Introduction
More than 900 scientists around the world visited the 12th Annual Meeting of the Association
for Cancer Immunotherapy (CIMT) in Mainz, Germany from 6–8 May, 2014. Recent advancements
in various specific fields of cancer immunotherapy were discussed in Europe`s largest
meeting of this kind under the motto “Next Waves in Cancer Immunotherapy,” the highlights
of which are summarized in this meeting report.
Therapeutic Vaccination
Willem W. Overwijk (MD Anderson Cancer Center, Houston, U.S.A.) started the session
by showing the pros (cheap, specific, safe and long-term protection) and cons (poor
efficacy, requirement of functional immune system) of therapeutic cancer vaccines.
Overwijk and colleagues focus on peptide vaccination against melanoma using gp100
peptide in Incomplete Freund's Adjuvant (IFA). He tried to answer the question why
many vaccinated cancer patients do not experience tumor regression despite increased
levels of cancer antigen-specific T cells. Overwijk stressed the point that one reason
for the failure might be the use of IFA-formulated vaccines. His group showed that
vaccination with peptides formulated in IFA led to accumulation of CD8 T cells at
the injection site, resulting in dysfunction and apoptosis of these T cells.
1
In contrast, water-based vaccines permitted T cell accumulation at the tumor site
and exhibited therapeutic anti-tumor effects while T cells induced upon IFA-formulated
vaccination became exhausted at the injection site. He also shed light on why the
IFA-formulated vaccine against gp100 did not synergize with anti-CTLA-4 therapy in
the clinical setting, showing that the vaccination site also traps other tumor antigen-specific
T cells which were activated by anti-CTLA-4 therapy. However, he highlighted that
this is not always the case since virus-based vaccination synergizes with anti-CTLA-4
therapy. He concluded that not all cancer vaccines are created equal; persistent vaccine
formulations can entrap tumor-specific T cells while short-lived formulations release
the T cells to traffic to tumor sites.
Long-peptide (LP) vaccines (∼20 amino acids) are a promising strategy as they require
processing prior to MHC class-I presentation by dendritic cells (DCs), which may mimic
tumor antigen presentation more accurately than short peptides that load directly
on not only APCs but also all nucleated cells without prior processing.
2
Yasuharu Nishimura (Kumamoto University, Kumamoto, Japan) presented the development
of LP-based cancer immunotherapy targeting both tumor antigen-specific CD4 and CD8
T cells. Using genome-wide cDNA microarray analyses, his group identified several
new genes encoding for cancer testis antigens strongly expressed in oral and esophageal
squamous cell carcinomas but not in many normal adult tissues. To select a candidate
LP encompassing both Th cell and CTL epitopes, they combined prediction of tumor-associated
antigen-derived HLA class II binding LPs with sequence data of known HLA-A2 or HLA-A24-restricted
CTL epitopes, stimulated patient PBMCs to assess specific Th1 responses and confirmed
cross-presentation of LPs in vitro and with HLA-I transgenic mice in vivo. They identified
an LP from LY6K, HLA-A24-restricted LY6K172–191, which naturally encodes the CTL epitope
LY6K177–186. LY6K172–191 LP elicited Th1 responses in human PBMCs and cross-primed
CTLs in HLA-A24 transgenic mice. In addition, the presence of LY6K172–191 LP-specific
Th cells in head and neck cancer patients vaccinated with the LY6K177–186 short peptide
(SP) suggests that tumor lysis induced by SP vaccination-activated CTLs accelerates
uptake and processing of LY6K protein by APCs which in turn present LY6K-derived LPs
to CD4 T cells. Nishimura observed a synergy between LY6K-specific Th and CTL responses
in vitro and suggested that the same phenomenon may occur in vivo. Similar LPs were
also identified in two other cancer testis antigens, CDCA1
3
and KIF-20A.
4
Taken together, TAA-derived LPs encoding both Th and CTL epitopes may be useful for
immunotherapy of various types of cancer.
5
Successful induction of T cell immunity by direct mRNA vaccination is associated with
several challenges. Ugur Sahin (TRON, Translational Oncology at the University Medical
Center of the Johannes Gutenberg University Mainz, Mainz, Germany) outlined his approach
of tackling these challenges and the clinical translation of personalized, mRNA-based
cancer immunotherapy as performed in his institute. Pharmacological optimization of
mRNA in-house
6-8
enabled intranodal delivery of naked mRNA, stimulating robust expansion of immunodominant
CTLs, indicated by preclinical studies
9
as well as by preliminary immune evaluation of a phase I/II clinical trial against
melanoma. Recently, Sahin and coworkers succeeded in translating this approach from
local to systemic DC targeting using liposomal formulations which resulted in superior
immunostimulation and rejection of established tumors in preclinical studies, providing
the basis for several planned phase I/II clinical trials (melanoma, breast cancer,
head and neck cancer) in the context of an RNA warehouse approach with pre-furnished
RNA portfolios to be individually composed for each patient. According to Sahin, targeting
tumor mutations is the key to successful personalized anti-cancer therapy. Using a
complex mutation identification process
10
including NGS-sequencing of expressed mutations in tumor biopsies, prioritization
and mutation immunogenicity testing with patient PBMCs and subsequent in-house product
manufacture, immunization with an oligo-epitopic RNA vaccine is feasible within three
months and is currently employed in a phase I/II clinical trial against melanoma (NCT01684241).
Sahin concluded with stressing the importance of the regulatory development for actively
personalized cancer immunotherapeutics and the active involvement of the CIMT regulatory
research group, to facilitate clinical translation of individualized anti-tumor immunotherapies.
