Expert Opinion on Biological Therapy

ISSN: 1471-2598 (Print) 1744-7682 (Online) Journal homepage: https://www.tandfonline.com/loi/iebt20

Avelumab: combining immune checkpoint

inhibition and antibody-dependent cytotoxicity

Gerhard Hamilton & Barbara Rath

To cite this article: Gerhard Hamilton & Barbara Rath (2017) Avelumab: combining immune
checkpoint inhibition and antibody-dependent cytotoxicity, Expert Opinion on Biological Therapy,
17:4, 515-523, DOI: 10.1080/14712598.2017.1294156

To link to this article: https://doi.org/10.1080/14712598.2017.1294156

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Published online: 22 Feb 2017.

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EXPERT OPINION ON BIOLOGICAL THERAPY, 2017
VOL. 17, NO. 4, 515–523
http://dx.doi.org/10.1080/14712598.2017.1294156

DRUG EVALUATION

Avelumab: combining immune checkpoint inhibition and antibody-dependent

cytotoxicity
Gerhard Hamilton and Barbara Rath
Department of Surgery, Medical University of Vienna, Vienna, Austria

ABSTRACT ARTICLE HISTORY

Introduction: Immune checkpoint inhibition holds great promise for selected tumors. The human Received 25 November 2016
monoclonal antibody (mAB) avelumab is directed to programmed death ligand-1 (PD-L1) and is Accepted 8 February 2017
supposed to inhibit the immunosuppressive PD-L1/PD-1 interaction and, furthermore, effect anti- KEYWORDS
body-dependent cytotoxicity (ADCC) lysis of tumor cells. Avelumab; immune
Areas covered: This article presents an overview of the current means to activate the antitumor checkpoints; programmed
immune defense by targeting PD-1 or PD-L1 with mABs and their possible role in ADCC-mediated death ligand-1;
tumor cell elimination. antibody-dependent
Expert opinion: Avelumab contains a Fc region which can bind cognate receptors on immune effector cytotoxicity
cells and induce ADCC-mediated tumor cell lysis, in contrast to other mABs directed to PD-1/PD-L1
which lack the ability to trigger ADCC due to belonging to the IgG4 subclass or possessing a mutated Fc
region. Preclinical and clinical data indicate that avelumab can be safely administered to cancer patients
with a toxicity profile comparable to other mABs and without lysis of PD-L1-positive activated immune
cells. This antibody yielded durable responses in a phase II trial in advanced Merkel cell carcinoma
patients. Tumor cell lysis by avelumab prevents cells from resorting to alternative checkpoints as shown
by targeting PD-1 and the upregulation of TIM-3.

1. Introduction by the immune system and imply that targeting negative

regulatory immune checkpoints would be beneficial for
A hallmark of the malignant phenotype of tumor cells
patients with preexisting T-cell-inflamed tumors [4]. For exam-
includes the ability to override the host immune system
ple, blockade of CTLA-4 or PD-1 has been associated with
and inactivate infiltrating cytotoxic effector cells [1,2]. The
significant clinical responses in metastatic melanoma patients
accumulation of tumor-infiltrating lymphocytes (TILs) within
[5]. CTLA-4 is homologous to the T-cell costimulatory protein,
primary and metastatic tumors could be demonstrated to
CD28, and both molecules bind to CD80/B7-1 and CD86/B7-2
comprise tumor antigen-specific T lymphocytes which can
on antigen-presenting cells (APCs), but CTLA-4 binds CD80
be expanded in vitro. Despite spontaneous antitumor immu-
and CD86 with greater affinity than CD28 and such impairs
nity in distinct tumor types, the disease progresses in most
the stimulatory signal of CD28 [3]. Similarly, binding of pro-
patients, thus indicating the failure of the immune response
grammed death-ligand 1 (PD-L1) to PD-1 or B7-1 transmits an
to control tumor growth and recurrence. Obviously, an
inhibitory signal which reduces the proliferation and survival
effective cellular immune response is blocked within the
of CTLs. Ipilimumab, a fully human monoclonal antibody
tumor microenvironment by inhibitory factors which would
(mAB) against CTLA-4, as well as nivolumab and pembrolizu-
have to be antagonized for a reconstitution of a successful
mab which target PD-1, have been shown to prolong overall
immune defense. Isolated TILs were shown to express a
survival (OS) in patients with advanced melanoma [5,6]. The
range of inhibitory receptors that under normal conditions
combination of a CTLA-4 and a PD-1 inhibitor seems to be
contribute to T-cell regulation. These receptors, including
superior to the monotherapies, especially in patients with PD-
cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), pro-
L1-negative tumors, but increases toxicity.
grammed cell death-1 (PD-1), lymphocyte-activation gene-3,
Metastatic lung cancer, including non–small-cell lung can-
and T-cell Immunoglobulin domain and Mucin domain 3
cer (NSCLC) and small-cell lung cancer, has a poor prognosis
(TIM-3), seem to inactivate the ongoing immune response
with a 5-year survival rate of less than 5% [7]. A subpopula-
and to prevent efficient elimination of the tumor cells under
tion of NSCLC patients whose tumors harbor specific muta-
attack [3].
tions in driver genes can be treated with molecularly
T-cell-inflamed tumors exhibit high expression of immuno-
targeted therapies resulting in improved OS. However, for a
suppressive mechanisms which follow the infiltration of cyto-
large group of NSCLC patients, therapeutic options have
toxic T lymphocytes (CTLs) as a negative feedback control.
been confined to surgery, chemotherapy, and irradiation
Thus, the immunosuppressive pathways are intrinsically driven
alone. Immunotherapies, which aim at eliciting immune-

