Gene therapy of lung cancer

Where we are

and where to go?

Gene therapy of lung cancer

Where we are
and where to go?

Gene therapy of lung cancer

Where we are
and where to go?

GT in TUMORS: TARGETS
Tumor cells
Immune system
Hematopoietic stem cells
(HSCs)

Target 1: Tumor cell

Major
Objectives

Inhibit
tumor cell
proliferation

Induce
tumor-cell
specific toxicity
(suicide genes)

Virus mutants
selectively replicate
and lyse
tumor cells

Target 2: Immune system

Major
Objectives

Anti-cancer
Vaccination
(DNA vaccines)

Increasing
the cytotoxic
response
against tumor-cell

Genetic
modifications
of T cells
(redirecting)

Target 3: HSCs
Transferring genes conferring their resistance to
high dose chemotherapy (HDC) into
hematopoietic stem cells
Supramaximal chemotherapy therapy possible in
clinical trials concerning

breast
ovary
testicle
small cell lung cancer because of specific drug prophile
1) ABC proteins (as MDR-1)
2) genes of antioxidant enzymes (GS-transferase)
are considered

Target 1: Tumor cell

Major
Objectives

Inhibit
tumor cell
proliferation

Induce
tumor-cell
specific toxicity
(suicide genes)

Virus mutants
selectively replicate
and lyse
tumor cells

Supplementary (Substitutive) GT
Clinical trial examples
Gene

Development
(clinical
phase)

Application

III

P53 (Ad5CMV-p53)

Planoepithelial cancer of head &

neck, NSCLC

III

Allovectin (HLAB7/microglobulin 2)

Advanced melanoma (adiuvant for

chemiotherapy)

III

TNF- (TNFerade)

Pancreatic cancer (adiuvant for

chemiotherapy)

I, II

Cycline G

Metastases of colon cancer to liver

IL-2

Transfer of autologous lymphocytes

in melanoma metastases

II

Suppressory GT of tumor
Triple helix target:
IGF-I, survivine
DNA

Antisens, siRNA, target:

mRNA

PKC, clusterin, BCL-2, c-erbB2,

EGF, VEGF

Aptamers, target:
target

protein

VEGF, MUC1,

Antisense therapy 1
triple-helix anti-IGF-I

First strand is a transcript of pMT AG vector (homopurine RNA);

2nd and 3rd strand represent typical genomic DNA (promoter of IGF-I gene)
IIIII
******

Watsona-Crick bonds
Hoogsteen bonds

Suppressory therapy 1

Clinical applications of Antisense ON

Target

Development

I, III
I,II,III

Bcl-2 (Genasense)

Application
SC lung cancer; melanoma,
myeloma multiplex

Protein Kinase C-alpha NSC lung cancer,

metastases of colon cancer,
(ISIS 3521)
prostate cancer (HR)

II

C-raf Kinase (ISIS

5132)

metastases of colon cancer;

prostate cancer (HR),
lung cancer

C-myb

Chronic myeloid leukemia

H-ras (ISIS 2503)

Breast cancer

Suppressory therapy 1

Clinical applications of Antisense ON

Target

Development

I, III
I,II,III

Bcl-2 (Genasense)

Application
SC lung cancer; melanoma,
myeloma multiplex

Protein Kinase C-alpha NSC lung cancer,

metastases of colon cancer,
(ISIS 3521)
prostate cancer (HR)

II

C-raf Kinase (ISIS

5132)

metastases of colon cancer;

prostate cancer (HR),
lung cancer

C-myb

Chronic myeloid leukemia

H-ras (ISIS 2503)

Breast cancer

Suppressory therapy 2

Clinical applications of Antisense ON

Development

Target

Application

I-III

Bcl-2 (Genasense,
SPC2996)

Melanoma, CLL (adiuvant for

chemiotherapy)

I, II

X-IAP (AEG35156103)

Recurrent AML, breast cancer,

NSCLC, different type tumor
dissemination (adiuvant for
chemiotherapy)

II

Clusterin (OGX-011) Neo-adiuvant for therapy of

HIF-1 (EZN-2968)

prostate cancer

Different tumors

Suppressory therapy 3
Ribozymes (bcr-abl; ras)

siRNA features
Native siRNA, products of nucleolytic dsRNA degradation driven by
ribonuclease Dicer have charakteristic composition.,,

2nt unpaired

2nt unpaired

RNAi phases
I siRNA duplex generation
II RISC silencing complex
formation
III
RISC activation and
recognition of mRNA
complementary to antisense
siRNA strand
IVFinal mRNA degradation
Consequence:
Gene silencing (no protein
expression)

Limitations of RNAi
RNAi efficiency is limited by ability of synthetic siRNA
molecules to bind mRNA and inhibit the target gene.
Different siRNA molecules directed against the same gene
fragment are characterized by different silencing potential.
Thats why efficiency of each siRNA type should be
evaluated separately.
It was also found, that RNAi specifity depends on the amount
of siRNA used in the assay. Its excess induces expression
of many genes associated with cell response to the stress /
viral RNA.

