human oncogenic viruses · human oncogenic viruses • epstein barr virus nasopharyngeal carcinoma,...
TRANSCRIPT
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Human oncogenic viruses
• Epstein Barr virus. Nasopharyngeal carcinoma, B cell Lymphomas, Hodgkin’s diseaseHerpes virus
• Human T lymphotropic virus I. Adult T cell leukemia lymphoma, retrovirus
• Hepatitis B virus. Liver cancer PV
• Hepatitis C virus. Liver cancer
• Human papilloma virus. Ano-genital cancer, H&N cancer PV
• Kaposi sarcoma virus. Sarcomas in immunodeficient patients. Herpes virus 8
• Merkel carcinoma virus. Skin cancer, polyoma virus
• Trichodysplasia Spinulosa virus (TSV). Polyoma virus
PV: preventive vaccine available
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Trichodysplasia Spinulosa-associated novel polyoma virus (TSV), isolated from immunocompromised patientFeltkamp and associates Plos pathogens 2010
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HPV infection
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HPV infection cycle is linked to keratinocyte differentiation program
Normal viral life cycle Viral protein expression
HPV virionEpisomal DNAEarly proteinsLate proteins
E1,
E2,
E5
E6,
E7
L1,
L2
E4
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clearance
>99% minority
CD4+ Th1/Th2 immunity to E2, E6, E7 & L1
CD8 immunity to E6 (E7?)
T cells Circulate & Migrate
immunity cervical neoplasiacervical neoplasiacervical neoplasia
No E6,E7 CD4+ immunityImpaired CD4+ T-cellsInfrequent CD8+ T-cellsRegulatory T-cells
immune failure?
Natural history of cell-mediated adaptiveimmune response to high risk HPV16
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Overview of different types oftherapeutic vaccines tried for HPV16
• Viral vector based vaccines: TA-HPV, MVAtremendous problems with antigenic competition by vector sequences
• DNA vaccinesInefficient way to achieve long-lived antigen expression in DC
• DC based vaccineslaborious and expensive. Direct in vivo DC targeting of antigen more attractive
• Protein vaccines : TA-CIN, E6E7 Iscomatrixrelatively inefficient CD8 CTL induction
• Peptide vaccines: Minimal HLA class I binding peptidesexogenous loading of MHC class I molecules tolerance. Lack of proper CD8memory responses due to lack of CD4 help
Synthetic Long Peptide vaccines
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Enhanced MHC I processing and presentation by
human MoDC of SLP HIV gag216-237 compared to gag protein
Source of DC: MoDC
HLA-B7 Ag presentation/CD8+ T cell activation
1 3 5 1 3 5 1 3 5 1 3 50
200
400
600
800
1000
SSP-GAG223-231
no Ag
SLP-GAG216-237GAG-protein
DC + Ag incubation time in hr
IFN
- b
y G
AG
-spe
cific
CD
8+ T
cel
ls (p
g/m
l)
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Long peptide vaccine in HPV16 Mouse Tumour Model
0 10 20 30 40 50 60 70 800.0
0.2
0.4
0.6
0.8
1.0
days after tumour challenge
surv
ival
Long
Short
naive
GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR
0
10
20
30
40
0
10
20
30
40
0
10
20
30
40
% T
etra
mer
+C
D8+
T-c
ells
0
10
20
30
40
0
10
20
30
40
CTL 1x CTL 2x
Long 1x Long 2x Long 2xin MHC II KO
Zwaveling et al, J. Immunol. ,2002
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CD40Lcostimulation
activation
TLR Ligands
mDC
T-killer
iDC
T-helper
CD40
IL-2
License to Kill
Bennett et al., Schoenberger et al., Nature, 1998
Synthetic long peptides
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Clinical grade HPV16 therapeutic vaccine consists of synthetic overlapping long peptides comprising all potential CTL and Th epitopes.
