I M M U N O T H E R A P Y F O RI M M U N O T H E R A P Y F O RC A N C E RC A N C E R

Copyright Notice:

All slides in this Slide Library on Immunotherapyare copyright by the individual lecturers.

Slides may not be duplicated without expresswritten permission.

Copyright 2002 All Rights Reserved

I M M U N O T H E R A P Y F O RI M M U N O T H E R A P Y F O RC A N C E RC A N C E R

A scientific activity sponsored byMontefiore Medical Center, the

University Hospital for the AlbertEinstein College of Medicine.

Faculty Chairperson:Nora Disis, MD

Associate Professor, University ofWashington, Associate Member,

Fred Hutchinson Cancer ResearchCenter Seattle, WA

Supported by an unrestricted Educational Grant from EMD Pharmaceuticals.

Michael Morse, MDAssistant Professor of

Medicine, Duke UniversityMedical Center

Durham, NC

Michael Bishop, MD, FACPSenior Investigator,

Experimental Transplantationand Immunology Branch,National Cancer Institute

Bethesda, MD

Jeffrey Weber, MD, PhDAssociate Professor of

Medicine, University ofSouthern California, Norris

Cancer CenterLos Angeles, CA

CME Course Director:Howard Kaufman, MD,

FACSAssistant Professor of Surgery,

Medical Oncology, &Microbiology & Immunology,

Albert Einstein College ofMedicine, & Chief, Division of

Surgical Oncology, AlbertEinstein College of Medicine &

Montefiore Medical CenterBronx, NY

W e l c om eW e l c om eHoward Kaufman, MD, FACS

Assistant Professor of Surgery, Medical Oncology, & Microbiology &Immunology, Albert Einstein College of Medicine, & Chief,

Division of Surgical Oncology, Albert Einstein College of Medicine& Montefiore Medical Center Bronx, NY

History Of Cancer Therapy

l 1600 BC Surgical excision

l 1809 Surgical oncology

l 1896 Radiation therapy

l 1942 Chemotherapy

l 1976 Immunotherapy

9 Year-old Girl with Ocular Melanoma

After 3 courses ofhigh-dose IL-2

1/31/01

Before IL-211/22/99

O v e r v i ew o f T u m o rO v e r v i ew o f T u m o rI m m u n o l o g yI m m u n o l o g y

Nora Disis, MDAssociate Professor, University of Washington, Associate

Member, Fred Hutchinson Cancer Research CenterSeattle, WA

Overview of Tumor Immunology

I. Immunity & Antigen Recognition

II. Human Tumors Are Immunogenic

III. Design of Immunotherapeutic Strategies

Immunity & AntigenRecognition

Immunity & Cancer

l Immunosuppression increases cancer risk

– Lymphoma (x 90), skin (x 29), cervix (x 14)

l Spontaneous regressions occur

– Melanoma (69), GI (34), lung (25), breast (22)

l Tumors can have infiltrating lymphocytes

l Human tumors are immunogenic

– Tumor antigens have been defined

*Challis JB, Stam HJ. Acta Oncol 1990;29:545-550.

Antibodies

• Soluble proteins• Produced by B cells• Bind intact proteins• Have Fc receptors• Can kill cells by binding

• Complement• Cytotoxic-APC

• Can block cell growth• Breast cancer

Cell

ANTIBODIES

Fc

Cytotoxic T Cells (CD8+)

• Peptide antigen• Class I MHC• Directly kills the cell

• Injects enzymes• Death signal

• Clonal expansion• Helped by T helper cells

Cell

CD8+ CELL DEATH

Class I MHC

T Helper Cells (CD4+)

IL-5 IL-10

IL-4

ANTIBODIES

CD8+

IL-2IFN

CD4+

CTL

• Peptide antigen• Class II MHC• Secrete cytokines

• Depending on many factors• T-helper 1: IL-2, IFN- CTL• T-helper 2: IL-10, 5, 4-Ab• Immunologic memory

Antigen Recognition

MHC II

MHC I

CD8+

Cell

DEATH

IL-5 IL-10

IL-4

IL-2IFN

ANTIBODIES

HELPER T CELLS

CD4+

CYTOTOXIC T CELLS

Immunotherapy for Cancer

Advantages

l Tumors are immunogenic

l Single cell kill

l Migrate to tissue

l Memory

l Life-long protection

Types of Immunotherapy

l Cytokines

l Antibody infusion

l Vaccines

l Adoptive immunotherapy

l Immunomodulators

Human Tumors AreImmunogenic

Human Cancer is Immunogenic

1

10

100

1000

10000

100000

I

gGA

nti

bod

ies

in µ

g/m

L Tetanus Tetanus

HER-2/neu

Donors HER2+Colon Ca

HER2+Colon Ca

Solid Tumor AntigensMage, 1,2,3

MARTgp100CEA

HER2MucinPSAPAP

(shared between multiple tumor types)

Ward, RL, et al. Hum Immunol. 1999;60:510-515.

Tumor Antigens

l Viral proteins: HBV, EBV, HPV

l Oncogenic proteins: p53, ras, HER2

l Glycosylated proteins and carbohydrates: mucin, Tn

l Antigens defined by immunity: MAGE

Immunity & Tumor Growth

l Proteins expressed by cancers elicit immunity

l Tumor-specific T cells and antibodies are foundin cancer patients

l Cancer grows despite existent tumor-specificimmunity

Inefficient Immune Response

• Cytokine environment• Ineffective antigen-presenting cells• Immunosuppressive factors

• Direct modulation, e.g. virus• Secreted factors

• Antigen weakly immunogenic• Tolerance

Design of ImmunotherapeuticStrategies

Immunotherapy for Cancer

Passive Immunity

l Supply the response

l Short lived

l Example: MoAb

Active Immunity

l Endogenous response

l Depends on the host

l Example: Vaccine

Cancer Patients Can Be Vaccinated

0

2

4

6

8

10

12

14

16

DT

H i

n m

m2

#1 #2 #3 #4 #5

DTH pre tt

DTH post tt

Breast Cancer Patients (Stage III/IV)

Advanced Stage Cancer Patients (n=32) Volunteer Donors (n=72/25)

Recall Antigen Neo-Antigen

0

10

20

30

40

50

60

70

80

90

100

% K

LH

imm

unit

y

KLH Ab KLH T cell

Donors

Cancer Patients

Schiffman et al, 2001

Make “Self” More Immunogenic

CD8+

Cell

DEATH

IL-5 IL-10

IL-4

IL-2IFN

ANTIBODIES

KILLER T CELLS

HELPER T CELLS

CD4+

PEPTIDES

IMMUNE SYSTEM ACTIVATORS

ANTIGEN-PRESENTING

CELLS

Disease Burden & Immunotherapy

Dis

ease

Bu

rden

ClinicallyUndetectable

LocalizedDisease

AdvancedDisease

1:10,000 T cells 1:50 T cells 1:2 T cells?

M O N O C L O N A LM O N O C L O N A LA N T I B O D I E SA N T I B O D I E S

Michael R. Bishop, MD, FACPSenior Investigator, Experimental Transplantation &

Immunology Branch, National Cancer InstituteBethesda, MD

Clinical Applications ofMonoclonal Antibodies

ll DiagnosisDiagnosis

ll Monitoring of disease progressionMonitoring of disease progression

ll TherapyTherapy

Considerations for the Use of MonoclonalAntibodies for Immunotherapy

ll Choice of target antigenChoice of target antigen

ll ImmunogenicityImmunogenicity of of MoAbMoAb

ll MoAbMoAb half-life half-life

ll ManufacturingManufacturing

ll UnconjugatedUnconjugated vsvs. Conjugated. Conjugated–– RadioisotopesRadioisotopes–– ToxinsToxins–– ChemotherapyChemotherapy

Therapeutic Effects ofMonoclonal Antibodies

lDirect Effects

– Induction of apoptosis

– Block growth factor receptors

– Anti-idiotype Ab formation

l Indirect Effects

– Ab-dependent cell-mediated cytoxicity (ADCC)

– Complement-mediated cellular cytotoxicity

Green MC, et al. Cancer Treat. Rev 2000; 26:269-286.

ADCC: Recruitment of natural killer (NK) cells, macrophagesand monocytes by MoAB through its binding to their Fcgreceptors

Anderson DR, et al. Biochem Soc Trans. 1997;25:705-708.; Clynes RA, et al. Nat Med. 2000;6:443-446.

Mechanisms of Action: ADCC

Reff ME, et al. Blood. 1994; 83:435-445.

