Cancer Immunology and Immunotherapy CenterSt. Savas Cancer Hospital

Towards new approaches for cancer immunotherapyTowards new approaches for cancer immunotherapy

Constantin N. Baxevanis

A hypothetical model of cancer therapies-induced tumor A hypothetical model of cancer therapies-induced tumor dormancy, elimination and recurrencedormancy, elimination and recurrence

The role of antitumor endogenous immunityThe role of antitumor endogenous immunity

Tumor

Growth inhibition

Non-immunogenicdormancy

Completion of therapy

Earlyrecurrences

Laterecurrences

METASTASES

Apoptosis

Cancertherapies

immunogenic

EndogenousimmunityPermanent

immunogenicdormancy

Th1high

Elimination

suppressiveless immunogenic

Endogenousimmunity immunoediting

Th1low

Immunosurveillance

EquilibriumNon-permanent

Immunogenic dormancy

Escape

Earlyrecurrences

Laterecurrences

Cellularhomeostasis

Naturaldormancy

Tumorescape

Epigenetic changes

Therapeutic management of cancer through activation ofTherapeutic management of cancer through activation ofendogenous antitumor immunityendogenous antitumor immunity

Tumor oriented therapiesTumor oriented therapies• chemo• radio• hormonal• targeted

Pre-existent Antitumor ImmunityPre-existent Antitumor Immunity

ImmunotherapiesImmunotherapies• immune Ab• vaccines• ACT

+ +++

++

Tumor dormancyTumor dormancy escapeescape

ClinicalClinicalmanifestationsmanifestations

Improvements in advanced cancer (2010-2014)Improvements in advanced cancer (2010-2014)

Targeted therapies: • Ipilimumab (a-CTLA4)• Vemurafenib• Dabrafenib• Trametinib MEK 1/2 kinase inhibitor• Nivolumab (anti-PD1)

Ser-Thr kinase inhibitors

FDA approval

Immunotherapies: Sipuleucel-T (autologous cellular vaccine)

To be added soon…• Lambrolizumab (anti-PD-1)• MPDL (anti-PDL1)

Intratumoral immune signatures as prognostic and predictive markersIntratumoral immune signatures as prognostic and predictive markers

Immune contexture

Location

Density

Type Adaptive immunity, cytotoxicity, memory T cells

Quantification (cells/mm2)

Tumor center, margin, tertiary lymphoid islets

Functionalorientation

IL12RB1

IL12RB2

CD28CCR5CXCR3TBX21

IRF1IFNγG7MBTAP1IL17GNLYPRF1CCL5CD8ASTAT1

IL23RCCL24RORCIL17A

IL5IL13IL4STAT6IL4RIL10RIFNγR2

STAT3IFNγR1

CCL22CCL17CTLA4FOXP3TGFβIL10

Th1 cytotoxic Th17 Th2 Tregs

Imm

unos

core

fibroblastsfibroblasts

Type 1microenvironment

CD8+

CD4+

Type 2microenvironment

Intratumoral immune signatures as prognostic and predictive markersIntratumoral immune signatures as prognostic and predictive markers

tumortumorcorecore

invasiveinvasivemarginmargin

tumortumorbedbed

CD8CD8

TH17TH17

CD8 HiHi

CD8 HiHi/IL17 LoLo

CD8 LoLo/IL17 HiHi

CD8 LoLo/IL17 LoLo

Tosolini M et al. Cancer Res 2011;71:1263

Therapeutic management of cancer through activation ofTherapeutic management of cancer through activation ofendogenous antitumor immunityendogenous antitumor immunity

Tumor oriented therapiesTumor oriented therapies• chemo• radio• hormonal• targeted

Pre-existent Antitumor ImmunityPre-existent Antitumor Immunity

ImmunotherapiesImmunotherapies• immune Ab• vaccines• ACT

+ +++

++

Targeted therapies to potentiate the Targeted therapies to potentiate the endogenous antitumor immunityendogenous antitumor immunity

Growth factors

RTK(e.g. KIT, PDGFR, IGF, MET, FGFR, EGFR)

RAS

RAF

MEK

ERK Cell proliferation, growth, survival

=mutations

Gajewski TF et al. Nature Immunol, 2013;14:1014

Anti-tumor therapies based on immune system pathways in the tumor microenvironmentAnti-tumor therapies based on immune system pathways in the tumor microenvironment

Therapeutic interventions that target immune inhibitory Therapeutic interventions that target immune inhibitory pathways in the tumor microenvironmentpathways in the tumor microenvironment

Gajewski TF et al. Nature Immunol, 2013;14:1014

Growth factors

RTK(e.g. KIT, PDGFR, IGF, MET, FGFR, EGFR)

