Cancer Immunol Immunother

DOI 10.1007/s00262-017-1974-2

MEETING REPORT

Mechanisms ofefficacy incancer immunotherapy: 14th Annual

Meeting oftheAssociation forCancer Immunotherapy (CIMT),
Mainz, Germany, May 1012, 2016
JudithTheelen1 JenniferBraun1 HelenHrzer2 BjoernPhilippKloke1
MartinaOtt2 IrenePizzitola3 UweSchirmer1 MatthiasMiller1

Received: 14 September 2016 / Accepted: 11 February 2017

Springer-Verlag Berlin Heidelberg 2017

Keywords CIMT 2016 Cancer immunotherapy
Personalized immunotherapy Combination therapy
Abbreviations
ADCC Antibody-dependent cellular cytotoxicity
AIPV Extended combination of adaptive-cen-
tric therapy (V:amph-vaccine), innate-
centric therapy (I:extended half-life IL-2),
antitumor antibody (A), and checkpoint
blockade antibodies (P)
ALL Acute lymphocytic leukemia
AML Acute myeloid leukemia
AREG Apoptosis regulator
ARG1 Arginase 1
BAIT Bispecific antibody immune therapeutic
BCMA B-cell maturation antigen
BM Bone marrow
CCR2 Chemokine (C-C motif) receptor 2
ccRCC Clear cell renal cell carcinoma
CDC Cellular-dependent cytotoxicity
CIMT Association for Cancer Immunotherapy
CLL Chronic lymphocytic leukemia
This meeting report is a summary of presentations from the
Fourteenth Annual Meeting of the Association for Cancer
Immunotherapy, CIMT 2016, published together with a series
of Focussed Research Reviews based on lectures given at the
conference.
* Matthias Miller
matthias.miller@biontech.de
1
BioNTech AG, Kupferbergterrasse 1719, 55131Mainz,
Germany
2
Immatics Biotechnologies GmbH, Tbingen, Germany
3
GlaxoSmithKline, Stevenage, UK
CMC Chemistry, manufacturing and control
CMS Consensus molecular subtypes
CR Complete response
DEN  N-Nitrosodiethylamine
FAP Fibroblast activation protein
Fc Fragment, crystallizable
FcR Fc -receptor
GBM Glioblastoma multiforme
GMP Good manufacturing practice
GvHD Graft versus host disease
GvL Graft versus leukemia
HSC Hematopoietic stem cell
ICI Immune checkpoint inhibitor
IFN Interferon
iNOS Inducible nitric oxide synthase
iONC Immuno-oncology platform (Merck)
Kras Kirsten rat sarcoma viral oncogene
homolog, GTPase
MDS Myelodysplastic syndrome
MDSC Myeloid-derived suppressor cell
ME Microenvironment
MM Multiple myeloma
M-MDSC Monocytic MDSC
MMTV Mouse mammary tumor virus
MSI Microsatellite instability
NAD Nicotinamide adenine dinucleotide
NKT Natural killer T-cell
NOS2 Nitric oxide synthase 2
NSCLC Non-small cell lung cancer
PEI Paul-Ehrlich-Institut
PK Pharmacokinetics
PMN-MDSC Polymorphonuclear MDSC
RFS Regression-free survival
SCS Subcapsular sinus
SLP Synthetic long peptide

13
Vol.:(0123456789)

snRNP200 Small nuclear ribonucleoprotein U5 subu-
nit 200
TAM Tumor-associated macrophage
TCM T central memory stem cell
TCR T cell receptor
tEV Tumor-derived extracellular vesicle
TGF- Transforming growth factor beta
TLS Tertiary lymphoid structure
TNF Tumor necrosis factor
TSCM T memory stem cell
viSNE Visual Stochastic Network Embedding
Introduction
The increasing numbers of immunotherapeutic regimens
that enter not only clinical testing but also the routine appli-
cation as standard of care in hospitals were well-reflected in
a record number of participants. The 2016 Annual Meeting
of the Association for Cancer Immunotherapy (CIMT) with
more than 1000 registered participants attracted more sci-
entists than ever before. More than 45 presentations com-
prehensively addressed intriguing data on many aspects of
tumor immunotherapy covering checkpoint blockade, dif-
ferent vaccination approaches and emerging concepts for
cellular therapies. Accompanying the presentations, over
320 posters were presented allowing for intense discussions
of the respective data. The overall topic was the fine-dissec-
tion of the effects of successful therapies and how to imple-
ment promising approaches even more efficient into treat-
ment protocols. This meeting report summarizes the most
important presented findings and discussions of this 14th
annual meeting of the CIMT with the title Mechanisms of
Efficacy in Cancer Immunotherapy.
Tumor microenvironment
The tumor microenvironment plays a critical role for both
tumor progression and tumor control by the immune sys-
tem. In particular, the nature of the tumor immune micro-
environment has been shown to influence disease outcome.
Therefore, novel therapeutic approaches try to change
the balance of immune responses from tumor protection
towards tumor rejection for an effective cancer immuno-
therapy. With his research, Mikael Pittet (Massachusetts
General Hospital and Harvard Medical School, Boston,
USA) focuses on discovering previously unrecognized
cancerhost cell interactions and identifying aspects of
immune cell dynamics that could establish new paradigms
for translational efforts.
