Not Only Chemotherapy Anti-Tumor Vaccines for Tumor Eradication Irit Avivi Department of Hematology and BMT Rambam Medical Health Care Campus Rambam Medical.

Not Only ChemotherapyNot Only Chemotherapy

Anti-Tumor Vaccines for Tumor Anti-Tumor Vaccines for Tumor EradicationEradication

Irit AviviIrit Avivi

Department of Hematology and BMTDepartment of Hematology and BMT

Rambam Medical Health Care CampusRambam Medical Health Care Campus

Haifa Haifa

Talk PlanTalk Plan The immune system in the healthy personThe immune system in the healthy person Why does the immune system fail to eradicate Why does the immune system fail to eradicate

cancer ?cancer ? Strategies to overcome this immunological Strategies to overcome this immunological

impairmentimpairment Vaccine studies in MyelomaVaccine studies in Myeloma Future directionsFuture directions

Current Approach to CancerCurrent Approach to Cancer Targeting tumor cellsTargeting tumor cells

chemotherapy – attacking divided cellschemotherapy – attacking divided cells

The tumor is a foreign entity

Is their any evidence for anti-tumor response?

Evidence for the Role of the Immune Evidence for the Role of the Immune System on Cancer GrowthSystem on Cancer Growth

Allogeneic stem cell transplantation Allogeneic stem cell transplantation

recipient donor

Evidence for the Role of the Immune Evidence for the Role of the Immune System on Cancer GrowthSystem on Cancer Growth

Allogeneic stem cell transplantation Allogeneic stem cell transplantation

recipient donor

• Graft-vs-host disease is associated with graft vs tumor effect•

More Evidence for the Role of the More Evidence for the Role of the Immune System on Cancer GrowthImmune System on Cancer Growth

T cells & Antibodies directed against tumor cellsT cells & Antibodies directed against tumor cells

exist in the circulationexist in the circulation

HoweverHowever

Immune responses are ineffective in Immune responses are ineffective in

preventing disease establishment and progressionpreventing disease establishment and progression

Why ?Why ?

The Immune ResponseThe Immune Response

B

DC

DCDC

T

FORIEGN

B

T

FORIEGN

T

T

T T

Dendritic Cells- The most Dendritic Cells- The most important Antigen Presenting Cells important Antigen Presenting Cells

T

T

T

T

DC maturation

iDC Monocytes mDC

Berzofsky et al JCI 2004

CD40L

MHC- peptide

Antigen PresentationAntigen Presentation

mDCT

T cell activation

How does the Tumor Escape the Immune System?How does the Tumor Escape the Immune System?

DCDC

DC

T

TT

T

Tumor Tumor

ActivationAnergy

VEGF IL6

Antigen Presentation

How does the Tumor Escape the Immune System?How does the Tumor Escape the Immune System?

DCDC

DC

T

TT

T

Tumor Tumor

Activation Anergy

VEGF IL6

CTLA-4

Trg

PDL1

PD1

PDL1

PD1

TT

How Does the Tumor Escape How Does the Tumor Escape the Immune System?the Immune System?

The tumor expresses Host AgsThe tumor expresses Host Ags

It impairs DC maturation and functionIt impairs DC maturation and function

Interferes with Antigen presentationInterferes with Antigen presentation

The tumor interacts with T cells AnergyThe tumor interacts with T cells Anergy

The tumor express inhibitory moleculesThe tumor express inhibitory molecules

The tumor induces accumulation of regulatory T cellsThe tumor induces accumulation of regulatory T cells

Strategies to Overcome Strategies to Overcome Tumor-Induced Immunological Defects Tumor-Induced Immunological Defects

Tumor escape OvercomingTumor escape Overcoming Host Ags Host Ags Tumor specific AgsTumor specific Ags

Immunogenic stimulatorsImmunogenic stimulators

Impaired DCs Maturation Impaired DCs Maturation Ex vivo DCs maturation Ex vivo DCs maturation

T cell Anergy T cell Anergy Interfere with Interfere with

mechanisms involvedmechanisms involved

in anergyin anergy

Ex vivo DC Maturation Results in Potent Mature DCs

DC

DC

DC

DC

T T

T

Ex vivo In Vivo= with Tumor

T

DC maturation

Strategies to Load Tumor Antigens onto DC

Tumor cell fusion

Loading of Tumor lysate

Loading of Tumor peptides

Individual Ag Whole Cell

Vaccination with Individual AntigensVaccination with Individual Antigens AdvantagesAdvantages

Tumor specificityTumor specificity FeasibilityFeasibility Monitoring response against defined AgMonitoring response against defined Ag Lower risk for autoimmunityLower risk for autoimmunity

