Final program of the 4th Waddensymposium organized by the
Department of Immunohematology and Blood Transfusion of the LUMC.
Location:
Grand Hotel Opduin
Ruijslaan 22
1796 AD De Koog (Texel)
The Netherlands
Department of Immunohematology and Blood Transfusion
Symposium secretariat: Amber Günthardt
Mail: a.n.gunthardt@lumc.nl
Phone: +31 71 526 38 27
Organizing committee:
Ferry Ossendorp
Sjoerd vd Burg
Wim Fibbe
John Haanen
Kees Melief
Jaap Oostendorp
Jaap Jan Zwaginga
th
4 Waddensymposium
on
Novel strategies in immunotherapy
of cancer
This symposium is organized in collaboration with:
`
www.waddensymposium.eu
Location: Grand Hotel Opduin, Texel, the Netherlands
www.opduin.nl
Final program, 26th of June till the 29th of June 2011
Sunday 26th of June
18.00
Arrival Hotel Opduin, Texel
20.00
Dinner
Monday 27th of June
07.30 - 08.15 Breakfast
08.15 - 08.30 Welcome, symposium in perspective: Willem Fibbe, Leiden [The Netherlands]
Molecular targeting
Chair: Ferry Ossendorp
08.30 – 09.05 Michel C. Nussenzweig [USA]
”DC routing”
09.05 – 09.40 Yvette van Kooyk [The Netherlands]
“Glycan modification of antigens for potentiating anti-tumor immunity”
09.40 – 10.15 Günther Hartmann [Germany]
“RIG-I mediated immune activation”
10.15 – 10.50 Jacques Neefjes [The Netherlands]
“How Doxorubicin alters the transciptome and MHC class I peptidome:
Consequences for tumor immunotherapy”
10.50 – 11.20 Coffee break
11.20 – 11.55 Maries van den Broek [Switzerland]
“Local radiotherapy promotes tumour-specific CD8-effector function
through DC activation”
11.55 – 12.55 Short abstracts
Wendy Unger, Jasper van den Boorn, Petra Paul, Thorbald van Hall,
Claudia Cunha Oliveira, Gijs Zom, Rodney Rosalia, Joost van den Berg
13.00 – 13.45 Lunch
14.00 – 18.00 Informal discussion between young talented scientists / invited
speakers (Transfer at 14.00h)
18.00 – 20.00 Dinner
20.00 – 21.00 Keynote lecture
Chair: Jaap Oostendorp
Axel Hoos [USA]
“Accelerating innovation of cancer immunotherapies”
Tuesday 28th of June
07.30 – 08.30 Breakfast
Preclinical models
Chair: Sjoerd van der Burg
08.30 – 09.05 Thomas Kündig [Switzerland]
“Intralymphatic Immunotherapy”
09.05 – 09.40 Per Thor Straten [Denmark]
“CD8 T cells in cancer; killers, supporters and suppressors”
09.40 – 10.15 Vincenzo Bronte [Italy]
“Chemokine nitration prevents intratumoral infiltration of antigen- specific
T cells”
10.15 – 10.45 Coffee break
10.45 – 11.20 Pam Sharma [USA]
“Targeting the ICOS/ICOSL pathway for improved anti-tumor responses”
11.20 – 11.55 Linda Sherman [USA]
“New roles for CD4 help in the tumor environment”
11.55 - 12.30 Frances Balkwill [UK]
“Inflammatory cytokines and autocrine tumour-promoting networks
in human ovarian cancer”
12.30 – 13.00 Short abstracts
Rinke Bos, Moniek Heusinkveld, Marleen van Loenen, Daniel Hirshhorn
13.00 – 13.30 Lunch
13.45 – 18.00 Informal discussion between young talented scientists / invited
speakers (Transfer at 13.45h)
18.00 – 20.00 Dinner
20.00 – 21.00 Keynote lecture
Chair: Kees Melief
Jim Allison [USA]
“Anti-CTLA-4 treatment in cancer”
Wednesday 29th of June
07.30 – 08.30 Breakfast
Clinical application
Chair: John Haanen
08.30 – 09.05 Glenn Dranoff [USA]
“Mechanisms of Protective Tumor Immunity”
09.05 – 09.40 Jolanda de Vries [The Netherlands]
“The potency of human plasmacytoid dendritic cells to induce immune
responses in melanoma patients”
09.40 – 10.15 Christian Ottensmeier [UK]
“DNA vaccination against cancer”
10.15 – 11.15 Short abstracts
Marij Welters-Schoenmaekers, Gerty Schreibelt, Stefan Nierkens, Pauline
Meij, Tetje van der Sluis, Marieke Fransen, Pia Kvistborg, Marcelo
Navarrete
11.15 – 11.45 Coffee break
11.45 – 12.45 General discussion and position paper
12.45 – 13.30 Lunch
13.35
Departure by bus to Harbour
14.00
Departure boat to Den Helder
END OF MEETING
ABSTRACTS
MONDAY
Glycan modification of antigens for potentiating anti-tumor immunity
Ingeborg Streng-Ouwehand, Manja Litjesn, Astrid van Beelen, Hakan Kalay, Sven
Bruijns, Wendy Unger, and Yvette van Kooyk
Dendritic cells (DC) are specialized in the recognition of pathogens and play a pivotal
role in the control of immunity. Yet DCs are also important for homeostatic control
recognizing self antigens and tolerizing its environment, indicating that the nature of
the antigen it recognizes may steer a DC towards immunity or tolerance. DC express
several C-type lectins, that function as innate receptors that recognize pathogens, and
facilitate antigen uptake and presentation. Yet many of these receptors also modify
responses through signalling interference with TLR. We and others have shown that
the C-type lectins DC-SIGN, MGL and Siglecs recognize specific glycan structures on
many pathogens, and modulate DC mediated responses. Our data demonstrate that the
glycan composition of the pathogens plays a dominating role in directing the immune
response of DC.
Recently we started to modify antigens with specific glycans to favour direction of
antigens to DC, in particular to these specific antigen uptake receptors to allow
processing of antigen for presentation to T cells. Glycan modification of antigen with
DC-SIGN or MGL binding glycans can strongly affect the antigen uptake and
presentation capacity of DC and instruct antigen specific CD4 and CD8 T cell
responses and Th1 differentiation. We have shown that uptake of specific glycosylated
antigen is differently routed intracellularly and favors cross presentation without the
need of any TLR signalling. We show that glycan modified antigens as protein/peptide
or particulate compositions targets these innate receptors on DC which leads to
tailored immune responses both in-vitro and in-vivo that may control immunity
against cancer and infectious diseases.
MONDAY
RIG-I-mediated immune activation
Günther Hartmann
The innate immune system detects viruses based on the recognition of viral nucleic
acids. Immunorecognition of viral nucleic acids leads to the initiation of early antiviral
immune responses that limit viral replication and are essential for eliciting acquired
immune responses to virus specific antigens. Detection of viral nucleic acids in the
cytosol is based on the family of the so-called RIG-I-like receptors (RLRs). RNA with
a triphosphate group at the 5´end (3pRNA) is the ligand for RIG-I (1). By using a new
chemical approach for 5´triphosphate oligoribonucleotide synthesis we found that
short double strand conformation with base pairing of the nucleoside carrying the
5´triphosphate is required for RIG-I binding. These results explain how RIG-I detects
negative strand RNA viruses which lack long double-stranded RNA but do contain
panhandle blunt short double-stranded 5´triphosphate RNA in their single-stranded
genome (2). The crystal structure of the RIG-I CTD domain bound to the blunt-end of
a 5’-ppp-dsRNA was resolved. The structure, supported by mutation and functional
studies, defines how a lysine-rich basic cleft within the RIG-I CTD domain sequesters
the 5’-ppp end of the bound RNA (3). We found that a conserved histidine in RIG-I
controls immune tolerance of 2′O-methylated cap1 self RNA. Furthermore we
demonstrate that RIG-I-RNA ligand interaction not only activates type I IFN, but also
induces inflammasome activation (4) and pro-apoptotic signaling (5). Based on these
activities, RIG-I ligands are promising candidates for the therapy of viral infection and
cancer. We developed short interfering RNA (siRNA) containing triphosphate groups
at the 5´ends (3p-siRNA) targeting oncogenes (6) and designed potent RIG-I selective
ligands for tumor therapy.
1. Hornung V, … and Hartmann G. 5'-Triphosphate RNA Is the Ligand for RIG-I. Science
2006; Nov 10; 314:994-7.
2. Schlee M, … and Hartmann G. Recognition of 5´-triphosphate by RIG-I helicase requires
short blunt double-stranded RNA as contained in panhandle of negative strand virus.
Immunity 2009, 31:25-34.
