7/16/2019

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Robert Ivkov, Ph.D.

Systemic exposure to nanoparticles effects anti-

cancer immune stimulation for metastatic breast

cancer: A study in mouse models

Acknowledgements

• Preethi Korangath

• Sara Sukumar

• Brian Simons

• Elizabeth Jaffee

• Todd Armstrong

Funding agency

• Cordula Gruettner

Micromod Partikeltechnologie GmbH

Germany

• Mohammed Hedayati

• Shu-Han Yu• James Barnett

• Anirudh Sharma

• Jacqueline Stewart

• Elizabeth Henderson

• Sri Kamal Kandala

• Chun-Ting Yang

Biostatisticians

• Chen Hu

• Xian C Zhou

• Wei Fu

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DisclosuresConsulting: Mosaic Research

Imagion Biosystems (SAB and consulting)

Honoraria: Taylor and Francis Publishing (International Journal of Hyperthermia)

- Robert Ivkov, Ph.D.

Nanoparticles are colloids that can resemble

biological agents

40-100 nm

Maeda, et al. Adv. Drug Del. Rev. 2013

Enhanced Permeability and Retention

(EPR)

Nanometer-scale anti-cancer agents can exploit aberrant

properties of cancer tumors

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Innate and adaptive immune systems intercept nano-scale

invaders

Nanoparticle blood circulation kinetics (PK) is decided by interactions with

blood proteins, with rapid clearance reducing access to tumor extravascular

spaces.

Immune cells survey blood and tissues to internalize or destroy

opsonized materials

The ‘Stealth’ nanoparticle paradigm

Current cancer drug paradigm motivates development of stealth

nanoparticles that evade detection by the immune system

By relying on a generalized biologic concept,

attention is focused on materials engineering

‘Stealthy’ particles exploit aberrant vascular properties of tumors

Need long circulation time to target tumor efficiently

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Targeting nanoparticles to tumors?

• Without adequate targeting – nanoparticles are unlikely

to be beneficial and toxicity profile may dominate.

• Mode of ‘targeting’ is determined by indication

• Local primary tumor or palliation – percutaneous or

local intravascular

• Metastatic disease (direct) – systemic treatment, e.g.

intravenous

• Metastatic disease (combination indirect) –

local/systemic nanoparticle treatment combined with

other local/systemic therapies.

Effect of size (morphology) on PK

Natarajan, et al. Bioconjugate Chem. 2008, vol 19, pp 1211-1218

Effect of size (morphology) on biodistribution

Natarajan, et al. Bioconjugate Chem. 2008, vol 19, pp 1211-1218

Pharmacokinetic biodistribution of the RINPs (20, 30, and 100 nm size), negative controls of RNP (20

nm) NP without targeting molecules, and RINP (20 nm) non-immunoreactive were measured as %ID/g

in various organs after 48 h injection. The difference between targeted (RINP) and nontargeted

particles (RNP) and non-immunoreactive RINP (5) should be noted. The mean ± SD (error bars) were calculated based on three animals (n = 3) in each study group. IR = immunoreactive.

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Nanoparticles interact with the tumor

immune microenvironment

• Is there a fundamental flaw in our approach

to develop nanoparticle therapies for

cancer?

• Unlike small molecules, nanoparticles are

engineered foreign particles that can

actively interact with the immune system

In vitro drug screening Xenografts in immunodeficient model

BNF-HERCEPTINBNF-Plain

100nm

Starch shell

HER2

Actin

Breast cancer cell lines

In vitro iron uptake by ferene assay

Spearman r=0.8929

p=0.0123

Antibody-labeled nanoparticles demonstrate activity with a positive

correlation between iron and HER2 protein expression

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Mouse models chosen to present varied host

immune status

Immune system

components

Syngeneic

FVB/N

(Intact immune

system)

Athymic nude

(Least

Immunodeficient)

NOD SCID

Gamma (NSG)

(Highly

Immunodeficient)

Mature B cells Present Present Absent

Mature T cells Present Absent Absent

Dendritic cells Present Present Defective

Macrophages Present Present Defective

Natural killer cells Present Present Absent

Complement Present Present Absent

Overall study design (xenografts)

Frozen to quantitate

absolute iron

content by ICP-MS

Mouse strains:

NSG (Highly immunodeficient)

NUDE (Least immunodeficient)

Human tumor models:

MDA-MB-231

MCF7/neo

MCF7/HER

HCC1954

BT474

HER2 -ve

HER2 +ve

Tumor vol.

