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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.