Increased TAM densities often correlate with decreased
patient survival, but the events that maintain or amplify
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Cancer Immunol Immunother
TAMs remain largely unknown. In the first part of his talk,
Pittet showed how some tumors produce long-range sig-
nals, such as the peptide hormone angiotensin II, which
act over extended distances in the body to continuously
amplify and mobilize TAM precursors, including hemat-
opoietic stem cells and CCR2+ monocytes. These findings
should help identify new therapeutic targets for manipulat-
ing TAMs.
Pittet also presented evidence that macrophages outside
the tumor stroma factor into cancer progression. He showed
that CD169+ subcapsular sinus (SCS) macrophages in
tumor-draining lymph nodes can suppress tumor outgrowth
by physically blocking the dissemination of tumor-derived
extracellular vesicles (tEVs). However, tumor progression
and therapeutic agents disrupt the SCS macrophage bar-
rier, which enables tEVs to enter the lymph node cortex,
interact with B cells, and foster tumor-promoting humoral
immunity.
The second part of Pittets talk focused on how to make
tumors responsive to immunotherapies. Using lung adeno-
carcinoma, mouse models resistant to current treatment
options, his group rationally selected drugs for their ability
to kill tumor cells in an immunogenic manner (e.g., oxali-
platin+cyclophosphamide against Kras/Trp53 mutant lung
tumors). These drugs induced tumor infiltration by CD8+
T cells and sensitized the tumors to checkpoint inhibition,
enabling durable cancer control. These findings clearly
underline the importance of a thorough selection of drugs
in combination trials.
Next, Ellen Pur (University of Pennsylvania, Phila-
delphia, USA) talked about the functional heterogeneity of
tumor stroma and its implications for modulating antitumor
immunity.
The gene expression signature of activated fibroblasts
is known to predict human cancer progression. Therefore,
the distinction between quiescent normal fibroblasts and
activated cancer-associated fibroblasts offers therapeutic
opportunities The fibroblast activation protein (FAP) here
represents a biomarker for carcinoma stromal cells, allow-
ing for distinction between quiescent and cancer-associ-
ated fibroblasts and has been shown to correlate with poor
prognosis in several types of human cancer (colon cancer,
non-small cell lung cancer, and pancreatic ductal adenocar-
cinoma). FAP+ cells can promote tumorigenesis by several
means making F AP+ cells a promising target for antican-
cer therapy. Depletion of FAP+ stromal cells by CAR (FAP
CAR) T cells can generate a more inflammatory environ-
ment and increase CD8 T cell infiltration in tumors thus
enhancing the efficacy of cancer vaccination in preclini-
cal experiments. In addition to this effect mediated by the
immune system, FAP CAR T cells can inhibit the growth
of tumors in an immune-independent fashion by disrupting
tumor-promoting desmoplasia.
Cancer Immunol Immunother
With his talk about major achievements in the charac-
terization of myeloid-derived suppressor cells (MDSCs),
Vincenzo Bronte (Verona University, Verona, Italy) closed
this exciting session about the tumor microenvironment.
The first part of his talk focused on cancer-induced
myelopoiesis and on the question how chemotherapy can
affect the immune suppression by myeloid-derived suppres-
sor cells (MDSC) in cancer tissue. This process of mye-
lopoiesis leads to the differentiation of monocytic MDSCs
(M-MDSCs, CD11b+Ly6GLy6Chigh) and polymorpho-
nuclear MDSCs (PMN-MDSCs, C D11b+Ly6G+Ly6Clow),
which potently inhibit antitumor immunity. In addition
to the direct suppression of antitumor immunity, MDSCs
also support tumor growth, angiogenesis, differentiation,
and metastasis. Accordingly, it could be shown that some
chemotherapeutic agents that eliminate M-MDSCs, such as
gemcitabine or 5-FU, exert an immunomodulatory effect.
In the second part of his talk, Bronte discussed the role
of l-arginine metabolism in tumor immunity. He and his
group could show that by inhibiting the activity of argin-
ase (ARG) and nitric oxide synthase (NOS) in the tumor,
the killing ability of tumor-infiltrating lymphocytes (TILs)
in prostate cancer organocultures could be rescued. These
results describe a novel and dominant mechanism by which
cancers induce immunosuppression. In conclusion, drugs
controlling ARG and NOS might be successfully used to
create a favorable tumor environment for TILs and thus
support immunotherapeutic approaches for the treatment of
cancer.
Improving Immunity
George Prendergast (Lankenau Institute for Medical
Research, Wynnewood, USA) presented data on IDO1 that
catalyzes the breakdown of tryptophan to NAD via kynure-
nine. As tryptophan is essential for T-cell proliferation,
IDO1 suppresses effector T-cell proliferation. In cancer,
toll-like receptor and IFN- signaling induce IDO expres-
sion and accordingly favor tumor immune escape.
