You are on page 1of 1

John T. Wilson, Chinonso C. Opara, Salka Keller, Hanna B.

Kern, Matthew Manganiello, Connie Cheng,
Chen-Chang Lee, Anthony J. Convertine and Patrick S. Stayton
Department of Bioengineering, University of Washington, Seattle, WA
Enhancement of CTL and Th1 Responses by Vaccination with
Stimuli Responsive Polymers for Delivery of Antigens and
Oligonucleotide Adjuvants

The limited availability of tumor-associated antigens and the inability to
deliver such antigens in a way that effectively stimulates CD8
+
cytotoxic
(CTL) and CD4
+
Type 1 helper (Th1) T cells has been a challenge to
tumor immunotherapy. The long-term goal of this research is to use pH
responsive carriers to deliver antigens and immunomodulatory
oligonucleotide adjuvants to antigen presenting cells, in order to promote
anti-tumor cellular immunity.
B
We would like to acknowledge funding from the Initiative for Maximizing
Student Diversity (CCO), Irvington Institute Fellowship Program of the
Cancer Research Institute (JTW), the State of Washington Life Sciences
Discovery Fund (PSS), and NIH (PSS).

“Smart” polymers mediate dual cytosolic delivery of ovalbumin and
oligonucleotides from a single carrier.
Polymeric delivery of CpG potentiates its adjuvant capacity to stimulate
CD8+ and Th1 T cell responses.
Polymeric delivery of siRNA induces gene suppression
This novel class of “smart” vaccines offers a reprogrammable delivery
platform with potential for improving the efficacy of cancer vaccines.
The pH-responsive, endosomolytic “smart” polymers we have designed
are capable of being conjugated to antigens and electrostatically
complexed to oligonucleotides for cytosolic delivery of biologic cargo.
Here, we describe the characterization and preliminary in vivo and in
vitro evaluation of this new class of vaccine delivery vehicle.
pH responsive “smart” core
cytosolic delivery of cargo
enhanced class I presentation
delivery to cytosolic PRRs
immunoregulatory siRNA
polycationic corona
electrostatic complexation of oligos
co-localization of oligos and antigen
enhanced cellular uptake
depot formation
Figure 6. Polymeric delivery of
ovalbumin and CpG increases IgG
titer and IgG2c/IgG1 ratio.
Polymeric Delivery of Antigen and CpG ODN
enhances CD8
+
and Th1 CD4
+
Responses

Figure 3. Immunization
regimens and treatment groups.
C57BL/6 mice were immunized
subcutaneously at the base of
the tail. 25 µg of ova and 28 µg
of CpG were administered.
Figure 1. Dual-loading of antigen and oligonucleotides on polymeric carriers.
Scheme for covalent conjugation of ovalbumin (ova) to polymers via a reducible
disulfide bond and electrostatic complexation of oligonucleotide therapeutics.
Nanocarriers for Dual-Delivery of Antigens and
Oligonucleotides
polymer
ovalbumin
(ova)
Traut’s
Reagent
S
NH2
+Cl-
ova-SH
~4 thiols/ova
NH2 SH
2-pyridinethione
oligo
antigen conjugation oligonucleotidecomplexation
b) a)
C
p
G
O
D
N
1
8
2
6
+/-
d
s
R
N
A
0 1 2 3 4 0 1 2 3 4
+
1
0
m
M
G
S
H
ova conj mix ova conj
Size (nm)
F
r
e
q
u
e
n
c
y

(
%
)
0 20 40 60 80
0
5
10
15
20
25
RESULTS
Polymeric Delivery of siRNA
induces Gene Suppression


Figure 7. Negative control
siRNA (Scrambled) or GAPDH
siRNA (50 nM) was complexed
with ova-polymer conjugates at
4:1 charge ratio and incubated
with DC2.4 cells for 24 hrs.
Relative GAPDH expression
levels were measured by qRT-
PCR and normalized as a
percentage of untreated cells.
0
0.2
0.4
0.6
0.8
1
1.2
Untreated GAPDH Scrambled

