Cell adoptive cancer Immunotherapy
Daniel Benítez Ribas, PhD
Dept of Immunology. Hospital Clínic Barcelona
SCI. Barcelona, 18/11/2016
Cancer Immunotherapy
Breakthrough of the year 2013
Tumor-specific T cell recognition in the periphery
Lymphocyte priming to tumor antigens
Block inhibitory pathways
Specificity
Only patients who have pre-existing tumor-specific T cells will benefit most from checkpoint blockade
WEAK IMMUNOGENIC
STRONG IMMUNOGENIC
Tumor immunogenicity
The cancer immunity cycle
Therapies that might affect the cancer-immunity cycle
Introduction
• Tumors can be immunogenic
• Recognition of tumor antigens can lead tumor elimination
• Immunotherapy aiming to stimulate T lymphocytes
• Antigen (tumor)-specific T lymphocytes eliminate tumor
• Memory responses minimal residual disease (metastasis)
The primary goal of cancer immunotherapy is to activate the
immune system in cancer patients
1. Products of mutated oncogenes and tumor suppressor genes
2. Product of other mutated genes
3. Over expressed or aberrantly expressed cellular proteins
4. Tumor antigens produced by oncogenic viruses
5. Oncofetal antigens
6. Altered glycolipids and glycoproteins
7. Cell type-specific differentiation antigens
Tumor antigens
Based on their molecular structure and source
Characteristics of an ideal cancer antigen
Criteria Top subcriteria
Therapeutic function Superb data controlled vaccines trial suggestive
Immunogenicity T-cell and/or antibody responses elicited in clinical trials
Oncogenicity Associated with oncogenic process
Specificity Absolutely specific
Expression level and % positive cells Highly expressed on all cancer cells in patients designated for treatment
Stem cell expression Evidence for expression on putative cancer stem cells
Nº patients with antigen-positive cancer High level of expression in many patients with a particular tumor type
Nº epitopes Longer antigen with multiple epitopes and thepotential to bind to most MHC-molecules
Cellular location of expression Normally expressed on the cell surface with no or little circulating antigen
Three ways for self antigens to become tumor antigens
OJ Finn. Cancer Immunology. N Engl J Med, 2008; 358:2704-15
Immunostimulatory and Immunosuppressive Forces in the Tumor Microenvironment
OJ Finn, Cancer Immunology. N Engl J Med, 2008; 358:2704-15
Therapeutic Approaches
NATURE OUTLOOK. CANCER IMMUNOTHERAPY. Nature 2013, 504: 7480, S1-S16
Generation/induction antigen (tumor)-specific T cells
• Cancer vaccines (tumor lysates, cells, peptides, DNA, mRNA)
• Dendritic cells (loaded with lysates, proteins, peptides, mRNA)
Indirect (Active immunization)
• TIL (Tumor-infiltrating lymphocytes)
• TCRT (TCR engineered T cells)
• haTCR (high affinity TCR engineered T cells)
• CAR-T (Chimeric antigen receptor engineered T cells)
Direct (ACT)
Generation/induction antigen (tumor)-specific T cells
• Cancer vaccines (tumor lysates, cells, peptides, DNA, mRNA)
• Dendritic cells (loaded with lysates, proteins, peptides, mRNA)
Indirect (Active immunization)
Therapeutic polivalent vaccine
Whole cell vaccineCancer vaccine:Heterologous Melanoma cell lines vaccine
1) heterogeneous expression of the cell surface antigens (melanoma-
associated antigens, common surface antigens and HLA antigens)
2) balanced representatives of primary, lymph node and metastatic
melanoma cells
3) heterogeneity with respect to their in vitro growth rate; and
4) absence of viral (HIV, hepatitis and HTLV), bacterial and fungal infectious
organisms in the donor serum
THERAPEUTIC POLIVALENT VACCINE PREPARATION
MELANOMA CELL LINES GENERATION, SELECTION AND EXPANSION (10 cell lines)
MICROBIOLOGICAL TESTING