Prendergast pointed out that IDO inhibitors, such as
Indoximod, Epacadostat, and GDC-0919, thus can enhance
anti-tumor immunity. Such agents were shown in the
MMTV-neu HER2+ breast cancer model to strengthen
T-cell responses. Furthermore, he referred to a phase IB
trial, combining taxanes and indoximod in stage IV breast
cancer patients, which revealed a very fast kinetic of the
response to IDO inhibitors. Notably, IDO inhibitors were
shown to increase the therapeutic effect of checkpoint
inhibitors, pointing towards synergistic mechanisms of the
two therapeutic strategies. IDO1 expression was mostly
found insitu in cancer cells, TAMs, MDSCs, mesenchymal
stem cells, and endothelial cells, whereas the expression on
APCs in lymph nodes, which could favor the generation of
regulatory T cells hampering tumor immune responses, is
controversially discussed.
In addition, Prendergast introduced that IDO1 activ-
ity is linked to productive IDO2 mRNA splicing and the
tryptophan metabolite kynurenine induces IDO2 expres-
sion. IDO2 also displays enzymatic activity, albeit on a
lower level than IDO1. His group found that chloroquine
inhibits IDO2 selectively and that in studies of low-dose
chloroquine treatment of brain metastasis patients, the sur-
vival was increased by about twofold, but only if the patient
lacked inactivating genetic polymorphisms in the IDO2
gene which lead to enzymatic incompetence.
Additional data on IDO2-deficient mice that show a
defective regulatory T-cell (Treg) compartment and that
are resistant to Kras-induced pancreatic cancer suggest a
unique role for IDO2 in tumor biology.
In closing, Prendergast concluded that IDO pathways are
key players in orchestrating inflammatory processes that
determine adaptive immune responses. His presentation
nicely underscored the importance of understanding the
mode of action of single drugs to create new combinatorial
regimens.
The next speaker, Hergen Spits (AIMM Therapeutics,
Amsterdam, the Netherlands), asked what can be learned
from the antibody repertoire from patients cured by immu-
notherapy. He and his lab developed an antibody discovery
platform that captures up to 85% of the B-cell repertoire of
a given patient. Therefore, B cells of three acute myeloid
leukemia (AML) patients with a potent graft versus leuke-
mia (GvL) reaction were screened for the identification of
AML specific antibodies, of which 17 were identified.
The first presented antibody called antibody (Ab)
AT1413 targets sialylated CD43, also known as sialo-
phorin. AT1413 binds all WHO defined types of AML
and myelodysplastic syndrome (MDS) cells. This antibody
can induce antibody mediated killing of tumor cells via
NK cells. The group also designed a bispecific antibody
immune therapeutic (BAIT) of the tumor specific antibody
and an anti-CD3 single chain variable fragment (scFv)
using sortase-mediated transpeptidation and click chemis-
try. The bispecific antibody effectively recruits CD3+ effec-
tor cells to the tumor tissue leading to the specific killing
of, e.g., the C D43+ AML cell line SH2. Since onco-sia-
lylated CD43 is also expressed on a majority of melanoma
cells, the AT1413 antibody can also induce ADCC against
melanoma cells.
In another study, the group treated a melanoma patient
with several doses of PBMCs stimulated with irradi-
ated tumor cells in vitro. After 9 years, the patient was
still in remission and his B cells were screened for mela-
noma specific antibodies. They discovered an IgG3 anti-
body (AT1412) recognizing CD9, a tetraspanin, which is
13

involved in tumor cell spreading. The antibody binds to cell
lines derived from melanoma, colon carcinoma, pancreatic
cancer, and multiple myeloma but only weekly to the cor-
responding normal tissue. In a mouse model, the antibody
was shown to inhibit melanoma metastasis. In addition, it
could be shown that the remission-free melanoma patient
had an elevated numbers of CD4+ and CD8+ T cells against
neoantigens and that this combined action of neoantigen-
specific T cells and elevated anti-CD9 antibody titer was
the cause for his tumor-free status. As discussed in previous
sessions, it is the combination of multiple strategies that is
likely to provide a high benefit to tumor patients, once more
underscoring the importance of a thorough understanding
of the modes of action of individual drugs.
Per thor Straten (University of Copenhagen, Copen-
hagen, Denmark) presented data about the suppression of
tumor growth and incidence by voluntary exercise. From
various tumor models, it is known that physical activ-
ity reduces the risk of different cancer types. In prostate
cancer, even therapeutic effects of exercise have been
described. Yet, the mechanisms how exercise interferes
with tumor progression remain unknown.
To elucidate the effects of physical activity on cancer, he
and his group employed different murine cancer models and
cages supplemented with running wheels which resulted
in voluntary exercise of the animals of approximately
210 km per night. Two weeks after tumor cell inocula-
tion mice were sacrificed and tumor burden was assessed.
He and his group could show that prior voluntary exercise
significantly suppressed tumor cell growth upon intrave-
nous (i.v.) or subcutaneous (s.c.) B16F10 cell inoculation.