3. Wang Y, Ludwig J, ...Tuschl T°, Hartmann G°, Patel DJ°. 2010. Structural and functional
insights into 5'-ppp RNA pattern recognition by the innate immune receptor RIG-I. Nat Struct
Mol Biol 2010;17:781-7. °equally contributed
4. Poeck H, ... Hartmann G°, Hornung V°, Ruland J°. Recognition of RNA virus by RIG-I
results in activation of CARD9 and inflammasome signaling for interleukin 1 beta production.
Nat Immunol. 2010;11:63-9. °equally contributed
5. Besch R, …and Hartmann G. Proapoptotic signalling by RIG-I and MDA-5 results in type
I interferon independent apoptosis in melanoma. J Clin Invest 2009, 119:2399-411.
6. Poeck H, …and G Hartmann. 5´-triphosphate-siRNA: turning gene silencing and RIG-I
activation against melanoma. Nature Medicine 2008; 14:1256-63.
MONDAY
How Doxorubicin alters the transciptome and MHC Class I peptidome:
Consequences for tumor immunotherapy
Jacques Neefjes
Tumor Immunotherapy responses can be boosted by combining treatment with
radiotherapy and/or chemotherapy. Radiotherapy activates locally the mTOR pathway
that enhances translation and generates more peptides for MHC class I molecules
(Reits et al., 2006). A frequently used drug in oncology is the topo-isomerase II
inhibitor doxorubicin that is more effective that etoposide, another topo-II inhibitor.
We will show how doxorubicin selectively alters the epigenetic code in areas of open
chromatin that are abundantly present in immune and in tumor cells. This has marked
effects on transcription over long periods and alters the peptidome in MHC class I
molecules such that selective elimination can be improved.
Understanding the biology of these cancer compounds will rationalize the application
in immunotherapy of cancer
MONDAY
Radiotherapy promotes tumour-specific effector CD8+ T cells via DC activation
Anurag Gupta, Hans Christian Probst, Van Vuong, Alexandro Landshammer, Sabine
Muth, Silvia Behnke, Reto Schwendener, Martin Pruschy, Alexander Knuth, Maries
van den Broek
Radiotherapy is an important treatment for cancer. The main mode of action is
thought to be the irreversible damage to tumour cell DNA, but there is evidence that
irradiation mobilizes tumour-specific immunity and recent studies showed that the
efficacy of ablative radiotherapy depends on the presence of CD8+ T cells. We show
here that the efficacy of ablative radiotherapy crucially depends on dendritic cells and
CD8+ T cells, whereas CD4+ T cells or macrophages are dispensable. We show that
local ablative irradiation results in activation of tumour-associated dendritic cells that
in turn support the development and local survival of tumor-specific effector CD8+ T
cells as well as the maintenance of their local effector function.
We propose that – in the absence of irradiation – the activation status of dendritic cells
rather than the amount of tumour-derived antigen or frequency of tumour-specific
CD8+ T cells is the bottleneck, which precludes efficient anti-tumour immunity.
MONDAY
Sialic acids on (tumor)antigens induce tolerance via modulation of dendritic cells
Wendy Unger, Maurizio Perdicchio, Marleen Verstege, Martino Ambrosini and
Yvette van Kooyk
Sialic acids (SA) are the most prevalent terminal monosaccharides on cell surface
glycoproteins and connected to the underlying glycan-core structure by different
linkages like 2,3 (SA-2,3) and 2,6 (SA-2,6). Increased expression of SA is
related to invasion and metastasis of tumors such as melanoma and colon cancer.
These tumors are also associated with high frequencies of tumorantigen-specific
regulatory T-cells (Treg). Since hypersialylation of certain bacteria is often linked to
immune evasion, we hypothesized that SA on tumor cells dampen anti-tumor immune
responses by modulation of antigen-sampling dendritic cells (DC). We set out to link
either SA-2,3 or SA-2,6 to the model antigen OVA to assess their effect on murine
BMDC as well as OVA-specific T-cell responses. Compared to native OVA, the
uptake of the glycoconjugates was increased; however, this did not result in increased
MHC class-II antigen presentation as proliferation of OT-II T-cells was similar.
Native OVA targeted BMDC induced Th1 development, especially when adjuvants
were co-administered. By contrast, OVA-SA-2,3 and OVA-SA-2,6 targeted
BMDC drive the differentiation of naïve T-cells into FoxP3+ Treg, which suppress the
proliferation of responder T-cells. We are currently investigating the phenotype of the
BMDC incubated with the different glyco-antigens and whether SA on murine
melanoma cells drive Treg generation via modulation of DC in-vivo.
MONDAY
Monobenzone-induced immune attack of pigmented cells
Jasper G. van den Boorn, Cornelis J. Melief, Rosalie M. Luiten
Monobenzone is a phenolic compound with the capacity to induce progressive skin
depigmentation that is clinically and histologically indistinguishable from vitiligo
vulgaris. The exact mode of action by which monobenzone induces vitiligo has
remained unknown. Recently we have shown that monobenzone specifically endows
exposed pigmented cells with immunogenicity. Using autologous healthy human
donor DC-T cell stimulations, we established that monobenzone-exposed melanoma
cells induced a robust melanoma-reactive CD8+ T cell response in vitro within 7
days, as compared to unexposed melanoma cells. Importantly, 78% of the melanoma
cell-reactive CD8+ T cell clones generated with monobenzone were found to
recognize monobenzone-treated and –untreated melanoma cells equally well.
Furthermore we have found that upon enzymatic conversion of monobenzone by the
melanosomal enzyme tyrosinase, the quinone metabolites resulting from this reaction
form haptens specifically to cysteine residues in proteins. Moreover, this enzymatic
conversion was found to generate reactive oxygen species, induce melanosome
autophagy and mediate the release of tyrosinase- and MART-1-containing CD63+
exosomes from monobenzone-exposed pigmented cells. Moreover, monobenzoneexposed pigmented cells activated dendritic cells. These data show that monobenzone,
by its specific effects on the enzyme tyrosinase, induces potent CD8+ T cell immunity
against auto-antigens expressed by melanocytes and melanoma cells. Monobenzone
thereby presents a powerful new drug in the field of melanoma immunotherapy.
This research was funded by an innovational research grant of the Netherlands
Organisation of Scientific Research (NWO-vidi).
MONDAY
A Genome-wide Multi-Dimensional RNAi Screen Reveals Pathways Controlling
MHC Class II Antigen Presentation
Petra Paul, Tineke van den Hoorn, Marlieke Jongsma and Jacques Neefjes
MHC class II molecules present antigenic peptides to T helper cells to facilitate
immunity against pathogens and are strongly linked to autoimmune diseases. To
unravel processes controlling MHC class II antigen presentation, we performed a
genome-wide flow cytometry-based RNAi screen detecting MHC class II expression
and peptide loading. The 276 hits were subjected to two additional high-throughput
assays. We identified nine regulators of MHC class II gene transcription acting in
complex feedback networks controlled by chromatin modification, pathogens, TGF
and other signalling events. Phenotypic clustering of the intracellular distribution of
MHC class II located a new GTPase, FLJ22595/ARL14, on MIICs. Integration of
functional and gene expression datasets identified five novel proteins, including
FLJ22595/ARL14, which control MHC class II transport in dendritic cells. The results
represent the first genome-wide functional analysis of a critical immunological
process – MHC class II antigen presentation – and identify new pathways and targets
for manipulating immune responses.
MONDAY
A novel category of tumor antigens enable CTL immunity to tumor escape
variants.
Thorbald van Hall
Loss of antigen presentation by MHC I constitutes a major escape mechanisms by
tumors and allows them to escape elimination by cytotoxic T-lymphocytes. Recently,
we identified a novel category of tumor antigens that are selectively presented by such
tumor variants and named them TEIPP (T cell epitopes associated with peptide
processing)*. TEIPP antigens are derived from housekeeping proteins, but are
surprisingly not presented in MHC I under normal conditions. Multiple defects in the
antigen presentation pathway, e.g. TAP or tapasin, drive the emergence of these
peptides at the cell surface. A broad and cytotoxic CD8+ T cell population recognizes
these as neo-epitopes in the context of classical MHC I molecules. Interestingly, we
found that a major fraction of TEIPP T cells are restricted by the non-classical MHC
Qa-1b, which is the mouse homolog of HLA-E. This non-polymorphic molecule
normally accommodates monomorphic signal peptides and functions as a ligand for
germ-line receptors CD94/NKG2 on NK cells. We now found that this conserved
peptide is replaced by a novel peptide repertoire of surprising diversity as a result of
impairments in the antigen processing pathway.
The surprising absence of TEIPP peptides from normal cells was not related to weak
binding or stability of the Trh4/Db complexes, or to limited availability of MHC-I
heavy chains, but rather to expression levels of the antigen. Strongly augmented
expression levels of Trh4 were needed to reach comparable peptide display as on
TAP-deficient cells, suggesting that the influx of TAP-transported peptides in the ER
forms an efficient barrier for peptides from alternative processing routes.