~150mm3

PBS, BNF particles

(5mgFe/animal)

24hrs

End points:• Prussian blue (iron distribution)

• ICP-MS

• Quantitative MRI

• IHC for HER2, CD31, IBA-1

Korangath, et al. in

review

Barnett, Sharma, Henderson, Stewart, Simons

MDAMB231 MCF7/neo MCF7HER2

BNF-Plain BNF-HER BNF-Plain BNF-HER BNF-Plain BNF-HER

Nude

BT474

BNF-Plain BNF-HER

HCC1954

BNF-Plain BNF-HER

Tumour ICP-MS from athymic nude mice

*

***

***

**

*** ******

******

******

PBS

BP

BH

µg iro

n/g

m d

ry w

eigh

t tiss

ue

3000

2000

1000

0

PBS

BP

BH

Herceptin-labeled nanoparticles are retained by tumors irrespective of

HER2 status; whereas much less of plain nanoparticles remain in tumors

unpublished

Korangath, Barnett, Henderson, Yang, Armstrong, Jaffee, Sukumar, Simons, et al.

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Histopathology analysis of tumors reveals antibody-

labeled nanoparticles localize to regions rich with

immune cells.

DAPI

IBA1

H & E PB

HER2

CD31

HER2

IBA-1

PB

PB

IBA-1

H & E HER2

Histopathology analysis of tumors reveals little or no correlation of

antibody-labeled nanoparticle localization to HER2 positive or CD31

positive regions.

Nude mice injected with BH NSG mice injected with BH

Nude mice injected with BHNSG mice injected with BH

Finkle et al Clin Can Res, 2004

Human HER2 overexpressing transgenic mouse model

Weeks to mammary tumor onset

Dis

ease f

ree s

urv

ival

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HuHER2 allograft model developmentFrom human HER2 overexpressing transgenic mouse (Genentech) to FVB/n animals

Subsequent transplantation to FVB/N

Confirmed tumor architecture by H&E and

HER2 protein by HER2 Immunohistochemistry in allograft tumors

Overall study designTransgenic

allografts grown in

mammary fat pad of

FVB/N mice (Donor)

Mouse strains:

NSG (Highly immunodeficient)

NUDE (Least immunodeficient)

FVB/N (Immune competent)

End points:

• Prussian blue

• IHC for Tumor marker (HER2)

Vasculature (CD31)

Immune cells

o CD45

o PAX5 (B cells)

o CD3 (T cells)

o IBA-1 (Mono/

macrophage)

Histology

• ICP-MS for iron content

Tumor vol.

~150mm3

PBS, BNF particles

(5mgFe/animal)

24hrs

BNF-particles

BNF-HERBNF-Plain

100nm

Starch shell

James Barnett, Anirudh Sharma, Elizabeth Henderson, Jacqueline Stewart, Simons

Host immune system influences nanoparticle uptakeFVB/n

BP

BH

Tumor 1 Tumor 2

Nude

Tumor 1 Tumor 2

NSG

Tumor 1 Tumor 2

PB

S

BN

F_

Pla

in

BN

F-H

ER

PB

S

BN

F_

Pla

in

BN

F-H

ER

PB

S

BN

F_

Pla

in

BN

F-H

ER

0

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

5 0 0 0

µg

iro

n/g

m d

ry w

eig

ht

tis

su

e

PBSBPBH

µg iro

n/g

m d

ry w

eight

tiss

ue

NudeFVB/N NSG

*

******

******

******

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In vitro data demonstrate that innate immune cells

displaying ‘inflammatory’ phenotype (TH1-type

induction) preferentially retained Herceptin

(antibody) labeled nanoparticles.

Un

ind

uced

BP

Un

ind

uced

BH

Ind

uced

BP

Ind

uced

BH

0

1 0

2 0

3 0

4 0

Iro

n u

pta

ke

(p

g/c

ell

)Ir

on u

pta

ke

(pg/c

ell)

BP BH BP BH

Uninduced Induced

Macrophages

p=0.01

0

2

4

6

8

1 0

Iro

n u

pta

ke

(p

g/m

l)Ir

on u

pta

ke

(pg/c

ell)

Neutrophils

BP BH BP BH

Uninduced

Bone marrow

derived

p=0.04

Induced Peritoneal

derived

unpublished

Korangath, Barnett, Henderson, Yang, Armstrong, Jaffee, Sukumar, Simons, et al.

• The tumor immune microenvironment preferentially retains antibody-

labeled nanoparticles over plain nanoparticles.

• EPR paradigm fails to account for observed ‘targeting’ of antibody-

labeled nanoparticles.

• A pro-inflammatory phenotype of phagocytic immune cells in the tumor

immune microenvironment preferentially take up antibody-labeled

nanoparticles.

• Is there any potential effect on tumor growth upon exposure?

Summary

What is the immune effect of systemic

exposure to nanoparticles, and how does

this affect cancer tumors?

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Flow cytometry of cells isolated from treated tumors

Transgenic allografts

grown in mammary

fat pad of FVB/N

mice (Donor) • T cells

• GD T cells

• B cells

• NK cells

• Macrophages

• Monocytes

• Neutrophils

• Dendritic cells

• Tumor cells

Flow cytometry

Tumor vol.

