gene therapy of liver cancer: experience from clinical trials · viral vectors for gene transfer...

Division of Division of HepatologyHepatology and Gene Therapy. and Gene Therapy. Center for Applied Medical Research (CIMA)Center for Applied Medical Research (CIMA)

University Clinic. University of University Clinic. University of NavarraNavarra

Gene Therapy of liver cancer:Experience from clinical trials

Falk SymposiumFreiburg

October 2006

Viral Vectors for Gene TransferViral Vectors for Gene Transfer

Short-term expression vectors• First generation adenovirus

Replicative cytopathic vectors• Conditioned replicating adenoviruses

Long-term expression vectors• Gutless or helper-dependent adenoviruses• Retrovirus (lentivirus)• Adeno-associated viruses (AAV)

Gene Gene Therapy of CancerTherapy of Cancer

Tumor cell

reversing thereversing themalignant phenotypemalignant phenotype

Interfering with Interfering with tumor tumor biologybiology::

-- antiangiogenic interventionantiangiogenic intervention-- blocking survival signalsblocking survival signals

geneticgeneticimmunotherapyimmunotherapy

molecularmolecularchemotherapychemotherapy

Treatment of established HCC with Ad.IL-12

Before treatment Ad.lacZ

Ad.IL-12

Barajas et al HEPATOLOGY 2000

University of University of Navarra Navarra Clinical Trial ofClinical Trial of Ad.IL12 in GI Ad.IL12 in GI TumorsTumors

•• PhasePhase II, non, non--controlled designcontrolled design•• 21 21 patients with advancedpatients with advanced GI GI tumorstumors

–– HepatocellularHepatocellular Carcinoma, Carcinoma, Liver Metastases of Liver Metastases of Colorectal Cancer or Pancreatic CancerColorectal Cancer or Pancreatic Cancer

•• Intratumor injectionIntratumor injection ofof Ad.IL12 (1stAd.IL12 (1st--generationgeneration, , nonnon--replicativereplicative adenovirusadenovirus

•• 3 3 monthly injections of the same dosemonthly injections of the same dose•• Dose escalation fromDose escalation from 2.52.5··10101010 vp tovp to 33··10101212 vpvp

• Main goals: feasibility and safetySangro et al Journal Clinical Oncology, 2004

EchoEcho--guidedguided gene gene therapy of therapy of HCC HCC withwith Ad.ILAd.IL--12:12:Needle at the edge of the lesionNeedle at the edge of the lesion

Journal Clinical Oncology, 2004

día 0 día 1 día 3 día 7 día 10

0

50

100

150

200

< 1012 vp≥ 1012 vp

Ch a

n ges

aft e

r th e

rap y

(pg /

ml ) *

*

* p < 0.05 vs. day 0

IL12/00/001 IL12/00/001 IFNIFN--γ γ InductionInduction


IL12/00/001 IL12/00/001 Tumor Infiltration by Tumor Infiltration by CD8+ T CD8+ T CellsCells

Patient 09/ Tumor: HCC/ Vector dose: 2.5·1011 pv

Before Therapy After Therapy


IL12/00/001:IL12/00/001:antitumorantitumor response response Dosestep 1 2 3 4 5 6 7

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

Tumor P H P C C C P H H H H C P C H H P P H Cc P

Response S Pr Pr S S Pr S P S S Pr Pr Pr Pr Pr Pr Pr S Pr

Status D A D D D D D A D A D D D D D D D D D D D

Numberof doses 1 3 2 2 3 3 3 3 3 3 3 1 1 1 3 2 1 1 1 1 1

Tumor - P: pancreas; H: hepatocellular carcinoma; C: colorectal;Cc: cholangiocarc.Respuesta - Pr: progression; S: stabilization; P: partial responseStatus - D: death; A: alive


IL12/00/001: IL12/00/001: antitumorantitumor responseresponse((patient 09 with HCC; retroperitoneal metastatic lymph nodes))

