Post on 18-Jul-2020
Multifunctional Exosomes Construction by Precision Cell EngineeringLeonid Gaidukov, Ke Xu, Kevin Dooley, Chang Ling Sia, Christine McCoy, Gauri Mahimkar, Palak Shah, Nuruddeen Lewis,
Aaron Sulentic, Shelly Martin, Sriram Sathyanarayanan, Jonathan Finn
Codiak Biosciences, 35 CambridgePark Drive, Suite 500, Cambridge, MA 02140
Abstract
Introduction: Codiak BioSciences has leveraged natural exosome biology to
develop a therapeutic platform based on precisely engineered exosomes. Our
engExTM platform utilizes the unique exosome scaffolds PTGFRN and BaspI that
allow surface display and luminal loading of an array of structurally and
biologically different proteins. Here we sought to expand the capabilities of the
engEx platform for the precise engineering of multifunctional exosomes by
constructing combinatorial extracellular vesicles (EVs) derived from our lead
therapeutic candidate exoIL-12.
Methods: We generated exoIL-12 cells and EVs using random and site-specific
integration (RI and SSI) and tested their expression stability and activity. Then, we
constructed double and triple exosomes of IL-12 with the immunomodulators
CD40L and FLT3L using a combination of RI and SSI in different safe-harbors. We
tested protein expression and biological activity of the combo EVs, and confirmed
co-expression of protein ligands by co-IP, Western blot, ELISA and activity assays.
Results: While SSI resulted in more homogenic and stable cell pools, both methods
yielded exosomes of similar protein levels and activity, and thus were both applied
to construct multifunctional EVs. Combinatorial exoIL-12/CD40L/FLT3L showed
similar protein levels and activity as their single EV counterparts with some
variability between different safe-harbors used for SSI. Most importantly, all proteins
were efficiently co-expressed in combo EVs, demonstrating the capability of
PTGFRN for high-density display of multiple ligands on exosome surface.
Summary/Conclusion: Future in vivo studies will investigate the potential synergistic
activity of these multifunctional exosomes when applied as combinatorial
therapeutic agents in mouse cancer models. Overall, this POC study demonstrates
the potential of our engExTM platform to engineer multifunctional exosomes and
has broad therapeutic applications for immuno-oncology, vaccines development
and targeted functional delivery.
Summary
Presented at the 23rd Annual Meeting of the American Society of Gene & Cell Therapy, May 12-15, 2020 in Boston, MA, USA. All inquiries can be directed to presenting authors or by visiting www.codiakbio.com.
engEx platform for the multifunctional EV engineering
Efficient multi-ligand co-expression in combo EVs
Co-IP confirms multi-ligand co-expression in combo EVs
Figure 4. Co-IP confirms efficient ligand co-expression in exoIL-12/CD40L/FLT3L. Co-immunoprecipitation
(co-IP) is achieved by coupling magnetic streptavidin-coated dynabeads with the biotinylated antibodies
against one of the exosome surface proteins (aCD81, aIL12, aCD40L). Specific exosome populations are then
immunoprecipitated with the coupled beads, co-labeled with aIL12-APC, aCD40L-FITC and aFLT3L-PE
antibodies, and detected by FACS (A). FACS diagrams of exoIL-12/CD40L/FLT3L tEV2 co-IP experiment. Note
>95% co-expression efficiency of all three proteins using any one of three IP methods.