Furthermore, he provided evidence for beneficial effects
of exercise in a spontaneous melanoma model [Tg(Grm1)
Epv mice] and in the N-nitrosodiethylamine (DEN)-
model of induced liver cancer, in which exercise signifi-
cantly reduced the lesion size. Analyses using tumors from
B16F10 subcutaneously inoculated mice with and without
exercise revealed an up-regulation of genes involved in
immunological pathways and an increased tumor infiltra-
tion by T cells, NK cells, and DCs in exercised mice. In
further experiments, the group could show by NK cell
depletion experiments that NK cells are essential to medi-
ate the exercise-induced therapeutic effect. They next found
a significant increase of both epinephrine and nor-epineph-
rine in the blood of exercised mice compared to controls.
The blockade of -adrenergic receptors by propranolol
completely abolished the beneficial effect of exercise in the
s.c. B16F10 inoculation model, whereas the administra-
tion of low-dose epinephrine to non-exercising mice mim-
icked the effect of physical activity partially. The depletion
of exercise-induced IL-6 also significantly reduced the
recruitment of NK cells to s.c. transplanted B16F10 tumors
and abolished the beneficial effect of exercise. Interestingly,
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Cancer Immunol Immunother
administration of IL-6 to non-exercised mice did not mimic
the effect of physical activity.
Keynote address
This years keynote address was delivered by Wolf H.
Fridman (Centre de Recherche des Cordeliers, Univer-
sity Paris Descartes, Paris, France). His presentation The
diversity of immune microenvironments in human cancers
summarized decades of his successful career as a scientist
and was, as announced by the chair Christoph Huber, edu-
cational and entertaining at the same time.
Starting his presentation, Fridman mentioned that inter-
national efforts should be made to join forces for research
and clinical testing and suggested to build a European
tumor immunology umbrella. Fridman next introduced the
high degree of organization in the tumor microenvironment
(ME) that also contains different types of immune cells.
Correlating the presence of certain immune cell subtypes to
the patients prognosis, he and his group could show, e.g.,
for colorectal carcinoma that in particular, the infiltration
of B cells, CD45RO+ and CD8+ cells can be correlated to a
good prognosis of the patients. The thereof developed local
immune score which for non-metastatic cancer (stage IIII)
correlated well with the clinical outcome indeed showed
that the localization, density, and type of several immune
cells insitu has tremendous impact on the clinical course of
cancer patients. For non-small cell lung cancer (NSCLC),
he further indicated that the localization of immune cells in
highly organized tertiary lymphoid structures (TLS) featur-
ing B-cell follicles and high endothelial venules within the
ME is an important site of priming and activation of tumor-
reactive T and B cells. Accordingly, correlations between
the density of DCs within tumor specimen and cytotoxic-
ity-related genes were indicative of a prolonged overall sur-
vival, in particular when high amounts of CD8 T cells were
present. Not surprisingly, the same held true for the den-
sity of tumor antigen-specific antibody-producing follicular
B cells within the TLS. Remarkably, for colorectal cancer
patients, this in situ immune contexture was shown to be
reproduced at different metastatic sites leading to the con-
clusion that there is an immune imprint on tumor cells in a
given patient regardless of the site of tumor growth.
As one remarkable exception to the rule that an infiltra-
tion of tumors with CD8+ T cells and the density of DCs
have a good prognostic impact, clear cell renal cell carci-
noma (ccRCC) was introduced. For this indication, it was
found that for a subgroup of tumors, an IFN-signature is
indicative of the expression of, e.g., PD-1, PD-L1, PD-L2,
and LAG3, the simultaneous expression of which alone
or in the presence immature DCs correlated with a poor
prognosis.
Cancer Immunol Immunother
Fridman next correlated the data from transcriptomic
data reads with immunohistochemical data leading to the
development of expression patterns for individual immune
cell subsets, the so-called immunome, which was repro-
duced in over 6000 different tumor specimens. Focusing
on the consensus molecular subtypes (CMS) of colorectal
carcinoma, his group could link the ME transcriptomic
data with individual CMS and identified two particular
groups with immunome-signatures being indicative of high
D8+ immune effector cells. Based on the
infiltration of C
immunome- and MSI-stratification, Fridman proposed a
personalized treatment schedule. Notably, the approach to
stratify patients with respect to the response to anti-PD-1
therapy according to the inflammatory and mesenchymal
signature could be expanded to other cancer indications as
well. Finally, Fridman again discussed ccRCC for which
the transcriptomic analysis could also be correlated to the
treatment response, in this case to sunitinib.
Concluding, Fridman proposed a classification of can-
cers based on transcriptomic signatures and the associated
immune contexture that could be able to advise a pharma-
ceutical intervention with a high probability for success.
Altogether, this keynote address nicely underscored the
theme of this years CIMT that existing therapies can be
further optimized if efforts are put into deciphering of the
underlying mechanisms and by selecting therapeutic regi-
mens based on new markers predicting the response of an
individual tumor.
Therapeutic vaccination
The therapeutic vaccination plenary session was opened
by Darrell J. Irvine (Massachusetts Institute of Technol-
ogy, Cambridge, USA) with his talk about the concept of
recruiting synergistic innate and adaptive immune cells into
tumor tissue.
Since small molecules (such as peptide vaccines) are
cleared from blood, whereas large molecules (like albumin)
are transported via the lymph system, Irvine and his group
added a lipophilic albumin-binding tail to both antigen and
adjuvant and promoted albumin-mediated lymph node tar-
geting of vaccines invivo, resulting in enhanced potency of
the vaccines.