CONTACT Gerhard Hamilton gerhard.hamilton@meduniwien.ac.at

© 2017 Informa UK Limited, trading as Taylor & Francis Group
516 G. HAMILTON AND B. RATH
mediated destruction of lung tumor cells, may be an alter-
native approach but has had minimal success in lung cancer
in the past, leading to the assumption that lung cancer is
poorly immunogenic [8]. However, the novel targeting of
immune checkpoint proteins by monoclonal antibodies in
NSCLC was found to prolong survival of suitable patients
significantly [9]. In general, application of immune check-
point-directed therapy has applied to a growing number of
tumor types so far [10].
2. Passive immunotherapy for cancer treatment
Passive immunotherapy is defined as use of agents, such as
monoclonal antibodies or adaptive cell therapy, that directly
target tumor in contrast to administration of cytokines or
vaccines [10]. CTLs are considered the effectors of immune
responses against tumors, and recognition of tumor antigens
by specific T cells is a prerequisite for mounting an immune
cell attack. Naive CD8+ T cells are cross-primed by APCs,
invoking a program leading to tumor-specific CTLs which
proliferate and invade the tumor sites. CTLs kill tumor cells
through release of granzymes, perforin, and ligands of the
tumor necrosis factor (TNF) superfamily, such as Fas ligand.
The Th1 subpopulation of CD4+ T cells enhances the expan-
sion, priming, and infiltration of CD8+ T by producing large
amounts of interferon-γ (IFNγ) and chemokines. Furthermore,
innate immunity effectors which comprise macrophages, gran-
ulocytes, mast cells, dendritic cells (DCs), and natural killer (NK)
cells can destroy tumor cells directly through several mechan-
isms, including antibody-dependent cellular cytotoxicity
(ADCC).
Several checkpoint molecules exist which attenuate the
T-cell immune response to tumor-associated antigens, and
mABs interacting with two of these molecular pathways,
namely CTLA-4 and PD-1, have shown clinical activity in sev-
eral tumors [3,10]. Some patients with advanced solid tumors,
like metastatic melanoma, exhibit a prolonged response to
immunotherapy which lasts for many years after anti-CTLA-4
antibody treatment [11]. CTLA-4 is a protein receptor
expressed by activated CTLs which binds to CD80 (B7-1) or
CD86 (B7-2) on APCs to delimit the activity of T cells under
normal conditions. Upregulation of CTLA-4 expression on T
cells can occur in response to transforming growth factor-β
(TGFβ) during the early stage of tumorigenesis. mABs directed
to this ligand inhibit CTLA-4-CD80/CD86 binding and activate
CTLs through abolishment of this inhibitory circuit. The main
side effects of an anti-CTLA-4 therapy included diarrhea, coli-
tis, transaminitis, and pituitary dysfunction [12]. A second
immune checkpoint molecule, namely programmed death-1
(PD-1), is a T-cell surface receptor expressed on B cells, NK
cells, activated monocytes, and DCs [13,14]. Normally, PD-1
limits autoimmunity as a coinhibitory immune checkpoint
but in tumors dampens the activity of TILs [15]. PD-1 binds
programmed PD-L1/B7-H1 and 2 (PD-L2/B7-DC), inducing
T-cell anergy by interacting with the former receptor on
APCs [16]. A range of cancers, including gastric cancer, renal
cell cancer, bladder cancer, hepatocellular cancer, cutaneous
cancer, breast cancer, and melanoma, glioblastoma as well as
NSCLC, express PD-L1 which provides protection against
Box 1. Drug summary.
Drug name Avelumab
Phase Pre-registration
Indication Cancer
Pharmacology description PD-L1 antagonist
Route of administration Injectable
Pivotal trial(s) [64]
immune system attacks [17,18]. Additionally, PD-L1 is
expressed on the surface of resting T cells, B cells, DCs, macro-
phages, vascular endothelial cells, heart, lung, placenta, liver,
and pancreatic islet cells [13]. Normally, PD-L1 is involved in
self-tolerance, such as protecting peripheral tissues from
excess of inflammation and autoimmune pathologies [19,20].
PD-L1 is induced by various pro-inflammatory cytokines like
IFNγ, tumor necrosis factor-α (TNF-α), vascular endothelial
growth factor, granulocyte-macrophage colony-stimulating
factor, and interleukin-10.
A range of mABs against the checkpoint proteins have been
developed, including the CTLA-4-directed ipilimumab and treme-
limumab, as well as the PD-1 inhibitors nivolumab and pembro-
lizumab and PD-L1 inhibitors atezolizumab (MPDL3280A),
durvalumab (MEDI4736), and avelumab (MSB0010718C) with
many more under development (Box 1) [10,21]. Detection of the
expression of PD-1/PD-L1 in tumors by immunohistochemistry is
used as predictive biomarker of response to immune checkpoint
therapy, but technical issues and clinical activity of antibodies in
antigen-negative patients indicate limited reliability of this assay.
Despite attempts for standardization of the immunohistochemical
techniques for confirmation of PD-1/PD-L1 expression, differing
reagents, protocols, and thresholds of positivity impede compar-
ison of different trials [22].
Furthermore, an important precondition for the signifi-
cance of the immune checkpoint inhibitor expression as pre-
dictor of a therapeutic response is the infiltration of the
respective tumors by immune effector cells. Tumor load of
neoantigens, intratumoral heterogeneity of checkpoint inhibi-
tor expression, and distinct mutations constitute additional
predictive markers under evaluation [23]. Anti-CTLA-4 antibo-
dies can be applied to stimulate immune cell infiltration in
combination with PD-1/PD-L1-directed agents. Thus, ipilimu-
mab (a CTLA-4 checkpoint inhibitor) and nivolumab (a PD-1
checkpoint inhibitor) have been shown to have complemen-
tary activity in metastatic melanoma [24]. However, treatment-
related adverse events (AEs) of grade 3 or 4 occurred in 16.3%
of the patients in the nivolumab group, 27.3% in the ipilimu-
mab group, but 55.0% in the antibody combination group,
demonstrating increased toxicity of the combined antibodies.
Through blockade of the immune checkpoints, the balance
between autoimmunity and immune tolerance is disturbed.
Thus, the new immunotherapies are associated with immune-
meditated AEs comprising immune-mediated pneumonitis, colitis,
hepatitis, endocrinopathies, rash, encephalitis among others [25].
Initiation of appropriate management, usually in the form of
immunosuppression, results in complete reversibility of side
effects, but inadequate treatment can lead to severe toxicity or
even death.