Complement Dependent Cytotoxicity(CDC)

CDC: Induced by MoAB binding to C1q, resulting in activation of thecomplement cascade and generation of the membrane attack complex

Obstacles to the Effectivenessof Monoclonal Antibodies

ll Heterogeneity of Heterogeneity of agag distribution on malignant cells distribution on malignant cells

ll Non-homogenous blood flow to tumorsNon-homogenous blood flow to tumors

ll High interstitial pressure in tumorsHigh interstitial pressure in tumors

ll Unbound Unbound agag-binding -binding AbAb

ll Human anti-mouse Human anti-mouse AbAb (HAMA) (HAMA)

ll Cross reactivity with normal tissue Cross reactivity with normal tissue agag

Green MC, et al. Cancer Treat. Rev 2000; 26:269-286.

Monoclonal Antibodies for Treatment ofB cell Malignancies

Antigen Antibody Type Investigational

Status Rituximab (Rituxan)

Chimeric FDA approved

Tositumomab (Bexxar)

131I-Murine Submitted CD20

Ibritumomab (Zevalin)

90Y-Murine Submitted

CD52 Alemtuzumab

(Campath) Humanized FDA approved

CD22 Epratuzumab (Lymphocide)

Humanized Phase II/III

Riley JK, Sliwkowski MX. Semin Oncol. 2000; 27(suppl 12):17-24.

CD20 StructureCD20 Structure

ll 297 amino acids297 amino acids

ll 4 4 transmembranetransmembrane domains domains

ll IntracellularIntracellularphosphorylationphosphorylation consensus consensussequences forsequences forserineserine//threonine kinasesthreonine kinases

–– Protein Protein kinasekinase C (orange) C (orange)

–– CalmodulinCalmodulin/calcium/calcium(green)(green)

–– Casein Casein kinasekinase II (yellow) II (yellow)

1Tedder TF, Engel P. Immunol Today. 1994; 15:450-454.; 2Bubien JK, et al. J Cell Biol. 1993;121: 1121-1121.; 3Deans JP, et al. J Biol Chem. 1995; 270:22632-22638.

CD20: A Putative Calcium Channell Structural homology

with calcium channels1

l Increased intracellularcalcium in stablytransfected cell lines1,2

l Calcium flux detectedupon stimulation ofCD20 with moAbscrosslinked withantimouse secondaryantibodies3

l Calcium chelators blockapoptosis induced byCD20 stimulation3

Riley JK, Sliwkowski MX. Semin Oncol. 2000; 27(suppl 12):17-24.

Effects of CD20 Crosslinking

l Increase in intracellular calcium

l Activation of

– Src family of tyrosine kinases

– Serine/threonine kinases

l Phosphorylation of

– CD20

– phospolipase Cg

l Upregulation of

– c-myc and b-myb mRNA

– adhesion molecule expression

– MHC II protein expression

CD20 Expression in B cell Malignancies

0 100 200 300 400 500 600

CLL

CLL/PLL

Small-Cleaved

Follicular Small Cell

Marginal Zone

Mantle Cell

Large Cell

Burkitt's Lymphoma

Hairy Cell

Mean Channel Fluorescence

Bcl-2 Protein

l Overproduced in most follicular lymphomas and many diffuselymphomas

l Localized mitochondria, endoplastic reticulum, nuclear envelope

l Physiologic function: confers longevity on memory B and T cells

l Prolongs cell survival by blocking apoptosis

l Implicated in chemoresistance

Reed JC, et al. J Cell Biochem. 1996;60:23-32.

Rituximab: An Anti-CD20Monoclonal Antibody

l Genetically engineered chimericmurine/human monoclonalantibody

– Variable light- and heavy-chain regions from murineanti-CD20 antibody IDEC-2B8

– Human IgGk constantregions

Rituximab: Mechanisms of Action

ll DirectDirect

–– Stimulation ofStimulation of apoptosis apoptosis by inducing Ca by inducing Ca2+2+ flux flux

•• CaspaseCaspase activation activation

•• DNA fragmentationDNA fragmentation

•• Cell deathCell death

ll IndirectIndirect

–– Antibody-dependent cell-mediated Antibody-dependent cell-mediated cytotoxicitycytotoxicity (ADCC) (ADCC)

–– Complement-mediated Complement-mediated cytotoxicitycytotoxicity

Hainsworth JD, et al. Blood. 2000; 95:3052-3056.; Hainsworth JD, et al. Proc ASCO. 2001; 20. Abstract 1175.

RituximabRituximab for Initial Treatment of for Initial Treatment ofLGNHL: Treatment SchemaLGNHL: Treatment Schema

Rituximab 375 mg/m2, slow I.V. infusion

1 2 3 4 5 6

Weeks

Reevaluate CRPRSD

PD Off Study

Rituximab MaintenanceRituximab 375 mg/m2

weekly x 4 every 6monthsx 2 years

Hainsworth JD, et al. Blood. 2000; 95:3052-3056.; Hainsworth JD, et al. Proc ASCO. 2001; 20. Abstract 1175.

RituximabRituximab for Initial Treatment of for Initial Treatment ofLGNHL: Response DataLGNHL: Response Data

Per

cen

t of

Pat

ien

ts

47

7

40

65

27

38

0

20

40

60

80

100

Overall ResponseRate

Complete ResponseRate

Partial ResponseRate

Week 6

After 1-2 Courses Rituximab

Hainsworth JD, et al. Blood. 2000; 95:3052-3056.; Hainsworth JD, et al. Proc ASCO. 2001; 20. Abstract 1175.

RituximabRituximab for Initial Treatment of for Initial Treatment ofLGNHL: Duration of ResponseLGNHL: Duration of Response

100

0Months

Per

cen

t Pro

gres

sion

Fre

e

Actuarial Progression-FreeSurvival1 Year 69%2 Years 67%

6 12 18 24 30 36

80

60

40

20

0

0

20

40

60

80

100

Months Posttherapy Initiation

Pro

por

tion

Su

rviv

ing

(%)

0 6 12 18 24 30 36 42

Reprinted with permission from Vose JM, et al. J Clin Oncol. 2001; 19:389-397.

Phase II Trial Rituximab Plus CHOP:Overall Survival

Coiffier B, et al. Blood. 2000;96:223a. Abstract 950.

GELA Phase III Trial of Rituximab/CHOP

l Stage II-IV diffuse large B cell lymphoma (DLCL)

l Previously untreated

l Elderly (60 to 80 years)

l ECOG PS ≤≤ 2

l No history of indolent NHL or CNS lymphoma

l No contraindication for doxorubicin or vincristine

GELA Phase III Trial of Rituximab/CHOPGELA Phase III Trial of Rituximab/CHOP

Coiffier B, et al. Blood. 2000;96:223a. Abstract 950.

UntreatedDLCLElderlyPatients

RANDOMIZATION

CHOPCyclophosphamide 750 mg/m2, day 1

Doxorubicin 50 mg/m2 , day 1Vincristine 1.4 mg/m2 , day 1

Prednisone 40 mg/m2, days 1-5G-CSF days 5-12

Every 3 weeks for 8 cycles

CHOP with RituximabSame CHOP schedule

plus Rituximab 375 mg/m2, day 1Every 3 weeks for 8 cycles

GELA Phase III Trial ofRituximab/CHOP: Survival

Coiffier B, et al. Blood. 2000;96:223a. Abstract 950.

83%

6 8 %

4 9 %

6 8 %

0%

20%

40%

60%

80%

100%

Overall Survival Event-Free Survival

C H O P

C H O P & Rituximab

P < 0.01 P < 0.0005

Per

cen

t of

Pat

ien

ts

Ongoing Phase III Trials:Intergroup Treatment Schema

Elderly;Elderly;DiffuseDiffuse

large-celllarge-cellNHLNHL

RANDOMIZATION

CHOPEvery 21 days (6-8 cycles)

CHOP + RituximabEvery 21 days (6-8 cycles)

RESTAGING

Stratify::CRCR vs vs.. PR PR

RANDOMIZATION

Rituximab MaintenanceRituximabRituximab 375 mg/m 375 mg/m22

weekly x 4 every 6 months xweekly x 4 every 6 months x2 years2 years

Observation

Stratify:Number ofNumber ofrisk factors:risk factors:

0, 10, 1vsvs..