HPV16 E6
HPV16 E7
158
98
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Phase I, end stage cervical cancer
Before vaccination After vaccination
Kenter, Clin Cancer Res, 2008
Interferon γ Elispot assay
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HPV16-induced premalignant lesion of vulva
Non-specific symptoms: pain, itching, burning
Diagnosis: vulvoscopy, biopsies
Non-treated: can progress to cancer
Therapy: surgery, laser vaporization (mutilating)
Chronic disease: recurrence following standard treatment: 30-50%
Chronic disease: Only 1.3% resolves spontaneously
Vaccination of 20 HPV16+ VIN3 patients with HPV16 SLP vaccine
Kenter et al. NEJM, 2009
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Trial Design, Phase II, HPV16+ Vulvar Intraepithelial Neoplasia (VIN III)
EndpointsImmunology
d0
week 1 week 4 week 7 week 10week 5 week 11week 2 week 8
• Proliferation assay
•IFNγ ELISPOT
•Cytokine analysis (CBA, ELISA)
• CD4/CD8 analyses (ICS)
On PBMC and Biopsies (VIN lesion, vaccination site)
•Symptoms
•Change in lesion size
•Change in histology
•Change in HPV detection
Clinical responses
300 µg per peptide sc in Montanide ISA-51
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pre-vacm
ediu
m
E6.
I
E6.
II
E6.
III
E6.
IV
E7.
I
E7.
II
MR
M
0
25000
50000
75000
100000
cpm
post-vac
med
ium
E6.
I
E6.
II
E6.
III
E6.
IV
E7.
I
E7.
II
MR
M
0
25000
50000
75000
100000
cpm
medium E6.1 E6.2 E6.3 E6.4 E7.1 E7.2 MRM0
1000
2000
3000
4000
5000 TNFaIL-10IL-5
IL-2
IFNg
IL-4
cyto
kine
s (p
g/m
l)
medium E6.1 E6.2 E6.3 E6.4 E7.1 E7.2 MRM0
1000
2000
3000
4000
5000 TNFaIL-10IL-5
IL-2
IFNg
IL-4
cyto
kine
s (p
g/m
l)
Lymphocyte Proliferation Test (ex-vivo 6 days)
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before After 4th vaccinationPre vax Post vax
PR
CR
HPV16-SLP vaccination in VIN3Clinical results at 3 months
Kenter et al., New Engl. J Med. 2009
> 50% CR and PR. Strong correlation of lesion size and clinical response with T cell immune responses to the vaccine (This paper and Welters et al. PNAS, 2010)
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Melief, Immunity, 2008, up-dated 2012
Subversion of effector T-cell responsesby cancerous cell growth
IDO, NO
IL-10 granulocytes
nitrosylasion (TCR,MHC) IL-6
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Melief, Immunity, 2008, updated 2012
Sites of action of Immunotherapyof Cancer
Anti-IL-10 (R), anti-TGFβ(R), anti-IL6 (R)
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Experimental setupchemo-immunotherapy
• TC-1 tumor expressing HPV-16 E6 and E7 oncoproteins
• SLP vaccine 35-mer long peptide (E743-77), provided in Montanide (slow release): prime-boost (s.c.)
• Chemotherapy provided systemically (i.p.)
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Optimal synergy between chemotherapy and peptide when both are provided on the same day
untreated
0 10 20 30 40 50 60 70 800
10
20
30
40
50
60
70
80
90
100
110
Days after tumor challenge
Tum
or s
ize
(mm
2 )
Pept/pept
0 10 20 30 40 50 60 70 800
10
20
30
40
50
60
70
80
90
100
110
Days after tumor challenge
Tum
or s
ize
(mm
2 )
Cis/Cis/Cis
0 10 20 30 40 50 60 70 800
10
20
30
40
50
60
70
80
90
100
110
Days after tumor challenge
Tum
or s
ize
(mm
2 )
Cis/pept/pept
0 10 20 30 40 50 60 70 800
10
20
30
40
50
60
70
80
90
100
110
Days after tumor challenge
Tum
or s
ize
(mm
2 )
Cis+pept/pept
0 10 20 30 40 50 60 70 800
10
20
30
40
50
60
70
80
90
100
110
Days after tumor challenge
Tum
or s
ize
(mm
2 )
cisplatin
peptide
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Types of chemotherapy tested
Clinically used chemotherapeutics for HPV-induced tumors tested:
• Gemcitabine (Gem)• Topotecan (Topo)• Cisplatin (Cis)• Carboplatin (Carbo)
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Cisplatin and Carboplatin showsynergy with vaccination
0 10 20 30 40 50 60 700
10
20
30
40
50
60
70
80
90
100
110untreated (n=18)
cisplatin orcarboplatinSLP
pept/pept (n=24)cisplatin (n=18)cisplatin + peptide (n=34)carbo (n=16)carbo + peptide (n=16)
days after TC-1 tumor challenge
Perc
ent s
urvi
