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7/16/2019
1
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
7/16/2019
2
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
7/16/2019
3
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
7/16/2019
4
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.
7/16/2019
5
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
7/16/2019
6
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.
7/16/2019
7
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
7/16/2019
8
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
*
******
******
******
7/16/2019
9
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?
7/16/2019
10
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.
7/16/2019
11
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
7/16/2019
12
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
800
1000
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