Finally, we demonstrated that TEIPP T cell responses can easily be detected in
humans and revealed the identity of the first human TEIPP.
Thus, therapeutic application of classical and non-classical TEIPP antigens might
enable the CTL-mediated elimination of tumors with MHC I presentation defects to
prevent this type of immune escape.
* Nat Med 12:417, 2006; J Immunol 178:657, 2007; Canc Res 67:8450, 2007; J Exp
Med 207:207, 2010; J Immunol 185:6508, 2010. Cur Op Immunol 23:293, 2011.
MONDAY
Peptide transporter TAP mediates between competing sources of MHC-I
peptides.
Cláudia C. Oliveira, Bianca Querido, Marjolein Sluijter, Sjoerd H. van der Burg and
Thorbald van Hall
Defects in the intracellular peptide transporter TAP in tumors are associated with
immune evasion. Recently, we described a category of TAP-independent peptides that
fail to be presented by normal processing-intact cells, but are only displayed on cells
with deficiency in the classical MHC-I pathway. Tumor cells presenting TAPindependent peptides can be targeted and eliminated by specific CD8+ T cells. We
studied the ubiquitously expressed ceramide synthase Trh4 as a prototypic example of
these neo-antigens and found that partial inhibition of TAP function allows for cell
surface presentation of Trh4 peptide/Db complexes. These partially inhibited cells
exhibited a mixture of TAP-dependent and TAP-independent peptide repertoires,
indicating that TAP operates as a lever of control that determines relative
contributions of both peptide repertoires. The absence of the Trh4 peptide from
normal cells was not related to binding or stability of Trh4/Db complexes, or to
limited availability of MHC-I heavy chains, but rather to expression levels of the
antigen, since increased expression of Trh4 induced peptide presentation on
processing-intact cells. However, high expression levels were needed to reach
comparable peptide display as on TAP-deficient cells, indicating that the ER influx of
TAP-transported peptides forms an efficient barrier for peptides from other processing
routes. Decreased TAP function, as commonly found in cancers and virus-infected
cells, alleviates this barrier and results in the co-presentation of this TAP-independent
peptide repertoire. This repertoire might constitute an important line of host defense
suitable to be exploited in therapeutic intervention strategies.
MONDAY
Efficient induction of anti-tumor immunity by Toll-like receptor ligand-peptide
conjugates
Zom GGP, Khan S, Britten CM, Sommandas V, Camps MGM, Loof NM,
Meeuwenoord NJ, Filippov DV, Van der Marel GA, Overkleeft HS, Melief CJM,
Ossendorp F
Chemical conjugates comprising synthetic Toll-like receptor ligands (TLR-L)
covalently bound to synthetic antigenic peptides are an attractive vaccine modality
that can induce a robust CD8+ T-cell immune response. We have previously shown
that the mechanism underlying the improved power of conjugates compared to
mixtures of peptides and TLR ligands, depends on improved delivery of the antigen
together with a dendritic cell (DC) activation signal. In the present study we show that
Toll-like receptor peptide conjugates (TPC) have superior capacity of T-cell priming
compared to free synthetic peptide injected together with a free TLR-ligand.
Importantly, we observed that prophylactic immunization with TPC leads to efficient
induction of tumor protection in mice challenged with aggressive tumors such as B16
F10 Ovalbumine transduced melanoma and RMA lymphoma. In addition, our data
show that both CD4 and CD8 T-cell responses are required for the anti-tumoral
effects. Collectively, these data show that TPC are promising, fully synthetic, vaccine
candidates.
MONDAY
Encapsulation of protein antigen in nanoparticles induces potent CD8+ T cells in
vitro with improved anti-tumor effector function and persistence in peripheral
blood after adoptive transfer.
Rodney A Rosalia, Ana Luisa Silva, Ahmed Allam, Selina Khan, Sjoerd van der
Burg, Wim Jiskoot, Ferry Ossendorp, Jaap Oostendorp
Potent effector CD8+ T cells are vital in anti-tumor immune responses as they are
capable of direct recognition and cytotoxicity of malignant cells. Protein antigens
aimed at activating CD8+ T cells have so far yielded disappointing results due to poor
efficiency of cross-presentation of soluble protein by Dendritic cells (DC). Deficient
cross-presentation of protein antigens results in sub-optimal priming and activation of
CD8+ effector T cells. Therefore, alternative strategies need to be developed to
improve specific cellular immune responses induced by protein antigens.
In the present study we have applied Poly-Lactic-co-Glycolic-Acid (PLGA)nanoparticles containing Ovalbumin (OVA) (PLGA-OVA) as an antigen-delivery
system to improve crosspresentation of soluble proteins by DC. Optimal Ag crosspresentation was achieved with 100-fold lower PLGA-OVA amounts compared to
soluble OVA (sOVA). Improved Ag crosspresentation by DC resulted in efficient
priming of CD8+ T cells inducing highly activated CD69-positive, IFN-γ and IL-2
producing CD8+ T cells. Adoptive transfer of in vitro primed CD8+ T cells showed
better antigen-specific in vivo cytotoxicity, longer persistence in peripheral blood,
higher memory T cell formation and ultimately better capacity to protect animals from
s.c. tumor challenge in comparison to T cells stimulated with DC loaded with sOVA.
Therefore, we conclude that PLGA-encapsulation of protein antigen is a very efficient
way to transfer protein antigen in cross-presentation pathways. Enhanced Ag crosspresentation led to potent CD8+activation with strong anti-tumor effector function
upon adoptive transfer.
MONDAY
Genetic manipulation of tumor infiltrating lymphocytes from melanoma
Joost H. van den Berg & Per Thor Straten
Adoptive cell transfer (ACT) is a promising immunotherapeutic treatment possibility
for disseminated cancer. ACT of high numbers of tumor infiltrating lymphocytes
(TIL) results in objective response rates above 50% in heavily pretreated melanoma
patients. Nevertheless, only a small fraction of adoptively transferred T lymphocytes
is able to reach the tumor site once re-infused into the body. This project aims to
improve the homing capacity of T lymphocytes towards the tumor site, which can
possibly make ACT more efficient in a larger fraction of patients.
Melanoma tumors secrete several chemokines which play an important role in tumor
growth and metastasis. These chemokines can be utilized to increase the homing
capacity of T lymphocytes towards the tumor micro-environment. In this project, in
vitro expanded TIL cultures will be equipped with the chemokine receptor CXCR2
using mRNA electroporation. CXCR2 is the receptor for IL-8 and gro-α, two
chemokines that are produced by the majority of melanomas. Electroporation will
lead to transient chemokine receptor expression, sufficient to improve homing
towards chemokine secreting tumors, without the risk of insertional mutagenesis
associated with viral transductions.
We have successfully established a mRNA electroporation protocol to express
CXCR2 on patient derived TIL cultures. Electroporation results in high transfection
efficiencies of the receptor (>90%) with no increase in cell death and preservation of
killing capacity against a tumor antigen. Using a trans-well assay, we showed that
CXCR2 transfected T lymphocytes have an improved in vitro migration capacity
towards recombinant IL-8 and gro-α. In the next phase of this project, the in vivo
homing capacity of transfected T cells towards melanoma will be evaluated in murine
models.
TUESDAY
Intralymphatic Immunotherapy
Thomas M. Kündig
The immune response is initiated by dendritic cells (DCs) and other antigenpresenting cells. These cells are present in nearly all organs and tissues of the body, so
that from an immunological perspective any organ or tissue could serve as a route for
vaccine administration. The choice of route is therefore typically based on practical
aspects. Using conventional needle and syringe the subcutaneous or intramuscular
route are standard. The dermis and especially the epidermis are technically more
difficult to target, but are likely to gain more interest due to the recent development of
micro-needle patches and needle free injection devices. Vaccine administration via
mucosal surfaces such as nasal or oral vaccination represents another option for
needle free vaccine administration. While all these routes of administration have been
proven to work and protect against childhood diseases, influenza and many other
infectious agents, the discussion and comparison of these different routes usually
focuses on convenience, reduction of pain and distress for children, cost and on the
possibility for mass vaccination.
Our research focuses on how the route of administration can enhance the efficacy of
vaccination. Especially in therapeutic vaccination, i.e., in a smaller patient number
that already suffers from a disease, vaccination efficiency rather than convenience is
the main issue. This is particularly the case in therapeutic cancer vaccines and in
allergen specific immunotherapy. Intralymphatic vaccination maximizes the efficacy
of vaccines based on peptides, proteins, DNA and RNA, and the efficacy of adjuvants
by several orders of magnitude. The presentation will focus on the history of this long
known vaccination route, the types of vaccines that profit the most from
intralymphatic administration, clinical applicability and results.
