Kevin Dooley, Ke Xu, Sonya Haupt, Nuruddeen Lewis, Rane Harrison, Shelly Martin, Christine McCoy, Chang Ling Sia, Su Chul Jang, Katherine Kirwin, Russell McConnell, Bryan Choi, Adam T. Boutin, Damian Houde, Jorge Sanchez-Salazar, Nikki Ross, Agata Villiger-Oberbek, Kyriakos D. Economides, John Kulman, Sriram Sathyanarayanan

Codiak BioSciences, Cambridge, MA

Development of a platform for exosome engineering using a novel and selective scaffold protein for surface display

Presented at the Annual Meeting of the International Society for Extracellular Vesicles • April 27, 2019 • Kyoto, Japan All inquiries can be directed to presenting authors or by visiting www.codiakbio.com.

Summary

• Optimized exosome purification protocol enabled research into and discovery of exosome-specific scaffold proteins, including surface glycoprotein PTGFRN.

• Overexpression of PTGFRN in a producer cell resulted in a 150-fold increase in exosome surface expression.

• Both termini of PTGFRN are amenable to genetic fusion with broad classes of proteins.

• High-density exosome surface display of bioactive molecules mediated by PTGFRN enables production of potent exosomes.

High purity is required for identification of exosome scaffold proteins

Figure 1. Proteomic analysis of highly purified exosomes and identification of novel scaffold proteins. A) Iodixanol density gradient centrifugation was used to purify exosomes from high density suspension cell culture. B) Transmission electron microscopy (TEM) images confirmed purity and morphology. C) Proteomic analysis by LC/MS-MS led to the identification of highly abundant and unique exosomal proteins, including a single-pass transmembrane glycoprotein, PTGFRN.

CD40L engineered exosomes targeted and activated B cells

Figure 8. Exosomes expressing CD40L extracellular domain were more potent than soluble CD40L and facilitated B cell targeting. A) A single-chain CD40L trimer was appended to PTGFRN. B) CD40L exosomes exhibited a 20-fold increase in potency compared with soluble CD40L protein by measuring CD69 expression on B cells in PBMC culture (left). These exosomes were 135-fold more potent than exosomes overexpressing CD40L in its native conformation (right), due to high-density display achieved with PTGFRN. C) CD40L exosomes demonstrated preferential uptake in B cells compared with overexpression of PTGFRN alone in PBMC culture using a C-terminal GFP tag. A concomitant suppression of general phagocytic uptake by antigen presenting cells was also observed.

PTGFRN packages fusion proteins into exosomes more efficiently than conventional scaffolds

Figure 5. Comparison of full length and truncated forms of PTGFRN to conventional scaffolds. A) Exosomes expressing full length (FL) and a truncated form (Δ687) of PTGFRN with C-terminal fusions to GFP were characterized. B) Interestingly, stable expression of the structural paralogue IGSF8-GFP does not result in similar levels of exosome enrichment as PTGFRN. C) Analysis of FL-GFP exosomes by nano flow cytometry showed a uniform population approximately 95% positive for GFP. D) Exosomes expressing GFP fusions to conventional scaffolds used for exosome engineering including CD81, pDisplay (pD), and LAMP2B were purified. The producer cell GFP expression was measured by flow cytometry (right Y axis) and the purified exosome fluorescence was measured spectrophotometrically at equal exosome concentration (left Y axis). FL PTGFRN demonstrated 30-fold improvement in packaging efficiency over pD and LAMP2B.

PTGFRN is a highly abundant, exosome-specific protein

Figure 2. PTGFRN structure and expression on exosomes derived from various cell lines. A) All members of the EWI-motif containing immunoglobulin superfamily (IGSF-EWI), including PTGFRN, are type-I transmembrane glycoproteins structurally composed of tandem extracellular IgV domains containing the characteristic Glu-Trp-Ile motif and a short cytoplasmic tail. B) PTGFRN enrichment in exosomes purified from a producer cell line was verified by SDS-PAGE and Western blot. C) Conditioned medium from a variety of cell types was purified and subjected to proteomic analysis by LC/MS-MS. The relative amounts of PTGFRN and the accessory ESCRT protein ALIX were quantified and normalized to HEK293 expression levels.

