Het immuunsysteem: gevaarlijke indringers buiten … · 2 Eindelijk erkenning… Tumor Immunology...

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Dendritische cellen in kankertherapie: immuunmonitoren van patiënten

Tanja de GruijlAfd. Medische Oncologie

Immuuntherapie en Immunomonitoring LabVU medisch centrum

Het immuunsysteem: gevaarlijke indringers buiten houden…

Bacteriën plakken aanepitheel (b.v. huid)

Locale infectie: penetratie van epitheel

Locale infectie van de weefsels

Specifieke afweercellengaan in de aanval

Bescherming tegen infectie

…maar óók tumoren?VUmc Medical Oncology

I M M U N O T H E R A P Y L A B

VUmc Medical Oncology


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Eindelijk erkenning…

Tumor Immunology

Hanahan and Weinberg Cell 2011

Een afweerreactie tegen kanker begint met Dendritis che Cellen eneindigt met T cellen

DC activate T cells in Lymph Nodes

Melanoma with Dendritic Cells

T cells migrate to tumor site

…and eradicate tumor cells

CD83CD83





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Het principe van tumor vaccinatie: afweer

opwekken tegen tumor-specifieke “vreemde”

eiwittenTumor

eiwitten





DC vaccines: limitations of the autologous approach

• Laborious• Time consuming • Costly• Poor availability of precursors• High inter- and intra-donor variability: standardiza tion issues

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Het alternatief: DC targeting

…but died before it was announced that he had won the Nobel Prize this year.

Ralph Steinman: the discoverer of DC, received the Heineken Prize from Prince

Willem Alexander last year…





DNA

RNA

viruses conjugated proteins

long peptides

nanoparticles

liposomes

immature DC

SKIN

mature DC T cell activation and expansion

LYMPH NODE

antibody

glycosylationmotif

DC targeting in vivo: approaches

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Lymphnode

DC

CTLCTL CTL

Kill of micrometastases

Th

Tumor

Skin

Lymphnode

DC

CTLCTL CTLCTL

Kill of micrometastases

ThTh

Tumor

Skin

In vivo DC targeting and immunisation efficacy depends on:1) Local DC concentration: cytokine mobilization2) DC accessibility: skin DC and/or LN-resident DC3) DC subset4) DC specificity of targeting: high efficiency load ing5) Ag uptake: endocytosis6) DC maturation induction7) Ag expression and/or processing: timing in relation to DC maturation and migration to LN 8) CTL and Th activation

Huid: het grootste immuunorgaan van ons lijf

i.d. injected DEX-FITC





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Het targeten en activeren van Dendritische cellen in de huidhet humane huidexplant model





Tumoren scheiden suppressieve stoffen af die DC ontwikkeling en activering remmen

DC differentiation, maturation, and functions

IL-10, VEGF, IL-6, PGE2, M-CSF, IDO

Tumor-conditioned tissues

immunogenicanti-angiogenic

Mature DCtolerogenicpro-angiogenic

Immature DC

Hoe kunnen we deze suppressie overwinnen?

Mature DC





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Migration and activation of human skin DC subsets the skin explant model

FACS AnalysisCytokine releaseEndocytic activityMLRT cell activation

intradermal injection of cytokines/TLR-L/Ad viruses

6 mm punch biopsies taken

Full-thickness or epidermal/dermal sheets cultured

Explants removed on d2DC harvested at d2 or d7

Skin emigration: intradermal cytokine-inducedchanges in DC subset composition

i.d. GM-CSF+IL-4

i.d. IL-10

De Gruijl et al J Immunol 2006

day 7 after migration

0

5000

10000

15000

20000

25000

30000

5000 2500 1250 625 313 156 78 0

DC per 10e5 PBL

Pro

lifer

atio

n [C

PM

]