TUESDAY
CD8 T cells in cancer; killers, supporters, and suppressors
Per Thor Straten
We have characterized peptide antigens derived from proteins associated with crucial
cancer traits, e.g., metastasis (RhoC), apoptosis (survivin, Bcl-2, Bcl-Xl, Mcl-1), and
drug resistance (TRAG-3 and CYP1B1). Several of these antigens are in clinical
testing (www.clinicaltrial.gov), however, vaccinations against cancer has so far not
lived up to its promise, in part due to regulatory - or counter-active - mechanisms of
the immune system. Heme-Oxygenase 1 (HO-1) and indoleamine 2,3-dioxygenase
(IDO) are immune suppressive molecules expressed by cancer cells or by cells in the
tumor microenvironment. We have characterized CD8 suppressor T cells specific for
an HO-1 derived peptide and show that these T cells inhibit cytokine production,
proliferation and cytoxicity. Importantly, some cancer patients harbour high
frequencies of HO-1 specific T cells in their blood. Studying CD8 T cell responses to
IDO derived peptides we demonstrate that IDO specific T cells can recognize and kill
tumor cells as well as IDO-expressing dendritic cells and thereby unleash suppressed
responses. A clinical trial is ongoing at our institution targeting IDO in patients with
non small cell lung cancer. Another approach for harnessing the immune system to
combat cancer is adoptive cell transfer (ACT) of in vitro expanded tumor infiltrating
lymphocytes (TIL) – this treatment show promising clinical results in melanoma.
However, the targets recognized by TIL is largely unknown. Using a combination of
recently developed technologies for high-throughput analyses of T-cell responses and
a compilation of all described melanoma antigens, we dissected the melanomarestricted T-cell responses found among TIL cultures. We show that different
melanoma fragment-based cultures from the same patient present with very diverse
characteristics in terms of T-cell specificities, and that tumor specific responses tend
to decrease in frequency during rapid expansion. Surprisingly, the majority of
detected responses are of very low frequency in percentage of CD8+ T cells. Thus, in
terms of frequencies of tumor specific CD8 T cells the frequencies obtained by ACT
seems not that different from vaccination induced responses.
TUESDAY
Targeting the ICOS/ICOSL pathway for improved anti-tumor responses
Padmanee Sharma and James Allison
Blockade of the T cell inhibitory molecule cytotoxic T lymphocyte antigen-4 (CTLA4) can lead to durable anti-tumor responses. A recent report documented an increase
in survival of patients with advanced melanoma treated with Ipilimumab in a
randomized placebo controlled Phase III trial, the first drug of any type to do so, with
~25% of patients alive for over 4 years after treatment. While this trial documents
that CTLA-4 blockade can lead to clinical benefit, there is clearly a need to increase
the numbers of patients who derive benefit from treatment.
To investigate the immunologic impact of CTLA-4 blockade on human immune
responses we conducted the first pre-surgical clinical trial with anti-CTLA-4 in
patients with localized urothelial carcinoma of the bladder. Immunological analyses
were conducted on tumor tissues and blood samples.
We found an increased frequency of CD4 and CD8 T cells expressing high levels of
the inducible costimulator (ICOS) molecule in tumor tissues of treated patients. The
CD4+ICOShi population contained effector T cells that produced IFN-
recognized the NY-ESO-1 antigen expressed on tumor cells. We therefore identified
ICOS as marker of a subset of effector T cells that is increased after anti-CTLA-4
therapy. We also detected changes in tumor tissues consisting of increased T-bet and
IFN- mRNA and an increased ratio of the Th1 cytokine IFN- to the Th2 cytokine
IL-10, which was consistent with an increased ratio of ICOS-expressing effector to
FOXP3-expressing regulatory T cells.
Immunologic changes within tumors correlated with changes within peripheral blood
in that an increased frequency of CD4+ICOShi T cells was also detectable in the
systemic circulation of treated patients. We retrospectively examined peripheral blood
samples from patients with metastatic melanoma who were treated with anti-CTLA-4
and found that sustained elevation of CD4+ICOShi T cells correlated with improved
survival.
These observations led us to test the possibility that engagement of ICOS could
enhance the efficacy of anti-CTLA-4 therapy. To this end we transduced mouse
B16F10 melanoma cells with a cDNA encoding ICOSL or a control construct. B16ICOSL+ cells (IVAX) and control B16 cells were irradiated and used alone or in
combination with anti-CTLA-4 to treat mice bearing established B16F10 tumors. We
found that combination of the IVAX with anti-CTLA-4 was markedly more effective
than the control vaccine plus anti-CTLA-4 or that of any single treatment alone. The
increase in therapeutic efficacy was accompanied by a marked in increase in the
density and functionality of CD4 and CD8 T cells within the tumor.
These results suggest a novel strategy for manipulating the immune system to
enhance anti-tumor responses: checkpoint blockade coupled with provision of agonist
signals to enhance costimulation mediated by ICOS.
TUESDAY
New Roles for CD4 help in the tumor environment
Linda A Sherman
CD4 help for CD8+ T lymphocytes prevents CD8 tolerance and promotes the survival
of effector and memory CD8+ T cells. Although much is known about the benefits of
CD4 help during CD8 priming, the role of CD4 help within the tumor
microenvironment is less explored. Using a model in which tumor eradication by
CD8+ T cells requires tumor specific CD4 help, we examined the basis for helper
dependence of tumor eradication by CD8+ T cells. Recruitment of CD8+ T cells was
greatly accelerated by production of IFN-
resulted in the production of multiple chemokines in the tumor environment.
Production of IL-2 by tumor resident CD4+ T cells enhanced CD8+ T cell proliferation
and significantly upregulated the production of granzyme B required for tumor cell
killing. These results highlight the importance of CD4 help in optimizing recruitment
and effector function of CD8+ T cells within targeted tissues.
TUESDAY
Inflammatory cytokines and autocrine tumour-promoting networks in human
ovarian cancer
Frances Balkwill
A complex network of inflammatory cytokines and chemokines regulates
communication between the malignant cells and supporting stroma in most
experimental and human cancers. There is ample evidence from animal models that
individual members of this cytokine network and their intracellular signalling
pathways can make malignant cells resistant to apoptosis, can stimulate a tumorpromoting leukocyte infiltrate that may also suppress anti- cancer immune responses,
and can regulate angiogenesis.
Of all the individual cytokines and chemokines that have been implicated in tumor
growth and spread, the most convincing and extensive animal model data relate to the
cytokines TNF-, IL-6 and the chemokine receptor CXCR4. These, and the CXCR4
ligand CXCL12, as well as the receptor Notch3 and its Jagged1 ligand, are coexpressed and co-regulated in ovarian cancer cell lines and human ovarian cancer
biopsies. We have named this co-regulation the TNF network. High levels of
expression of TNF network gene pathways in cell lines and ovarian cancer biopsies is
significantly associated with pathways and process important to cancer growth and
spread including angiogenesis, cell adhesion, stromal development, Notch signaling
and the immune cell infiltrate. In ovarian cancer biopsies high levels of malignant cell
staining for some TNF network members related to poor prognosis.
To translate these observations to clinical practice, we have focused on IL-6. IL-6 is a
major mediator of cancer-related inflammation in several cancers and promotes tumor
growth and angiogenesis as well as evasion of apoptosis. We investigated the
therapeutic activity of CNTO328 (siltuximab), an anti-human IL-6 antibody, in preclinical and clinical experiments.
There was evidence of clinical activity of the anti-IL-6 antibody, with one patient of
eighteen demonstrating a partial response and seven showing disease stabilisation for
up to nine months. In terms of mechanism of action, the clinical, pre-clinical and in
silico experiments showed that antibodies to IL-6 can have multiple actions within the
tumor microenvironment in ovarian cancer including reductions in cytokine
production, tumor angiogenesis and tumor macrophage infiltrate. We conclude that
IL-6 is an important regulator of the tumour microenvironment in ovarian cancer and
is a therapeutic target.
TUESDAY
CD4+ T cell help in the tumor milieu
Rinke Bos and Linda Sherman
CD4 help for CD8+ T lymphocytes prevents CD8 tolerance and promotes the survival
of effector and memory CD8+ T cells. Using a murine model of spontaneous tumor
formation in the pancreatic islets, RIP-Tag2HA, in which tumors express the
hemagglutinin (HA) protein of influenza as a surrogate tumor antigen, we have
described additional helper functions of tumor specific CD4+ T cells that require the
presence of CD4 help within the tumor environment. Tumor resident CD4+ T cells
accelerated the recruitment of CD8+ T cells through the IFN-
of chemokines. Production of IL-2 by the tumor resident CD4+ T cells enhanced CD8+
T cell proliferation and also significantly upregulated the production of granzyme B.
Elimination of either of these cytokines reduced tumor eradication and when both
cytokines were absent, CD4 help was completely abrogated. We are now examining
whether CD4 help can be replaced by systemic IL-2 treatment and what the effects are
on CD8+ T cells in the tumor environment. All of these experiments have been
performed with low-avidity CD8+ T cells and we are currently investigating whether
high avidity CD8+ T cells will also benefit from CD4 help or whether the IL-2
production by the CD8+ T cells themselves is sufficient for efficient tumor killing.
