Figure 1: Generation and functional analysis of trastuzumab-gelonin:

(A) Schematic diagram for generation of trastuzumab-gelonin conjugate. Free gelonin amine groups are modified with 2-IT on ice under a nitrogen blanket. Trastuzumab was reacted with SPDP at room temperature. After 30 minutes reaction, both reactions were stopped by buffer exchange. Then amine-modified gelonin and SPDP-linked trastuzumab were combined and allowed to react overnight on ice under a nitrogen blanket. Iodoacetamide was added to block unreacted sulfhydryl groups. Protein G purification was performed to remove excess gelonin. (B) Shown is an image of an SDS-PAGE gel with trastuzumab (Tmab), gelonin (rGel) and purified trastuzumab-gelonin (Tmab-rGel). Under non-reducing conditions, the trastuzumab-gelonin conjugate presents as several bands at a higher molecular weight than trastuzumab (~150 kDa). Under reducing conditions, the purified trastuzumab-gelonin conjugate lane has three bands that correspond to the light and heavy chains of trastuzumab in addition to free gelonin. The averaged DAR was 1.84 determined by SDS-PAGE. (C) An ELISA plate was coated with anti-gelonin antibody, incubated with PBS, trastuzumab, gelonin and trastuzumab-gelonin and subsequently incubated with an anti-human Fab AP conjugated secondary antibody. No signal is observed for PBS, trastuzumab or gelonin, whereas trastuzumab-gelonin treated wells were observed to have a high positive signal, consistent with a successful conjugation of trastuzumab to gelonin. (D) Shown is the cell viability of NCI-N87 cells treated with gelonin (circles) or trastuzumab-gelonin (squares) for 72 hours. Points represent the mean of triplicate wells with standard deviation error bars.