Modified Hematopoietic Stem Cell-Derived Dendritic Cell Therapy Retained Tumor-Inhibitory Function and Led to Regression of Primary and Metastatic Pancreatic Tumors in Humanized Mouse Models

<b>Background/Objectives</b>: Dendritic cell (DC)-based immunotherapies offer a promising strategy for cancer treatment but are limited by inefficient activation of cytotoxic T cells and, in turn, the host immune system. This report demonstrated that CD34⁺ hematopoietic stem cell (HSC)-derived allogeneic DCs engineered by an optimized lentiviral vector (LVV) expressing CD93, CD40-ligand (CD40L), and Chemokine (C-X-C motif) ligand-13 (CXCL13) significantly enhanced the host immune system, activated tumor-specific cytotoxic T cells, and led to complete regression of both primary and metastatic pancreatic tumors in humanized mouse models. This LVV shows comparable pre-clinical efficacy compared to the first-generation vector, in addition to being compliant for clinical use, which allows further pre-clinical development towards the human trials. <b>Methods</b>: This 2nd generation (Gen) LVV incorporates codon-optimized transgenes (CD40L, CD93, and CXCL13) with rearranged sequence to enhance expression, driven by a strong EF1α promoter. CD34⁺ HSCs were transduced with this modified 2nd Gen LVV and differentiated to Engineered DCs. Therapeutic efficacy of the DC therapy with the modified vector was tested on humanized mouse models of pancreatic tumors. This was accomplished by establishing an early-stage disease model (using MIA PaCa-2 (MP2)-tumors) and late-stage metastatic disease model of the pancreatic tumors to mimic the clinical setting using luciferase-expressing MP2-(Luc)-pancreatic tumor-bearing humanized mice. <b>Results</b>: The modified lentiviral construct had 6-fold greater expression of CD40L, 2% less toxicity, 4.5-fold greater CD40L, and 2.2-fold greater CXCL13 secretion than its predecessor. In vitro, Engineered DCs induced robust T cell proliferation in up to 20% of T cells, up to 4-fold greater interferon-gamma (IFN-γ) secretion than controls, and showcased antigen-specific cytotoxicity by CD8⁺ T cells. In vivo, two intradermal doses of the 2nd Gen DCs led to complete regression of primary pancreatic tumors and metastases. Treated mice exhibited prolonged survival, indicating the induction of durable anti-tumor immunity. <b>Conclusions</b>: Vector optimization retained the efficacy of DC-based therapy, achieving curative responses in pancreatic tumor models. These findings support the clinical development of this 2nd Gen DC immunotherapy for pancreatic and potentially other tumors.