2.1. Overview of the market for cancer checkpoint
immunotherapies
High response rates and prolonged OS in cancer patients
treated with checkpoint inhibitors led to an exponential
growth of the respective drug development, clinical trials,
and use of this class of anticancer agents. In 2014, the
NSCLC market was dominated by the chemotherapeutic
drug pemetrexed, erlotinib, and bevacizumab with costs of
$5.4 billion, but expense is expected to increase exponentially
due to the novel immunotherapeutic drugs [26]. So far, three
immune checkpoint inhibitors, namely ipilimumab (Yervoy;
Bristol-Myers Squibb, New York, NY, USA), nivolumab
(Opdivo; Bristol-Myers Squibb/Ono Pharmaceuticals, Osaka,
Japan), and pembrolizumab (Keytruda; Merck Co., Kenilworth,
NJ, USA) are marketed for the treatment of malignant mela-
noma [27]. The two PD-1-specific mABs nivolumab and pem-
brolizumab yield high responses (rates of 24–32%) which are
durable for the majority of patients and are associated with a
low number of AEs, consisting mainly of fatigue, anemia, and
dyspnea. The estimated cost of treating all American patients
with nivolumab or pembrolizumab as calculated for data avail-
able in 2016 is $1.57 and $0.97 billion per year, respectively
[28]. The immuno-oncology market will grow to >$27 billion in
2021, fueled by the administration of a range of checkpoint
inhibitors across multiple solid tumor indications [29]. In 2022,
the anti-PD-1 agents are expected to hold the highest market
share at 72% and anti-CTLA-4 agents and anti-PD-L1 agents
are expected to account for 20% and 8% of the market,
respectively.
2.2. Antibodies targeting PD-1
Immunomodulation with the PD1-targeting mABs has shown
to mediate tumor shrinkage and to extend OS in several
pivotal phase I/II studies in melanoma, renal cell carcinoma
(RCC), and NSCLC [9,30]. Nivolumab (BMS-936558/MDX1106b,
Opdivo) is a human IgG4 antibody against PD-1, lacking
detectable ADCC, which has been approved by the FDA for
the application in unresectable or metastatic melanoma, meta-
static NSCLC, and advanced RCC. Long-term data on 129
NSCLC patients showed an overall response rate of 17.2%
with a median duration of the responses of 18.5 months
[31]. Drug-related AEs (any grade) occurred in 71% of patients
with NSCLC, with grade 3 to 4 drug-related AEs reported in
14%. However, the drug failed to outperform chemotherapy
for first-line treatment in a lung cancer trial in patients not
preselected for PD-1 expression (Checkmate 026).
Pembrolizumab (MK-3475, lambrolizumab, Keytruda) is a
highly specific anti-PD-1 humanized IgG4 antibody that con-
tains a mutation at S228P designed to further suppress Fc-
mediated ADCC. First-line treatment of NSCLC has cut progres-
sion and the risk of death in half. The median progression-free
time was 10.3 months versus 6 months for patients on che-
motherapy alone [32].
2.2.1. Resistance to anti-PD1 immunotherapy
Despite high antitumor activity of antibodies directed to PD-1
immune checkpoint in lung cancer, resistance to these
EXPERT OPINION ON BIOLOGICAL THERAPY 517
therapies has increasingly been visible, similarly to the situa-
tion observed with the targeted small-molecule inhibitors. In
tumors progressing after initial response to anti-PD-1 therapy,
upregulation of TIM-3, an alternative immune checkpoint
receptor, in PD-1 antibody-coated T cells was found [33].
Anti-PD-1 antibodies were still active at the time of resistance,
but the new blockade could only be reversed by addition of a
TIM-3-blocking antibody. Upregulation of TIM-3 expression
after prolonged exposure to PD-1-blocking antibody was con-
fined to CTLs in the tumor microenvironment. Cancers seems
to possess the ability to escape immune checkpoint inhibition
by invoking pathways involving a set of alternative immune
receptors. Genomic and transcriptomic investigations may
help to identify further positive correlates of response or
resistance, such as a high mutational/neoantigen load of the
melanomas and NSCLC, as well as low intratumoral heteroge-
neity, among others. Resistant tumors have been shown to
exhibit distinct alterations, such as loss of Phosphatase and
Tensin homolog (PTEN), modification of the Januskinase/
Signal Transducers and Activators of Transcription (JAK/STAT)
pathway, and elevated expression of coinhibitory molecules
[23]. Additionally, the cancer–immunity cycle requires the
release of tumor antigens, presentation of these antigens to
invoke effector cell activation, trafficking, and infiltration of
such primed cells into the tumor as well as overcoming an
immunosuppressive microenvironment, and thus, tumor cell
kill is impaired by any failures to successful progress through
this sequence of events, especially the infiltration step [34].
2.3. Antibodies directed to PD-L1
Therapeutic PD-L1 antibodies comprise MPDL-3280/Tecentriq,
BMS-936559/MDX-1105 (discontinued), MEDI4736/durvalu-
mab, and MSB0010718C/avelumab [10,30]. So far, Tecentriq
was the first checkpoint inhibitor approved for advanced
urothelial carcinoma [35]. The PD-L1 gene promoter region
has a response element to the IFN regulatory factor, and upon
IFNγ stimulation, PD-L1 is not only expressed on tumor cells,
but likewise on T cells, NK cells, macrophages, myeloid DCs, B
cells, epithelial cells, and vascular endothelial cells [36].
Atezolizumab/MPDL3280A/Tecentriq is a phage-derived
human IgG1 mAB which was engineered with a mutation in
the Fc domain (298 Asn to Ala) to prevent N-linked glycosyla-
tion and ADCC activity [37,38]. In stage IIIB/IV or recurrent
NSCLC, 19% of objective response rate (ORR) was recorded
when atezolizumab used as a first-line therapy compared with
17% when it was a second-line or subsequent therapy. The
most commonly reported AEs were fatigue and nausea. In a
second phase II study, the OS was 12.6 months in patients
who received atezolizumab, compared with 9.7 months in
those who received docetaxel but overall responses (OR)
with atezolizumab were durable, with a median duration of
14.3 months versus 7.2 months for docetaxel. About 40% of
patients in the atezolizumab group experienced grade 3 to 4
AEs versus 53% in the docetaxel group [10,38,39]. The most
common atezolizumab-related grade 3 AEs were pneumonia
and increased aspartate aminotransferase in serum.
BMS-936559 is a PD-L1-specific, fully human, and high-affi-
nity IgG4 mAB that inhibits the interaction of PD-L1 with PD-1
518 G. HAMILTON AND B. RATH