DisadvantagesDisadvantages Limited number of antigensLimited number of antigens HLA restrictionHLA restriction Tumor evasion through down regulation of antigen Tumor evasion through down regulation of antigen

expressionexpression

Vaccination with Whole Cell Derived AntigensVaccination with Whole Cell Derived Antigens AdvantagesAdvantages

Broad response limits risk of tumor evasionBroad response limits risk of tumor evasion Presentation of unidentified & patient’s -tumor Presentation of unidentified & patient’s -tumor

specific Agsspecific Ags Presence of helper (DC4) and CTL (CD8) Presence of helper (DC4) and CTL (CD8)

response crucial for maintenance of long term response crucial for maintenance of long term immune responseimmune response

DisadvantagesDisadvantages Technical challenge of manipulating whole cells for Technical challenge of manipulating whole cells for

multi-center settingmulti-center setting Risk of auto-immunityRisk of auto-immunity

Fusion Vaccines

Why did we Choose Myeloma?Why did we Choose Myeloma?

7 year survival Relapse rate

Khaled Y et al, BMT 44:325, 2009

Myeloma- Myeloma- Immunomodulated DiseaseImmunomodulated Disease

““Vaccination” with Vaccination” with DC/MM Fusions DC/MM Fusions

Vaccination with DC/MM Fusions: Vaccination with DC/MM Fusions: Trial DesignTrial Design

Phase I dose escalation trialPhase I dose escalation trial

EligibilityEligibility Patients with advanced stage myelomaPatients with advanced stage myeloma Presence of measurable diseasePresence of measurable disease > 20% plasma cells in bone marrow for > 20% plasma cells in bone marrow for

vaccine generationvaccine generation

Vaccination Schedule: Vaccination Schedule: 3 subcutaneous vaccinations in conjunction 3 subcutaneous vaccinations in conjunction

with GM-CSF administered every 3 weekswith GM-CSF administered every 3 weeks

Adherent PBMCs cultured for 5-7 days with GM-CSF & IL-4; TNF- added for 48-72 hours

Myeloma cells isolated

DCs assessed for DC & tumor specific markers

Myeloma cells assessed for tumor & DC specific markers

DC & myeloma fused with 50% PEG at DC: tumor, 3:1 to 10:1

Fusion cells quantified by measuring dual expression ofunique DC & tumor markers

Doses prepared & frozen microbiology testing sent

Leukapharesis

Bone marrow

aspiration

CD38

MUC1

DR

CD86

CD86

CD38

CD40

CD83

CD80

CD138

CD138

GM-CSF 100ugat vaccine site for 4 days

Monocytesn=18

Myeloma Cells CD-38 Dendritic Cells CD86

Vaccine CharacterizationVaccine Characterization

Fusion cell

Adverse EventsAdverse Events Treatment associated events, transient grade I-II:Treatment associated events, transient grade I-II:

Injection site reactions 37Injection site reactions 37 Edema 6Edema 6 Muscle Aches 5Muscle Aches 5 Fatigue 2Fatigue 2 Fever 1Fever 1 Chills/sweats 2Chills/sweats 2 Diarrhea 1Diarrhea 1 Pruritis 1Pruritis 1 Rash 2Rash 2 Anorexia 1Anorexia 1 No Autoimmune disordersNo Autoimmune disorders

Episode of DVT/PE with antecedent history of DVT Episode of DVT/PE with antecedent history of DVT

Vaccine site reaction: Skin Biopsy

Immunological Response: Vaccine Site Reaction Recruitment of “ attacking” CD8 T cells

Recruitment of antigen- presenting DCs (CD1a)

0

10

20

30

40

50

60

DCs

Tumor

Fusions

Stim

ulat

ion

Inde

x (S

I)

(n=16) (n=16)(n=16)

Fusion Cells Stimulate Fusion Cells Stimulate Allo – CD8 +T cellAllo – CD8 +T cell

Tumor Lysate Induced Tumor Lysate Induced

IFNIFNγγ Expression by CD8+ T cells Expression by CD8+ T cells

100 101 102 103 104

FL1-H

Pre-vaccination#3

55.8

10.8

100 101 102 103 104

FL1-H

1 month postvaccination

57

4.5

100 101 102 103 104

FL1-H

Pre-mobilization

51

1.78

100 101 102 103 104

FL1-H

Post-transplantation

72

4.1

IFN

CD8 FITC

Vaccination Induced Immune Response

Persistent Response against Persistent Response against Tumor Specific Antigen Tumor Specific Antigen

0

1

2

3

4

5

6

% MUC1 CD8

Pre vaccine 1 month post 3 months post

Immunologic response associated with disease stabilization

Vaccination with DC/Myeloma Fusions: Vaccination with DC/Myeloma Fusions: Conclusions Conclusions

Vaccination is feasible and well toleratedVaccination is feasible and well tolerated Majority of patients show immunologic Majority of patients show immunologic

responseresponse Disease stabilization in a majority of patientsDisease stabilization in a majority of patients

How to Improve the Efficacy of How to Improve the Efficacy of Fusion Vaccines?Fusion Vaccines?