■ GM-CSF+IL-4□ IL-10 + GM-CSF+IL-4○ IL-10

-1500 -1000 -500 0 500 1000 1500

GM-CSF+IL-4

IL-10 +GM-CSF/IL-4

IL-10

medium

pg/ml

IL-10

IL-12

-1500 -1000 -500 0 500 1000 1500

GM-CSF+IL-4

IL-10 +GM-CSF/IL-4

IL-10

medium

pg/ml

IL-10

IL-12

8

Skin emigration: intradermal cytokine-inducedchanges in DC subset composition

i.d. GM-CSF+IL-4

i.d. IL-10

19%

58%

23%CD1a+CD14- LC/DDC

DN DDC

CD14+ DDC

Mediumn=19

IL-10n=15

GM-CSF+IL-4n=13

57%28%

15%

Breast Cancern=6

Breast Cancerpost-chemo n=4

45%

41%

14%

72%

10%

18%28%

54%

18%

P<0.001

P<0.01

P<0.001

P<0.05

P<0.01

P<0.00119%

58%

23%CD1a+CD14- LC/DDC

DN DDC

CD14+ DDC

Mediumn=19

IL-10n=15

GM-CSF+IL-4n=13

57%28%

15%

Breast Cancern=6

Breast Cancerpost-chemo n=4

45%

41%

14%

72%

10%

18%28%

54%

18%

P<0.001

P<0.01

P<0.001

P<0.05

P<0.01

P<0.001

Tumor-conditioned tissues frustrate DC differentiatio nand maturation

DC differentiation, maturation, and functions

IL-10, VEGF, IL-6, PGE2, M-CSF, IDO

Tumor-conditioned tissues

immunogenicanti-angiogenic

Mature DC

tolerogenicpro-angiogenic

Immature DC

can the balance be shifted by targeting and activation?

Mature DC

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Allogeneic tumor cell or DC lines and DC targeting

off-the-shelf alternatives?

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DC-targetedadenoviruses

DC-progenitorcell line

HLA-A2-matched allogeneic vaccine DCOne

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GM-CSF-transducedtumor cell lines

GVAX

GVAX DCOne CD40L-Ad5

Gerichte infectie en gelijktijdige activatievan DC door CD40-redirected

Adenoviruses

Ad-TAA

+

CD40 targeted Ad-TAA

sCAR mCD40L

Ad-TAA

+


sCAR mCD40L

Mature TAA-expressing DCin LN activates specific T cells

Immature DC in skin orLN is transduced

Ad-TAA

+


sCAR mCD40L

Ad-TAA

+


sCAR mCD40L

Mature TAA-expressing DCin LN activates specific T cells

Immature DC in skin orLN is transduced





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CD40-Ad targeting skin DC: a stable mature phenotype

dermis

epidermis

No response CTL activation

Adenovirus, encoding Influenza

M1 antigen

CD40 conjugate

dermis

epidermis

No response CTL activation

Adenovirus, encoding Influenza

M1 antigen

CD40 conjugate

day 7

medium

Ad

CD40-Ad

CD83GFP

1.5%

49%

82%

medium

Ad

CD40-Ad

CD83GFP

1.5%

49%

82%

De

Gru

ijl e

t alJ

Imm

unol

2002

DC-targeted Adenovirus vaccines

CD40-targeted Ad: superior DC transduction in melanoma-draining LN and CTL activation

Melanoma SLN1

Melanoma SLN2

1.6%

0.5%

0.45%

6.07%

6.18%

15.45%

MART1-Tm

CD

8+

MART1-Tm

MART1-Tm

CD

8+

CD

8+

MART1 T cell frequency

DC-targeted Adenovirus vaccines

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A B

C D

Intradermaal ingespoten CD40-Ad :DC targeting in muis lymfeklieren





CD40-targeted Ad in huid en lymfeklieren: tumor bescherming

0 3 6 9 12 15 18 200

200

400

600

800

1000 Ad-GFP

Ad-gp100CD40-Ad-gp100

***

***

*

CFm40L

Days post tumor inoculation

Tum

or v

olum

e (m

m3 )





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1999

2000

2002

2003

2005

2006

2008

20092011

2010

… het is een lange weg van het lab naar de kliniek!







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GVAX


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Myeloid leukemic cell lines as a source of dendritic cells

CD34+ progenitor

CD83+ mature DC

myelomonocytic cell lines(e.g. THP-1, KG-1)

cytokines orcalcium ionophores

• AML blasts share the ontogeny of professional myeloid APC

• AML cell lines can acquire DC characteristics

• instant differentiation and activation in low percentages (5-10%)

DCOne: a DC line closely matching primary DC differentiation

• Myelomonocytic (AML) cell line from FAB M4• AML-associated translocation (12;22)(p13;q11-q12)• Non-adherent lobular cells• Cytokine dependent growth (GM-CSF/M-CSF/SCF/IL-3)• CD34+/CD14+

• HLA-A2, A3, B44; HLA-DR10, 11, 52, HLA-DQ5, 7• HLA class-I matches >70% of caucasian population

