bystander effects elicited by targeted radionuclide therapy · targeted radionuclide therapy ......

Bystander Effects elicited by

targeted radionuclide therapy

Marie Boyd and Rob Mairs

Targeted Therapies Group, CRUK Beatson Labs, Glasgow

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Targeted RadiotherapyTargeted Radiotherapy

131131II--conjugateconjugate

�euroblastoma�euroblastoma

• 2nd commonest solid

tumour in children

• peak age < 2 years

• mostly disseminated at

1st presentation: poor

prognosis

• rapid growth

• chemo/radiosensitive

Meta-iodobenzylguanidine

(MIBG)

�H

�H

�H2

I

HO �H2

Adrenaline

HO

�oradrenaline

HO �H2

HO

OH

Guanethidine

�H2

H�

�H

�

CatecholaminesCatecholamines, adrenergic neurone blockers , adrenergic neurone blockers

and MIBG and MIBG

�oradrenaline transporter

Preoperative [Preoperative [131131I]MIBG treatmentI]MIBG treatment

3 year old neuroblastoma stage IV patient(PA Voute, 2001)

• 3 consecutive treatments; interval = 4 weeks

• 1st scan: bone and bone marrow invasion + large retroperitoneal tumour

• 2nd scan: decreased uptake in bone, bone marrow and primary tumour

• 3rd scan: bone and bone marrow cleared; primary tumour resectable

For mets but..

Transfer of the Transfer of the noradrenalinenoradrenaline

transporter (�AT) gene transporter (�AT) gene

noradrenaline transporter

for [131I]MIBG targeted radiotherapy of

- glioma

- urological tumours

- neuroblastoma

�

H

�H

�H2

I131

�AT�AT

RSV

Introduction of �AT gene into human UVW Introduction of �AT gene into human UVW gliomaglioma cells cells

induces active uptake of [induces active uptake of [131131I]MIBG I]MIBG

SK-�-BE(2c) UVW UVW/�AT

0

100

200

300

MIBG uptake (cpmx 103per 106cells)

no desmethylimipramine

+ desmethylimipramine

UVW/�AT-

surviving fraction

0

1

2

3

4

5

6

7

V/V

0

0 5 10 15 20 25

time (days)

0

0.5

1

2

3

4

5

Growth Delay

MBq/ml

0.0001

0.001

0.01

0.1

1

[131I]MIBG (MBq/ml)

Clonogenic assay

UVW spheroids

UVW/�AT spheroids

UVW/�AT

monolayers

Toxicity of Toxicity of [[131131I]MIBGI]MIBG to �AT to �AT transfectantstransfectants

2.5 7.5 100 5 12.5

T.I. = 1000

Expression of the NAT transgene in malignant

tissue using tumour-specific elements

- telomerase promoters

- radiationradiation--inducible WAF1 promoterinducible WAF1 promoter

Viral deliveryViral delivery

Replication selective Herpes Virus (HSV 1716)[Prof Moira Brown, Institute of �eurological Sciences, Glasgow]

• HSV1716 deleted in RL1 gene - replicates and lyses only

dividing tumour cells

• avirulent in normal brain

• viral replication and oncolysis enhanced by radiation?

• MRC trial:

Patients with relapsed high grade glioma

No toxicity

Some long term survivors

GLIOMAGLIOMA

CELLCELL

Viral therapy for Viral therapy for gliomaglioma

Cell kill by lysis +

MIBG targeted radiotherapy

�AT�AT

�AT�AT

�AT�AT

�AT�AT �AT�AT

�AT�AT

�AT�AT

�AT�AT

[[131131I]MIBGI]MIBG








HSV1716-�AT

Images of mouse tumours 1 and 7 days after intravenous injection of HSV1716

Uninfected tumour cells stain blue. HSV-infected cells stain brown

X 5 magnification

Areas of virus

concentration are

indicated by

arrows

A

B

C

Area A at x 20

magnification

Area C at x 40

magnification

These cells show

classic signs of

HSV infection:

multi–nucleated

and necrotic

x 5 magnification

There are more

and larger areas of

positive staining

compared with

Day 1 D

EClassic signs of

HSV infection:

multi-nucleation;

giant cells; holes

due to cell death

Highly necrotic;

most cells have

lysed due to HSV

infection

Day 1 Day 1 Day 1

Day 7 Day 7Day 7

Area E at x 20

magnification

Area D at x 40

magnification

[131I]MIBG uptake (%

of injected dose per gram of tissue)

