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The promise of IL12 as a cancer treatment has yet to be fulfilled with multiple tested approaches being limited by unwanted systemic exposure and unpredictable pharmacology. To address these limitations, we generated exoIL12, a novel, engineered exosome therapeutic that displays functional IL12 on the surface of an exosome. IL12 exosomal surface expression was achieved via fusion to the abundant exosomal surface protein PTGFRN resulting in equivalent potency in vitro to recombinant IL12 (rIL12) as demonstrated by IFNγ production. Following intratumoral injection, exoIL12 exhibited prolonged tumor retention and greater antitumor activity than rIL12. Moreover, exoIL12 was significantly more potent than rIL12 in tumor growth inhibition. In the MC38 model, complete responses were observed in 63% of mice treated with exoIL12; in contrast, rIL12 resulted in 0% complete responses at an equivalent IL12 dose. This correlated with dose-dependent increases in tumor antigen–specific CD8+ T cells. Rechallenge studies of exoIL12 complete responder mice showed no tumor regrowth, and depletion of CD8+ T cells completely abrogated antitumor activity of exoIL12. Following intratumoral administration, exoIL12 exhibited 10-fold higher intratumoral exposure than rIL12 and prolonged IFNγ production up to 48 hours. Retained local pharmacology of exoIL12 was further confirmed using subcutaneous injections in nonhuman primates. This work demonstrates that tumor-restricted pharmacology of exoIL12 results in superior in vivo efficacy and immune memory without systemic IL12 exposure and related toxicity. ExoIL12 is a novel cancer therapeutic candidate that overcomes key limitations of rIL12 and thereby creates a therapeutic window for this potent cytokine.

Mean Cytokine Values in Cynomolgus Plasma After Subcutaneous Dosing

Fold-change Cytokine Values in Cynomolgus Plasma After Subcutaneous Dosing

Figures S1 through S6

20201229pdtedSupplementaryFigures.pdf from Exosome Surface Display of IL12 Results in Tumor-Retained Pharmacology with Superior Potency and Limited Systemic Exposure Compared with Recombinant IL12

Extracellular vesicles (EVs) are an important intercellular communication system facilitating the transfer of macromolecules between cells. Delivery of exogenous cargo tethered to the EV surface or packaged inside the lumen are key strategies for generating therapeutic EVs. We identified two "scaffold" proteins, PTGFRN and BASP1, that are preferentially sorted into EVs and enable high-density surface display and luminal loading of a wide range of molecules, including cytokines, antibody fragments, RNA binding proteins, vaccine antigens, Cas9, and members of the TNF superfamily. Molecules were loaded into EVs at high density and exhibited potent in vitro activity when fused to full-length or truncated forms of PTGFRN or BASP1. Furthermore, these engineered EVs retained pharmacodynamic activity in a variety of animal models. This engineering platform provides a simple approach to functionalize EVs with topologically diverse macromolecules and represents a significant advance toward unlocking the therapeutic potential of EVs.

Cyclic dinucleotide (CDN) agonists of the STimulator of InterferoN Genes (STING) pathway have shown immune activation and tumor clearance in pre-clinical models. However, CDNs administered intratumorally also promote STING activation leading to direct cytotoxicity of many cell types in the tumor microenvironment (TME), systemic inflammation due to rapid tumor extravasation of the CDN, and immune ablation in the TME. These result in a failure to establish immunological memory. ExoSTING, an engineered extracellular vesicle (EV) exogenously loaded with CDN, enhances the potency of CDN and preferentially activates antigen presenting cells in the TME. Following intratumoral injection, exoSTING was retained within the tumor, enhanced local Th1 responses and recruitment of CD8+ T cells, and generated systemic anti-tumor immunity to the tumor. ExoSTING at therapeutically active doses did not induce systemic inflammatory cytokines, resulting in an enhanced therapeutic window. ExoSTING is a novel, differentiated therapeutic candidate that leverages the natural biology of EVs to enhance the activity of CDNs., Su Chul Jang et al. develop exoSTING, consisting of an engineered extracellular vesicle loaded with a potent cyclic dinucleotide (CDN) agonist of the STING pathway. They find that exoSTING shows more than 100-fold increased potency in in vivo tumor models and has increased tumor retention and lower levels of systemic inflammatory cytokine production as compared to free CDN.