Stable cellular expression of PTGFRN resulted in 150-fold enrichment of PTGFRN on exosome surface

Figure 3. Analysis of exosomes isolated from producer cells following overexpression or deletion of PTGFRN. A) SDS-PAGE of purified exosomes show enrichment or absence of PTGFRN following overexpression (++) or deletion (-/-). The number of PTGFRN molecules per exosome was quantitated using an AlphaLISA assay developed with antibodies raised against the soluble ectodomain of PTGFRN. B) Cryo-electron micrographs show 25 nm projections densely packed on the surface of exosomes overexpressing PTGFRN. C) Cellular overexpression or deletion of PTGFRN did not alter the broader protein composition of secreted exosomes.

PTGFRN overexpression enhanced activity of exosome-mediated delivery of STING agonist

Figure 4. Exosome PTGFRN expression levels positively correlate with IFN-β production and tumor growth inhibition. A) Representative in vitro activity of exosomes displaying varying levels of PTGFRN including overexpressed (++), wild type (WT), and knockout (-/-). Maximum IFN-β production in PBMCs correlated with PTGFRN expression level. B) Similar trends were observed in a subcutaneous B16F10 efficacy model using 3 intratumoral (IT) doses of exosomes loaded with a minimally efficacious dose of small molecule STING agonist on Days 6, 9, and 12 (indicated in red).

IL-7-PTGFRN decorated exosomes exhibited 1,500-fold potency improvement over pDisplay

Figure 7. In vitro potency characterization of exosomes functionalized with IL-7. A) IL-7 was tethered to the surface of exosomes using PTGFRN or pD. B) IL-7-PTGFRN exosomes cleared IL-7 receptor (IL-7R) expression on CD8+ T cells in a dose-dependent manner 24 hours post-treatment, with a 1,500-fold improvement in EC50 compared to IL-7-pD exosomes.

IL-12 engineered exosomes elicited a potent anti-tumor response and exhibited superior PK/PD to soluble IL-12

Figure 9. IL-12-PTGFRN exosomes induced durable IFN-γ response in vitro and in vivo. A) Exosomes expressing a single-chain version of IL-12 fused to PTGFRN (IL-12-FL) exhibited similar potency to soluble IL-12 by measuring IFN-γ response in PBMC culture. B) In vivo efficacy was assessed using mouse orthologs in a subcutaneous B16F10 melanoma model. Three IT doses of 200 ng of IL-12-FL exosomes or soluble IL-12 were administered on Days 5, 6, and 7 (indicated in red). At Day 16, IL-12-FL exosomes demonstrated superior tumor growth inhibition compared with soluble IL-12 (**P < 0.01). C) Furthermore, a single IT dose of IL-12-FL exosomes resulted in enhanced tumor retention and sustained IFN-γ production in the tumor compared with soluble IL-12 (D).

PTGFRN enables high-density surface display of therapeutic proteins

Figure 6. PTGFRN enables high-density surface display of structurally and biologically diverse proteins on exosomes. Proteins of interest, including reporters, peptides, cytokines, antibody fragments, tumor necrosis superfamily members, and multi-domain blood coagulation factors, were fused to the surface exposed N-terminus of either full-length or truncated forms of PTGFRN. Exosomes displaying all of the above proteins were purified and characterized. In most cases, fusion proteins were clearly visible by SDS-PAGE (callout). 170 kDa B-domain deleted FVIII represents the largest protein successfully anchored to the exosome surface by PTGFRN fusion.

rCD40L

scCD40L-FL CD40L(native)