RAS

RAF

MEK

ERK Cell proliferation, growth, survival

=mutations

VemurafenibDabrafenibLGX818

TrametinibMEK162CobimetinibSelumetinib

Effects of tumor oriented therapies on the endogenousEffects of tumor oriented therapies on the endogenousantitumor immunity: III kinase inhibitorsantitumor immunity: III kinase inhibitors

Increased direct antigenpresentation (IL-12)

Decreased immuneSuppressive factor release(IL-10, IL-6)

Anti-angiogenetic effects

Decreased infiltrationof tumors by regulatory

T cells

Activation of T cells (IFNγ, TNFα)and increased homing (VCAM-1, CXCL9,10)

Increased antigencross-presentation

Conventional therapies to potentiate the Conventional therapies to potentiate the endogenous antitumor immunityendogenous antitumor immunity

Effects of tumor oriented therapies on the endogenousEffects of tumor oriented therapies on the endogenousantitumor immunity: I chemotherapiesantitumor immunity: I chemotherapies

Antineoplastic agents

Cancer cells

Increasedantigenicity

Tumorpeptides IFNβ

MHCclass I

Tumorantigens

Increasedimmunogenicity

CRTATP

HMGB1 HSP90

Increasedsusceptibility

to immune attacks

TRAIL-R1TRAIL-R2 NKG-2DL

Fas DNAM-1

Effects of tumor oriented therapies on the endogenousEffects of tumor oriented therapies on the endogenousantitumor immunity: I chemotherapiesantitumor immunity: I chemotherapies

Antineoplastic agents

Immune system

Activation ofinnate effectors

DCsIL-1β

IL-12 IL-18

Activation ofAdaptive effectors

CD8: Granzyme B

DCs: CD40, CD80, CD86, MHC II

Inactivation ofsuppressors

VEGFVEGFR MDSC

FoxP3 M2

NKNKT

γ/δ T IFNγ

Effects of tumor oriented therapies on the endogenousEffects of tumor oriented therapies on the endogenousantitumor immunity: II Radiotherapyantitumor immunity: II Radiotherapy

Formenti SC and Demaria S, J Natl Cancer Inst, 2013; 105:256-265

Promote priming of antitumor T cells by dendritic cells

PromotPromotee effector phase effector phase

Facilitate tumor infiltration by T cells

Improve tumor recognition and killing by T cells

Th1 responseTh1 response

cytotoxiccytotoxicside effectsside effects

Immunotherapies to potentiate the Immunotherapies to potentiate the endogenous antitumor immunityendogenous antitumor immunity

T-cellreceptor

antigen

MHC

CTLA-4

CD28

TAMTAM

B7PD-L1

PD-1anti-CTLA-4

anti-PD-1

Ipilimumab5 yr OS in advanced melanoma pts(progressed after chemo or treatment naive)12-50% (Lebbe C et al. J. Clin. Oncol. 2013;31/suppl.)[abstr. 9053]

Nivolumab• 62% and 43%, 1 yr and 2 yr OSin ipi-resistant melanoma pts(Topalian S et al. J Clin Oncol 2014;32:1020]

• 43% and 32%, 1 yr and 2 yr OS in NSCLC 70% and 52%, 1 yr and 2 yr OS in RCC(Robert C et al. Eur. J. Cancer 2013;49:2968)

• Lambrolizumab 50% OS (1 yr) in ipi-resistant advanced melanoma pts(Hamid O et al. N. Engl. J. Med. 2013;369:134)

Ipilimumab+Nivolumab53% of pts with advanced, treatment-resistant melanoma had objective tumor response with tumor regression in 80% of them(Wolchok JD et al. N. Engl. J. Med. 2013;369:122)

Immune checkpoint blockadeImmune checkpoint blockade

TTHH/CTL/CTL

cell proliferationgrowthsurvival NF-κB activation

IL-2 productionmTOR activation

Mechanisms of action of cancer vaccinesMechanisms of action of cancer vaccines

IL-1β, -12, -15, -18TNF-αIFNs

CXCL 2, 3, 7, 9, 10, 11CCL 2, 3, 4, 7, 8, 19 Specific activated

CD4+ T cell

IL-2IFNγCCL5

IFNγTNFα

Cancer immunoediting reoccurs during immunotherapiesCancer immunoediting reoccurs during immunotherapies

vacstart

vacend

Generate IR

Equilibrium

Anti-tumor response

Reduced tumor growth ratesReduced tumor growth rates IncreasedIncreasedOSOS

M e m o r yM e m o r y

Anti-tumor response

Elimination

tumortumorburdenburden conventional therapyconventional therapy

Note1: Effect of immunotherapy on tumor growth

A common vaccine may initially induce no significant reduction in tumor size → clinical benefit delayed