Combination Therapy
For decades combination therapy has been an important treatment modality in various
diseases including infectious diseases, cardiovascular diseases and cancer to maximize
therapy responses.
11
In cancer immunotherapy, combinatorial therapy approaches are still at the early stage
of development, nevertheless already displaying great potential for future application
as demonstrated during the “Combination Therapy” session.
The introduction of the checkpoint modulators Ipilimumab (anti-CTLA-4 antibody) and
Nivolumab (anti-PD-1 antibody) revolutionized the field and had a tremendous impact
for cancer immunotherapy,
12-14
but as pointed out by the first speaker of the session, Michael Curran (MD Anderson
Cancer Center, Houston, U.S.A.), there are still opportunities to augment their therapeutic
potential by joint action. The observation that blocking CTLA-4 leads to reciprocal
upregulation of PD-1 on activated T cells suggested that a dual blockade of these
non-redundant pathways might produce greater anti-tumoral activity than that observed
for blockade of CTLA-4 alone. Curran was able to confirm that the joint action of
CTLA-4 and PD-1 blockade in conjunction with a B16-Flt3-ligand vaccination in a preclinical
B16-BL6 melanoma model augmented tumor infiltration of effector T cells, led to a
favorable T effector to T regulatory (Treg) cell ratio and increased IFN-γ production
in the tumor, resulting in rejection of 50% of tumors, compared with 10% achieved
with CTLA-4 blockade alone.
15
Motivated by these promising preclinical results, a clinical phase I study was initiated
combining Ipilimumab and Nivolumab in patients with advanced melanoma. In accordance
with preclinical data, the trial revealed a superior outcome of the concurrent treatment
with Ipilimumab and Nivolumab in comparison to Ipilimumab alone, but at an expense
of a higher incidence of grade 3 and 4 adverse events.
16
However, combining two checkpoint inhibitors as demonstrated above, is only one out
of a plethora of possibilities. Whereas CTLA-4/PD-1 blockade focused on “releasing
the brakes”, direct T cell stimulation is another interesting therapeutic approach.
Targeting co-stimulatory receptors might additionally “press the gas pedal” for optimal
T cell effector function and is currently under investigation at different stages.
17,18
In a B16-BL6 mouse model, concurrent administration of an agonistic 4–1BB antibody
and CTLA-4 blockade, combined with a B16-Flt3-ligand vaccination, resulted in the
rejection of 57% of the implanted tumors in comparison to 13% for 4–1BB activation
and 20% for CTLA-4 blockade alone.
19
Despite the encouraging preclinical data for such a regimen, a planned phase I study
combining Urelumab (anti-4–1BB antibody) and Ipilimumab has been withdrawn prior to
enrollment due to multiple incidences of high grade hepatitis induced by Urelumab
in a prior phase I study (NCT00803374), followed by the termination of another phase
I/II trial (NCT00309023). In this regard, Curran stated that the metaphor “Effective
tumor immunotherapy: start the engine, release the brakes, step on the gas pedal and
get ready to face autoimmunity” nowadays still seems to hold true.
17
Nevertheless, it is worth mentioning that Kocak and colleagues provided first evidence
that the combination of anti-4–1BB and anti-CTLA-4 therapy ameliorates their immune
related side effects.
20
Curran demonstrated that the ability of 4–1BB activation to potently suppress Th17
responses explains its ability to attenuate anti-CTLA-4 induced autoimmunity.
21
Curran concluded that the clinical investigation should be initiated as therapeutic
synergy was demonstrated in multiple preclinical tumor models. Given powerful combinations
of immunotherapeutic antibodies like CTLA-4/PD-1 blockade or CTLA-4 blockade and 4–1BB
agonist, Curran postulated that future combinations should involve vaccines to generate
more tumor-specific T cells, and/or should utilize drugs which help break down the
physical and suppressive barriers to T cell infiltration of tumors.
In addition to aforementioned targets which are partially approved or in late clinical
trials, there are also potential new therapeutic targets on the horizon. Mark Smyth
(QIMR Berghofer Medical Research Institute, Brisbane, Australia) and his colleagues
identified a new checkpoint molecule to augment anti-tumoral natural killer (NK) cell
function. CD96 is a member of the immunoglobulin-superfamily interacting with ligands
of the nectin and nectin-like family and is expressed constitutively on resting NK
cells.
22
So far little was known about its function, except that it shares the ligand CD155
with CD226 and TIGIT.
23
Using CD96-deficient mice, the group was able to demonstrate that CD96 competes with
CD226 for CD155 binding and therefore limits CD226-driven secretion of pro-inflammatory
mediators like IFN-γ by NK cells. Consequently, blocking of CD96 via a monoclonal
antibody resulted in higher resistance against MCA-induced fibrosarcomas and fewer
lung metastases after intravenous challenge with B16F10.
22
The last speaker of this session, Yutaka Kawakami (Keio University, Tokyo, Japan)
emphasized the importance of seeking for combinations outside the field of immunotherapy
to intensify tumor immunity. Combining targeted therapies with immunotherapy can ideally
lead to complementary response kinetics by rapidly inducing decrease of tumor burden
and release of multiple endogenous tumor antigens. Moreover, he mentioned that the
inhibition of the MAPK pathway in human melanoma cell lines by mutant BRAF inhibitors
reduced the production of immunosuppressive cytokines (e.g., IL-6, IL-10 and VEGF),
restoring the ability of DCs to produce high levels of IL-12 and TNF-α and subsequently
to stimulate T cells.
