Jeffrey Schlom, Ph.D.Laboratory of Tumor Immunology and BiologyCenter for Cancer ResearchNational Cancer Institute, NIH

Development of Recombinant Vaccines for the Therapy of Carcinomas

Monotherapy and Combination Therapy

STRATEGIC PLAN

Immunologic Platform:

– Combination immune therapies immune stimulation strategies reduction of immune inhibitory entities

– Combination Therapies: Vaccine plus: conventional therapies conventional therapies in novel strategies other experimental therapies

Cancer Vaccine Development:– Focus on human carcinoma– Focus on development of vaccines that can be widely evaluated

Ultimate Use:– Early in disease process/low tumor burden– Survival as the endpoint– Minimal toxicity

Jorge CarrasquilloC.H. Park

Translational Research Programmatic EffortPRECLINICAL STUDIES:

Laboratory of Tumor Immunologyand Biology (LTIB)

James HodgeAl TsangClaudia PalenaJack Greiner

Connie RogersBenedetto FarsaciSofia GameiroMatteo VergatiMary LitzingerKen Hance

Laboratory of Molecular Biology

Vaccine Branch

Ira Pastan

Jay Berzofsky

CLINICAL STUDIES:

LTIB/Medical Oncology BranchJames GulleyPhilip ArlenRavi MadanMary Pazdur

Medical Oncology Branch_______

William Dahut Tito FojoWilliam Figg

Radiation Oncology____

Urologic Oncology_______________Kevin Camphausen

Marston Linehan Peter Pinto

Biostatistics and Data Management SectionSeth Steinberg

NIH Nuclear Medicine

Translational Research Programmatic Effort

CLINICAL STUDIES — EXTRAMURAL:Georgetown – John MarshallDana Farber Cancer Center – Donald Kufe, Paul Eder, Philip KantoffColumbia – Howard KaufmanCancer Institute of New Jersey – Edward Lattime, Robert DiPaolaOhio State – William Carson

Eastern Cooperative Oncology Group (ECOG) – Robert DiPaola, Howard Kaufman, Louis Weiner

PRIVATE SECTOR:• GlobeImmune – Alex Franzusoff, David Apelian• BN ImmunoTherapeutics – Wayne Godfrey, Reiner Laus

CANCER THERAPY EVALUATION PROGRAM (CTEP):Howard Streicher Jan Casadei

NCI Technology Transfer Center: Kevin Brand, Karen MaureyNIH Office of Technology Transfer: Mojdeh Bahar

Strategies to Enhance Vaccine Potency

1. Mode of Delivery of the Vaccine– place the gene for the tumor antigen into a vector

3. T-cell Costimulation– these molecules are essential for vigorous T-cell

activation– place costimulatory molecule into vaccine

vector

4. Alter the a.a. sequence of the tumor antigen to enhance the immune response “epitope enhancement”

2. Diversified Vaccine Prime and Boost

5. Combination therapies

Vaccine Platforms

Recombinant poxviruses• vaccinia; (MVA)• fowlpox

Recombinant saccharomyces (yeast)

Chitosan / nanoparticles

• Pox vectorsVaccinia (rV-) elicits a strong immune response

– host induced immunity limits its continuous use– MVA (replication defective)

Avipox (fowlpox rF-, ALVAC)– derived from avian species– safe; does not replicate– can be used repeatedly with little if any host neutralizing

immunity

• Can insert multiple transgenes

• Do not integrate into host DNA

• Efficiently infect antigen presenting cells including dendritic cells

Recombinant Vaccine Vectors

Major Costimulatory Effect must be on the T-cell No Overlap of T-cell Ligands No Redundancy of Costimulatory Mechanisms

Costimulatory Molecule Candidates:

CD2(Region 1)

CD28CTLA-4

Ligand

TCRT-Cell

LFA-1

Tyrosine Kinase,Ca2+ MobilizationcAMP Production

IL-2-R upregulation,IL-2 secretion

CostimulatoryMechanism

Tyrosine Kinase,Phospholipase C

LFA-3(CD58)

B7-1(CD80)

CostimulatoryMolecule

APC

ICAM-1(CD54)

MHC + Peptide

Costimulatory MoleculeT-

cell

Act

ivat

ion

(CPM

x 1

05 )None LFA-3 ICAM-1 B7-1 TRICOM

TRICOMTRIad of COstimulatory Molecules

TRICOM = B7-1/ICAM-1/LFA-3

CEA/TRICOM = CEA/B7-1/ICAM-1/LFA-3

CEA/MUC-1/TRICOM = CEA/MUC-1/B7-1/ICAM-1/LFA-3 (PANVAC)