Background Exosomes are natural, abundant extracellular vesicles capable of transferring complex molecules between neighboring and distant cell types. Translational research efforts have focused on co-opting this communication mechanism to deliver exogenous payloads to treat a variety of diseases. Important strategies to maximize the therapeutic potential of exosomes therefore include payload loading, functionalization of the exosome surface with pharmacologically active proteins, and delivery to target cells of interest. Methods Through comparative proteomic analysis of purified exosomes, we identified several highly enriched and exosome-specific proteins, including a transmembrane glycoprotein (PTGFRN) belonging to the immunoglobulin superfamily. Leveraging PTGFRN as a scaffold for exosome surface display, we developed our engExTM platform to generate engineered exosomes functionalized with a variety of structurally and biologically diverse proteins.Systemically administered exosomes are primarily taken up by macrophages in the liver and spleen. To redirect exosome uptake to other cell types, we employed our engineering platform to display functional targeting ligands, including single domain antibodies, single chain variable fragments, single chain Fabs (scFabs), and receptor ligands, on the exosome surface at high density. To demonstrate that exosome surface modifications can alter cellular tropism, we generated exosomes displaying anti-Clec9A scFabs to target conventional type 1 dendritic cells (cDC1s), anti-CD3 scFabs to target T cells, and CD40 ligand to target B cells. The engineered exosomes exhibited functional antigen binding that led to greater association with the cell types expressing the cognate receptor both in vitro and in vivo. Results In mice, systemic administration of exosomes engineered to display scFabs targeting Clec9A resulted in a 4-fold increase in the percentage of cDC1 cells in the blood that had taken up exosomes over controls, and a 6-fold increase in the number of exosomes taken up per cell. We further showed that compared to untargeted exosomes, those with altered tropism achieved increased functional payload delivery to the target cell of interest. In primary mouse dendritic cells, anti-Clec9A exosomes loaded with a cyclic dinucleotide STING agonist achieved greater pathway induction, 2.3-fold greater as measured by IFNβ production, 2-fold by IFNα, and 15-fold by IL-12, when compared to an untargeted control. Preliminary in vivo data show that intra-tumorally administered anti-Clec9A exosomes reduce the required STING agonist dose 10-fold to achieve single-agent tumor control and induce immune responses against tumor-associated antigen, compared to controls. Conclusions These results demonstrate the potential of our engExTM platform to generate targeted exosome therapeutics capable of immune cell stimulation and tumor growth inhibition in vivo. Ethics Approval All experimental animal studies were performed according to Codiak BioSciences IACUC approved AUP CB2020-001.

Background The promise of Interleukin-12 as a cancer treatment has yet to be fulfilled with multiple tested approaches being limited by unwanted systemic exposure and unpredictable pharmacology. To address these limitations, we generated exoIL-12™, a novel, engineered-exosome therapeutic that displays functional IL-12 on the surface of an exosome. Methods IL-12 exosomal surface expression was achieved via fusion to the abundant exosomal surface protein PTGFRN. Potency was assessed in vitro using human PBMCs or murine splenocytes and in vivo using mouse subcutaneous tumor models. Local versus systemic pharmacology was determined with intratumoral injection in mice and subcutaneous injection in monkeys. All studies were benchmarked against recombinant IL-12 (rIL-12). Results Exosomes engineered to express either murine or human IL-12 had equivalent potency in vitro to rIL-12 as demonstrated by IFNγ production. Following intratumoral injection, exoIL-12 exhibited prolonged tumor retention and greater antitumor activity than rIL-12. Moreover, exoIL-12 was 100-fold more potent than rIL-12 in tumor growth inhibition. In the MC38 tumor model, complete responses were observed in 63% of mice treated with exoIL-12; in contrast, rIL-12 resulted in 0% complete responses at an equivalent IL-12 dose. This correlated with dose-dependent increases in tumor antigen-specific CD8+ T cells. Re-challenge studies of exoIL-12 in complete responder mice showed no tumor regrowth. Moreover, depletion of CD8+ T cells completely abrogated the antitumor activity of exoIL-12. Following intratumoral administration, exoIL-12 exhibited 10-fold higher intratumoral exposure than rIL-12 and prolonged IFNγ production up to 48 hr. Retained, local pharmacology of exoIL-12 was further confirmed using subcutaneous injections in non-human primates. Conclusions This work demonstrates that tumor-restricted pharmacology of exoIL-12 results in superior in vivo efficacy and immune memory without systemic IL-12 exposure and related toxicity. exoIL-12 is a novel cancer therapeutic candidate that has the potential to overcome key limitations of rIL-12 and thereby create a therapeutic window for this potent cytokine. Ethics Approval All animals were maintained and treated at the animal care facility of Codiak Biosciences in accordance with the regulations and guidelines of the Institutional Animal Care and Use Committee (CB2017-001).