val
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CTL’s are the key mediators incisplatin-SLP induced anti-tumor responses
0 150
10
20
30
40
50
60
70
80
90
100
110
15 20 25 30 35 40 45 50
pep+cis/pep/cispep+cis/pep/cis + anti CD4pep+cis/pep/cis + anti CD8pep+cis/pep/cis + anti CD4+CD8untreated
days after TC-1 tumor challenge
Perc
ent s
urvi
val
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23
1 vaccination of ISA-HPV-SLP® given after 2nd chemotherapy course (carbotaxol) induces robust immune responses in
patients with HPV16+ positive cervical carcinoma
Synergy SLP® vaccination-chemotherapyInitial data exploratory clinical trial (CHDR)
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Synergy SLP® vaccination-chemotherapyInitial data exploratory clinical trial Proliferative T cell response to HPV16 E6/E7 before and after a single SLP vaccine dose, given two weeks after completion of two cycles of chemotherapy for metastatic HPV16+ cervical cancer
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Conclusions chemo-immunotherapy
• Clinically used chemotherapeutics for HPV-induced tumors do not impair T-cell responses• Recent clinical data confirm this observation
• Carboplatin and Cisplatin synergize with the HPV16 SLP vaccine in therapeutic vaccination protocols
• Our data shows that in addition to immunogenic cell death, chemotherapy can have other immunostimulatory effects
• Low dose cisplatin treatment is associated with • An increase in the % of leukocytes in the tumor• A reduction in the percentage of macrophages
• Combination of carboplatin and paclitaxel causes a decrease inmyeloid cells and improved T cell responses in both mouse andhuman observations
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Long peptide containing a CTL or Th epitopeTLR-L
Fundamental study:
* Cell biology of TLR-L conjugates in DCs(Uptake, routing, antigen presentation)
* Immunological response (T-cell induction and Tumor protection)
Next generation of synthetic vaccinesKhan et al. J. Biol. Chem`2008
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Overall conclusions
• Concentrated antigen delivery (DNA, RNA, SLP) with appropriate adjuvants is crucial. Synthetic vaccines allow rational vaccine design
• Favoured cancer target antigens are involved in cancer initiation, progression and/or metastasis. Example: oncogenic proteins E6 and E7 of high risk HPV
• Long peptide vaccines harboring both CD4 and CD8 T cell epitopes and requiring DC processing are efficient. DNA prime/long peptide boost may be considered. Processing route of SLP appears to differ from that of proteins
• Further improvements seen by adding pegylated type I interferon or TLR ligands but especially by conjugating TLR ligands to the long peptides
• For maximally effective cancer treatment develop combination treatment such as long peptide vaccination with chemotherapy or irradiation and inhibitors of checkpoint control monoclonal antibodies (CTLA-4 blocker, PD-1, PD-L1 blockers, anti-IL6 (R), anti-IL10 (R), anti-TGFβ (R) and other immunomodulators)
• Reduce toxicity of the monoclonal antibody treatments by local delivery in slow release formulation close to tumor-draining lymph nodes
• Adoptive transfer of cancer-specific T cells is best combined with optimal vaccination
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Acknowledgements
Dept. of GynaecologyGemma Kenter
Peggy de Vos-v. SteenwijkMuriel van den Hende
Margriet LöwikDorien Berends-van der Meer
Mariette van Poelgeest
PharmacyJan Wouter Drijfhout
Jaap OostendorpRob Valentijn
Lorraine Fathers
Dept of Clinical Oncology Renske Goedemans
Jeanette van den HulstTamara Ramwadhdoebe
Lien van der Minne Marij Welters
Thorbald van HallSjoerd van der Burg
Recent Alumni Cedric Britten
Sander ZwavelingAnnemieke de Jong
Martijn BijkerRienk Offringa
Dept of IHBSelina Khan
Linda StijnenboschMarieke Fransen
Kees FrankenRamon Arens
Tetje van der SluisGijs Zom
Rodney RosaliaEsther QuakkelaarFerry Ossendorp
Dept. of PathologyGert Jan FleurenKatja JordanovaHans Morreau