Multhoff [Mode de compatibilité] · • Release of interferones (i.e. IFN, TNF) • Release of inflammatory cytokines (i.e. IL1, IL6) • Release of T chemokines (i.e. CXCL9, 10,

Klinik und Poliklinik für Strahlentherapie und Radiologische Onkologie

© Multhoff

Prof. Dr. Gabriele MulthoffKlinik für Strahlentherapie und Radiologische Onkologie Klinikum rechts der Isar, Technische Universität München

Immunomodulatory effects of irradiation


© Multhoff

Time scale of irradiation effects

Ionization, radicals, heat <sec

DNA damage, repair min - hours

Cell death hours - days

Immune effects hours - days

Mutations days - months

Cancer years - decades


© Multhoff

Major form of cell death after irradiation is apoptosis

2.5µm 2.5µm

Apoptosis Necrosis


© Multhoff

Apoptosis: Switch of phosphatidylserine (PS) from the inner to the outer membrane leaflet

0 Gy 2 Gy 10 Gy

outer leaflet

inner leaflet

NormalPS

PS

ApoptosisAnnexin V

green: Annexin V and PS antibodies detects PS in outer leafletblue: nucleus (DAPI)


© Multhoff

Characteristics of apoptosis and necrosis

Apoptosis NecrosisProgrammed cell death Accidental

Active Passive

DamageDNA

DamageMembrane

Cell shrinkage Cell swelling (osmosis)

Apoptotic bodies (Blebbing)Membrane intactPS in outer leaflet

Cell burstMembrane damaged PS in inner leaflet

No inflammation Inflammation


© Multhoff

RadiationRadiationRadiationRadiation

DNADNADNADNA----DamageDamageDamageDamage

ProliferationProliferationProliferationProliferation

CellCellCellCell----CycleCycleCycleCycle

Apoptosis Apoptosis Apoptosis Apoptosis

Annexin VAnnexin VAnnexin VAnnexin VCaspases

RepairRepairRepairRepair

Hsp70

HMGB1

Danger signals of dying cells: HMGB1, survivin, Hsp70

Rödel, Frey, Multhoff, Gaipl 2013


© Multhoff

Hsp70 is also released by viable tumor cells in exosomes

Acetylcholinesterase (AChE) activity at characteristic density of 1.17g/ml

enzy

mat

ic a

ctiv

ity

0

100

200

AChE activity

[g/m

l]

1.08

1.11

1.14

1.15

1.17

1.19

1.21

1.22

1.24

1.08

1.11

1.14

1.15

1.17

1.19

1.21

1.22

1.24

CX+Colo+

silver stain

cyto

solTubulin

Hsp70

Hsc70

Bag-4

Colo - -+ +lys exos

CX - -+ +lys exos

Calnexin

Grp94 ER

Western Blot

Hsp70 in exosomes Gastpar Canc Res 2005


© Multhoff

Preparation of few ml of whole blood

Tumor patient for the

detection of

immune and

tumor markers

HSP70

HMGB1

Survivin

Monitoring of therapy response

Design of a kit

Clinical trial

Survivin

Hsp70

HMBG1

Danger signals serve as biomarkers in the serum of tumor patients to detect cancer and monitor outcome

• Gabriele Multhoff (TU München)

• Udo Gaipl (University Erlangen)• Franz Rödel (University Frankfurt)


© Multhoff

Non-targeted immunomodulatory effects induced by irradiation

• Bystander effects

• Abscopal effects Tumor

Irradiation

Abscopal

Effect

Bystander

Effect


© Multhoff

Principle of bystander effects

Bystander effects are defined as effects on tumor cells in close proximity to the irradiated tumor or to the tumor micromilieu that were not exposed to radiation

• Therapeutic intervention (irradiation) of tumors causes the release of “danger signals”, pro-inflammatory cytokines/chemokines, or reactive oxygen species in close proximity to the irradiated tumor

• These secreted factors induce locally a non-specific immune stimulation that can result in control of non-irradiated tumor or impact the tumor micromilieu

Tumor

Irradiation

Bystander

Effect


© Multhoff

Principle of abscopal effects

Abscopal effects are defined as systemic effects on tumors that are locally distinct from the irradiated tumor