24
Tumor Microenvironment
The session was opened by George Coukos (Ludwig Center for Cancer Research, Lausanne,
Switzerland) who presented an overview of his major discoveries implicated in novel
anti-tumor therapies in cancer patients. He showed his important contributions in
the characterization of tumor vasculature endothelial cells in cancer patients. His
first study described the mechanism of paracrine dialog among tumor-located endothelial
cells and T cells in ovarian cancer. Interestingly, he linked the short ovarian cancer
patient survival time and absence of TIL to high expression of endothelin B receptor
(ETBR) on tumor endothelial cells. This discovery enabled the usage of new pharmacological
compounds to enhance the efficacy of cell immunotherapies in cancer patients.
25
Moreover, Coukos showed a new molecular mechanism to disable tumor T cell infiltration
due to a specific phenotype of tumor vasculatures.
26
In this recent discovery, the death mediator, Fas ligand (FasL) is described as a
new factor on tumor endothelial cells involved in hijacking T cell effector functions.
Selective expression of FasL on the vasculature of human and mouse solid tumors but
not in normal vasculature might allow endothelial cells to kill tumor-infiltrating
effector CD8 T cells but not Tregs. Interestingly, Tregs can survive despite this
death mechanism thanks to constitutive expression of c-FLIP at high levels. He elegantly
demonstrated that the overexpression of this transcription factor in CD8 T cells renders
CD8 T cells resistant to FasL-mediated cell death. In addition, pharmacologic inhibition
of VEGF (anti-VEGF antibody) and PGE2 (Aspirin), factors responsible for the induction
of FasL expression on endothelial cells, produced a marked infiltration of CD8 T cells
at the tumor site over Tregs. Combinations of pharmacological attenuation of FasL
expression on tumor endothelial cells led to potent CD8-dependent tumor rejection.
Sergio Quezada (UCL Cancer Institute, London, U.K.) summarized his important findings
linked to the success of Ipilimumab in melanoma patients. His preclinical work deeply
dissected the mechanism of the mode of action and the potency of anti-CTLA-4 antibody
therapy in the context of GVAX vaccination (irradiated B16F10 tumor cell–based vaccine
that secretes GM-CSF) in melanoma mouse tumor models. His results clearly showed that
the effectiveness of this therapy is linked to the antibody isotype, which drove intratumoral
Treg cell depletion
27
by antibody-dependent cell-mediated cytotoxicity (ADCC). Quezada's team also demonstrated
that intratumoral macrophages expressing Fcγ receptor IV are essential for the elimination
of Treg cells targeted by anti-CTLA-4 antibody.
27
In addition, Quezada showed preliminary results from a study using patient samples
to validate the potential relevance of his findings for the in vivo mode of action
of Ipilimumab in melanoma patients and to characterize the expression pattern of Fc
receptors in the tumor environment. In this regard, several immunomodulatory surface
markers (such as PD-1, OX40, 4-1BB, ICOS and GITR) on human CD4 and CD8 T as well
as Treg cells were characterized in TILs from melanoma patients. This study might
be beneficial for screening potential responder patients for this type of immunotherapy
28
and allow intelligent combination with other immune modulatory antibodies.
Priti Hegde (Genentech, San Francisco, U.S.A.) showed a very interesting ongoing study
to characterize the immune-cancer signature of the tumor environment across six different
human tumors: colorectal cancer, melanoma, bladder cancer, non-small cell lung cancer
(NSCLC), renal cell cancer and triple-negative breast cancer. The major aim is to
clearly dissect the complexity of the tumor environment and to find key factors which
can be targeted specifically for successful anti-tumor therapy. The work is based
on immunohistochemical analysis run in parallel with highly sensitive immune gene
expression assays (iCHIP) using the Fluidigm Biomark platform to interrogate the quality
of the immune response across these six cancer types. The large amount of processed
data showed how the complexity of the immune signature (based on factors such as effector,
suppressor, Th1, Th2, Th17, immune check point markers) in relation to the vasculature
and milieu (chemokines and cytokines components) is integrated specifically for each
type and stage of tumor. Indeed, the analyzed tumors showed distinct immunoscores
with respect to suppressor players, T effector/Treg ratio as well as chemoattractants.
This large scale analysis will hopefully allow the planning of intelligent combination
therapies in order to target specific components of the immune system dependent on
the precise type and stage of human tumors.
Immunoguiding
The history of cancer immunotherapy is still short and so is the history of monitoring
antigen-specific T cell responses. Pedro Romero (Ludwig Center for Cancer Research,
Lausanne, Switzerland) reminded the audience of this fact by recalling that the IFN-γ
ELISpot was invented in the late 80s and intracellular cytokine staining as well as
MHC-tetramers for flow cytometry-based immune monitoring not until the 90s of the
20th century. He therefore stressed the need for further efforts in the direction
of standardization and harmonization as it is, among others, also pursued by the CIMT
Immunoguiding Program (CIP).
29-31
One important determinant of T cell function which is not yet regularly included in
T cell immune monitoring is the micro RNA (miRNA) profiling of T cells. miRNAs are
∼22 nucleotide, non-coding RNAs involved in the post-transcriptional regulation of
gene expression. They can exert their effects through multiple mechanisms, including
translational repression and mRNA degradation. Romero and coworkers already analyzed
the miRNA profile of human CD8 T cells and found differentiation-associated patterns
of miRNA expression.
32
Continuing this line of investigation, they set out to investigate the role of miRNA-155
for CD8 T cell differentiation and effector function utilizing the LCMV model system.
33
Having revealed that miRNA-155 is upregulated in effector CD8 T cells, they observed
a massive reduction of T cell expansion in miRNA-155-negative animals while T cell
differentiation was unaltered. In accordance with this finding, miRNA-155-negative
T cells exhibited decreased proliferation accompanied by increased apoptosis. Importantly,
Romero demonstrated the relevance of this regulator for efficient anti-tumoral T cell
immunity in miRNA-155 knockout as well as overexpression studies. As target mRNA,
the group was able to identify SOCS-1 that diminishes γ-chain cytokine signaling.
