cell adoptive cancer immunotherapy · 2016-11-30 · • tumors can be immunogenic • recognition...

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


perc

enta

ge

6050403020100

100

80

60

40

20

0




perc

enta

ge

6050403020100

100

80

60

40

20

0



Stage III melanoma patients


perc

enta

ge

6050403020100

100

80

60

40

20

0



Stage IV melanoma patients


perc

enta

ge

6050403020100

100

80

60

40

20

0




perc

enta

ge

6050403020100

100

80

60

40

20

0




perc

enta

ge

6050403020100

100

80

60

40

20

0



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.



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



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


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


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

cell adoptive cancer immunotherapy · 2016-11-30 · • tumors can be immunogenic • recognition...

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