MIXED OF MELANOMA LINES
STORING FROZEN AT N2
• Discarded microbiological contamination• Growing • No production of IL-10 and TGF-b, FasL• Phenotype• Surgical localization
Whole cell vaccineCancer vaccine: Heterologous Melanoma cell line vaccine
• THAWED
• IRRADIATION
• DNFB
• ADITION OF BCG
Vaccine Administration
• INTRADERMAL INOCULATION CLOSE TO
LYMPH NODES
•MONTHLY (FIRST YEAR), EVERY THREE
MONTHS (SECOND YEAR) AND EVERY SIX
MONTHS (THIRD YEAR)
Whole cell vaccine
CLINICAL RESULTS
PACIENTS
AVALUABLE DISEASE: 23
RESPONSES: 6 (26%)
3 COMPLETES (18, 10+, 20+ months)
2 PARTIALS (8, 16+)
1 MIXES (30+)
WITHOUT DISEASE: 15
5 (33%) STABLE
Whole cell vaccine
(Treatment of patients with progressive unresectable metastatic melanoma with a heterologouspolyvalent melanoma whole cell vaccine. Int J Cancer 2003)
Complete remission of the multiple subcutaneousmetastasis after 1.5 year of treatment
Complete remission of the multiple subcutaneous metastasisafter 8 months of treatment
Stage MM
Patients Follow up Median survival Mean survival SD
Stage IIIC 23 13 years 22.5 month 30.7 months 27.7 months
Stage IV 41 12 years 13.0 months 22.1 months 30.9 months
Update
10 years extension
Conclusions
• It is feasible and safe to apply allogeneic tumor cells (irradiated)
• No side effects, the vaccine is well tolerated
• Inactivated whole cell vaccines have an impact in clinical responses in
melanoma patients (Overall survival and complete responses)
Therapeutic vaccines
How to boost antigen (tumor) presentation?
Natural Immune Response
Cellular response (adaptive)
Clinical application of DCs
Tolerogenic DCs
• Allergy
• Transplantation
• Autoimmunity (RA)*
• Immune-based
disorders (IMID)*
• MS/NMO
ü Diabetis
In human
Immunogenic DCs
Cancerü Melanoma
ü Colon
ü Breast
ü Prostate
ü Glioma
ü RCCHIV
Map from clinicaltrials.gov
Dendritic cell based clinical trials (2016 active)
• Move to specific sites in the body
• Cells can sense diverse signals (sense surroundings)
• Integrate inputs to make decisions
• Execute complex response behaviors
Why to use cells as therapeutic agents?
Small molecules Biologics Cells
Therapeutic agents
DC (moDCs, circulating pDCs, mDCs, CD34+ derived) application in
human patients is safe and well tolerated
DCs induce Ag-specific T cells and humoral responses modifying the
type of cytokines and antibody switching
Some patients (tumor) develop efficient clinical responses (room for
improvement and optimization)
DC clinical application. General considerations
Immature DC
Growth factorsMaturation factorsTumor antigens
Apheresis Administration
CD34+ cells Monocytes Natural DCs
Clin Cancer Res; 22(8) April 15, 2016
Ex vivo culture of DCs. Natural DCs or their precursor cells
DC-based therapy in stage IV melanoma patients
“Pilot study of treatment of biochemotherapy-refractory stage IV
melanoma patients with autologous dendritic cells pulsed with a
heterologous melanoma cell lines lysate“
CD14 -MHC II +CD40 +/-CD80 +/-CD83 -CD86 +
CD14 -MHC II +++CD40 +CD80 ++CD83 +CD86 +++
IL-1b, IL-6 TNF-a, PGE2
Tumor cell lines lysate
Cancer Immunol Immunother. 2004 Jul;53(7):651-8
dendritic cell therapy:
• is well tolerated,
• induces an immune response
• shows promising antitumoral activity in stage IV melanoma patients
11 patients stage IV MM (post-biochemotherapy)
• 1 PR (partial response lasting 5 months)
• 2 MR
• 8 PD
DC-based therapy
Clinical results
DC vaccination: status
After 20 years of DC vaccination:
• We can now induce an immune response in 40% of stage IV melanomapatients
• Patients with immune responses show increased progression freesurvival, but long-term clinical responses are still limited (25%)
