IL-12 Engineered Exosomes Elicited a Potent Anti-Tumor Response And
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engEx™: A novel exosome engineering platform enabling targeted transfer of pharmacological molecules 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, Agata Villiger-Oberbek, Kyriakos D. Economides, John Kulman, Sriram Sathyanarayanan Codiak BioSciences, Cambridge, MA 2150 What are exosomes? Stable cellular expression of PTGFRN resulted in 150-fold enrichment PTGFRN enables high-density surface display of therapeutic proteins • Exosomes are extracellular vesicles (30-200 nm) that convey complex molecules and of PTGFRN on exosome surface ∆687 FL biological signals between cells. A PTGFRN C Comparative Proteomics WT ++ -/- Exosome Signaling 900 » Convey and protect complex 120 12 nm macromolecules that alter the 300 function of recipient cells 35 5000 0 ALIX MoleculeExo Membrane Cytoplasm » Intrinsically non-immunogenic 200 1. nm Nucleus SDCBP WT PTGFRN WT (PSM) ALIX Peptide FKBP VHH IL7 GFP scFv scCD40L scFab scIL12 BDD-FIII B PTGFRN PTGFRN SDCBP a 12 a 1 a 1 a 2 a 2 a 0 a 1 a 0 a 10 a • Codiak BioSciences is developing a therapeutic platform utilizing exosome biology, 100 known as engEx™. Figure 6. PTGFRN enables high-density surface display of structurally and biologically diverse proteins on exosomes. Exosomes can be engineered to carry specific biologically active entities, including Proteins of interest, including reporters, peptides, cytokines, antibody fragments, tumor necrosis superfamily members, and multi-domain • High-density exosome surface 0 0 100 200 300 900 0 100 200 300 900 small molecules, proteins, antibodies, peptides, and nucleic acids, individually or in display visible by cryo-EM blood coagulation factors, were fused to the surface exposed N-terminus of either full-length or truncated forms of PTGFRN. Exosomes combination, on the exosome surface, in the lumen, or both. PTGFRN++ (PSM) PTGFRN -/- (PSM) 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. • We believe our ability to alter tropism by precisely modifying the exosome surface may Figure 3. Analysis of exosomes isolated from producer cells following overexpression or deletion of PTGFRN. allow us to target many cell types in the immune system. 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 CD40L engineered exosomes targeted and activated B cells High purity is required for identification 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. A B B cell Activation C B cell Uptake 100 100 1000 of exosome scaffold proteins scCD40L-FL scCD40L-FL scCD40L-FL-GFP rCD40L CD40L FL-GFP ) 00 75 75 WT 00 ( ) A B C Established Exosome Protein I rCD40L ive IGF-EWI PTGFRN overexpression enhanced activity t 600 MF si Exo Luminal Protein ALIX 0 0 ( FP Po ) Contaminant of exosome-mediated delivery of STING agonist G 00 Fraction M S P ( 200 C6 2 2 PBMC IFN-β Production B16F10 SC Tumor 200 on i A B 5 ct 2.0 10 2500 a PTGFRN FA Control 0 0 0 PTGFRN -- PTGFRN -/- scCD40L-FL CD40L 10-2 10-1 100 101 102 103 104 107 108 109 1010 1011 109 1010 1011 1012 ) 3 2000 5 WT WT (native) C0L Concentration (ngmL) Exosome Concentration (pmL) Concentration (pmL) 200 nm ome Fr s 100 1. 10 SDCBP PTGFRN + PTGFRN ++ ) xo E 1500 Figure 7. Exosomes expressing CD40L extracellular domain were more potent than soluble CD40L and facilitated B cell targeting. (RL 1.0 105 Figure 1. Proteomic analysis of highly purified - A) A single chain CD40L trimer was appended to PTGFRN. B) CD40L exosomes exhibited a 20-fold increase in potency compared with TNC LGALS3BP FN exosomes and identification of novel scaffold proteins. NID2 I 1000 soluble CD40L protein by measuring CD69 expression on B cells in PBMC culture (left). This can be attributed to trimeric CD40L presentation ™ 0 A) OptiPrep density gradient centrifugation was 0 100 200 00 4 .0 10 (mm Volume Tumor in a biological membrane coupled with oligomerization mediated by PTGFRN. These exosomes were 135-fold more potent than 500 used to purify exosomes from high density suspension Gradient Input (PSM) exosomes overexpressing CD40L in its native conformation (right), due to high-density display achieved with PTGFRN. C) CD40L exosomes cell culture. B) Transmission electron microscopy (TEM) images confirmed purity and 