PSA/TRICOM = PSA/B7-1/ICAM-1/LFA-3 (PROSTVAC)

All vaccines contain: rV- as a prime vaccine avipox (fowlpox, rF-) as multiple booster vaccines

CEA, MUC-1, and PSA transgenes all contain enhancer agonist epitopes

Costimulatory Molecule Ligand on T cell

B7-1 (CD80) CD28/CTLA-4

ICAM-1 (CD54) LFA-1

LFA-3 (CD58) CD2

CEA-specific Lymphoproliferation of T Cells from CEA-Tg Mice Vaccinated with TRICOM Vectors

0

2000

4000

6000

8000

020406080

[CEA], (g/ml)

0

100000

200000

300000

400000 Ovalbumin

Con-A

Prol

ifer

atio

n (C

PM)

CEA/TRICOM

CEA/B7-1

CEA

TRICOM

None

VAAA Regimen

CEA

CEA/B7-1

CEA/TRICOM

All groups withGM-CSF and

low dose IL-2

TRICOM

Aarts WM, Schlom J, Hodge JW. Cancer Res. 62:5770-7, 2002

Therapy of 14-Day Established CEA+ Experimental Metastases in CEA-Tg Mice Using CEA/TRICOM Vectors

VaccineTumor

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Weeks Post Tumor Transplant

% S

urvi

val

VAAA Regimen

CEA

CEA/TRICOM

All groups withGM-CSF and

low dose IL-2

CEA/TRICOM

CEA

Buffer Control

(P<0.02 vs CEA)

Aarts WM, Schlom J, Hodge JW. Cancer Res. 62:5770-7, 2002

The Next Frontier: Combinatorial Therapies

The use of cancer vaccines in combination with conventional therapies

Chemotherapy Hormone therapy Local radiotherapy of tumor Small molecule targeted therapeutics

Vaccine Combination Therapies

1. Vaccines Induce Minimal Toxicity – can act independently of concomitant therapy

2. Do NOT confusemultiple therapies used prior to vaccine

vs.therapies used with vaccine or following vaccine

Vaccine Combination Therapies

3. The vaccine induction of a dynamic host immune response can be boosted by

– concomitant or subsequent therapies

(a) can alter the phenotype of tumor cells, rendering them more susceptible to T-cell killing

(b) can lyse populations of tumor cells which, in turn, act as a booster for tumor-specific immune cells

(c) can kill or inhibit regulatory T cells and thus boost the immune response

16Hodge et al, Oncology 2008

Potential Multiple Effects of Local Irradiation of Tumors

Combination Therapy: Vaccine + External Beam Radiation

Tumor(MC38- CEA+ SQ)

0Day

V1

8

rV-CEA/TRICOMrF-GM-CSF

15

V2

22

V3

29

V4

rf-CEA/TRICOMrF-GM-CSF

14

8 Gy (2 Gy x 4)

Chakraborty M, Abrams SI, …, Schlom J, Hodge JW. Cancer Res. 15:4328-37, 2004

% Fas +

.

Days Post Tumor Transplant

0 7 14 21 28 35 42 49 0 7 14 21 28 35 42 49 0 7 14 21 28 35 42 49

0

100

200

300

400

500

n=5n=0 n=0n=0

No Treatment Vaccine + IrradiationVaccine Irradiationn=12 n=13 n=9n=1211 (41)12 (36) 54 (415)66 (435)

0 7 14 21 28 35 42 49

Tum

or v

olum

e (m

m3 )

FAS-L FAS

CD8+

T-cellTarget

Cell

Radiation-Enhanced Antigen-Specific Lysis of Tumor Cells

Radiation

101

103

102

104

101 103102 104100 101 103102 104100 101 103102 104100 101 103102 104100 101 103102 104100

CEA-FITC

Fas

-PE

Fas

Iso

typ

eH

&E

His

toch

emis

try

0 Day 11 Day7 Day4 Day2 Day

Time Post Tumor Irradiation (8 Gy)

5 870 5

5 800 15

6 340 60

80 89

2 100 88

3

Persistence of Fas Upregulation on MC38-CEA+ Tumors After External-Beam Irradiation

153Sm is currently FDA approved and clinically utilized for palliation of bone metastasis in multiple tumor histologies.

