Scalable production and anti-tumor efficacy of iPSC-derived immortalized anti-CD19 CAR macrophages

Abstract

Abstract Chimeric antigen receptor (CAR) -T cell therapy has shown remarkable efficacy in patients with relapsed or refractory mature B cell lymphoma and multiple myeloma. However, durable responses remain limited due to several factors, including antigen escape, T-cell exhaustion, and insufficient tumor infiltration. Therefore, relapse following CAR-T cell therapy is associated with poor prognosis, and practical strategies for treating these cases have not yet been established. Moreover, the autologous nature of CAR-T cell manufacturing leads to logistical delays, preventing immediate administration and requiring interim treatments for disease management during the manufacturing window. To overcome these problems, induced pluripotent stem cell (iPSC)-derived immune cells have emerged as a promising off-the-shelf alternative. Recent developments in iPSC-derived CAR-macrophages have demonstrated remarkable progress, with encouraging therapeutic efficacy. However, clinical translation remains hindered by the need of a considerable number of cells, highlighting the challenge of scalable production. We developed a novel immortalized precursor cell line by introducing a doxycycline-inducible system for c-MYC, BMI1, and BCL-XL, enabling robust and scalable cell expansion. This approach allows for more efficient large-scale production compared to conventional iPSC-derived differentiation strategies. We have established immortalized macrophages (imMac), which can proliferate for an extended period in the presence of doxycycline (dox-on imMac). Upon doxycycline removal, these cells undergo terminal differentiation into mature macrophages (dox-off imMac) within one week. In this study, we generated a CD19-targeted CAR-imMac (CAR-imMac) by transducing imMac with a lentiviral vector encoding a CD19-specific CAR. This CAR-imMac has the potential to expand to 1×10²⁵ cells over a 3-month culture period, allowing for the delivery of up to 1×10¹⁵ doses. We assessed the anti-tumor activity of CD19-CAR-imMac in vitro using K562 cells engineered to overexpress CD19 via lentiviral transduction. Flow cytometry analysis revealed that CAR-imMac exhibited significantly enhanced phagocytosis of CD19-overexpressing K562 cells compared to CD19-negative K562 cells (16.87% vs. 5.61%, p < 0.0001). Upon M1 polarization with interferon-γ (IFN-γ) and lipopolysaccharide (LPS), CAR-imMac exhibited further increased phagocytic capacity (non-M1 vs. M1: 16.87% vs. 26.20%, p < 0.0001) and significantly stronger anti-tumor activity against CD19-overexpressing K562 cells (p = 0.047). M1-polarized CAR-imMac also demonstrated superior tumor killing efficacy compared to non-polarized CAR-imMac (p = 0.041). Additionally, cytokine bead array (CBA) analysis revealed elevated secretion of inflammatory cytokines, such as TNF, IL-1β and IL-6, in M1 CAR-imMac co-cultured with tumors. In a xenograft mouse model injected with CD19-overexpressing K562 cells, we assessed the anti-tumor efficacy of imMac cells at different stages of differentiation. Mice received either dox-on CAR-imMac, consisting of macrophage progenitors cultured with doxycycline until just prior to transplantation, or dox-off CAR-imMac, comprising mature macrophages generated by doxycycline withdrawal seven days before transplantation. No doxycycline was administered to mice following cell transplantation in either group. We compared three experimental groups: (1) untreated control, (2) dox-on CAR-imMac combined with anti-CD47 antibody, and (3) dox-off CAR-imMac combined with anti-CD47 antibody. The dox-on CAR-imMac group showed substantially greater inhibition of tumor growth on day 13 (p = 0.0449) and day 20 (p = 0.0386), along with prolonged survival compared to the untreated control group (p < 0.01). Notably, a trend toward improved survival was also observed in the dox-on group relative to the dox-off CAR-imMac group (p = 0.0982). We speculated that the superior efficacy of dox-on CAR-imMac cells may be due to their floating, progenitor-like state and enhanced migratory capacity, which may enhance their ability to engage tumor cells within the peritoneal cavity. Collectively, our study demonstrated the therapeutic potential of CAR-imMac against B-cell lymphoma and highlighted the feasibility of off-the-shelf CAR-imMac production. The efficient and high-yield manufacturing process supports its scalability and sustainability for industrial-scale production and broad clinical deployment.