D
• engEx platform is effective for the precision engineering of multifunctional exosomes
• PTGFRN can efficiently co-express multiple proteins on the exosome surface, with no
drop in expression level and activity of individual proteins
• RI and SSI are both valid methods for the engineering of stable and potent exosomes
• SSI in different safe-harbors allows combo EV construction via precision cell engineering
magnetic
beads
Immunoprecipitation (IP) Detection
aIL12-APC
aFLT3L-PE
aCD81
aIL12
aCD40L
aCD40L-FITC
exoIL-12/CD40L/FLT3L (tEV2)
Random and site-specific integration for EV engineering
Random Integration (RI)
• Single integration events
• Stable genomic sites (safe harbors)
• Lower expression levels
• Homogenic and stable expression
Site-Specific Integration (SSI)
Triple EVs (tEVs)Double EVs (dEVs)
0
10
20
30
40
50
EV
su
rface
pro
tein
(ng
/1e1
0 p
art
icle
s) IL12
CD40L
sE
V1
sE
V2
dE
V1
FLT3L
sE
V3
exoIL-12/
CD40L/FLT3L
dE
V4
tEV
2
exoIL-12/CD40Lsingle EVs
dE
V2
dE
V3
exoIL-12/
FLT3L
B Protein quantitationA Western blot
A
B
C
D
E
F
30-200 nm vesicles released and taken up by all cells
Crucial mechanism for intercellular communication
Convey and protect complex macromolecules which can alter the function of recipient cells
Intrinsically non-immunogenic
Natural or engineered tropism to specific cells and tissues
Exo Engineering Platform (engEx)
PTGFRN is a novel EV scaffold for protein surface display
Figure 1. Identification of PTGFRN as a novel EV scaffold protein. OptiPrep density gradient centrifugation
was used to purify exosomes from high density suspension cell culture (A). Transmission electron microscopy
(TEM) images confirmed purity and morphology (B). Proteomic analysis led to the identification of a highly
abundant exosomal protein PTGFRN, highly enriched in exosomes purified from the producer cells. Cryo-
electron microscopy analysis showed ~25 nm projections densely packed on the surface of exosomes
overexpressing PTGFRN (C). PTGFRN is a single-pass type-I transmembrane glycoprotein composed of six
tandem extracellular IgV domains and a short cytoplasmic tail. Recombinant proteins, like 60 kDa single chain
hIL-12 (scIL-12) can be efficiently anchored to the exosome surface by PTGFRN fusion (D).
PTGFRN++
CPTGFRN -
D
PTG
FR
N
IL-1
2
Exo
fra
ctio
n
A B
200 nm
Multifunctional exosomes
EXOSOME MEMBRANE
Exterior
Lumen
• Proteins
• Peptides
• Nucleic acids
• Proteins
• Peptides
PTGFRN
BASP1
Combo EVs bear multiple functional moieties (proteins, peptides, nucleic acids) luminally or on the surface
Expand exosome engineering toolkit and demonstrate novel capabilities of EngEx platform
Combo EVs may have synergism of action, broader functionality and increased potency
Broad applications, including combinatorial therapy,
vaccines development, targeted functional delivery
engEx utilizes exosome scaffolds PTGFRN and BaspI for surface display and luminal loading
Stable and potent exoIL-12 engineered by SSI and RI
0 30 6060
70
80
90
100
Days of culture (post selection)
IL12-A
PC
(%
)
SSI RI
0
1
2
3
4
105 106 107 108 109 1010 1011
EV Concentration (P/mL)
Ab
so
rban
ce 6
40
nm
0
SSI (0 m)
RI (0 m) RI (2 m)
SSI (2 m)
RI (1 m)
SSI (1 m)
0 30 60
108
109
1010
1011
EC
50
(P
/mL
)
SSI RI
Days of culture (post selection)
0 10 20 300
50
100
Days of selection
Via
bilit
y (
%)
RISSI
0 20 40 600
1
2
3
4
5
Days of culture (post selection)
VC
D (
e6
cells
/mL
)
RISSI
exoIL-12
Figure 2. Comparison of exoIL-12 vesicles generated by random and site-specific integration. scIL-12-
PTGFRN is expressed from RI and SSI vector. SSI targets integration to AAVS1 genomic locus and is selected
with puromycin expressed from an endogenous promoter through splice acceptor (SA) and 2A self cleaving
peptide. Stable cell pools were generated by puromycin selection of HEK producing cells. Note a significantly
quicker selection of SSI pools (A). Stable cell pools were cultured for 60 days post selection and showed similar
stability, growth rate and viable cell density (B). Expression stability of stable pools was assayed by cell surface
labeling with aIL12-APC antibody at 0, 30 and 60 days of cell culturing. Note higher homogeneity and stability
of SSI pools (C, D). Purified exosomes were assayed for IL-12 activity by HEK IL12 reporter assay (E) from which
EC50 values were derived (F). Note stable and similar activity of exoIL-12 from RI and SSI pools.