QUADRAMET is a therapeutic agent consisting of radioactive samarium (153Sm) and chelator.

It preferentially binds to osteoblastic metastatic tumor deposits in bone.

Low Dose Radiation (25 Gy) ofLnCaP Human Prostate Cell Line

Tumor Antigen Genes 0 Gy 25 Gy PSA 1 2.79

PSMA 1 4.14 PAP 1 29.0

CEA 1 10.3 MUC-1 1 3.67

101 102 103 104100

FA

SM

HC

-I

0 Gy

25 gy

0 Gy25 gy

0 Gy 25 gy

MFI

Cel

l Num

ber

Treatment of LnCaP prostate cancer cells with low dose radiation results in the upregulation of MHC and Fas

Gene Expression in LnCaP cells RT-PCR

Treatment of LnCaP Prostate Cells with Palliative Levels of 153Sm (Quadramet) Modulates Phenotype, Upregulates TAA, and

Increases Sensitivity to Antigen-specific CTL Killing

101 102 103 104100

FA

SM

HC

-I

0 Gy

25 gy

0 Gy25 gy

0 Gy 25 gy

MFI

Cel

l N

umbe

r

Treatment of LnCaP prostate cancer cells with Palliative doses of 153Sm results in the upregulation of MHC class I and Fas

Treatment of LnCaP prostate cancer cells with Palliative doses of 153Sm results in increased sensitivity to multiple CTLs

Treatment of LnCaP prostate cancer cells with Palliative doses of 153Sm results in the upregulation of TAAs

Gene Expression in LNCaP cells after Sm-153 treatment Accessory Genes 0 Gy 25 Gy Fas 1 1.96 ICAM-1 1 29.1 Tumor Antigen Genes 0 Gy 25 Gy PSA 1 2.79 MUC-1 1 3.67

.

0

20

40

60

80

100

Day 4 Sm-153 delivered Dose (Gy)

0 (Mock) 25 50

p<0.001 p<0.001

CTL: PSA-Specific

% L

ysis

% L

ysis

.

0

10

20

30

40

50

Day 4 Sm-153 delivered D ose (Gy)

0 (Mock) 25 50

p<0.001

CTL: MUC-1-Specific

Chakraborty, Wansley…Schlom, Hodge, NCI, Clin Cancer Res., 2008. Collaboration with Nuclear Medicine Branch.

PSA-TRICOM + 153Sm

RANDOMIZE

Arm A: PSA-TRICOM + 153Sm (n=34)

Arm B: 153Sm (n=34)

Vaccine: rV-PSA/TRICOM s.c. d 1rF-PSA/TRICOM s.c. d 15, 29, q 4 wksAll vaccines given with GM-CSF 100μg s.c. x 4 d

153Sm: 1 mCi/kg d 8, may be repeated q 12 wks upon hematologic recovery.

Patient Population: Metastatic Androgen Independent Prostate Cancer

PI Gulley NCI# 7678

Effect of the pan Bcl-2 Inhibitor GX15-070 on the Immune System:

Preclinical Studies

Activated mature CD8 T lymphocytes are more resistant to GX15-070 than very early activated

1/29/2009 Benedetto Farsaci 25

A ○ 24h

▲ 72hB ○ 24h

▲ 72hDensitometry 1 0.33

0 1GX µM

IP: MCL1IB: BAK

IP: MCL1IB: MCL1

C

D ○ 24h

▲ 72hE

○24h▲ 72h Densitometry 1 0.76

0 1GX µM

IP: MCL1IB: BAK

IP: MCL1IB: MCL1

F

Very early activated

CD8 T lymphocytes

Activated mature CD8

T lymphocytes

- NON SELF-CEA LUNG TUMOR MODEL (C57BL/6 MICE)

- SELF-CEA LUNG TUMOR MODEL (CEA-TG MICE)

Assays

• IFN-γ production from splenocyte bulk cultures

• Pulmonary tumor nodules count

26

Experimental setup• Mice: C57BL/6 or CEA-Tg, female• Tumor cells: LL2-CEA 3x105 cells/muse (i.v.)• Type of tumor model: Pulmonary tumor nodules• Vaccine prime: PFU 1x108 rV CEA-TRICOM + PFU 1x107 rF GM-CSF /

mouse (s.c.)• Vaccine boost: PFU 1x108 rF CEA-TRICOM + PFU 1x107 rF GM-CSF /

mouse (s.c.)• Inhibitor: GX15-070 0.5 or 2 mg/Kg/mouse (i.v.)• Groups (6 mice/group):