Before therapy After 3 doses of Ad-IL12


Gene therapy of HCC with Adenovirus encoding thymidin kinase (HSV-tk)

- Phase I/II

-Dose escalation (1011- 3x1012 vp)

- 3 monthly intratumoral injections

GENE THERAPY WITH ADENOVIRAL VECTORS CONTAINING THYMIDIN-KINASE

2 days

GCVx 14 days

Antitumor Antitumor effecteffect

Necrosis, apoptosis, inflammation

Ad-tk

AdlacZ Adtk

PET monitoring of PET monitoring of tktk activityactivity ((using using 1818FHBGFHBG as as substrate for substrate for tktk))in in rats injected withrats injected with Ad.Ad.tk ortk or Ad.Ad.LacZLacZ

kidneys

bladder

liver

Ad.LacZ Ad.tk

Día 9

Día 16

PET

PETDAY 0

PET

DAY 2

AdCMVtk

Penuelas et al Gastroenterology, 2005

Ad.tk, 1012 vpGastroenterology, 2005

501636501636Gastroenterology, 2005

First generation adenoviral vectors:

• given by i.t. injection are well tolerated and infect very efficiently HCC tumor nodules

• however, duration of the expression is very short,repeated tumor transduction is not possible andantitumor efficacy is very limited.

LESSONS FROM GENE THERAPY CLINICAL TRIALS FOR LIVER CANCER

TUMORMALIGNANT

CELLS

LYMPH NODE

EFFECTORCTLs

Th CTLPRECURSOR

NKs

DIRECT

ULTRASOUND-

GUIDED INJECTION

TRANSFECTEDCYTOKINE

MIGRATION

DCsIN

VIT

RO

DIFF

EREN

TIAT

ION

IMMUNOSTIMULATINGFACTOR

VIRAL VECTORENCODING

PERIPHERALBLOOD OR

LEUKOAPHERESISPRODUCT

AG TRANSFER

1110987654321011109876543210

302520151050

Non-treatedside

Mea

n tu

mor

dia

met

er(m

m)

11109876543210

302520151050

DC AdCMVIL-12

7/8

6/8 0/8

11109876543210

Treatedside

0/8DC AdCMVLacZ

SYSTEMIC THERAPEUTIC IMMUNITY

Week after tumor injection.

Journal Clinical Oncology 2005

-1w

Leukapheresis

Tumor biopsy

PBMCs

DTH

1st dose

0 3w 6w

2nd dose 3rd dose

Thaw CD14+ cells

Thaw CD14+ cells

DTH DTH

1 year

Clinical response

Immune monitoring Follow-up

8w

Safety evaluation

Immune monitoring

Immune monitoring

Immune monitoring

Tumor biopsy

AFIL-12

DC culture

AFIL-12

DC culture

AFIL-12

DC culture


PatientNum.

Sex/AgeTumortype

ECOG Previous therapySite of

metastases

01 M / 44 PC 0 RT/Chemo L, P, LN

02 F / 72 HCC* 0 TACE/Surg/EI L

03 M / 62 CRC 1 Surg/Chemo/EI L, P

04 M / 63 CRC 0 Surg/Chemo L, Lu

05 M / 72 HCC 0 PEI/TACE L, sc

06 M / 21 HCC 2 Chemo L, Lu, LN

07 M / 49 CRC 2 Surg/Chemo/EI L, Lu

08 M / 39 CRC 2 Surg/Chemo L, B

09 F / 55 PC 1 RT/Chemo P, L, LN

10 M / 73 HCC * 0 TACE L

11 M / 56 CRC 0 Surg/Chemo L, LN

12 M / 49 PC 1 RT/Chemo P, L, LN

13 M / 69 HCC * 1 TACE/Surg/EI L, B, st

14 F / 68 HCC* 1 Chemo L, B

15 F/ 73 HCC * 0 TACE L

16 M / 39 HCC * 1 None L, LN

17 M / 53 ChC 2 Chemo/RT L, B, LN

*: Concurrent chronic hepatitis C viral infection


Dose(numberof DC)