In the last years, combinations of immunotherapies
have been increasingly discussed. It has been shown that
the combination of an anti-tumor antigen antibody and
an untargeted IL-2 fusion protein with delayed systemic
clearance is able to control tumor growth in aggressive
isogenic tumor models. This tumor control is mediated
via a concerted innate and adaptive response involving
neutrophils, NK cells, macrophages, and C D8+ T cells.
An extended combination (AIPV) of adaptive-centric
therapy (A: antitumor antibody), innate-centric ther-
apy (I: extended half-life IL-2), checkpoint blockade
antibodies (P), and a vaccine (V) results in substantially
improved survival rates in melanoma, cervical cancer,
and breast cancer models compared to adaptive-centric
or innate-centric therapy alone. This AIPV combina-
tion therapy dramatically reprograms the tumor microen-
vironment, especially affecting expression of chemokines
and cytokines important for immune cell recruitment. In
addition, it creates a positive feedback loop for the innate
immune system by inducing a strong de novo antibody
response.
In the second talk of the session, Gerold Schuler
(University Medical Center Erlangen, Erlangen, Ger-
many) reported on the experiences of the University
Medical Center in Erlangen with trials investigating
dendritic cells. He presented the adjuvant multicenter
phase III Dendritic Cells plus Autologous Tumor RNA
in Uveal Melanoma trial (NCT01983748) which aims
at preventing later fatal metastatic disease by adjuvant
vaccination. Two hundred patients suffering from uveal
melanoma typed positive for monosomy 3 (a proven pre-
dictor of metastasis) are planned to be included and vac-
cinated at eight timepoints over a period of 2years with
DCs loaded with autologous tumor RNA to explore the
total antigenic repertoire of the individual tumor. As of
May 2016, 55 patients have been screened and 27 patients
have been enrolled in the trial.
The session was closed by Cornelis Melief (Leiden Uni-
versity Medical Center and ISA Pharmaceuticals, Leiden,
The Netherlands) who highlighted that for an effective can-
cer treatment combinatorial treatments such as therapeutic
vaccination with chemotherapy or with monoclonal Abs
that work as checkpoint inhibitors or that bind to immuno-
suppressive cytokines are essential.
In the last years, ISA Pharmaceuticals successfully
developed synthetic long peptides (SLPs) for vaccination
against oncoproteins of HPV16 and was able to show clini-
cal proof-of-concept in high grade vulva intra-epithelial
neoplasia patients. A current pilot study investigates the
effect of therapeutic vaccination with HPV16 SLPs dur-
ing ongoing chemotherapy with carboplatin and pacli-
taxel in patients with recurrent or metastatic cervical can-
cer. MDSC numbers decreased to normal levels in these
patients during chemotherapy with carboplatin and pacli-
taxel. Furthermore, adequately timed HPV16 SLP vaccina-
tion sustained T-cell reactivity in these patients, showing
that vaccination during myeloid cell depletion by cancer
chemotherapy fosters robust T-cell responses. This syner-
gism of a selected chemotherautic drug combination with
therapeutic vaccination is mainly performed by promoting
tumor cell apoptosis by TNF (platinum compounds) or by
depletion of MDSCs (carboplatin and paclitaxel).
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CIMT Endeavour
In course of the CIMT annual meeting, the 4th CIMT
endeavour workshop took place. The workshop presented
a lively education and networking platform that addressed
the translation of cancer immunotherapies from preclini-
cal proof of concept to products for patients. The focus
of CIMT endeavour is to encourage entrepreneurship and
commercialization by focusing on the right skills, mind-
sets, and attitude. The two scientists Nadia Mensali (Oslo
University Hospital, Oslo, Norway) and Remko Schotte
(AIMM Therapeutics, Amsterdam, The Netherlands) pre-
sented their work and were challenged regarding a possi-
ble translation and product commercialization by an expert
panel consisting of Stefan Ries (Roche, Penzberg, Ger-
many), Georg Schnappauf (Zwicker Schnappauf & Part-
ner, Munich, Germany) and David Phillips (SR One Ltd,
San Francisco, USA). In the presentations of the workshop,
Lothar Germeroth (Juno Therapeutics, Gttingen, Ger-
many) and Derek Jantz (Precision BioSciences, Durham,
USA) presented their view of the story which hurdles need
to be taken to successfully translatscience into products.
Cellular therapies
In recent years, the number of clinical trials in the field of
cancer T-cell therapy was increasing constantly. Several
trials demonstrated the anti-tumoral efficacy not only in
patients with advanced leukemia but also in patients with
solid tumors like melanoma.