1. No treatment2. GX15-070 alone3. Vaccine alone4. Vaccine + GX15-070

1/29/2009 Benedetto Farsaci

Day 0

Tumor cell injection

Day 12

Vaccine boost

Day 19

GX15-070

Day 23Day 35

Sacrifice

Day 5

Vaccine prime

GX15-070

Pulmonary tumor meta-analysis

1/29/2009 Benedetto Farsaci 27

n = 13 n = 20

n = 12

n = 21

No treatment GX aloneVaccine alone Vaccine + GX

P < 0.001*

P = 0.027*P = 0.531

P = 0.044*P = 0.030*

P < 0.001*

Closed symbols: CEA-Tg mice. Open symbols: C57BL/6 mice. * = Statistical significance from two-tailed Mann-Whitney test, 95% confidence interval.

Closed symbols: CEA-Tg mice. Open symbols: C57BL/6 mice. * = Statistical significance from two-tailed Mann-Whitney test, 95% confidence interval.

GX15-070 inhibits Treg function

1/29/2009 28

p = 0.002

CD8 alone

CD8 + Untreated

Tregs

CD8 + GX-treated

Tregs

p = 0.002

p = 0.002

p = 0.002

NP68 µg/mL

10-4

CD8:Treg ratio

10-4

10-5

10-5

1:1

1:2

1:1

1:2

Ability of Docetaxel to Alter Tumor-Cell Phenotype:Enhanced Sensitivity to Antigen-Specific T-Cell Lysis

No treatment Taxotere

Fas-PE

MHC 1-PE

100 101 102 103 104Fas-PE

M1

30.54%(37)

100 101 102 103 104

Fas-PE

M1

90.36%(196)

100 101 102 103 104Db-PE

M1

99.51%(117)

100 101 102 103 104Db-PE

M1

67.68%(721)

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100:1 Media

E:T

% L

ysis

0 ng/ml Tax

2.5 ng/ml Tax25 ng/ml Tax

250 ng/ml Tax

Tumor cell (MC38) Lysisby CEA-Specific T Cells

Garnett, Schlom, Hodge, Clin Cancer Res., 2008

Docetaxel +/- PANVAC

RANDOMIZE

Arm A: Weekly Docetaxel+ PANVAC (rV, rF-CEA-MUC-1-TRICOM)

Arm B: Docetaxel alone

Patient Population: Metastatic Breast Cancer (Docetaxel Naïve) n=48

Primary endpoint: TTP

Preliminary Data: 14 patients enrolledArm A Arm B

Median TTP 10.5 months 2 months# on >6 months 5/6 (1 too early) 1/7

Prostate Cancer Vaccine Program

Prostate Cancer and Vaccine Therapy

• Long interval from primary diagnosis to metastaticdisease

• Serum PSA (doubling time/velocity) as a surrogate for therapeutic benefit or disease recurrence

• Nomogram (Halabi) at metastatic disease– can predict more indolent vs more aggressive

disease

Vaccine/Androgen Receptor Antagonist Therapy

RANDOMIZE

Arm A: Vaccine* (n=21)rV-PSA + rV-B7-1 prime, rF-PSA boosts monthly IL-2 low dose x 5 days, recombinant GM-CSF x 4 days

Arm B: Nilutamide* (n=21)(Androgen Receptor Antagonist)

*If patient progressed by PSA but still NED radiographically, they could add in the therapy of the other arm

Patient Population: Androgen Independent Prostate Cancer with Rising PSA and No Radiographic Evidence

of Disease (D = 0.5)

Arlen et al. J. Urol. 174:539-46, 2005

Time to Treatment FailureRegimen n Median time to treatment failure

Vaccine 21 9.9 months

Nilutamide 21 7.6 months

Time to Treatment Failure After Cross-OverRegimen n Median time to treatment failure

Vaccine Vaccine + nilutamide 12 13.9 months (after cross-over)*25.9 months (from initiation of therapy)

Nilutamide Nilutamide + vaccine 8 5.2 months (after cross-over)

15.5 months (from initiation of therapy)

* Median time to cross over was 12.0 months

Overall Survival: Randomized Trial in Patients with Nonmetastatic HRPC Receiving Vaccine (rV-PSA/B7.1, rF-PSA) vs. Androgen Receptor

Antagonist (Nilutamide) with Crossover at Progression

At progression, patients continued initial therapy and crossed over to also receive other therapy.