Road map to clinical responses

Immune systemactivation

Anti-tumoreffects

Effect on survival

some weeks few months several months

Note2: Patient selection in immunotherapy trials

Phase III trials have shown that immunotherapies are

less effective in patients with heavy disease burden

providing a biologic rationale for using

therapeutic vaccines earlier in disease process

Patients with progressive cancers may not have time to

develop a significant immune response and derive

benefit from treatment

Note3: Identifying intermediate endpoints in immunotherapy

Biomarkers to evaluate biologic response in the

absence of clinical response are needed• Cellular immune responses• Humoral immune responses

Intratumoral cellular events in A2.1/DR1/neuT mice vaccinatedIntratumoral cellular events in A2.1/DR1/neuT mice vaccinatedwith a HER-2(435-443) (CTL) + HER-2(776-790) (AE36) (Twith a HER-2(435-443) (CTL) + HER-2(776-790) (AE36) (THH) peptide vaccine) peptide vaccine

{Gritzapis AD et al, {Gritzapis AD et al, Cancer Res. 2006Cancer Res. 2006, , 66(10):545266(10):5452;Gritzapis AD et al, Cancer Res, 2010; ;Gritzapis AD et al, Cancer Res, 2010; 70(7):268670(7):2686}}

APC

AE36+DR1DR1

HER-2(435-443)HER-2(435-443)+A2+A2

iDC

CD8+ CTLIFN-γ+

CD107+

CD4+ effIFN-γ+

++

FasLFasLFasFas

TregsIL-10+

TGF-β+

Foxp3+

CD25high

mDC

IFN-γ

++

CCR7+

A2.1/DR1/neuT+

TUBO.A2

BALB-NeuT HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-KO

HER-2(435-443)HER-2(435-443)+ HER-2(776-790) (AE36)

apoptosis

HER-2(435-443)

0 25 50 75 1251000

50

100

150

200

250

Days

Tu

mo

r si

ze (

mm

2)

2

HER-2(435-443) + AE36

00

50

100

150

200

250

Days

Tu

mo

r si

ze (

mm

)

25 50 75 125100 150 175 200

rechallengeTUBO.A2

Important features of HER-2/neu• various signal cascades• survival, proliferation, differentiation invasion, adhesion, angiogenesis• overexpression associated with aggressive disease

ECD

TM

ICD

kinasedomain

sites fortyrosine P

GVGSPYVSRLLGICLHER-2(776-790) (AE36)

HLA-A3, -A2.1, -A24, -A11

HLA-DR1, -DR3, -DR4, -DR11 -DR7, -DR8, -DR15,

-DR51, -DR52, -DR53

Sotiriadou NN et al. Brit J Cancer 2001;85:1527Salazar LG et al. Clin Cancer Res 2003;9:5559

CD4+

CD8+

IL-2IL-2

IFN-IFN-γγ

+

perforinperforin

GZGZ

XX

HER-2 xenograftsin SCID mice

% s

urv

ival

0

Days after treatment

0

20

40

60

80

100

10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170

APC HER-2(776-790)

Preclinical studies with AE36Preclinical studies with AE36

CD8+CD4+ (AE36)CD8+CD4+ (AE36)

CD8+CD8+

Sotiriadou NN et al. Cancer Immunol Immunother 2007;56:601Voutsas IF et al. Int J Cancer 2007;121:2031Gillogly ME et al. Cancer Immunol Immunother 2004;53:490

Vaccinating prostate cancer patients with AE37Vaccinating prostate cancer patients with AE37

vac DTH

Vaccination schedule with AE37Vaccination schedule with AE37

Toxicity profileToxicity profile

Dermal reactions during vaccinations

vac 1 vac 2 vac 3 vac 4 vac 5 vac 6

Immunological responses and suppressor elementsImmunological responses and suppressor elementsduring vaccination with AE37during vaccination with AE37

Perez SA et al. Clin. Cancer Res. 2010,16:3495

THE AE37 CANCER VACCINETHE AE37 CANCER VACCINE

Retrospective analyses from the prostate Ca trialRetrospective analyses from the prostate Ca trial

Identification of immunologic biomarkers to predict treatmentactivity and overall survival

Perez SA et al. Cancer Immunol. Immunother. 2013, 62:1599Perez SA et al. Cancer Immunol. Immunother. 2014, in press

R0 R6 LTLTB

LTI0

10

20

30

4040

80

TG

Fβ

(n

g/m

l)

