; Marozin, S.; Schmid, R.M.; Ebert, O. Engineered newcastle disease virus as an improved oncolytic agent against
hepatocellular carcinoma. Mol. Ther. J. Am. Soc. Gene Ther. 2010,18, 275–284. [CrossRef]
109.
Andtbacka, R.H.I.; Collichio, F.; Harrington, K.J.; Middleton, M.R.; Downey, G.; Öhrling, K.; Kaufman, H.L. Final analyses of
OPTiM: A randomized phase III trial of talimogene laherparepvec versus granulocyte-macrophage colony-stimulating factor in
unresectable stage III-IV melanoma. J. Immunother. Cancer 2019,7, 145. [CrossRef] [PubMed]
Viruses 2021,13, 1420 29 of 29
110.
Joos, S.; Nettelbeck, D.M.; Reil-Held, A.; Engelmann, K.; Moosmann, A.; Eggert, A.; Hiddemann, W.; Krause, M.; Peters, C.;
Schuler, M.; et al. German Cancer Consortium (DKTK)—A national consortium for translational cancer research. Mol. Oncol.
2019,13, 535–542. [CrossRef]
111.
Angelova, A.L.; Barf, M.; Geletneky, K.; Unterberg, A.; Rommelaere, J. Immunotherapeutic Potential of Oncolytic H-1 Parvovirus:
Hints of Glioblastoma Microenvironment Conversion towards Immunogenicity. Viruses 2017,9, 382. [CrossRef]
112.
Geletneky, K.; Bartsch, A.; Weiss, C.; Bernhard, H.; Marchini, A.; Rommelaere, J. ATIM-40. High rate of objective anti-tumor re-
sponse in 9 patients with glioblastoma after viro-immunotherapy with oncolytic parvovirus H-1 in combination with bevacizumab
and PD-1 checkpoint blockade. Neuro-Oncology 2018,20, vi10. [CrossRef]
113.
Moehler, M.; Heo, J.; Lee, H.C.; Tak, W.Y.; Chao, Y.; Paik, S.W.; Yim, H.J.; Byun, K.S.; Baron, A.; Ungerechts, G.; et al. Vaccinia-based
oncolytic immunotherapy Pexastimogene Devacirepvec in patients with advanced hepatocellular carcinoma after sorafenib
failure: A randomized multicenter Phase IIb trial (TRAVERSE). Oncoimmunology 2019,8, 1615817. [CrossRef] [PubMed]
114.
Ribas, A.; Dummer, R.; Puzanov, I.; VanderWalde, A.; Andtbacka, R.H.I.; Michielin, O.; Olszanski, A.J.; Malvehy, J.; Cebon, J.;
Fernandez, E.; et al. Oncolytic virotherapy promotes intratumoral t cell infiltration and improves anti-pd-1 immunotherapy. Cell
2017,170, 1109–1119. [CrossRef] [PubMed]
115.
Yost, K.E.; Satpathy, A.T.; Wells, D.K.; Qi, Y.; Wang, C.; Kageyama, R.; McNamara, K.L.; Granja, J.M.; Sarin, K.Y.; Brown, R.A.; et al.
Clonal replacement of tumor-specific T cells following PD-1 blockade. Nat. Med. 2019,25, 1251–1259. [CrossRef] [PubMed]