Five-Year Overall Survival:38%: Nilutamide first59%: Vaccine first

0

25

50

75

100

% S

urvi

val

12 24 36 48 60 720Months (from diagnosis date)

(n = 21)

(n = 21)

Vaccine +/– Nilutamide

Nilutamide +/– Vaccine

Madan, Gulley, Schlom et al.Clin. Cancer Res. 14:4526-4531, 2008

Randomized Multicenter Placebo-controlled Vaccine Therapy Trial in Castrate-resistant

Metastatic Prostate Cancer PatientsPatients (n = 125)

– Metastatic prostate cancer (CT or bone scan +)– Gleason score ≤ 7; no visceral disease– Chemotherapy naïve

Vaccine: rV, rF-PSA-TRICOM (PROSTVAC) + GM-CSF

Control arm: empty vector

Randomization: 2:1 (double blind)

P.I.: P. Kantoff, Dana-Farber Cancer Center

Analyses: W. Godfrey, BNITB. Blumenstein, statistician

Kantoff PW, Schuetz TJ, Blumenstein BA, Glode LM, Billhartz DL, Gulley JL, Schlom J, Laus R, Godfrey WR. Overall survival (OS) analysis of a phase II randomized controlled trial (RCT) of a poxviral-based PSA targeted immunotherapy in metastatic castration-resistant prostate cancer (mCRPC). 2009 ASCO Annual Meeting, Orlando, FL, May 29-June 2, 2009.

0.006

Randomized Multicenter Placebo-controlled Vaccine Therapy Trial in Castrate-resistant

Metastatic Prostate Cancer Patients

Observations:

A. Time to Progression: no difference in arms

B. Median survival at 4 yearsPlacebo: 16.6 monthsVaccine: 25.1 months (p=0.006)

C. 40% reduction in death rate in vaccine arm

Phase III Trial Planned

Overall survival analysis of a Phase II study of PSA-TRICOM in the treatment of

metastatic, castrate-resistant prostate cancer

Ravi A. Madan1, James L. Gulley1, William L. Dahut2, Kwong Y. Tsang1, Seth M. Steinberg3, Jeffrey Schlom1 and Philip M. Arlen1

1Laboratory of Tumor Immunology and Biology, 2Medical Oncology Branch, and 3Biostatistics and Data Management Section, Center for Cancer

Research, National Cancer Institute, NIH, Bethesda, Maryland

HalabiP

redicted S

urvival of ≥18 M

onthsH

alabiPred

icted Survival of <

18 Months

Pat

ient

Num

ber

0 10 20 30 40 50

Months From On-Study Date

123456789

101112131415

n=15

123456789

1011121314151617

n=17

Halabi Predicted Survival

Survival greater than Predicted

Survival less than Predicted

StillAlive

6 7

31 8

13 16 10

9 22 21 28

5 30 14 15

2 18

23 32 24 17 19 25

4 11 20

1 27 26 12 29

3

Predicted vs. Actual Survival of PSA-TRICOM PatientsUpdated with March 10, 2008 data

Actual survival

Halabi predicted survival

PERC

ENT SU

RVIVAL

Actual survival (*) compared with the Halabi predicted survival (o).

Median Overall SurvivalPhase III

Mitoxantrone 16.4 moDocetaxel (weekly) 17.3 mo Δ 0.9 moDocetaxel (3 weekly) 18.9 mo Δ 2.5 mo

NCI Phase IIDocetaxel (HPS 16.5 mo) 15.5 mo (Δ 1.0 mo)

Randomized Phase IIVector control 16.3 mo PSA-TRICOM (p = 0.006) 24.4 mo Δ 8.1 mo

(HR = 0.6)

NCI Phase IIPSA-TRICOM 26.6 mo

(HPS 17.4 mo) Δ 9.2 mo

(ave HPS 12.3 mo) 14.6 mo Δ 2.2 mo(ave HPS 20.9 mo) ≥37.3 mo Δ ≥16.4 mo

Phase II/III Trials: Metastatic Prostate Cancer

Vaccine Combination Therapies

The vaccine induction of a dynamic host immune response can be boosted by

– concomitant or subsequent therapies

(a) can alter the phenotype of tumor cells, rendering them more susceptible to T-cell killing

(b) can lyse populations of tumor cells which, in turn, act as a booster for tumor-specific immune cells

(c) can kill or inhibit regulatory T cells and thus boost the immune response