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DCOne

DCOne

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Pharmaceutical development process of DCOne

Working Cell Bank

Master Cell Bank

DCOneVaccine DCOneVaccine

Proliferation

ImmatureDCOne

ImmatureDCOne

ProgenitorDCOne

ProgenitorDCOne

MatureDCOneMatureDCOne

Differentiation Maturation Irradiation/Final Formulation

DCOne progenitor cell

development and validation of potencyassays

A Phase I vaccination study in AML patients: Clinical Trial Design

postpre

MRD

BM biopsy

Immune monitoring

DTH test

Clinical assays

w0 w2 w4 w6

vaccine

1

vaccine

2

vaccine

3

vaccine

4

MRD MRD MRD MRD

Immune assays

MRD

BM biopsy

Immune monitoring

DTH test

minimal residual disease (MRD) monitoring (WT-1 qPCR)

bone marrow biopsy (myelogram, FCM and molecular analysis, pathological analysis)

MRD

BM biopsy

Immune

monitoring

DTH test

cytokine responses in MLR against DCOne, LAA specific T-cell response in biopsies and peripheral blood lymphocytes

delayed-type hypersensitivity (DTH) skin test, ex vivo T cell expansion and LAA specific T cell reactivity testing

time point of DCOne immunization

� Cohort 1: n=3; 4 bi-weekly vaccinations with 1x107 DCOne (d0, d14, d28 and d42)� Cohort 2: n=3; 4 bi-weekly vaccinations with 2.5x107 DCOne� Cohort 3: n=3; 4 bi-weekly vaccinations with 5x107 DCOne� + Additional 3 patients with the optimal or DLT dose

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The next step: DCOne Plus

DCOnePlus Approach

Immature DCOne

Progenitor cell

Mature DCOne

Peptide loadedDCOne: DCOnePlus

TAA Peptide A, B or C

Can be combined in fixed ratios.For different tumor indications,

different combinations will be applicable.



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GVAX


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• Most common malignancy in elderly men• Second leading cause of cancer deaths in western countries• 1 in 6 men affected• 192,280 new cases and 27,360 deaths in 2009 in the US (www.cancer.org)

Prostate cancer





Prostaatkanker: ziektebeloop

Clinicallylocalized

Relapsedand

Metastatic

HormoneRefractory

AsymptomaticRising PSA

HormoneRefractory

symptomatic

Local treatment

Endocrine InvestigationalChemo-therapy

Progression of disease

Clinicallylocalized

Relapsedand

Metastatic

HormoneRefractory

AsymptomaticRising PSA

HormoneRefractory

symptomatic

Local treatment

Endocrine InvestigationalChemo-therapy

Progression of disease

immunotherapy





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Vaccination and/or immune modulation

Provenge

Exciting times!

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DC activatie en belading in vivo : het Prostaat GVAX vaccin





Exciting times!

Yervoy

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CTLA-4

X

Unrestrained T cellproliferation

Blocking negative checkpoints

Jim Allison

Ipilimumab (MDX-010)*anti-human CTLA-4 Antibody*high affinity and specificity*fully human IgG1k antibody *blocks the binding of CTLA-4 to B7*does not mediate ADCC

Drake Nat Rev Immnol 2010

Prostate GVAXVaccine consisting of twoAAV-GM-CSF transduced, irradiated prostate cancercell lines (LNCaP, PC-3)

CELL GENESYSCELL GENESYS

Ipilimumab (Yervoy)*anti-human CTLA-4 Antibody*high affinity and specificity*fully human IgG1k antibody *blocks the binding of CTLA-4 to B7*does not mediate ADCC

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Treatment and sampling scheme

* * *

* blood collection forimmunomonitoring

GVAX every 2 weeks for a total of 13 i.d. doses

anti-CTLA4 mAb (Ipilimumab) every 4 weeks for a total of 6 infusio ns

* ** * *w0v1

w4v3

w8v5

w16v9

w20v11

w24v13

w12v7

fu1

Dose level patient #Cohort 1 1-3 Cohort 2 4-6Cohort 3 7-9Cohort 4 10-12

Cohort 5 13-28

anti-CTLA4 dose0.3 mg/kg1.0 mg/kg3.0 mg/kg5.0 mg/kg

3.0 mg/kg

Clinical results

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0

50

100

150

200

250

0

30

60

90

120

150

0

10

20

30

40

50

Clinical results

Partial Response (PR); >50% PSA decline

Stable Disease (SD); No PR or PD

Progressive Disease (PD); >25% PSA increase

Ser

um P

SA

(ng

/ml)

11 / 28

12 / 28

5 / 28

Number of patients

n.a.