or CD80 [40]. IgG4 antibodies poorly activate ADCC, and,
additionally, its stabilizing S228P Fc mutation prevents Fab
exchange and further impairs ADCC [41]. In a phase I trial,
13% of the patients with NSCLC achieved a partial response
and 12% had stable disease. Drug-related AEs were observed
in 39% of patients and included rash, hypothyroidism, hepati-
tis, and one case each of sarcoidosis, endophthalmitis, dia-
betes mellitus, and myasthenia gravis. BMS-936559 was
discontinued.
Avelumab (MSB0010718C) represents a fully human anti-
PD-L1 IgG1 mAB [42]. Through the blockade of PD-L1, avelu-
mab reactivates CTLs and, furthermore, can induce ADCC due
to the native Fc-region contained [43]. This mAB is discussed
in detail in the following sections.
MEDI4736 (durvalumab) is a human IgG1 mAB with high
affinity and specificity to PD-L1, which had also been engi-
neered to prevent ADCC [44]. In pretreated bladder cancer
patients, the ORR was 31.0% in 42 response-evaluable
patients, with 46.4% in the PD-L1-positive subgroup and 0%
in the PD-L1-negative subgroup [45]. The most common treat-
ment-related AEs of any grade were fatigue (13.1%), diarrhea
(9.8%), and decreased appetite (8.2%).
3. Role of ADCC in antitumor therapy
The Fc receptor FcγRIII (CD16) provides NK cells with the
capacity to mediate ADCC upon recognition of the Fc segment
of selected IgGs bound to cell surfaces [46,47]. CD16 (FcγRIII),
contains an immunotyrosine-activating motif in the cytoplas-
mic domain which upon recognition of an antibody-coated
target cell results in rapid activation and degranulation [48].
Tumor eradication by a range of mAB anticancer therapeutics,
such as anti-CD20 (rituximab, IgG1), anti-Her2 (trastuzumab,
IgG1), anti-CD52 (alemtuzumab, IgG1), anti-EGFR (cetuximab,
IgG1), and others, relies on this antigen-specific NK cell-
targeting mechanism [46,49]. The third-generation therapeutic
mABs are both humanized, and their Fc regions have been
modified for increased binding to FcγRIII [50]. Although each
of the antigens targeted by these therapeutics is expressed on
non-target cell populations, all of the MAbs have demon-
strated safety and clinical benefit [51].
NK cells lyse target cells by granzyme B- and perforin-
mediated apoptosis or death receptor ligands [52]. NK cells
are therefore able to lyse target cells without priming and
secrete cytokines like IFNγ to recruit and activate adaptive
immune cells. However, other effector cells, such as macro-
phages, may be involved in the activity of many anticancer
antibodies by their expression of all classes of Fcγ receptors
which mediate antibody-dependent phagocytosis. In all tumor
types, the macrophages constitute most of the infiltrating cells
while hardly any NK cells were identified [53,54]. Combining
tumor-targeting mABs and ADCC-promoting agents that sti-
mulate effector cells may hold great clinical potential.
However, most of the fully human or humanized antibodies
directed to PD-1 or PD-L1 are of the IgG4 isotype which
exhibits low activity in ADCC assays. Furthermore, the immune
checkpoint antibodies of IgG1 isotype are genetically engi-
neered to eliminate activity in ADCC in order to protect PD-
1/PD-L1-positive immune cells [55]. Thus, avelumab is the
single exception of a therapeutic antibody which exploits
immune checkpoint inhibition and ADCC-mediated killing of
tumor cells simultaneously (Figure1.).
Another mechanism involving ADCC affects regulatory T
cells (Treg), which express CTLA-4 constitutively. Inhibition of
CTLA-4 results in reduced Treg function which may also con-
tribute to antitumor responses by anti-CTLA-4 treatment. For
example, anti-CTLA-4-IgG2a mediates a rapid reduction of
Tregs at the tumor site in experimental animals [56].
Ipilimumab engages ex vivo FcγRIII (CD16)-expressing, non-
classical monocytes resulting in ADCC-mediated lysis of Tregs