SELEX

Apoptosis

TRAIL receptors activation

Apoptosis

Inhibition of anti-apoptotic Bcl-2

protein family

Inhibition of anti-apoptotic Bcl-2

protein family

Inhibition of tumor cell proliferation

PROBLEMS

We need to reach and kill all tumor cells

The problems exist to gain solid tumor interior
tissue
Continous oligonucleotide infusion is difficult to
provide
Alternative plasmid in vivo therapy is limited due
to
Low transfection efficacy
Lack of lung cancer specific promoter
Low transcription level

Safety Study of an Antisense Product in

Prostate, Ovarian, NSCLC, Breast or
Bladder Cancer (OncoGenex Technologies, USA)
Phase II
OGX-427 is a second-generation ASO that inhibits expression of Hsp27.
Hsp27 increases with

cell stress,
cytotoxic chemotherapy,
radiation therapy
and hormone therapy
and has been shown to inhibit cell death.

Patients (major inclusion criteria): >18 years; metastases

GX-427 injections at 200mg, 400mg, 600mg, 800mg or 1000mg once a
week until withdrawn

Drug: Docetaxel

Angiogenesis: VEGF as GT target

Early effects

Regression of small vessels

growth

Late effects

Vascularisation
inhibition

Bevacizumab binds VEGF

Anti-VEGF
antibody
(Bevacizumab)

VEGF

P
P

P
P

VEGFR-1

P
P

P
P

VEGFR-2

Endothelial Cell

Anti-VEGF Strategies
MAbs

TKIs

Ligand
conjugates

Ligand

Ligand

Ligand

K K

K K

Cell
death

Protein
synthesis

Antisense
oligonucleotides

Ligand

K K

Signal
transduction

K K TKI

Signal
transduction

Target 2: Immune system

Major
Objectives

Anti-cancer
vaccination

Increasing
the cytotoxic
response
against tumor-cell

Genetic
modifications
of T cells
(redirecting)

TUMOR
CELL
Irradiated tumor
cells

Dendritic
cells

Plasmids

Peptides
Viral vectors
coding neo-antigens

TUMOR
CELL
Irradiated tumor
cells

Dendritic
cells

Plasmids

Peptides
Viral vectors
coding neo-antigens

TUMOR
CELL
Irradiated tumor
cells

Dendritic
cells

Plasmids

Peptides
Viral vectors
coding neo-antigens

Specific ways to generate

dendritic cells (DC)

Immune response against tumors

Frequent lymphocyte infiltrates (Tumor infiltrating lymphocytes,
TILs) in tumor tissue
TIL reveal phenotype of cytotoxic effector cells, they are able to
kill tumoer cells
It has been demonstrated that TIL cells present specific receptors
(TCR) against known neo-antigens (usually tumor associated
antigens, TAA)

Lung cancer antigens of determined amonoacide sequence:

MUC1
MAGE-A3
EGFR
TGF-2
hTERT

Dendritic cell vs naive T (CD4

or CD8) lymphocyte
Antigen (TAA, TSA)
MHC I / MHC II

TCR
CD8 / CD4

CD80 / CD86

CD28

Fas (CD95)
ICAM-1 (-2)
LFA-3
CD40

FasL (CD178)
LFA-1
CD2
CD40L

Dendritic cell vs naive T (CD4

or CD8) lymphocyte
Antigen (TAA, TSA)
MHC I / MHC II

TCR
CD8 / CD4

CD80 / CD86

CD28

Fas (CD95)
ICAM-1 (-2)
LFA-3
CD40

FasL (CD178)
LFA-1
CD2
CD40L

Dendritic cell vs naive T (CD4

or CD8) lymphocyte
Antigen (TAA, TSA)
MHC I / MHC II

TCR
CD8 / CD4

CD80 / CD86

CD28

Fas (CD95)
ICAM-1 (-2)
LFA-3
CD40

FasL (CD178)
LFA-1
CD2
CD40L

Dendritic cell vs naive T (CD4

or CD8) lymphocyte
Antigen (TAA, TSA)
MHC I / MHC II

TCR
CD8 / CD4

CD80 / CD86

CD28

Fas (CD95)
ICAM-1 (-2)
LFA-3
CD40

FasL (CD178)
LFA-1
CD2
CD40L

CYTOKINES, e.g. IL-2

2
3

Tumor cell vs primed T (CD4 or

CD8) lymphocyte
Antigen (TAA, TSA)
MHC I / MHC II
CD80 / CD86
Fas (CD95)
ICAM-1 (-2)
LFA-3
CD40