• Therapeutic intervention (irradiation) of tumors causes the release of “danger signals” such as HMGB1, HSP, cell death signals, …,pro-inflammatory cytokines or reactive oxygen species

• These secreted signals derived from dying tumor cells can cause a stimulation of the immune system, predominately of the innate system

• Activated effector cells cause regression of distant non-irradiated tumors

Tumor

IrradiationAbscopal

Effect


© Multhoff

Summary: immune modulations caused by irradiationof the tumor

• DNA damage response• Induction of MHC and NKG2D ligands on tumor cells• Release of danger signals (i.e. HSP, HMGB1, survivin)• Release of interferones (i.e. IFN, TNF)• Release of inflammatory cytokines (i.e. IL1, IL6)• Release of T chemokines (i.e. CXCL9, 10, 11,16) ) that induce the expression of chemokine receptors (CXCR3) on T cells• Up-regulation of adhesion molecules on tumor ECs (i.e. ICAM1, VCAM, E-selectin)• Maturation of dendritic cells (DC)• Lymphocyte infiltration (T and NK cells)• Release of cytotoxic mediators (i.e. IFN, TNF, Granzyme, Perforin) by effector lymphocytes• Immune-mediated tumor cell kill

Burnette & Weichselbaumer Rad Oncol 2013


© Multhoff

• Monoclonal antibodies Gramatzki et Valerius1997 (i.e. anti-CD20 Maloney et al 1997, CEA Meyer et al 2009, Ras et al 1997, Kass et al 1999, EGFR Chung et al 2008, HER2 Goldenberg 1999,CO17-1A (EpCam) Riethmüller et al 1994, CTLA4 blockade Hodi et al 2003)

• Tumor vaccines Pardoll 1998 (i.e. irradiated tumor cells, tumor cell lysates Nestle

et al 1998, danger signals Matzinger 1994, Zitvogel et al 2008 i.e. HSP Multhoff et al 1995, HSP-peptide complexes Binder et Srivastava 2005, HMGB1 Gaipl et al 2011, Apetoh et al 2007, Annexin A5, survivin Rödel et al 2007, Kroemer et al 2005, mucin 1 Rammanthan et al 2005)

• Cell based therapies (i.e. T, NK cells Herberman et al 1989, Schaue et McBride 2010, Dendritic cells Schuler et al 1997, Banchereau et al 1998, Aicher et al 1997)

• Small molecule inhibitors (i.e. EGFR Krause et al 2005)

Immunological approaches that could be combined with irradiation (Oettgen et al 1991)


© Multhoff


© Multhoff

Effects of Heat shock protein 90 (Hsp90) inhibition (Schilling et al 2008, Kabakov et al 2010, Camphausen et Tofilon 2007, Chiosi 2004, Neckers 1997)

• Degradation of oncogenic client proteins• EGFR endocytosis• Decrease in insulin-like growth factor 1(IGF-1R)• Cell cycle checkpoint activation (CDK4)• Tumor growth reduction• Blocking of DNA damage repair• Suppression of angiogenesis (HIF1, VEGF blocking)• Suppression of p-glycoprotein (MDR)• Decrease in S phaseBut• Induction of the expression of Hsp70 (inhibitor of apoptosis)

Clinical use of Hsp90 inhibitorsProstate, cervical, pancreatic, lung, ENT, breast, bladder, oesophagal carcinomas, glioblastoma , melanoma


© Multhoff

Multhoff JI 1997; Vega JI 2008; Gehrmann Plos One 2008; Falguieres MCB 2008, Staubacher Proteomics 2009

Normal

Tumor

Hsp70 Normal Tumor

Intracellular Low High

Membrane No Yes

Extracellular No Yes

Hsp70 localization in normal and tumor cells

Tumors differ from normal cells in their Hsp70 expression levels and localization


© Multhoff

• AML, ALL, MDS (Yeh LR 2009, accepted)

• Squamous cell carcinoma lung (Pfister Cancer 2007)

• Lower rectal carcinoma (Pfister Cancer 2007)

• Colon carcinoma (Kocsis CSC 2009, accepted)

• Sarcoma (unpublished)

• Prostate carcinoma ?