~150mm3

PBS, BNF particles

(5mgFe/animal)

24hrs

FVB/N (Immune competent n=8)

Dissociate in digestion

media to get single cells

for 1 hr

Put on magnet for 1hr

Nanoparticle associated

cells

Separated supernatant

B c e lls

N K c e lls

TAM

M o no

N e u tro

D e n d ri

T ce lls

G D T c e lls

B c e lls

N K c e lls

TAM

M o no

N e u tro

D e n d ri

T ce lls

G D T c e lls

B c e lls

N K c e lls

TAM

M o no

N e u tro

D e n d ri

T ce lls

G D T c e lls

PBS

BP

BHB c e lls

N K c e lls

TAM

M o no

N e u tro

D e n d ri

T ce lls

G D T c e lls

Nanoparticles are associated with Immune cells - not tumor cells

unpublished

Korangath, Barnett, Henderson, Yang, Armstrong, Jaffee, Sukumar, Simons, et al.

ii) NK cells

**

Rat

io

v) Neutrophils

**

**

**

vi) Dendritic cells

Rat

io

*

**

iii) Monocytes

**

**

iv) TAMs

Rat

io

i) tumour cells

0

5 0

1 0 0

1 5 0

Ra

tio

P B S

B P S u p

B P n a n o

B H S u p

B H n a n o

H e r

0

5 0

1 0 0

1 5 0

Ra

tio

P B S

B P S u p

B P n a n o

B H S u p

B H n a n o

H e r

0

5 0

1 0 0

1 5 0

Ra

tio

P B S

B P S u p

B P n a n o

B H S u p

B H n a n o

H e r

Days after injection

T cells

Days after injection

Days after injection

Dendritic cells

NK cells

Days after injection

(GD) T cells

In vivo data demonstrate that immune reaction to

nanoparticles displays ‘infection-like’ response.

unpublished

Korangath, Barnett, Henderson, Yang, Armstrong, Jaffee, Sukumar, Simons, et al.

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In vivo systemic exposure to NPs inhibits tumor

growth and leads to CD8+ T cell infiltration

Exposure to plain NPs induces tumor growth suppression similar to Herceptin-

labeled NPs

13.8 20.2 19.3

PBS BP BH

HuHER2 allograft growth

Tu

mo

rv

ol u

me

mm

3

Init

ial 4 7

11

14

17

21

24

28

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

D a y s

Tu

mo

r v

olu

me

mm

3

P B S

B P

BHPBS

BP

BH &

&

0

200

400

600

800

1000

Initial 4 7 11 14 17 21 24 28

Days

unpublished

Korangath, Barnett, Henderson, Yang, Armstrong, Jaffee, Sukumar, Simons, et al.

Summary and Conclusion

➢ Nanoparticles present an interesting and unique class of materials for

biomedical research and clinical applications.

➢ Their size may predispose them to interact with host immune systems,

raising the question that small-molecule drug PK/BD models are valid and

providing an opportunity to ‘reprogram’ the immune system to recognize

malignant tumors as ‘foreign’.

➢ Systemic exposure to nanoparticles induces a complex systemic host

immune responses that can lead to tumor growth suppression.

➢ Conversely, the host immune system in tumor microenvironment influences

retention of antibody conjugated nanoparticles, particularly when displaying

an inflammatory phenotype - but this retention may not be associated with

therapeutic activity.

➢ Nanoparticle characterization requires multiple techniques to measure their

properties.

Thank you for your attention

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Analysis of nanoparticle delivery to tumors, Nature Rev, 2016

0

200

400

600

800

1000

1200

1400

1600

1800

Tu

mo

r v

olu

me

mm

3

n=8

0

200

400

600

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1200

1400

1600

1800

Tu

mo

r v

olu

me

mm

3

n=6

0

200

400

600

800

1000

1200

1400

1600

1800

Init

ial 4 7 11 14 17 21 24 28 31

Tu

mo

r v

olu

me

mm

3 n=6

0

200

400

600

800

1000

1200

1400

1600

1800

Tu

mo

r v

olu

me

mm

3

n=3

PBS

HERBH

BP

Autochthonous HER2+ tumors respond equally to BP, BH, or free Herceptin

unpublished

Korangath, Barnett, Henderson, Yang, Armstrong, Jaffee, Sukumar, Simons, et al.

MDAMB231 MCF7/neo MCF7HER2

BNF-Plain BNF-HER BNF-Plain BNF-HER BNF-Plain BNF-HER

Nude

NSG

BT474

BNF-Plain BNF-HER

HCC1954

BNF-Plain BNF-HER

Tumour ICP-MS from athymic nude mice

*

***

***

**

*** ******

******

******

PBS

BP

BH

µg iro

n/g

m d

ry w

eight

tiss

ue

3000

2000

1000

0

Tumour ICP-MS from NSG mice

**

***

******

***

***

***

***

***PBS

BP

BH

Herceptin-labeled nanoparticles are retained by tumors irrespective of

HER2 status; whereas much less of plain nanoparticles remain in tumors