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BackgroundTumor associated macrophages (TAMs) play a critical role in tumor immunosuppression and resistance to immune checkpoint blockade. Reprogramming ‘M2’ TAMs to a proinflammatory ‘M1’ phenotype by selectively silencing M2 phenotype-driving transcription factors, such as STAT6, is a promising strategy to relieve TAM-induced immunosuppression. We have developed exoASO-STAT6™, an investigational therapeutic candidate consisting of exosomes loaded with antisense oligonucleotides (ASOs) targeting STAT6. By leveraging the TAM tropism of exosomes, exoASO-STAT6™ is the first systemically administered exosome designed to selectively silence STAT6 in TAMs. Preclinical biodistribution studies demonstrated that the liver is the main organ targeted by exoASO after intravenous (IV) dosing.MethodsWe evaluated the translational potential of exoASO-STAT6 to treat hepatocellular carcinoma through pharmacokinetics (PK), pharmacodynamic (PD) and anti-tumoral efficacy studies in preclinical models, as well as computational analysis of human HCC datasets.ResultsPK/PD were evaluated in naïve mice dosed IV with a single dose of exoASO-STAT6. PK analysis showed that the STAT6-ASO is rapidly cleared from plasma. Retention of the ASO in liver was dose-dependent and observed for at least 21 days. exoASO-STAT6 induced significant Stat6 mRNA knockdown (KD) in liver tissue with maximum KD at day 7 (70% KD at the 30 ug dose). IV administration of exoASO-STAT6 in an orthotopic, CPI resistant HCC model attenuated tumor growth and induced complete remission of tumor lesions in 50% of mice, while combination therapy with anti-PD1 antibodies further enhanced anti-tumor activity (75% complete remissions). Gene expression and histological analysis of the liver showed effective remodeling of the tumor microenvironment including a significant increase in interferon and cytotoxic T-cell gene signatures and iNOS expression. PD studies were also performed in cynomolgus monkeys that demonstrated a dose-dependent and durable silencing of STAT6 mRNA (50% and 31% at 1- and 3- weeks post-dose, respectively). STAT6 knockdown correlated with a reduction in STAT6 target genes, IL4R and EGR2, confirming modulation of the STAT6 pathway. Finally, we identified a STAT6 macrophage transcriptional signature and show high expression in human HCC tumors, both in immune cell-rich and TAM-rich/CD8 T-cell low tumors that correlates with worse survival.ConclusionsIn summary, we demonstrate that exoASO-STAT6 has a durable PK/PD profile in the liver of several species and potent antitumoral efficacy in a preclinical model of HCC. Furthermore, we identify an inverse correlation between the STAT6 macrophage signature and survival in human HCC tumors. Altogether our data support the systemic administration of exoASO-STAT6 as a promising therapy for liver cancer.Ethics ApprovalFor MiceMice 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).Animal housing and experimental procedures (mice) were conducted according to the French and European Regulations and the National Research Council Guide for the Care and Use of Laboratory Animals and Institutional Animal Care and Use Committee of Oncodesign (Oncomet) approved by French authorities (CNREEA agreement N° 91).For cynomolgus monkeys:All animals were maintained and treated at the animal care facility of Altasciences in compliance with the Animal Welfare Act and recommendations set forth in the Guide for the Care and Use of Laboratory Animals (National Research Council 2011).