The data warrant further studies to investigate the relevance of miRNA-155 in human
cancer-reactive T cells as well as possibilities of active therapeutic intervention
based on the presented findings.
Complementing the strong focus on adaptive immunity, Eric Vivier (Centre d’ Immunologie
de Marseille-Luminy, Marseille, France) introduced NK cells as a subset of cytotoxic
innate lymphoid cells (ILC) that exhibits promising features for cancer immunotherapy.
34
He gave a comprehensive overview about the fundamentals of NK cell immunology and
the current immunotherapeutic concepts linked to them. The important role of NK cells
in defense against infections was clarified by a study showing that NK lymphopenia
caused by MCM4 deficiency is connected to recurrent childhood viral infection as well
as respiratory tract diseases.
35
But what are the fundamental mechanisms by which NK cell activity and target recognition
is governed? The speaker rolled out the scenario of the “missing self” and the “stress-induced
self” leading to NK cell activation. In the former case, the loss of MHC molecules
on tumor cells resulting in a lack of inhibitory signaling causes NK cell activation.
In the latter case, the upregulation of stress-induced ligands (e.g., by malignant
transformation, infection, physical or chemical injury) is the functional trigger.
36
Based on the understanding of NK cell biology and interactions in the living organism,
Vivier proposed different approaches allowing the harvest of the anti-tumoral potency
of NK cells in immunotherapy. Currently being tested is the approach of allogeneic
donor lymphocyte infusion (DLI). Preclinical data from the Vivier laboratory proved
the feasibility of effective tumor therapy utilizing NK cells lacking inhibitory MHC
class-I receptors. The concept awaits further development into the direction of clinical
testing. Lirilumab, an antibody interfering at the same functional axis by blocking
the binding of HLA molecules to killer inhibitory receptor KIR2DL3 is already in clinical
testing.
37,38
Early clinical data have been promising and justify further clinical development.Vivier
also pointed out that albeit in wild-type mice, NK cells reject an MHC-deficient bone
marrow graft, there is no autoimmunity in MHC class-I−/− mice. Starting from the question
how this observation could be explained, he diligently dissected the underlying mechanisms.
It is known that in MHC class-I-deficient hosts, NK cells are hyporesponsive, which
demonstrates their adaptive nature and highlights the relevance of inhibitory MHC
class-I receptors for the tuning of NK cell reactivity.
39
As the phosphatase SHP-1 is responsible for downstream signaling of MHC class-I inhibitory
signaling, Vivier and his colleagues created a Cre/lox-regulated transgenic mouse
model allowing the deletion of the PTPN6 gene encoding SHP-1 in NK cells. In broad
functional studies, the group proved that lack of functional SHP-1 induces a state
of hyporesponsiveness in NK cells. SHP-1 is the decisive signal transduction modifier
mediating the functional tuning of NK cells via MHC class-I receptors. Vivier resolved
the apparently conflicting initial observation by the phrase “time matters, also for
NK cells”. He further proposed that NK cells detect sudden alterations of their environment,
but if these alterations persist, they adapt by ceasing to react. The speaker put
this notion into a broader context by stating that the concept of discontinuity can
be applied to the immune system in general.
40
Immunoinformatics and Genomics
Tumors exhibit reoccurring drivers and antigens as well as patient-specific mutations.
Next Generation Sequencing (NGS) is able to identify both and thus represents a “game-changer”
in cancer immunotherapy. John Castle (TRON - Translational Oncology at the University
Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany) presented
the establishment of a genomics and bioinformatics platform for identifying and prioritizing
somatic mutations for individualized cancer vaccines. An essential part was setting
up a complete infrastructure including the establishment of a secure and fast way
to transfer and process sequencing data, high performance computing, a labor information
management system
41
and a clinical specimen biobank. The core platform developments comprise methods for
RNA-Seq profiling of formalin-fixed, paraffin-embedded samples, statistical algorithms
for NGS mutation detection, virus detection and HLA typing using standard RNA-Seq
reads,
42
single cell genomics, T cell receptor profiling and the TRON Expression Atlas. With
the genomics and bioinformatics platform established, Castle and coworkers were able
to identify the mutanome of B16F10 murine melanoma cells and prioritize mutations
producing neo-epitopes for the elicitation of immune responses.
10
Furthermore, immunomic, genomic and transcriptomic characterization of CT26 colorectal
carcinoma cells revealed that these tumor cells are primarily triploidy and tetraploidy.
43
In addition, they found that mutated tumor alleles are expressed according to their
DNA frequency,
44
which has important implications in the design of individualized tumor vaccines. Clinical
processes have been audited and defined in standard operating procedures. The established
genomics and bioinformatics platform for individualized mutation-targeting cancer
vaccines is being used in a first-in-human clinical trial for melanoma (NCT01684241).
Once somatic mutations are found and validated, which ones produce a neo-antigen which
is (a) presented on a patient's HLA molecules and (b) likely to elicit an immune response?
Morten Nielsen (Technical University of Denmark, Lyngby, Denmark) addressed this question
by presenting his work on immunoinformatic methods for the prediction of peptide-MHC
interactions and T cell epitopes. The primary principle of his algorithms is to learn
underlying rules and patterns of experimentally validated MHC-binding peptide sequences
by fitting mathematic models. In case of MHC class-I binding prediction, Nielsen showed
that the field has reached a plateau where the accuracy does not improve with more
data. The NetMHC algorithm covers more than 100 human and animal MHC class-I molecules.
