DC vaccination… - ESCCAAnna Borodovsky Dept. of Hematology, Leiden UMC Michel Kester Mirjam...
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23/10/2017 1 Application of siRNA technology in dendritic cell vaccines ESCCA workshop Advanced Immunology Willemijn Hobo 25-09-2017 Dendritic cells (DCs) • Nobel prize 2011: Ralph Steinman • Bridge between innate and adaptive immunity • Potent antigen-presenting cells and activators of T cells BDCA1 + (CD1c + ) BDCA3 + (CD141 + ) CD123 ++ BDCA2 + mDC pDC moDC Hematopoietic stem cell Monocyte Dendritic cell (DC)-based vaccines • Few hundred clinical trials • Advantage • More specific • Less toxic • Longer-lasting therapy • Source • Generated from • Monocytes moDCs • CD34 + HSPCs • Primary DCs from blood (pDC, BDCA1 + mDC) • Result in expansion and activation of tumor-specific CD4 and CD8 T lymphocytes • However, limited numbers of clinical responses Monocyte HSPC Immature DC Antigen-loaded Mature DC Improvements of DC vaccines needed Plantinga et al. frontiers in Immunology 2014 Figdor et al. Nature medicine 2004 Improvements of DC-based vaccines 2004 2014 T cell activation requires antigen recognition & co-stimulation Inhibitory ligand Inhibitory receptor APC CD80/86 CD28 T cell TCR signal transduction Proliferation Cytokine production T cell activation TCR signal transduction Proliferation Cytokine production T cell inhibition Melero et al Nature Rev. Cancer 2007 Monocyte-derived DCs matured with IL-4, GM-CSF, IL-1ß, IL-6, TNF-α, PGE2 Dendritic cell phenotype CD14 CD83 HLA-ABC HLA-DR PD-L1 PD-L2 HVEM B7-H3 CD80 CD86 ICOS-L CCR7
Transcript of DC vaccination… - ESCCAAnna Borodovsky Dept. of Hematology, Leiden UMC Michel Kester Mirjam...
23/10/2017
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Application of siRNA technology in dendritic cell vaccines
ESCCA workshop Advanced ImmunologyWillemijn Hobo25-09-2017
Dendritic cells (DCs)
• Nobel prize 2011: Ralph Steinman
• Bridge between innate and adaptive immunity
• Potent antigen-presenting cells and activators of T cells
BDCA1+
(CD1c+)
BDCA3+
(CD141+)CD123++
BDCA2+
mDCpDC moDC
Hematopoietic stem cell Monocyte
Dendritic cell (DC)-based vaccines
• Few hundred clinical trials
• Advantage
• More specific
• Less toxic
• Longer-lasting therapy
• Source
• Generated from
• MonocytesmoDCs
• CD34+ HSPCs
• Primary DCs from blood (pDC, BDCA1+ mDC)
• Result in expansion and activation of tumor-specific
CD4 and CD8 T lymphocytes
• However, limited numbers of clinical responses
MonocyteHSPC
Immature DC
Antigen-loadedMature DC
Improvements of DC vaccines needed
Plantinga et al. frontiers in Immunology 2014 Figdor et al. Nature medicine 2004
Improvements of DC-based vaccines
2004 2014
T cell activation requires antigen recognition &
co-stimulation
Inhibitory ligand
Inhibitory receptor
APC
CD80/86
CD28
T cell
TCR signal transduction
Proliferation
Cytokine production
T cell activation
TCR signal transduction
Proliferation
Cytokine production
T cell inhibition Melero et al Nature Rev. Cancer 2007
Monocyte-derived DCs matured with IL-4, GM-CSF, IL-1ß, IL-6, TNF-α, PGE2
Dendritic cell phenotype
CD14 CD83 HLA-ABC HLA-DR
PD-L1 PD-L2 HVEM B7-H3
CD80 CD86 ICOS-LCCR7
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Enhancing DC immune stimulatory capacity
CD80/86
PD-L1/L21. Silencing of inhibitory molecules using
RNAi technology
immature DC stage
2. Pulsing with antigen peptide or mRNA
mature DC stageAntigen pulsing
DC with super immune stimulatory phenotype stronger activation of T cells
Gene silencing mechanisms
Lam et al. Mol Ther Nucleic Acids 2015
Gene silencing mechanisms of siRNA and miRNA
Lam et al. Mol Ther Nucleic Acids
2015
No protein expression
Selection of siRNA sequences
Chemical modifications
Used in our laboratory: Stealth Select siRNAs (Thermo Fisher)
• Limited off-target effects (only anti sense strand can enter RISC)
