Mouse iPSC-derived Endothelial Cells

Cd64-expressing Mouse iECs are Protected from Antibody-mediated Killing

Allogeneic cell therapies often fail because the recipient's antibodies recognize and destroy the transplanted cells (via ADCC or CDC), significantly limiting their survival and efficacy. To develop a "cloaking" strategy by engineering cells to overexpress the IgG receptor CD64. This allows cells to capture host antibodies and block their killing mechanisms, enabling allogeneic grafts-including iECs, beta cells, and CAR T cells-to evade antibody-mediated destruction.

Mouse C57BL/6 (B6) induced pluripotent stem cells (iPSCs) were differentiated into B6 iECs, and the cells were transduced with lentiviral particles to express the mouse Cd64 transgene. These B6 iECs^Cd64 bound free IgG2a Fc in a dose-dependent manner (Fig. 1a, b). In impedance-based killing assays, Cd64 expression fully protected B6 iECs against ADCC and CDC mediated by anti-H-2b antibodies (Fig. 1c, d).

The strategy was then applied to B6 "hypoimmune" (HIP) iECs (*B2m^-/-Ciita^-/-*Cd47), which evade cellular immunity but remain vulnerable to antibodies (Fig. 2a). After adding human CD64 and the target antigen CD52 (Fig. 2b), these cells demonstrated robust human IgG1 Fc capture (Fig. 2c). While B6 HIP iECs^CD52 were susceptible to alemtuzumab-mediated killing, B6 HIP iECs^CD52,CD64 remained entirely resistant to both ADCC and CDC (Fig. 2d, e). In vivo, Luc+ grafts were transplanted into Rag1^-/- mice (Fig. 2f). Following alemtuzumab treatment, B6 HIP iECs^CD52 grafts were eliminated within one week, whereas B6 HIP iECs^CD52,CD64 grafts survived, confirming that CD64 provides critical antibody protection in vivo (Fig. 2g, h).