CTL NK Cell
Tumor Cell
Other Anti-PD-1
or Anti-PD-L1 AB Avelumab
PD-1 PD-L1 Fc γ RIII

Figure 1. Schematic presentation of tumor cell – CTL – NK cell interactions.

Tumor cells express PD-L1 receptors which are blocked by the anti-PD-L1 avelumab mAB (green). CTL effector cells recognise the MHC class I molecule of tumor cells
via T-cell receptor (left, top) and the CTL PD-1 receptor is blocked by avelumab to inhibit PD-1/PD-L1 binding and generation of immune checkpoint inhibition.
Furthermore, avelumab links NK cells via their Fc receptor to PD-L1 ligands of the tumor cell and induces tumor cell lysis via ADCC. Avelumab may also bind to PD-
L1 of CTLs but, in this case, ADCC seems not to be triggered. Other antibodies directed to PD-1 or PD-L1 (grey) provide blockade of checkpoint inhibition but lack
the ability to invoke NK cell lysis. Full color available online.

[57]. Accordingly, patients responding to ipilimumab displayed
higher counts of peripheral nonclassical monocytes compared
with nonresponder patients.
4. Avelumab: preclinical studies
Avelumab/MSB0010718C is a fully human IgG1 anti-PD-L1
mAB which can lyse a range of PD-L1-positive human
tumor cells in the presence of peripheral blood mononuc-
lear cells (PBMC), or NK effector cells (Figure1) [42,43]. In
preclinical experiments, avelumab-mediated ADCC lysis of
tumor cells was comparable for NK cells purified from
either healthy donors or cancer patients [43]. In contrast,
avelumab-mediated lysis of whole PBMCs, including PD-L1-
positive subsets as targets, was marginal, corroborating
analyses of PBMC subsets of patients treated with avelu-
mab [43,51]. However, IFNγ is induced at sites of tumor
cell–immune effector cell contact which results in
increased PD-L1 expression of both tumor cells and lym-
phocytes, thus possibly enhancing ADCC-mediated lysis of
both cell types [43]. For example, PD-L1 expression was
markedly upregulated by IFNγ in chordoma cell lines,
which significantly increased their sensitivity to ADCC [58].
In detail, in respect to immune effector cells, addition of
avelumab to an antigen-specific in vitro stimulation assay
using normal PBMCs markedly reduced the proliferation
CD4+ T cells but increased the frequency of activated CD8
+ T lymphocytes, which are characterized by the production
of IFNγ [43]. An association between low CD4+ T cells and
an increased magnitude of the CD8+ CTL responses to
tumors was reported for an animal model [59]. The mechan-
isms of the resistance of PD-L1-positive immune cells to
avelumab-mediated ADCC are not clear. However, the
IFNγ-induced PD-L1 expression of tumor cells showed no
clear correlation to their sensitivity to ADCC-mediated lysis,
and IFNγ upregulates major histocompatibility complex
(MHC) class I expression which is a negative costimulatory
signal for NK cells and may protect cells from lysis [43]. In
conclusion, the IgG1 mAB avelumab did not induce lysis of
activated PD-L1-positive human immune cells, but increased
antigen-specific immune activation and, therefore, seemed
suitable for PD-L1 blockade in patients.
ADCC of avelumab towards primary mesothelioma cell
lines was evaluated using autologous and allogeneic NK cells
[60]. The lymphocytes present in malignant effusions of malig-
nant mesothelioma induced IFNγ-mediated PD-L1 expression
on the tumor cell surface. Of three mesothelioma cell lines
tested, two were lysed by avelumab-mediated ADCC in pre-
sence of autologous NK cells. Additionally, avelumab
enhanced antigen-specific immune activation in PBMCs from
metastatic breast cancer (MBC) patients. In conclusion, the
preclinical experiments demonstrated checkpoint inhibition
and ADCC without significant negative effects on immune
cells for avelumab.
5. Avelumab in clinical studies
Avelumab is in an early stage of clinical evaluation, and, there-
fore, the Merkel carcinoma phase II trial is the first study
EXPERT OPINION ON BIOLOGICAL THERAPY 519
reported in the literature, and data of all other trials is avail-
able in abstract form.
5.1. Phase I study of avelumab
A phase I dose-escalation study of avelumab (1–20 mg/kg) in
117 patients has been completed at the National Institutes of
Health’s Clinical Center (NCT01772004), with responses and
prolonged disease stabilization observed in several tumor
types [61]. The toxicity profile for avelumab appeared compar-
able to that of other anti-PD1/PD-L1 mABs. Only minor
