in part due to new antibody display and other library screening techniques, which are now being exploited to isolate human antibodies with high affinity and specificity. The microbial surface display technologies for screening antibody libraries include phage, yeast and bacteria. Phage-display is widely used due to its simplicity, versatility and ability to be adapted to many specific conditions, including selection on whole cells and tissues. Yeast and bacteria display platforms have several advantages over the phage system including use of flow-cytometry and sorting techniques to enable finer affinity discrimination of selected antibodies. Among the non-microbial systems is ribosomal display that has the capacity to screen libraries of greater size as well as facilitating diversity and efficient antibody maturation in vitro. Although isolation of human antibodies from the above mentioned systems has been successful, there can be unexpected problems with subsequent 23727046 therapeutic mAb development due to constraints of protein expression, correct folding and posttranslational modifications. This has been particularly true for antibodies isolated by phage-display technology. There has been great interest in screening antibodies directly from mammalian Antibody Display and Discovery cells due to their ability to provide proper posttranslational modification, as well as the existence of the natural chaperones that assist in antibody folding. Animal cells have been used for the direct screening of hypermutating antibodies and during antibody selection from a retroviral-antibody display library. Transient antibody 
expression on the surface of human 293T has also been recently reported as a system to perform in vitro affinity maturation of human antibodies. Furthermore, sulfation of tyrosine residues in the CDR residues of human antibodies can markedly affect antigen recognition and contribute bidirectionally to the binding activity of antibodies. These latter findings suggest that antibody selection and expression on the surface of human cells may not only 21927650 identify a population of antibodies that would be difficult or even impossible to detect in other microbial or cell-free display systems, which lack the ability to sulfonate CDR tyrosines, but may also be able to select against antibodies that may otherwise loose activity upon transferring to mammalian expression systems. In this report, we show that bivalent functional human scFvFc fusion proteins can be efficiently expressed on surface of lentiviral transduced human cells, as well as incorporated onto the surface of lentiviral particles. The displayed scFvFc antibodies can undergo post-translational CDR tyrosine sulfation. Combined magnetic bead and FACS selections on transduced human cells have provided, proof-in-principle, that 106-fold enrichments of specific antibodies can be achieved in a single, rapid selection step. In addition, scFvFc displaying human cells could be used directly in functional KPT-9274 biological screens with remarkable sensitivity. Results Optimization of scFv surface expression in mammalian cells PS11 scFv, an antibody targeting the Tat-recognition motif of cyclin T1, was chosen as a model for optimizing functional expression of scFv on the surface of mammalian cells. To gain bivalency and increase the sensitivity of detecting antigens bound to surface antibody, the PS11 scFv was expressed as an scFvFc fusion protein. For anchoring to the cell membrane, PS11 scFvFc protein