2, 3, 42, 3, 4

Habermann, Rituximab Investigator’s Meeting, September 2000Rituximab Investigator Newsletter 2000; 2:6-9

Monoclonal Antibodies Used forImmunotherapy in Solid Tumors

Humanizedmurine IgG1

Mammarycancer

HER2oncoprotein

Trastuzumab (Herceptin®)

Murine IgG2aColon / rectalcancer

17 – 1A antigenEndrecolomab (Panorex®)

Type(chimerized, etc)

Target cell /disease

Specificity(antigen)

Antibody

White, Weaver, Grillo-Lopez. Ann Rev Med 52:125, 2001

Herceptin®® (Trastuzamab):Humanized Anti-HER2 Antibody

Targets HER2•• High affinity High affinity ((KKdd=5 =5 nMnM))

and specificityand specificity•• Humanized Humanized

95% human95% human5% 5% murinemurineIgG1

HerceptinHerceptin®®: Potential: PotentialMechanism of ActionMechanism of Action

ll Down regulates HER2 receptor expressionDown regulates HER2 receptor expression

ll Inhibits proliferation of human tumor cells thatInhibits proliferation of human tumor cells thatoverexpress overexpress HER2 proteinHER2 protein

ll Enhances immune recruitment and antibody-Enhances immune recruitment and antibody-dependent cellular dependent cellular cytotoxicitycytotoxicity (ADCC) against HER2 (ADCC) against HER2protein protein overexpressingoverexpressing cancer cells cancer cells

ll Down regulates Down regulates angiogenesis angiogenesis factorsfactors

* Independent Response Evaluation Committee

Design: single-arm, open-label

Week 2

Herceptin4 mg/kg loading

Herceptin2 mg/kg qw maintenance

Week 1 Week 3 Week 4, 5, 6, 7, 8…

Assessmentsl Primary end points

– Tumor assessments at weeks 8, 12, 24, then q12w thereafter– Objective tumor response (REC)*

l Secondary end points

– Duration of response, TTP, time to treatment failure assessments,survival

Until progression

Herceptin®® Monotherapyin Relapsed MB: Schema

Cobleigh MA, et al. J Clin Oncol. 1999;17:2639-2648.

% of Patients

CR 3.6

PR 12

ORR 15

SD (6 mos) 4

Clinical benefit† 19

* Response Evaluation Committee.† CR + PR + SD ≥ 6 mo.

Herceptin® Monotherapy in Relapsed MBC:Response Rates by REC*

Cobleigh et al. J Clin Oncol. 1999;17:2639.

Pro

gre

ssio

n-f

ree

resp

on

der

s

0 5 10 15 20 25Time from initial response (mo)

1.0

0.8

0.6

0.4

0.2

0.0

Median time to disease progression = 9.1 mo

* Response Evaluation Committee.

Herceptin® Monotherapy in Relapsed MBC:Median Duration of Response in Responders by

REC*

Cobleigh et al. J Clin Oncol. 1999;17:2639.

Herceptin®® First-Line Monotherapyin MBC: Schema

Vogel et al. Vogel et al. J J Clin OncolClin Oncol. 2001. In press.. 2001. In press.

Primary end point: ORR Secondary end points: RD, TTP and survival

• Eligible patients (n = 114)• MBC• HER2 overexpression (2+/3+)• No prior CT for MBC• Measurable disease• KPS > 60%

Weekly Herceptin

4 mg/kg loading dose

2 mg/kg/wk maintenance

Weekly Herceptin

8 mg/kg loading dose

4 mg/kg/wk maintenance

RANDOMIZE

Herceptin®® First-Line Monotherapy inMBC: Evaluable Population

Vogel et al. J Vogel et al. J Clin OncolClin Oncol. 2001. In press.. 2001. In press.

Herceptin®

* CR + PR + SD ≥≥ 6 mo.

22 (42)20 (34)Clinical Benefit*

6 (11)6 (12)SD > 6 mo

16-4013-3595% CI

15 (28)14 (24)ORR

11 (21)11 (19)PR

4 (8)3 (6)CR

53 (100)58 (100)No. of patients

4 mg/kgn (%)

2 mg/kgn (%)Parameter

Vogel, et al. Proc Amer Soc Clin Oncol. 2001;20:22a. Abstract 86.

HerceptinHerceptin®® MonotherapyMonotherapy in Relapsed in RelapsedMBC: FISH Clinical Outcome AnalysisMBC: FISH Clinical Outcome Analysis

No. of patients No. of patients evaluableevaluable 173 (100)173 (100) 36 (100)36 (100)

CRCR 88 00PRPR 2525 22ORR (%)ORR (%) 33 (19)33 (19) 0095% CI95% CI 14–2014–20 0–10 0–10

Clinical Benefit*Clinical Benefit* 41 (24)41 (24) 00

FISH–n (%)

FISH+n (%)

IHC 2+/3+ combined

* CR + PR + SD > 6 mo.

Nonresponders (n = 71) 1.8Stable disease < 6 mo (n = 13) 15.3Responders (n = 30) 18.8

TTP (mo post-randomization)

Pro

bab

ilit

y of

Pro

gres

sion

0 10 20 30 40

1.0

0.8

0.6

0.4

0.2

0.0

Herceptin®® First-Line Monotherapy inMBC: Time to Disease Progression

TTP (mo)

Vogel et al. J Clin Oncol. 2001. In press.

* Intent-to-treat population.† CR + PR + MR + SD > 6 mo.‡ 2 mg/kg and 4 mg/kg dose.

No. of patients (n) 114 222

ORR (%) 26 15*

SD > 6 mo (%) 11 4

Clinical benefit† (%) 38 19

Median time to disease progression (mo) 3.5–3.8‡ 3.1

Median DR (mo) > 12 9.1

Median survival (mo) 24.4 13

First-Line RelapsedParameters

Herceptin®® Monotherapy in MBCStudies: Summary

Cobleigh MA, et al. J Clin Oncol. 1999;17:2639-2648.Vogel, et al. J Clin Oncol. 2001. In press.

PriorPrior MedianMedian Median TTPMedian TTPTherapyTherapy NN RR (%)RR (%) DR (DR (momo)) ( (momo))

> 2 > 2 4646 1111 6.6 6.6 NANA

1–21–2 222222 1515 9.1 9.1 3.13.1

NoneNone 114114 2626 ~18.0 ~18.0 3.5–3.83.5–3.8

Baselga J, et al. J Clin Oncol. 1996;14:737-744.; Cobleigh MA, et al. J Clin Oncol. 1999;17:2639-2648.;Vogel, et al. Proc Am Soc Clin Oncol. 2000;19:71a. Abstract 275.

Summary of Summary of MonotherapyMonotherapy Trials: Trials:ResponseResponse

Slamon DJ, et al. N Engl J Med. 2001;344:783-792.

HerceptinHerceptin®® Combination Pivotal Trial Combination Pivotal Trialin First-Line MBC (H0648g): Schemain First-Line MBC (H0648g): Schema

• Eligible patients (n = 469)• MBC• HER2-positive• No prior CT for MBC• Measurable disease• KPS � 60%

No prior adjuvant AC Prior adjuvant AC

Taxol(n = 96)

Herceptin + Taxol® (n = 92)

AC(n = 138)

Herceptin + AC (n = 143)

Stratification

Randomization Randomization

AC = doxorubicin (60mg/m2) or epirubicin (75mg/m2) + cyclophosphamide(600 mg/m2) q3w for 6 cycles;Taxol® (175 mg/m2 x 3 h)q3w for 6 cycles; Herceptin(4 mg/kg IV) loading dose, 2mg/kg qw until progression.

Slamon DJ, et al. N Engl J Med. 2001;344:783-792.

HerceptinHerceptin®® Combination Pivotal Combination PivotalTrial: End PointsTrial: End Points

Primary

l Time to disease progression (TTP)

Secondary

l Objective response rate (ORR)

l Duration of response (DR)

l Time to treatment failure (TTF)

l 1-year survival

l Quality of life (QOL)

CR 8 3

PR 43 28

ORR (%) 50 32

95% CI 44–57 26–38

p value = < 0.001

Herceptin + CT(n = 235)

CT(n = 234)Parameter

Slamon DJ, et al. N Engl J Med. 2001;344:783-792.

Herceptin®® Combination Pivotal Trial:Objective Response Rate

H + CT 235 152 63 15CT 234 103 25 6

1.0

0.8

0.6

0.4

0.2

0.0

p < 0.001Median follow-up: 30 mo

Herceptin + CT (n = 235)CT alone (n = 234)

0 5 10 15 20 25 30

Pro

gres

sion

-fre

e su

rviv

al

Months

Slamon DJ, et al. N Engl J Med. 2001;344:783-792.

Herceptin®® Combination Pivotal Trial:Time to Progression

Herceptin®® Combination Pivotal Trial:Overall Survival*

0 10 20 30 40 50

1.0

0.8

0.6

0.4

0.2

0.0

RR = 0.80p = 0.046

Pro

gres

sion

-fre

e su

rviv

al

Herceptin + CT (n = 235)CT (n = 234)†

MonthsH + CT 235 192 134 96 11

CT 234 160 116 76 13

25.1 mo Month %

5 2415 6225 6540 72

% of patients crossing overto Herceptin at progression*

** Median follow-up: 35Median follow-up: 35 mo mo (range: 30–51) (range: 30–51)†† These patients are still reported in the These patients are still reported in theCT arm.CT arm.

Slamon DJ, et al. N Engl J Med. 2001;344:783-792.