45
Building models for MHC class-II predictions is more complicated, as the peptide binding
groove is open at both ends, making it crucial to identify the core of an MHC class-II
binding motif.
46
NetMHCII currently offers MHC class-II binding prediction models for more than 25
human and murine MHC-II alleles.
47
For the majority of MHC alleles, the binding specificity has not been characterized
as there exists no or little peptide data for training. Pan-specific MHC prediction
methods fill this gap as they are designed to deal with the immense MHC polymorphisms.
NetMHCpan is trained on binding data covering more than 150 MHC class-I alleles, and
allows prediction of peptide binding to any HLA molecule with known protein sequence.
48
NetMHCIIpan predicts binding for all HLA-DR, HLA-DP and HLA-DQ alleles.
49
To be loaded onto the MHC complex, proteins are first cleaved by the proteasome before
entering the endoplasmatic reticulum via TAP molecules. Although both steps are modeled
in prediction tools, the data suggest that adding those predictions has only a slight
benefit
50
due to MHC class-I pathway co-evolution.
51
To be recognized by T cells, a peptide must be presented on MHC molecules. Thus, binding
is a necessary but not sufficient step. What defines a T cell epitope? A predicted
MHC binding affinity of smaller than 500 nM is routinely used as a threshold for likely
immunogenic T cell epitopes. However, a recent study suggests that different alleles
vary in epitope repertoire size and in binding affinity, both thresholds associated
with immunogenicity.
52
Nielsen showed that peptide-MHC class-I stability is a better predictor of immunogenicity
than binding affinity alone.
53,54
Integrating both predictors, combined with a recently described T cell propensity
model
55
, leads to a significant increase in T cell epitope prediction accuracy.
56
As more experimental data are generated, an improvement in prediction performance
is anticipated. In summary, rational T cell epitope discovery, especially beyond MHC
binding, is indeed feasible.
With a continuous decrease in costs of high-throughput techniques, a plethora of data,
such as genomic and transcriptomic profiles, is emerging, often referred to as “big
data”. Joel Dudley (Icahn School of Medicine at Mount Sinai, New York, U.S.A.) started
with the phrase “Let the data tell you about the biology” and showed examples of the
chances and challenges of integrating and interpreting the digital universe of information
for better models of disease. Given the rapid technological progress, it is possible
to measure more than is known, which forms the basis of data-driven science. Taking
advantage of existing data, combining bioinformatic tools and embracing complexity
enables the full understanding of patient disease physiology and is leading to new
paradigms redefining disease. Using existing disease-related clinical literature and
disease-specific gene expression profiles, Dudley and coworkers recently integrated
large public data sets to understand systematic patterns connecting regulatory variation
with disease functional genomics.
57
A data-driven approach to connect drugs and disease using gene expression data profiles
predicted new indications for established drugs (compound repositioning).
58
This approach identified tricyclic antidepressants as potential inhibitors of small-cell
lung cancer and other neuroendocrine tumors.
59
One of the next steps will be to explore how approved drugs modulate networks within
the complex immune system. Dudley concluded with emphasizing the large opportunity
for systems biology, bioinformatics and systems medicine approaches
60
for the vast amount of big data.
Cellular Therapy
Adoptive Cell Transfer (ACT) therapies are among the most promising treatment strategies
in the field of cancer immunotherapy. In general one can differentiate between adoptive
transfer of autologous ex vivo expanded TILs and the reinfusion of genetically engineered
leukocytes. In the latter case, T cells are either redirected by transfer of antigen-specific
T cell receptors (TCRs) or by chimeric antigen receptors (CARs).
61
Most commonly, αβTCRs are used for this approach but there are also attempts to employ
γδTCR-redirected T cells for ACT.
62
CARs, which recently came under the spotlight as a novel technology, recognize antigens
via the antigen-binding site of a monoclonal antibody fused to intracellular signaling
domains like the CD3-ζ chain.
63
CAR-expressing T cells are thereby able to recognize and kill tumor cells that express
the cognate surface antigen without the need for its presentation on MHC molecules.
Carl June (Perelman School of Medicine at the University of Pennsylvania, Philadelphia,
U.S.A.) presented his work on “CARs in the clinic for leukemia and beyond.” He reported
on different generations of CARs from CD4/8 CARs
64
and single-chain (sc) Fv CARs against CD19 depending on the signaling of the CD3-ζ
chain, to second and third generation CARs typically including signaling domains derived
from costimulatory molecules like CD28, CD137 (4–1BB), CD134 (OX40) or ICOS. June
pointed out that CAR-expressing T cells are long-lived and can be found after more
than 11 years in treated patients.
65
The inclusion of costimulatory signaling domains permits the survival and proliferation
in response to antigen in the absence of exogenous factors.
66
June mentioned that human T cells expressing CARs including 4–1BB costimulatory domains
show a central memory phenotype, superior anti-tumoral efficacy and prolonged survival
compared with CD28 CAR-expressing T cells.
67
CD19-targeting CARs that contain 4–1BB costimulatory domains also showed powerful
effects in patients with CLL
68,69
and ALL.
70
Engineered T cells expanded more than 1000-fold and one CAR-expressing T cell killed
∼1000 tumor cells. Treatment induced a complete remission in 2/3 patients with CLL
and in 27/30 patients with ALL. Responders showed a strong proliferation of CAR-expressing
T cells compared with non-responders. June also pointed out that engineered T cells
were found in the central nervous system. This opens the opportunity to use CAR-transgenic
T cells for the treatment of neuro-oncological diseases. CAR-engineered T cells recognizing
targets like overexpressed mesothelin might also be used for the treatment of solid
tumors
71
but this might raise potential on-target off-tumor immunotoxicity due to the expression
of this antigen on healthy tissues.