The promise of IL12 as a cancer treatment has yet to be fulfilled with multiple tested approaches being limited by unwanted systemic exposure and unpredictable pharmacology. To address these limitations, we generated exoIL12, a novel, engineered exosome therapeutic that displays functional IL12 on the surface of an exosome. IL12 exosomal surface expression was achieved via fusion to the abundant exosomal surface protein PTGFRN resulting in equivalent potency in vitro to recombinant IL12 (rIL12) as demonstrated by IFNγ production. Following intratumoral injection, exoIL12 exhibited prolonged tumor retention and greater antitumor activity than rIL12. Moreover, exoIL12 was significantly more potent than rIL12 in tumor growth inhibition. In the MC38 model, complete responses were observed in 63% of mice treated with exoIL12; in contrast, rIL12 resulted in 0% complete responses at an equivalent IL12 dose. This correlated with dose-dependent increases in tumor antigen–specific CD8+ T cells. Rechallenge studies of exoIL12 complete responder mice showed no tumor regrowth, and depletion of CD8+ T cells completely abrogated antitumor activity of exoIL12. Following intratumoral administration, exoIL12 exhibited 10-fold higher intratumoral exposure than rIL12 and prolonged IFNγ production up to 48 hours. Retained local pharmacology of exoIL12 was further confirmed using subcutaneous injections in nonhuman primates. This work demonstrates that tumor-restricted pharmacology of exoIL12 results in superior in vivo efficacy and immune memory without systemic IL12 exposure and related toxicity. ExoIL12 is a novel cancer therapeutic candidate that overcomes key limitations of rIL12 and thereby creates a therapeutic window for this potent cytokine.

Background: Tumor-associated macrophages (TAMs) promote tumor progression and resistance to immune checkpoint inhibitors and are thus attractive targets for cancer immunotherapy. The STAT6 transcriptional network is an important driver of the immune-suppressive M2 macrophage program in the tumor microenvironment (TME). Previous attempts to therapeutically target these transcriptional networks have not been successful. Exosomes serve as an efficient, natural, intercellular communication system that can deliver nucleic acids and other macromolecules. Leveraging the potential of exosomes, we have developed a novel, engineered exosome therapeutic candidate loaded with antisense oligonucleotides (ASO) targeting STAT6 (exoASO-STAT6), that effectively silences STAT6 expression in TAMs. Results: In vitro and in vivo studies demonstrate an enhanced delivery of ASO to M2 macrophages. exoASO showed a 2x improvement in uptake vs free ASO in human M2 macrophages in vitro. Following IV administration, exoASO demonstrated up to 11x increase in uptake in monocytes and MDSCs in the blood, Kupffer cells in liver and TAMs and MDSCs in the tumor. In vitro treatment with exoASO-STAT6 resulted in 90% target gene KD in human, mouse and cynomolgus monkey M2 macrophages, which was persistent for up to 10 days. Additionally, exoASO-STAT6 demonstrated greater potency than free ASO. STAT6 KD resulted in a 7x decrease in M2 marker CD163 and a 25x increase in pro-inflammatory cytokines such as IL-12 or TNFα, demonstrating effective macrophage reprogramming. In vivo efficacy studies in CT26 showed potent dose-dependent single agent activity of exoASO-STAT6, with a cumulative dose of 36 μg of ASO resulting in 94% TGI and 80% complete responses. CD8 T-cell depletion abrogated anti-tumor activity and the complete responders were resistant to tumor cell re-challenge, demonstrating a CD8-T cell mediated immunological memory response. In an orthotopic model of HCC that is resistant to anti-PD-1 or anti-CSF1R therapy, IV administration of exoASO-STAT6 significantly attenuated tumor growth, as observed by a 61% reduction in tumor mass and complete elimination of tumor lesions in 50% of treated mice. exoASO-STAT6 therapy resulted in a decrease in M2 markers such as Tgfb1 and Ccl17 and an increase in M1 markers such as IL1b. A significant increase in interferon and cytotoxic T-cell gene signatures was also observed, demonstrating effective reprogramming of the TME. Conclusion: exoASO-STAT6 is a novel therapeutic that selectively targets STAT6, a key transcription factor in TAMs. This therapy results in effective macrophage reprogramming to a pro-inflammatory M1 phenotype and potent single agent anti-tumor activity in multiple checkpoint refractory tumor models. In sum, exoASO-STAT6 represents a first-in-class strategy to target TAMs in a highly selective manner. Citation Format: Sushrut Kamerkar, Charan Leng, Olga Burenkova, Su Chul Jang, Christine McCoy, Kelvin Zhang, William Dahlberg, Marie Leblanc, Hugo Quillery, Sylvie Maubant, Olivier Duchamp, Kyriakos Economides, Timothy Soos, Dalia Burzyn, Sriram Sathyanarayanan. Engineered exosome- mediated STAT6 knockdown in tumor associated macrophages (TAMs) results in potent single agent activity in a hepatocellular carcinoma (HCC) model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1792.