Vac(id): 0.5 ml AE37 (500 μg)+GM-CSF (60 μg) x 2

vac DTH

R0 R6 LT LTB

TGF IFN specific spots

0

20

40

60

80

100100150200250300

p=0.073 p=0.202 p=0.299 p=0.277

IFN

γ E

lisp

ot

(sp

ecif

ic s

po

ts/1

0^

6 P

BM

C)

PrevaccinePrevaccine TGFTGFββ levels predict frequencies levels predict frequencies of of IFNIFNγγ producing T cells producing T cells before and duringbefore and during vaccinations vaccinations

IFNγTGFβ

IFNγ

Vac(id): 0.5 ml AE37 (500 μg)+GM-CSF (60 μg) x 2

vac DTH

R0 R6 LTLTB

LTI0

10

20

30

4040

80

TG

Fβ

(n

g/m

l)

0

10

20

30

40

50

100

p=0.1263 p=0.0709 p=0.0330

DT

H

mm

(ort

ho

go

nal

mean

)

R6 LT LTB

PrevaccinePrevaccine TGFTGFββ levels predict levels predict DTHDTH responses responses duringduring vaccinations vaccinations

TGFβDTH

1.1. Inverse correlation between Inverse correlation between pre-existingpre-existing TGF- TGF-ββ and IFN and IFNγγ levels levels

2. Patients with 2. Patients with low TGFlow TGFββ levels levels beforebefore vaccinations vaccinations

develop develop positivepositive AE37-induced immunityAE37-induced immunity both in vitro and in vivo both in vitro and in vivo

ConclusionsConclusions

0

50

100

150

200

IFN

-γ s

pots

/10

6 P

BM

Cs

0

10

20

30

40

50

DT

H

mm

(o

rth

og

on

al m

ean

)

Vac(id): 0.5 ml AE37 (500 μg)+GM-CSF (60 μg) x 2

vac DTH

R6 LT LTB

p=0.0384 p=0.0380

PrevaccinePrevaccine frequencies of frequencies of IFN IFNγγ producing T cells in responseproducing T cells in responseto stimulation with AE37, predict to stimulation with AE37, predict DTHDTH responses responses duringduring vaccinations vaccinations

IFNγDTH

p=0.0904

Patients with higher frequencies of Patients with higher frequencies of AE37-specific IFNAE37-specific IFNγγ producing T cells producing T cells

beforebefore vaccinations develop vaccinations develop positive AE37-induced DTH positive AE37-induced DTH reactions reactions

ConclusionsConclusions

Pre-existing TGFPre-existing TGFββ levels and/or DTH as prognostic factors for OS levels and/or DTH as prognostic factors for OS

R0>median (n=12)R0<median (n=11)

TGFβ

0 25 50 75 100 125 150 1750

25

50

75

100

Per

cent

sur

viva

l

p=0.0495

0 50 100 1500

50

100

p=0.0155 DTH5/TGF<med.(n=8)

DTH<5/TGF>med.(n=5)

follow up (mo) (diagnosis)

p=0.0214

DTH5mm(n=15)

DTH < 5mm(n=8)

0 25 50 75 100 125 150 1750

50

100

DTH

TGFβ/DTH

Pre-existing and induced IFNPre-existing and induced IFNγγ immunity as immunity as

prognostic and predictive factor for OSprognostic and predictive factor for OS

IFNmedian, (n=15)IFN<median, (n=8)

IFNmedian, (n=11)IFN<median, (n=12)

IFNγ levels 6 months post vaccinations

Prevaccination IFNγ levels

0 25 50 75 100 125 150 1750

50

100

follow up (months from diagnosis)

Pe

rce

nt

ov

era

ll s

urv

iva

lp=0.0848

0 25 50 75 100 125 150 1750

50

100

follow up (months from diagnosis)

Pe

rce

nt o

ve

rall

su

rviv

al

p=0.0022

HLA-A24/HLA-DR11

0 25 50 75 100 125 150 1750

25

50

75

100

p=0.0570

follow up (months from diagnosis)

% O

ve

rall

su

rviv

al

0 25 50 750

25

50

75

100

A24+/DR11+ (n=9)A24-/DR11+/- ή A24+/DR11- (n=14)

p=0.1000

follow up (months from 1st vac)

% O

ve

rall

su

rviv

al

Criteria for defining high responders among AE37-vaccinated patientsCriteria for defining high responders among AE37-vaccinated patients