Administer to patients with Administer to patients with

minimal residual disease minimal residual disease

Administer after eliminating Regulatory T cellsAdminister after eliminating Regulatory T cells

High Dose Therapy (HDT)High Dose Therapy (HDT)

Provides minimal residual disease Provides minimal residual disease

Induces a significant reduction in Induces a significant reduction in

suppressive regulatory T cellssuppressive regulatory T cells

“ A vaccine supportive environment ”

Induction

Therapy

Cohort 2

Cohort 1 and 2

Inclusion: ASCT ; Exclusion: Autoimmunity

Study UpdateStudy Update

41 patients enrolled to date41 patients enrolled to dateAll underwent successful vaccine generationAll underwent successful vaccine generation

21 - completed vaccinations, Med FU21 - completed vaccinations, Med FU

22 - ongoing study22 - ongoing study

Vaccine details:Vaccine details:

Mean percent of fusion: 40%; Fusion viability: 78%Mean percent of fusion: 40%; Fusion viability: 78%

Mean dose: 3.9 x 10Mean dose: 3.9 x 1066 fusion cells fusion cells

Safety: unchanged. 2 ANA without symptomsSafety: unchanged. 2 ANA without symptoms

Perc

enta

ge o

f CD

4+ T

cel

ls

expr

essi

ng IF

N

Perc

enta

ge o

f CD

8+ T

cel

ls e

xpre

ssin

g IF

N

Mean Percentage of Tumor Reactive T Cells

Pre Tx Post Tx Peak PostVaccine

Peak PostVaccine

Post TxPre Tx

CD4 CD8

Immunological Response to Vaccine

Clinical Response Clinical Response

Transplant VaccinationPost-tx 1 month

Post-tx4 months

Post-tx9 months

CR/nCR 67%

Response RateResponse RatePercentage of Patients Achieving CR Over Time Percentage of Patients Achieving CR Over Time

68% -complete response or very good partial response >>>> 40% with HDT only

Vaccination with DC/MM Fusions Following SCTVaccination with DC/MM Fusions Following SCT

Conclusions Conclusions

Feasible & well toleratedFeasible & well tolerated

Induces a significant anti-myeloma Induces a significant anti-myeloma immune responseimmune response

Results in a high response rateResults in a high response rate

Strong correlation between immunological Strong correlation between immunological and clinical responsesand clinical responses

How to improve?

How does the Tumor Escape the Immune System?How does the Tumor Escape the Immune System?

DCDC

DC

T

TT

T

Tumor Tumor

Activation Anergy

VEGF IL6

CTLA-4

Trg

PDL1

PD1

PDL1

PD1

TT

PD-L1/PD-1 Pathway•PD-L1 -an inhibitory ligand expressed by APCs •Signals via the costimulatory complex

•promotes

•Induces apoptosis of memory effector cells & disrupts CTL mediated lysis

•Inhibits T cell activation

PDL-1 Expressed by Tumor PDL-1 Expressed by Tumor is Associated with Toleranceis Associated with Tolerance

Shin & Rehermann Nature Medicine 2006

100 101 102 103 104

FL2-H100 101 102 103 104

FL2-H

IgG PDL1

100 101 102 103 104

FL2-H100 101 102 103 104

FL2-H

DC

MM

PDL-1 is Expressed by DCs and Myeloma Cells

100 101 102 103 104

FL1-H

100 101 102 103 104

FL1-H

100 101 102 103 104

FL1-H

100 101 102 103 104

FL1-H

17.3%4.8%

Control Myeloma PatientsIg

G

IgG

PD

-1

PD

-1

CD 4

PD-1 Expression on T cells

p=0.004

Mean PD-1 Expression on CD4 T cells : Increase with Advanced Disease

% E

xpre

ssio

n (

CD

4 T

Cel

ls)

CD4 T cell expressing PD-1 Increase Following Fusion Stimulation

% E

xpre

ssio

n of

P

D-1

(C

D 4

T c

ells

)