Objective: To feed individual house flies a specific amount of bacteria in order todetermine bacteria “fate” (persistence, via enumeration; spatiotemporal location, viamicroscopy) and house fly immune response (whole fly or tissue-specific, via downstream mRNA or protein expression analyses)

Objective: To feed individual house flies a specific amount of bacteria in order todetermine bacteria “fate” (persistence, via enumeration; spatiotemporal location, viamicroscopy) and house fly immune response (whole fly or tissue-specific, via downstream mRNA or protein expression analyses)

Objective: To feed individual house flies a specific amount of bacteria in order todetermine bacteria “fate” (persistence, via enumeration; spatiotemporal location, viamicroscopy) and house fly immune response (whole fly or tissue-specific, viadownstream mRNA or protein expression analyses).

Exosomes are natural and abundant nanoscale vesicles for intercellular communication, capable of transferring biological instructions between neighboring and distant cell types. Translational research efforts have focused on exploiting this communication mechanism to deliver exogenous pharmacologic payloads to treat a variety of diseases including cancer. Functionalization of the exosome surface with proteins and peptides is an important strategy to maximize the potential of exosomes as therapeutics. Comparative proteomic analysis (LC/MS) of stringently purified exosomes led to the identification of several highly enriched and unique proteins, including a transmembrane glycoprotein (Protein X, PrX), belonging to the immunoglobulin superfamily. Stable expression of PrX in a producer cell line resulted in a 200-fold increase of PrX on the secreted exosomes. Protein X was extensively characterized and the minimum structural requirements for exosome enrichment were determined. With our engExTM platform, we developed precision engineered exosome therapeutics using PrX as a scaffold to enable high-density exosome surface display of an array of structurally and biologically diverse proteins, including enzymes, antibodies, type I cytokines, and TNF superfamily members. These proteins were genetically fused to PrX and overexpressed in a producer cell. Significantly higher transgene expression on secreted exosomes was achieved compared to conventional scaffolds, including the tetraspanins CD9/CD63/CD81 and LAMP2B. Oligomerization of PrX coupled with avidity effects inherent in exosome surface display resulted in a clear activity advantage compared to free protein. Protein X-mediated display of CD40L on exosomes resulted in a 20-fold potency increase in B cell activation over recombinant CD40L. Furthermore, expression of CD40L redirected exosome uptake from phagocytic antigen presenting cells (APCs) to B cells, demonstrating exosome surface modifications can alter cellular tropism. We also evaluated the functionality of IL-12 tethered to the exosome surface and demonstrated superior tumor retention compared to free cytokine, resulting in robust anti-tumor activity in anti-PD-1 refractory B16F10 tumor models. These results demonstrate the potential of the engExTM platform to generate novel exosome therapeutics. Citation Format: 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 D. Kulman, Sriram Sathyanarayanan. engEx: A novel exosome engineering platform enabling targeted transfer of pharmacological molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2150.