The first speaker was Carl June (University of Penn-
sylvania, Philadelphia, USA) who performed pioneer-
ing work in developing ag-specific CAR modified T cells
(CAR T cells) for treatment of late stage leukemia. In his
presentation, he summarized four current trials investigat-
ing relapsed and refractory C D19+ chronic lymphocytic
leukemia (CLL) and acute lymphocytic leukemia (ALL) as
well as B-cell maturation antigen (BCMA) positive B-cell
malignancies [multiple myeloma (MM)]. The therapy with
CD19 CAR T cells (construct CTL019) yielded strong
clinical responses. In the phase I trial for CLL, eight of 14
patients had a clinical response with a complete response
(CR) observed in four patients. In pediatric ALL, 55 out
of 59 children had a CR at 1month after infusion and the
12month regression free survival (RFS) was 55%. Notably,
CLL patients with a durable remission had an evidence for
longer persistence of CTL019 in the circulation. A robust
CAR T cell expansion was also observed for BCMA CAR
T cells (CART-BCMA) in multiple myeloma patients. In
this pilot trial, two of three treated patients had a clear-
ance of tumor burden after infusion of CART-BCMA
cells without lymphodepletion. To further improve the
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Cancer Immunol Immunother
anti-tumor effect of immunotherapies for relapsed C D19+
B lymphoma, sequential treatment with CTL019-transgenic
cells and an anti-PD-1 antibody was tested. Until the end
of 2015, only one patient has been recruited but this indi-
vidual showed massive clearance of the tumor which indi-
cates the feasibility of sequential administration of check-
point antagonists and CAR T cells. These successes might
get even more pronounced by modifying the CAR signaling
domains by incorporating, e.g., the intracellular domains of
CD28, 4-1BB, or ICOS which might affect the persistence
and functional mode of a CAR T cell.
Another way to improve safety as well as efficacy of cel-
lular based immunotherapies by the means of T-cell recep-
tor (TCR) gene editing was reported by Chiara Bonini
(Vita-Salute San Raffaele University, Milan, Italy). A proof
of principle study in immune competent mice model of
MM revealed that even the disruption of a single chain of
the endogenous TCR, and substitution with an entire tumor
specific TCR can improve the safety of engineered T cells.
These cells showed the same efficacy as TCR transduced
T cells, but pathologic events and fatal graft versus host
disease (GvHD) were not visible in mice that received T
cells with single-edited TCRs, whereas more than 50% of
the control mice that received the normal TCR developed
GvHD. Bonini also addressed the question of long-term
persistence of transferred T cells. Persistence and in vivo
expansion is associated with the T-cell phenotype and a
high number of T memory stem cells (TSCM) and central
memory cells (TCM). But also the phenotype of TSCM and
TCM cells has a clear impact on the clinical outcome. In
particular, BM infiltrating TCM and T SCM cells from relaps-
ing AML patients express higher levels of PD-1, 2B4, and
Tim-3 already early after transplantation and display a
T-cell exhausted phenotype that is less functional.
Robert Hawkins (University of Manchester and Christie
Hospital, Manchester, England) reported about the adoptive
T-cell therapy with TILs. TILs are supposed to target that
only tumor specific antigens, harbor high affinity TCRs, are
truly personalized and safer due to thymic selection. The
quality of TIL treatment is critical in terms of viability and
T-cell purity as well as the quantity of TILs that can be har-
vested after expansion. Currently, the group around Robert
Hawkins established a GMP grade manufacturing process
using WAVE-Bioreactors for T-cell expansion. The final
TIL product shows cell viability and purity of about 95%.
In a representative trial, TIL harvesting from 44 patients
with refractory and relapsed melanoma was completed
with a success rate of more than 90%. Isolated TILs were
returned to 22 patients and no side effects related to the
cell transfer as well as stable invivo expansion of admin-
istered TILs could be observed. For some selected patients,
the treatment had a clear anti-tumor effect with a complete
remission of metastases and durable benefits. Nevertheless,
Cancer Immunol Immunother
Hawkins also reported on several strategies to improve cel-
lular therapies with TILs. One thinkable approach was the
introduction of IL-2 cytokine switches for IL-2 independ-
ent growth. Engineered TILs transduced with a molecular
growth switch expand independent from IL-2 and may have
a longer persistence in the periphery.
Novel technologies forimmune assessment
Despite tremendous recent successes in the field of tumor
immunotherapy, technical limitations can hinder scientists
from performing more robust or comprehensive experi-
ments. This session, therefore, presented recent develop-
ments that tackle some of the existing limitations.
Timothy Tree (Kings College London, London,
United Kingdom) presented an update on the isolation of
Tregs. These cells are usually identified using a combina-
tion of surface markers, such as CD4 and CD25, plus the
transcription factor FoxP3, making them impossible to
be isolated viable. Studies that focus on T reg biology thus
rely on their identification based on the expression of
CD4+CD25highCD127low; however, they are phenotypi-
cally complex and heterogeneous. Classical flow cytometry
using fluorochrome-conjugated antibodies allows the meas-
urement of few parameters at single-cell-level resolution.
To address this limitation, Tree developed and validated
a new method to identify and understand the phenotypic
complexity of Treg compartment using mass cytometry.