• Objective clinical responses (5-10%)
• Monocyte derived DC vaccines are not yet optimal:limited survival, migration, co-stimulation, activation, Ag presentation, DCsubsets
• Most patients treated are late stage cancer patients (2-3 line)
Dendritic cells in cancer immunotherapy
Clinical Response Overall Survival
Melanoma 8.5 % 50 % - 377 %
Prostate Cancer 7.1 % 56 % - 175 %
Malignant Glioma 15.6 % 49 % - 150 %
Renal Carcinoma 11.5 % 108 %
Data extracted from: Clinical use of dendritic cells for cancer therapy. Lancet Oncology 2014Sébastien Anguille, Evelien L Smits, Eva Lion, Viggo F van Tendeloo, Zwi N Berneman
More than 3000 patients have been treated with DCs
DTH 6 mm Biopsies
no specific T cells (n=7)
perc
enta
ge
6050403020100
100
80
60
40
20
0
Progression free survival (months)
specific T cells (n=24)
Stage III melanoma patients
no specific T cells (n=7)
perc
enta
ge
6050403020100
100
80
60
40
20
0
Progression free survival (months)
specific T cells (n=24)
no specific T cells (n=7)
perc
enta
ge
6050403020100
100
80
60
40
20
0
Progression free survival (months)
specific T cells (n=24)
Stage III melanoma patients
Progression free survival (months)
perc
enta
ge
6050403020100
100
80
60
40
20
0
no specific T cells (n=18)
specific T cells (n=8)
Stage IV melanoma patients
Progression free survival (months)
perc
enta
ge
6050403020100
100
80
60
40
20
0
no specific T cells (n=18)
specific T cells (n=8)
Progression free survival (months)
perc
enta
ge
6050403020100
100
80
60
40
20
0
no specific T cells (n=18)
specific T cells (n=8)
Progression free survival (months)
perc
enta
ge
6050403020100
100
80
60
40
20
0
no specific T cells (n=18)
specific T cells (n=8)
Stage IV melanoma patients
Clinical response and DTHSkin-derived specific T cells
De Vries et al. J Clin Oncol 2005
Dendritic Cell Immunotherapy: Mapping the way
Figdor et al Nature Med. 2004
Plasmacytoid DC are
• antigen-presenting cells.
• scarce (less than 0,1% of peripheral blood leukocytes).
• the major type I IFN producers.
• critical for anti-viral immunity.
• not yet well understood.
PlasmacytoidDC
CD123
BDC
A-2
Plasmacytoid DC are major type I IFN producers
DC vaccination: pDCs ?
• Immature pDCs infiltrate solid tumors
• Type I IFN seems to yield more potent DCs in terms of secretion of IL-
12 and induction of tumor-specific CTLs and Th1 in vitro
• pDCs create the appropriate environment for efficient CTL response
against viruses
• Activated and injected together with mDCs, pDC may improve the anti-
tumor responses (animal models)
TLR-ligand stimulation (6h)Peptide loading (last 2h)
Quality control:Phenotype IFN-α productionT cell stimulation
pDC isolation BDCA-4 (6h)
O/N with rhIL-3 (10 ng/ml)
Injection (intranodal)
Apherese (4h)
pDC: Clinical grade purification
Quality control:PhenotypePurity YieldViability
• Prophylactic vaccines mimic synthetic CpG oligonucleotides in their ability to modulate immune responses. Mol Immunol. 2011 • Commonly used prophylactic vaccines as an alternative for synthetically produced TLR ligands to mature monocyte-derived dendritic cells. Blood, 2010
Jurjen Tel et al. Cancer Res 2013;73:1063-1075
©2013 by American Association for Cancer Research
Schematic overview of the pDC culture protocol and vaccination strategy.
Activated pDCs are mature and migrate to distinct lymph nodes in vivo.
Jurjen Tel et al. Cancer Res 2013;73:1063-1075
©2013 by American Association for Cancer Research
pDCs induce immune responses to FSME in vivo
T cell responses Humoral responses
FSME - IgG in Blood
Before
Vacc.
After 1
Vacc.
After 2
Vacc.
After 3
Vacc.
0
100
200
300
ns
Arb
itrar
y un
its (
U/m
l)
***
FSME specific T cell response
Before
vacc
.