Hiroshi Shiku (Mie University Graduate School of Medicine, Tsu Mie, Japan) reported
on the ACT of TCR gene-transduced lymphocytes. He pointed out that the quality, specificity
and quantity of transferred TCR-transgenic (TCRtg) T cells determines the efficacy
of the treatment. In a TCRtg murine study, he and colleagues showed that ACT is more
efficient in combination with vaccination or anti-GITR monoclonal antibodies. In a
phase I clinical trial of TCR-engineered T cells that recognize MAGE-A4143–151 on
HLA-A24:02, the most common HLA haplotype in Japan, ten refractory esophageal cancer
patients without previous lymphodepletion were treated with 2 × 108, 2 × 109 or 5
× 109 engineered T cells in combination with vaccination of the cognate peptides on
day 14 and 28 after ACT. All patients showed transduced T cells in their peripheral
blood 14 days after transfer and in four patients for more than 150 days. Three of
the monitored patients showed stable disease or extended tumor-free status. Shiku
and colleagues observed that the number of engineered T cells in the blood rather
declined after administration of the short peptide vaccine. In murine studies they
were able to show that this was not the case when a DNA or a long peptide vaccine
was administered, and therefore plan to replace the intended peptide vaccine with
a long peptide-containing alternative. Finally, Shiku presented a new generation of
retroviral vectors for the transfer of TCRs that encode siRNA targeting the constant
regions of endogenous TCR-α and TCR-β genes. This approach prevents the miss-pairing
of transferred TCR chains with endogenous ones, increases the number of tumor antigen-specific
TCRs and thereby the anti-tumoral effect, and reduces the risk of GvHD.
Jürgen Kuball (University Medical Center Utrecht, Utrecht, The Netherlands) presented
his pioneering work on γδT cells. γδT cells only comprise a small subset of peripheral
CD3 T cells but are abundant in epithelial tissue of the gastrointestinal and genital
tract. Although γδT cells are able to rearrange TCR genes, the recognition largely
resembles pattern recognition receptors of the innate immune system. For example,
a subpopulation of Vγ2− T cells is able to recognize CMV infected cells but also cancer
cells including primary leukemic blasts. This property might substantially contribute
to the improved control of leukemia in patients with CMV infections.
72
It was shown that in leukemia patients that received T cell-depleted allogeneic stem
cell transplantation, the risk for CMV infection is very high due to the lack of a
T cell response needed for the control of CMV-infected cells. The transfer of CMV-reactive
T cells on the other hand might lead to severe GvHD. Kuball and colleagues demonstrated
that the transfer of αβT cell-depleted cells prevents from CMV reactivation without
substantial GvHD due to the transfer of CMV-reactive Vγ2− T cells. Furthermore, he
presented data on the transfer of γδTCRs into αβT cells. γ9δ2T cells recognize mevalonate
metabolites like isopentenyl pyrophosphate (IPP) which are frequently overproduced
in a broad range of tumor cells. This recognition depends on the expression of the
surface antigen-presenting molecule BTN3A1 on target cells.
73
Kuball not only showed that it is possible to transfer γ9δ2TCRs into αβT cells but
also explained that these T cells are able to specifically kill various leukemia cell
lines in vitro and in vivo. He pointed out that there are differences in the anti-tumor
reactivity among γ9δ2T cell clones explaining the so far limited tumor control of
γ9δ2T cells in clinical studies.
74
Furthermore, he reported on a rho-GTPase that is involved in IPP presentation on BTN3A1
molecules. He and colleagues could show that this small GTPase co-localizes with BTN3A1
and is altered in leukemic but not in healthy stem cells. To evaluate the potency
of the γδTCR approach, a clinical gene therapy trial with δ2+ TCRs is on its way.
Improving Immunity
Gunther Hartmann (University Hospital Bonn, Bonn, Germany) described the versatile
use of blunt, short, double-stranded 5`triphosphate RNA (3pRNA), the ligand for retinoic-acid
inducible gene 1 (RIG-I),
75
for the induction of immune responses against cancer. His group showed that RIG-I
activation triggers cell death in human melanoma cells in vitro while endogenous Bcl-XL
rescued non-melanoma cells from apoptosis induction.
76
3pRNA also can exert its effects on NK cells. In human PBMCs, direct contact of RIG-I
activated monocytes with NK cells is necessary for NK cell activation. Repetitive
injections of 3pRNA were also effective in suppressing tumor growth in the B16 model
of melanoma via secretion of IFN-γ by activated NK cells. Interestingly, RIG-I-activated
melanoma cells secrete exosomes, small cell-derived vesicles, which are internalized
by monocytes and can act as carriers of immunostimulatory molecules.
77
Hartmann and his group demonstrated that these exosomes contain 3pRNA and RIG-I as
well as costimulatory molecules like CD80 and CD86. Moreover, exosomes can also directly
affect melanoma cells via direct apoptosis. In different murine tumor models, a nuclease-resistant
3pRNA which does not have TLR activity was delivered to tumor cells, epithelial cells
and tissue resident DCs via formulation with PEI without signs of toxicity. Intratumoral
injection of such formulated 3pRNA was effective in controlling melanoma growth and
synergized with check-point inhibitors like anti-PD-1 antibody. Mice were also protected
against lung metastases when the 3pRNA-PEI complexes were administered systemically.
In addition, intraperitoneal injection of this formulation exhibited anti-tumoral
activity in an ovarian cancer model where rechallenge of the surviving mice with ovarian
cancer cells led again to rejection, indicating the existence of an effective T cell
memory response. Such immunostimulatory agents activating RIG-I may be used as novel
immunotherapeutic strategies against cancer.