Here, the antibodies are conjugated with metals, which
allow to analyze up to 40 parameters without losing single-
cell resolution. Based on a panel of 26 markers, a differ-
ent Treg subset based on the expression of CD161, CD73,
CD45RA, and CCR3 was identified. They implemented
their analysis using Visual Stochastic Network Embedding
(viSNE), a tool that allows mapping of high-dimensional
cytometry data onto a two-dimensional plot in which cells
are divided into clusters. A cluster analysis was performed
using FLOCK (FLOwing without K), and the group could
show that mass cytometry in combination with viSNE and
FLOCK can overcome current limitations of established
methods and picked up 22 distinct subpopulations of Tregs,
which are reproducibly and objectively identified within
multiple donors.
Tree next discussed a new assay to measure Treg sup-
pressive function. In the classical Shevach assay, the
inhibition of T-cell proliferation by T regs is measured. Here,
the suppression of proliferation is dependent on the ratio
of the cells. However, the strength of the stimulation and
the presence of APCs can influence the final results, high
numbers of fresh cells are required, and only low through-
put with low reproducibility is possible. To overcome these
restrictions, the group developed a new flow cytometry
sorter-assisted invitro co-culture assay, reducing the num-
ber of cells and allowing for higher throughput at high
reproducibility.
In the last part of his talk, Tree touched on the antigen-
specificity of Treg. The possibility to identify antigen-spe-
cific Treg can open a new area in cell based therapies, since
unwanted immune reactions can be targeted by avoiding the
risk of a more general immune suppression.
Sine Reker Hadrup (Technical University of Den-
mark, Copenhagen, Denmark) presented an update about
a new technology to detect antigen-specific T cells, which
is a key step for the development of novel cancer vaccines
and immunotherapy. The current gold standard to detect a
single ag-specific T cell is based on the MHC multimers.
Using various either fluorescent or metal-conjugated anti-
bodies, the current limitation is 100 individual T-cell spe-
cificities per sample, which poorly matches the large com-
plexity and diversity of the T-cell repertoire in humans. To
provide a solution to these unmet needs, the group devel-
oped a novel technology based on unique DNA-barcode
labelled MHC multimers, where it is possible to stain up
to 1000 T-cell specificities in a single small size biologi-
cal sample that could be few mL of blood or enzymatically
digested material. The respective T cell-specificity can be
identified after a PCR reaction and the respective cells can
then be sorted by classical MHC multimer staining. This
technology now allows to conclusively address how the
TCR repertoire looks in a given sample or how it is altered
by for instance pharmaceutical intervention. The high-
throughput screening and profiling of, e.g., cancer respon-
sive T cells can provide a more complete picture of what
specificities are key players for instance in tumor tissue.
The last speaker of this session, Giuliano Elia (Philo-
chem, Otelfingen, Switzerland), presented updates on
a phase II trial in which immunocytokines have been
applied intralesionally for the treatment of advanced stage
melanoma patients. Immunocytokines are recombinant
fusion proteins in which cytokines of interest are fused
with an antibody fragment, which targets the drug to the
site of disease. In particular, Philogen used the combina-
tion of L19IL2 (Darleukin) and L19TNF (Fibromun),
called Daromun in which the L19 fragment recognizes the
alternatively spiced extra-domain B of fibronectin, selec-
tively expressed on newly formed tumor vasculature. This
fragment allows prolonged residence of cytokines at the
lesion site, resulting in fewer administrations necessary
to get an effect. Moreover, in contrast to systemic treat-
ment, local injection of the drugs resulted in a higher con-
centration of the cytokines insitu, thus limiting toxic side
effects. In terms of efficacy, the combination of immuno-
cytokines has a strong synergistic effect when compared
to single agents, leading to 5% of CR and 50% of PR rate
at 12week post injection. Compared to historical controls,
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the intralesional administration of Daromun slowed the
progression of the disease to stage IV melanoma, which is
fatal in most cases, with a significant improvement of the
overall survival at 1year post treatment. Anticancer activ-
ity of immunocytokines depends on T cells and immuno-
histochemistry accordingly demonstrated the presence of
CD8+ TIL, with no significant modulation of C D4+, Treg,
or NK cells. Besides a local control of disease, Daromun
was shown to mediate systemic anticancer activity, since in
7/13 non-injected lesions (neighbouring and distal), com-
plete responses as a bystander effect were observed.
Based on these very encouraging results, last January,
the Philogen group has announced the launch of a ran-
domised Phase III clinical trial, enrolling more than 200
patients in an international multi-centre study. Recurrence-
free survival rate at 1year post surgery might confirm the
efficacy of the intralesional injection of Daromun as a neo-
adjuvant in combination with surgery.
Antibodies
Rony Dahan (Rockefeller University, New York, USA)
opened the last session which focused on the role of anti-
bodies in immunotherapy. On the one hand, it has previ-
ously been shown that type I Fc receptors (FcR) like
FcRIII are required to mediate ADCC (antibody-depend-
ent cellular cytotoxicity). In addition, the engagement of
FcRIIA expressed by DCs can generate a potent vaccinal
anti-tumor effect by inducing a cellular memory forma-
tion. In this context, Dahan presented the specific FcR-
requirements for the immunomodulatory antibodies against
PD-1/L1, CTLA-4, and CD40.