Background: The Stimulator of Interferon Genes (STING) pathway is an attractive target in immuno-oncology. Selective activation of the STING pathway in antigen presenting cells (APCs) is essential for eliciting a potent and specific anti-tumor immune response. STING is ubiquitously expressed in normal cells including T cells and endothelial cells. Direct intra-tumoral administration of a free STING agonist results in activation of the STING pathway in all cells, resulting in loss of viability of immune cells, tissue damage, and systemic immune activation due to vascular leakage. Exosomes are an efficient natural messenger system that delivers macromolecules between cells. Leveraging exosome biology, we have developed a novel engineered exosome therapeutic, exoSTING, to selectively target the STING pathway in tumor resident APCs. Results: exoSTING is composed of exosomes engineered to express high levels of Protein X (PrX), a transmembrane glycoprotein naturally occurring on exosomes, and loaded with a STING agonist. Following intra-tumoral injection in checkpoint refractory B16F10 tumors, exoSTING selectively activates STING in APCs, leading to anti-tumor immunity with greater than 100-fold improvement in potency compared to free STING agonist. The activity of exoSTING is substantially diminished in the PrX knock out exosomes, highlighting the importance of this surface glycoprotein for preferential activation of APCs. In contrast to the free STING agonist, exoSTING is retained within the injected tumor, minimizing systemic exposure. Furthermore, exoSTING administration preserves the viability of T cells and APCs, reduces collateral tissue damage, and does not induce systemic cytokine production, resulting in a broader therapeutic window in contrast to free STING agonist. exoSTING produced an increased systemic tumor-specific T cell response as demonstrated by elimination of non-injected abscopal tumors. The specificity of exoSTING activity was demonstrated using a STING knockout mouse (Tmem173gt/J). T cells and myeloid cells play a critical role in exoSTING mediated anti-tumor immunity, in contrast to NK cells which are not required. exoSTING treatment results in significant induction of PD-L1 expression (P Conclusion: exoSTING is an engineered exosome therapeutic that leverages exosome biology and specifically targets the STING pathway in APCs, resulting in greater potency with preserved viability of T cells and APCs, greater systemic tumor antigen specific immune response, reduced systemic cytokine production, and enhanced efficacy. Citation Format: Su Chul Jang, Raymond J. Moniz, Chang Ling Sia, Rane A. Harrison, Damian Houde, Nikki Ross, Ke Xu, Nuruddeen Lewis, Raymond Bourdeau, Christine McCoy, Tong Zi, Agata Villiger-Oberbek, Scott Estes, Jorge Sanchez-Salazar, Kevin Dooley, William K. Dahlberg, Sriram Sathyanarayanan, Kyriakos D. Economides. exoSTING: An engineered exosome therapeutic that selectively delivers STING agonist to the tumor resident antigen-presenting cells resulting in improved tumor antigen-specific adaptive immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 944.

Fishing for Energy (FfE) is a partnership of Covanta Energy Corporation, the National Fish and Wildlife Foundation (NFWF), the National Oceanic and Atmospheric Administration’s (NOAA) Marine Debris Program, and Schnitzer Steel Industries, Inc. The purpose of the FfE partnership is to provide fishermen with a no-cost disposal option for old or derelict fishing gear and to convert it into clean, renewable energy, using state-of-the-art Energy-from-Waste technology. To date, nearly 270 tons of gear has been collected, metals are recovered for recycling, and the rest has generated approximately 330 MWh of electricity.Copyright © 2010 by ASME

All values reported as means±SD. P value was calculated by repeated measures ANOVA. Pair-wise comparisons were done using a significance criterion of 0.017 (0.05/3) in order to account for multiple comparisons. aP,0.017 when comparing the variable between "before IPAA" and "0-12 m after IPAA"; bP,0.017 when comparing the variable between "0-12 m after IPAA" and "12 m+ after IPAA"; cP,0.017 when comparing the variable between "before IPAA" and "12 m+ after IPAA"