TUESDAY
M2 macrophages induced by PgE2 and IL-6 from cervical carcinoma are
switched to activated M1 macrophages by CD4+ Th1 cells
Moniek Heusinkveld, Peggy J. de Vos van Steenwijk†, Renske Goedemans, Tamara
H. Ramwadhdoebe, Arko Gorter‡, Marij J.P. Welters, Thorbald van Hall, Sjoerd H.
van der Burg
Monocytes attracted by tumor-induced chronic inflammation differentiate to antigenpresenting cells (APC), the type of which depends on cues in the local tumor milieu.
Here, we studied the influence of human cervical cancer cells on monocyte
differentiation and showed that the majority of cancer cells either hampered monocyte
to DC differentiation or skewed their differentiation towards M2-like macrophages.
Blocking studies revealed that M2-differentiation was caused by tumor-produced
PgE2 and IL-6. TGF, IL-10, VEGF or M-CSF did not play a role. Notably, these
CD14+CD163+ M2-macrophages were also detected in situ. Activation of cancer cellinduced M2-like macrophages by several TLR-agonists revealed that when compared
to DC these M2-macrophages displayed a tolerogenic phenotype reflected by a lower
expression of co-stimulatory molecules, an altered balance in IL-12p70 and IL-10
production and a poor capacity to stimulate T-cell proliferation and IFNγ production.
Interestingly, upon cognate interaction with Th1 cells these tumor-induced M2macrophages could be switched to activated M1-like macrophages that expressed high
levels of co-stimulatory molecules, produced high amounts of IL-12 and low amounts
of IL-10, as well as acquired the lymphoid homing marker CCR7. The effects of the
interaction between M2-macrophages and Th1 cells could partially be mimicked by
activation of these APC via CD40 in the presence of IFNγ. Our data on the presence,
induction and plasticity of tumor-induced tolerogenic APC in cervical cancer suggest
that tumor-infiltrated Th1 cells can stimulate a tumor-rejecting environment by
switching M2-macrophages to classical pro-inflammatory M1 macrophages.
TUESDAY
Designing T-cells with desired T-cell receptor make-up for adoptive
immunotherapy
M.M. van Loenen, R. de Boer, R.S. Hagedoorn, A.L. Amir, H.M. van Egmond, V.
Jankipersadsing, J.H.F. Falkenburg, M.H.M. Heemskerk
T-cell receptor (TCR) gene transfer is an attractive strategy to equip T-cells with
defined antigen-specific TCRs using short-term in vitro procedures. Selection of host
cells with a known specificity and introduction of a well characterized TCR may
result in an off-the-shelf therapy that combines high anti-tumor reactivity with a
minimal risk of off-target toxicity. For efficient anti-tumor reactivity, high expression
of the introduced TCR is required.
We have explored several strategies to optimize expression of the high affinity minor
histocompatibility antigen specific HA-1-TCR and limit the expression of mixed TCR
dimers, consisting of the endogenous TCR chains pairing with introduced TCR
chains, that could harbor potentially harmful specificities. Of different strategies
explored, the most marked improvement in HA-1-TCR expression and functionality
was observed after TCR transfer of a cysteine modified and codon optimized HA-1TCR. Furthermore, results demonstrate that next to increased HA-1-specific
reactivity, potentially harmful neoreactivities due to mixed dimer formation were
dramatically reduced after cysteine modified HA-1-TCR gene transfer. To
demonstrate the feasibility and study the efficacy of TCR gene transfer, we aim to
start a clinical trial beginning 2012. In this trial, refractory leukemia patients that
received allogeneic stem cell transplantation (SCT) will be treated with virus-specific
donor T-cells engineered with the modified HA-1-TCR.
To increase the number of patients that can be treated with TCR-modified T-cells,
tumor associated antigens (TAA) are potential interesting target antigens. However,
since most TAAs are self antigens, TCRs specific for TAAs presented in self-HLA
are of low affinity. Recently, we isolated from a patient experiencing severe graft
versus host disease (GvHD) after HLA-mismatched SCT several high avidity T-cells
directed against TAAs presented in allo-HLA. These T-cells were demonstrated to
exert single peptide specificity and recognition was strictly correlated with PRAME
expression. However, next to high reactivity against multiple different PRAME
positive melanoma and leukemic cells, limited on-target reactivity against matured
dendritic cells and kidney epithelial cells was observed. Inclusion of a suicide gene as
safety switch may be necessary to enable in vivo elimination of the engineered T-cells
if adverse events occur. Human CD20 was proposed as a non-immunogenic suicide
gene targeted by the widely used clinical-grade antibody rituximab and can
additionally functions as a selection marker. However, transduction of T-cells with a
bi-cistronic vector encoding both TCR and CD20 resulted in poor co-expression, due
to unstable expression of CD20.don optimization of CD20 has recently demonstrated
to allow high co-expression of both the PRAME-TCR and CD20. This co-expression
allowed selection of engineered T-cells as well as efficient elimination of the cells in
vitro. These results demonstrate that codon optimized CD20 can be a useful safety
switch.
TUESDAY
Potent anti-tumor strategies based on OX40 engagement
Daniel Hirschhorn
Expansion and recruitment of CD4(+) Foxp3(+) regulatory T (T reg) cells are
mechanisms used by growing tumors to evade immune elimination. In addition to
expansion of effector T cells, successful therapeutic interventions may require
reduction of T reg cells within the tumor microenvironment. We report that the
combined use of the alkylating agent cyclophosphamide (CTX) and an agonist
antibody targeting the co-stimulatory receptor OX40 (OX86) provides potent
antitumor immunity capable of regressing established, poorly immunogenic B16
melanoma tumors. CTX administration resulted in tumor antigen release, which after
OX86 treatment significantly enhanced the antitumor T cell response. We
demonstrated that T reg cells are an important cellular target of the combination
therapy. Paradoxically, the combination therapy led to an expansion of T reg cells in
the periphery. In the tumor, however, the combination therapy induced a profound T
reg cell depletion that was accompanied by an influx of effector CD8(+) T cells
leading to a favorable T effector/T reg cell ratio. Closer examination revealed that
diminished intratumoral T reg cell levels resulted from hyperactivation and T reg cellspecific apoptosis. Thus, we propose that CTX and OX40 engagement represents a
novel and rational chemoimmunotherapy.
TUESDAY
Anti-CTLA-4 treatment in cancer
James P. Allison and Padmanee Sharma
Over the past several years it has become apparent that cell intrinsic and extrinsic
regulatory pathways that act in concert to minimize harm to normal tissues have
limited the effectiveness of active immunologic strategies for cancer therapy. We
conducted extensive pre-clinical studies in mouse models which showed that blockade
of the inhibitory signals mediated by CTLA-4 in T cells, either alone or in
combination with a variety of immunologic and conventional therapies, led to tumor
eradication and long-lived immunity. This work led to the generation of antibodies to
human CTLA-4 and the conduct of an extensive series of clinical trials in human
cancer. Over 6,000 patients have been treated with the CTLA-4 antibody Ipilimumab
(Bristol-Meyers Squibb). Objective responses have been observed in metastatic
melanoma, castrate resistant prostate cancer, as well as renal, lung, and ovarian
cancer. In a recent Phase III trial, Ipilimumab was shown to prolong survival of stage
IV metastatic melanoma patients, with 25% alive and ongoing at 4 years. This is the
first drug of any type to show a survival benefit in metastatic melanoma in a placebo
controlled randomized trial. In March 2011 Ipilimumab was approved by the FDA
for both first and second line therapy of metastatic melanoma. A Phase III
registration trial in castrate resistant prostate cancer is now underway.
Recent studies have shown that the genetic instability inherent in cancer results in an
extraordinary number of coding mutations in cancer. Many of these give rise to
neoantigens which can provide multiple avenues for attack of tumor cells. It seems
logical to begin to combine conventional therapies, or the new “targeted” therapies,
that can cause tumor cell destruction with immune checkpoint blockade in order to
obtain effective immune responses to these neoantigens, thereby effectively
increasing the valency of therapy and minimizing the chances of acquistion of tumor
resistance and escape. We have begun to explore the effects of targeted therapies on
immune responses and whether the combination of anti-CTLA-4 and targeted therapy
in pre-clinical models. The ultimate goal is to determine whether we can take
advantage of the high response rate to genetically targeted agents with the durability
of immunotherapy.