FISH+

Herceptin + CT (n = 176)CT (n = 169)

RR = 0.71p = 0.007

RR = 1.11p = NS

Herceptin + CT (n = 50)CT (n = 56)

Months

0.2

0

0.4

0.6

0.8

1.0

Months

0.2

0

0.4

0.6

0.8

1.0

Pro

bab

ilit

y of

su

rviv

al

0 10 20 30 40 50

20.0 m

26.2 mo

FISH–

0 10 20 30 40 50

24.0 mo

Herceptin®® Combination Pivotal Trial:Overall Survival

Mass. Proc Am Soc Clin Oncol. 2001;20:22a. Abstract 85.

Monoclonal Antibodies Used for Immunotherapy

Humanized Murine MabAcute / chronicmyelogenous leukemia

CD-33 antigenAnti-CD33 (Hu-M195) a

Chimeric human / murineIgG1

NHLCD20 antigenIbritumomab Tiuxetanb,c

(ZevalinTM IDEC-Y2B8)

Murine IgG2aNHLCD22 antigenLL2 (Epratuzumab) a, b

Murine IgG2aNHLHLA-DR antigenLYM-1a

MouseNHLB1 antigenAnit-B1 (Tositumomoab)a

Chimeric human / murineIgG1

NHLCD20 antigenRituximab(Rituxan ®)

Humanized IgG1CLLCD52 antigenCAMPATH-1

Customized human MabB-cell lymphomaIndividual patients’ B-cell tumor antigens

Anti-idiotype antibodies

Humanized murine IgG1Mammary cancerHER2 oncoproteinTrastuzumab (Herceptin®)

Murine IgG2aColon / rectal cancer17 – 1A antigenEdrecolomab(Panorex ®)

Type (Chimerized, etc.)Target Cell / DiseaseSpecificity (Antigen)Antibody

a131I-conjugated b90Y-conjugated c111In-conjugatedMab: monoclonal antibody; CLL; chronic lymphocytic leukemia; NHL; non-Hodgkin’s lymphoma

White, Weaver, Grillo-Lopez, Ann Rev Med 52:125

C y t o k i n e sC y t o k i n e s a n d C a n c e r a n d C a n c e rT h e r a p yT h e r a p y

Jeffrey Weber, MD, PhDAssociate Professor of Medicine, Universityof Southern California, Norris Cancer Center

Los Angeles, CA

What are Cytokines?

l Cytokines are substances (generally proteins andglycoproteins) which are secreted by immune cells and haveautocrine and paracrine functions; they may function locally,or they may function at a distance, either to suppress oraugment immunity.

l Cytokines function to regulate the innate (inbornNK/macrophage/neutrophil) immune response and adaptive(flexible, T and B cell) immune response

What Cytokines Don’t Workas Anticancer Agents?

l IL-1 beta had minimal anticancer activity and in fact mayaccount for the severe side effects of fever, hypotensionand anorexia with IL-2

l TNF caused severe hypotension used systemically

l IL-4 had little anticancer activity and was toxic

l IL-6 had minimal anticancer activity and is a growth factorfor myeloma cells

l GM-CSF and IL-3 have no anticancer activity

What Cytokines are Importantfor Cancer Therapy?

l IL-2 and IFN-alfa 2b are FDA approved

l IL-2 has known activity in renal cell, melanoma, lymphoma,leukemia; IFN also has activity in the above diseases plus CML,KS and Hairy Cell Leukemia

l IL-12 and GM-CSF are likely effective adjuvants

l TNF and IL-4 may play a “niche” role in therapy in vitro

l IL-1 and IL-6 play a role in cancer-related toxicity

l IL-2 substitutes like BAY-50-4798 are designed to reduce its“capillary leak” toxicity

What is Alfa-Interferon?

l A superfamily of closely related genes on chromosome 9that encode variably glycosylated proteins in the 150-160amino acid range; they bind type I IFN receptors

l All interferons have profound and pleiotropic effects ongene expression

l Gamma interferon binds a type II IFN receptor andmediates different effects than type I IFNs

l Gamma interferon has no anticancer effects, but is FDA-approved for Chronic Granulomatous Dis.

What Does Alfa-Interferon Do?

l Gene upregulation MHC class I

Tumor antigensAdhesion molecules

l Anti-angiogenesis

l ImmuneB cell activityT cell activityMacrophagesDendritic cellsFc receptors

l Antiviral

Alfa-Interferon: Renal Cell Cancer

l Small but consistent response rates in the 5-10% range in anumber of studies

l Randomized controlled trial of IFN-alfa vs. Megace®

showed a survival of 8.5 for IFN vs. 6 months in 335patients with PFS <5% at 2 years

l Not clear what the optimal dose, route and schedule forIFN are

Alfa-Interferon: Renal Cell Cancer

l In a compilation of 551 patients on phase II IFNtrials given IM or SC, overall response rates were8-29% with a median of 11%

l Interferon is the second choice treatment for RCC,and is intended for palliation only; eligiblepatients should be offered FDA approved IL-2 ifthey agree and are eligible

Does Interferon-alfa 2b HaveAnti-Melanoma Activity?

l Kirkwood: 10-100 MU IV daily X 28 days with 23 pts; 22% RR

l Dorval: 10 MU/M2 T.I.W. SC with 22 pts; 27% RR

l Robinson: 30 MU/M2 T.I.W. SC X 12 weeks with 40 pts; 25% RR

l Sertoli: 10 MU/M2 T.I.W. IM with 21 pts; 14% RR

l Overall, 106 eligible pts; 22.6% RR

Dorval T, et al. Invest New Drugs. 1987;5:S61-S63. Sertoli MR, et al. Oncology. 1989;46:96-98.

Does Interferon-alfa 2b HaveAnti-Melanoma Activity with DTIC?

l Five trials of IFN-alfa 2b with cimetidine; 19/111 or17% RR; no advantage seen

l Eight trials of IFN-alfa with DTIC; 613 total pts;144/613 = 23% RR

l Median survival of groups receiving IFN+TAM,DTIC+IFN, DTIC+TAM or DTIC in a 2X2 matrixtrial was 9.1 months with median time toprogression of 3.7 months; no advantage for IFNplus DTIC

Kirkwood JM, et al. J Clin Oncol. 1996;14:7-17.

Adjuvant Interferon for Melanoma

l The ECOG 1684 trial of 280 randomized patientswho were observed or received one year ofinterferon-alpha 2b suggested a disease-free andoverall survival advantage for high-dose IFNleading to FDA approval.

l IFN yielded a 3.8 year versus a 2.78 year OS and 0.8vs. 1.8 year RFS; the advantage was 27% survivalprolongation at p<0.04

Kirkwood JM, et al. J Clin Oncol. 2000;18:2444-2458.

Adjuvant Interferon for Melanoma

l The SWOG 9111/ECOG 1690 trial of 642 patientswho received one year of HDI, two years of LDI orOBS showed no overall survival differences, withHR=1.0 for HDI vs. OBS but DFS was improved inthe HDI arm

l Median OS in the observed arm was 6.0 years, betterthan the treatment arm of the prior study

l The HR for RFS in HDI vs. OBS arm was 1.28

l Was high-dose IFN still the standard of care?

Adjuvant Interferon for Melanoma

l The post relapse survival in the 1690 trial was 4.3years vs. 1.8 years in the 1684 trial. Why?

l Post relapse survival in the two HDI arms wassimilar: 2.6 yrs for 1690 vs. 2.1 yrs for 1684.

l More patients in the OBS arm receivedbiochemotherapy than HDI pts (17% vs. 6 %) afterrelapse. Intensity of treatment was same.

l Does this suggest that IFN works, but can be givenearly or late after relapse?

Kirkwood JM, et al. J Clin Oncol. 2001;19:2370-2380.

Adjuvant Interferon for Melanoma

l Was adjuvant IFN ineffective and too toxic?

l The recent SWOG/ECOG 1694 IFN vs. GM-Kvaccine trial showed a clear DFS and OS advantagefor the IFN arm early after accrual was finished andwas prematurely halted.

l 774 eligible patients accrued

l RFS was prolonged with HR=1.6; OS prolongedwith HR=1.28; stage IIB patients also benefited; OSp value was = 0.04

E1694/S9512/C509801Relapse-Free Survival, Eligible Patients

0.0

0.2

0.4

0.6

0.8

1.0

0 5 10 15 20 25 30 35 40 45

Months

Pro

bab

ilit

y

GMKIFN

p = 0.0027

E1694/S9512/C509801Overall Survival, Eligible Patients

1.0

0.0

0.2

0.4

0.6

0.8

0 5 10 15 20 25 30 35 40 45

Months

Pro

bab

ilit

y

GMKIFN

p = 0.015

Relapse-Free Survival for IFN TrialsE1684, E1690, E1694 High-Dose IFN Arms

0.0

0.2

0.4

0.6

0.8

1.0

0 1 2 3 4 5 6 7 8 9 10Years

Pro

bab

ilit

y

E1684E1690E1694

Kirkwood JM, et al. J Clin Oncol. 1996;14:7-17.; Kirkwood JM, et al. J Clin Oncol. 2000;18:2444-2458.; Kirkwood JM, et al. J Clin Oncol. 2001;19:2370-2380.; Eggermont AM. Melanoma Res.1997;7:S127-S131.