changes were found in analyses of immune cell subsets in
PBMCs of patients in response to avelumab at 15, 43, and
127 days after initiation of treatment. Thus, avelumab seem
to efficiently mediate ADCC of human tumor cells that express
PD-L1, but only minor levels of avelumab-mediated lysis were
noted among unstimulated PBMCs.
5.2. Pharmacokinetics of avelumab
The pharmacokinetics of avelumab and the PD-L1 receptor
occupancy (RO) were determined in samples collected from
the phase I dose-escalation trial (NCT01772004) [62]. In vitro
data using blood samples from healthy donors had indi-
cated that 1 µg/ml avelumab was sufficient for >95% satura-
tion of the cognate antigen. For the avelumab trial, dosage
was escalated in four steps ranging from 1 and 3 to 10 and
20 mg/kg antibody. About 50 patients with advanced solid
tumors were treated with avelumab, every two weeks (q2w),
at the four dose levels and showed a linear increase of Cmax
and area under curve (AUC) with dosage. Half-lives for the
antibody were 66, 86, 92, and 115 h for the four dose levels,
respectively, with statistically significant differences
between the lowest and the three higher doses. The phar-
macokinetics data were consistent with a two-compartment
model with linear elimination of the antibody. It could be
demonstrated that trough levels at a dose of 10 mg/kg, but
not at lower doses, were sufficient for >95% RO. Absolute
lymphocyte count was stable, and immune cell subsets
evaluated exhibited no significant changes due to possible
avelumab-induced ADCC. Thus, the 10 mg/kg dose of ave-
lumab was recommended for the ongoing phase II and III
trials.
5.3. Avelumab treatment of Merkel cell carcinoma
Merkel cell carcinoma (MCC) is a rare and aggressive neuroen-
docrine tumor of the skin [63]. Early-stage disease can be
treated with surgical resection and radiotherapy, but minimal
residual disease is frequently identified. In addition, responses
to chemotherapy of advanced MCC are short lived, and immu-
notherapy targeting the PD-1/PD-L1 checkpoint holds great
promise as oncogenesis in MCC is linked to polyomavirus
integration and frequent ultraviolet-radiation-induced muta-
tions. Avelumab was studied in patients with stage IV MCC
(NCT02155647) that had progressed after cytotoxic che-
motherapy [64]. The mAB was administered intravenously at
a dose of 10 mg/kg every 2 weeks. The proportion of patients
who achieved an objective response was 28/88 patient
520 G. HAMILTON AND B. RATH
(31.8%) including eight complete and 20 partial responses.
Serious treatment-related AEs were reported in five patients
(6%): one case of enterocolitis, infusion-related reaction (IRR),
increased aminotransferases, chondrocalcinosis, synovitis, and
interstitial nephritis each. Five grade 3 treatment-related AEs
occurred in four (5%) patients: lymphopenia in two patients,
blood creatine phosphokinase, aminotransferase, and blood
cholesterol increases in one patient each. There were no treat-
ment-related grade 4 AEs or treatment-related deaths. Thus,
avelumab showed significant clinical activity in this difficult-to-
treat tumor type.
5.4. Avelumab for the treatment of chemoresistant
ovarian cancer
A total of 124 relapsed ovarian cancer patients were treated
with avelumab, resulting in a response rate of 9.7% comprised
of 12 partial responses with 6 ongoing [65]. Stable disease was
observed in 55 patients (44.4%) yielding a disease control rate
of 54%. Thus, single-agent avelumab showed an acceptable
safety profile and clinical activity in these heavily pretreated
patients. Treatment-related AEs occurred in 66.1% of patients
with most common fatigue (13.7%), IRR (12.1%), and diarrhea
(11.3%), all of grade 1 to 2. Grade 3 to 4 side effects were
reported in eight patients (6.5%), and there were no treat-
ment-related deaths.
5.5. Avelumab treatment of NSCLC
Patients with advanced NSCLC progressing after platinum-
based chemotherapy (NCT01772004) were treated with avelu-
mab at 10 mg/kg q2w [66]. Histology was adenocarcinoma
(62%), squamous cell carcinoma (29%), or other (9%). Any
grade AEs occurred in 139 (75.5%) patients including fatigue,
nausea, IRRs, chills, decreased appetite, and diarrhea. Drug-
related AEs grade ≥3 occurred in 22 (12%) patients and three
drug-related deaths were reported (radiation pneumonitis,
acute respiratory failure, and disease progression).