WEDNESDAY
Mechanisms of Protective Tumor Immunity
Glenn Dranoff
Efficacious cancer immunotherapies will likely require combinations of strategies that
enhance tumor antigen presentation and antagonize negative immune regulatory
circuits. We demonstrated that vaccination with irradiated, autologous melanoma
cells engineered to secrete GM-CSF followed by antibody blockade of CTLA-4
accomplishes clinically significant tumor destruction with minimal toxicity in a
majority of stage IV metastatic melanoma and some advanced ovarian carcinoma
patients. The extent of tumor necrosis in post-treatment biopsies was linearly related
to the natural logarithm of the ratio of infiltrating CD8+ effector T cells to FoxP3+
Tregs, suggesting that further Treg inhibition might increase the frequency of clinical
responses. Through an analysis of cytokine deficient mice, we delineated a critical
role for GM-CSF in Treg homeostasis. GM-CSF is required for the expression of the
phosphatidylserine binding protein MFG-E8 in antigen presenting cells, whereas the
uptake of apoptotic cells by phagocyte-derived MFG-E8 maintains peripheral Treg
activity. The pharmacologic inhibition of MFG-E8 function through genetic or
engineering-based approaches blocks Treg induction, which intensifies vaccineinduced responses, leading to the regression of established tumors in mice. The
clinical translation of these therapeutic strategies to Phase I testing in humans is
underway.
The detailed analysis of patients achieving sustained clinical benefits from irradiated,
autologous GM-CSF secreting tumor cell vaccines and CTLA-4 antibody blockade
also affords a rich opportunity to identify antigens associated with immune-mediated
tumor destruction and to delineate mechanisms of therapeutic immunity. We
elucidated several of the molecular pathways that underlie these anti-tumor effects,
including the NKG2D system and multiple secreted/or cell surface proteins that
contribute to tumor promoting inflammation. The therapy-induced antibodies
manifest functional activity in vitro, antagonizing tumor cell survival, invasive
potential, and angiogenesis. These findings support a key role for humoral immunity
in tumor destruction, and highlight interest in more detailed characterization of the
anti-tumor B cell repertoire in vaccinated patients.
WEDNESDAY
The potency of human plasmacytoid dendritic cells to induce immune responses
in melanoma patients
Jurjen Tel, Daniel Benitez-Ribas, Erik Aarntzen, Gregor Winkels, Gerty Schreibelt,
Cornelis J.A. Punt, Carl G. Figdor, I. Jolanda M. de Vries
Plasmacytoid dendritic cells (pDCs) represent a highly specialized naturally occuring
dendritic cell subset and are the main producers of type I interferons in response to
viral infections. Combined with the capacity to mature and participate in the adaptive
immune response this DC subset might represent a powerful candidate for DC-based
immunotherapy. The clinical application of this specialized DC subset was long
hampered by the lack of a clinical grade isolation procedure and an appropriate
maturation stimulus. Here, we show the first phase I clinical trial using pDCs purified
according to good manufacturing practice guidelines with the CliniMACS-based
isolation procedure. In this clinical trial 5 stage IV melanoma patients received
FSME-IMMUN® matured tumor peptide-loaded pDCs in escalating doses and were
tested for vaccine related toxicity. Whereafter 10 more stage IV melanoma patients
received tumor peptide-loaded pDCs. We characterized the immunological response
induced by the pDCs in terms of FSME specific T-cell proliferation and antibody
responses, and the decetion of tumor-peptide specific T-cells in blood and delayed
type hypersensitiviy reactions. Moreover, we show the tracking of 111In-labeled pDC
in vivo after vaccination and demonstrate that pDCs upon injection migrate to distant
lymph nodes.
In summary, mature tumor peptide-loaded pDCs were used, and tracked in vivo by
111
In labeling, in a clinical trial to induce antitumor responses in melanoma patients.
WEDNESDAY
Immune correlates of success and failure of therapeutic cancer vaccination.
M.J.P. Welters, G.G. Kenter, C.J.M. Melief, S.H. van der Burg
Persistent infection with oncogenic human papillomavirus type 16 (HPV16) results in
anogenital lesions, such as vulvar intraepithelial neoplasia (VIN), and their
subsequent progression to carcinoma. The development of HPV16-induced diseases is
dependent on the oncoproteins E6/E7 and is associated with immune failure at three
different levels. Patients either fail to induce an HPV16 E6/E7-specific response or
mount a weak T-cell response. When present HPV-specific effector T cells may fail to
infiltrate the lesions and last but not least the immune response can be suppressed at
both the induction and effector level by HPV16 E6/E7-specific CD4+ regulatory Tcells.
To overcome this immune deficit we have developed an immunotherapeutic vaccine
strategy based on the use of synthetic long overlapping HPV16 E6/E7 peptides
(HPV16-SLP). In a phase II clinical trial HPV16-SLP vaccination of patients with
HPV16+ high-grade VIN lesions (VIN3) resulted in an objective clinical response rate
of 79% and a complete and durable (>24 months) regression (CR) of the lesion in
47% of the patients. The clinical outcome was related to the size of the lesion at study
entry. Examination of HPV16-specific immunity in relation to lesion size revealed
that the patients with smaller lesions displayed much stronger vaccine-prompted
HPV16-specific effector T-cell responses (already ex vivo detectable) with higher
IFNγ (P=0.0003) and IL-5 (P<0.0001) levels than patients with large lesions.
Characteristically, this response was accompanied by a distinct peak in cytokine
levels after the first vaccination. Additionally, the group of CR patients harboured
earlier and significant higher levels of HPV16 E6/E7-specific IgG levels as a result of
a well induced Th2 response upon vaccination compared to patients with no CR
(P<0.05). In contrast, the patient group with larger lesions displayed a weaker effector
response and a stronger HPV16-specific regulatory immune response, as witnessed by
a significantly lower IFNγ/IL-10 ratio and a significantly higher frequency of HPV16specific CD4+CD25+Foxp3-positive T cells (P=0.005) upon vaccination.
In conclusion, the HPV16-SLP vaccine-induced immunity in patients, who were or
were not successfully treated, revealed that therapeutic vaccination can be a truly
effective therapy but it also teaches us that the HPV-specific CD4+CD25+Foxp3+ T
cells expanded by vaccination mainly in patients with larger lesions at study entry can
play a role in blunting vaccine-induced T-cell reactivity and clinical responses.
Therefore, progressive disease may deregulate the specific immune response to such
an extent that for those patients the current immunotherapy strategy needs further
adjustment.
WEDNESDAY
Vaccination of melanoma patients with monocyte-derived dendritic cells
matured with commonly used prophylactic vaccines
Gerty Schreibelt, Erik Aarntzen, Kalijn Bol, Cornelis J.A. Punt, Carl G. Figdor,
Gosse J. Adema, I. Jolanda M. de Vries
Currently dendritic cell (DC)-based immunotherapy is explored in clinical trials,
predominantly in cancer patients. Murine studies showed that only maturation with
Toll like receptor (TLR) ligands generates mature DC that produce interleukin-12 and
promote optimal T cell help. Unfortunately, the limited availability of clinical grade
TLR ligands significantly hampers the translation of these findings into DC-based
immunotherapy for cancer patients. We recently developed an alternative maturation
cocktail containing the preventive vaccines BCG-SSI, Act-HIB and Typhim. This
vaccine-cocktail contains TLR ligands and, when supplemented with prostaglandin
E2, gives rise to highly mature, interleukin 12-producing DC that are able to migrate
towards CCL21. In vitro, DC matured with the vaccine-cocktail are potent inducers of
tumour antigen-specific T cells and Th1 polarization. We recently started a clinical
trial in melanoma patients to investigate whether vaccine-matured DC improve antitumour responses in vivo. Thirteen patients were vaccinated intradermally and
intravenously with increasing doses of DC. After vaccination, intradermally injected
DC migrated to the lymph nodes and all patients developed a T cell response to the
monitoring molecule KLH. However, the first clinical results suggest that toxicity is
higher than after vaccination with DC matured with a cytokine cocktail (IL-1,
TNF, IL-6, PGE2). Patients suffer grade I-II fever, fatigue, flu-like symptoms and
injection site reactions. Some patients developed up to grade III liver toxicity and in
two patients vaccine-specific T cells infiltrated in the lungs. However, these were
temporary phenomena. Follow-up time is not sufficient to show whether the use of a
vaccine-cocktail as maturation stimulus results in enhanced anti-tumour responses as
compared to the cytokine cocktail.
WEDNESDAY
Plasmacytoid dendritic cells determine the in vivo function of specific subsets of
conventional dendritic cells in tumor immunotherapy
Nierkens S, den Brok MH, Garcia Z, Togher S, Wagenaars J, Boon L, Ruers TJ,
Figdor CG, Schoenberger SP, Adema GJ,†, and Janssen EM.