What Randomized Controlled Trials HaveBeen Done with Postoperative

Adjuvant Interferon-Alfa?l ECOG 1684 alfa-2b 287 DFS and OS

l NCCTG 83-7052 alfa-2a 273 DFS stage III

l EORTC 18871 alfa-2a 900 negative low dose

l WHO 16 alfa-2a 426 negative low dose

l EORTC 18952 alfa-2b 1000 closed ?????

l ECOG 1690 alfa-2b 660 DSF not OS

l ECOG 1694 alfa-2b 771 DSF and OS (stopped early)

Does Interferon-Alfa 2b Have an Effect onConcurrent Vaccinations?

l ECOG 3694 trial: 107 patients randomized to GMKvaccine, IFN + GMK concurrent, or GMK started 5weeks after hi-dose IFN

l Primary end point was anti-GM2 IgG titre

l There were no differences in probability of or the peakvalue of anti-GM2 IgG or IgM between arms

l No correlation between RFS and antibody titre

l At 23 mo. median follow-up, RFS was prolonged in theIFN groups vs no IFN!

Adjuvant Interferon for Melanoma

l Once again, IFN-alpha is the standard adjuvant treatmentfor stage III melanoma, and should be the standardagainst which future adjuvant therapies are measured.

l Prophylactic Paxil may decrease IFN toxicity, as can IVhydration during hi-dose therapy.

l The current SWOG trial randomizes stage IIB/III patientsto receive HDI versus three cycles of a modified Leghachemobiotherapy

Quesada JR, et al. Blood. 1986;68:493-497.; Foon KA, et al. Am J Med. 1986;80:351-356. 

1010719264TOTAL

135784195Golomb, et al.

048242825Rai, et al.

7086714Foon, et al.

010603030Quesada, et al.

NoneMRPRCRPt #Study

Conclusion: IFN is an active, although second line,agent for Hairy Cell Leukemia.

Alfa Interferon for Hairy Cell Leukemia

Alfa Interferon for CML

l At MD Anderson, there is a large experience with IFN for CML:

– Patients treated: 274

– Complete hematologic response: 219 (80%)

– Cytogenetic responses: 159 (58%)

• Complete: 72 (26%)

• Partial <35%: 32 (12%)

• Minor: 55 (20%)

Median survival is greater than 89 months with a significant numberof projected 10 year survivors.

Allan NC, et al. Lancet. 1995;345:1392-1397.; Ohnishi K, et al. Blood. 1995;86:906-916.

Alfa Interferon for CMLFour randomized trials of alfa-interferon versus

chemotherapy have been performed indicatingsuperior survival for alfa-IFN:

Alfa-IFN is an established nontransplant therapy for CML

0.0293254159Ohnishi, et al.

0.0014161587Allan, et al.

0.0084566513Hehlmann, et al.

0.0025272322ItalianCooperative

p valueChemoIFN# pts

Interleukin-2: T Cell Growth Factor

l A glycoprotein T-cell growth factor that binds to a receptor on T-cells

l First tested at the NCI in the early 1980s, it was found thatsurprisingly RCC responded to IL-2

l Higher doses of IL-2 appeared to induce significant clinicalresponses, with severe toxicity

l A trial of IL-2 at high doses intravenously was expanded andresulted in a 24% response rate

Side Effects: Interleukin-2

l Toxicities of IL-2 stem from a capillary leak

l IL-2 therapy in high dose is like a controlled state of septic shockwith low BP, low SVR, high CO

– Grade III/IV hypotension present in 74%

– Grade III/IV cardiac toxicity present in 11%

– Grade III/IV hematologic present in 39%

– Grade III/IV hepatic in 39%

– Grade III/IV renal in 80%

– Grade III/IV pulmonary in 19%

Interleukin-2: Renal Cell Cancer

l High-dose bolus IL-2 in renal cell CA:

– Cycle 1 rIL-2 720,000 IU/kg I.V. over 15 minutes every 8hours for a maximum of 14 doses

– Then a 5-9 day rest period

– Cycle 2 rIL-2 720,000 IU/kg I.V. over 15 minutes every 8hours for a maximum of 14 doses

Interleukin-2: Renal Cell Cancer

High dose rIL-2 for renal cell CA: 255 patients

# of PatientsECOG Performance Status

112More than 1 year from diagnosis to first IL-2

143Less than 1 year from diagnosis to first IL-2

218Prior Nephrectomy

92

8011660

Interleukin-2: Renal Cell Cancer

l High dose bolus IL-2 for RCC

l Complete responses 7%, 80+ month duration

l Range of complete response durations: 7-131+

l Partial responses 8%, 20 month duration

l Range of partial response durations: 3-126+

l Overall 37/255 or 15% response rate

IL-2 And LAK Cells: Renal Cell Cancer

l 1985 trial showed that LAK cells plus high dose IL-2 gave a 35%response rate in RCC

l LAK cells were grown from pheresis specimens of PBMCincubated for 72 hours in IL-2 ex vivo

l Adoptive transfer of LAK cells caused profound pulmonarytoxicity and hypotension

l No compelling evidence that IL-2 + LAK better than IL-2 alone

Renal Cell Cancer: Interleukin-2l Management of IL-2 toxicities:

– Dopamine used for low urine output; dosingcontinues until creatinine is 3.5-4.0

– Neosynephrine® for hypotension resistant to fluid

– Indocin® and Tylenol® around the clock for fevers andchills induced by IL-2

– Cimetidine used for increased acidity and prophylacticOxacillin for neutrophil dysfunction that led to a 27%rate of sepsis in one series before antibiotics were used

Lissoni P, et al. Arch Ital Urol Androl. 1997;69:41-47.; Tourani JM, et al. Ann Oncol. 1996;7:525-528.; Negrier S, et al. Cancer J Sci Am. 2000;6:S93-S98.

Low Dose Interleukin-2: Renal CellCancer

l Lissoni, et al.: 91 patients;, 23% RR, 2% CR

l Buter, et al.: 47 patients; 19% RR, 3% CR

l Tourani, et al.: 39 patients; 18% RR, 3% CR

l Yang & Rosenberg: 53 patients; 11% RR, 6% CRl Crecy study: Negrier, et al.: 425 patients; got SC IL-2 vs. SC IFN

vs. SC IL-2+IFN; there was a 6.5% vs. 8% vs. 18.6% RR.No difference in overall survival was observed.

Interleukin-2 for Renal Cell Cancer:Conclusions

l Response rate N=255 is 15%, 7% CR, 8% PR

l Median overall duration of response is 54 months

l Responders can have a substantial tumor load

l Sites of regression include lungs, lymph nodes, bone, boweldisease

l Side effects manageable and virtually always reversible uponstopping therapy

l Low-dose IL-2 has fewer CRs and lower OS

High-Dose IL-2 for Melanoma

l Retrospective data from 266 patients in 7 trials from 17institutions

l 17% overall response rate; 16 (6%) CR and 30 (11%) PR

l At a median 5-year follow-up, 69% of CRs are alive, 47% ofall responders

l Median duration of response: 6.5 mos.

l 10/16 CRs, and 6/30 PRs remain progression-free 5-15 yearslater

Interleukin-2 in CombinationTreatment for Melanoma

l 7 trials of IL-2 with anti-GD3 antibodies:

– 145 total patients with only 5 responses, 1 CR and 4PR or 3% RR

– 16 trials of bolus, continuous or low-dose SC IL-2with alfa-interferon: 217 got bolus, 224 got CIV, 41got SC

– 48/217=22% RR for bolus, 28/224=12% RR for CIV,and 3/41=8% RR for SC IL-2

l Conclusion: IFN does not add to IL-2

What Correlates with SurvivalAfter IL-2 Treatment?

l Performance status

l Development of vitiligo and/or autoimmune thyroiditis

l Amount of IL-2 given during first course

l Height of the rebound lymphocytosis

l Vitiligo seen in 11/74 melanoma pts with response, in nonon-responders, and in no RCC patients receiving highdose IL-2

High-Dose IL-2 for Lymphoma

IL-2 has activity in stage IV non-Hodgkin’s lymphoma.