Figure 3. Construction of exoIL-12/CD40L/FLT3L combo EVs with preserved protein expression levels.Single, double and triple EVs of IL12, CD40L and FLT3L were engineered by a combination of RI and SSI in
different safe-harbors. Protein expression on EV surface was measured by Western blot (A), ELISA and FACS (B).
Triple exosomes tEV2 showed nonreduced expression levels of all three proteins compared to their single EV
counterparts. Overall, the data shows that multiple proteins can be simultaneously anchored to the EV surface
by PTGFRN fusion with no reduction in their expression levels.
A
B
Preserved biological activities of multi-functional EVs
Figure 5. Multifunctional EVs show preserved biological activity. Selected multifunctional EVs with
preserved expression levels were applied for in vitro activity assays. IL-12 activity was assayed with HEK-blue IL-
12 reporter cells by measuring SEAP reporter activity in response to IL-12 binding to IL-12 receptor and
activation of the STAT-4 pathway (A). CD40L activity was measured by measuring B cell activation by CD69
expression in human PBMC culture (B). FLT3L activity was assayed by measuring ERK phosphorylation in THP-1
monocytes following their stimulation by FLT3L binding or PMA/ionomycin (full stimulation) (C). Shown are
activities normalized to protein concentration (top raw), EV concentration (middle raw), and the derived
EC50 values (bottom raw). Note the preserved of activity of multifunctional EVs.
0
1
2
3
106 107 108 109 1010 1011 1012
EV Concentration (P/ml)
Ab
so
rba
nce 6
40n
m
0
exoIL-12/CD40L
(dEV3)
exoIL-12 (sEV1)
exoIL12/CD40L/FLT3L
(tEV1)
IL-12 (activity per EV)
0
20
40
60
80
100
120
105 106 107 108 109 1010 1011
EV Concentration (P/ml)
B c
ell, C
D69 (
% p
osit
ive)
0
exoCD40L (sEV2)
exoIL-12/CD40L
(dEV3)
exoIL-12/CD40L/
FLT3L (tEV1)
CD40L (activity per EV)
0
20
40
60
80
100
107 108 109 1010 1011
200
250
EV Concentration (P/mL)
pE
RK
(%
+)
No
rmali
zed
to
rh
FL
T3L
0
exoFLT3L (sEV3)
exoIL-12/FLT3L (dEV4)
PMA/ionomycin
unstimulated
FLT3L (activity per EV)
sEV1 dEV2 tEV1
109
1010
EC
50
(P
/mL
)
IL-12 (EC50 per EV)
sEV2 dEV2 tEV1
108
109
EC
50
(P
/mL
)
CD40L (EC50 per EV)
sEV3 dEV4
109
1010
1011
EC
50
(P
/mL
)
FLT3L (EC50 per EV)
0
1
2
3
4
10 -3 10 -2 10 -1 100 101 102 103
IL-12 Concentration (ng/ml)
Ab
so
rba
nce 6
40n
m
rhIL-12 (ng/mL)
0
exoIL-12 (sEV1)
exoIL-12/CD40L/
FLT3L (tEV1)
exoIL-12/CD40L (dEV3)
IL-12 (activity per ng)A
0
20
40
60
80
100
120
10 -3 10 -2 10 -1 100 101 102 103 104
CD40L Concentration (ng/mL)
B c
ell, C
D69 (
% p
osit
ive)
rCD40LexoCD40L (sEV2)
0
exoIL-12/
CD40L/
FLT3L
(tEV1)
exoIL-12/CD40L
(dEV3)
CD40L (activity per ng)B
FLT3L (activity per ng)
0
20
40
60
80
100
10 -3 10 -2 10 -1 100 101 102 103 104
200
250
FLT3L Concentration (ng/mL)
pE
RK
(%
+)
No
rmali
zed
to
rh
FL
T3L
0
exoIL12/FLT3L
(dEV4)
exoFLT3L (sEV3)
rhFLT3L
PMA/ionomycin
unstimulated
C
• Multiple integration events
• High expression levels
• Heterogeneous growth and expression
• Possible genomic instability and silencing
Construction of exoIL-12/CD40L/FLT3L combo EVs
IL-12
FLT3L
3xCD40L
Tetra-spanins
PTGFRN