PatientNum

TreatmentCourses

Procedure ofGuidance

Clinicalresponse

01 3 US PR

02 3 US PD

03 3 US PD

04 3 US PD

10x106

05 3 US PD

06 2 US NE

07 2 US NE

08 1 US NE

09 3 EUS PD

25x106

10 3 US SD

11 3 US PD

12 2 CT NE

13 3 US PD

14 2 US NE

15 3 US SD

16 3 US PD

50x106

17 1 US NE

PR: partial responsePD: progressive diseaseSD: stable diseaseNE: not evaluable


CD8+ lymphocytes

Variation in the number oftumor infiltrating lymphocytes

following therapy


B) After treatmentA) Before treatment

A case of partial response to therapy


30 min

24h

48 h

111In-Labelled DCs

Lung

Lung

Sequestration of DCs inside the tumor


IL-8

ser

um c

once

ntra

tion

(pg/

ml)

800

600

400

200

0

Patients Controls

P < 0.001

0

50

100

150

200

250

ASPC-1 HEPG2 CaCo2 HT29 SW48

Tumor cell lines

IL-8

(pg/

ml)

~ 39 kDa

iDCNeg NP mDC iDCNeg NP mDC

CXCR1 CXCR2

~ 40 kDa

International Journal of Cancer, 2006

Liver

time

conc

entra

tion

Intrahepatic levels

Blood concentration

Long-term expression vector with therapeutic gene

Multifocal HCC

normal mice

HCC in TG mice

HCC in HCC in PK/cPK/c--mycmyc transgenic mice

Colony of Colony of PK/cPK/c--myc myc transgenicstransgenics

C-C-myc heterozygous

1 month of age

carbohydrate-rich diet

8 months of age

70% develop HCC

Modified TetModified Tet--on regulatory systemon regulatory system

rtTA2s-M2 EalbPa1AT rTetR VP16

Zabala y cols., Cancer Res, 2004

+ Dox

tetO7

Palb IL-12

P. Inducible

tetO7

Palb IL-12

Liver-specificconstitutive promoter

Repeated induction ofRepeated induction of ILIL--12 12 expressionexpression in in the liver ofthe liver of PK/cPK/c--myc myc transgenic mice with multifocaltransgenic mice with multifocal HCCHCC

Four treatment rounds and 3 cycles of induction each round

Laparotomy

Hydrodynamic injection of the plasmid

2ª 3ª1ª Induction

( 3 treatment ROUNDS)

Day 0 Day 1 Day 51 y 112

Sacrifice

Day 168

4th ROUND

3-5 weeks

Laparotomy

Tumor Tumor size and survivalsize and survival

0

5

10

15

20

25

30

Día 0 Día 168 Día 0 Día 168

Tum

or s

ize

(mm

)

Saline IL12

Repeated induction ofRepeated induction of ILIL--12 12 expressionexpression in in the liver ofthe liver of PK/cPK/c--myc myc transgenic mice with multifocaltransgenic mice with multifocal HCC: HCC: antitumor efficacyantitumor efficacy

Day 0 Day 168 Day 0 Day 168

Tumor regression

Saline 0/10 0/10 10/10 50

IL12 2 /10 2/10 6/10 70

RespondersP<0,05

Tumor stabilization

No response % Survival

Salin

oIL

12Día 0 Day 168Sa

line

IL12

Repeated induction ofRepeated induction of ILIL--12 12 expressionexpression in in the liver ofthe liver of PK/cPK/c--myc myc transgenic mice with multifocaltransgenic mice with multifocal HCC: HCC: antitumor efficacyantitumor efficacy

T

H

T

HSalin

e

T

T

H

IL12

T

Lymphocyte infiltrationLymphocyte infiltration in in cc--mycmyc transgenic mice subjected to transgenic mice subjected to repeated cycles ofrepeated cycles of ILIL--12 12 inductioninduction in in the liverthe liver