0

0.2

0.4

0.6

The effect of scheduling of viral delivery on tumour uptake of [131I]MIBG

106HSV1716 NAT

virus + MIBG simultaneously - 24 h uptake

virus + MIBG simultaneously - 48 h uptake

virus 24 hours before MIBG - 24 h uptake

virus 24 hours before MIBG - 48 h uptake

106HSV1716

106 1716/NAT + [131I]MIBG 24 h later

106 1716/NAT + [131I]MIBG simultaneously

105 1716/NAT + [131I]MIBG 24 h later

105 1716/NAT + [131I]MIBG simultaneously

105 1716/NAT + PBS 24 h later

106 1716/NAT + PBS 24 h later

Tumor volume (V/V

0)

Time after treatment (days)

0

5

10

15

20

25

0 5 10 15 20 25 30

Effect on tumor growth of HSV1716/NAT + [131I]MIBG delivered

simultaneously or sequentially

N=12, mean and sd

HSV1716/GFPGFP plasmid/Effecteine

250 m250 m

TransfectionTransfection of of multicellularmulticellular tumour spheroids with the tumour spheroids with the

gene encoding the jellyfish green fluorescent protein (GFP)gene encoding the jellyfish green fluorescent protein (GFP)

UVW UVW gliomaglioma cells infected cells infected with HSV1716 / with HSV1716 / laclac--ZZ

[131I]MIBG

[211At]MABG

beam

beam

Herpes virus

for tumour cell lysis

+

transfer of NAT gene,

controlled by WAF1

or telomerase

promoter

Targeted

radiotherapy

Gene

therapy

GENETICALLY GENETICALLY

ENHANCED TARGETED ENHANCED TARGETED

RADIOTHERAPYRADIOTHERAPY

FOR GLIOMAFOR GLIOMA

[123I]MIBG

3 types of therapeutic decay particle3 types of therapeutic decay particle

Radionuclide decay particle range

123I Auger electrons 10 nm

131I beta particles 800 m

211At alpha particles 60 m

Incorporation of the Auger

electron emitter 123I

into D�A using the

thymidine analogue

[123I]IUdR

131I -particle bombardment

of D�A using the noradrenaline

analogue [131I]MIBG

211At -particle bombardment

of D�A using the noradrenaline

analogue [211At]MABG

Bystander Effects

• Killing/damage of un-irradiated cells due to irradiation of adjacent cells

• Physical: radiation cross-fire, i.e., direct traversal

• Biological: direct traversal not required

Transfer of medium

Transfer of serum

cell death

chromosomal

aberrations

mutations

genomic

instability

Manifestation of radiation-induced

biological bystander effects (RIBBE)

unirradiatedunirradiated

cellscells

Esp low dose

& low dose

rate

p53

Are RIBBEs significant in targeted radiotherapy?

- transfectant mosaic spheroids

- media transfer experiments

How do we investigate RIBBE in our gene

therapy/targeted radiotherapy scheme?

NonNon--mosaic mosaic multicellularmulticellular spheroidsspheroids

100% GFP-expressing cells 100% GFP-non-expressing cells

TransfectedTransfected mosaic mosaic

spheroids derived from spheroids derived from

the human the human gliomaglioma cell line cell line

UVWUVW

The spheroids, ranging in size from

100 to 500 m diameter, are

composed of mixtures of cells

transfected with the GFP gene

(green) and cells transfected with

the �AT gene (red).

[211At]astatine

[211At]MABG

meta-astatobenzylguanidine

[131I]MIBG

meta-iodobenzylguanidine

NH

NH

NH2

131I

NH

NH

N H2

211At

Transfer of mediumTransfer of medium

NonNon--irradiated cellsirradiated cellsRemove targeted radionuclideRemove targeted radionuclide

Replace medium andReplace medium and

Incubate for Incubate for 1hr1hr

After 1hr incubation, gamma count the

transferred medium and add activity,

equivalent to that of radiopharmaceutical

which leaked from cells, to a third flask -

[= Activity control]

Clonogenic Assay

24h24h

DONOR RECIPIENT ACTIVITY

CONTROL

Media transfer protocol

Direct + Indirect: incubate with MIBG 2h; wash; incubate to allow generation of “bystander poisons” (direct + bystander effect)

Donor: as Direct + Indirect but remove medium then replace with fresh medium (~ direct effect only)

Recipient: receive medium from Donor (~ bystander effect only)

Activity control: to correct for small amount of [*I]MIBG leaked from Donor cells and transferred to Recipients

Untransfected: wild-type UVW cells (do not express �AT) recipients of medium from MIBG-treated wild-type UVW donors

Groups evaluated

RIBBE after RIBBE after -irradiation of UVW/NAT cells

- This cell line demonstrates RIBBE after external beam irradiation

- Direct irradiation is more cytotoxic than RIBBE at high dose

- RIBBE are most significant at low doses

Recipient

Donor

Direct + indirect

surviving fraction

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

0 1 2 3 4 5 6 7 8 9

dose (Gy)

0.01

0.1

1.0

surviving fraction

0 1 2 3 4 5 6 7 8 9

dose(Gy)

RIBBE after treatment with RIBBE after treatment with --emitter [emitter [131131I]MIBG I]MIBG

Untransfected cells

Activity control

Direct + indirect

Donor

Recipient

� medium from irradiated cells is very cytotoxic - dose response

� significant effect at high doses; RIBBE cell kill almost as potent as direct

kill in cells treated with radiopharmaceutical

D/R

surviving fraction

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

0 1 2 3 4 5 6 7 8

[131I]MIBG activity conc. (MBq/ml)

RIBBE elicited by treatment with Auger electron emitter ([RIBBE elicited by treatment with Auger electron emitter ([123123I]MIBG) I]MIBG)

and and --emitter ([emitter ([211211At]MABG) At]MABG) -- high LEThigh LET

- U-shaped survival curves for RIBBE-kill - i.e. dose-related cytotoxicity at low activity

concentration and diminishing bystander kill at higher activity concentration

- RIBBE elicited by high LET targeted radionuclides result in cell kill of magnitude similar

to that caused by direct irradiation - hence potentially powerful therapeutic effect

surviving fraction

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

0 1 2 3 4 5 6 7 8

Untransfected

Activity control

Recipient

Donor

Direct + Indirect

[123I]MIBG activity conc (MBq/ml)

surviving fraction

[211At]MABG activity conc (kBq/ml)

35302520151050

0.1

0.20.3

0.40.50.6

0.70.80.9

1.01.1

0

RIBBE in a second cell line - EJ138 bladder carcinoma

- Cell line shows RIBBE in response to external beam irradiation

- Recipient cells - dose response at low doses then plateau

0.01

0.1

1.0

surviving fraction

0 1 2 3 4 5 6 7 8 9

Dose (Gy)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

surviving fraction

0 1 2 3 4 5 6 7 8 9

Dose (Gy)

RIBBE after RIBBE after -irradiation

Recipient

Donor

Direct + indirect

RIBBE following exposure of EJ138 cells to --emitter emitter [131I]MIBG

Effect similar to that observed in UVW cell line:

- no U-shaped survival curve

- RIBBE cell kill less than in donor cells

surviving fraction

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1


0 1 2 3 4 5 6 7 8 9 10 11

Untransfected cells

Activity control

Direct + indirect

Donor

Recipient

RIBBE following exposure of EJ138 cells to Auger electron emitter ([123I]MIBG) and -emitter ([211At]MABG) - high LET

Similar to effect observed in UVW cell line:

- U-shaped survival curves for RIBBE-killsurviving fraction

00.1

0.2

0.30.40.5

0.6

0.7

0.8

0.91.0

1.1

0 40

sur vivi ng fr act ion

[123I]MIBG activity conc (MBq/ml)

EJ138 parental

Activity control

Recipient

Donor

Direct + Indirect

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.91.0

1.1

0 1 2 3 4 5 6 7 8 9 10 11453530252015105

[211At]MABG activity conc (kBq/ml)

RIBBE elicited by treatment with RIBBE elicited by treatment with --emitter ([emitter ([211211At]MABG) At]MABG)

and Auger electron emitter ([and Auger electron emitter ([123123I]MIBG) I]MIBG) -- high LEThigh LET

Radiation-induced biological bystander

effects may contribute significantly to the

efficacy of targeted radionuclide therapy

Conclusion

Conclusions

RIBBE from targeted radionuclides appear distinct from

external beam irradiation:

- dose response

- LET dependent

- U-shaped survival curves

RIBBE from high LET radionuclides at low doses are more

cytotoxic than direct irradiation

Only cells which take up radiopharmaceutical produce RIBBE

AUDR

MABG

Is subcellular localisation of radiopharmaceutical important ?