(17%)21/121147121TOTAL

(17%)7/395239IL-2 + LAK

(17%)14/829582IL-2

(%)CR+PRPRCRPts

EvaluableRegimen

Interleukin-2 for Lymphoma & LeukemiaPost Stem Cell Transplant

l 25 patients post-ASCT for NHL at Seattle:

l 80% completed induction IL-2; all tolerated maintenance well.

l Disease-free survival is 60% at 30 months- compares favorably ifnot better than historical controls at Seattle

l 46 patients with AML in CR1 received IL-2 median of 36 days posttransplant; at 2 years, DSF is 82%, which is quite favorablecompared to historical controls

l Currently there are large SWOG trials underway of ASCT +/- IL-2for NHL and AML

Interleukin 2 as an Adjunctin Acute Leukemia

Seattle regimen:Induction 9 X 106 IU/M2/day X 4d CIV

Rest 4 daysMaintenance 1.6 X 106 IU/M2/day X 10 d

In a phase II trial of AML patients in relapse, 13/24 CR were observedin relapses 2-4.

l Several trials of post CR1 IL-2 treatment have been done: In one 18patient trial, 6/18 pts (33%) were in CR at 4 years vs. 5/36 pts (14%)without IL-2 retrospectively

l Median CR duration for 10/23 patients in CR1 who stayed on IL-2post-remission was 22 months at Dana Farber.

IL-2 Plus Histamine

l In animal models, histamine compounds decrease theinhibition of the action of IL-2 by macrophages

l A randomized open label trial was conducted of 305 stage IVmelanoma patients who received low-dose IL-2 vs. low-doseIL-2 + histamine

l No differences in response rate were seen; overall survivalwas increased in the subgroup of patients with livermetastases with p<0.004; overall survival for all pts. increasedfrom 5.0 to 9.1 months but was not significantly different(unadjusted p=0.052)

l The FDA’s ODAC declined to approve Maxamine

BAY 50-4798: IL-2 Without Toxicity?

l A genetically engineered IL-2 molecule with a two amino-acid change to eliminate the “toxic” region.

l In animal models induces less capillary leak whichsuggests it will have fewer side effects.

l Now being evaluated in a phase I trial; the dose escalationcontinues and we are now at roughly a dose equivalent toIL-2 without dose limiting toxicity, and will less need forpressors.

Is GM-CSF Effective as Adjuvant Therapyfor Stages III/IV Melanoma?

l 48 patients with resected stages III/IV melanoma receivedGM-CSF at 125 µg/M2 days 1-14 every 28d X 12 cycles

l Median survival was 37.5 mos. vs. 12.2 mos. in the matchedUAB database from 1960-1988

l 14 Stage III pts had 35.8 mos. median survival, worse thanthat seen with IFN (46 mos.) but 4 or more LNs or > 3 cm LNwas required in this trial.

l Rationale? What are the pitfalls in interpreting this trial?

Spitter LE, et al. J Clin Oncol. 2000;18:1614-1621.; Atkins MB, et al. Clin Cancer Res. 1997;3:409-417.

IL-12: Clinically Active Cytokine?

l A heterodimeric protein of 70,000 MW that promotes bothNK and cytolytic T cell activity

l It has antitumor activity systemically in mouse models andwhen delivered as a gene therapy

l In phase I, of 40 patients 1 CR, 1 PR, 4 SD with IV IL-12

l In another phase I, of 50 pts 1 CR, 34 SD with SC IL-12

l IL-12 is being tested with IL-2 and with IFN in melanoma,and in T cell leukemia-lymphoma; it also has preclinicalactivity as a adjuvant in murine models

gp100/tyrosinase Peptides/IFA Vaccine +IL-12 and +/- GM-CSF: Schema

l There was a rationale for testing both cytokines as adjuvants sotwo trials were done with a peptide vaccine

l All patients were HLA-A201 positive with completely resectedstages II or III/IV melanoma

l Intervals were q 2 weekly X 4, then q 4 weekly X 3 then q 8weekly X 1; 8 injections over 26 weeks

l Pheresis and skin testing were performed prior to and after 6months of vaccine therapy

l IL-12 was given intradermally once and GM-CSF subcutaneouslyfor 5 days both at the vaccine site

T-Cell Immune Response to gp100-2Mat Day 10 by Cytokine Release

pg/

ml

of G

amm

a I

nte

rfer

onp

er 1

05 E

ffec

tors

0

500

1000

1500

2000

2500

3000

Treatment

pre post pre postno IL-12 with IL-12

T cell Immune Response to GP100 Over Time

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

pre after 2 after 4 after 6 after 8

Number of Vaccinations over Time

% C

D8+

T c

ells

Each line represents an individual patient. Solid lines indicate treatment with IL-12;dashed lines indicate no IL-12.

(0 weeks) (4 weeks) (10 weeks) (18 weeks)(26 weeks)

Conclusions: gp100/tyrosinasePeptides/IFA Vaccine +/- IL-12:

l Well tolerated: only grade III local toxicity, no grade IV, 3patients of 48 with ulceration

l The IL-12 group so far has had greater DTH reactivity togp100; none seen to tyrosinase

l The IL-12 group seems to have greater across the boardcytokine release reactivity to both gp100-WT and -2M butnot tyrosinase.

l 85% of patients had an immune response

T cell Immune Response to gp100-2Mat Day 10 by Cytokine Release

0

500

1000

1500

2000

2500

Treatment

pre post pre postno GM- CSF with GM-CSF

pg/

ml

of G

amm

a I

nte

rfer

onp

er 1

05 E

ffec

tors

Conclusions: gp100/tyrosinase Peptides/IFAVaccine +/- GM-CSF:

l Well tolerated: only grade II local toxicity, no grade III/IV,no patients of 43 with ulceration

l The GM group so far has had similar DTH reactivity togp100; none seen to tyrosinase

l The GM group seems to have increased across the boardcytokine release reactivity to gp100-WT and -2M but nottyrosinase.

l 85% of patients had an immune response

Conclusions: Cytokine Therapyfor Cancer

l IL-2 has modest antitumor activity in metastatic renal celland melanoma, is FDA approved for both, primarilybecause of the 5-10% long term survival “tail on the curve,”but is quite toxic

l IL-2 at low doses may have promising activityposttransplant as an immune “restorative”

l Alfa-interferon had a modest effect on survival when usedas adjuvant therapy for resected melanoma, but is toxic

l IL-12 and GM-CSF are promising adjuvants for vaccines

D e n d r it icD e n d r it ic C e l l V a c c i n e C e l l V a c c i n e

A p p r o a c h e s A g a in s tA p p r o a c h e s A g a in s t

C a n c e rC a n c e rMichael Morse, MD

Assistant Professor of MedicineDuke University Medical Center

Durham, NC

Acknowledgments

l H. Kim Lyerly

l Paul Mosca

l Tim Clay

l Amy Hobeika

l Ian Cumming

l Michelle St. Peter

l Shubi Khan

l Tracey Kerby

l Terry Kruger

l Sharon Peplinski

l NCI– Jeff Schlom– Larry Kwak

l Immunex, Inc.– Dania Caron– Elaine Thomas

l Schering Plough, Inc.– Mary Ellen Ryback

l Coulter, Inc.– Pat Roth

Funding from the National Cancer Institute.

l Becton Dickinson– Skip Maino

l Duke– Eli Gilboa– Smita Nair– Dave

Boczkowski

I would like to thank the following fortheir contributions to this research:

Processing and presentation of class I antigens

Endoplasmic Reticulum

Golgi

Class I MHC and beta 2 micoglobulin synthesized

Protein synthesized

ProteinFragmented

Class I MHC bind peptides

MHC-peptide complex transported

Tumor Cell

MHC-peptide complex recognized byantigen specific T cells

Accessory molecules produced

Dendritic cell

Class II MHC-peptide

Peptide & Protein Vaccines

Protein/peptideDendritic cell

T cell

Adjuvant

(QS21, GM-CSF)

Peptide & Protein Vaccines

Vaccine Immune Response

Clinical Response Reference

gp100, tyrosinase +IL12 33/38 + ELISA

24/48 relapses at 20mo (mel.)

J Clin Oncol. 2001;19:3836-3847.

gp100+ tetanus tox + Montanide ISA-51

or QS-21 14% with gp100+

CTL

75% survival at 4.5yrs; (resected

melanoma) Clin Cancer Res.

2001;7:3012-3024.

gp100 , IL-2, IL-12

7/7 alone 4/5 with IL12 1/11 with IL-2

Greater tumor regression with

IL-2 J Immunol.

1999;163:6292-6300.

MAGE-3, PADRE, IFA

5/14 MAGE+ immunity

NR (resected mel.)

J Immunother. 1999;22:431-440.

MART-1 + IFA 13/25 DTH+

10/22 Elispot+ 9/25 relapsed at

16mo Clin Cancer Res.

1999;5:2756-2765.

