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Background Myeloid cells are found in virtually all tumors and play diverse roles in the tumor microenvironment (TME), with the ability to contribute to both pro-tumor and anti-tumor functions depending on their phenotype and environment. During tumor progression, bone marrow cells are activated and mobilized to tumor and pre-metastatic sites where they rapidly differentiate into immunosuppressive myeloid cells. We hypothesized that we can take advantage of myeloid biology and infiltration into tumors to deliver therapeutic cargo to reprogram the TME in favor of anti-tumor immunity. In mouse models, we demonstrated that Genetically Engineered Myeloid cells (GEMys) engineered to express interleukin 12 (IL-12) reverse the immunosuppressive signature and activate effective anti-tumor immunity.1 In combination with cyclophosphamide/fludarabine (Cy/Flu) pre-conditioning, IL-12 GEMys cure mice of established solid tumors and provide protection from subsequent rechallenge with the same tumor line, indicating formation of immunological memory against the tumor. We are translating these promising pre-clinical findings into the clinic and have developed a manufacturing protocol for human GEMys which will provide a platform for modular cell therapy development.Methods We generated a lentiviral vector encoding truncated human epidermal growth factor receptor (tEGFR) and single chain human IL-12. The tEGFR serves as a marker of transduction efficiency for product release and can also be targeted using anti-EGFR antibodies for depletion of transduced cells in vivo in case of toxicity. We obtained granulocyte colony-stimulating factor mobilized apheresis product from three healthy donors and isolated CD34+ cells using the CliniMACS Plus system. Cryopreserved CD34+ cells were thawed, transduced, expanded, and differentiated. We characterized the final IL-12 GEMy cell product by measuring transduction efficiency, IL-12 secretion, IFN-gamma production in co-culture with donor matched T cells, and described the cell phenotype in vitro and in vivo in immunocompromised mouse models.Results We successfully manufactured human CD34+ cell-derived IL-12 GEMys that express tEGFR on the surface and secrete IL-12. The phenotype of the final cell product is consistent with a myeloid progenitor phenotype that rapidly differentiates into myeloid cells following injection into mice or additional time in culture.Conclusions We have demonstrated the feasibility and reproducibility of manufacturing human IL-12 GEMys. These data will enable an NCI-sponsored first-in-human clinical trial of IL-12 GEMys in patients with relapsed and refractory solid tumors. Patients will be mobilized, apheresed for cell product manufacturing, treated with a standard preconditioning regimen of Cy/Flu, and receive IL-12 GEMys intravenously in a 3+3 dose escalation trial design.Reference Kaczanowska S, Beury DW, Gopalan V, Tycko AK, Qin H, Clements ME, Drake J, Nwanze C, Murgai M, Rae Z, Ju W, Alexander KA, Kline J, Contreras CF, Wessel KM, Patel S, Hannenhalli S, Kelly M, Kaplan RN. Genetically engineered myeloid cells rebalance the core immune suppression program in metastasis. Cell. 2021;184:1-20.