85 (82-190)

305 (51-919)

Duration of response (median and range in days)

PSA Progressive Disease (PD)

PSA Stable Disease (SD)

PSA Partial Response (PR)

Category:

>25% on-study PSA increase

No PR or SD

>50% on-study PSA decline

Response

Veelbelovende klinische resultaten!

Ser

um P

SA

ng/

mL

0

10

20

30

40

50

60

Pt 8

15-9-2005 29-3-2006

PSA en tumor respons gingen samen met

T cel activering maar ook met

autoimmuniteit:

hypofyse/schildklier/bijnier afwijkingen!

PSA

Botscan





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Prostate Cancer as a learning model

Can we identify lymphoid and myeloid immune parameters that correlate with

clinical activity and may be useful for clinical response prediction?

Immunomonitoring: principal question

…or treatment resistance prediction? >>avoid autoimmune side effects

1. Serology- tumor-specific antibodies

2. Peripheral blood T eff-/Treg cells- frequency- activation status- effector/memory phenotype

3. T cell Functionality- TAA-specific reactivity- suppression assayssuppression assayssuppression assays- cytokine profiles

4. Peripheral Blood DC (PBDC) and Myeloid Derived Su ppressor Cells (MDSC) - frequency- activation status

PSMA antilichaamresponsen in serum

P#1

P#2

P#3

pre FU-1

P#4

P#5

P#6

pre FU-1

P#7

P#8

P#9

pre FU-1

P#10

P#11

P#12

pre FU-1

100kDa

0.3 mg/kg (D1) 1.0 mg/kg (D2) 3.0 mg/kg (D3) 5.0 mg/kg (D4)

Alle patiënten met anti-tumorresponsen ontwikkelden antilichamen tegen het tumoreiwit PSMA na vaccinatieen dit was geassocieerd met langere overleving!


0 10 20 30 40 50 60 700

25

50

75

100 PSMA+

PSMA-

p = 0.035

N=12; med. survival 52 mths

N=16; med. survival 21 mths

cut-off: 2.0

months

Per

cent

sur

viva

l





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Bad news?

Good news

2. T cell cytokine profiles

� Th17: relation with response,

survival and autoimmune events

T cell profiles

� meting van CTLA4 op de T cellenvan patiënten vóór behandeling zouselectie mogelijk kunnen maken van patiënten die baat zullen hebben bijipilimumab behandeling

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Een groot succes maar met ernstige autoimmuunbijwerkingen: b.v. colitis.

Ook hier selectie van belang!





Ipilimumab monitoring: óókbij melanoompatiënten!





DC vaccinaties :

Van autoloog

naar allogeen

naar DC targeting in vivo!

Nóg effectiever met T cel checkpoint remming

Maar immunomonitoring essentieel om patienten met kans op clinical benefit te selecteren

Group 1 Group 2 Group 3 Group 4Group 1 Group 2 Group 3 Group 4

Patient codes

CD4+CD25intFoxP3+ increaseTreg preNon- naive CD4+ T cell preNon-naive CD8+ T cell preCD4+CTLA4+ preCD4+PD-1+ precDC1 CD40 activationcDC3 CD40 activation

Non-naive CD4+ T cell increasegrMDSC increaseTreg increasemMDSC pre

Monocyte CD40 activation

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Immunotherapy lab

Saskia Santegoets

Anita Stam

Sinéad Lougheed

Petra Scholten

Martine Reijm

Mary von Blomberg

Rik Scheper

Tanja de Gruijl

Medical Oncology Clinic

Helen Gall

Fons van den Eertwegh

Winald Gerritsen

Karin Jooss

Natalie Sacks

Kristen Hege

Israel Lowy


Jean-Marie Cuillerot





Sandra van Wetering

Dinja Oosterhoff

Jorn Kaspers

Michelle van Meerendonk

Pepijn Wijnands

Anneke Reurs

Ada Kruisbeek

Het immuunsysteem: gevaarlijke indringers buiten … · 2 Eindelijk erkenning… Tumor Immunology...

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Transcript of Het immuunsysteem: gevaarlijke indringers buiten … · 2 Eindelijk erkenning… Tumor Immunology...