Background: Tumor-associated macrophages (TAMs) and myeloid derived suppressor cells (MDSCs) promote an immunosuppressive milieu by inhibiting T-cell activation and recruitment, leading to resistance to immune checkpoint therapies. Interleukin 10 (IL10) and prostaglandin E2 (PGE2) promote tumor initiation and spread, in part, through TAM polarization and MDSC recruitment. C/EBPβ is a critical transcription factor that regulates the immunosuppressive state of TAMs and MDSCs and is activated by IL10 and PGE2 signaling. We developed a proprietary engineered exosome loaded with antisense oligonucleotides (ASOs) targeting C/EBPβ (exoASO-C/EBPβ), that selectively delivers ASO to TAMs and MDSCs, thereby inhibiting C/EBPβ expression and inducing reprogramming from an immunosuppressive (M2) to a pro-inflammatory (M1) phenotype. Results: exoASO-C/EBPβ demonstrated dose-dependent target gene knockdown (KD) in primary human M2 macrophages in vitro with a 2-fold higher potency (IC50) compared to free ASO. C/EBPβ KD resulted in profound changes in gene expression and cytokine secretion profile consistent with reprogramming to a M1 phenotype. exoASO-C/EBPβ induced a significant induction of pro-inflammatory cytokines, in M2 macrophages generated with IL10 or PGE2, both of which show prominent activation of the C/EBPβ pathway. Biodistribution in tumor-bearing mice (IT and IV) demonstrate selective uptake by MDSCs and TAMs. Intra-tumoral (IT) administration of exoASO-C/EBPβ in CT26 tumors resulted in a significantly higher KD of C/EBPβ in tumor-associated CD11b+ cells, as compared to CD11b- cells. Additionally, exoASO-C/EBPβ induced changes in Nos2, CD206 and Csf1r mRNA expression in CD11b+ cells, demonstrating in vivo M2 to M1 reprogramming. Furthermore, IT microinjections of exoASO-C/EBPβ in YUMM1.7 tumor-bearing mice resulted in a significant induction of pro-inflammatory M1 markers TNFα and iNOS, in contrast, free C/EBPβ ASO showed limited increase in M1 markers. exoASO-C/EBPβ administered IT as a single agent demonstrated significant anti-tumor activity in multiple tumor models including: the CT26-colorectal cancer (60% complete responses (CRs)), MB49-bladder cancer (70% CRs) and B16F10-melanoma (30% CRs). Combination treatment with anti-PD1 in all these models further increased the anti-tumor efficacy. Finally, we used a myeloid cell specific C/EBPβ/PGE2/IL10 gene signature to identify cancer indications where exoASO-C/EBPβ therapy might have the most therapeutic significance. Conclusion: exoASO-C/EBPβ is a novel therapeutic candidate that selectively targets and attenuates a critical transcription factor in immunosuppressive myeloid cells, resulting in reprogramming of TAMs and potent anti-tumor activity across multiple TAM-rich mouse models as a monotherapy. Citation Format: Sushrut Kamerkar, Charan Leng, Olga Burenkova, SuChul Jang, Kelvin Zhang, Yanyan Liu, William Dahlberg, Kyriakos Economides, Timothy Soos, Dalia Burzyn, Sriram Sathyanarayanan. Exosome-mediated delivery of antisense oligonucleotides targeting C/EBPβ reprograms tumor-associated macrophages and induces potent single agent anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3508.

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.

The mutant form of the GTPase KRAS is a key driver of pancreatic cancer but remains a challenging therapeutic target. Exosomes are extracellular vesicles generated by all cells, and are naturally present in the blood. Here we show that enhanced retention of exosomes, compared to liposomes, in the circulation of mice is likely due to CD47-mediated protection of exosomes from phagocytosis by monocytes and macrophages. Exosomes derived from normal fibroblast-like mesenchymal cells were engineered to carry short interfering RNA or short hairpin RNA specific to oncogenic KrasG12D, a common mutation in pancreatic cancer. Compared to liposomes, the engineered exosomes (known as iExosomes) target oncogenic KRAS with an enhanced efficacy that is dependent on CD47, and is facilitated by macropinocytosis. Treatment with iExosomes suppressed cancer in multiple mouse models of pancreatic cancer and significantly increased overall survival. Our results demonstrate an approach for direct and specific targeting of oncogenic KRAS in tumours using iExosomes.

