Correction of cross-linker sensitivity of Fanconi anemia group F cells by CD33-mediated protein transfer

Studies have previously described the feasibility of receptor-mediated prot ein transfer in a cell culture model of Fanconi anemia (FA) group C. This s tudy explores the versatility of this approach by using an antibody single- chain fusion protein to correct the phenotypic defect in FA group F cells. A 68.5-kd chimeric protein (His-M195FANCF) was expressed, consisting of a H is tag, a single-chain antibody to the myeloid antigen CD33, and the FANCF protein, as well as a 43-kd His-FANCF fusion protein lacking the antibody m otif, in Escherichia coli. The nickel-agarose-purified His-M195FANCF protei n bound specifically to the surface of HeLa cells transfected with CD33 and internalized through vesicular structures. The fusion protein, but not CD3 3, sorted to the nucleus, consistent with the known nuclear localization of FANCF. No similar binding or internalization was observed with His-FANCF. Pretreatment of the transfected cells with chloroquine abolished nuclear ac cumulation, but there was little change with brefeldin A, indicating a mini mal if any role for the Golgi apparatus in mediating transport from endosom es to the cytosol and the nucleus. The intracellular half-life of His-M195F ANCF was ap-proximately 160 minutes. Treatment of CD33-transfected FA group F lymphoblastoid cells with 0.1 mg/mL His-M195FANCF conferred resistance t o mitomycin C. No similar protection was noted in CD33-parental cells or CD 33(+) FA cells belonging to groups A and C. These results demonstrate that antibody-directed, receptor-mediated protein transfer is a versatile method for the delivery of biologically active proteins into hematopoietic cells. (C) 2001 by The American Society of Hematology.