1. IFNγ (SI)≥1.55

R1/R2R2/R3R3/R4R4/R5

and R6 or LT or LTB

2. DTH (mm)≥5R6/LTR6/LTBLT/LTB

AND

Pt no IFNγ DTH classification HLA alleles

8 R1-R5 + R6/LT - A24/DR11

10 R4-R5 + R6/LT R6-LTB HR DR11

11 R1-R4 + R6/LT -

12 R1-R3 + R6-LTB LT/LTB HR DR11

13 -

14 R2/R3 + LT/LTB R6-LTB HR A24/DR11

15 R3-R5 + R6-LTB R6-LTB HR A24/DR11

16 R1-R5 + R6-LTB R6-LTB HR A24/DR11

17 R2/R3 + R6-LTB -

19 - -

20 R1-R5 + R6-LTB R6-LTB HR -

21 R1-R5 + LT/LTB R6-LTB HR -

22 - R6-LTB

23 - R6-LTB

24 R2-R5 + R6-LTB -

25 - LT/LTB A24/DR11

26 R1-R3 + LT R6-LTB

27 R1-R5 + R6/LTB R6-LTB HR A24

28 R4/R5 + LT/LTB LT/LTB HR A24/DR11

29 R4/R5 + LTB R6-LTB HR A24/DR11

30 R1-R5 + LTB R6-LTB HR A24/DR11

31 R2-R5 + R6/LTB R6-LTB HR A24/DR11

HLA-A2

0 25 50 75 100 125 150 1750

25

50

75

100

p=0.0800

follow up (months from diagnosis)

% O

ve

rall

su

rviv

al

HLA-A2- (n=15)

HLA-A2+ (n=8)

Reduced overall survival in HLA-A2+ prostate cancer patients Reduced overall survival in HLA-A2+ prostate cancer patients

0

0.2

0.4

0.6

0.8

1

1.2

0 12 28 156 0 12 24 28 156 0 12 24 28 0 12 28 156

% C

D8+

/De

xtr

am

er+

ce

lls

Boosting pre-existent immunity to PSABoosting pre-existent immunity to PSA153-161153-161 during vaccinations with AE37 during vaccinations with AE37

pat#29 pat#30 pat#32pat#31

CD8+/Dex+(HLA-A24/PSA153-161)CD8+/Dex+(HLA-A2/PSA146-154)

0.41 0.16 0.26 0.63

weeks

0.28 0.26 0.42 0.33

pat#29 pat#30 pat#31 pat#32

156

Biomarkers for clinical outcomes in AE37-vaccinated prostate cancer patientsBiomarkers for clinical outcomes in AE37-vaccinated prostate cancer patients

clinicallylocalizeddisease

biochemicalrecurrence

hormone sensitivedisease

castration resistantdisease

(non-metastatic)

metastatic disease castration resistant

metastatic disease hormone sensitive

death

death

Vaccine-boosting pre-existing immunity HER-2

death

IFNγ

IFNγ/DTH

HLA-A24+HLA-DR11+

HLA-A2+HLA-A2+TGFTGFββ

death

Pre-existing immunity HER-2

HLA-alleles

Endogenous immunity and HLA-allelic expression dictateEndogenous immunity and HLA-allelic expression dictateimmunological and clinical responses to AE37 vaccinationimmunological and clinical responses to AE37 vaccination

in prostate cancer patientsin prostate cancer patients

HLA Endogenous vaccine induced survival immunity immunity

HLA-A24HLA-DR11HLA-A2

TGFβ

TPSA

IFNγ

LOW

HIGHsup

pre

sse

d

no

n-s

up

pre

sse

d

HLA-A2

0 25 50 75 100 125 150 1750

25

50

75

100

p=0.0800

follow up (months from diagnosis)

% O

vera

ll su

rviv

al

What to do next: Retrospective analysis for validating What to do next: Retrospective analysis for validating a six parameter signaturea six parameter signature

diagnosis CRNM CRM Death

HLA-DR11HLA-A24HLA-A2TPSA

IFNγ-HER2TGFβ

AcknowledgementsAcknowledgements

Dr. S.A. Perez, PhD, Research coordinator at CIICDr. A. Thanos, MD, Director of the Urology Clinic at St Savas HospitalDr. M. Papamichail, PhD, MD, Honorary Director at CIICDr. E. von Hofe, PhD, President of Antigen Express Inc.Dr. I.F. Voutsas, PhDDr. L. Mahaira, PhDDr. O. Katsara, PhDDr. E. Iliopoulou, PhDP. Tzonis, MDE. Anastasopoulou, MScI. Kalogeropoulou, BScE. Pappou, BScT. Keramitsoglou, PhD, Immunobiology dept. Helena Venizelou HospitalM. Varla-Eleftherioti, MD, PhD, Immunobiology dept. Helena Venizelou Hospital