After 3
vacc
.128
256
512
1024
2048
4096
8192
16384
T ce
ll P
rolif
erat
ion
- C
PM
ns
Frühsommer-Meningoenzephalitis (FSME; tick-borne encephalitis) activates pDCs via TLR9 and TLR7
Tumor specific T-cells in 8 vaccinated patients
gp100154
gp100280
tyrosinase
CD8 FITC
Tetr
amer
APC 0.4 % 0.2 %
0.1 %0.2 %
0.3 % 0.4 %
gp100 tyrosinase
10-5
10-6
10-7
gp100154 gp100280 tyrosinase
Freq
uenc
y of
ant
igen
spec
ific
CD3+ C
D8+
T ce
lls
DTH MLPC
nsns**
pDC vaccination improves OS
Jurjen Tel et al. Cancer Res 2013;73:1063-1075
©2013 by American Association for Cancer Research
Clinical outcome to pDC vaccination was compared with a group ofcarefully matched historical control patients who received dacarbazine asfirst-line treatment
Clinical outcome after vaccination with pDCOverall survival stage IV melanoma patients
Perc
ent s
urvi
val
Overall survival (months)
100-
80-
60-
40-
20-
10-0 12 24 36 48
1-year survival: - historical matched controls 35 % - pDC group 60%
2-year survival: - historical matched controls 10 % - pDC group 45%
matched controls (n=72)
P=0.001
pDC (n=15)
Natural human plasmacytoid dendritic cells induce antigen-specific T-cell responses in melanoma patients. Cancer Res. 2013 Feb 1 2013;73(3)
Conclusions
• Human pDC can be isolated and stimulated according to GMP
• FSME is a good substitute for CpG-C to trigger pDC via TLR, IFN-α,
migration in vitro and in vivo, Ag presentation
• Commonly used vaccines are safe, good and cheap substitutes for
synthetic GMP TLR ligands
• Clinical trials with peptide-loaded pDC are feasible (natural circulating
DCs)
• No severe side effects nor toxicity has been observed
• pDC vaccine increase OS in stage IV melanoma patients
OUTLOOK: SEARCHING FOR SYNERGY
NATURE OUTLOOK. CANCER IMMUNOTHERAPY. Nature 2013, 504: 7480, S1-S16
1. Potentiate Ag-specific T cells
2. Block checkpoints inhibitors
3. Block immunosuppressive tumors
environment
Therapeutic neoantigen-based vaccination
Opportunities for immunotherapy in microsatellite instable colorectal cancer.Cancer Immunol Immunother. 2016 Apr 8. Westdorp H, et al.
Opportunities for immunotherapy in microsatellite instable colorectal cancer.Cancer Immunol Immunother. 2016 Apr 8. Westdorp H, et al.
Therapeutic approach for Lynch syndrome
Protocol PEI NCT Eudra-CTCrohn intralesional 08-049 NCT02622763 2014-001083-35
MS*/NMO* 14-089 NCT02283671 2013-005165-39
CCR*” 09-133 NCT01413295
DIPG*-DC 15-215 NCT02840123 2015-003362-84
CCR*+ Avelumab 2016-003838-24
•MS/NMO: multiple sclerosis; neuromielitis optica, •CCR: colorectal cancer, •DIPG: difuse pontine glioma
Clinical trials ongoing (based on DC)Hospital Clinic de Barcelona
“ DCs Phase II randomized trial of autologous tumour lysate dendritic cell (ADC) plus best supportive care (BSC) compared with BSC, in pre-treated advanced CRC patients colon. Eur J Cancer. 2016 Sep;64:167-74.