Squamouscell carcinoma

Hsp70-FITC100 101 103102 104

Cou

nts

200

4060

Adenocarcinoma

Hsp70-FITC

100 101 103102 104

Cou

nts

20

104

68

Low rectal carcinoma

Hsp70-FITC

100 101 103102 104

Cou

nts

50

2510

1520

Coloncarcinoma

Cou

nts

100

2030

Hsp70-FITC

100 101 103102 104

Gastriccarcinoma

Hsp70-FITC

100 101 103102 104

50

1015

20

Cou

nts


Hsp70-FITC100 101 103102 104

Cou

nts

200

4060

Adenocarcinoma

Hsp70-FITC

100 101 103102 104

Cou

nts

20

104

68


Hsp70-FITC100 101 103102 104

Cou

nts

200

4060


Hsp70-FITC100 101 103102 104

Hsp70-FITC100 101 103102 104100 101 103102 104

Cou

nts

200

4060

Cou

nts

200

4060

200

4060

Adenocarcinoma

Hsp70-FITC

100 101 103102 104

Cou

nts

20

104

68

Adenocarcinoma

Hsp70-FITC

100 101 103102 104

Hsp70-FITC

100 101 103102 104100 101 103102 104

Cou

nts

20

104

68

20

104

68


Hsp70-FITC

100 101 103102 104

Cou

nts

50

2510

1520

Coloncarcinoma

Cou

nts

100

2030

Hsp70-FITC

100 101 103102 104

Gastriccarcinoma

Hsp70-FITC

100 101 103102 104

50

1015

20

Cou

nts


Hsp70-FITC

100 101 103102 104

Cou

nts

50

2510

1520

Coloncarcinoma

Cou

nts

100

2030

Hsp70-FITC

100 101 103102 104

Low rectalcarcinoma

Hsp70-FITC

100 101 103102 104

Cou

nts

50

2510

1520

Low rectalcarcinoma

Hsp70-FITC

100 101 103102 104

Hsp70-FITC

100 101 103102 104100 101 103102 104

Cou

nts

50

2510

1520

50

2510

1520

Coloncarcinoma

Cou

nts

100

2030

Hsp70-FITC

100 101 103102 104

Coloncarcinoma

Cou

nts

100

2030

100

2030

Hsp70-FITC

100 101 103102 104

Hsp70-FITC

100 101 103102 104100 101 103102 104

Gastriccarcinoma

Hsp70-FITC

100 101 103102 104

50

1015

20

Cou

nts

Gastriccarcinoma

Hsp70-FITC

100 101 103102 104

Hsp70-FITC

100 101 103102 104100 101 103102 104

50

1015

205

010

1520

Cou

nts

Multhoff Exp Hem 1999; Multhoff CSC 2001; Gastpar JI 2004; Staubacher Proteomics 2009

Membrane Hsp70 is a negative prognostic marker


© Multhoff

Hsp70-based therapeutic approaches

• Antibody (naked) therapy: Hsp70 antibody mediated ADCC

• Nanoparticle therapy: Doxorubicin-, anti-survivin loaded nanoparticles conjugated to Hsp70 antibody

• NK cell-based therapy: Hsp70 peptide activated NK cells

• Enzyme therapy: Granzyme B mediated apoptosis


© Multhoff

1048

FACS ADCC assay

Antibody therapy: cmHsp70.1 mAb initiates ADCC in membrane Hsp70 positive CT26 tumor cells, in vitro


© Multhoff

Media 1xAb Ab+TKD

***

cmHsp70.1 mAb initiates ADCC in membrane Hsp70 positive CT26 tumor cells, in vivo

CT26 tumor reduction in BALB/c mice


© Multhoff

p=0.310p<0.0001

days after Ab treatment days after Ab treatment

surv

ival

[%]

Hsp70 positive CT26 tumors

Hsp70 negative A20 lymphoma

Survival curves after 3 injections of cmHsp70.1 mAb

cmHsp70.1 mAb enhances survival of BALB/c mice bearing membrane Hsp70 positive CT26 tumors (i.p.)