0.2

0.4

0.6

0.8

1

1.2

0 10 155 20 25 30 35

[211At]AUDR conc. (kBq/ml)

recipient

donor

control

surviving fraction

surviving fraction

[211At]MABG activity conc. (kBq/ml)

35302520151050

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

0

40

[131I]IUdR effect similar to [131I]MIBG

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

Surviving fraction

0 1 2 3 4 5 6 7 8

[131I]IUdR activity conc. (MBq/ml)

Recipient

Donor

Direct + indirect

surviving fraction

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

0 1 2 3 4 5 6 7 8


What are the bystander toxins?

Do Reactive Oxygen Species

play a role in RIBBE elicited by

targeted radionuclides?

- low mol. wt. chemical scavengers

perturbed bystander effects following X-irradiation

or -beam irradiation

- In our system:

- media transfer expts - using cells stably transfectedwith the SUPEROXIDE DISMUTASE gene

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0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

0 1 2 3 4 5 6

dose (Gy)

RIBBE after RIBBE after -irradiation of untransfected cells

and SOD-transfectants

SOD transfection abolishes bystander effect

SOD-transfected

untransfected

Recipient cells’surviving fraction

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

0 1 2 3 4 5 6 7 8

Dose ([131I]IUdR (MBq/ml)

SOD-transfected

untransfected

RIBBE after RIBBE after [131I]IUdR treatment of untransfected cells


SOD transfection abolishes bystander effect


[123I]IUdR - SOD abolishes bystander effect only at low activity conc

SOD-transfected

RIBBE after RIBBE after [123I]IUdR treatment of untransfected cells


untransfected

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

0 1 2 3 4 5 6 7 8 9 10 11 12

Dose [123I]IUdR (MBq/ml)


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Is a protein involved in

RIBBE elicited by

targeted radionuclides?

- Boil medium before transfer

to recipient cultures

Effect on RIBBE RIBBE of boiling the medium from -irradiated cells before transfer to recipients

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2


0 Gy no treatm

ent

2 Gy no treatm

ent

0 Gyboil -> RT

0 Gyboil -> 4

0C

2 Gy boil -> RT

2 Gyboil -> 4

0C

0 Gy boil -> RT

0 Gyboil -> 4

0C

2 Gy boil -> RT

2 Gy boil -> 4

0C

Controls

Treatment groups

Medium only controls

boil -> room

temp

boil -> 40C

Effect on RIBBE RIBBE of boiling the medium from

[131I]MIBG-treated cells before transfer to recipients

Controls

Treatment groups

Medium only controls

boil -> room

temp

boil -> 40C

0 MBqno treatm

ent

4 MBqno treatm

ent

0 MBqboil -> RT

0 MBqboil -> 4

0C

4 MBqboil -> RT

4 MBqboil -> 4

0C

0 MBqboil -> RT

0 MBqboil -> 4

0C

4 MBqboil -> RT

4 MBqboil -> 4

0C

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2Recipient cells’surviving fraction

Staff

Nazia Baig

Marie Boyd

Jenny Campbell

Anne Marie Clark

Tony McCluskey

Rob Mairs

Teeny Ross

Annette Sorenson

Lesley Wilson

Sources of funding

Cancer Research Campaign

British Urological Foundation

Urology Dept., Glasgow

European Community

Neuroblastoma Society

Scottish Office

Department of Health

Dr Hadwen Trust

Scottish Hospitals Endowment

Research Trust

North Glasgow NHS Trust

Clerk Maxwell Foundation

Ian Sunter Trust

Targeted Therapy GroupTargeted Therapy Group

Cancer Research UK Cancer Research UK BeatsonBeatson Laboratories Laboratories

Glasgow, ScotlandGlasgow, Scotland

CollaboratorsWilson Angerson

John Babich

Darrel Bigner

Heinz Bonisch

Moira Brown

Mark Gaze

David Hirst

Sissy Jhiang

David Kirk

Carmel Mothersill

Jonathan Owens

Kevin Prise

Tracy Robson

Mike Zalutsky

bystander effects elicited by targeted radionuclide therapy · targeted radionuclide therapy ......

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