100 101 102 103 104

FL1-H

100 101 102 103 104

FL2-H

PDL-1 Expression on DC/Myeloma Fusions

CD

86

CD 38

PDL-1

PD-1 BlockadePD-1 Blockade CT-011: clinical humanized PD1 antibodyCT-011: clinical humanized PD1 antibody

Binds effector memory Tcs, resulting in their activation Binds effector memory Tcs, resulting in their activation and inhibition of apoptosis and inhibition of apoptosis

In animal models, administration induced anti-tumor In animal models, administration induced anti-tumor immune responses & tumor regressionimmune responses & tumor regression

Clinical Trials:Clinical Trials: Phase I trial : tolerated with evidence of clinical benefitPhase I trial : tolerated with evidence of clinical benefit Administration post ASCT results in increased CD4+ Administration post ASCT results in increased CD4+

effector memory T cells effector memory T cells (Rotem-Yehudar et al., A1216; Ash 2009)(Rotem-Yehudar et al., A1216; Ash 2009)

% E

xpre

ssio

n

of

PD

-1 (

CD

4 T

cel

ls)

PD-1 Blockade Results in Downregulation of PD-1Expression on CD 4 T Cells following Fusion Stimulation

p=0.048

% E

xp

ress

ion

of

IFN

γ (

CD

4 T

ce

lls)

PD-1 Blockade Results in Increased Interferon-gamma Following Fusion Stimulation

100 101 102 103 104

FL1-H100 101 102 103 104

FL1-H100 101 102 103 104

FL1-H

0.210.03 0.55

2.510.90.01

CD8/IFNgCD8/IgG CD8/IFNg

In Vivo Vaccine Primed Tumor Reactive T cells are Increased in the Presence of Ex-vivo PD-1 Blockade

)

Pre-Vaccine #1

1 month post vaccines

100 101 102 103 104

FL1-H100 101 102 103 104

FL1-H100 101 102 103 104

FL1-H

0.03 0.21 0.55

CD8 CD8 INFG CD8INFG + AntiPD1

PD-1 Blockade Results in Increased Lysis of Autologous Tumor by Fusion Stimulated T Cells

Granzyme

Tu

mo

r

+ Anti-PD-1 Control

100 101 102 103 104

FL1-H100 101 102 103 104

FL1-H

23% 32%

PD1-PDL1 pathway in MyelomaPD1-PDL1 pathway in Myeloma Strong in vitro data supporting the potential role Strong in vitro data supporting the potential role

of PD1-PDL1 pathway in myelomaof PD1-PDL1 pathway in myeloma

- High expression of PDL1 on DCs & MM cells- High expression of PDL1 on DCs & MM cells

- High expression of PDL on T cells- High expression of PDL on T cells PD1-PDL1 blockade improved anti-tumor PD1-PDL1 blockade improved anti-tumor

response in vitroresponse in vitro The in vitro data provide strong platform for The in vitro data provide strong platform for

combining anti PD1-PDL1 hrapy with combining anti PD1-PDL1 hrapy with

DirectionsDirections

Choose the right tumor: Choose the right tumor:

Immunogenic vs “Non Immunogenic?Immunogenic vs “Non Immunogenic?

Improve the immunogenic target: Improve the immunogenic target:

specific peptides vs whole tumor ?specific peptides vs whole tumor ?

Control the inhibitory environment Control the inhibitory environment

Potential ApproachesPotential Approaches

DCDC

DC

T

TT

T

Tumor Tumor

Activation Anergy

VEGF IL6

CTLA-4

Trg

PDL1

PD1

PDL1

PD1

TT

The Rambam- Harvard Vaccine Program The Rambam- Harvard Vaccine Program

DC/MM – anti PD1L Myeloma vaccine studyDC/MM – anti PD1L Myeloma vaccine study DC/MM- immuno-mudulatory drug studyDC/MM- immuno-mudulatory drug study DC/AML- anti PD1L Myeloma vaccine study DC/AML- anti PD1L Myeloma vaccine study DC/RCC – Sunitinib StudyDC/RCC – Sunitinib Study

Sunitinib -kinase inhibitor of Flt3, Kit, VEGFSunitinib -kinase inhibitor of Flt3, Kit, VEGF

and PDGF receptors and PDGF receptors Pre-clinical study for vaccination for CLL Pre-clinical study for vaccination for CLL

AcknowledgmentsAcknowledgments

Hematology Department, RMC Hematology Department, RMC

Tami Katz, Tami Katz,

Lina Bisharat, Lina Bisharat,

Jacob M RoweJacob M Rowe

Hematology Department, BIDMCHematology Department, BIDMC

Jacalyn Rosenblat Jacalyn Rosenblat

David AviganDavid Avigan

ThanksThanks