HER2/neu helper peptides

14/18 with prolif to one Ag NR

J Clin Invest. 2001;107:477-484.

Idiotype + GM-CSF (lymphoma)

19/20 tumor+ T cells

8/11 became PCR- in PB

Nat Med. 1999;5:1171-1177.

Genetically

Modifiedor

Virally-infected

Tumor cell

IL-2

GM-CSF

HLA moleculeCostmulatory molecule

T cell

Monocyte

DC

Tumor Cell Vaccines

Viral proteins

Tumor Cell Vaccines

Vaccine Immune Response Clinical Response Reference

Melanoma transduced with Ad-GM-CSF 5/9 + CTL activity 1/9 minor response

Cancer Immunol Immunother.

2001;50:373-381.

Allogeneic panc. tumor secreting GM-CSF 3/14 DTH+

3 patients with DFS >25mo

J Clin Oncol. 2001;19:145-156.

CancerVax (melanoma)

82% + complmnt dependent cytotox

(CDC)

Median survival 54 mo, if deltaCDC >

10% Ann Surg Oncol. 1998;5:595-602.

Autologous colon CA + BCG

Increased DTH in all patients

No overall survival benefit

Vaccine 2001;19:2576-2582.

Autologous GBM+ Newcastle Virus

DTH increased from 1.67 to 4.05

cm2 Median survival was

46 weeks J Neurooncol.

2001;53:39-46.

Viral Vector & Plasmid Vaccines

DC

Expressed antigen

Muscle cell

Expressed antigen

Viral Vector & Plasmid Vaccines

Vaccine Immune Response Clinical

Response Reference

ALVAC-CEA B7.1

5/9 CEA+ T cell precursor frequency

0 PR; 4/23 with decreased CEA

Clin Cancer Res. 2001;7:1181-1191.

Vaccinia CEA/ avipox-CEA +GM-CSF

Greater immune responses than reverse order Minimal

J Clin Oncol. 2000;18:3964-

3973.

Vaccinia CEA No CEA+ responses

4/20 with stable disease

Clin Cancer Res. 1999;5:2330-2337.

PSMA/CD86 plasmid 67-100% DTH+ NR

Eur Urol. 2000;38:208-217.

Dendritic Cells

l Considered professional antigen-presenting cells

l Derived from bone marrow precursors, but multiplelineages can generate antigen-presenting cells

l Comprise approximately 0.3%-0.5% of the total circulatingperipheral blood mononuclear cells

l Clinical utility limited by low numbers of dendritic cells

Variables Associated with DC Vaccination

l Source- lineage– DC1 (myeloid, CD11c+, CD123 dim)

– DC2 (lymphoid, CD11c-, CD123 bright)

– CD8a in murine system

l Type of antigen loading

l Maturation and/or activation

l Route of administration

l Clinical evaluation– Clinical endpoints

– Immunologic endpoints

Clinical Sources of Dendritic Cells

l Peripheral blood- direct isolation

l CD34+ progenitors- BM or PBST

l Monocyte progenitors following in vitro expansion(GM-CSF and IL-4, etc.)

l Mobilization of progenitors (G-CSF, GM-CSF, G-CSF +GM-CSF, GM-CSF+ IL-4, Flt3L, or Flt3L-G-CSF)

l Converting non-DC to DC-like cells

Immature DC

o ↑↑ intracellular MHC

o ↑↑ endocytosis

o ↑↑ CCR1, CCR5, CCR6

o ↓↓ CCR7

o ↓↓ CD54, CD58, CD80, CD86

o ↓↓ CD40

o ↓↓ CD83

o Tolerize?

Mature DC

o ↑↑ surface MHC

o ↓↓ endocytosis

o ↓↓ CCR1, CCR5, CCR6

o ↑↑ CCR7

o ↑↑ CD54, CD58, CD80, CD86

o ↑↑ CD40, DC-LAMP

o ↑↑ CD83

o Activate

Cytokines- such as GM-CSF+IL-4, GM-CSF+IL-13,GM-CSF+TNFa, GM-CSF+IFNa, etc.

PathogensCytokines

T Cells

DC Precursors

Days to weeks

HoursHours

Antigen Loading of Dendritic Cells

l Peptides that can bind to MHC directly: – often defined, often class I, commonly HLA A*0201

(can use unfractionated peptides)

l Antigens that require intracellular processing:

– Protein (endocytosed and processed)

– DNA/RNA - (protein expressed and processed)

– Viral vectors- (protein expressed and processed)

– Cell lysate- (endocytosed and processed)

– DC - tumor cell fusions

– Apoptotic bodies, exosomes

Maturation of DC: Effects on Phenotypeand Antigen Loading & Processing

Morse M, et al. Cancer Res. 1998;58:2965-2968.

0

15

30

5

10

20

25

7.5:1 15:1 30:160:17.5:1 15:1 30:160:1

CE

A-s

pec

ific

lysi

s (

%)

Stimulators

DC+CEA RNA+TNF-aDC+TNF-a+CEA pepDC+TNF-a+CEA RNA

DC+CEA ep+TNF-a

CD80 CD83

CD80

Post-TNF-a treatment

Pre- TNF-a treatment

Cou

nts

0

200

CD83

101 103 104102100 101 103 104102100

101 103 104102100 101 103 104102100

Cou

nts

0

200C

oun

ts

0

200

Cou

nts

0

200

Mosca RJ, et.al. Blood. 2000;96:3499-3504.

LPS CD40L/IFN-y CD40L/IFN-y/LPSCD83

IL-12

CD40L IFN-y TNF-ααAim V Only

Only a Subset of CD83+ DCcan be Matured to Produce IL-12

Intravenous Administration ofIn 111-labeled DC

Adapted from Fig 1. Morse MA, et al. Cancer Res. 1999;59:56-58.

Subcutaneous & Intradermal Administrationof In 111-labeled DC

Adapted from Fig 2. Morse MA, et al. Cancer Res. 1999;59:56-58.

Subcutaneous Intradermal

Lymph node

Tumor Antigens for Clinical Trials

l Lymphoma—targeting Id protein

l Melanoma—targeting melanoma antigens- Mage-1, Mart,gp 100, Mage-3

l GI—targeting CEA, ras, p53

l Breast—targeting CEA, Her-2/neu, p53

l Prostate—targeting PSA, PSA-M

l Lung- targeting CEA, Mage-3

l Telomerase

Recognition of Malignant Myeloma Cells byId Specific T cells

Li Y, et.al. Blood. 2000;96:2828-2833.

ScreenPheresisIn vitro generation of DCAdministration of DCRepeat pheresis

0 1 2 3 4 5 6 7 8

X

XX

X X X X

X

ScreenEntryLeukapheresis

DCInjection

DCInjection

DCInjection

DCInjection

Direct comparison ofpre- and post-

immunization bloodsamples

Study Week

Phase I Trial of DC in Patients withMetastatic Disease

Clinical Responses

l Tumor response/shrinkage

l Tumor marker response- ?PSA, etc.

l Other clinical markers- ? vitiligo

l Delay in time to progression

l Delay in time to recurrence

l Increase in overall survival

l Adverse effects — ? autoimmunity

Immunologic Responses

In Vivo

l DTH

l Vitiligo

l Tumor infiltration

In Vitro

l T cell proliferation

l T cell cytokine release

l ELISPOT (cytokine release)

l Intracellular cytokine

l Peptide-MHC tetramer

l Cytotoxic T-lymphocytefunction

l CTLp frequency

Magnitude and Duration of Antigen-SpecificImmune Response May Affect Clinical Response

“Resting Immune Response

CTLp 1 in 5,000 by LDA”

“Clinically effective” Cell response

“Clinically ineffective”Cell responseDid not achieve sufficientfrequency of antigen-specificT cells

Did not achieve Sufficient duration of

Antigen-specificT cells

Immunotherapy

Time (Months)

Cel

lula

r Im

mu

ne

Fun

ctio

n

Results of Phase I Study of DC Loadedwith CEA Peptide (CAP-1)

l Safe and feasible to administer up to 1 x 10E8 DCIV

l 1 minor response

l T cell infiltration of DC injection sites butminimal DTH response

l Minimal detectable CEA specific T cell activityusing CTL activity as read-out

0

5000000

10000000

15000000

20000000

25000000

5 7 9 11 13 15

EBV 0.1 µg/ml

EBV 0.01 µg/ml

Flu-MP 0.1 µg/ml

Flu-MP 0.01 µg/ml

Nu

mb

er o

f C

ells

Days in Culture

Direct Assessment of Peripheral Blood ImmuneFunction Minimizes In Vitro Artefact

In Vitro Expansion of Antigen-specific T cells

(EBV specific CD8+ T cells)