© Multhoff

4°C 37°C

cmHsp70.1-FITC cmHsp70.1-FITC

Membrane (4°C) Cytosol (37°C)

Proteomic and lipidomic profiling of Hsp70 associated compounds in lipid rafts

Subcellular distribution of Hsp70 in the endo-lysosomal compartment

Nanoparticle therapy: Elevated temperature results in uptake of Hsp70 from the membrane into the cytosol

Stangl et al JCMM 2011; Gaca et al JCR 2013


© Multhoff

cmHsp70.1

PEG-linker

Nanoparticle unmodified(NP-PEG)

Adapted and modified from Spänkuch et. al., Neoplasia. 2008 Mar;10(3):223-34. ; Gaca et al JCR 2013

DoxorubicinAnti-survivin

Nanoparticle with Hsp70-mAb(NP-Hsp70)

Doxo-Nanoparticle unmodified(Doxo-NP-PEG)

Doxo-Nanoparticle with Hsp70-mAb

(Doxo-NP-Hsp70) cmHsp70.1

PEG-linker

PEG-linker

PEG-linker

Nanoparticle therapy: Hsp70 mAb-nanoparticle (NP)


© Multhoff

NK cell therapy: TKD/IL-2 activates NK cells

Enhances the migratory capacity of NK cellsGastpar CR 2005

ctrl IL-2 IL-2/ TKD

CD94 177 198 707

NKG2D 72 86 208

NKp30 37 47 199

NKp44 1 132 399

NKp46 233 290 393

Up-regulates activating, NK receptorsGross CSC 2003, Gross Bio Chem 2003

Tumor

NK

NK

Cx+

20/110/

1 5/1 2/1

% s

peci

fic ly

sis

0

10

20

30

40

50

60

70

80

90controlHsp70 mAb

Cx-

20/110/

1 5/1 2/1

days after tumor injection

5 15 25 35 45 55 65 750 10 20 30 40 50 60 70su

rviv

al (

%)

0

20

40

60

80

100

control (n=21)T (IL-2/TKD) (n=14)NK (IL-2/TKD) (n=17)NK (IL-2) (n=5)

TKD/IL-2 activated NK cellsenhance survival of mice

Kill Hsp70 positive tumor cells, in vitroMulthoff Exp Hem 1999, Gastpar J Immunol 2004

Successful phase I clinical trialKrause Clin Can Res 2004


© Multhoff

Clinical phase II trial with IL-2/TKD activated NK cells after RCT

* Immunological assessment: 4 weeks after last treatment day of RCTTumor assessment: 4 weeks after last treatment day of RCT Eligible patients will be randomised to either study or control group after tumor assessment

Scr

eeni

ng fo

r H

sp70

RA

ND

OM

ISA

TIO

NRCT4 mo

4 wks*

CRSDPR

NK NK NK NK NK NK

0 1284 16 20 wks24

0 1284 16 20 wks24

NK Infusion of TKD/IL-2-activated NK cells

RCT4 mo

Radiotherapy: 60-66 Gy Chemotherapy:Cisplatin/ Vinorelbine

Study group: NK therapy Best Supportive Care

Control group: no NK therapy Best Supportive Care

Follow-up period(18 months after start of NK

treatment)

Hsp70 positive patients entering standard therapy

End of NK treatment

further cycles possible

Pre-study part Interventional study part

Start of NK treatment

Biological responses

Clinical responses (PFS, OS)


© Multhoff

Molecular therapy: granzyme B mediated tumor killing

Trapani Genome Biol 2(12):3014.1 (2001)

Granzyme B initiates perforin-independent apoptosis selectively in Hsp70 positive tumor cellsGross JBC 2003; Nylandsted J Exp Med 2004

Mammalian expression system for human granzyme B

Mechanism of killing by human granzyme B


© Multhoff

Induction of apoptosis in Hsp70+tumors by granzyme B

untre

ated

Granz

yme

BCam

ptot

hecin

Cas

pase

-3 p

ositi

ve c

ells

[%]

0

10

20

30

40

50

60

70

12h24h48h

Granzyme B

Camptothecin

Control

Granzyme B [µg]0.0 0.1 0.2 0.3 0.4 0.5 0.6

Abs

orpt

ion

[405

nm

]

0.0

0.1

0.2

0.3

0.4

0.5

# 2# 3# 4

Yield: 10-30 mg GrB per 2 l

supernatant with a purification yield

of 64%

Apoptosis induction in CT26 cells

Gehrmann JIM 2011


© Multhoff

NK cell therapy

(IL-2/TKD)

Molecular therapy (Granzyme B)