G
A
P
D
H

E
x
p
r
e
s
s
i
o
n

Figure 5. As measured by IFNg a) ICS and b) ELISPOT, delivery of CpG ODN
electrostatically complexed to polymer-ova conjugates significantly increases
CD8
+
INFg
+
T cell responses. c) IFNg ELISPOT of splenocytes restimulated with the
class II epitope ova
323-339
(ISQAVHAAHAEINEAGR) demonstrates a similar
enhancement in Th1 CD4
+
responses associated with polymeric delivery of CpG.
*p<0.05 by Mann-Whitney U test.
CONCLUSIONS
Figure 2. a) Shows ova-polymer conjugation and reduction with intracellular
concentrations of glutathione, liberating ova from polymer. b) Agarose gel
electrophoresis of conjugates incubated with double stranded RNA (siRNA) and
CpG ODN1826 at various +/- charge ratios
Moving forward, using our polymeric delivery platform, we are
characterizing the level of gene knockdown with siRNA across immune
cells and genes of interest. In dendritic cells, we have preliminarily
demonstrated that polymer-ova conjugates facilitate siRNA delivery and
gene suppression of glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), a model housekeeping gene. We hope to later target genes
that serve as regulatory checkpoints in immune response, such as IL-
10 and A20
ACKNOWLEDGEMENTS
e
n
d
p
o
i
n
t


t
i
t
r
e
(
l
o
g
1
0
)
o
v
a
m
i
x
c
o
n
j
C
p
G
m
i
x
+
C
p
G
c
o
n
j
+
C
p
G
0
2
4
6
8
IgG1
IgG2c
Antigen-Polymer Conjugates augment CD8
+
Response
%

I
F
N
g
+
o
f

C
D
8
+
o
v
a
c
o
n
j
m
i
x
0.0
0.5
1.0
1.5
2.0
*
*
a) b)
Figure 4. Immunization with ova-polymer conjugates enhances CD8
+
IFNg
+
T cell
responses. Splenocytes were restimulated with SIINFEKL and IFNg production
detected via intracellular cytokine staining (ICS). a) Representative flow cytometry
dot plot demonstrating increase in CD8
+
IFNg
+
cell population from mouse
immunized subcutaneously with conjugate. b) Conjugates (conj) enhanced ova-
specific CD8
+
responses over both free ova (ova) or a physical mixture of ova and
polymer (mix). *p<0.05 by Mann-Whitney U test.
INTRODUCTION
IgG1 corresponds to the humoral
Th2 response, while IgG2c is
indicative of the critical cellular Th1
response, which we are striving for .
Alone, delivery of antigen biases
Th2, but addition of CpG enhances
Th1. We hope to further increase
Th1 response by using siRNA to
silence genes that promote Th2
and/or inhibit Th1.
%

I
F
N
g
+
o
f

C
D
8
+
c
o
n
j
C
p
G
c
o
n
j+
C
p
G
m
ix
+
C
p
G
0
5
10 *
* *
S
F
C
s
/
1
E
6

c
e
l
l
s
c
o
n
j
C
p
G
c
o
n
j+
C
p
G
m
ix
+
C
p
G
0
500
1000
1500
2000 *
* *
S
F
C
s
/
1
E
6

c
e
l
l
s
c
o
n
j
C
p
G
c
o
n
j+
C
p
G
m
ix
+
C
p
G
0
500
1000
1500
*
* *
%

I
F
N
g
+
o
f

C
D
8
+
c
o
n
j
C
p
G
c
o
n
j+
C
p
G
m
ix
+
C
p
G
0
5
10 *
* *
S
F
C
s
/
1
E
6

c
e
l
l
s
c
o
n
j
C
p
G
c
o
n
j+
C
p
G
m
ix
+
C
p
G
0
500
1000
1500
2000 *
* *
S
F
C
s
/
1
E
6

c
e
l
l
s
c
o
n
j
C
p
G
c
o
n
j+
C
p
G
m
ix
+
C
p
G
0
500
1000
1500
*
* *
ova conj mix CpG conj+CpG mix+CpG
a) b) c)