TCR
CD8 / CD4
CD28
Fas Ligand (CD178)
LFA-1
CD2
CD40L

IMMUNE TOLERANCE IN TUMORS 1

Low or altered expression of TAA antigens on tumor cells
Decreased or absent MHC I molecules
Atypic MHC II molecules (?)
Lack of B7 costimulatory molecules
Lack of adhesive molecules as LFA-1,-3 or ICAM-1
Superficial anti-adhesive molecules (mucin) on tumor
cells

IMMUNE TOLERANCE IN TUMORS 2

Immunosupressive factors secretion:
TGF-beta, IGF-I L-6, IL-10, IL-13
PGs
Tumor cells are not susceptible to cytotoxic activity of cytokines, as
for example soluble FasL or TNFs
Proapoptotic molecules (FasL) on tumor cells wich can kill TILs
Active tumor participation in local inflammatory cell/ mediator
network in order to
a) modify Th1/Th2 ballance towards Th2 domination;
b) improve cancer cell survival inter alia with use of proinflammatory
cytokines (e.g. TNF- of anti-tumor immunity versus TNF receptor type 2);
c) Reprogramme DCs towards immune tolerance of TAA (regulatory T cells)

Metastases generation

Przerzut
metastasis

metastasis
Przerzut

Przerzut
metastasis

TUMOR Progression

Heterogenous solid tumor

DNA Vaccines 1
Phase of
clinicla trial

Commercial
name

Therapeutic tool/ Notes

II

1650-G

Allogenic NSCLC cells

II

Ad-CCL23

MoDC modified with AdV vector encoding

CCL23 (chemokine with strong T cell
activation properties), administered directly
to the tumor

II

Mel
Cancer Vac

MoDC fused (Hybridoma) with melanoma

cells (immunogenic, neo-antigens crossreactivity with lung cancer )
Advanced non-operative NSCLC
+ Cox inhibitors+ IL-2

II

INGN-225

MoDC modified with AdV encoding mutant

p53 (as mutated it serves as neo-antigen )

DNA Vaccines 2
Phase of
clinicla
trial

Commercial
name

Therapeutic tool/ Notes

III

BLP-25/
START

Vaccination with MUC1 antigen in liposomal

career, MUC1 expression on lung cells is necessary,

III

TG4010

Avian vaccinia virus vector, transgene: MUC1 +

IL2 ; MUC1 expression on lung cells is necessary,

FowlpoxCEA/
TRICOM

Autologous MoDC transfected with Avian vaccinia virus

vector; transgene: TAA (CEA*) + TRICOM**
Different tumors, including lung cancer; CEA expression
on tumors cells necessary

*
**

significant increase in 3 years survival

Carcinoembryonal antigen
TRICOM = B7.1 (CD80) + ICAM-1 + LFA-3

Target 2: Immune system

Major
Objectives

Anti-cancer
vaccination

Increasing
the cytotoxic
response
against tumor-cell

Genetic
modifications
of T cells
(redirecting)

Target 2: Immune system

Major
Objectives

Anti-cancer
vaccination

Increasing
the cytotoxic
response
against tumor-cell

Genetic
modifications
of T cells
(redirecting)

Generations of CARs

TAA-specific TCR receptors &

Chimeric CAR receptors
(Acc. Nature Reviews/Immunology 2011, zmodyfikowane)

Note: TAA antigen sequence must be defined

Limitations of GT 1
Short term character of GT
DNA and/or RNA introduced to cells must be active for a
longer time
Transfected cells should live and function normally
However, in supplementation GT transgene integrates with
genome with difficulties; the site of integration is
completely unpredictable
Cell divisions often eliminate effects of transfection patients need the repetitive therapies

Limitations of GT 2
Immune response
In GT in vivo patient exposition to foreign
antigens (DNA, protein career) may
induce immune reaction
In supplementation GT, immune system
response is more and increased with
consecutive trangene exposition

Limitations of GT 3
Troubles with viral vectors
Shortcomings:

toxicity,
Immune and inflammatory response,
Infection of unwanted cells,
problems with control of transgene expression.

There is a a risk of virus pathogenic features recovery,

despite assumed defective character of wirus used
for vector contruction (onkogenesis not excluded!).

Limitations of GT 4
Multifactor ethiopathogenesis of
diseases
In some cases only, the disease could be cured
with approach directed again one single gene
GT systems based on multi-gene simultaneous
correction in the same cell are, up to date, not
sufficient enough

Two individual patient stories

Ashanti da Silva / Jessi Gelsinger

Where are we?

Perspectives of tumor GT
Clinical trials by
phase
2010: only circa 30
trials directed against
tumors reached the
phase III
Delivery of immune
stimulating cytokines
or anti-cancer genetic
vaccines

Where we go?
Perspectives of GT in tumors
Gene Therapy + Immune therapy = immune gene
therapy?
GT vaccinations as adjuvant therapy?
GT as chemotherapy assistance?
go in all?
Improved vectors and transfection systems?
Can we really mimic full succesful anti-tumor immune
responses?
Turning point or step by step progress?

Thank you for

your attention