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