NK

• DFG MU1238 7/2• Helmholtz Gemeinschaft “Präsidentenfonds“

• SFB-824• multimmune GmbH

• EU-CELLEUROPE• BMBF-Innovative Therapies

Antibody therapy (cmHsp70.1)

Radiation/ Hypoxia

Lipidomic and proteomic

profiling

Definition of tumor markers

in the serum

• BMBF Spitzencluster m4• TU-München

• Wilhelm Sander Stiftung• BMBF Kompetenzverbund Strahlenforschung

Funding


© Multhoff

Collaborations

Alexzander Asea (Scott & White Hospital Texas, USA)

Mike Atkinson, Michaela Nathrath (HMGU)

Günther Dollinger (UBW)

Anna Friedl , Kirsten Lauber (LMU)

Antonio DeMaio (UCSD, San Diego, USA)

Ralf Dressel, Frauke Alves, Christian Dullin (University Göttingen)

Franz Pfeifer (TUM)

Udo Gaipl, Benjamin Frey (University Erlangen)

Carmen Garrido (University Dijon), Marja Jäättelä (University Copenhagen)

Mike Horseman (University Aarhus)

Graham Pockley, Gemma Foulds (University Nottingham, UK)

Franz Rödel (University Frankfurt)

Gerd Schmitz (University Regensburg)

Arne Skerra, Lars Friedrich (WZW)

Vasilis Ntziachristos (HMGU)

Laszlo Vigh (University of Szeged)

Axel Walch, Isabel Winkelmann (HMGU)

Scientific team

Christine Bayer, PhD

Stephanie Ertl

Stephanie Erl

Mathias Gehrmann, PhD

Katharina Ilicic

Daniela Schilling, PhD

Thomas Schmid, PhD

Wolfgang Sievert

Stefan Stangl

Dieter Walsh


© Multhoff

Modified from Shresta et al. (1998), Curr. Opin. Immunol. 10: 581

NK cell therapy: NK cells kill tumor cells perforin-dependent / independent by granzyme B mediated apoptosis


© MulthoffDaugaard et al., 2007; Fouchaque et al 1999

C-terminus (aa 540-646)

Substrate-bindingdomain (aa 385-540)

N-ATPasedomain (aa 1-385)

Multhoff Exp Hem 1999; Multhoff CSC 2001; Gastpar JI 2004; Staubacher Proteomics 2009

Heat shock protein 70 (Hsp70 / HSPA1A)

Hsp70

Major stress-inducible member of the HSP70 family

Stress factors: heat, ionizing irradiation, UV, chemotherapeutics, hypoxia, heavy metals, oxygen radicals, …

Localization: intracellular (cytosol, ER), membrane, extracellular

Gene: chromosome 6p (TNFα - Hsp70 - complement)

Protein: 641aa, 72 kDa, gobular, conserved, no transmembrane domain

Domains: ATPase (44kDa), substrate binding (18kDa), C-terminal helix (10kDa)

Moonlighting function: molecular chaperone (folding, unfolding, transport, anti-apoptotic, antigen processing)

3D structure


© Multhoff

NK cells T cellsPeripheral blood 5-20% 60-80%

Origin Lymphoid progenitor in bone marrow

Lymphoid progenitor in bone marrow

Function First line of defenseViral/bacterial infectedcells, tumors

Adaptive immunityViral/bacterial infectedcells, tumors

Receptor Non-clonal, multiple Clonal

Phenotype TCR/CD3-, CD16/56+, KIR+, ILT+, Lectin-type+

TCR/CD3+, CD4/CD8

Killing ADCC, FAS/FASL, Granzymes, Perforin

FAS/FASL, Granzymes, Perforin

Cytokines IL-2, IL-15, IL-12, IL18 IL-2, IL-15, IL-12, IL18

Killer Immunoglobulin-like Receptors (KIRs)

Lectin-like receptors(CD94, NKG2A/C/D)


© Multhoff

Radiated

tumor

Gehrmann et al (2005) Cell Death Differentiation 12: 38-51; Gehrmann et al (2010) Int J CPT 48: 492

Irradiation increases the Hsp70 membrane expression

Therapies

•y-Irradiation

•Vincristin sulfate

•Taxoides

•Hsp90 inhibitors

•COX2 Inhibitors

Isotyp-FITC Hsp70-FITC

10 Gy

ControlFSC/SSC

Increase in membrane Hsp70 positive colon carcinoma cells following irradiation (10 Gy)