Objective responses were observed in 22 (12%) patients
(1complete response (CR) and 21 partial responses (PR)), and
stable disease was found in 70 patients (38%). Median pro-
gression-free survival was 11.6 weeks and the response in PD-
L1-positive patients was 14.4% and 10.0% in PD-L1-negative
patients, respectively. Median progression-free survival in PD-
L1-positive patients was 11.7 weeks vs. 5.9 weeks in PD-L1-
negative patients. In conclusion, avelumab was administered
to pretreated NSCLC patients safely with a toxicity profile
similarly to other anti-PD-1/anti-PD-L1 agents.
In a second trial, 145 patients received avelumab and were
monitored for a median duration of 13 weeks (range 0–31)
[67]. Median age was 70 years, and performance status was 0
(31.0%) or 1 (69.0%), with histology of mostly adenocarcinoma
(63.4%) and squamous (26.9%) carcinoma. Treatment-related
AEs occurred in 82 patients (56.6%; all grades); those occurring
in ≥ 10% were IRR in 24 patients (16.6%) and fatigue in 21
patients (14.5%). Grade ≥3 treatment-related AEs were
reported in 13 patients (9.0%). There were no treatment-
related deaths. Among 75 patients, unconfirmed overall
response rate was 18.7% (1 CR and 13 PRs; 12 were ongoing).
Stable disease was reported in 34 patients (45.3%), resulting in
a disease control rate of 64.0%, and a median PFS was
11.6 weeks for all treated patients.
5.6. Avelumab treatment of breast cancer
Avelumab was studied in 168 patients with locally advanced
breast cancer (BC) or MBC refractory to or progressing after
standard-of-care therapy (NCT01772004) [68]. Patients had
received a median of three prior therapies with taxane and
anthracycline. Any grade treatment-related AEs occurred in
120 patients (71.4%) with the most common comprising fati-
gue, nausea, and IRRs. Treatment-related grade ≥3 AEs
occurred in 24 patients (14.3%) and included (≥1%) fatigue,
anemia, increased gamma-glutamyltransferase (GGT), and
autoimmune hepatitis (each 3 [1.8%]) and arthralgia (2
[1.2%]). There were two treatment-related deaths (acute liver
failure and respiratory distress). Response rate in the entire
cohort was 5.4% (nine patients), with one complete and eight
partial responses. Stable disease was observed in additional 40
patients (23.8%), yielding an overall disease control rate of
29.2%. Among all patients with PD-L1-expressing immune
cells within the tumor, 33.3% (4 of 12) had partial responses.
In patients with triple-negative BC who had PD-L1-positive
immune cells within the tumor, 44.4% (four of nine) had
partial responses, compared to 2.6% for triple-negative BC
and PD-L1-negative immune cells, respectively. In conclusion,
avelumab showed an acceptable safety profile and had clinical
activity in a subset of patients with MBC.
6. Conclusion
Cancer immunotherapy has already yielded promising results
in several tumor types, and new mABs for immune checkpoint
inhibition arrive at an accelerated pace. Comparable to the
situation experienced with small-molecule inhibitors directed
to mutated kinases in NSCLC, questions addressing the best
mABs, possible combinations of agents, selection of the most
responsive patients, and mechanisms of resistance to the
therapeutics are discussed. Targeting of checkpoint inhibitor
has been expanded from CTLA-4 to the PD-1/PD-L1 pathway
and yielded significant anticancer activity in heavily pretreated
patients. For the immunotherapies, duration of response is
suggested as alternative parameter to disease-free survival
and OS. Tumor PD-L1 expression can be constitutive or induce
by IFNγ, resulting in different levels of expression and intratu-
moral heterogeneity. In general, intrinsic expression is mostly
homogeneous throughout the tumors and is better suited for
anti-PD-L1 antibody treatment in contrast to the patchy posi-
tivity in response to induction by IFNγ [69].
Most of the therapeutic mABs directed to tumor antigens
exploit ADCC as an important mechanism for highly efficient
cancer cell eradication. In the past, ADCC was avoided in
immune checkpoint inhibition for the concern to damage
and eliminate immune cells which express the relevant target
antigen in their activated state. Nevertheless, avelumab was
the first mAB to capitalize both on immune checkpoint inhibi-
tion via PD-1/PD-L1 and direct-cell attack via ADCC. Preclinical
and clinical data support the use of this ADCC-competent