0.0 0 demonstrated preferential uptake in B cells compared with overexpression of PTGFRN alone in PBMC culture using a C-terminal GFP tag. morphology. C) Proteomic analysis by LC/MS-MS led to the identification of highly 10- 10- 10- 10-2 10-1 100 101 102 103 6 8 9 10 12 14 16 18 20 22 24 A concomitant suppression of general phagocytic uptake by antigen presenting cells was also observed. abundant and unique exosomal proteins, including a single-pass transmembrane STING Agonist ( M) Day glycoprotein, PTGFRN, belonging to the EWI motif-containing immunoglobulin Figure 4. Exosome PTGFRN expression levels positively correlate with IFN-β production and tumor growth inhibition. superfamily (IGSF-EWI). 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. IL-12 engineered exosomes elicited a potent anti-tumor response B) Similar trends were observed in a subcutaneous B16F10 efficacy model using 3 intratumoral (IT) doses of exosomes and exhibited superior PK/PD to soluble IL-12 PTGFRN is a highly abundant, exosome-specific protein loaded with a minimally efficacious dose of small molecule STING agonist on Days 6, 9, and 12 (indicated in red). A IL-12-FL B B16F10 SC Tumor C D 2000 103 104 Untreated IL-12-FL rIL-12 Untreated IL-12-FL rIL-12 Cell Exo PTGFRN ALIX Control ) A B 220 C rIL-12 3 IL-12-FL 1600 3 MSC PTGFRN packages fusion proteins into exosomes rIL-12 10 102 120 1200 /tumor 100 γ Raji more efficiently than conventional scaffolds 102 0 PTGFRN 60 800 MB231 omain Boundaries Nano FCM 101 LOQ pg IFN- 0 10 pg IL-12/tumor WT Eng. 1 ** 10 LOQ 0 A ∆1 B C WT (mm Volume Tumor 400 K562 120 0 ∆26 5% 100 0.036 0.037 0 100 100 HT1080 ∆395 t 0 3 12 24 48 20 IGSF8-GFP n FL-GFP 5 6 7 9 11 13 15 0 3 12 24 48 u 0.12 ∆537 EC (ngmL) 0.12 o 0 Day Time (h) Time (h) HEK C ∆6 FL-GFP 0 0.0 0.5 1.0 1.5 120 Figure 8. IL-12-PTGFRN exosomes induced durable IFN- response in vitro and in vivo. A) Exosomes expressing a single chain version of PTGFRN IGSF3 IGSF8 γ PTGFRN Exosome Expression -20 TG reduced IL-12 fused to PTGFRN (IL-12-FL) exhibit similar potency to soluble IL-12 by measuring IFN-γ response in PBMC culture. B) In vivo efficacy was 0 101 103 105 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 Figure 2. Structure of EWI-IGSF proteins and PTGFRN expression on exosomes derived FITC- from various cell lines. A) All members of the EWI subfamily are type-I transmembrane FL-GFP ∆687-GFP IGSF8-GFP were administered on Days 5, 6, and 7 (indicated in red). At Day 16, IL-12-FL exosomes demonstrated superior tumor growth inhibition Figure 5. Comparison of full length and truncated forms of PTGFRN glycoproteins structurally composed of tandem extracellular IgV domains containing the 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 to conventional scaffolds. A) Exosomes expressing full length (FL) and sustained IFN-γ production in the tumor compared with soluble IL-12 (D). characteristic Glu-Trp-Ile (EWI) motif and a short cytoplasmic tail. B) PTGFRN and IGSF8 5 D 10 a truncated form (Δ687) of PTGFRN with C-terminal fusions to GFP were identified as major molecular partners of the canonical exosome tetraspanins Exosome Cell were characterized. B) Interestingly, stable expression of the structural CD9 and CD81. The extracellular domain of PTGFRN forms stable oligomers on the 100 paralogue IGSF8-GFP does not result in similar levels of exosome cell surface, making it an attractive scaffold to display antigens that require high- ) Summary Cell enrichment as PTGFRN. C) Analysis of FL-GFP exosomes by nano flow FC density clustering for activity. Exosomes purified from a producer cell were purified and ( 4 G cytometry showed a single, monodisperse population 95% positive analyzed by SDS-PAGE alongside producer cell lysate. The 120 kDa PTGFRN band is FP 10 FP G for GFP. D) Exosomes expressing GFP fusions to commonly reported • Optimized exosome purification protocol enabled research into and discovery of exosome-specific scaffold visible in exosome lysate and was confirmed by Western blotting.C) At equal protein (M proteins, including surface glycoprotein PTGFRN. F ome scaffolds used for exosome engineering including CD81, pDisplay s 0 I concentration, PTGFRN is not detectable by Western blotting in producer cell lysate, ) xo (pD), and LAMP2B were purified.