The differences in function, location and migratory pattern of conventional dendritic
cells (cDCs) and plasmacytoid DCs (pDCs) may not only point to specialized roles in
specific immune responses but also signify additive and interdependent relationships
required to clear pathogens and boost immunity. Here we studied the in vivo
requirement of cross-talk between cDCs and pDCs for eliciting anti-tumor immunity
against in situ released tumor antigens in the absence or presence of the Toll-like
receptor (TLR) 9 agonist CpG. Previous data indicated that CpG boosted tumorspecific T cell responses after in vivo tumor destruction and increased survival after
tumor re-challenges. The present study shows that cDCs are indispensable for crosspresentation of tumor antigens released by ablation, while pDCs are essential to
benefit from the immune stimulating effects of CpG. Depletion of pDCs inhibited
CpG-stimulated priming of antigen-specific CD8+ T cells in blood, spleen and lymph
nodes and significantly diminished the induction of long-term anti-tumor immunity.
In the absence of pDCs, CD80 expression on cDCs was lower upon CpG
administration. The efficacy of CpG was completely dependent on signaling through
MyD88 and type I IFN. Moreover, by transferring wild type pDCs or cDCs into
TLR9-deficient mice, we showed that TLR9 expression in pDCs is required to benefit
from CpG as an adjuvant. Ex vivo characterization and DC-vaccination studies
+
revealed that CD11band the recently identified merocytic DCs were most
+
DCs were potent stimulators of CD8 T
cells against antigens derived from dying cells, merocytic DCs strongly stimulated
both CD4 and CD8 T cells. Hence merocytic DCs may be an even better target for DC
+
DCs. Fms-like tyrosine kinase 3 ligand
(FLT3L) primarily expanded these specific subsets in vivo showing an association
between the beneficial anti-tumor responses upon FLT3L treatment and the induction
of merocytic DCs. These studies indicate that the efficacy of CpG to stimulate antitumor immunity is dependent on cross-talk between TLR9-expressing pDCs and
specific subsets of cDCs.
WEDNESDAY
Coordinated isolation of CD8+ and CD4+ T cells recognizing a broad repertoire of
cytomegalovirus pp65 and IE1 and adenovirus hexon epitopes for highly specific
adoptive immunotherapy
Maarten L. Zandvliet, Ellis van Liempt, Inge Jedema, Louise A. Veltrop-Duits, Henk-Jan
Guchelaar, Arjan C. Lankester, Marco W. Schilham, J.H. Frederik Falkenburg, and
Pauline Meij
Adoptive transfer of specific cytomegalovirus (CMV)- and adenovirus (AdV)-specific
memory T cells can be used for treatment viral complications after allogeneic stem cell
transplantation (alloSCT). Since coordinated CD8+ and CD4+ T cells specific for a broad
repertoire of viral-specific epitopes may be most effective for adoptive immunotherapy, the
aim of this study was to isolate these cells from peripheral blood irrespective of the HLA
type of the patient and donor. CMV pp65, CMV IE-1 and AdV hexon protein-spanning 15mer peptide pools were used to induce simultaneous activation of both CD8+ and CD4+
CMV-specific T cells. Isolation of IFNg-secreting cells resulted in efficient enrichment of
CMV- and AdV-specific T cells. The T cell lines contained high frequencies of CD8+ and
CD4+ T cells recognizing multiple CMV pp65, CMV IE1 and AdV epitopes. This study
provides a feasible strategy for the rapid generation of clinical grade CD8+ and CD4+ T
cell lines with high specificity for multiple CMV pp65, CMV IE1 and AdV hexon epitopes,
and currently both methods are implemented into the clinic for the treatment of CMV or
AdV induced complications after alloSCT. The generation of the CMV and AdV-specific T
cell products has been translated to a clinical grade method, scaled up and validated.
Furthermore specifications of the T cell products have been determined for the quality
control and the release of the product before administration to the patient.
WEDNESDAY
Eradication of Human Papilloma Virus-induced tumors by combined treatment
of low-dose chemotherapy and long peptide vaccination
T.C. van der Sluis, S. van Duikeren, A. Sloots, F. Ossendorp, R. Arens, S.H. van der
Burg and C.J.M. Melief.
About 50% of the patients with Human Papilloma Virus (HPV) induced neoplasia
display clinical benefit after treatment with Synthetic Long Peptide (SLP) vaccination.
The success or failure of these vaccinations correlates clearly with the vaccineinduced T-cell immunity. Remarkably, some patients did not respond to vaccination
until subsequent treatment with chemotherapeutic agents, suggesting that
immunotherapy and chemotherapy may work synergistically.
In a preclinical mouse model of HPV16-induced cervical cancer we tested
experimentally whether chemotherapy could improve tumor eradication induced by a
long peptide vaccine containing a cytotoxic T-cell epitope and a T-helper epitope. In
most mice, peptide vaccination or a well tolerated dose of chemotherapy as a standalone therapy induced only a temporal decrease of the tumor size. Conversely, a
combination of these therapies induced full tumor eradication in nearly all mice.
Especially, the platinum anti-cancer drugs Cisplatin and Carboplatin work
synergistically with SLP-vaccination. Importantly, tumor-specific T-cell responses
appear not to be negatively effected by chemotherapy. Instead, the percentages of
effector-memory like tumor-specific T cells are increased in mice receiving the
combination therapy. The results of combination therapy with SLP vaccination and
low dose chemotherapy have important implications for the rational design of the
immunotherapy to cancer.
WEDNESDAY
Dissection of T cell therapy- and anti-CTLA4-induced cytotoxic T cell responses
in melanoma
Kvistborg P, Shu J, Heemskerk B, Thrue CA, Blank C, Besser M, Dudley ME,
Hadrup S, Straten Pt, Haanen J, Schumacher T.
There is strong evidence that melanoma-reactive T cell responses induced by
immunotherapeutic interventions such as anti-CTLA4 (Ipilimumab) treatment or
T cell therapy with tumor-infiltrating lymphocytes (TIL) can exert
clinically meaningful effects. However, at present we have very little
information on how these therapies influence tumor-specific T cell
responses. Furthermore, as the number of potential melanoma-associated
antigens to which these responses can be directed is very high, classical
strategies to map cytotoxic T cell reactivity do not suffice. Knowledge of
such reactivities would be useful to design more targeted strategies that
selectively aim to induce immune reactivity against these antigens.
In the past years we have aimed to address this issue by designing MHC class
I molecules occupied with UV-sensitive ‘conditional’ peptide ligands,
thereby allowing the production of very large collections of pMHC complexes
for T cell detection. Secondly, we have developed a ‘combinatorial coding’
strategy that allows the parallel detection of dozens of different T cell
populations within a single sample. The combined use of MHC ligand exchange
and combinatorial coding allows the high-throughput dissection of diseaseand therapy-induced CTL immunity. We have now used this platform to monitor
immune reactivity against a panel of over 200 melanoma-associated epitopes.
Data on the composition of TIL products used for adoptive cell therapy and
on the effect of TIL therapy or Ipilimumab treatment on the tumor-reactive T
cell repertoire in melanoma patients will be presented.
WEDNESDAY
Idiotype Vaccination for indolent B-cell non-Hodgkin lymphomas
Marcello Navarrete
The clonal tumor immunoglobulin expressed on the surface of malignant B cells,
termed idiotype (Id), has been used as a tumor-specific antigen in therapeutic
vaccination strategies for indolent B-cell non-Hodgkin lymphomas. A number of
phase 1 and phase 2 clinical trials have established the safety and immunogenicity of
Id vaccination in follicular lymphoma (FL) and other indolent B-cell non-Hodgkin
lymphomas. However, recently reported controlled randomized trials of idiotype
vaccination for FL have yielded conflicting results. An improved understanding of the
Id capable of acting as an antigen but also as an antigen receptor as well as a deeper
insight into the mutual influences between the immune system and lymphoma are
crucial for the future development of this non-toxic therapy.
Id vaccination for follicular lymphoma was primarily being developed as remission
consolidation after chemotherapy. We have recently reported data on Id vaccination
as primary intervention for treatment-naive indolent B-cell lymphoma. Effective
immunity against untreated lymphoma appears to be dependent on idiotype-specific T
cells and sustained remissions in patients with vaccination-induced cellular immunity
suggest clinical benefit. In addition, we have recently identified and characterized
numerical and functional defects in circulating lymphocytes from patients with
untreated chronic B-cell malignances in a prospective fashion. This data, together
with the results of ongoing projects indicating that different modes of Id vaccination
could differ in their propensity to generate the various T-cell subsets, may allow the
development of strategies aimed for reprogramming the immune system from
lymphoma-promoting effects toward adverse conditions for the malignant B cells.