• Stabilized against nuclease degradation for greater longevity and stability
• No/minimal induction of non-specific cellular stress response pathways
siRNA delivery tools
• Viral vectors
lentiviruses, adeno(-associated) viruses
• Electroporation
• Transfection polymers
PLGA, glycol chitosan
• Transfection lipids
lipofectamine, polyethylenimine (PEI), SAINT-RED, liposomes
Roeven and Hobo et al. J Immunother, 2015; Hobo et al. Cancer Immunol Immunother. 2013; Hobo et al. Blood. 2010
Non-viral siRNA delivery reagent for ex vivo use
Composition (1:1 molar ratio):
• Saint-18 cationic amphiphilic lipid (1-methyl-4-(cis-9-
dioleyl)methyl-pyridinium-chlorid)
• DOPE neutral helper lipid (1,2-dioleoyl-sn-glycero-3-
phosphoethanolamine)
Characteristics:
• High transfection efficacy
• Relatively non-toxic
• Heat-sterilized without loss of function
• Highly stable (> 3 years at 2-8oC)
• Ex vivo grade production is feasible
SAINT-RED
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Dendritic cell transfection with PD-L siRNA
Roeven and Hobo et al. J Immunother, 2015; Hobo et al. Cancer Immunol Immunother. 2013; Hobo et al. Blood. 2010
300V150μF
PD-L siRNA
immature DC mature DCmonocytes
GM-CSF, IL-4GM-CSF, IL-4TNF-a, IL-1b
IL-6, PGE2
2 days
Blood product
6 days
PD-L siRNA/SAINT-RED
siRNA sequences were obtained via Invitrogen/Thermo scientific (Stealth select) or
licensed from Alnylam pharmaceuticals (eventual GMP batches)
Compare siRNA sequence for silencing efficiency
Roeven and Hobo et al. J Immunother, 2015; Hobo et al. Cancer Immunol Immunother. 2013; Hobo et al. Blood. 2010
Determine silencing efficacy and off-target effects
Roeven and Hobo et al. J Immunother, 2015; Hobo et al. Cancer Immunol Immunother. 2013; Hobo et al. Blood. 2010
Knockdown Phenotype Viability
> 80% knockdown
Evaluate duration of silencing
Roeven and Hobo et al. J Immunother, 2015; Hobo et al. Cancer Immunol Immunother. 2013; Hobo et al. Blood. 2010
Investigating DC immune stimulatory capacity in
vitro
CD80/86PD-L1/L2
DC with super immune stimulatory phenotype stronger activation of T cells
++
–
Biotinylated recombinant MHC molecules loaded with specific peptides bound to
fluorochrome-labeled straptavidine
MHC peptide tetramers are bound by T cellsexpressing the TCR of the appropriate
specificity
1.6%
CD8
MiH
A t
etra
mer
Peripheral blood
PE
Detection of antigen-specific T cells using tetramer
technology
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Dual color tetramer staining of tumor-reactive
CD8+ T cells
Combination with: phenotypic and/or functional markers (e.g. IFNγ/TNFα, CD107a)
PD-L silenced DC augment expansion of tumor-reactive memory T cells
Patient PBMC: <0.01% tumor-reactive CD8+ memory T cells
1 week culture with PD-L silenced or control DC loaded with HA1 mRNA
Roeven and Hobo et al. J Immunother, 2015; Hobo et al. Cancer Immunol Immunother. 2013; Hobo et al. Blood. 2010
PBMC: 5 allo-SCT patients, low percentages of tumor-reactive T cells
% D
egra
nu
lati
on
Student t-test, *P<0.05, **P<0.01
PD-L silenced DC augment expansion of tumor-reactive memory T cells
Roeven and Hobo et al. J Immunother, 2015; Hobo et al. Cancer Immunol Immunother. 2013; Hobo et al. Blood. 2010
Blood Spleen
Tetramer analysis tumor-reactive T cells
D0 D7 D14 D21
+
IL2, IL15
rhIL15 (every 2-3 days, 0.5µg/injection)
Primed MiHACD8+ T cells
NSG mice
Van der Waart et al, Cancer Immunol Immunother 2015
PD-L silenced DC are efficient stimulators of tumor-reactive T cell responses
Van der Waart et al, Blood 2015
DC boosted tumor-reactive T cells exert anti-tumoreffect in mice Prophylactic vaccination with PD-L silenced
MiHA mRNA-loaded moDC after alloSCT
(PSCT19 study)
EudraCT: 2014-003537-26
Study Design: Single center phase I proof-of-concept study in series
of 10 patients treated with HLA-matched allogeneic stem cell
transplantation.