ELISPOT Response After Vaccination withDC Based Vaccines

Normal Donor PBMC (EBV+)ELISPOT Analysis

Antigen

IaCu (Tetramer #7) ELISPOT(MSPs values)

0

50

100

150

200

250

300

PWM EBV CMV Mart-1 Media w/cells

Media w/ocells

Well 1Well 2Well 3Well 4Well 5Well 6Mean

IFN

g se

cret

ing

spot

s/20

0KR

esp

ond

ers

Peptide-MHC Tetramer Detection of EBV specificCD8+ T cells in PBMC EBV+

Antigen specificT cell

CMV EBV Mart-1

CD8

Tet

ram

er

0.02% 0.47% 0.01%

TCR

PE Labeled, peptideMHC tetramer

CytotoxicT cellCD8+

TCR

0.03% 0.07%

0.08% 6.12%

untreated CMV 1 µg/ml

Mart-1 1 µg/ml PWM 5 µg/ml

0.86%

EBV 1 µg/ml

IFNγγ

CD69

Intra-cellular Cytokine Staining of Antigen-specific T cells in PBMC EBV+ (CD8+ T cells)

0.03 %

0.05 %

0.05 %

0.41 %

IFN-γγ

CD69

Post-VaccinationPre-Vaccination

Tet Tox

HBVc HBVc

Tet Tox

Intracellular IFN-gamma Analysis of Vaccine Response

% o

f T

Cel

ls

CD

69+

/ IN

F- γγ

+

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 1 2 3 4

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 1 2 3 4

CD8

CD4

Intra-cellular IFN-g Analysis of CD4+ and CD8+ T cells in PBMC

Tet Tox Cntrl

% o

f T

Cel

ls C

D69

+/ I

NF

- γγ+

Wks

Wks

vacc

ine

vacc

ine

CD4+Response

CD8+Response

Phase I Trials of Her2/neu ICD Protein-pulsed DCin Patients with Metastatic Disease

ScreenPheresisIn vitro generation of DCAdministration of DCRepeat pheresis

0 1 2 3 6 9 12 13

X

XX

X X X X

X

ScreenEntryLeukapheresis

FreshDC

InjectionDC

InjectionDC

InjectionDC

Injection

Direct comparison of pre-and post immunization

blood samples

Study Week

Time of analysis 0 1 3 106

Generation of Her2/neu ICD specific T cells In Vitrowith Her2/neu ICD Protein-pulsed DC

5:1 10:1 20:1 40:10

5

10

15

20

25

30

5:1 10:1 20:1 40:10

5

10

15

20

25

30Experiment #1 Experiment #2

Effector to target ratio

Exp #1 and #2: Targets

l = DC+CMV lysate

n = DC+HER2/neu ICD

7.5:1 15:1 30:1 60:10

5

10

15

20

25

30

35

7.5:1 15:1 30:1 60:1

TargetDC+Her2/neu ICD protein

TargetDC+varicella lysate

Effector to target ratio

Experiment #3

Exp #3: Stimulators

l = DC+varicella lysate

n = DC+HER2/neu ICD

0

10

20

30

40

50

60

PHA Tet Tox KLH HER2/ neuICD

CMV CMVc Media

ELISPOT Response after Vaccination withHer2/neu ICD DC Vaccines

Pre-immunization Her2/neu ICD= 7 in 100,000 PBMC

0

20

40

60

80

100

120

PHA Tet Tox KLH HER2/ neuICD

CMV CMVc Media

ELISPOT Response after Vaccination withHer2/neu ICD DC Vaccines

Post immunization Her2/neu ICD= 22 in 100,000 PBMC

Spo

ts p

er 6

7,00

0 C

ells

Spo

ts p

er 6

7,00

0 C

ells

0

20

40

60

80

-2 0 1 3 5 9

0

20

40

60

80

-2 0 1 3 5 9

Flt3L-mobilized DC-ELISPOT ResultsFlt3L-mobilized DC-ELISPOT Results

Tet Tox Cntrl

KLH Cntrl

Spo

ts p

er 6

7,00

0 C

ells

Spo

ts p

er 6

7,00

0 C

ells

Wks

Wks

Flt3L

vacc

ine

Flt3L

vacc

ine

% o

f CD

4s C

D69

+/ I

NF

- γγ+

0

0.1

0.2

0.3

0.4

0.5

-2 0 1 3 5 9

0

0.1

0.2

0.3

0.4

0.5

-2 0 1 3 5 9

Intra-cellular IFN-gamma Analysis of

PBMC after Flt3L DCImmunization

Tet Tox Cntrl

KLH Cntrl%

of C

D4s

CD

69+/

IN

F-

γ+γ+

Wks

Wks

Flt3L

vacc

ine

Flt3L

vacc

ine

t s p e r 1 0 0 , 0 0 0B M C

0

20

40

60

80

100

120

- 2 0 1 3 6 10

No Ag

0

20

40

60

80

10 0

12 0

- 2 0 1 3 6 1 0

Tet To x

0

20

40

60

80

1 00

1 20

- 2 0 1 3 6 10

K L H

0

20

40

60

80

10 0

12 0

- 2 0 1 3 6 1 0

Cap- 1

Inj .: #1 #2 #3 #4(W k. 9)

IFN- γ EL ISP O T

In j.: #1 #2 #3 #4( Wk. 9)

F l t-3L Fl t-3L

SP

OT

S

WeekWeek

Dendritic Cell Vaccines

Vaccine Immune Response

Clinical Response Reference

CD34 + DC +gp100 MART -1, MAGE-3,

tyrosinase, gp100 16/18 with Ag+ T cell activity

Regression of >1 melanoma met in 7/18

Cancer Res. 2001;61:6451.

DC+ MAGE-3 peptide (GI tumors)

4/8 MAGE-specific CTL

3/12 with minor responses

Clin Can Res. 2001;7:2277.

Flt3L mobilized DC + CAP1-6D (CEA+)

5/12 tetramer+ T cells

2/12 PR, 2/12 M R

PNAS. 2001;98:8809.

DC+ MAGE1,3 12/24 IFN-g

secreting PBL 5/17 with stable

disease J Leukoc Biol. 2001;69:937.

DC + tumor RNA, KLH (colon CA)

11/13 KLH+ responses

7/13 decreased CEA; 0 PRs

Hepatogastro. 2001;48:347.

DC + mouse PAP (prostate Ca)

100% IFN-g+ T cells N R

J Immunol. 2001;166:4254.

Dendritic Cell Vaccines (continued)

V a c c i n e I m m u n e

R e s p o n s e Cl in ica l

R e s p o n s e Reference

D C + g l i o m a p e p t i d e s 4 / 7 w i t h A g -spec i f i c CTL

T cel l inf i l t ra t ion in to tumor of 2 /4

C a n c e r R e s . 2 0 0 1 ; 6 1 : 8 4 2 -8 4 7 .

D C + M A G E 3 ( b l a d d e r C a ) 1 pat ient evaluable 3 / 4 w i t h P R

Cl in Cancer Res . 2001 ;7 :23 -3 1 .

B lo o d D C + P A P / G M -C S F f u s i o n

3 8 % i m m u n e r e s p o n s t o P A P

6 w i t h P S A dec l ine

J C l in Onco l . 2 0 0 0 ; 1 8 : 3 8 9 4 .

M a t u r e D C p l u s M A G E -3

8 / 8 M A G E + E L I S P O T

1/8 s table d isease ( m e l a n o m a )

J I m m u n o l . 2 0 0 0 ; 1 6 5 : 3 4 9 2 -

3 4 9 6 . D C + M A R T -1 a n d

G P -100 iv 1 /5 w i th Ag

spec i f i c CTL 1 /7 P R J I m m u n o t h e r .

2 0 0 0 ; 2 3 : 4 8 7 -4 9 8 .

C D 3 4 + D C + I d prote in

4 /10 Id -spec i f ic T ce l l responses

1 /10 dec reased B M p l a s m a c e l l s

B r J H a e m a t o l . 2 0 0 0 ; 1 0 8 : 8 0 5 -

8 1 6 .

D C + I d ( m y e l o m a ) 2 4 / 2 6 K L H + 4 / 2 6

Id+ T ce l l s 17 /26 a l ive a t 30 m o p o s t A B M T

Bio l B lood M a r r o w T r a nspl . 2 0 0 0 ; 6 : 6 2 1-6 2 7 .

Induction and Expansion of Antigen-specificImmune Response

0.01%

“Resting Immune Response”

“Clinically Ineffective”Immunotherapy

Time (Months)

Cel

lula

r Im

mu

ne

Fun

ctio

n “Clinically Effective”

“Responding Immune Response”

Expa

nsio

n“Clinically Detectable”

67%

0.51%

P a n e l D i s c u s s i o nP a n e l D i s c u s s i o n

Q u e s t i o n / A n s w e rQ u e s t i o n / A n s w e r