© Multhoff

1863 Virchow „Tumors are infiltrated by lymphocytes“

1893 Coley „Severe bacterial infections result in tumor regression“

1909 Ehrlich „Virulent capacity of tumors“: Immune system restricts cancer growth

1958 Burnet „Hypothesis of immune surveillance of cancer“

1958 De Vries Adoptive transfer of allogeneic lymphocytes in tumor patients

1965 Mathe & Amiel Graft vs host disease (GvHD) and graft vs leukemia reactions (GvL)

20022007

Dunn, OchsenbeinSwann, Smyth

„Immune editing“: Elimination, Equilibration, Escape

2007 Stewart Innate immune system is the first line of defense: inflammation induced by NK cells, Mp, coordinated by DCs

2004 Dunn RAG-2 deficient mice (no T, B cells) develop epithelialtumors (50%)

2006 Malmberg, Ljunggren

IFNγ deficient mice develop tumors

Immune system and cancer


© Multhoff

2002 Diefenbach, Raulet NK cells, DCs, Makrophages (Mp), neutrophils secrete dangersignals to stimulate the innate immune system

2008 Lin Innate cytokines: IL-2, IL-12, IL-18, IL-23Innate danger signals: HSPs, HMGB1, Annexin A5

20032008

DiefenbachZitvogel

Innate ligands: NKG2D ligands (MICA/B, RAE-1, ULBP1,2,3), HSPs > activate NK cells

2008 Prestwich Secretion of pro-inflammatory cytokines by DCs

2007 Raman Innate chemokines: CXCL10, CXCL9, CXCL11

2004 Houghton Adaptive Immunity: T cells recognize TAA presented by DCs in context with co-stimulators: B7-1 (CD80), B7-2 (CD86), CD40/CD40L

19981999

LordNishimura

Th1: IL-2, IFN γ, TNFα, GM-CSF > Mp, T (CD8) cellsTh2: IL-4, IL-5, IL-10 > naive B cells

2004 Dranoff Kill mechanims of T cells: perforin, granzyme, Fas ligandT cell cytokines: IFNγ, TNFα, TNFβ activate Mp

Mechanisms of tumor elimination by the immune system


© Multhoff

Hypoxia

Resistance to therapy

Cell growth/differentiation

Genomicinstability

Programmed cell death/apoptosis

Tumor progression/ metastasis

Neoangiogenesis/vascularisation

Sutherland et al. 1998


© Multhoff

Arterial end

Höckel et al. 1999

Reoxygenation effects on overall survival of patients


© Multhoff

Indirect / direct effects of irradiation

Indirect: e- interacts with mediator (e.g. H2O)

H2O � H2O+ + e-

H2O+ + H2O�H3O+ + OH-

O2 fixation of DNA damage Production of RO2*

Direct: e- interacts directly with DNA

Abbreviations:e- : fast electron RO2* : Reactive Oxygen Species


© Multhoff

Immune system

Granulocytespolymorphonuclearleukos

Agranulocytesmononuclearleukos

Neutrophils Lymphocytes(T, B, NK cells)

Basophils Monocytes

Eosinophils Macrophages / DCs

Multipotent Human Stem Cells (BM)

Myeloid Progenitors Lymphoid Progenitors

Mega- Erythrocyte Mastcell Myeloblast T cell B cell NK cellkaryocyte

Baso Neutro Eosino Monocyte Plasma cell


© Multhoff

• Interferones (IFN α, IFNγ, IFNβ, TNFα) Guilot et al 1997, Quesada et al 1984, Ludwig et al 1995

• Chemokines (CXCL8, CCl7, CCL8, CCl11, CCl13, CCL17, CCL19, CCL20)

• Cytokines (IL-2 Rosenberg et al 1998, Keilholz et al 1997, IL-15 Handgretinger)• Enzymes (granzyme, perforin)• Bacillus Calmette-Guerin (BCG) Herr et al 1992

Non-specific immunotherapeutic approaches (Oettgen et al 1991)

Documents

Multhoff [Mode de compatibilité] · • Release of interferones (i.e. IFN, TNF) • Release of inflammatory cytokines (i.e. IL1, IL6) • Release of T chemokines (i.e. CXCL9, 10,