78
Utilizing another type of RNA, in this case long antigen-encoding mRNA, RNA-based
vaccines hold promise as a new class of drug for the immunotherapy of cancer.
79
Several preclinical studies have shown that direct vaccination with naked antigen-encoding
RNA can elicit efficient B and T cells responses as well as therapeutic immunity without
causing toxicity, enabling clinical translation of this strategy.
9,80,81
Ulrike Gnad-Vogt (CureVac, Tuebingen, Germany) presented the clinical development
of intradermally administered self-adjuvanted RNActive® vaccines which contain free
and protamine-complexed mRNA for antigen expression and adjuvanticity, respectively.
82
In a phase I/IIa clinical study with castration-resistant prostate cancer patients,
the CV9103 vaccine comprising PSA, PSMA, PSCA and STEAP1 encoding mRNAs was well-tolerated
and induced antigen-specific immune responses in 79% of 33 immunologically evaluable
patients (NCT00831467). Importantly, immune responses against multiple antigens were
found to be associated with longer survival. Another phase IIb trial with CV9104 which
includes PAP and MUC1 antigens in addition to the ones in CV9103 is ongoing to test
the clinical efficacy for the treatment of prostate cancer (NCT02140138). After revealing
that RNActive® vaccination induces chemokines like CXCL-10, CXCL-9 and CCL5 which
play roles in the recruitment of immune cells to the tumor site,
83
analysis of tumor tissue for immune cell infiltration and cytokines/chemokines profiling
is also planned within this study. The RNActive® platform was tested alone against
NSCLC with a 65% immune response rate in a phase I/IIa CV9201 (NY-ESO1, MAGE C1, MAGE
C2, Survivin and 5T4) trial with the addition of MUC1 (NCT00923312), and a phase Ib
CV9202 trial is underway to test the combination of the RNActive® platform with irradiation
(NCT01915524). Based on preclinical studies, Gnad-Vogt concluded that this platform
can also be combined with other treatments including check-point blocking antibodies
against CTLA-4 and PD-1 which opens new opportunities for clinical testing.
Looking at immune-mediators for improving immunity, Qing Yi (Lerner Research Institute,
Cleveland, U.S.A.) investigated the role of IL-9 in the context of tumor immunotherapy.
IL-9 is a pleiotropic cytokine that can act as a mediator of inflammation in autoimmune
diseases as well as in allergic inflammation while it protomes a tolerant environment
via enhancing the immunosuppressive functions of Tregs and mast cells. Yi presented
data showing that endogenous IL-9 contributes to reduced tumor growth in a metastatic
B16 model of mouse melanoma, as tumor growth was enhanced in the absence of IL-9.
84
Interestingly, adoptive transfer of tumor antigen-specific Th9 cells secreting IL-9
promoted a better tumor clearance than Th1 cells in vivo. Moreover, adoptively transferred
Th9 cells persisted longer and differentiated into functional cytotoxic T cell-like
effector cells with self-renewal capacity.
85
The transfer of Th9 cells also led to effector cell activation in tumor-draining lymph
nodes and to their recruitment to the tumor where they exerted their cytotoxic effects.
Yi and his group also explained the mechanism of action for this effect such that
IL-9 induces tumor and lung epithelial cells to secrete CCL20 which attracts DCs to
the tumor microenvironment through CCR6 in order to internalize tumor antigens.
86
These DCs then migrate to tumor-draining lymph nodes where they prime host effector
cells and especially CD8 cytotoxic T cells which migrate to tumor sites via CCL20
chemoattraction to lyse tumor cells.
The microbial metabolome is comprised of the symbiotic microbes co-existing in close
interaction with the immune system. Laurence Zitvogel (Institute Gustave Roussy, Villejuif,
France) showed diligently how the gut microbiota help shape the immune response against
cancer. Her group recently demonstrated that a commonly used chemotherapeutic agent,
cyclophosphamide (CTX), disrupts mucosal integrity and induces translocation of Gram+
gut-resident bacteria such as L. johnsonii and E. hirae to secondary lymphoid organs
such as the spleen where they induce pathogenic Th17 (pTh17)-type T cell priming in
a Myd88-dependent fashion.
87
Treatment of mice with the antibiotic Vancomycin led to reduced efficiency of CTX
in the treatment of mastocytomas. In addition, the anti-tumoral effect of CTX in a
sarcoma mouse model was found to be reduced in germ-free (GF) mice compared with specific
pathogen-free (SPF) mice as a result of reduction in pTh17 responses. Interestingly,
adoptive transfer of ex vivo differentiated polyclonal pTh17 cells reversed the Vancomycin-induced
resistance against CTX. In line with these results, Zitvogel suggested that L. johnsonii
and E. hirae can be used as probiotics to restore chemosensitivity in case of microbial
dysbalance in the body.
Keynote Lecture
Josef Penninger (Institute of Molecular Biotechnology, IMBA, Vienna, Austria) presented
two examples of how immune-related pathways can be explored to prevent and treat cancer.
Emphasized by knockout studies in mice, the TNF-family molecule RANKL (Receptor Activator
of Nuclear Factor κ B Ligand, also known as osteoprotegrin ligand (OPGL)) and its
receptor RANK are well-known as essential regulators for the development and activation
of osteoclasts and thus play a crucial role in bone remodeling.
88
Furthermore, RANK/RANKL signaling has been initially associated, among others, with
DC-T cell interaction,
89
lymph node formation,
90
body temperature regulation
91
and formation of lactating mammary glands.
92
Dysfunction of this pathway explained bone-related pathologies like osteoporosis and
arthritis
93
but also bone loss in leukemia or chronic obstructive pulmonary disease (COPD).