A

Exo Fraction

C D

B

200 nm

A

A

IL-7-pDIL-7-FL

A

B

C

B cell Activation

B cell Uptake

C Comparative Proteomics

C

0 100 200 3000

100

200

300

Gradient Input (PSM)

Exo

som

e F

rac

tion

(PS

M)

ALIX

PTGFRNMARCKSL1

MARCKS

BASP1LGALS3BPTNC

NID2

SDCBP

IGSF-EWILuminal Protein

Established Exosome Protein

Contaminant

A

PTGFRN IGSF3 IGSF8

B220

Cell Exo

100120

80605040

30

20

�PTGFRN

C

0.0 0.5 1.0 1.5

HEK

HT1080

K562

MB231

Raji

MSC

Exosome Expression

ALIXPTGFRN

0 100 200 300 9000

100

200

300

900

PTGFRN++ (PSM)

WT

(PSM

)

ALIX

SDCBP

PTGFRN

0 100 200 300 900

PTGFRN -/- (PSM)

ALIX

SDCBPPTGFRN

A

B

120

Molecule/Exo

PTGFRNWT ++ -/-

> 500035 0

High-density exosome surfacedisplay visible by cryo-EM

PTGFRN++WT

A B

0

500

1000

1500

2000

2500

Day

Tum

or V

olu

me

(m

m3 )

Control

PTGFRN ++WTPTGFRN -/-

6 8 9 12 14 16 18 20 2210 2410-5 10-4 10-3 10-2 10-1 100 101 102 1030.0

5.0 104

1.0 105

1.5 105

2.0 105

STING Agonist ( M)

IFN

- (

RLU

) PTGFRN ++WT

FSAPTGFRN -/-

PBMC IFN-β Production B16F10 SC Tumor

WT Eng.120

120

IGSF8-GFP

FL-GFP

4-20% TGX, reduced

BA

C 150

101 103 105

50

100

0

95%

FITC-A

Co

un

t FL-GFP

WT

PTGFRN Domain Boundaries

∆149

∆269

∆395

∆537

∆687

IGSF8-GFPFL-GFP ∆687-GFP

0

0.12 3 12 24 48

100

101

102

103

Time (h)

pg

IFN

-γ/t

um

or

LOQ

Untreated IL-12-FL rIL-12

D

PTGFRN 687 CD81 IGSF8 Palm pD LAMP2B0

50

100

103

104

105

Exo

som

e G

FP (

FC)

Ce

ll GFP (M

FI)

Exosome Cell

0

0.12 3 12 24 48

100

101

102

103

104

Time (h)

pg

IL-1

2/tu

mo

r

Untreated IL-12-FL rIL-12

LOQ

107 108 109 1010 10110

25

50

75

100

Exosome Concentration (p/mL)

WT

scCD40L-FLCD40L

10-2 10-1 100 101 102 103 1040

25

50

75

100

CD40L Concentration (ng/mL)

CD

69 P

osit

ive

(%

) rCD40LscCD40L-FL

109 1010 1011 10120

200

400

600

800

1000

Concentration (p/mL)

GFP

(M

FI)

scCD40L-FL-GFPFL-GFP

12nm

1.5nm

scIL-1270 kDa

BDD-FVIII170 kDa

scFab51 kDa

scFv27 kDa

scCD40L50 kDa

GFP27 kDa

IL-717 kDa

Peptide3 kDa

FKBP12 kDa

FL∆687

VHH15 kDa

B

106 107 108 109 1010 1011 10120

25

50

75

100

Concentration (p/mL)

IL-7

R E

xpre

ssio

n (

%)

IL-7-FL

WTIL-7-pD

EC50 (ng/mL)

rIL-12

0.036

IL-12-FL

0.037

BB16F10 SC Tumor

0

400

800

1200

1600

2000

Day

Tum

or V

olu

me

(m

m3 )

Control

rIL-12IL-12-FL

**

5 96 7 11 13 15