Background: Tumor-associated macrophages (TAMs) are potent drivers of an immunosuppressive tumor microenvironment by reducing T cell recruitment and activation, promoting resistance to immune checkpoint inhibition. Experimental therapies blocking monocyte differentiation or eliminating TAMs show minimal anti-tumor activity. STAT6 and C/EBPβ have key roles in regulating the immunosuppressive state of TAMs however previous attempts to selectively target these transcription factors were unsuccessful. We have developed proprietary engineered exosome therapeutic candidates loaded with antisense oligos (ASOs) targeting STAT6 and C/EBPβ (exoASO). exoASO is designed to selectively deliver ASOs to TAMs and decrease STAT6 and C/EBPβ expression, leading to reprogramming of M2-like TAMs to a M1-like immune stimulatory phenotype. Results: exoASO-STAT6 and exoASO-C/EBPβ induced dose-dependent knockdown (KD) of target genes in primary human M2 macrophages with greater potency (IC50) than free ASO. Gene expression analysis and cytokine assays showed that exoASO-mediated KD resulted in a marked reprogramming to a proinflammatory (M1) phenotype. IV administration of exoASO in a TAM-rich model (CT-26) demonstrates selective uptake by myeloid derived suppressor cells and macrophages. We evaluated efficacy of exoASO-STAT6 or exoASO-C/EBPβ alone or in combination with anti-PD1 therapy. Both exoASO-STAT6 and exoASO-C/EBPβ were efficacious as monotherapies (50% and 60% complete responses (CR) respectively), whereas anti-PD1 or anti-CSFR1 mAb were not effective (0% CR). exoASO-C/EBPβ in combination with anti-PD1 mAb resulted in greater efficacy (80% CR), and enhanced survival (70% at day 55) compared to exoASO-C/EBPβ monotherapy (50% survival at day 55). In contrast, combination of anti-PD1 with exoASO-STAT6 did not show improved efficacy as compared to monotherapy. Durable anti-tumor responses were observed with both exoASO therapies as mice achieving CR against the primary tumor had no tumor growth upon re-challenge. We evaluated anti-tumor efficacy using an orthotopic hepatocellular carcinoma (HCC) model. Mice were dosed IV using: exoASO-C/EBPβ; free C/EBPβ ASO; anti-PD1 or; anti-CSF1R mAbs. exoASO-C/EBPβ treatment attenuated HCC-mediated increase in liver to body weight ratios (≤ 10%) and showed little to no observable tumor lesions in 50% of treated mice. In contrast, increased liver to body weight ratios (≥ 10%) and observable tumor lesions occurred in all mice (100%) treated with either free C/EBPβ ASO or anti-CSF1R mAb. Conclusion: exoASO-STAT6 and exoASO-C/EBPβ are efficacious as monotherapies in TAM-rich tumors when administered locally or systemically. exoASO-C/EBPβ has increased potency when administered in combination with checkpoint antibodies. Collectively, exoASOs against STAT6 and C/EBPβ are a first-in-class strategy to target TAMs in a highly selective manner. Citation Format: Sushrut Kamerkar, Dalia Burzyn, Olga Burenkova, Charan Leng, Raymond Yang, Katherin Kirwin, Tong Zi, Su Chul Jang, William Dahlberg, Eric Zhang, Scott Estes, Sylvie Maubant, Olivier Duchamp, Kyriakos Economides, Timothy Soos, Sriram Sathyanarayanan. Genetic reprogramming of TAMs by engineered exosomes results in potent single agent anti-tumor activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5696.

Exosomes are extracellular vesicles produced by all cells with a remarkable ability to efficiently transfer genetic material, including exogenously loaded siRNA, to cancer cells. Here, we report on a bioreactor-based, large-scale production of clinical-grade exosomes employing good manufacturing practice (GMP) standards. A standard operating procedure was established to generate engineered exosomes with the ability to target oncogenic Kras (iExosomes). The clinical-grade GMP iExosomes were tested in multiple in vitro and in vivo studies to confirm suppression of oncogenic Kras and an increase in the survival of several mouse models with pancreatic cancer. We perform studies to determine the shelf life, biodistribution, toxicology profile, and efficacy in combination with chemotherapy to inform future clinical testing of GMP iExosomes. Collectively, this report illustrates the process and feasibility of generating clinical-grade exosomes for various therapies of human diseases.

Toxoplasma gondiiis a prevalent obligate intracellular parasite which chronically infects more than a third of the world’s population. Key to parasite prevalence is its ability to form chronic and nonimmunogenic bradyzoite cysts, which typically form in the brain and muscle cells of infected mammals, including humans. While acute clinical infection typically involves neurological and/or ocular damage, chronic infection has been more recently linked to behavioral changes. Establishment and maintenance of chronic infection involves a balance between the host immunity and parasite evasion of the immune response. Here, we outline the known cellular interplay betweenToxoplasma gondiiand cells of the central nervous system and review the reported effects ofToxoplasma gondiion behavior and neurological disease. Finally, we review new technologies which will allow us to more fully understand host-pathogen interactions.