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PD-L silenced MiHA mRNA moDC vaccination
CsA
MiHA tetramerscreening
MiHA+ patientMiHA- donor
Monocyte Immature DC Mature DC
MiHA mRNA
QC testing &product release & vaccination
CD14+
CliniMACS
KLH protein
PD-L siRNA/Saint-red
GMP manufacturing of reagents
Primary:
• Evaluate safety and toxicity
• Evaluate of efficacy to induce in vivo expansion of MiHA-reactive CD8+ T cells
• Evaluate immune response to KLH (i.e. control antigen)
Secondary:
• Evaluate clinical efficacy in case of detectable mixed chimerism and/or minimal
residual disease
Objectives
• Clinical protocol
• Investigator’s Brochure
• Investigational Medicinal Product Dossier
• Patient / donor information
• Patient / donor informed consent forms
• Registration in clinical trial registry
• Monitoring plan
• Electronic case report form (eCRF)
• Risk analysis of critical raw materials and excipients (grondstoffen / hulpmiddelen)
• Risk analysis of culture procedure
• Validation of culture procedure
• …
Paper work before start study
Day:
pre 0 7 14 21 28 42 63 84
Tetramer analysis
T / B / NK analysis
Material stored for immunological monitoring (PBMC, serum)
Chimerism / disease monitoring
PB&BM PB PB PB PB PB PB&BM PB PB&BM
Dose:
max 0.25x106 c/kg
DC vaccination & follow-up
Currently 4 patients vaccinated (and 4 to follow the upcoming months):0/4 GVHD & 3/4 boosting of tumor-reactive T cell response!
Response to PD-L silenced MiHA-DC vaccination
Patient 1 Patient 1
Hobo and Dolstra, unpublished data
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siRNA technology is an efficient and successfull approach to
favorably modulate DC immunogenicity in order
to boost anti-tumor immunity
ConclusionsLaboratory of Hematology,
RadboudumcHanny Fredrix
Frans MaasAnniek van der Waart
Marij LeendersMenno WinkelCarel Trilsbeek
Jos PaardekooperMarianne BakkerEugenie RuttenHans Veenstra
Marion MassopPaul Ruijs
Rob WoestenenkFrank Preijers
Bert van der ReijdenJoop Jansen
Harry Dolstra
Laboratory of Medical Immunology,Radboudumc
Arnold van der MeerIrma Joosten
Dept. of Hematology,RadboudumcMieke Roeven
Suzanne van DorpOlga Huber
Nori WijngaardenElles Strijbosch
Marian LucassenNicole BlijlevensMichel Schaap
Dept. of Clinical Pharmacy, Radboudumc
Anna de GoedeJanine van der Linden
Alnylam Pharmaceuticals,Cambridge, USA
Tanya NovobrantsevaAnna Borodovsky
Dept. of Hematology,Leiden UMC
Michel KesterMirjam HeemskerkMarieke GriffioenFred Falkenburg
Synvolux therapeutics,Groningen
Marcel RuitersGerben Zondag
Dept. of Hematology,UMC GroningenMarco de Groot
Gerwin Huls
Dept. of Hematology,UMC Utrecht
Moniek de WitteJürgen Kuball
Acknowledgements
Financial support:
