7142015

1

HIFU Drug Delivery and

Immunotherapy

Katherine W Ferrara

UC Davis

Minimally toxic activatable particle

Treat accessible lesions Big hairy audacious goal (BHAG)

Immune therapy is effective for

melanoma emerging as effective for

many cancers

bull FDA-approved

ndash Interleukin-2 (RCC melanoma)

ndash CTLA-4 antibody (melanoma)

ndash PD-1 Ab (melanoma)

bull Investigational

ndash CpG as adjuvant to radiotherapy (melanoma

companion animals)

ndash

Durable response in

significant (15) of patients

7142015

2

Complexation of Cu(II) and Dox within

liposomes

Ammonium sulfate

method

Copper gluconate

TEA method

6

3

7

5 4

pH

Problem Even liposomal doxorubicin has substantial

cardiac toxicity and dose cannot exceed 500 mgm2 in

lifetime

Solution Create a doxorubicin salt that is very stable in

circulation

Kheirolomoom et al Molecular Pharmaceutics

0

50

100

3 5 7 8

Do

x r

ela

tive

Fl

()

pH

Outline

Activatable particles

+ hyperthermia

Particles + ablation

+ adjuvants

Activatable particles +

hyperthermia +

adjuvants

Temperature sensitive particles

paired with mild heating

Achieve hyperthermia Inject and release drug 0-40 minutes later Alter microenvironment Cell death M1 macrophages- 24 hours and later

7142015

3

CuDox-LTSLs

40 min US

US+

US+

CuDox-LTSLs

20 min US

Heart

Heart

Tumor

Thermally-activated vehicles

Below Tc Above Tc

DSPE-PEG-2000 DPPC Lyso-palmitoyl PC Drug

bull Direct and local release can

increase concentration and

decrease systemic toxicity

bull Small molecule can penetrate

tissue

Kheirolomoom et al JCR 2013

US-

US-

Tissue

Autofl

Dox

CuDox-lipo

100 nm

Copper-doxorubicin

temperature-sensitive

particles

Tissue

Autofl

Increasing

Dox

Particles US

C u D o x -L T S L s AS D o x -L T S L s

0

1

2

3

4

Do

xo

ru

bic

in f

luo

re

sc

en

ce

ra

tio

M u s c le + U S T u m o r

C u D o x -L T S L s AS D o x -L T S L s

0

2

4

6

Do

xo

ru

bic

in f

luo

re

sc

en

ce

ra

tio

T u m o r + U S M u s c le

Cu-dox temperature-

sensitive particles Conventional temperature-

sensitive particles

plt005 compared to control

Plt0001 compared to control

CuDox-TSL (8 treatments)

0

20

40

60

80

100

120

0 50 100 150 200 250

Su

rviv

al

Day post treatment

Control

CuDox-LTSLs

CuDox-LTSLs+US

-500

500

1500

2500

3500

4500

5500

0 10 20 30 40 50

Tu

mo

r g

row

th

Day post treatment

Control

Control+US

CuDox-LTSLs

CuDox-LTSLs+US

200 250

~

~

US

Treat 2xweek 4 weeks

6 mgkg

Kheirolomoom et al JCR 2013

7142015

4

1 mm

100 microm

Days

Kheirolomoom et al JCR 2013

2 Weeks

100 microm

1 mm

(Fe)

100 microm

(F480)

4 Weeks

1 mm

100 microm

001

01

1

10

100

03 07 10 20 40

ID

Time insonation (hrs)

05 cm

1 cm

2 cm

4 cm

Assumes 5 blood volume in tumor

10 sec tumor blood refresh

5 L blood volume

Diameter

ID delivered

depends on volume insonified

time of insonation

Why do we favor thermally-sensitive

nanoparticles

-10

-5

0

5

10

15

20

25

30

0 5 10 15 20 25 30We

igh

t c

ha

ng

e

Day post treatment

Control

CuDox-LTSLs

CuDox-LTSLs+US

0

5

10

15

20

25

WBC KmicroL RBCMmicroL

Blo

od

ce

ll c

ou

nt

CuDox-LTSLs

Control

0

2

4

6

8

10

Albumin Total protein

Pro

tein

g

dL

CuDox-LTSLs

Control

0

400

800

1200

1600

Org

an

we

igh

t m

g

CuDox-LTSLs

Control

Toxicity is low

7142015

5

Outline

Activatable particles

+ hyperthermia

Particles + ablation

+ adjuvants

Activatable particles +

hyperthermia +

adjuvants

Use of TLR agonists to enhance Teffector

cell function

adapted from Immunotherapy 2009 November 1 1(6) 949ndash964

DC Dendritic cell iDC Immature dendritic cell MDSC Myeloid-derived suppressor cell Macro Monocytemacrophage TAM Tumor-associated macrophage Teff Effector T cell Treg Regulatory T cell US ultrasound

TLR

activation

iDC

MDSC TAM Treg

Tumor

Teff

Teff

Macro

Th1

Mature

DC

IL-12

IFN-a TNF-a

Tumor Tumor

antigen

IL-12

IFN-g

IL-2

Copper-doxorubicin temperature-sensitive

particles + CpG (3 treatments)

0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0

0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

1 0 0 0 0

1 2 0 0 0

1 4 0 0 0

1 6 0 0 0

1 8 0 0 0

2 0 0 0 0

D a y p o s t tre a tm e n t

ND

L T

um

or

gro

wth

(

)

C o n tro l

C p G

C p G C o n tra la te ra l

C u D o x + U S + C p G

C u D o x + U S + C p G C o n tra la te ra l

7142015

6

CpG CpG

Contralateral

CuDox+US+CpG

Contralateral

CuDox+US+CpG

Control

2 mm

200 microm

CuDox

Abscopal response

Immune cell

populations

0

04

08

12

16

2

I

FN

-g+

C

D8

+ T

ce

lls

of

Le

uk

oc

yte

s

0

1

2

3

MD

SC

s

Outline

Activatable particles

+ hyperthermia

Particles + ablation

+ adjuvants

Activatable particles +

hyperthermia +

adjuvants

7142015

7

Long circulating

nanoparticles

Ablation + 64Cu-LCL

20

0 IDcc

25 IDcc

1cm

6 hours 20 hours 48 hours

0

1

0 2 4 6 8Op

tical

Den

sit

y

(Arb

itra

ry U

nit

s)

Location (mm)

Circle 1 Circle 2

Circle 3 No Tx

7142015

8

Ablate fraction of tumor + drug

0

02

04

06

08

1

12

14

0 50 100 150

Tu

mo

r V

olu

me (

cc)

Days after First Treatment

No Tx LCL

Single Single + LCL

Circle Circle + LCL

2 mm

4 mm

4 Protocols 100 Curative for

local disease

bull Hyperthermia +

TSL in 8 treatments

bull Hyperthermia+TSL

+ CpG in lt3

treatments

bull Hyperthermia +

Ablation + TSL in 3

treatments

bull Ablation + LCL

Wong et al in review

0

25

50

75

100

0 100 200

Su

rviv

al

Days After First Treatment

Hyp+TSL+Circle

TSL+Circle

TSL+Single

No Tx

0

25

50

75

100

0 40 80 120 160

Surv

ival

Time (days)

NT

LCL

Single

Single + LCL

Circle

Circle + LCL

None

TSL

LCL

Drug

US None Comb Hypertherm Ablation

lt100 cure

lt100 cure wCpG

100 cure wo CpG

100 cure w CpG

NA

NA

Curative Protocols

7142015

9

1 Protocol- extending survival in

systemic disease needs

optimization

bull Hyperthermia+TSL

+ CpG in lt3

treatments

Kheirolomoom et al in review

0 4 8 1 2 1 6 2 0 2 4 2 8 3 2 3 6 4 0 4 4 4 8 5 2 5 6 6 0 6 4

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

D a y p o s t tre a tm e n t

Su

rv

iva

l (

)

C o n tro l

C p G

C u D o x + U S + C p G

p=002

Conclusion

bull Imaging essential in developing new

nanotherapies identifying cell populations

evaluating efficacy

bull Activatable nanotherapies can cure local

disease enhance systemic therapy

bull Addition of immune adjuvants may allow

the accessible lesions to be treated

resulting in systemic benefits

Charles Caskey James Chomas Paul Dayton Lisa Even Brett Fite Karen Gagnon Xiaowen Hu Elizabeth Ingham Sarah Johnson Azadeh Kheirolomoom Dustin Kruse Jiro Kusunose Chun-Yen Lai Yu Liu Aaron Lum Karen Morgan Eric Paoli Shengping Qin Jai Woong Seo Susanne Stieger Kate Watson Andrew Wong Guodong Zhang Hua Zhang

U Bergen Frits Thorsen Cecilie Brekke Rolf Reed

UCD John Boone Sandy Borowsky Bob Cardiff

Nipavan Chiamvimonvat Roy Curry Greg Farwell

Bruce Hammock C Lebrilla Claude Meares Jinyi Qi

Tingrui Pan Scott Simon C Tepper So Yamada

LLNL Paul Hoeprich

G TechEmory Hanjoong Jo

Northwestern Tom Meade

BurnhamUCSB Juliana Hamzah Erkki Ruoslahti

UCB He Dong Ting Xu

Cedars Sinai Gadi Palled Dan Gazit Galina Shapiro

CMGI (Dave Kukis)

Pfizer Oncology

Targeson (Jack Rychak)

Image-Guided Therapy (Erik Dumont)

Siemens Medical Solutions

NCI and NIBIB

7142015

10

Manzoor AA et al Cancer Res 2012 Nov 172(21)5566-75

Dewhirst laboratory Duke University

Free dox

Dox-TSL

0 2 5 min

1 5 15 min of heat

Before

10 min after

heat ends

Intensity map (0 0 55) mm in x-y plane

Intensity map (0 5 55) mm in x-y plane

Thermal effects are very significant

7142015

11

Fite BZ et al PLoS ONE 7(4) e35509

Thermal and mechanical

oversight are feasible

MRgFUS insert from

Image Guided Therapy

7T 3 MHz ultrasound in

Bruker magnet

Heating is ON

from 0 to 20 s

With probe motion

05 1 15 2 25 3 35 4 45

05

1

15

2

25

3

35

4

45 1

15

2

25

3

35

4degC

Foiret et al submitted

US+ temperature sensitive

liposomes (TSL) or long circulating

liposomes (LCL)

bull US hyperthermia+TSL= local cure (100 in 8

treatments)

bull US hyperthermia+CpG+TSL= local cure (100

in 3 treatments) + slow systemic disease

ndash Needs optimization many alternatives

bull US ablation + LCL or TSL slow disease or cure

7142015

12

200 microm 200 microm

4mm 200 microm

After ablation

Canine melanoma - Combined radiotherapy

and immunotherapy using CPG

oligodeoxynucleotides and indolamine 23

dioxygenase (IDO) blockade

Monjazeb et al J Immunother Cancer 2013 1(Suppl 1) P256

Why nanoparticles Why ultrasound

Target accumulation

0

2

4

6

8

10

12

14

16

18

20

IDg

Paoli et al JCR 2010

7142015

2

Complexation of Cu(II) and Dox within

liposomes

Ammonium sulfate

method

Copper gluconate

TEA method

6

3

7

5 4

pH

Problem Even liposomal doxorubicin has substantial

cardiac toxicity and dose cannot exceed 500 mgm2 in

lifetime

Solution Create a doxorubicin salt that is very stable in

circulation

Kheirolomoom et al Molecular Pharmaceutics

0

50

100

3 5 7 8

Do

x r

ela

tive

Fl

()

pH

Outline

Activatable particles

+ hyperthermia

Particles + ablation

+ adjuvants

Activatable particles +

hyperthermia +

adjuvants

Temperature sensitive particles

paired with mild heating

Achieve hyperthermia Inject and release drug 0-40 minutes later Alter microenvironment Cell death M1 macrophages- 24 hours and later

7142015

3

CuDox-LTSLs

40 min US

US+

US+

CuDox-LTSLs

20 min US

Heart

Heart

Tumor

Thermally-activated vehicles

Below Tc Above Tc

DSPE-PEG-2000 DPPC Lyso-palmitoyl PC Drug

bull Direct and local release can

increase concentration and

decrease systemic toxicity

bull Small molecule can penetrate

tissue

Kheirolomoom et al JCR 2013

US-

US-

Tissue

Autofl

Dox

CuDox-lipo

100 nm

Copper-doxorubicin

temperature-sensitive

particles

Tissue

Autofl

Increasing

Dox

Particles US

C u D o x -L T S L s AS D o x -L T S L s

0

1

2

3

4

Do

xo

ru

bic

in f

luo

re

sc

en

ce

ra

tio

M u s c le + U S T u m o r

C u D o x -L T S L s AS D o x -L T S L s

0

2

4

6

Do

xo

ru

bic

in f

luo

re

sc

en

ce

ra

tio

T u m o r + U S M u s c le

Cu-dox temperature-

sensitive particles Conventional temperature-

sensitive particles

plt005 compared to control

Plt0001 compared to control

CuDox-TSL (8 treatments)

0

20

40

60

80

100

120

0 50 100 150 200 250

Su

rviv

al

Day post treatment

Control

CuDox-LTSLs

CuDox-LTSLs+US

-500

500

1500

2500

3500

4500

5500

0 10 20 30 40 50

Tu

mo

r g

row

th

Day post treatment

Control

Control+US

CuDox-LTSLs

CuDox-LTSLs+US

200 250

~

~

US

Treat 2xweek 4 weeks

6 mgkg

Kheirolomoom et al JCR 2013

7142015

4

1 mm

100 microm

Days

Kheirolomoom et al JCR 2013

2 Weeks

100 microm

1 mm

(Fe)

100 microm

(F480)

4 Weeks

1 mm

100 microm

001

01

1

10

100

03 07 10 20 40

ID

Time insonation (hrs)

05 cm

1 cm

2 cm

4 cm

Assumes 5 blood volume in tumor

10 sec tumor blood refresh

5 L blood volume

Diameter

ID delivered

depends on volume insonified

time of insonation

Why do we favor thermally-sensitive

nanoparticles

-10

-5

0

5

10

15

20

25

30

0 5 10 15 20 25 30We

igh

t c

ha

ng

e

Day post treatment

Control

CuDox-LTSLs

CuDox-LTSLs+US

0

5

10

15

20

25

WBC KmicroL RBCMmicroL

Blo

od

ce

ll c

ou

nt

CuDox-LTSLs

Control

0

2

4

6

8

10

Albumin Total protein

Pro

tein

g

dL

CuDox-LTSLs

Control

0

400

800

1200

1600

Org

an

we

igh

t m

g

CuDox-LTSLs

Control

Toxicity is low

7142015

5

Outline

Activatable particles

+ hyperthermia

Particles + ablation

+ adjuvants

Activatable particles +

hyperthermia +

adjuvants

Use of TLR agonists to enhance Teffector

cell function

adapted from Immunotherapy 2009 November 1 1(6) 949ndash964

DC Dendritic cell iDC Immature dendritic cell MDSC Myeloid-derived suppressor cell Macro Monocytemacrophage TAM Tumor-associated macrophage Teff Effector T cell Treg Regulatory T cell US ultrasound

TLR

activation

iDC

MDSC TAM Treg

Tumor

Teff

Teff

Macro

Th1

Mature

DC

IL-12

IFN-a TNF-a

Tumor Tumor

antigen

IL-12

IFN-g

IL-2

Copper-doxorubicin temperature-sensitive

particles + CpG (3 treatments)

0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0

0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

1 0 0 0 0

1 2 0 0 0

1 4 0 0 0

1 6 0 0 0

1 8 0 0 0

2 0 0 0 0

D a y p o s t tre a tm e n t

ND

L T

um

or

gro

wth

(

)

C o n tro l

C p G

C p G C o n tra la te ra l

C u D o x + U S + C p G

C u D o x + U S + C p G C o n tra la te ra l

7142015

6

CpG CpG

Contralateral

CuDox+US+CpG

Contralateral

CuDox+US+CpG

Control

2 mm

200 microm

CuDox

Abscopal response

Immune cell

populations

0

04

08

12

16

2

I

FN

-g+

C

D8

+ T

ce

lls

of

Le

uk

oc

yte

s

0

1

2

3

MD

SC

s

Outline

Activatable particles

+ hyperthermia

Particles + ablation

+ adjuvants

Activatable particles +

hyperthermia +

adjuvants

7142015

7

Long circulating

nanoparticles

Ablation + 64Cu-LCL

20

0 IDcc

25 IDcc

1cm

6 hours 20 hours 48 hours

0

1

0 2 4 6 8Op

tical

Den

sit

y

(Arb

itra

ry U

nit

s)

Location (mm)

Circle 1 Circle 2

Circle 3 No Tx

7142015

8

Ablate fraction of tumor + drug

0

02

04

06

08

1

12

14

0 50 100 150

Tu

mo

r V

olu

me (

cc)

Days after First Treatment

No Tx LCL

Single Single + LCL

Circle Circle + LCL

2 mm

4 mm

4 Protocols 100 Curative for

local disease

bull Hyperthermia +

TSL in 8 treatments

bull Hyperthermia+TSL

+ CpG in lt3

treatments

bull Hyperthermia +

Ablation + TSL in 3

treatments

bull Ablation + LCL

Wong et al in review

0

25

50

75

100

0 100 200

Su

rviv

al

Days After First Treatment

Hyp+TSL+Circle

TSL+Circle

TSL+Single

No Tx

0

25

50

75

100

0 40 80 120 160

Surv

ival

Time (days)

NT

LCL

Single

Single + LCL

Circle

Circle + LCL

None

TSL

LCL

Drug

US None Comb Hypertherm Ablation

lt100 cure

lt100 cure wCpG

100 cure wo CpG

100 cure w CpG

NA

NA

Curative Protocols

7142015

9

1 Protocol- extending survival in

systemic disease needs

optimization

bull Hyperthermia+TSL

+ CpG in lt3

treatments

Kheirolomoom et al in review

0 4 8 1 2 1 6 2 0 2 4 2 8 3 2 3 6 4 0 4 4 4 8 5 2 5 6 6 0 6 4

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

D a y p o s t tre a tm e n t

Su

rv

iva

l (

)

C o n tro l

C p G

C u D o x + U S + C p G

p=002

Conclusion

bull Imaging essential in developing new

nanotherapies identifying cell populations

evaluating efficacy

bull Activatable nanotherapies can cure local

disease enhance systemic therapy

bull Addition of immune adjuvants may allow

the accessible lesions to be treated

resulting in systemic benefits

Charles Caskey James Chomas Paul Dayton Lisa Even Brett Fite Karen Gagnon Xiaowen Hu Elizabeth Ingham Sarah Johnson Azadeh Kheirolomoom Dustin Kruse Jiro Kusunose Chun-Yen Lai Yu Liu Aaron Lum Karen Morgan Eric Paoli Shengping Qin Jai Woong Seo Susanne Stieger Kate Watson Andrew Wong Guodong Zhang Hua Zhang

U Bergen Frits Thorsen Cecilie Brekke Rolf Reed

UCD John Boone Sandy Borowsky Bob Cardiff

Nipavan Chiamvimonvat Roy Curry Greg Farwell

Bruce Hammock C Lebrilla Claude Meares Jinyi Qi

Tingrui Pan Scott Simon C Tepper So Yamada

LLNL Paul Hoeprich

G TechEmory Hanjoong Jo

Northwestern Tom Meade

BurnhamUCSB Juliana Hamzah Erkki Ruoslahti

UCB He Dong Ting Xu

Cedars Sinai Gadi Palled Dan Gazit Galina Shapiro

CMGI (Dave Kukis)

Pfizer Oncology

Targeson (Jack Rychak)

Image-Guided Therapy (Erik Dumont)

Siemens Medical Solutions

NCI and NIBIB

7142015

10

Manzoor AA et al Cancer Res 2012 Nov 172(21)5566-75

Dewhirst laboratory Duke University

Free dox

Dox-TSL

0 2 5 min

1 5 15 min of heat

Before

10 min after

heat ends

Intensity map (0 0 55) mm in x-y plane

Intensity map (0 5 55) mm in x-y plane

Thermal effects are very significant

7142015

11

Fite BZ et al PLoS ONE 7(4) e35509

Thermal and mechanical

oversight are feasible

MRgFUS insert from

Image Guided Therapy

7T 3 MHz ultrasound in

Bruker magnet

Heating is ON

from 0 to 20 s

With probe motion

05 1 15 2 25 3 35 4 45

05

1

15

2

25

3

35

4

45 1

15

2

25

3

35

4degC

Foiret et al submitted

US+ temperature sensitive

liposomes (TSL) or long circulating

liposomes (LCL)

bull US hyperthermia+TSL= local cure (100 in 8

treatments)

bull US hyperthermia+CpG+TSL= local cure (100

in 3 treatments) + slow systemic disease

ndash Needs optimization many alternatives

bull US ablation + LCL or TSL slow disease or cure

7142015

12

200 microm 200 microm

4mm 200 microm

After ablation

Canine melanoma - Combined radiotherapy

and immunotherapy using CPG

oligodeoxynucleotides and indolamine 23

dioxygenase (IDO) blockade

Monjazeb et al J Immunother Cancer 2013 1(Suppl 1) P256

Why nanoparticles Why ultrasound

Target accumulation

0

2

4

6

8

10

12

14

16

18

20

IDg

Paoli et al JCR 2010

7142015

3

CuDox-LTSLs

40 min US

US+

US+

CuDox-LTSLs

20 min US

Heart

Heart

Tumor

Thermally-activated vehicles

Below Tc Above Tc

DSPE-PEG-2000 DPPC Lyso-palmitoyl PC Drug

bull Direct and local release can

increase concentration and

decrease systemic toxicity

bull Small molecule can penetrate

tissue

Kheirolomoom et al JCR 2013

US-

US-

Tissue

Autofl

Dox

CuDox-lipo

100 nm

Copper-doxorubicin

temperature-sensitive

particles

Tissue

Autofl

Increasing

Dox

Particles US

C u D o x -L T S L s AS D o x -L T S L s

0

1

2

3

4

Do

xo

ru

bic

in f

luo

re

sc

en

ce

ra

tio

M u s c le + U S T u m o r

C u D o x -L T S L s AS D o x -L T S L s

0

2

4

6

Do

xo

ru

bic

in f

luo

re

sc

en

ce

ra

tio

T u m o r + U S M u s c le

Cu-dox temperature-

sensitive particles Conventional temperature-

sensitive particles

plt005 compared to control

Plt0001 compared to control

CuDox-TSL (8 treatments)

0

20

40

60

80

100

120

0 50 100 150 200 250

Su

rviv

al

Day post treatment

Control

CuDox-LTSLs

CuDox-LTSLs+US

-500

500

1500

2500

3500

4500

5500

0 10 20 30 40 50

Tu

mo

r g

row

th

Day post treatment

Control

Control+US

CuDox-LTSLs

CuDox-LTSLs+US

200 250

~

~

US

Treat 2xweek 4 weeks

6 mgkg

Kheirolomoom et al JCR 2013

7142015

4

1 mm

100 microm

Days

Kheirolomoom et al JCR 2013

2 Weeks

100 microm

1 mm

(Fe)

100 microm

(F480)

4 Weeks

1 mm

100 microm

001

01

1

10

100

03 07 10 20 40

ID

Time insonation (hrs)

05 cm

1 cm

2 cm

4 cm

Assumes 5 blood volume in tumor

10 sec tumor blood refresh

5 L blood volume

Diameter

ID delivered

depends on volume insonified

time of insonation

Why do we favor thermally-sensitive

nanoparticles

-10

-5

0

5

10

15

20

25

30

0 5 10 15 20 25 30We

igh

t c

ha

ng

e

Day post treatment

Control

CuDox-LTSLs

CuDox-LTSLs+US

0

5

10

15

20

25

WBC KmicroL RBCMmicroL

Blo

od

ce

ll c

ou

nt

CuDox-LTSLs

Control

0

2

4

6

8

10

Albumin Total protein

Pro

tein

g

dL

CuDox-LTSLs

Control

0

400

800

1200

1600

Org

an

we

igh

t m

g

CuDox-LTSLs

Control

Toxicity is low

7142015

5

Outline

Activatable particles

+ hyperthermia

Particles + ablation

+ adjuvants

Activatable particles +

hyperthermia +

adjuvants

Use of TLR agonists to enhance Teffector

cell function

adapted from Immunotherapy 2009 November 1 1(6) 949ndash964

DC Dendritic cell iDC Immature dendritic cell MDSC Myeloid-derived suppressor cell Macro Monocytemacrophage TAM Tumor-associated macrophage Teff Effector T cell Treg Regulatory T cell US ultrasound

TLR

activation

iDC

MDSC TAM Treg

Tumor

Teff

Teff

Macro

Th1

Mature

DC

IL-12

IFN-a TNF-a

Tumor Tumor

antigen

IL-12

IFN-g

IL-2

Copper-doxorubicin temperature-sensitive

particles + CpG (3 treatments)

0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0

0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

1 0 0 0 0

1 2 0 0 0

1 4 0 0 0

1 6 0 0 0

1 8 0 0 0

2 0 0 0 0

D a y p o s t tre a tm e n t

ND

L T

um

or

gro

wth

(

)

C o n tro l

C p G

C p G C o n tra la te ra l

C u D o x + U S + C p G

C u D o x + U S + C p G C o n tra la te ra l

7142015

6

CpG CpG

Contralateral

CuDox+US+CpG

Contralateral

CuDox+US+CpG

Control

2 mm

200 microm

CuDox

Abscopal response

Immune cell

populations

0

04

08

12

16

2

I

FN

-g+

C

D8

+ T

ce

lls

of

Le

uk

oc

yte

s

0

1

2

3

MD

SC

s

Outline

Activatable particles

+ hyperthermia

Particles + ablation

+ adjuvants

Activatable particles +

hyperthermia +

adjuvants

7142015

7

Long circulating

nanoparticles

Ablation + 64Cu-LCL

20

0 IDcc

25 IDcc

1cm

6 hours 20 hours 48 hours

0

1

0 2 4 6 8Op

tical

Den

sit

y

(Arb

itra

ry U

nit

s)

Location (mm)

Circle 1 Circle 2

Circle 3 No Tx

7142015

8

Ablate fraction of tumor + drug

0

02

04

06

08

1

12

14

0 50 100 150

Tu

mo

r V

olu

me (

cc)

Days after First Treatment

No Tx LCL

Single Single + LCL

Circle Circle + LCL

2 mm

4 mm

4 Protocols 100 Curative for

local disease

bull Hyperthermia +

TSL in 8 treatments

bull Hyperthermia+TSL

+ CpG in lt3

treatments

bull Hyperthermia +

Ablation + TSL in 3

treatments

bull Ablation + LCL

Wong et al in review

0

25

50

75

100

0 100 200

Su

rviv

al

Days After First Treatment

Hyp+TSL+Circle

TSL+Circle

TSL+Single

No Tx

0

25

50

75

100

0 40 80 120 160

Surv

ival

Time (days)

NT

LCL

Single

Single + LCL

Circle

Circle + LCL

None

TSL

LCL

Drug

US None Comb Hypertherm Ablation

lt100 cure

lt100 cure wCpG

100 cure wo CpG

100 cure w CpG

NA

NA

Curative Protocols

7142015

9

1 Protocol- extending survival in

systemic disease needs

optimization

bull Hyperthermia+TSL

+ CpG in lt3

treatments

Kheirolomoom et al in review

0 4 8 1 2 1 6 2 0 2 4 2 8 3 2 3 6 4 0 4 4 4 8 5 2 5 6 6 0 6 4

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

D a y p o s t tre a tm e n t

Su

rv

iva

l (

)

C o n tro l

C p G

C u D o x + U S + C p G

p=002

Conclusion

bull Imaging essential in developing new

nanotherapies identifying cell populations

evaluating efficacy

bull Activatable nanotherapies can cure local

disease enhance systemic therapy

bull Addition of immune adjuvants may allow

the accessible lesions to be treated

resulting in systemic benefits

Charles Caskey James Chomas Paul Dayton Lisa Even Brett Fite Karen Gagnon Xiaowen Hu Elizabeth Ingham Sarah Johnson Azadeh Kheirolomoom Dustin Kruse Jiro Kusunose Chun-Yen Lai Yu Liu Aaron Lum Karen Morgan Eric Paoli Shengping Qin Jai Woong Seo Susanne Stieger Kate Watson Andrew Wong Guodong Zhang Hua Zhang

U Bergen Frits Thorsen Cecilie Brekke Rolf Reed

UCD John Boone Sandy Borowsky Bob Cardiff

Nipavan Chiamvimonvat Roy Curry Greg Farwell

Bruce Hammock C Lebrilla Claude Meares Jinyi Qi

Tingrui Pan Scott Simon C Tepper So Yamada

LLNL Paul Hoeprich

G TechEmory Hanjoong Jo

Northwestern Tom Meade

BurnhamUCSB Juliana Hamzah Erkki Ruoslahti

UCB He Dong Ting Xu

Cedars Sinai Gadi Palled Dan Gazit Galina Shapiro

CMGI (Dave Kukis)

Pfizer Oncology

Targeson (Jack Rychak)

Image-Guided Therapy (Erik Dumont)

Siemens Medical Solutions

NCI and NIBIB

7142015

10

Manzoor AA et al Cancer Res 2012 Nov 172(21)5566-75

Dewhirst laboratory Duke University

Free dox

Dox-TSL

0 2 5 min

1 5 15 min of heat

Before

10 min after

heat ends

Intensity map (0 0 55) mm in x-y plane

Intensity map (0 5 55) mm in x-y plane

Thermal effects are very significant

7142015

11

Fite BZ et al PLoS ONE 7(4) e35509

Thermal and mechanical

oversight are feasible

MRgFUS insert from

Image Guided Therapy

7T 3 MHz ultrasound in

Bruker magnet

Heating is ON

from 0 to 20 s

With probe motion

05 1 15 2 25 3 35 4 45

05

1

15

2

25

3

35

4

45 1

15

2

25

3

35

4degC

Foiret et al submitted

US+ temperature sensitive

liposomes (TSL) or long circulating

liposomes (LCL)

bull US hyperthermia+TSL= local cure (100 in 8

treatments)

bull US hyperthermia+CpG+TSL= local cure (100

in 3 treatments) + slow systemic disease

ndash Needs optimization many alternatives

bull US ablation + LCL or TSL slow disease or cure

7142015

12

200 microm 200 microm

4mm 200 microm

After ablation

Canine melanoma - Combined radiotherapy

and immunotherapy using CPG

oligodeoxynucleotides and indolamine 23

dioxygenase (IDO) blockade

Monjazeb et al J Immunother Cancer 2013 1(Suppl 1) P256

Why nanoparticles Why ultrasound

Target accumulation

0

2

4

6

8

10

12

14

16

18

20

IDg

Paoli et al JCR 2010

7142015

4

1 mm

100 microm

Days

Kheirolomoom et al JCR 2013

2 Weeks

100 microm

1 mm

(Fe)

100 microm

(F480)

4 Weeks

1 mm

100 microm

001

01

1

10

100

03 07 10 20 40

ID

Time insonation (hrs)

05 cm

1 cm

2 cm

4 cm

Assumes 5 blood volume in tumor

10 sec tumor blood refresh

5 L blood volume

Diameter

ID delivered

depends on volume insonified

time of insonation

Why do we favor thermally-sensitive

nanoparticles

-10

-5

0

5

10

15

20

25

30

0 5 10 15 20 25 30We

igh

t c

ha

ng

e

Day post treatment

Control

CuDox-LTSLs

CuDox-LTSLs+US

0

5

10

15

20

25

WBC KmicroL RBCMmicroL

Blo

od

ce

ll c

ou

nt

CuDox-LTSLs

Control

0

2

4

6

8

10

Albumin Total protein

Pro

tein

g

dL

CuDox-LTSLs

Control

0

400

800

1200

1600

Org

an

we

igh

t m

g

CuDox-LTSLs

Control

Toxicity is low

7142015

5

Outline

Activatable particles

+ hyperthermia

Particles + ablation

+ adjuvants

Activatable particles +

hyperthermia +

adjuvants

Use of TLR agonists to enhance Teffector

cell function

adapted from Immunotherapy 2009 November 1 1(6) 949ndash964

DC Dendritic cell iDC Immature dendritic cell MDSC Myeloid-derived suppressor cell Macro Monocytemacrophage TAM Tumor-associated macrophage Teff Effector T cell Treg Regulatory T cell US ultrasound

TLR

activation

iDC

MDSC TAM Treg

Tumor

Teff

Teff

Macro

Th1

Mature

DC

IL-12

IFN-a TNF-a

Tumor Tumor

antigen

IL-12

IFN-g

IL-2

Copper-doxorubicin temperature-sensitive

particles + CpG (3 treatments)

0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0

0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

1 0 0 0 0

1 2 0 0 0

1 4 0 0 0

1 6 0 0 0

1 8 0 0 0

2 0 0 0 0

D a y p o s t tre a tm e n t

ND

L T

um

or

gro

wth

(

)

C o n tro l

C p G

C p G C o n tra la te ra l

C u D o x + U S + C p G

C u D o x + U S + C p G C o n tra la te ra l

7142015

6

CpG CpG

Contralateral

CuDox+US+CpG

Contralateral

CuDox+US+CpG

Control

2 mm

200 microm

CuDox

Abscopal response

Immune cell

populations

0

04

08

12

16

2

I

FN

-g+

C

D8

+ T

ce

lls

of

Le

uk

oc

yte

s

0

1

2

3

MD

SC

s

Outline

Activatable particles

+ hyperthermia

Particles + ablation

+ adjuvants

Activatable particles +

hyperthermia +

adjuvants

7142015

7

Long circulating

nanoparticles

Ablation + 64Cu-LCL

20

0 IDcc

25 IDcc

1cm

6 hours 20 hours 48 hours

0

1

0 2 4 6 8Op

tical

Den

sit

y

(Arb

itra

ry U

nit

s)

Location (mm)

Circle 1 Circle 2

Circle 3 No Tx

7142015

8

Ablate fraction of tumor + drug

0

02

04

06

08

1

12

14

0 50 100 150

Tu

mo

r V

olu

me (

cc)

Days after First Treatment

No Tx LCL

Single Single + LCL

Circle Circle + LCL

2 mm

4 mm

4 Protocols 100 Curative for

local disease

bull Hyperthermia +

TSL in 8 treatments

bull Hyperthermia+TSL

+ CpG in lt3

treatments

bull Hyperthermia +

Ablation + TSL in 3

treatments

bull Ablation + LCL

Wong et al in review

0

25

50

75

100

0 100 200

Su

rviv

al

Days After First Treatment

Hyp+TSL+Circle

TSL+Circle

TSL+Single

No Tx

0

25

50

75

100

0 40 80 120 160

Surv

ival

Time (days)

NT

LCL

Single

Single + LCL

Circle

Circle + LCL

None

TSL

LCL

Drug

US None Comb Hypertherm Ablation

lt100 cure

lt100 cure wCpG

100 cure wo CpG

100 cure w CpG

NA

NA

Curative Protocols

7142015

9

1 Protocol- extending survival in

systemic disease needs

optimization

bull Hyperthermia+TSL

+ CpG in lt3

treatments

Kheirolomoom et al in review

0 4 8 1 2 1 6 2 0 2 4 2 8 3 2 3 6 4 0 4 4 4 8 5 2 5 6 6 0 6 4

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

D a y p o s t tre a tm e n t

Su

rv

iva

l (

)

C o n tro l

C p G

C u D o x + U S + C p G

p=002

Conclusion

bull Imaging essential in developing new

nanotherapies identifying cell populations

evaluating efficacy

bull Activatable nanotherapies can cure local

disease enhance systemic therapy

bull Addition of immune adjuvants may allow

the accessible lesions to be treated

resulting in systemic benefits

Charles Caskey James Chomas Paul Dayton Lisa Even Brett Fite Karen Gagnon Xiaowen Hu Elizabeth Ingham Sarah Johnson Azadeh Kheirolomoom Dustin Kruse Jiro Kusunose Chun-Yen Lai Yu Liu Aaron Lum Karen Morgan Eric Paoli Shengping Qin Jai Woong Seo Susanne Stieger Kate Watson Andrew Wong Guodong Zhang Hua Zhang

U Bergen Frits Thorsen Cecilie Brekke Rolf Reed

UCD John Boone Sandy Borowsky Bob Cardiff

Nipavan Chiamvimonvat Roy Curry Greg Farwell

Bruce Hammock C Lebrilla Claude Meares Jinyi Qi

Tingrui Pan Scott Simon C Tepper So Yamada

LLNL Paul Hoeprich

G TechEmory Hanjoong Jo

Northwestern Tom Meade

BurnhamUCSB Juliana Hamzah Erkki Ruoslahti

UCB He Dong Ting Xu

Cedars Sinai Gadi Palled Dan Gazit Galina Shapiro

CMGI (Dave Kukis)

Pfizer Oncology

Targeson (Jack Rychak)

Image-Guided Therapy (Erik Dumont)

Siemens Medical Solutions

NCI and NIBIB

7142015

10

Manzoor AA et al Cancer Res 2012 Nov 172(21)5566-75

Dewhirst laboratory Duke University

Free dox

Dox-TSL

0 2 5 min

1 5 15 min of heat

Before

10 min after

heat ends

Intensity map (0 0 55) mm in x-y plane

Intensity map (0 5 55) mm in x-y plane

Thermal effects are very significant

7142015

11

Fite BZ et al PLoS ONE 7(4) e35509

Thermal and mechanical

oversight are feasible

MRgFUS insert from

Image Guided Therapy

7T 3 MHz ultrasound in

Bruker magnet

Heating is ON

from 0 to 20 s

With probe motion

05 1 15 2 25 3 35 4 45

05

1

15

2

25

3

35

4

45 1

15

2

25

3

35

4degC

Foiret et al submitted

US+ temperature sensitive

liposomes (TSL) or long circulating

liposomes (LCL)

bull US hyperthermia+TSL= local cure (100 in 8

treatments)

bull US hyperthermia+CpG+TSL= local cure (100

in 3 treatments) + slow systemic disease

ndash Needs optimization many alternatives

bull US ablation + LCL or TSL slow disease or cure

7142015

12

200 microm 200 microm

4mm 200 microm

After ablation

Canine melanoma - Combined radiotherapy

and immunotherapy using CPG

oligodeoxynucleotides and indolamine 23

dioxygenase (IDO) blockade

Monjazeb et al J Immunother Cancer 2013 1(Suppl 1) P256

Why nanoparticles Why ultrasound

Target accumulation

0

2

4

6

8

10

12

14

16

18

20

IDg

Paoli et al JCR 2010

7142015

5

Outline

Activatable particles

+ hyperthermia

Particles + ablation

+ adjuvants

Activatable particles +

hyperthermia +

adjuvants

Use of TLR agonists to enhance Teffector

cell function

adapted from Immunotherapy 2009 November 1 1(6) 949ndash964

DC Dendritic cell iDC Immature dendritic cell MDSC Myeloid-derived suppressor cell Macro Monocytemacrophage TAM Tumor-associated macrophage Teff Effector T cell Treg Regulatory T cell US ultrasound

TLR

activation

iDC

MDSC TAM Treg

Tumor

Teff

Teff

Macro

Th1

Mature

DC

IL-12

IFN-a TNF-a

Tumor Tumor

antigen

IL-12

IFN-g

IL-2

Copper-doxorubicin temperature-sensitive

particles + CpG (3 treatments)

0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0

0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

1 0 0 0 0

1 2 0 0 0

1 4 0 0 0

1 6 0 0 0

1 8 0 0 0

2 0 0 0 0

D a y p o s t tre a tm e n t

ND

L T

um

or

gro

wth

(

)

C o n tro l

C p G

C p G C o n tra la te ra l

C u D o x + U S + C p G

C u D o x + U S + C p G C o n tra la te ra l

7142015

6

CpG CpG

Contralateral

CuDox+US+CpG

Contralateral

CuDox+US+CpG

Control

2 mm

200 microm

CuDox

Abscopal response

Immune cell

populations

0

04

08

12

16

2

I

FN

-g+

C

D8

+ T

ce

lls

of

Le

uk

oc

yte

s

0

1

2

3

MD

SC

s

Outline

Activatable particles

+ hyperthermia

Particles + ablation

+ adjuvants

Activatable particles +

hyperthermia +

adjuvants

7142015

7

Long circulating

nanoparticles

Ablation + 64Cu-LCL

20

0 IDcc

25 IDcc

1cm

6 hours 20 hours 48 hours

0

1

0 2 4 6 8Op

tical

Den

sit

y

(Arb

itra

ry U

nit

s)

Location (mm)

Circle 1 Circle 2

Circle 3 No Tx

7142015

8

Ablate fraction of tumor + drug

0

02

04

06

08

1

12

14

0 50 100 150

Tu

mo

r V

olu

me (

cc)

Days after First Treatment

No Tx LCL

Single Single + LCL

Circle Circle + LCL

2 mm

4 mm

4 Protocols 100 Curative for

local disease

bull Hyperthermia +

TSL in 8 treatments

bull Hyperthermia+TSL

+ CpG in lt3

treatments

bull Hyperthermia +

Ablation + TSL in 3

treatments

bull Ablation + LCL

Wong et al in review

0

25

50

75

100

0 100 200

Su

rviv

al

Days After First Treatment

Hyp+TSL+Circle

TSL+Circle

TSL+Single

No Tx

0

25

50

75

100

0 40 80 120 160

Surv

ival

Time (days)

NT

LCL

Single

Single + LCL

Circle

Circle + LCL

None

TSL

LCL

Drug

US None Comb Hypertherm Ablation

lt100 cure

lt100 cure wCpG

100 cure wo CpG

100 cure w CpG

NA

NA

Curative Protocols

7142015

9

1 Protocol- extending survival in

systemic disease needs

optimization

bull Hyperthermia+TSL

+ CpG in lt3

treatments

Kheirolomoom et al in review

0 4 8 1 2 1 6 2 0 2 4 2 8 3 2 3 6 4 0 4 4 4 8 5 2 5 6 6 0 6 4

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

D a y p o s t tre a tm e n t

Su

rv

iva

l (

)

C o n tro l

C p G

C u D o x + U S + C p G

p=002

Conclusion

bull Imaging essential in developing new

nanotherapies identifying cell populations

evaluating efficacy

bull Activatable nanotherapies can cure local

disease enhance systemic therapy

bull Addition of immune adjuvants may allow

the accessible lesions to be treated

resulting in systemic benefits

Charles Caskey James Chomas Paul Dayton Lisa Even Brett Fite Karen Gagnon Xiaowen Hu Elizabeth Ingham Sarah Johnson Azadeh Kheirolomoom Dustin Kruse Jiro Kusunose Chun-Yen Lai Yu Liu Aaron Lum Karen Morgan Eric Paoli Shengping Qin Jai Woong Seo Susanne Stieger Kate Watson Andrew Wong Guodong Zhang Hua Zhang

U Bergen Frits Thorsen Cecilie Brekke Rolf Reed

UCD John Boone Sandy Borowsky Bob Cardiff

Nipavan Chiamvimonvat Roy Curry Greg Farwell

Bruce Hammock C Lebrilla Claude Meares Jinyi Qi

Tingrui Pan Scott Simon C Tepper So Yamada

LLNL Paul Hoeprich

G TechEmory Hanjoong Jo

Northwestern Tom Meade

BurnhamUCSB Juliana Hamzah Erkki Ruoslahti

UCB He Dong Ting Xu

Cedars Sinai Gadi Palled Dan Gazit Galina Shapiro

CMGI (Dave Kukis)

Pfizer Oncology

Targeson (Jack Rychak)

Image-Guided Therapy (Erik Dumont)

Siemens Medical Solutions

NCI and NIBIB

7142015

10

Manzoor AA et al Cancer Res 2012 Nov 172(21)5566-75

Dewhirst laboratory Duke University

Free dox

Dox-TSL

0 2 5 min

1 5 15 min of heat

Before

10 min after

heat ends

Intensity map (0 0 55) mm in x-y plane

Intensity map (0 5 55) mm in x-y plane

Thermal effects are very significant

7142015

11

Fite BZ et al PLoS ONE 7(4) e35509

Thermal and mechanical

oversight are feasible

MRgFUS insert from

Image Guided Therapy

7T 3 MHz ultrasound in

Bruker magnet

Heating is ON

from 0 to 20 s

With probe motion

05 1 15 2 25 3 35 4 45

05

1

15

2

25

3

35

4

45 1

15

2

25

3

35

4degC

Foiret et al submitted

US+ temperature sensitive

liposomes (TSL) or long circulating

liposomes (LCL)

bull US hyperthermia+TSL= local cure (100 in 8

treatments)

bull US hyperthermia+CpG+TSL= local cure (100

in 3 treatments) + slow systemic disease

ndash Needs optimization many alternatives

bull US ablation + LCL or TSL slow disease or cure

7142015

12

200 microm 200 microm

4mm 200 microm

After ablation

Canine melanoma - Combined radiotherapy

and immunotherapy using CPG

oligodeoxynucleotides and indolamine 23

dioxygenase (IDO) blockade

Monjazeb et al J Immunother Cancer 2013 1(Suppl 1) P256

Why nanoparticles Why ultrasound

Target accumulation

0

2

4

6

8

10

12

14

16

18

20

IDg

Paoli et al JCR 2010

7142015

6

CpG CpG

Contralateral

CuDox+US+CpG

Contralateral

CuDox+US+CpG

Control

2 mm

200 microm

CuDox

Abscopal response

Immune cell

populations

0

04

08

12

16

2

I

FN

-g+

C

D8

+ T

ce

lls

of

Le

uk

oc

yte

s

0

1

2

3

MD

SC

s

Outline

Activatable particles

+ hyperthermia

Particles + ablation

+ adjuvants

Activatable particles +

hyperthermia +

adjuvants

7142015

7

Long circulating

nanoparticles

Ablation + 64Cu-LCL

20

0 IDcc

25 IDcc

1cm

6 hours 20 hours 48 hours

0

1

0 2 4 6 8Op

tical

Den

sit

y

(Arb

itra

ry U

nit

s)

Location (mm)

Circle 1 Circle 2

Circle 3 No Tx

7142015

8

Ablate fraction of tumor + drug

0

02

04

06

08

1

12

14

0 50 100 150

Tu

mo

r V

olu

me (

cc)

Days after First Treatment

No Tx LCL

Single Single + LCL

Circle Circle + LCL

2 mm

4 mm

4 Protocols 100 Curative for

local disease

bull Hyperthermia +

TSL in 8 treatments

bull Hyperthermia+TSL

+ CpG in lt3

treatments

bull Hyperthermia +

Ablation + TSL in 3

treatments

bull Ablation + LCL

Wong et al in review

0

25

50

75

100

0 100 200

Su

rviv

al

Days After First Treatment

Hyp+TSL+Circle

TSL+Circle

TSL+Single

No Tx

0

25

50

75

100

0 40 80 120 160

Surv

ival

Time (days)

NT

LCL

Single

Single + LCL

Circle

Circle + LCL

None

TSL

LCL

Drug

US None Comb Hypertherm Ablation

lt100 cure

lt100 cure wCpG

100 cure wo CpG

100 cure w CpG

NA

NA

Curative Protocols

7142015

9

1 Protocol- extending survival in

systemic disease needs

optimization

bull Hyperthermia+TSL

+ CpG in lt3

treatments

Kheirolomoom et al in review

0 4 8 1 2 1 6 2 0 2 4 2 8 3 2 3 6 4 0 4 4 4 8 5 2 5 6 6 0 6 4

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

D a y p o s t tre a tm e n t

Su

rv

iva

l (

)

C o n tro l

C p G

C u D o x + U S + C p G

p=002

Conclusion

bull Imaging essential in developing new

nanotherapies identifying cell populations

evaluating efficacy

bull Activatable nanotherapies can cure local

disease enhance systemic therapy

bull Addition of immune adjuvants may allow

the accessible lesions to be treated

resulting in systemic benefits

Charles Caskey James Chomas Paul Dayton Lisa Even Brett Fite Karen Gagnon Xiaowen Hu Elizabeth Ingham Sarah Johnson Azadeh Kheirolomoom Dustin Kruse Jiro Kusunose Chun-Yen Lai Yu Liu Aaron Lum Karen Morgan Eric Paoli Shengping Qin Jai Woong Seo Susanne Stieger Kate Watson Andrew Wong Guodong Zhang Hua Zhang

U Bergen Frits Thorsen Cecilie Brekke Rolf Reed

UCD John Boone Sandy Borowsky Bob Cardiff

Nipavan Chiamvimonvat Roy Curry Greg Farwell

Bruce Hammock C Lebrilla Claude Meares Jinyi Qi

Tingrui Pan Scott Simon C Tepper So Yamada

LLNL Paul Hoeprich

G TechEmory Hanjoong Jo

Northwestern Tom Meade

BurnhamUCSB Juliana Hamzah Erkki Ruoslahti

UCB He Dong Ting Xu

Cedars Sinai Gadi Palled Dan Gazit Galina Shapiro

CMGI (Dave Kukis)

Pfizer Oncology

Targeson (Jack Rychak)

Image-Guided Therapy (Erik Dumont)

Siemens Medical Solutions

NCI and NIBIB

7142015

10

Manzoor AA et al Cancer Res 2012 Nov 172(21)5566-75

Dewhirst laboratory Duke University

Free dox

Dox-TSL

0 2 5 min

1 5 15 min of heat

Before

10 min after

heat ends

Intensity map (0 0 55) mm in x-y plane

Intensity map (0 5 55) mm in x-y plane

Thermal effects are very significant

7142015

11

Fite BZ et al PLoS ONE 7(4) e35509

Thermal and mechanical

oversight are feasible

MRgFUS insert from

Image Guided Therapy

7T 3 MHz ultrasound in

Bruker magnet

Heating is ON

from 0 to 20 s

With probe motion

05 1 15 2 25 3 35 4 45

05

1

15

2

25

3

35

4

45 1

15

2

25

3

35

4degC

Foiret et al submitted

US+ temperature sensitive

liposomes (TSL) or long circulating

liposomes (LCL)

bull US hyperthermia+TSL= local cure (100 in 8

treatments)

bull US hyperthermia+CpG+TSL= local cure (100

in 3 treatments) + slow systemic disease

ndash Needs optimization many alternatives

bull US ablation + LCL or TSL slow disease or cure

7142015

12

200 microm 200 microm

4mm 200 microm

After ablation

Canine melanoma - Combined radiotherapy

and immunotherapy using CPG

oligodeoxynucleotides and indolamine 23

dioxygenase (IDO) blockade

Monjazeb et al J Immunother Cancer 2013 1(Suppl 1) P256

Why nanoparticles Why ultrasound

Target accumulation

0

2

4

6

8

10

12

14

16

18

20

IDg

Paoli et al JCR 2010

7142015

7

Long circulating

nanoparticles

Ablation + 64Cu-LCL

20

0 IDcc

25 IDcc

1cm

6 hours 20 hours 48 hours

0

1

0 2 4 6 8Op

tical

Den

sit

y

(Arb

itra

ry U

nit

s)

Location (mm)

Circle 1 Circle 2

Circle 3 No Tx

7142015

8

Ablate fraction of tumor + drug

0

02

04

06

08

1

12

14

0 50 100 150

Tu

mo

r V

olu

me (

cc)

Days after First Treatment

No Tx LCL

Single Single + LCL

Circle Circle + LCL

2 mm

4 mm

4 Protocols 100 Curative for

local disease

bull Hyperthermia +

TSL in 8 treatments

bull Hyperthermia+TSL

+ CpG in lt3

treatments

bull Hyperthermia +

Ablation + TSL in 3

treatments

bull Ablation + LCL

Wong et al in review

0

25

50

75

100

0 100 200

Su

rviv

al

Days After First Treatment

Hyp+TSL+Circle

TSL+Circle

TSL+Single

No Tx

0

25

50

75

100

0 40 80 120 160

Surv

ival

Time (days)

NT

LCL

Single

Single + LCL

Circle

Circle + LCL

None

TSL

LCL

Drug

US None Comb Hypertherm Ablation

lt100 cure

lt100 cure wCpG

100 cure wo CpG

100 cure w CpG

NA

NA

Curative Protocols

7142015

9

1 Protocol- extending survival in

systemic disease needs

optimization

bull Hyperthermia+TSL

+ CpG in lt3

treatments

Kheirolomoom et al in review

0 4 8 1 2 1 6 2 0 2 4 2 8 3 2 3 6 4 0 4 4 4 8 5 2 5 6 6 0 6 4

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

D a y p o s t tre a tm e n t

Su

rv

iva

l (

)

C o n tro l

C p G

C u D o x + U S + C p G

p=002

Conclusion

bull Imaging essential in developing new

nanotherapies identifying cell populations

evaluating efficacy

bull Activatable nanotherapies can cure local

disease enhance systemic therapy

bull Addition of immune adjuvants may allow

the accessible lesions to be treated

resulting in systemic benefits

Charles Caskey James Chomas Paul Dayton Lisa Even Brett Fite Karen Gagnon Xiaowen Hu Elizabeth Ingham Sarah Johnson Azadeh Kheirolomoom Dustin Kruse Jiro Kusunose Chun-Yen Lai Yu Liu Aaron Lum Karen Morgan Eric Paoli Shengping Qin Jai Woong Seo Susanne Stieger Kate Watson Andrew Wong Guodong Zhang Hua Zhang

U Bergen Frits Thorsen Cecilie Brekke Rolf Reed

UCD John Boone Sandy Borowsky Bob Cardiff

Nipavan Chiamvimonvat Roy Curry Greg Farwell

Bruce Hammock C Lebrilla Claude Meares Jinyi Qi

Tingrui Pan Scott Simon C Tepper So Yamada

LLNL Paul Hoeprich

G TechEmory Hanjoong Jo

Northwestern Tom Meade

BurnhamUCSB Juliana Hamzah Erkki Ruoslahti

UCB He Dong Ting Xu

Cedars Sinai Gadi Palled Dan Gazit Galina Shapiro

CMGI (Dave Kukis)

Pfizer Oncology

Targeson (Jack Rychak)

Image-Guided Therapy (Erik Dumont)

Siemens Medical Solutions

NCI and NIBIB

7142015

10

Manzoor AA et al Cancer Res 2012 Nov 172(21)5566-75

Dewhirst laboratory Duke University

Free dox

Dox-TSL

0 2 5 min

1 5 15 min of heat

Before

10 min after

heat ends

Intensity map (0 0 55) mm in x-y plane

Intensity map (0 5 55) mm in x-y plane

Thermal effects are very significant

7142015

11

Fite BZ et al PLoS ONE 7(4) e35509

Thermal and mechanical

oversight are feasible

MRgFUS insert from

Image Guided Therapy

7T 3 MHz ultrasound in

Bruker magnet

Heating is ON

from 0 to 20 s

With probe motion

05 1 15 2 25 3 35 4 45

05

1

15

2

25

3

35

4

45 1

15

2

25

3

35

4degC

Foiret et al submitted

US+ temperature sensitive

liposomes (TSL) or long circulating

liposomes (LCL)

bull US hyperthermia+TSL= local cure (100 in 8

treatments)

bull US hyperthermia+CpG+TSL= local cure (100

in 3 treatments) + slow systemic disease

ndash Needs optimization many alternatives

bull US ablation + LCL or TSL slow disease or cure

7142015

12

200 microm 200 microm

4mm 200 microm

After ablation

Canine melanoma - Combined radiotherapy

and immunotherapy using CPG

oligodeoxynucleotides and indolamine 23

dioxygenase (IDO) blockade

Monjazeb et al J Immunother Cancer 2013 1(Suppl 1) P256

Why nanoparticles Why ultrasound

Target accumulation

0

2

4

6

8

10

12

14

16

18

20

IDg

Paoli et al JCR 2010

7142015

8

Ablate fraction of tumor + drug

0

02

04

06

08

1

12

14

0 50 100 150

Tu

mo

r V

olu

me (

cc)

Days after First Treatment

No Tx LCL

Single Single + LCL

Circle Circle + LCL

2 mm

4 mm

4 Protocols 100 Curative for

local disease

bull Hyperthermia +

TSL in 8 treatments

bull Hyperthermia+TSL

+ CpG in lt3

treatments

bull Hyperthermia +

Ablation + TSL in 3

treatments

bull Ablation + LCL

Wong et al in review

0

25

50

75

100

0 100 200

Su

rviv

al

Days After First Treatment

Hyp+TSL+Circle

TSL+Circle

TSL+Single

No Tx

0

25

50

75

100

0 40 80 120 160

Surv

ival

Time (days)

NT

LCL

Single

Single + LCL

Circle

Circle + LCL

None

TSL

LCL

Drug

US None Comb Hypertherm Ablation

lt100 cure

lt100 cure wCpG

100 cure wo CpG

100 cure w CpG

NA

NA

Curative Protocols

7142015

9

1 Protocol- extending survival in

systemic disease needs

optimization

bull Hyperthermia+TSL

+ CpG in lt3

treatments

Kheirolomoom et al in review

0 4 8 1 2 1 6 2 0 2 4 2 8 3 2 3 6 4 0 4 4 4 8 5 2 5 6 6 0 6 4

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

D a y p o s t tre a tm e n t

Su

rv

iva

l (

)

C o n tro l

C p G

C u D o x + U S + C p G

p=002

Conclusion

bull Imaging essential in developing new

nanotherapies identifying cell populations

evaluating efficacy

bull Activatable nanotherapies can cure local

disease enhance systemic therapy

bull Addition of immune adjuvants may allow

the accessible lesions to be treated

resulting in systemic benefits

Charles Caskey James Chomas Paul Dayton Lisa Even Brett Fite Karen Gagnon Xiaowen Hu Elizabeth Ingham Sarah Johnson Azadeh Kheirolomoom Dustin Kruse Jiro Kusunose Chun-Yen Lai Yu Liu Aaron Lum Karen Morgan Eric Paoli Shengping Qin Jai Woong Seo Susanne Stieger Kate Watson Andrew Wong Guodong Zhang Hua Zhang

U Bergen Frits Thorsen Cecilie Brekke Rolf Reed

UCD John Boone Sandy Borowsky Bob Cardiff

Nipavan Chiamvimonvat Roy Curry Greg Farwell

Bruce Hammock C Lebrilla Claude Meares Jinyi Qi

Tingrui Pan Scott Simon C Tepper So Yamada

LLNL Paul Hoeprich

G TechEmory Hanjoong Jo

Northwestern Tom Meade

BurnhamUCSB Juliana Hamzah Erkki Ruoslahti

UCB He Dong Ting Xu

Cedars Sinai Gadi Palled Dan Gazit Galina Shapiro

CMGI (Dave Kukis)

Pfizer Oncology

Targeson (Jack Rychak)

Image-Guided Therapy (Erik Dumont)

Siemens Medical Solutions

NCI and NIBIB

7142015

10

Manzoor AA et al Cancer Res 2012 Nov 172(21)5566-75

Dewhirst laboratory Duke University

Free dox

Dox-TSL

0 2 5 min

1 5 15 min of heat

Before

10 min after

heat ends

Intensity map (0 0 55) mm in x-y plane

Intensity map (0 5 55) mm in x-y plane

Thermal effects are very significant

7142015

11

Fite BZ et al PLoS ONE 7(4) e35509

Thermal and mechanical

oversight are feasible

MRgFUS insert from

Image Guided Therapy

7T 3 MHz ultrasound in

Bruker magnet

Heating is ON

from 0 to 20 s

With probe motion

05 1 15 2 25 3 35 4 45

05

1

15

2

25

3

35

4

45 1

15

2

25

3

35

4degC

Foiret et al submitted

US+ temperature sensitive

liposomes (TSL) or long circulating

liposomes (LCL)

bull US hyperthermia+TSL= local cure (100 in 8

treatments)

bull US hyperthermia+CpG+TSL= local cure (100

in 3 treatments) + slow systemic disease

ndash Needs optimization many alternatives

bull US ablation + LCL or TSL slow disease or cure

7142015

12

200 microm 200 microm

4mm 200 microm

After ablation

Canine melanoma - Combined radiotherapy

and immunotherapy using CPG

oligodeoxynucleotides and indolamine 23

dioxygenase (IDO) blockade

Monjazeb et al J Immunother Cancer 2013 1(Suppl 1) P256

Why nanoparticles Why ultrasound

Target accumulation

0

2

4

6

8

10

12

14

16

18

20

IDg

Paoli et al JCR 2010

7142015

9

1 Protocol- extending survival in

systemic disease needs

optimization

bull Hyperthermia+TSL

+ CpG in lt3

treatments

Kheirolomoom et al in review

0 4 8 1 2 1 6 2 0 2 4 2 8 3 2 3 6 4 0 4 4 4 8 5 2 5 6 6 0 6 4

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

D a y p o s t tre a tm e n t

Su

rv

iva

l (

)

C o n tro l

C p G

C u D o x + U S + C p G

p=002

Conclusion

bull Imaging essential in developing new

nanotherapies identifying cell populations

evaluating efficacy

bull Activatable nanotherapies can cure local

disease enhance systemic therapy

bull Addition of immune adjuvants may allow

the accessible lesions to be treated

resulting in systemic benefits

Charles Caskey James Chomas Paul Dayton Lisa Even Brett Fite Karen Gagnon Xiaowen Hu Elizabeth Ingham Sarah Johnson Azadeh Kheirolomoom Dustin Kruse Jiro Kusunose Chun-Yen Lai Yu Liu Aaron Lum Karen Morgan Eric Paoli Shengping Qin Jai Woong Seo Susanne Stieger Kate Watson Andrew Wong Guodong Zhang Hua Zhang

U Bergen Frits Thorsen Cecilie Brekke Rolf Reed

UCD John Boone Sandy Borowsky Bob Cardiff

Nipavan Chiamvimonvat Roy Curry Greg Farwell

Bruce Hammock C Lebrilla Claude Meares Jinyi Qi

Tingrui Pan Scott Simon C Tepper So Yamada

LLNL Paul Hoeprich

G TechEmory Hanjoong Jo

Northwestern Tom Meade

BurnhamUCSB Juliana Hamzah Erkki Ruoslahti

UCB He Dong Ting Xu

Cedars Sinai Gadi Palled Dan Gazit Galina Shapiro

CMGI (Dave Kukis)

Pfizer Oncology

Targeson (Jack Rychak)

Image-Guided Therapy (Erik Dumont)

Siemens Medical Solutions

NCI and NIBIB

7142015

10

Manzoor AA et al Cancer Res 2012 Nov 172(21)5566-75

Dewhirst laboratory Duke University

Free dox

Dox-TSL

0 2 5 min

1 5 15 min of heat

Before

10 min after

heat ends

Intensity map (0 0 55) mm in x-y plane

Intensity map (0 5 55) mm in x-y plane

Thermal effects are very significant

7142015

11

Fite BZ et al PLoS ONE 7(4) e35509

Thermal and mechanical

oversight are feasible

MRgFUS insert from

Image Guided Therapy

7T 3 MHz ultrasound in

Bruker magnet

Heating is ON

from 0 to 20 s

With probe motion

05 1 15 2 25 3 35 4 45

05

1

15

2

25

3

35

4

45 1

15

2

25

3

35

4degC

Foiret et al submitted

US+ temperature sensitive

liposomes (TSL) or long circulating

liposomes (LCL)

bull US hyperthermia+TSL= local cure (100 in 8

treatments)

bull US hyperthermia+CpG+TSL= local cure (100

in 3 treatments) + slow systemic disease

ndash Needs optimization many alternatives

bull US ablation + LCL or TSL slow disease or cure

7142015

12

200 microm 200 microm

4mm 200 microm

After ablation

Canine melanoma - Combined radiotherapy

and immunotherapy using CPG

oligodeoxynucleotides and indolamine 23

dioxygenase (IDO) blockade

Monjazeb et al J Immunother Cancer 2013 1(Suppl 1) P256

Why nanoparticles Why ultrasound

Target accumulation

0

2

4

6

8

10

12

14

16

18

20

IDg

Paoli et al JCR 2010

7142015

10

Manzoor AA et al Cancer Res 2012 Nov 172(21)5566-75

Dewhirst laboratory Duke University

Free dox

Dox-TSL

0 2 5 min

1 5 15 min of heat

Before

10 min after

heat ends

Intensity map (0 0 55) mm in x-y plane

Intensity map (0 5 55) mm in x-y plane

Thermal effects are very significant

7142015

11

Fite BZ et al PLoS ONE 7(4) e35509

Thermal and mechanical

oversight are feasible

MRgFUS insert from

Image Guided Therapy

7T 3 MHz ultrasound in

Bruker magnet

Heating is ON

from 0 to 20 s

With probe motion

05 1 15 2 25 3 35 4 45

05

1

15

2

25

3

35

4

45 1

15

2

25

3

35

4degC

Foiret et al submitted

US+ temperature sensitive

liposomes (TSL) or long circulating

liposomes (LCL)

bull US hyperthermia+TSL= local cure (100 in 8

treatments)

bull US hyperthermia+CpG+TSL= local cure (100

in 3 treatments) + slow systemic disease

ndash Needs optimization many alternatives

bull US ablation + LCL or TSL slow disease or cure

7142015

12

200 microm 200 microm

4mm 200 microm

After ablation

Canine melanoma - Combined radiotherapy

and immunotherapy using CPG

oligodeoxynucleotides and indolamine 23

dioxygenase (IDO) blockade

Monjazeb et al J Immunother Cancer 2013 1(Suppl 1) P256

Why nanoparticles Why ultrasound

Target accumulation

0

2

4

6

8

10

12

14

16

18

20

IDg

Paoli et al JCR 2010

7142015

11

Fite BZ et al PLoS ONE 7(4) e35509

Thermal and mechanical

oversight are feasible

MRgFUS insert from

Image Guided Therapy

7T 3 MHz ultrasound in

Bruker magnet

Heating is ON

from 0 to 20 s

With probe motion

05 1 15 2 25 3 35 4 45

05

1

15

2

25

3

35

4

45 1

15

2

25

3

35

4degC

Foiret et al submitted

US+ temperature sensitive

liposomes (TSL) or long circulating

liposomes (LCL)

bull US hyperthermia+TSL= local cure (100 in 8

treatments)

bull US hyperthermia+CpG+TSL= local cure (100

in 3 treatments) + slow systemic disease

ndash Needs optimization many alternatives

bull US ablation + LCL or TSL slow disease or cure

7142015

12

200 microm 200 microm

4mm 200 microm

After ablation

Canine melanoma - Combined radiotherapy

and immunotherapy using CPG

oligodeoxynucleotides and indolamine 23

dioxygenase (IDO) blockade

Monjazeb et al J Immunother Cancer 2013 1(Suppl 1) P256

Why nanoparticles Why ultrasound

Target accumulation

0

2

4

6

8

10

12

14

16

18

20

IDg

Paoli et al JCR 2010

7142015

12

200 microm 200 microm

4mm 200 microm

After ablation

Canine melanoma - Combined radiotherapy

and immunotherapy using CPG

oligodeoxynucleotides and indolamine 23

dioxygenase (IDO) blockade

Monjazeb et al J Immunother Cancer 2013 1(Suppl 1) P256

Why nanoparticles Why ultrasound

Target accumulation

0

2

4

6

8

10

12

14

16

18

20

IDg

Paoli et al JCR 2010

7142015

13

Ultrasound used to guide hyperthermia

-eg Multi-frequency arrays

Therapy

Imaging

Therapy 128 elements at 5 - 7 MHz 027 mm pitch 384mm 4mm element

64 elements at 15 MHz 08 mm pitch 384 mm 5mm element

43 mm

24 mm

Stephens

et al

PMB

2008

IEEE

UFFC

2011

MRfUS

Image-Guided Therapy (IGT) system

bull 16 element annular array

bull Diameter 45mm

bull Radius of curvature 35mm

bull Aperture 85 degrees

bull Operating frequency 3MHz

bull Acoustic efficiency gt 65

bull Power rating 50Wac

bull Bandwidth 300KHz

bull Focal spot size (approx) 15mm x 15mm x 2mm

Temperature-sensitive particles

+ ultrasound= local cure

Temperature-sensitive particles

+ ultrasound + adjuvant = systemic cure

CuDox-lipo

100 nm

Copper-doxorubicin

temperature-sensitive

particles + CpG

Particles

US

Adjuvant

7142015

14

Day 7 Foxp3+ (Treg) cells

Contralateral tumors- in rim

Directly treated-

None in rim or tumor

Lymph node

Tumor

7142015

14

Day 7 Foxp3+ (Treg) cells

Contralateral tumors- in rim

Directly treated-

None in rim or tumor

Lymph node

Tumor