Figure 4.

Analysis of the specificity of affinity matured and humanized mAb hX3.12.6 by cell microarray technology. Using Retrogenix's custom cell microarray technology, mAb hX3.12.6 in scFv-Fc format was screened against 5,647 human plasma membrane proteins expressed on the surface of human HEK 293 cells. A, image of Retrogenix's custom cell microarray ZsGreen1 spotting pattern for ∼300 human plasma membrane proteins in duplicate on one of 16 slides. The array also included untransfected HEK 293 cells as a control. B, humanized scFv-Fc hX3.12.6 was screened at 20 μg/ml and was visualized using Alexa Fluor 647–conjugated goat anti-human IgG Fcγ pAbs. FcγRIIa is an expected nonspecific hit due to Fc binding. C, summary of the cell microarray screen where 20 μg/ml hX3.12.6 scFv-Fc against 5,647 human antigens arrayed in duplicate revealed ROR2 as the only specific hit. A rituximab biosimilar (1 μg/ml) was screened in parallel as control.