Macrophage Engineered Vesicles for Pediatric Osteosarcoma

Prior to the development of effective chemotherapy regimens, osteosarcoma (OS) was nearly universally fatal. Currently, the five-year survival is approximately 66%, but metastasis remains the most important fatal complication of OS and a therapy preventing OS metastases would be the single most significant improvement in outcome since the advent of chemotherapy in the 1960s. Micro-metastasis are undetectable but generally present at diagnosis. They are often in the G0 or resting phase of the cell cycle and insensitive to chemotherapy. Micro-metastasis exist in a microenvironment populated with tumor associated macrophages (TAMs) that support cancer growth and survival allowing the micro-metastasis to grow to overt metastatic disease after chemotherapy is completed. A strategy that can convert TAMs to the M1 or anticancer phenotype, has the potential to eradicate micro-metastatic disease and prevent the development of pulmonary metastases, and ultimately improve survival for children with OS. The overall goal of this project is to prevent metastasis in children with OS. To accomplish this goal, we have developed and tested M1 Macrophage-derived Engineered Vesicles (MEVs) from murine, bone marrow- derived monocytes and human monocytes. As further described in preliminary data, MEVs specifically interact with tumors in vivo and MEVs are able to convert TAMs to the M1 macrophage phenotype which leads to anticancer activity. In addition, an osteosarcoma cancer animal model system demonstrated that both cisplatin loaded MEVs and empty MEVs have 1) no observed or laboratory adverse effects after 12 weekly doses; 2) improved efficacy over saline and cisplatin controls on the primary tumor; 3) prevent the development of pulmonary metastasis. Given impressive preclinical activity, no toxicity and clear unmet medical need, the overarching goal of this proposal is to further advance MEVs as a therapeutic strategy by completing crucial IND enabling studies. We have already run one clinical manufacturing batch to establish feasibility and have established the majority of release criteria. We need to establish one additional release criteria (TNF), complete 3 additional test batches and conduct a dose response study in mice. Our central hypothesis is that MEVs repolarize TAMs, which will eradicate micro-metastatic disease and ultimately prevent the development of metastasis and improve survival in OS. Aim 1. Transfer manufacturing process of MEVs to a GMP facility. Production criteria including manufacture of ~100 nM diameter MEVs from a variety of cell types with yields of ~ 5 x 1012 particles per batch, drug concentrations of ~125 ug/mL, a batch-to-batch CV% of less than 15% with a less than 10% failure rate has been completed. An additional functional assay, TNF release, needs to be established. In addition, to support a phase 1 clinical trial, a critical step is to transfer manufacturing to a GMP facility with CLIA capability, which will be the Cell Therapy Manufacturing Lab at the University of Kentucky. Aim 1a. Escalating doses of MEVs will be incubated with M2 macrophages and TNF production is measured by ELISA to determine the dose response of macrophage repolarization and establish a functional release criteria. Aim 1b. Apheresis collections from healthy volunteers will be obtained from commercial vendors. Complete preparation of MEVs from 3 normal volunteers that meet the acceptance criteria outlined above will be performed. Successful completion of these milestone will establish the GMP manufacturing process that will support our phase 1 clinical trial. Aim 2. Conduct animal dose escalation trial. In a proof of concept study using an osteosarcoma cancer model (143B OS luciferase labeled in BALB/c scid mouse), the mean weekly MEV particle number and cisplatin dose administered was 4.21x1012 and 115 μg (4.6 ug/g), respectively. Aim1a. To determine the effect of MEV dose on clinical activity in a human 143B OS model, a three dose study will be conducted. A high (4.2x1012 particle number), intermediate (2.1 x1012 particle number) and low dose (1.1x1012 particle number) will be assessed. Mice will be dosed weekly for up to 10 doses and changes in tumor volume will be assessed by luciferase imaging. The primary comparison is the development of pulmonary metastasis between doses levels, however regression in tibial tumor burden and adverse effects will also be assessed. Aim 1b. Toxicity via weight, laboratory parameters and pathological assessment of mouse tumors. The proposed studies will establish GMP manufacturing and determine the starting dose for our phase 1 clinical trial, which will establish the recommended phase 2 dose of MEVs. In addition, we anticipate engineered vesicles are a platform technology for use as highly specific drug delivery nanoparticles as well as modulators of biological response for a variety of diseases and hypothesize specific delivery can both improve efficacy and eliminate adverse effects.