mAB in checkpoint inhibition, and clinical trials revealed side
effects comparable to ADCC-disabled IgG therapeutics.
Advantages of ADCC in immunotherapy directed to check-
points may be increased efficacy and, in particular, preclusion
of upregulation of alternative immune checkpoint effectors,
resulting eventually in resistance to therapy. The full potency
of avelumab is expected to be revealed in subgroups of
patients positive for expression of PD-L1 and for those dis-
playing better immunocompetence than heavily pretreated
patients. Further trials of avelumab for mesothelioma, gastric
cancer, urothelial and adrenocortical carcinoma, renal cancer
as well as in combination with small-molecule inhibitors are
ongoing [70].
7. Expert opinion
Immunotherapy for cancer was of limited efficacy until inhibi-
tion of the immune defense system by tumor-activated sup-
pressive checkpoints became clear. Accordingly,
administration of an mAB to CTLA-4 prolonged survival in
melanoma patients. Attempts for immune checkpoint inhibi-
tion by agents directed to PD-1/PD-1L has generated a range
of mABs which were found to have clinical activity in mela-
noma, NSCLC, breast cancer, and other tumor types. Avelumab
is a human IgG1 antibody capable of triggering ADCC as
powerful additional means to eliminate tumor cells. The
other mABs directed to PD-L1 have inactivated Fc regions or
are of IgG4 subclass and unable to trigger ADCC. In addition to
the inhibition of the checkpoint blockade, ADCC guided by
anti-PD-L1 antibodies may lyse antigen-positive activated
immune effector cells known to be induced by IFNγ in the
tumor infiltrate. Despite ADCC triggering, preclinical and clin-
ical testing of avelumab revealed no increased toxicity to
immune cells or elevated toxic side effects compared to simi-
lar therapeutic mABs. Avelumab-guided cancer cell lysis by
ADCC may constitute an important advantage in precluding
upregulation of alternative suppressive immune checkpoint
proteins by tumors, as demonstrated for TIM-3 in case of
inhibition of anti-PD-1. Clinical results indicate that enrich-
ment of the patient populations for PD-L1 positivity and dis-
tinct tumor-specific phenotypes select for the patients with
highest chance of responses.
Clinical activity of avelumab in the trials for NSCLC, breast
cancer, and ovarian cancer patients was limited, as found for
other immune checkpoint-inhibiting mABs directed to the
same receptors. Most of the initial studies of immune check-
point inhibitors recruit patients which were heavily pretreated
with chemotherapy. Whereas patients are tested extensively
for expression of PD-1/PD-L1 antigens, there is obviously
hardly any attempt to check their immunocompetence
which may constitute an important predictor of successful
response to checkpoint inhibition and ADCC immune cell
attack. Recent data indicate that breast cancer patients have
not recovered from deleterious effects of chemotherapy on
the immune system even after 9 months following end of
therapy [71]. Phenotypes of repopulating B and CD4+ T cells
were significantly different from pre-chemotherapy profiles
and showed no signs of recovery. Many treatments adversely
affect NK cell function, and the optimal synergy between
EXPERT OPINION ON BIOLOGICAL THERAPY 521
chemoradiotherapy and innate as well as adaptive immunity
in the treatment of cancer is not clear [72]. Chemotherapy,
biological therapies, and radiotherapy can temporarily weaken
immunity in cancer patients. A panel of in vitro tests, including
flow cytometric measurements of PBMC subpopulations, T-cell
responsiveness to antigenic stimuli, and tests for NK activity
would provide measures of the immunocompetence of the
patients and possibly explain part of failure of immune check-
point inhibition. Research on stimulating the immune system
in general or individual cell populations, like NK cells, is
ongoing, and such methods may be employed as co-medica-
tion. The best modes of application of immune checkpoint
inhibitors and their most efficient combinations are studied in
a host of clinical trials. For example, avelumab will be studied
in ovarian cancer in a phase Ib/II trial in combination with
entinostat, a benzamide histone deacetylase inhibitor under-
going clinical trials for treatment of various cancers [73]. In
conclusion, clinical data obtained in studies of administration
of avelumab suggests the feasibility to combine immune
checkpoint inhibition with ADCC-mediated eradication of
tumor cells to increase anticancer activity and prevent a pos-
sible switch to alternative checkpoint inhibitors.
Funding
This manuscript has not been funded.
Declaration of interest
The authors have no relevant affiliations or financial involvement with any
organization or entity with a financial interest in or financial conflict with
the subject matter or materials discussed in the manuscript. This includes
employment, consultancies, honoraria, stock ownership or options, expert
testimony, grants or patents received or pending, or royalties.
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