94
The importance of re-establishment of the physiological balance of the RANK/RANKL
pathway is underlined by the fact that Denosumab, an IgG2-anti-RANKL-antibody mimicking
the interceptive role of the molecular decoy OPG, has already been approved for osteoporosis,
skeletal-related events in cancer and giant cell tumors.
95
The diagnosis of osteoporosis is most commonly made in postmenopausal women. The observed
bone loss in females was attributed to the finding that sex hormones control the RANK/
RANKL pathway. While estrogen levels decreasing postmenopausally led to less OPG expression
and therefore augmented osteoclastogenesis and osteoporosis,
96
progesterone was found to induce RANKL expression in mammary epithelial cells eliciting
their proliferation.
97,98
Hence, the potential involvement of the RANK/ RANKL pathway in breast cancer and its
associated bone metastasis was investigated. Penninger and his colleagues showed that
the synthetic progesterone MPA, by activation of the RANK/ RANKL pathway, has a tumor
promoting effect as it leads to increased mammary epithelial cell proliferation and
survival, and also affects mammary cell stems. Moreover, his group provided the first
genetic evidence that inactivation of RANK on mammary epithelial cells markedly delays
the incidence and onset of MPA/DMBA-driven breast cancer.
99
These findings are of high interest as especially women receiving hormone replacement
therapy (HRT) or taking hormonal contraceptives manifest an increased risk for the
development of breast cancer
100
, and anti-RANKL therapy might be used in these patients to prevent or treat disease.
101
Further benefit of this therapeutic approach may be expected for bone metastasis,
as data indicate that the inhibition of RANKL results in reduced tumor burden in bone
and abolishment of paralysis in a mouse model of melanoma metastasis.
102
Penninger also talked about the role of RING finger E3 ubiquitin-protein ligase Cbl-b
(Casitas B cell lymphoma (CBL)-proto-oncogene-b); in immune system activation and
tolerance. Cbl-b is a key regulator of activation thresholds in mature T lymphocytes,
underlined by numerous studies in Cbl-b−/− mice, which develop spontaneous, albeit
mild, autoimmunity
103
or arthritis even in the absence of microbacterial adjuvant stimulation.
104
Cbl-b is involved in signaling pathways that determine T cell activation vs. T cell
tolerance. Since immune suppression and insufficient activation of T cells are central
limitations of tumor immunotherapy, the question was raised whether Cbl-b displayed
any role in anti-tumor immunity. Indeed, it was found that Cbl-b−/− mice spontaneously
reject TC-1 tumors and UVB-induced skin tumors. This tumor rejection is controlled
by CD8 T cells which kill tumor cells in a perforin-dependent way. Strikingly, proven
by therapeutic transfer, naïve Cbl-b−/− CD8 T cells are sufficient to mediate rejection
of established TC-1 tumors. Furthermore, these CD8 T cells display impaired proliferative
suppression by Tregs, and long-term anti-tumoral immunity is established in mice lacking
functional Cbl-b.
105
However, Cbl-b is not exclusively controlling T cell anti-cancer immune responses
but also possesses functions in innate immune cells, as in a control experiment, TC-1
tumor growth was significantly delayed compared with Cbl-b−/− Rag−/− control mice.
It appears that in these mice, the absence of Cbl-b in NK cells licenses these cells
to spontaneously reject tumor mass.
In the B16F10 melanoma model, Cbl-b−/− mice showed significantly reduced lung metastases,
while depletion of NK cells led to a large increase in metastases count. The therapeutic
transfer of Cbl-b−/− NK cells into B16F10 tumor-bearing mice conveyed fewer metastases
in the lungs of wild-type mice. Remarkably, within the NeuT+ mouse model where female
mice develop spontaneous metastatic breast tumors, the cross-bred Cbl-b−/− NeuT+ mice
were able to control tumor growth with reduced metastatic tumor/lung ratios. This
effect was absent when NK cells were depleted, implying that NK cells defective in
Cbl-b are sufficient to inhibit the progression of tumor and distant metastases.
106
It appears that Cbl-b affects multiple regulatory circuits in anti-tumor immunity,
making it an interesting target for tumor immunotherapy. Indeed, Penninger's group
further explored molecular Cbl-b-mediated ubiquitylation targets resulting in the
identification of the TAM (Tyro3, Axl, Mer) family of tyrosine kinase receptors. Utilizing
a novel, selective TAM inhibitor LDC1267 (blocking Cbl-b ubiquitylation and thus its
activity) to treat mice challenged with B16F10 melanoma or 4T1 mammary tumor cells
markedly reduced the number of metastases. Furthermore, adoptive transfer of wild-type
NK-cells treated with LDC1267 ex vivo significantly improved the anti-metastatic responses
to levels observed in mice transplanted with Cbl-b−/− NK cells. Interestingly, in
a last set of experiments, it was shown that the long-known anti-metastatic activity
of the anti-coagulation drug warfarin, a known inhibitor of the TAM receptor ligand
Gas6, depends on NK cells and their functional Cbl-b.
106
Penninger concluded that various signaling pathways are regulated by Cbl-b in innate
and adaptive immune cells and therefore an efficient manipulation of Cbl-b might give
hope for emerging cancer immunotherapies.
Conclusion
The promise of cancer immunotherapy has recently been recognized as “breakthrough
of the year” at the end of 2013.
107
Topics covered at CIMT2014 and summarized in this report represent various novel anti-cancer
approaches and their clinical translation as next waves of cancer immunotherapy. We
anticipate that the advancements in the field will come more close to clinical application
and lead to approval of novel immunotherapeutic drugs in the coming year, from which
we will hear at the 13th Annual CIMT Meeting (May 11–13 2015, Mainz, Germany).