PARTICIPANTS
Dr. James Allison
Memorial Sloan-Kettering Cancer Center
1275 York Avenue
Box 470, New York
NY 10065
allisonj@mskcc.org
Prof. Frances Balkwill
Barts Cancer Institute
Centre for Cancer and Inflammation
London EC1M 6BQ
United Kingdom
f.balkwill@qmul.ac.uk
Dr. Jasper van den Boorn
Institut für Klinische Chemie und
Pharmakologie
Universitätsklinikum Bonn
Sigmund‐Freud‐Str. 25
53127 Bonn
Germany
jvdboorn@uni-bonn.de
Dr. Rinke Bos
The Scripps Research Institute
Dept. of Immunology/ Microbial Science
10550 North Torrey Pines Road
La Jolla, CA 92037-1000
United States
rinkebos@scripps.edu
Prof. Vincenzo Bronte
University Hospital
Dept. of Pathology/Immunology Section
P.le Aldo Scuro, 10, Verona (37134)
Verona, Italy
vincenzo.bronte@univr.it
Prof. Günther Hartmann
Institut für Klinische Chemie und
Pharmakologie
Universitätsklinikum Bonn
Sigmund‐Freud‐Str. 25
53127 Bonn
Germany
gunther.hartmann@uni-bonn.de
Dr. Thorbald van Hall
LUMC
Department of Clinical Oncology
Albinusdreef 2, E3-Q
2333 ZA Leiden
The Netherlands
T.van_Hall@lumc.nl
Dr. Ramon Arens
LUMC
Dept. of Immunotherapy and Blood Transfusion
Albinusdreef 2, E3-Q
2333 ZA Leiden
The Netherlands
R.Arens@lumc.nl
Dr. Joost van den Berg
Center for Cancer Immune Therapy (CCIT)
University Hospital Herlev
Herlev Ringvej 75
DK-2730 Copenhagen
Denmark
Jvan0008@heh.regionh.dk
Prof. Maries van den Broek
University Hospital Zurich
Department of Oncology
Rämistrasse 100/ Wagistrasse 14
8091 Zurich/ 8952 Schlieren
Switzerland
maries@van-den-broek.ch
Claudia da Cunha Oliveira Msc
LUMC
Department of Clinical Oncology
Albinusdreef 2, E3-Q
2333 ZA Leiden
The Netherlands
c.cunha-oliveira@lumc.nl
Dr. Glenn Dranoff
Dana-Farber Cancer Institute
450 Brookline Avenue, Dana 520C
Boston, MA 02115
United States
glenn_dranoff@dfci.harvard.edu
Marieke Herbert-Fransen BSc
LUMC
Department of Immunotherapy and
Blood Transfusion
Albinusdreef 2, E3-Q
2333 ZA Leiden
The Netherlands
M.F.Herbert-Fransen@lumc.nl
Dr. Moniek Heusinkveld
LUMC
Department of Clinical Oncology
Albinusdreef 2, E3-Q
2333 ZA Leiden
The Netherlands
m.heusinkveld@lumc.nl
Daniel Hirschhorn-Cymerman
Memorial Sloan-Kettering Cancer Center
Tumor Immunology Laboratory
1275 York Avenue
Box 470, New York
NY 10065
hirschhd@mskcc.org
Dr. Axel Hoos
Bristol-Myers Squibb
5 Research Parkway
Wallingford CT 06492
USA
axel.hoos@bms.com
Dr. Yvette van Kooyk
VU University Medical Center
Department MCBI
Van der Boechorststraat 7
1081BT Amsterdam
The Netherlands
y.vankooyk@vumc.nl
Dr. Thomas Kündig
University Hospital Zürich
Rämistrasse 100
8091 Zürich
Switzerland
Thomas.Kuendig@usz.ch
Dr. Pia Kvistborg
The Netherlands Cancer Institute
Plesmanlaan 121
1066 CX Amsterdam
The Netherlands
p.kvistborg@nki.nl
Dr. Marleen van Loenen
LUMC
Department of Hematology
Albinusdreef 2, E3-Q
2333 ZA Leiden
The Netherlands
M.M.van_Loenen@lumc.nl
Dr. Pauline Meij
Dr. Carla Milagre
Barts Cancer Institute
Centre for Cancer and Inflammation
Charterhouse Square
London EC1M 6BQ
United Kingdom
c.s.milagre@qmul.ac.uk
Dr. Marcelo Navarrete
LUMC
Department of Hematology
Albinusdreef 2, E3-Q
2333 ZA Leiden
The Netherlands
m.a.navarrete@lumc.nl
Prof. Sjaak Neefjes
The Netherlands Cancer Institute
Plesmanlaan 121
1066 CX Amsterdam
The Netherlands
Dr. Stefan Nierkens
Nijmegen Centre for Molecular Life Sciences
Department of Tumor Immunology
Geert Grooteplein 28
6525 GA Nijmegen
s.nierkens@ncmls.ru.nl
j.neefjes@nki.nl
Prof. Michel C. Nussenzweig
The Rockefeller University
Laboratory of Molecular Immunology
1230 York Avenue New York, NY 10065
United States
nussen@mail.rockefeller.edu
LUMC
Dept. of Clinical Pharmacy/ Toxicology
Albinusdreef 2, E3-Q
2333 ZA Leiden
The Netherlands
p.meij@lumc.nl
Prof. Christian Ottensmeier
Institute University of Southampton
Somers Cancer Research Building
MP 824, Tremona Road, Southampton
SO16 6YD United Kingdom
cho@soton.ac.uk
Petra Paul MSc
The Netherlands Cancer Institute
Plesmanlaan 121
1066 CX Amsterdam
The Netherlands
p.paul@nki.nl
Dr. Gertie Schreibelt
Nijmegen Centre for Molecular Life
Sciences
Department of Tumor Immunology.
Geert Grooteplein 28
6525 GA Nijmegen
The Netherlands
Rodney Rosalia MSc
LUMC
Department of Clinical Pharmacy and
Toxicology
Albinusdreef 2, E3-Q
2333 ZA Leiden
The Netherlands
r.a.rosalia@lumc.nl
g.schreibelt@ncmls.ru.nl
Dr. Pam Sharma
MD Anderson Cancer Center
1515 Holcombe Boulevard
Houston TX 77030
USA
padsharma@mdanderson.org
Prof. Linda Sherman
The Scripps Research Institute
Department of Immunology and Microbial
Science
10550 North Torrey Pines Road
La Jolla, CA 92037-1000
United States
lsherman@scripps.edu
Tetje van der Sluis MSc
LUMC
Department of Immunotherapy and Blood
Transfusion
Albinusdreef 2, E3-Q
2333 ZA Leiden
The Netherlands
T.C.van_der_Sluis@lumc.nl
Prof. Per Thor Straten
Center for Cancer Immune Therapy
(CCIT)
Department of Hematology
University Hospital Herlev
Herlev Ringvej 75
DK-2730 Herlev, Denmark
thorstraten01@heh.regionh.dk
Dr. Wendy Unger
VU University Medical Center
Department MCBI
Van der Boechorststraat 7
1081BT Amsterdam
The Netherlands
w.unger@vumc.nl
Dr. Jolanda de Vries
Nijmegen Centre for Molecular Life
Sciences
Department of Tumor Immunology
Geert Grooteplein 28
6525 GA Nijmegen
The Netherlands
J.deVries@ncmls.ru.nl
Dr. Marij J.P. Welters
LUMC
Department of Clinical Oncology
Albinusdreef 2, E3-Q
2333 ZA Leiden
The Netherlands
m.j.p.schoenmakers-welters@lumc.nl
Gijs Zom MSc
LUMC
Department of Immunotherapy and Blood
Transfusion
Albinusdreef 2, E3-Q
2333 ZA Leiden
The Netherlands
G.G.P.Zom@lumc.nl
ORGANIZING COMMITTEE
Prof. Ferry Ossendorp
LUMC
Dept. of Immunotherapy and Blood
Transfusion
Albinusdreef 2, E3-Q
2333 ZA Leiden
The Netherlands
F.A.Ossendorp@lumc.nl
Dr. Sjoerd van der Burg
LUMC
Dept. of Clinical Oncology
Albinusdreef 2, E3-Q
2333 ZA Leiden
The Netherlands
S.H.van_der_Burg@lumc.nl
Prof. Wim Fibbe
LUMC
Dept. of Immunotherapy and Blood
Transfusion
Albinusdreef 2, E3-Q
2333 ZA Leiden
The Netherlands
W.E.Fibbe@lumc.nl
Prof. John Haanen
LUMC
Dept. of Clinical Oncology
Albinusdreef 2, E3-Q
2333 ZA Leiden
The Netherlands
J.B.A.G.Haanen@lumc.nl
Prof. Kees Melief
LUMC
Dept. of Immunotherapy and Blood
Transfusion
Albinusdreef 2, E3-Q
2333 ZA Leiden
The Netherlands
C.Melief@lumc.nl
Dr. Jaap Oostendorp
Dr. Jaap Jan Zwaginga
LUMC
Dept. of Immunotherapy and Blood
Transfusion
Albinusdreef 2, E3-Q
2333 ZA Leiden
The Netherlands
J.J.Zwaginga@lumc.nl
LUMC
Dept. of Clinical Pharmacy and
Toxicology
Albinusdreef 2, E3-Q
2333 ZA Leiden
The Netherlands
J.Oostendorp@lumc.nl