PhD Student University College London (UCL) London, United Kingdom
Abstract: Chimeric antigen receptor macrophages (CAR-M) represent a promising immunotherapy candidate for solid tumours, leveraging macrophages’ ability to infiltrate and remodel the tumour microenvironment. Yet CAR-M manufacturing is hindered by donor variability, limited scalability, and low transduction efficiency, posing challenges for clinical translation. We have established a standardized bioprocess to generate CAR-M from induced pluripotent stem cells (iPSCs) and umbilical cord blood-derived CD34+ hematopoietic stem cells (CB-HSCs), providing a reproducible alternative cell source to monocyte-derived macrophages. CB-HSCs were expanded 51-fold over 7 days with 97.47% viability using a defined cytokine cocktail (hSCF, hFLT3L, hTPO, IL-3), followed by differentiation into macrophages over 10 days, yielding cells with characteristic morphology and ~90% expression of CD45, CD14, and CD11b. Flow cytometry analysis of transduced HSCs demonstrated a transduction efficiency of up to 50% via GFP expression. The sorted GFP⁺ population maintained high cell viability and 95% of CD34 expression. Genetic modification with a lentiviral vector encoding an anti-CEA CAR with dual signalling domains successfully generated functional CAR-Ms. Live-cell imaging confirmed progressive, antigen-dependent phagocytosis of MC-38-CEA cells, increasing over time and peaking at 48 hours, accompanied by rising pro-inflammatory cytokine secretion. The dual-signalling CAR conferred M1 polarization and superior tumour cell engulfment compared to non-transduced HSCs-derived macrophages. This process establishes a reproducible framework for CAR-M production, addressing key challenges in standardisation and manufacturing for next-generation macrophage-based immunotherapies.
Funding Source: Government of the Kingdom of Saudi Arabia