T cell-mediated immunosuppression

• The main obstacle to cancer immunotherapy

• T cell-mediated immunosuppression is executed by∗ Treg: CD4+ CD25+ Foxp3+ T cells∗ Tr1 : CD4+ IL-10+ Foxp3- T cells∗ CD8+ IL-10+ regulatory T cells

• Treg in tumor microenvironment∗ Tumors induce Treg trafficking, differentiation and expansion∗ Treg block immune response against tumor-associated antigens

RegulatoryRegulatory T T cellscells ((TregTreg) ) and the suppression of antitumor and the suppression of antitumor immunityimmunity

CD8+CD8+

CD8+CD8+

CD8+CD8+

CD8+CD8+

CD8+CD8+

CD8+CD8+

CD4+CD4+

CD4+CD4+

CD4+CD4+

CD8+CD8+

CD8+CD8+

CD8+CD8+

CD8+CD8+

CD8+CD8+

CD8+CD8+

CD8+CD8+

CD4+CD4+

CD4+CD4+

CD4+CD4+

CD8+CD8+

CD8+CD8+

CD8+CD8+

CD8+CD8+

CD8+CD8+

CD8+CD8+

CD8+CD8+

CD4+CD4+

CD4+CD4+

CD4+CD4+

CD8+CD8+

T

CD4+CD25+

Treg

CD4+CD25+

CD4+CD25+

CD4+CD25+CD4+CD25+CD4+CD25+

CD4+CD25+CD4+CD25+CD4+CD25+

Tumor infiltrating lymphocytes

Induction ofInduction of Foxp3 Foxp3 andand ILIL--10 in IL10 in IL--1212--treatedtreated cc--myc transgenic micemyc transgenic mice

Foxp3

0

40

80

120

IL12-D50 R IL12-D50 NR Sal-D50

tumorno tumor

2(ΔC

t)*1

05

P<0,05

n.s.

IL12 (R) IL12 (NR) Saline

0

5

10

15

IL12-D50 R IL12-D50 NR Sal-D50

tumorno tumor

2(ΔC

t)*1

05

P<0,05

n.s.

IL10

IL12 (R) IL12 (NR) Saline

TGFβ and T cell-mediated immunosuppression

• TGFβ is present at high levels in tumor microenvironment

• TGFβ plays a key role in the induction of Foxp3+ T reg

• TGFβ mediates the suppressive effect of regulatory T cells on adaptive immunity and NK cell function

Tumor volume after 10 days induction of IL-12 expression in the liver of rats with metastatic colon cancer:

The effect of TGFβ blocking peptide P17

0

1000

2000

3000

4000

5000

6000

Control GL-IL12 GL-IL12+P17

Volu

me

(mm

3)

Day

50 o

rfin

al v

olum

e

Animal Survival

0102030405060708090

1000 7 14 21 28 35 42 49 56 63 70 77 84 91 98

ControlGL-IL12GL-IL12+P17

Days

% s

urvi

val

Double inducible system in a single vector

Aims in cancer gene therapy

1. To increase the duration of transgene expression

2. To control transgene expression using inducible promoters

3. To identify the effective transgene or combination of transgenes to achieve curative antitumor effects

4. To monitor trangene expression by in vivo imaging techniques

Collaborators

Lab. Cancer Gene TherapyCheng QianM.Gabriela KramerIgnacio MeleroRuben H-AlcocebaLin WangMin GuanIñigo NarvaizaMiguel BarajasCristina OlagüeHelena VillanuevaCelia Gomar

International collaborationStefan KochanekVolker Schmitz

RadiologyAlberto BenitoJavier LarracheJesús Pueyo

Nuclear MedicineJosé RichterIván PeñuelasJosé Martí

NursingBlanca Larrea

Clinical PharmacologyBelén SádabaJosé Azanza

Liver UnitJuan RuizGuillermo MazzoliniJorge QuirogaIgnacio Herrero Bruno SangroJesús Prieto

GIMaite HerraizJosé Carlos SubtilMiguel Muñoz

gene therapy of liver cancer: experience from clinical trials · viral vectors for gene transfer...

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