Engineering of QBEnd10- and rituximab-binding domains. (A) Coding sequences for the 31 extreme amino-terminal residues of mature full-length CD34 (flCD3) were cloned in-frame to the CD8 stalk and transmembrane domain via a serine-glycine linker (LSTK) or without this linker (STK) or connected directly to the CD8 transmembrane domain (flush). These open-reading frames were coexpressed with eGFP within a bicistronic retroviral vector. (B) Flow cytometric analysis of eGFP and QBEnd10 staining in T cells transduced with flCD34, LSTK, STK. QBEnd10 binding equivalent to that of flCD34 was seen in constructs containing the CD8 stalk, but not with the flush construct. The serine glycine linker did not improve QBEnd10 binding. (C) Further epitope minimization was performed by sequential amino- and carboxy-terminal deletion of the 31 residues of CD34 until binding of QBEnd10 was abrogated. In this way, we established a final minimal epitope-binding construct containing only 16-amino-acid residues from the 385 present in the native antigen. (D) Binding of rituximab to T cells transduced with full-length CD20 (CD20), the major extracellular loop of CD20 with some flanking residues on the CD8 stalk (dCD20-loop v2), the major extracellular loop of CD20 delineated precisely at the constraining cysteine on a CD8 stalk (dCD20-loop v1), and the circular CD20 mimotope described by Perosa et al²⁶  on a CD8 stalk.