Take home message
ü Cancer immunotherapies can eradicate tumours leading
to complete and durable responses
Acknowledgements
• Department of Immunology
• TSF/BST (Transplant Service Foundation)
• Dept. Dermatology
• Radiology
• Surgery
• Microbiology
• Medical oncology
• Hematology
Hospital Clínic de Barcelona NCMSL. Radboud University
Department of Tumor Immunology
DCs Phase II randomized trial of autologous tumour lysate
dendritic cell (ADC) plus best supportive care (BSC) compared
with BSC, in pre-treated advanced CRC patients colon
61 patients assessed for eligibility
52 patients randomizedITT population
9 patients excluded8 did not meet the inclusion criteria
6 positive virus (VIH, HBV) 2 no biopsiable disease
1 declined to participate
28 patients allocated toADC+BSC
24 patients allocated toBSC
27 patients receivedtreatment
8 patients receivedpotential active treatment+BSC
1 patient did not initiatetreatment
16 patients received BSC only
0 100 200 3000
25
50
75
100
ADC + BSCBSC
Days after randomization
Prog
ress
ion-
free
sur
viva
l (%
)
0 200 400 600 800 10000
25
50
75
100BSCADC + BSC
Days after randomization
Ove
rall
Surv
ival
(%)
No clinical benefit
PFS
OS
PRE
POST 40
POST 120
0
5000
10000
15000
p<0,0001
p=0,0067
CPM
DC expands/induce tumor specific T-cells
PRE
POST 40
POST 120
0
5
10
15
20
p<0,0001
% c
ells
Th1
(Ifn
-g +
)
OS
0 200 400 600 800 10000
20
40
60
80
100ATMLR increaseNon-ATMLR increase
p<0,02
Days after randomization
Ove
rall
Sur
viva
l (%
)OS is increased in patients with Ag-specific T cells
Exogenous Ag presentation
The poor ability to induce specific CD4+ T cell responses
against exogenous antigens has been correlated with the lack of
protein uptake by pDCs
AIM
To study the capacity of human pDCs to take up and present
exogenous antigen
Vaccination protocol: monocyte-derived DC
immature DC
KLH Maturation
monocytes
mature DC
IL-4GM-CSF
- Metastatic melanoma- HLA-A2.1+
- gp100+ tyrosinase+
peptides
• KLH specific T cells (PBMC)• KLH specific antibodies
(post-vacc serum)
controlKLH
KLH-Alexa
No humoral response to KLH
KLH-Alexa
Humoralresponse to KLH
pDCs are able to present KLH. Specific T cell proliferation
1/20 1/1000
2
4
6
8
10
12
14
16
3 H in
corp
orat
ion
(cpm
* 10
4 )
0
2
4
6
8
10
12
14
16
1/20 1/100
*
**
3 H in
corp
orat
ion
(cpm
* 10
4 )
pDC/PBL ratio
J Exp Med. 2006 Jul 10;203(7):1629
Endocytic receptors on freshly isolated pDCs
Isotype
DC-SIGN DCIR Dectin-1MR
CD32a
100 101 102 103 104
DEC 205
10 0 10 1 10 2 10 3 10 4
BDCA-2
10 0 10 1 10 2 10 3 10 4
MHC class I
100 101 102 103 104
CD36
100 101 102 103 104
• DEC-205 mediates antigen uptake and presentation by both resting and activated human plasmacytoid dendriticcells. Eur J. Immunol 2011• Targeting DCIR on human plasmacytoid dendritic cells results in antigen presentation and inhibits IFN-α production. Blood. 2008
KLH-Alexa 488
Mono-DC (KLH) Vaccinated
patients
100 101 102 103 104
36 %
Non-vaccinated
100 101 102 103 104
1.5 %
10 10 10 10 100 1 2 3 4
Normal donor
pDCs take up exogenous antigen
CD123
BD
CA-
2
• Magnetic isolation BDCA-4 beads
• Purity (>95%)
• pDCs from blood of:
NO NO YESSpecific antibodies
against KLH
pDCs are able to take up KLH (immune complexes post-vaccination serum dependent)
Before vaccinationAfter vaccination
1 % serum 5 % serum 20 % serum
55 %23 % 58 %
KLH-Alexa 488
Are FcR involved ?
pDCs from normal donors + Serum from same patient (before/after vaccination)
CD32a expression
Anti-CD32 + KLH-Alexa
101 102 103 104
20 %
Isotype + KLH-Alexa
101 102 103 104
45 %
KLH-Alexa
101 102 103 104
48 %
Irrelevant protein
101 102 103 104
5 %
KLH uptake is mediated by FcγRII/CD32
Are pDCs able to process and present KLH to specific T cells?
Blockade of PD-1 or CTLA-4 Signaling in Tumor Immunotherapy
Antoni Ribas. Tumor Immunotherapy Directed at PD-1. N Engl J Med, 2012 366;26
Checkpoints inhibitors and new therapeutic opportunities
• CTLA-4• PD-1
New therapeutic approaches
Anti-CTLA-4 Anti-PD-1• Hard wired• Targets CD28 pathway• Expands clonal diversity• Can move T cells into tumor• Disease recurrence afterresponse is rare
• Induce resistance• Targets TCR pathway• Does not expand clonal diversity• Does not move T cells into tumors• Disease recurrence after response is significant