He demonstrated that there are distinct FcR-
dependencies and mechanisms for anti-PD-1 and anti-PD-
L1 antibodies. While the anti-PD-L1 anti-tumor activity is
augmented by FcR binding, the opposite is shown for anti-
PD-1. The anti-PD-L1 anti-tumor activity is exerted via the
targeting of PD-L1-positive monocytes and thus the modu-
lation of the tumor microenvironment, whereas the binding
of anti-PD-1 to FcR leads to an elimination of activated
CD8+ cells.
For anti-CTLA-4 antibodies, it has been shown in mouse
models that they lead to an FcR-dependent intratumoral
depletion of Tregs. First, hints are available that this also
holds true for humans as shown with a humanized FcRs
mouse model (MC38).
Furthermore, Dahan reported that an FcRIIB-
dependent activity has been shown for anti-CD40 agonis-
tic human antibodies, even though this is an unexpected
requirement as this is an inhibitory FcR, whereas FcRIIA
engagement reduces the invivo activity.
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Cancer Immunol Immunother
Taken together, there are several FcR-mediated mecha-
nisms by which immunomodulatory antibodies act. Thus,
manipulation of the FcR-interaction by Fc-engineering
might allow for the design of a next wave of optimized anti-
bodies that can achieve better clinical responses as the cur-
rent generation of antibodies.
The second talk of this session was given by Paul Par
ren (Genmab, Utrecht, the Netherlands) about the molecu-
lar mechanism of complement activation by IgG-type anti-
bodies and the application of this insight to the generation
of a novel antibody format with enhanced complement
activation. Antibodies were shown to cluster via intermo-
lecular FcFc contacts after opsonizing their target struc-
tures on cells, resulting in the activation of the complement
system. Complement activation not only leads to comple-
ment-dependent cytotoxicity (CDC), but also induces to
the release of chemoattractants that attract and activate
effector cells of the immune system. Parren presented that
optimal C1q binding and hence complement activation is
optimally induced by antibody hexamers. Thus, the mod-
ulation of FcFc interactions to enhance hexamerization
has a direct effect on complement activation. To this end,
the HexaBody platform has been developed by Genmab.
This platform is designed for potentiation of therapeutic
antibodies by introducing single point mutations into the
Fc-backbone that enhances hexamerization as well as CDC
while retaining the developability characteristics, pharma-
cokinetics (PK), and safety properties of regular antibod-
ies. Two mutations that have been shown to enhance CDC
activation are E345K and E340G are applicable to a wide
range of IgG antibodies including antibodies to CD20 and
CD38. The results show that the H exaBody platform is a
promising tool for the generation of therapeutic antibodies
with enhanced properties.
The last talk was presented by Kin-Ming Lo (EMD
Serono/Merck KGaA, Billerica, USA). Merck KGaAs
IONC (Immuno-Oncology) translational innovation
platform focuses on tumor immune tolerance including
immune checkpoint inhibitors (ICI), immunocytokines,
fusion protein/bispecific antibodies, therapeutic cancer vac-
cines, and CAR T cells. In his talk, Lo described an inno-
vative bifunctional antibody fusion protein anti-PD-L1/
TGF- Trap (M7824), which is a fast follower of the anti-
PD-L1 antibody Avelumab. Avelumab, Merck KGaAs first
checkpoint blockade molecule that entered into the clinic,
is a fully human IgG1 therapeutic antibody blocking the
PD-L1/PD-1 interaction. More than 1800 patients have
already been treated with Avelumab, which is currently in
phase III trials.
M7824 is a novel construct comprising an anti-PD-L1
moiety and soluble TGF receptor II. It was designed to
block both cell intrinsic and extrinsic pathways as it targets
the PD1/PD-L1 immune checkpoint in the tumor as well
Cancer Immunol Immunother
as the immunosuppressive cytokine TGF in the tumor
microenvironment. Pre-clinical models demonstrated the
enhanced anti-tumor activity of M7824 compared to anti-
PD-L1 or TGF Trap alone. Pharmacodynamic studies
showed neutralization of systemic TGF correlated with
enhanced T-cell infiltration into tumors. Hence, dual target-
ing of PD-L1 and TGF pathways may provide effective
anticancer immunotherapy. M7824 has recently completed
phase I dose escalation studies, with expansion cohorts
planned to begin shortly in multiple indications.
Conclusions
The increasing pace at which novel cancer immunothera-
pies are reaching clinical testing and the approved ones
get more and more routinely applied is remarkable and has
been maintained for several years by now. However, along
with successes in the treatment of many cancer indications,
phenomena, such as immune escape mechanisms, often
get described thus maintaining an unmet medical need for
many patients. This years CIMT meeting, therefore, set
out to dive into the mechanisms of these regimens to pos-
sibly refine the treatments and lead to even more targeted
approaches. Throughout the sessions, fascinating new data
were shared and therapeutic strategies taking novel targets
into account were conclusively discussed. The personaliza-
tion of existing approaches based on a sound scientific basis
and the development of new concepts such as the emerg-
ing field of cell-based therapies are highly exciting and a
new wave of tumor immunotherapies is on the advent. We
all look forward to the next meeting of the CIMT in 2017
when first results from these research efforts will most
likely be presented.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict
of interest. Bjoern-Philipp Kloke has been co-organizer of the CIMT
endeavour workshop.
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