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Metastatic triple negative breast cancer (TNBC) includes poor prognoses and limited effective therapies. Metastasis is the major barrier to durable clinical responses as many patients appear cured of their disease, yet return to clinic with recurrences from dormant micro-metastases. Immune activating agents, such as Interleukin 12 (IL-12) have attracted attention as anti-cancer adjuvants due to their ability to elicit systemic anti-cancer immunity. Although targeting distributed metastases can be accomplished by intravenous (IV) administration, administration of IL-12 into patient vasculature is highly inefficient due to its short half-life and poor toxicity profile. There is a major unmet need to develop biocompatible carriers that improve targeted delivery of IL-12 by specifically homing to metastatic lesions. Mesenchymal stem cells (MSCs) are considered ideal biodelivery vehicles as they display innate tumor-trophism and can deliver biomolecules specifically to systemic metastases. However, major issues need to be addressed before MSCs can be used to deliver IL-12 to patients: MSC tumor homing needs to be improved, and unmodified MSCs can engraft and contribute to tumor growth and metastasis, raising legitimate safety concerns. To address these major limitations, our laboratory developed a MSC-derived biodelivery vehicle with enhanced homing abilities to deliver biomolecules directly to tumors. Our approach involved using scRNA-seq to identify chemoattractant receptor/ligand pairs (CXCR4/CXCL12, CCR2/CCL2) and endothelial adhesion molecules (PSGL-1/P-E-Selectins) that mediate leukocyte homing and extravasation. We then lentivirally expressed these receptors in MSCs and removed nuclei using Ficoll gradient ultracentrifugation. These bioengineered enucleated MSCs (termed Cargocytes™) have a defined lifespan (4 days) and robustly home to TNBC. This is an important breakthrough as TNBC-trophic Cargocytes can be scaled for clinical applications and are safe for in vivo use as described in our recent publication (Nature Biomedical Engineering, in press). By using syngeneic mouse models of metastatic TNBC, we have observed IV-injected Cargocytes robustly extravasate and precisely home to metastatic lung nodules, suggesting Cargocytes act as remarkable biodelivery vehicles. Indeed, the tumor-trophism of Cargocytes includes both integrating into large macro-metastases and homing to broadly distributed micro-metastases. Cargocytes effectively produce and deliver high doses of IL-12 to mouse TNBC, which induces strong immune activation in tumors. This immune activation involved a potent anti-metastatic response, evidencing strong therapeutic efficacy. These findings suggest Cargocyte-delivered IL-12 could constitute a safe, potent, and highly specific anti-metastatic therapy for TNBC and other metastatic cancers sharing similar chemoattractant profiles. Citation Format: Jeffrey White, Huawei Wang, Flavia Franco Da Cunha, Maureen Ruchhoeft, Richard Klemke. Bioengineering enucleated cell vehicles for targeted delivery of Interleukin 12 to metastatic tumors [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 3519.

Metastatic triple negative breast cancer (TNBC) includes poor prognoses and limited effective therapies. The prevalence of systemic micro-metastases is a major barrier to achieving durable clinical outcomes. Oncolytic viruses (OVs) have garnered attention as anti-cancer adjuvants due to their potential for systemic delivery and ability to lyse tumor cells while leaving healthy cells unaffected. Clinical trials have been conducted testing the efficacy of OVs on diverse cancers. Although targeting distributed metastases can be accomplished by intravenous (IV) administration, administration of virus into patient vasculature is inefficient due to the clearance of unprotected viruses by host immunity. There is a major unmet need to develop biocompatible carriers that improve delivery of OVs by circumventing host anti-viral immunity while homing to metastatic lesions. Mesenchymal stem cells (MSCs) are ideal OV carriers as they display innate tumor-trophic properties, propagate OVs, and shield virions from immunity until reaching their target. However, issues need to be addressed before MSCs can deliver OVs to patients: tumor homing needs improved and unmodified MSCs can engraft and contribute to tumor growth and metastasis, raising safety concerns. To address these limitations, our laboratory developed a MSC-derived biodelivery vehicle with enhanced homing abilities to deliver biologics, such as OVs, directly to tumors. Our approach involved using scRNA-seq to identify chemoattractant receptor/ligand pairs (CXCR4/CXCL12, CCR2/CCL2) and endothelial adhesion molecules (PSGL-1/P-E-Selectins) that mediate leukocyte tumor homing and extravasation. We then lentivirally expressed these receptors in MSCs and removed all DNA using ultracentrifugation and Ficoll density gradients. These bioengineered enucleated MSCs (Cargocytes™) have a defined lifespan (4 days) and robustly home to TNBC. This is an important breakthrough as TNBC-trophic Cargocytes can be scaled for clinical applications and are safe for in vivo use as described in our recent publication (Nature Biomedical Engineering, in press). OVs replicate in and lyse interferon-deficient tumor cells while having negligible effects on healthy cells. As the interferon response is absent in enucleated cells, Cargocytes support robust replication of OVs, including oncolytic Herpes Simplex Virus, Measles Virus, and Vesicular Stomatitis Virus (oVSV), allowing IV delivery of high titer OVs to tumors. In in vivo mouse models of metastatic TNBC, carrier-free oVSV fails to infect lung metastases. However, Cargocytes efficiently deliver oVSV to distributed micro-metastases and integrate deep into macro-metastases, evidencing a remarkable improvement in tissue distribution over intra-tumoral injections. Our findings suggest Cargocytes host and target oVSV directly to TNBC, constituting a highly specific OV delivery vehicle. Citation Format: Jeffrey White, Huawei Wang, Flavia Franco Da Cunha, Maureen Ruchhoeft, Richard Klemke. Bioengineering enucleated cell vehicles for tumor targeted intravenous delivery of oncolytic viruses [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 378.